Compositions and methods for silencing gene expression.

(54) Title: COMPOSITIONS AND METHODS TO SILENCE GENETIC EXPRESSION. (54) Title: COMPOSITIONS AND METHODS FOR SILENCING GENE EXPRESSION.

(57) Summary

The present invention relates to a method of regulating the expression of a target gene in a seed by introducing a naked dsRNA.

(57) Abstract

A method of introducing naked dsRNA into a seed is provided. The method comprising contacting the seed with the naked dsRNA under conditions which allow penetration of the dsRNA into the seed, thereby introducing the dsRNA into the seed.

PATENT TITLE No. 360866

Headlines):

Home:

Denomination:

Classification:

AB SEEDS LID,

Hagolan Street PO Box 1061,7111001, Lod, ISRAEL

COMPOSITIONS AND METHODS TO SILENCE GENETIC EXPRESSION,

CIP:

CPC:

C12N15 / 113 C12N15 / 8201

Inventor (s):

C12N15 / 8218; C12N15 / 8266; C12N15 / 8267;

, .........., ...... ......... i i ............. and ....... .........

AMIR AVNIEL; EFRAT LIDOR-NILI; RUDY MAOR; OFIR MEIR; ORLY NOIVIRT-BRIK: OSNAT YANAI-AZULAY tional:

country

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lundamento orí tos arti or Industrial Property Law.

Number:

MX / a / 2014/014313

Validity: Twenty years

Expiration Date Issue Date: Y of n

Reference patent

In accordance with article 2 from the filing date

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May 2033 —2018 III, and 59 of the Ley di; e has

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Twenty years of non-revocable years, counted to keep the rights in force, g ..r / y »'. Vi» · ........

I and 7 ° bis 2 of the Industrial Property Law 5/1999, 01/26/2004, 06/16/2005, articles 1, 3, fraction V, subsection a), 4, 12/1999, amended on 01 / 07/2002, 15/67/2004,

Organic Institute of the Mexican Institute of t / 2007); 1st, 3rd and 5th paragraph a) of the Agreement that delegates powers to the Deputy General Directors, Coordinator, Titular Divisional Directors of the Regional Offices, Divisional Subdirectors, Departmental Coordinators and other subordinates of the Mexican Institute of Industrial Property, (DOF , 1: 5/12/1999, amended on 02/04/2000, 07/29/2004. 08/04/2004 and 09/13/2007).

This letter is signed with an advanced electronic signature (FIEL), based on articles 7 BIS 2 of the Industrial Property Law; 3rd of its Regulations, and 1 section III, 2 section V, 28 BIS and 26 TER of the Agreement establishing the ineffective for the use of the Electronic Payment and Services Portal (PÁSE) of the Mexican Institute of Industrial Property, in the procedures indicated.

Who subscribes to the present title (Official Gazette of the Federation 25/01/2006, 66/05/2009, 06/01/2016, 06/18/2010. 2I and 12 · sections I and III of the Regulation of Instit 28 / 07/2004 and 7/09/2007); articles 1, 3 '4' 5 °

Industrial property (DQF 12/27/1999, reformed

THE DIVISIONAL DIRECTOR OF PATENTS

NAHANNY CANAL REYES

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Arenal No, 550, Floor: 1, Pueblo Santa Mana Tepopan, Xochímíico, '16020, Mexico City, (55) 63340700 www.gob.rnx / irnpi

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COMPOSITIONS AND METHODS TO SILENCE THE EXPI

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FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some of its embodiments, relates to methods for introducing dsRNA into plant seeds to modulate gene expression.

With a growing world population, growing demand for food, fuel and fiber, and a climate undergoing change, agriculture faces unprecedented challenges. The development of plants with improved characteristics is highly desirable, being among the main characteristics of primary interest for farmers and seed companies the improvement in tolerance to abiotic stress, efficiency in the use of fertilizers, resistance to diseases, and productivity, among others.

Improvement in plant characteristics is typically accomplished through either genetic engineering or classical hybridization. It is highly desirable to have new methods to improve the characteristics of plants through specific genetic alterations. These include methods for gene overexpression or gene silencing. A powerful technique for specific genetic silencing &lt;

sequences is the interference of RNA (RNAi). Initially discovered for the nematode C. elegans (Fire et al 1998,

Nature, 391: 806-811), RNAi is a mechanism in which the expression of a single gene can be specifically silenced by introducing into cells a double-stranded RNA (dsRNA) homologous with the selected gene.

Inside the cell, dsRNA molecules are cut into small fragments of 21-27 nucleotides by an enzyme related to RNase III (Dicer). These fragments, called small RNA interference fragments (siRNAs), are incorporated into the RNA-induced silencer complex (RISC). After further processing, the siRNAs are transformed into single-stranded RNAs that act as guide sequences to finally cleave the target messenger RNAs.

By using RNAi to specifically silence relevant target genes, the basic characteristics of an organism can be altered. Specifically in the case of plants, it has incredible potential to make modifications that can lead to improved resistance to stress and higher crop productivity.

In plants, RNAi is typically carried out by producing transgenic plants that overexpress a DNA fragment that is transcribed to produce dsRNA. This dsRNA is then processed to obtain siRNA:

cleavage and silencing of the target genes.

The main technical limitation for this technology is that it is difficult or impossible to transform many important crop plant species, preventing the constitutive expression of the constructs that direct the production of dsRNA. On the other hand, to date, its use has been significantly limited due to the questioning of the potential ecological impacts of transgenic virus resistant plants [Tepfer, 2002, Annu.

Rev. Phytopathol. 40, 467-491].

An additional impediment to obtaining transgenic plants is attributed to the difficulty of causing transformation and regeneration events to occur in the same cell types.

Therefore, the development of a method for obtaining transformed seeds that is independent of the methods inherent in tissue culture procedures is at the forefront of research in plant molecular biology.

Additional information on the background of the technique includes:

US 20040055041 discloses the transformation of seeds using the sonication system followed by Agrobacterium infection.

Chee et al. 1989 Plant Physiol. 91: 1212-1218 discloses soybean transformation by inoculating the seedling, cotyledon nodule, and adjacent cotyledon tissues of the germinating soybean using a

Agrobacterium containing a binary vector that expresses a transgene.

More information on the background of the technique can be found in: W02011 / 001434, US

20080022423, WO 2011112570, WO 2007/080127 WO 2007/080126,

US 20060272049, US 2010068172, US 20070250947,

WO9926467, US 20030154508, WO 02/14472, US 20030150017,

US 201000154083.

SUMMARY OF THE INVENTION

In accordance with one aspect of some embodiments of the invention, a method for introducing naked dsRNA into a seed is disclosed, the method comprising contacting the seed with the naked dsRNA under conditions that allow penetration of the dsRNA into the seed, thereby introducing dsRNA into the seed.

In accordance with one aspect of some embodiments of the invention, an isolated seed comprising an exogenous naked dsRNA is disclosed, the seed being devoid of a heterologous promoter to promote expression of the dsRNA in a plant.

In accordance with one aspect of some embodiments of the invention, an isolated seed comprising an exogenous naked dsRNA is disclosed.

In accordance with one aspect of some embodiments of the invention, an isolated seed is disclosed comprising an exogenous dsRNA that is present at a similar concentration in an embryo and a seed endosperm.

In accordance with one aspect of some embodiments of the invention an isolated seed is disclosed comprising an exogenous dsRNA that is spatially distributed in an embryo and an endosperm of the plant seed in a spatial distribution that differs from the special distribution of the exogenous dsRNA in a seed derived from a transgenic plant that expresses exogenous dsRNA recombinantly.

In accordance with one aspect of some embodiments of the invention an isolated seed comprising an exogenous dsRNA is disclosed, where a ratio of concentrations of exogenous dsRNA to siRNA maturing therefrom is higher in the seed compared to a transgenic seed which recombinantly expresses exogenous dsRNA.

In accordance with one aspect of some embodiments of the invention, a seed isolated from an exogenous dsRNA is disclosed, where the plant seed is devoid of a heterologous promoter to promote expression of the exogenous dsRNA, where the special distribution of the exogenous dsRNA and / or the siRNA maturing therefrom is altered in the seed compared to that in a transgenic seed that expresses the exogenous dsRNA recombinantly.

In accordance with one aspect of some embodiments of the invention, a plant or part of a plant is disclosed which comprises a naked exogenous dsRNA and which is devoid of a heterologous promoter to promote expression of the dsRNA in the plant.

In accordance with one aspect of some embodiments of the invention, a seed storage device is disclosed comprising a plurality of the seeds.

In accordance with one aspect of some embodiments of the invention a seeded field is disclosed comprising a plurality of any of the seeds.

According to some embodiments of the invention, the method also comprises drying said seed after said contacting.

In accordance with some embodiments of the invention, the method also comprises cultivating said plant under abiotic or biotic stress after said generator.

In accordance with some embodiments of the invention, naked dsRNA is designed to down-regulate the expression of a plant gene.

In accordance with one aspect of some embodiments of the invention, a method of producing a plant is disclosed, the method comprising:

(a) provide any of the seeds; and (b) germinate the seed in order to produce the plant.

In accordance with one aspect of some embodiments of the invention, a method for modulating gene expression is disclosed, the method comprising:

(a) contacting a seed of a plant with naked dsRNA, under conditions that allow penetration of the dsRNA into the seed, thereby introducing the dsRNA into the seed; and optionally (b) generate a plant from seed.

In accordance with some embodiments of the invention, the naked dsRNA is designed in such a way that it downregulates the expression of a plant gene.

In accordance with some embodiments of the invention, naked dsRNA is designed in such a way that it downregulates the expression of a gene for a viral pathogen.

According to some embodiments, penetration is performed in an endosperm and alternatively or additionally, a seed embryo.

In accordance with some embodiments of the invention, naked dsRNA is not integrated into the seed genome.

According to some embodiments of the invention, the conditions generate the presence of dsRNA in the plant for at least 10 days after germination.

In accordance with one aspect of some embodiments of the invention a method is disclosed for inhibiting the expression of a target gene in a plant virus, the method comprising providing the plant virus with the plant or part of a plant, thereby way by inhibiting the expression of a target gene in the plant virus.

In accordance with some embodiments of the invention, the method also comprises observing the decreased infectivity or replicability of the viral pathogen after delivery.

In accordance with one aspect of some embodiments of the invention a kit for introducing naked dsRNA into seeds is disclosed comprising;

(i) naked dsRNA; and (ii) a conditioning solution.

In accordance with some embodiments of the invention, the naked dsRNA and the conditioning solutions contained in separate containers.

In accordance with some embodiments of the invention, the dsRNA comprises siRNA.

In accordance with some embodiments of the invention, the dsRNA comprises siRNA and dsRNA.

In accordance with some embodiments of the invention, contacting is carried out by inoculating the seed with the dsRNA.

In accordance with some embodiments of the invention, the method also comprises conditioning the seed prior to contacting.

According to some embodiments of the invention, the conditioning is carried out by:

(i) wash the seed prior to contacting; and (ii) drying the seed after step (i).

According to some embodiments of the invention, the washing is carried out in the presence of doubly deionized water.

According to some embodiments of the invention, washing is carried out for 2-6 hours.

According to some embodiments of the invention, washing is carried out at 4-28 ° C.

In accordance with some embodiments of the invention, drying is carried out at 25-30 ° C for 10 In accordance with some embodiments of the invention, contacting is carried out in the presence of the naked dsRNA at a final concentration of 0.001- 100 pg / pl.

In accordance with some embodiments of the invention, contacting is carried out in the presence of the naked dsRNA at a final concentration of 0.001-0.5 pg / μΐ.

In accordance with some embodiments of the invention, the method also comprises treating the seed with an agent selected from the group consisting of a pesticide, a fungicide, an insecticide, a fertilizer, a coating agent and a coloring agent after contacting .

In accordance with some embodiments of the invention, the treatment comprises coating the seed with the agent.

In accordance with some embodiments of the invention, the seed is free of an agent selected from the group consisting of a pesticide, a fungicide, an insecticide, a fertilizer, a coating agent, and a coloring agent.

According to some embodiments of the invention, dsRNA is down-regulating the expression of a coding gene.

In accordance with some embodiments of the invention, dsRNA is down-regulating the coding expression.

In accordance with some embodiments of the invention, the seed is from the Viridiplantae superfamily.

In accordance with some embodiments of the invention, the conditions allow for the accumulation of dsRNA in the endosperm and alternatively or additionally the seed embryo.

According to some embodiments of the invention, a concentration of the naked dsRNA is adjusted according to a parameter selected from the group consisting of, seed size, seed weight, seed volume, seed surface area, density of the seed and seed permeability.

According to some embodiments of the invention, the contacting is carried out prior to the interruption of the seed dormancy and the emergence of the embryo.

In accordance with some embodiments of the invention, the seed is a conditioned seed.

In accordance with some embodiments of the invention, the seed or plant comprises RNA-dependent RNA polymerase activity for amplification of dsRNA expression.

In accordance with some embodiments of the invention, the seed is a hybrid seed.

In accordance with one aspect of some embodiments of the invention, a seed obtainable in accordance with the methods described herein is disclosed.

Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning commonly understood by a professional skilled in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In the event of a conflict, the patent specification, including definitions, shall prevail. Furthermore, the materials, methods and examples are illustrative only and are not necessarily to be construed as limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing made in colors. Copies of this patent publication or patent application with color drawing (s) are available from the Office upon request and after payment of the required amount.

Some embodiments of the invention are described herein, by way of example only, with reference to the accompanying drawings. With regard specifically to the drawings in detail, it is noted that the particular data is provided by way of example and for the purpose of providing an illustrated analysis of the embodiments of the invention. In this regard, the description provided with the drawings illustrates to the person skilled in the art how the embodiments of the invention can be practiced.

In the drawings:

FIGs. 1A-C show the stability of dsRNA for CGMMV in rice seedlings up to 3 weeks after germination. Figure 1A: Identification of dsRNA by RT-PCR, 1 week after germination. Upper panel: L 100 base pair DNA ladder, lanes 1-6: untreated control seeds, 7-16: treated seeds, lane 17:

negative control (mix) and path 18: c &lt;

(plasmid). The lower panel serves as a positive control for cDNA quality, PCR for the constitutive gene tubulin. Figure IB: Identification of dsRNA by PCR, 2 weeks after germination. Upper panel: L DNA ladder, 1-2 control seeds, 3-12 dsRNA treated seeds, 13 - positive control (plasmid), 14-15 negative controls (DDW). The lower panel shows the results for the constitutive tubulin gene. Figure 1C: dsRNA stability 3 weeks after germination. Upper panel: L DNA ladder, 1-4 control seeds, 5-9 dsRNA treated seeds, 11 - positive control (plasmid) and 12 is negative control (mix). The lower panel shows the results for the constitutive tubulin gene.

FIGs. 2A-B show the identification of CGMMV dsRNA by RT-PCR in tomato and sorghum seedlings, 10 days and 4 weeks after germination, respectively. Figure 2A: CGMMV dsRNA is stable in tomato plantins 10 days after germination. L- DNA ladder, 1-4: control seeds, 5-17:

seeds treated with dsRNA, 18-19: negative controls (DDW), 20 - PCR positive control (plasmid). Figure 2B: CGMMV dsRNA is stable in sorghum plantins 4 weeks after germination. 1-2: control seeds, 3-6: treated seeds, 7

- negative control (DDW).

FIG. 3 shows that the derived dsRNA

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integrates into the genome of treated rice seeds 5 weeks after germination. Three different DNA PCR reactions were carried out: (1) tubulin PCR (lanes 1-5); 1-2 are control plants, 3-4 are plants treated with dsRNA, 5 is a negative control (ddW), (2)

First PCR for DNA CGMMV (lanes 6-10); 6-7 are control plants, 8-9 are dsRNA-treated plants, 10 is a positive control (plasmid carrying the CGMMV sequence), (3)

Second PCR for DNA CGMMV (lanes 11-16); 11-12 are control plants, 13-14 are dsRNA-treated plants, 15 is a negative control (ddW), and 16 is a positive control (plasmid carrying the CGMMV sequence). L - DNA ladder from

100 Base pairs.

FIGs. 4A-C show the stability of GUS dsRNA in maize seedlings by RT-PCR, 7 and 15 days after germination. Figure 4A: GUS dsRNA is stable in shoots of maize seedlings 1 week after germination. L is a DNA ladder, 1-5 are control plants, 6-8 and 11-18 are plants treated with dsRNA, 9 is a negative control (ddW) and is a positive control (plasmid). Figure 4B: GUS dsRNA is stable in roots of maize seedlings 1 week after germination. 1-5 are control plants, 6-10 are plants treated with dsRNA, 11 is a negative control (ddW), and 12 is a positive control (plasmid). Figure 4C:

stable on roots of corn seedlings 15 days after germination. L - DNA ladder, 1 is a positive control (plasmid), 2 is a control plant, 3 is a negative control (ddW), and 4 is a dsRNA treated plant.

FIGs. 5A-B show that GUS dsRNA does not integrate into the genome of treated corn seeds 1 week after germination. The upper gel shows a DNA PCR in the GUS gene: 1-3 are control plants, 4-6 are plants treated with dsRNA and 7 is a positive control (plasmid). The lower gel is a positive control for DNA extraction showing a PCR of DNA in the ubiquitin gene: 1-3 are control plants, 4-6 are plants treated with dsRNA and 7 is a negative control (ddW).

FIG. 6 shows the siGLO gel electrophoresis analysis before the experiment. L - 100 base pair ladder, 1 - 5 µΐ of solution 2 µ solución of siGLO, 2 - 15 µΐ of solution 2 µΜ of siGLO, 3 - 30 µΐ of solution 2 µΜ of siGLO.

Bands corresponding to the expected size of 20-24 base pairs of fluorescent siRNA molecules can be observed.

FIGs. 7A-C are images showing the penetration of fluorescent siRNA molecules into various plant seeds. Seeds treated with fluorescent siRNA are s

t show in the two images on the left,

<td>control no</td><td>treated are shown in</td><td>two images of</td><td>the</td>

<td>right. The</td><td>fluorescent images are</td><td colspan="2">took 24 hours</td>

<td>after the</td><td>seed treatment</td><td>with siRNA to</td><td>a</td>

<td>5 concentration</td><td>end of 2 μΜ. Figure</td><td>7A - seeds</td><td>of</td>

Arabidopsis observed under a target with multiplication of

10X, Figure 7B - rice seeds observed under a 5X multiplication objective, Figure 7C - tomato seeds observed under a 5X multiplication objective.

FIGs. 8A-C show rice seeds 24 hours after treatment with siGLO dsRNA. The figures show treated seeds to the left of each image next to the untreated control seeds, at various levels of magnification.

FIGs. 9A-F are light and fluorescence images of sectioned rice seeds 48 hours after treatment with siGLO dsRNA. Century-treated and control rice seeds were sectioned to visualize the interior distribution of the fluorescent dsRNA using a fluorescent binocular. Figure 9A - Light mode of 4 rice seeds: 2 seeds treated with century down and 2 untreated seeds above. Figure 9B - Fluorescence image of the seeds shown in Figure

9A. Figure 9C - Increased fluorescence image of a rice seed treated with siGLO (shown below at

Left MPW in Figures 9A and B). Figur. · Ϊ *

INDUSTRIAL ^ ιίβΠ * '' Increased fluorescence of an untreated rice seed. Figure 9E - Augmented fluorescence image of a century treated seed showing a more absolute staining pattern compared to the seed shown in Figure 9C. Figure 9F - Increased fluorescence image of an untreated seed for comparison with Figure

9E.

FIGs. 10A-E are fluorescence images of sectioned tomato seeds 48 hours after treatment with siGLO dsRNA. SiGLO-treated and control tomato seeds were sectioned to visualize the interior distribution of the fluorescent dsRNA using a fluorescent binocular. Figure 10A - Fluorescence image of the outer surface of an untreated tomato seed. Figure 10B - Fluorescence image of the outer surface of a tomato seed treated with siGLO. Figure

10C - Enlarged image of a section (see box in the image of Figure 10B) of the outer surface of a tomato seed treated with siGLO. Figure 10D - Image of

<td>fluorescence</td><td>of</td><td>the</td><td>surface</td><td>inside</td><td>of a</td><td>seed</td><td>of</td>

<td>tomato without</td><td colspan="2">try</td><td>sectioned.</td><td>Figure</td><td>10E -</td><td>Image</td><td>of</td>

<td>fluorescence</td><td>of</td><td>the</td><td>surface</td><td>inside</td><td>of a</td><td>seed</td><td>of</td>

tomato treated with sectioned siGLO, the outline of the embryo can be clearly seen.

FIGs. 11A-H are fluorescent images of cucumber seeds sectioned 48 hours after treatment with siGLO dsRNA. Century-treated and control cucumber seeds were sectioned to visualize the internal distribution of the fluorescent dsRNA using a fluorescent binocular. Figure 11A - Fluorescence image of the inner surface of a cucumber seed treated with siGLO. Figure 11B - Fluorescence image of the inner surface of an untreated cucumber seed. Figures 11C-E Enlarged images of the anterior, middle and posterior sections (respectively) of the inner surface of a cucumber seed treated with siGLO. The embryonic contour can be visualized in the middle section (Figure 11D).

Figures 11F-H - Enlarged images of the anterior, middle, and posterior sections (respectively) of the inner surface of an untreated cucumber seed.

FIGs. 12A-D are fluorescent images of sectioned seeds of various plant species, including beans, tomato, sorghum and wheat, 48 hours after treatment with siGLO dsRNA. The siGLO-treated and control seeds were sectioned to visualize the interior distribution of the fluorescent dsRNA using a fluorescent binocular.

Light images were also taken for each seed and displayed alongside the reference fluorescent image. The dsRNA treated seeds are shown in the two images on the left and the untreated control seeds are shown in the two images on the right. Figure

12A shows two examples of dsRNA-treated and control bean seeds. Figure 12B shows tomato seeds treated with dsRNA and control. Figure 12C shows sorghum seeds treated with dsRNA and control. Figure 12D shows dsRNA-treated and control wheat seeds.

FIG. 13 shows the course of time in the results for century treatment of rice seeds. The effect of incubation time with siGLO dsRNA on fluorescence intensity was tested, indicating the amount and quality of dsRNA penetration. Images of the control seeds that remained untreated (1) were obtained together with the seeds treated with siGLO dsRNA during four different incubation times; 10 min (2), 3.5 hours (3), 5.5 hours (4), and 24 hours (5).

FIGs. 14A-D show silencing of the PDS-1 gene in rice using a dsRNA / siRNA mixture. Figure 14A Analysis of PDS-1 dsRNA on 2% agarose gel. From left to right: 100 base pairs, dsDNA and product dsRNA 1, dsDNA and dsRNA after DNase turbo, dsDNA and dsRNA after DNase turbo and RNaselII, space, and the same for product 2. Figure 14B -Image of the germinated 5 days after treatment, left control. Figure 14C - Image of rice seeds germinated 7 days after treatment, control below.

Figure 14D - Image of rice seeds planted 14 days after treatment, the red x represents dead seedling, control on the left.

FIG. 15 is a photograph showing that the PDS-1 silencing treatment results in chlorophyll bleaching and growth inhibition.

Control plants (on the left) show normal coloration and growth rate while PDS-silencing plants (on the right) appear paler in color and smaller, indicating signs of chlorophyll bleaching and growth inhibition 30 days after treatment.

FIGs. 16A-C show PDS-1 expression levels determined by real-time PCR. Figure 16A is an image of the rice seeds germinated 7 days after treatment, control below. Figure 16B - Image of rice seeds planted 5 weeks after treatment, the control plant is on the left and has a darker green color compared to that of the PDS-1 silencing plant. Figure 16C - RNA was extracted from control and i-silenced plants. 'verified PDS-1 expression levels by real-time PCR. UBQ5 expression levels served as normalizers and PDS-1 expression levels in control plants served as calibrators, and were assigned the value of 1.

FIG. 17 shows that there are no phenotypic differences in the root development of control germinated seeds (left) and treated with Hap2e dsRNA (right) 5 days after treatment. This shows that the seed treatment did not have negative effects on seed germination and initial development.

FIGs. 18A-C show success in achieving dsRNA-derived changes in Hap2e expression (miR169 target gene) by RT-PCR assay of 3 different primer sets on RNA extracted from rice seedling leaves 5 days later of germination. Figure 18A - RTPCR using a first set of primers in which both primers were located within the dsRNA molecule itself. Figure 18B - Second set of primers in which the 5 'part of the forward primer is in the dsRNA and the 3' part is in the mRNA, the reverse primer was located outside the dsRNA. Figure 18C - Third set of primers in which the primers were located outside the dsRNA molecule on the Hap2e gene. There were c results obtained for the three primer sets, which provided similar qualitative data. The average change (number of times) in the Hap2e expression of 5 control plants was used as a reference and plotted by assigning a value of 1. The treated samples were plotted separately and their respective changes in gene expression were calculated. Hap2e target in relation to expression in control plants.

Five of nine dsRNA-treated plants showed down-regulation of Hap2e compared to control (5/9 of treated plants, 55.5% efficiency).

FIG. 19 shows the dsRNA-derived changes in Hap2e expression (miRl69 target gene) detected by RTPCR, using the third set of primers in Figure

18C above, on RNA extracted from rice seedling leaves 7 days after germination. The average in the change (number of times) in the expression of

Hap2e in 4 control plants was used as a reference threshold and was plotted assigning a value of 1. The treated samples were plotted separately and their respective changes (number of times) in the expression of the Hap2e target gene were calculated in relation to the expression in control plants. Six treated plants were selected from a total of 16 plants and juni control were graphed, with 4 treated plants showing down regulation of Hap2e of approximately 50% and more compared to the control (4/16 is equivalent to an efficiency of 25

%), including complete silencing in a plant.

FIG. 20 shows the down regulation of Hap2e dsRNA (miR169 target gene) detected by RT-PCR, on RNA extracted from leaves of rice seedlings 18 days after germination. The average change (number of times) in Hap2e expression in 4 control plants was used as the reference threshold and was plotted with a value of 1 (shown by a red bar). The treated samples were plotted separately and their respective changes (number of times) in the expression of the Hap2e target gene were calculated in relation to the expression in control plants.

Eleven of 16 treated plants showed some down regulation of Hap2e (shown by blue bars), and 8 of them down regulation of Hap2e of more than 25% compared to the control (8/16 equals an efficiency of 50% ).

FIG. 21 shows the RT-PCR results on the RNA extracted from control corn seeds and treated with NFY dsRNA 10 days after germination. The expression level of the NFY target gene in the control plants was averaged and assigned a value of 1 as a reference for comparison with the value observed in the j with dsRNA. Four of the 8 treated plants shown in the graph showed down regulation of 50% NFY and more (4/8, 50% efficiency).

FIG. 22 shows the RT-PCR results on the RNA extracted from control tomato seeds and treated with NFY dsRNA 3 weeks after germination. The expression level of NFY target gene in 8 control plants (shown in red) was averaged and assigned a value of 1 as a reference for comparison with the value observed in plants treated with dsRNA (shown by blue bars). Down regulation of 40% and more was achieved with 23% efficiency (6 out of 26) in the treated plants in relation to the control plants.

FIGs. 23A-B show the height distribution of control and NFY dsRNA-treated tomato plants 55 days after inoculation. Figure 23A shows the height distribution of control plants (blue bars) and Figure 23B shows the height distribution of 20 treated plants (yellow bars).

FIG. 24 shows the main phenotypic differences between control tomato plants and those treated with NFY dsRNA 55 days after inoculation. Control plants are shown on the left and treated plants on the right of each image. A challenge is observed:

MPl

GAVE! INDUSTBIAL INJURY

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in smaller treated plants compared to control plants. The upper figure is a side view and the lower figure is a top view of the same plants.

FIG. 25 shows the RT-PCR results on the RNA extracted from control corn seeds and treated with dsRNA NAC 10 days after germination. The expression level of the NAC target gene in the control plants was averaged and assigned a value of 1 as a reference for comparison with that observed in the plants treated with dsRNA. All 14 plants treated with dsRNA showed downregulation of the NAC gene, with one plant showing complete silencing of the gene (# 8).

FIGs. 26A-B show the results of RT-PCR, using cDNA prepared either with random primers (Figure 2 6A) or oligo-dT (Figure 2 6B), on RNA extracted from control rice seeds and treated with ARF-8 dsRNA 18 days after germination. The expression level of the target ARF-8 gene in 9-10 control plants was averaged and assigned a value of 1 as a reference for comparison with that observed in plants treated with dsRNA. In the

Figure 26A, four plants showed down regulation of the ARF-8 gene of more than 26%, and in Figure 26B, seven plants showed down regulation of more than 50%.

FIG. 27 shows that there are no phenotypic differences in the germinated rice seeds of control (top row) and treated with SPL17 dsRNA (bottom row) 5 days after treatment.

FIGs. 28A-B show the results of the RT-PCR assay of 2 different sets of primers on the RNA extracted from the leaves of control rice seedlings and treated with SPL17 (miR156 target gene) dsRNA 5 days after germination. Figure 28A - RT-PCR using the first set of primers in which both primers were located within the dsRNA molecule itself. Figure 28B Second set of primers in which the primers were located on SPL17 ORF, outside the dsRNA molecule. The average change (number of times) in the expression of SPL17 in 5 control plants was used as the reference threshold and was plotted assigning the value of 1.

The treated samples were plotted separately and their respective changes (number of times) in the expression of the target gene SPL17 were calculated in relation to the expression in control plants. It is suggested that primer set # 1 will amplify both the endogenous sequence and dsRNA molecules introduced into the plant, while primer set # 2 will only amplify the sequence in

FIG. 29 shows the RT-PCR results on RNA extracted from leaves of control rice plants and treated with SPL17 (miR156 target gene) dsRNA 14 weeks after germination. The average change (number of times) in the expression of SPL17 in 4 control plants was used as the reference threshold and was plotted assigning the value of 1.

The treated samples were plotted separately and their respective changes in the expression of the target gene SPL17 in relation to the expression in control plants were calculated. Down regulation of up to 90% of SPL17 gene expression was observed. Seven of 10 dsRNA-treated plants showed SPL17 down-regulation of over 65% (7/10, efficiency 70%).

FIGs. 30A-B show the RT-PCR results on the

RNA extracted from control tomato seed leaves and treated with ARF8 (miRl67 target gene) dsRNA in plants 3 weeks and 8 weeks (Figure 30A and 30B, respectively) after germination. The expression level of target ARF-8 gene 20 in 8 or 20 control plants (Figure 30A and 30B, respectively) was averaged and assigned a value of 1 as a reference for comparison with that observed in plants treated with dsRNA Figure 30A - The change (number of times) in the expression of ARF8 in control tomato plants (shown by a red bar) dsRNA (shown by blue bars) 3 weeks after germination was plotted for each individual plant to demonstrate the large variation in ARF-8 expression in plants treated with dsRNA. Five of the 8 treated plants shown in the graph showed down regulation of ARF-8 of more than 40% (5/8, efficiency of 62.5%), Figure 30B - Same as in Figure 30A for tomato plants 8 weeks after germination.

Six of 9 treated plants shown in the graph showed down regulation of ARF-8 of more than 30% (6/9, efficiency of 66.7%).

FIGs. 31A-D show the specific distribution of heights in control tomato plants (blue bars) and treated with ARF8 dsRNA (dark red bars) 55 (Figure

31A), 62 (Figure 31B) and 72 days (Figure 31C) after treatment. Figure 31D shows the average height of the control plants compared to the treated plants 62 days after treatment.

FIGs. 32A-B show the phenotypic differences between control plants and plants treated with ARF8 dsRNA, (Figure 32A) and 72 days (Figure 32B) after seed treatment. Control plants are shown on the left and treated plants on the right of each image. In Figure 32A, the side view image and the bottom image is a top view of the same plants. The treated plants are shorter and more branched compared to control plants of the same age.

FIGs. 33A-B show the RT-PCR results on the

RNA extracted from leaves of control tomato plants and treated with FW2.2 dsRNA 9 weeks after germination.

Figure 33A shows the change (number of times) in FW2.2 expression in control plants (shown by red bars) and treated with dsRNA (shown by blue bars), which is plotted for each individual plant to demonstrate the variation in the expression level of the FW2.2 gene in the two groups of plants. Figure

33B shows the average expression of FW2.2 in control plants (red bar) compared to the treated plants (blue bar). Downregulation in the expression level of the FW2.2 gene is evident in treated plants compared to control plants.

FIG. 34 shows that there are no phenotypic differences between the control plants and those treated with FW2.2 dsRNA 72 days after treatment. Control plants (on the left) and dsRNA-treated plants (on the right) have the same average height and similar physical properties.

FIGs. 35A-B show more extensive and developed root systems in rice seedlings grown from rice seeds treated against the Della gene (Figure 35B) compared to control plants (Figure 35A).

FIGs. 36A-B show more extensive and developed root and shoot systems in rice seedlings grown from rice seeds treated against the NRR gene (Figure 36B) compared to control plants (Figure

36A) when the seedlings are grown in nitrogen-free medium.

FIGs. 37A-C show the results of RT-PCR (using oligo dT) on RNA extracted from control rice seed leaves and seeds treated with a mixture containing dsRNA molecules for down-regulation of three endogenous genes: Hap2e , Della and SQS, 18 days after germination. The expression level of each individual gene was averaged over 8 control plants (shown by a red bar) and used as the reference threshold (assigned a value of 1) for expression in treated plants. The treated samples (shown by blue bars) were plotted separately and their respective changes in the level of expression for each target gene were calculated in relation to the expression in control plants. Figure 37A - RT-PCR results of ex Hap2e gene, Figure 37B - RT-PCR expression results for the Della gene, Figure 37C - RT-PCR expression results for the SQS gene. Downregulation of all genes is evident in dsRNA-treated plants, ranging from 30% to 100% (complete silencing) of reduction in expression.

FIGs. 38A-D are confocal images showing the penetration of fluorescent siRNA molecules (red, siGlo) into tomato seeds. The nuclei of the cells were stained with Hoechst 33342 (blue). The seeds treated with fluorescent siRNA are shown in the Figures

38A and 38C, while the untreated control seeds are shown in Figures 38B and 38D. Figures

38A-B are fluorescent images, while Figures

38C-D are transmitted light images. The images were taken 24 hours after treatment of the seeds with siRNA at a final concentration of 1 µΜ.

FIGs. 39A-D are graphs of HPLC analyzes of SPL (SEQ ID NO: 126, Figures 39A and B) and GUS (SEQ ID NO: 21,

Figures 39C and D) dsRNAs before (Figures 39A and C) and after (Figures B and D) of seed treatment. Arrows indicate ssRNA and dsRNA.

FIGs. 40A-B are bar graphs showing real-time PCR analysis of the expression of 17-day-old tomato plants germinated from seeds treated with 50 pg / ml dsRNA for 24 hours. Figure

40A shows the change (number of times) in the expression of

SPL mRNA after SPL treatment (blue bars, SEQ

ID NO: 126), GUS (red bars, SEQ ID NO: 21) and FW2.2 (green bars, SEQ ID NO: 114) dsRNAs. Each bar represents a plant. The expression values ​​for the individual plants were normalized to the median expression of all the plants treated with GUS dsRNA.

Figure 40B shows the values ​​of the medians corresponding to the data shown in Figure

40A. p value = 0.02 for the difference in the level of expression of SPL in relation to the GUS control and p value = 0.07 for the difference in the level of expression of SPL in relation to the FW2.2 control. Error bars represent one standard deviation of the data.

FIGs. 41A-B are bar graphs showing real-time PCR analyzes of SPL mRNA expression in 18-day-old tomato plants germinated from seeds treated with 50 pg / ml dsRNA for 6 hours (dsRNAs are the of Figures 40A-B). Figure 41A shows the change (number of times) in SPL mRNA expression after SPL dsRNA treatment, while GUS dsRNA treatment was used as the baseline &lt;.

barrra represents a plant. The expression values ​​for the individual plants were normalized to the median expression of all the plants treated with GUS dsRNA.

Figure 4 IB shows the median values ​​corresponding to the data shown in Figure

41A. The change in expression in relation to the control group was significant (p value = 0.012). Error bars represent one standard deviation of the data.

FIGs. 42A-B are bar graphs showing real-time PCR analyzes of SPL mRNA expression in 18-day-old tomato plants germinated from seeds treated with 50 pg / ml dsRNA for 2 hours (dsRNAs are the of Figures 40A-B). Figure 42A shows the change (number of times) in SPL mRNA expression after SPL dsRNA treatment, while GUS dsRNA treatment was used as baseline contol. Each bar represents a plant. The expression values ​​for the individual plants were normalized to the median expression of all the plants treated with GUS dsRNA.

Figure 42B shows the values ​​of the medians corresponding to the data shown in Figure

42A. The change in expression in relation to the control group was significant (p value = 0.0015). Error bars represent a standard deviation of 1

FIGs. 43A-B are bar graphs showing real-time PCR analysis of SPL mRNA expression in 18-day-old tomato plants germinated from seeds treated with 50 pg / ml dsRNA for 10 minutes (dsRNAs are the of Figures 40A-B). Figure 43A shows the change (number of times) in SPL mRNA expression after SPL dsRNA treatment, while GUS dsRNA treatment was used as baseline contol. Each bar represents a plant. The expression values ​​for the individual plants were normalized to the median expression of all the plants treated with GUS dsRNA.

Figure 43B shows the values ​​of the medians corresponding to the data shown in Figure

43A. The change in expression in relation to the control group was significant (p value = 0.0035). Error bars represent one standard deviation of the data.

FIGs. 44A-B are bar graphs showing real-time PCR analysis of SPL mRNA expression in 13-day-old tomato plants germinated from seeds dipped in 50 pg / ml dsRNA solution (dsRNAs are those of Figures 40A-B). Figure 44A shows the change (number of times) in SPL mRNA expression after SPL dsRNA treatment, while GUS dsRNA treatment was used as baseline i

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barrra represents a plant. The expression values ​​for the individual plants were normalized to the median expression of all the plants treated with GUS dsRNA.

Figure 44B shows the values ​​of the medians corresponding to the data shown in Figure

44A. The change in expression in relation to the control group was significant (p value = 0.017). Error bars represent one standard deviation of the data.

FIGs. 45A-B are bar graphs showing real-time PCR analysis of SPL mRNA expression in 17-day-old tomato plants germinated from seeds treated with 25 pg / ml dsRNA for 24 hours (dsRNAs are the of Figures 40A-B). Figure 45A shows the change (number of times) in the expression of SPL mRNA after treatment with SPL dsRNA, while treatment with GUS dsRNA was used as baseline contol. Each bar represents a plant. The expression values ​​for the individual plants were normalized to the median of the expression of all the plants treated with GUS dsRNA.

Figure 45B shows the median values ​​corresponding to the data shown in Figure

45A. The change in expression in relation to the control group was significant (p value = 0.049). Error bars represent a standard deviation of 1

FIGs. 46A-B are bar graphs showing real-time PCR analyzes of SPL mRNA expression in 18-day-old tomato plants germinated from seeds treated with 25 pg / ml dsRNA for 2 hours (dsRNAs are the of Figures 40A-B). Figure 46A shows the change (number of times) in the expression of SPL mRNA after treatment with SPL dsRNA, while treatment with GUS dsRNA was used as baseline contol. Each bar represents a plant. The expression values ​​for the individual plants were normalized to the median expression of all the plants treated with GUS dsRNA.

Figure 46B shows the median values ​​corresponding to the data shown in Figure

46A. The change in expression in relation to the control group was significant (p value = 0.0062). Error bars represent one standard deviation of the data.

FIGs. 47Ά-Β are bar graphs showing real-time PCR analyzes of SPL mRNA expression in 20 18-day-old tomato plants germinated from seeds treated with 25 pg / ml dsRNA for 10 minutes (dsRNAs are those of Figures 40A-B). Figure 47A shows the change (number of times) in SPL mRNA expression after SPL dsRNA treatment, while GUS dsRNA treatment was used as baseline &lt;

barrra represents a plant. The expression values ​​for the individual plants were normalized to the median expression of all the plants treated with GUS dsRNA.

Figure 47B shows the values ​​of the medians corresponding to the data shown in Figure

47A. Error bars represent one standard deviation of the data.

FIGs. 48A-B are bar graphs showing real-time PCR analyzes of SPL mRNA expression in 17-day-old tomato plants germinated from seeds treated with 1 pg / ml dsRNA for 24 hours (dsRNAs are the of Figures 40A-B). Figure 48A shows the change (number of times) in the expression of SPL mRNA after treatment with SPL dsRNA, while treatment with GUS dsRNA was used as baseline contol. Each bar represents a plant. The expression values ​​for the individual plants were normalized to the median of the

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48A. Error bars represent one standard deviation of the data.

FIGs. 49A-B are bar graphs showing the

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real-time PCR analysis of expression

INDUSTRIAL 18-day-old tomato plants germinated from seeds treated with 1 pg / ml dsRNA for 2 hours (the dsRNAs are those of Figures 40A-B). Figure 49A shows the change (number of times) in the expression of SPL mRNA after treatment with SPL dsRNA, while GUS dsRNA treatment was used as baseline contol. Each bar represents a plant. The expression values ​​for the individual plants were normalized to the median expression of all the plants treated with GUS dsRNA. Figure 49B shows the values ​​of the medians corresponding to the data shown in Figure

49A. Error bars represent one standard deviation of the data.

FIGs. 50A-B are bar graphs showing real-time PCR analysis of SPL mRNA expression in 18-day-old tomato plants germinated from seeds treated with 1 pg / ml dsRNA for 10 minutes (dsRNAs are the of Figures 40A-B). Figure 50A shows the change (number of times) in SPL mRNA expression after SPL dsRNA treatment, while GUS dsRNA treatment was used as baseline contol. Each bar represents a plant. Expression values ​​for individual plants were normalized to the median expression of all treated plants.

Figure 50B shows the values ​​of the medians corresponding to the data shown in Figure

50A. Error bars represent one standard deviation of the data.

FIGs. 51A-B are bar graphs showing real-time PCR analyzes of SPL mRNA expression in 13-day-old tomato plants germinated from seeds treated with 50 pg / ml siRNA for 2 hours (dsRNAs are the of Figures 40A-B). Figure 51A shows the change (number of times) in the expression of SPL mRNA after treatment with SPL siRNA while treatment with GUS siRNA was used as baseline contol. The expression values ​​for the individual plants were normalized to the median expression of all the plants treated with GUS siRNA. Figure 51B shows the values ​​of the medians corresponding to the data shown in Figure 51A. The change in expression in relation to the control group had a p value of 0.12. Error bars represent one standard deviation of the data.

FIG. 52 is a graph showing HPLC analyzes of a mixture of FW2.2 ssRNA / dsRNA (SEQ ID NO: 114) before treatment. Arrows indicate the ssRNA and dsRNA fractions.

FIGs. 53A-B are bar graphs showing real-time PCR analyzes of FW2.2 mRNA expression in 17-day-old tomato plants germinated from seeds treated with 50 pg / ml dsRNA for 24 hours. Figure

53A shows the change (number of times) in the expression of

FW2.2 mRNA after treatment with FW2.2 dsRNA (SEQ ID

NO: 114), while treatment with GUS dsRNA (SEQ ID NO: 21) was used as contol baseline. Each bar represents a plant. The expression values ​​for the individual plants were normalized to the median expression of all the plants treated with GUS dsRNA.

Figure 53B shows the median values ​​corresponding to the data shown in Figure

53A. The change in the expression of FW2.2 in relation to the control group was significant (p value = 0.024). Error bars represent one standard deviation of the data.

FIGs. 54A-B are bar graphs showing real-time PCR analysis of FW2.2 mRNA expression in 18-day-old tomato plants germinated from seeds treated with 50 pg / ml dsRNA for 6 hours (the dsRNAs are those of Figures 53A-B). Figure 54A shows the change (number of times) in expression of FW2.2 mRNA after treatment with FW2.2 dsRNA, while treatment with GUS dsRNA was used as. Xjg contolled. Each bar represents a plant. The expression values ​​for the individual plants were normalized to the median expression of all the plants treated with GUS dsRNA. Figure 54B shows the values ​​of the medians corresponding to the data shown in Figure

54A. The change in FW2.2 expression in relation to the control group had a p value of 0.1. Error bars represent one standard deviation of the data.

FIGs. 55A-B are bar graphs showing real-time PCR analyzes of FW2.2 mRNA expression in 18-day-old tomato plants germinated from seeds treated with 50 pg / ml dsRNA for 2 hours (the dsRNAs are those of Figures 53A-B). Figure 55A shows the change (number of times) in FW2.2 mRNA expression after treatment with FW2.2 dsRNA, while GUS dsRNA treatment was used as baseline contol. Each bar represents a plant. The expression values ​​for the individual plants were normalized to the median expression of all the plants treated with GUS dsRNA. Figure 55B shows the median values ​​corresponding to the data shown in Figure 55A. The change in FW2.2 expression in relation to the control group was significant (p value = 0.049). Error bars represent a standard deviation of 1

FIGs. 56A-B are bar graphs showing real-time PCR analyzes of DELLA mRNA expression in 13-day-old rice plants germinated from seeds treated with 142 pg / ml dsRNA for 24 hours.

Figure 56A shows the change (number of times) in expression of DELLA mRNA expression after treatment with DELLA dsRNA (SEQ ID NO: 123), while treatment with GUS dsRNA (SEQ ID NO: 21) was used as contol baseline. Each bar represents a plant. The expression values ​​for the individual plants were normalized to the median expression of all the plants treated with GUS dsRNA. Figure 56B shows the median values ​​corresponding to the data shown in Figure 56A. The change in DELLA expression in relation to the control group was significant (p value = 6.28x10-4). Error bars represent one standard deviation of the data.

FIG. 57 is an image showing germinated wheat seeds three days after treatment. Top control seeds treated with O.lmM EDTA, medium - control seeds treated with GUS dsRNA (SEQ ID NO: 21), bottom seeds treated with PDS dsRNA (SEQ ID NOs: 44 and 45).

FIGs. 58A-B are bar graphs showing real-time PCR analysis of the expression of 7.5 week old corn plants germinated from seeds treated with 25 pg / ml dsRNA for 24 hours. Figure 58A shows the change (number of times) in the expression of

TBl mRNA after treatment with TB1 dsRNA (SEQ ID NO:

5) and CGMMV dsRNA (SEQ ID NOs: 8 and 11) as control. Each bar represents a plant. The expression values ​​for the individual plants were normalized to the median of the expression of all the plants treated with CGMMV dsRNA. Figure 58B shows the median values ​​corresponding to the data shown in Figure

58A. The change in TBl expression in relation to the CGMMV control had a p value of 0.065. Error bars represent one standard deviation of the data.

FIGs. 59A-C are bar graphs showing real-time PCR analyzes of NAC mRNA expression in five-day-old corn shoots and 12-day-old leaves germinated from seeds treated with 50 pg / ml dsRNA during 24 hours. Figure 59A - Seeds put in germination boxes after treatment with dsRNAs (SEQ

ID NOs; 83 and 21 for NAC and GUS, respectively). Figures 59B

- Washed and dried seeds after treatment with dsRNAs. GUS dsRNA was used as a control. Each point represents a plant. Figure 59C - Real-time PCR analysis of NAC mRNA expression in old leaves germinated from seeds treated in the same way as in Figures 59A and 59B, and planted in soil.

Observe upregulation of NAC mRNA 5 days after germination and downregulation thereafter.

FIGs. 60A-D are graphs showing the analyzes of

Real-time PCR of HY5 mRNA expression in one week old and two week old lettuce plants germinated from seeds treated with 50 pg / ml dsRNA for 24 hours. Figure 60A shows the expression levels of HY5.5 mRNA in one week old plants after treatment with HY5.5 dsRNA (SEQ ID NO: 156), HY5.6 dsRNA (SEQ ID NO: 160) or a mixture of both (1: 1, together 50 pg / ml). GUS dsRNA (SEQ ID NO: 21) or 0. lmM EDTA (buffer) were used as controls. Each point represents a plant.

Figure 60B shows the expression levels of HY5.6 mRNA in one week old plants after treatment with

HY5.5 dsRNA, HY5.6 dsRNA or a mixture of both. GUS dsRNA or

O.lmM EDTA (buffer) were used as controls. Each point represents a plant. Figure 60C shows the expression levels of HY5.5 mRNA in two week old plants after treatment with HY5.5 dsRNA (SEQ ID NO: 156),

HY5.6 dsRNA (SEQ ID NO: 160) or a mixture of both (1: 1,

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together 50 pg / ml). GUS dsRNA (SEQ ID

INDUSTRIAL dsRNA (SEQ ID NO: 167) or 0. lmM EDTA (buffer) were used as controls. Each point represents a plant. Figure 60D shows expression levels of HY5.6 mRNA in two-week-old plants after treatment with HY5.5 dsRNA (SEQ ID NO: 156), HY5.6 dsRNA (SEQ ID NO: 160) or a mixture of both (1: 1, together 50 pg / ml). GUS dsRNA (SEQ ID NO:

21), DHFR dsRNA (SEQ ID NO: 167) or 0. lmM EDTA (buffer) were used as controls. Each point represents a plant.

FIG. 61 is a graph showing real-time PCR analysis of DHFR mRNA expression in one-week-old lettuce plants germinated from seeds treated with 50 pg / ml dsRNA (SEQ ID NO: 167) for 24 hours. GUS dsRNA (SEQ ID NO: 21) or 0. lmM EDTA (buffer) were used as controls. Each point represents a plant.

FIGs. 62A-B shows the effect of treating cucumber seeds with DND1 dsRNA. Figure 62A is a graph showing real-time PCR analysis of the expression of

DND1 mRNA in 15-day-old cucumber plants germinated from seeds treated with 100 pg / ml dsRNA (SEQ ID

NOs: 171 and 172) for 24 hours. GUS dsRNA (SEQ ID NO: 21) or

0. lmM EDTA (formulation) were used as controls. Each point represents a plant. Figure 62B shows the gall percentage score of cucumber roots 11 days after

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seed treatment with DNDl dsRNA.

mM EDTA (formulation) are control treatments.

FIG. 63 is a graph showing real-time PCR analysis of PMR5 mRNA expression in 15-day-old cucumber plants germinated from seeds treated with 100 pg / ml dsRNA (SEQ ID NOs: 180 and 181) for 24 hours.

GFP dsRNA (SEQ ID NO: 17 6) or 0. lmM EDTA (formulation) were used as controls. Each point represents a plant.

FIG. 64 is a graph showing real-time PCR analysis of TubG mRNA expression in 15-day-old cucumber plants germinated from seeds treated with 100 pg / ml dsRNAs (SEQ ID NO: 185 and 186) for 24 hours.

GFP dsRNA (SEQ ID NO: 176) or 0. lmM EDTA (formulation) were used as controls. Each point represents a plant.

FIG. 65 is a graph showing real-time PCR analysis of DNDl mRNA expression in 15-day-old tomato plants germinated from seeds treated with 100 pg / ml dsRNA (SEQ ID NOs: 24 and 25) for 24 hours.

GFP dsRNA (SEQ ID NO: 176) or 0. lmM EDTA (formulation) were used as controls. Each point represents a plant.

FIG. 66 is a graph showing real-time PCR analysis of PMR5 mRNA expression in 15-day-old tomato plants germinated from seeds treated with 100 pg / ml dsRNA (SEQ ID NO: 32) for 24 hours. GFP dsRNA (SEQ ID NO: 176) or O.lmM EDTA (:

they used as controls. Each point represents a plant.

FIG. 67 is a graph showing real-time PCR analysis of MLO mRNA expression in 15-day-old tomato plants germinated from seeds treated with 100 pg / ml dsRNA (SEQ ID NOs: 37 and 38) for 24 hours.

GFP dsRNA (SEQ ID NO: 176) or O.lmM EDTA (formulation) were used as controls. Each point represents a plant.

FIG. 68 is a bar graph showing the average percentage of Powdery mildew disease in 15-day-old tomato plants germinated from seeds treated with Bil (SEQ ID NOs: 42 and 43) and PMR5 (SEQ

ID NO: 198) dsRNAs. GFP dsRNA was used as a control.

FIG. 69 is a graph showing real-time PCR analysis of PHYAE3 mRNA expression in 1-week-old soybean plants germinated from seeds treated with 50 pg / ml dsRNAs (SEQ ID NOs: 190 and 194) for 24 hours.

GUS dsRNA (SEQ ID NO: 21) was used as a control. Each point represents a plant.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some of its embodiments, relates to methods for introducing dsRNA into plant seeds to modulate gene expression.

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Before I explain in detail at least

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MPl of the invention, it is to be understood that the invention is not necessarily limited in its application to the details provided in the following description or exemplified by the Examples. The invention is capable of other embodiments or it can be practiced or carried out in various ways.

With the extensive growth of the world population and the limitation of habitats for the growth and cultivation of plants, there is a pressing need to improve plant yields under the present conditions in the process of change.

RNAi has emerged as a powerful tool to modulate gene expression that can be used to generate plants with improved stress tolerance.

In plants, RNAi is typically carried out by producing transgenic plants that overexpress a DNA fragment that is transcribed to produce a dsRNA.

This dsRNA is then processed to give siRNAs that mediate silencing of the target genes, typically by targeting cleavage of the target gene by means of an RNA-induced silencing complex (RISC) or by translational repression.

The main technical limitation for this technology is that it is difficult or impossible to transform many important crop plants, preventing the constitutive expression of the constructs that direct the production of dsRNA. On the other hand, to date, its use has been significantly limited due to the questioning of the potential ecological impacts of transgenic virus resistant plants [Tepfer, 2002, Annu.

Rev. Phytopathol. 40, 467-491].

The present inventors have now designed a novel technology to introduce dsRNA molecules directly into the seed of the plant. They enter the seeds and begin a silencing process, which is continued throughout the life cycle of the plant, leading to a plant with an improved characteristic of interest. The dsRNA that is introduced is naked and as such, exogenous transcriptional regulatory elements are not introduced into the plant, thus reducing the environmental concerns associated with transgenic plants. Furthermore, the modified seed can be germinated to generate a plant without the need to go through a series of laborious and cumbersome tissue culture regeneration steps.

As illustrated below and in the subsequent Examples section, the present inventors managed to configure the necessary conditions for 1 nude in the seeds (see Example 1). Bare dsRNA is not integrated into the genome and is highly stable in the plant and in solution (Examples 2-4). Bare dsRNA penetrates through the seed coat (ie, the seed coat) of both monocot and dicot plants and is distributed in the endosperm and the seed embryo (Examples 5-6). The present inventors managed to alter the expression of endogenous genes (Examples 7-14) as well as exogenous viral genes (Example 2). These results were reproduced for a number of plants in both monocot and dicot groups. Therefore, the present inventors were able to disclose a wide range of doses and kinetics that led to a significant alteration in gene expression (see, for example,

Examples 23-29). These results were also confirmed with wheat and substantiated with a biological effect showing delayed germination (Example 30). Genetic expression was altered in corn after the introduction of dsRNA for various targets (Examples 31 and 32).

Other vegetables that were treated with dsRNA include cucumber and lettuce (Examples 33-37). The present inventors also managed to show altered gene expression in other commercial crops, i.e. soybeans (see Example 42).

Therefore, the present results are to demonstrate that the present disclosure discloses a cost-efficient treatment for the treatment of plant seeds to achieve a desired agricultural or horticultural phenotype. Without limiting itself to theory, it is suggested that the suggested new mode of transformation and modulation of gene expression depends on and is associated with:

(i) Introduction of naked dsRNA inside the seeds (as opposed to merely covering the seeds). The introduction is done by soaking the seeds in a solution comprising the dsRNA in such a way that the dsRNA penetrates through the seed coat or by immersion such that the dsRNA covers the seed and penetrates through the coating after sowing;

(ii) Amplification of the dsRNA signal; and (iii) Propagation of the dsRNA signal throughout the plant.

The first step takes place once, during, and briefly after initial seed treatment, while the second and third steps occur in a repetitive loop for as long as the silencing signal remains active in the plant.

A suggested non-restrictive mode of action for

JLIV1 ia W * '^5,The described invention is based on each step:

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Introduction of dsRNA in seeds

A typical mature seed consists of an embryo encapsulated within a seed coat and an abundant layer of endosperm tissue between the embryo and the seed coat. The endosperm serves as a source of nutrients for the embryo during seed development, germination and seedling establishment.

Germination of seeds typically begins with exposure of the seeds to water, which is absorbed, or the embryo and endosperm. The endosperm therefore expands its volume, and in particular the endosperm of some plant species is capable of growing several times its original volume. The embryo, which was dormant during this stage, is now released from dormancy and cell division, expansion and differentiation begin. The endosperm feeds the developing embryo until it has developed enough to begin photosynthesis and autotrophic growth.

Based on these known mechanisms for seed germination, two possible modes of action are suggested for the initial step of introducing dsRNA into seeds:

The dsRNA molecules enter the embryo directly, carried in a solution that is used for seed treatment.

DsRNA molecules enter the endosperm as part of the endosperm's water absorption process. These molecules then feed the embryo as it develops as part of the nutrient flow from the endosperm during germination and seed development.

Based on the results described in Figures 713, it is estimated that a combination of both options occurs, that is, a certain amount of dsRNA enters the embryo directly and a certain amount is retained in the endosperm and feeds the developing embryo during seed germination.

DsRNA signal amplification

Once dsRNA molecules enter the embryo, they are recognized and processed by RNase III type enzymes such as Dicer or Dicer type enzymes (DCL). DCL enzymes process long dsRNA molecules into short double-stranded RNAs (known as siRNAs or shRNAs), which typically are 21-24 nucleotides (nt) in length. One of the siRNA strands typically breaks down rapidly and the second strand can be incorporated into RISC (RNA-induced silencing complex) protein complexes, which are Argonaute protein (AGO). AGO proteins contain a PIWI domain for binding to siRNAs and a PAZ domain with RNase activity. The siRNA / AGO complex then identifies an mRNA molecule, which is complementary to the siRNA and results in its silencing by cleavage or translational repression.

If siRNA is then released from the RISC complex and can act as a primer for RNA Dependent RNA Polymerase

RNA (RDRP), which is an enzyme found only in the plant kingdom and that can generate the amplification of the silencing signal generating new dsRNA molecules (secondary siRNA). These new synthesized dsRNAs can be reprocessed as described above as well, maintaining and amplifying the silencing signal.

Squelch signal propagation

Propagation of the silencing signal in plants is a well-known and well-understood phenomenon. Propagation across short distances, from cell to cell, is believed to occur through plasmodesms. This process is thought to be mediated by a 21 nucleotide long siRNA, product of the DCL enzyme. Additionally, systemic propagation throughout the plant is accomplished through the phloem, from the source to the sink.

<img file="MX360866B_D0015.tif" />

In the described methodology, it is assumed that n

INDUSTRIAL squelch occurs once the squelch signal begins and it is amplified as described above. It can involve both short-distance propagation and systematic propagation through various siRNA signaling molecules.

Therefore, in accordance with one aspect of the invention, there is disclosed a method for introducing a double-stranded bare RNA (dsRNA) into a seed, such method comprising contacting the seed with the bare dsRNA under conditions that allow the penetration of dsRNA, thereby introducing dsRNA into the seed.

As used herein, the phrase "naked dsRNA" refers to a dsRNA molecule that is not transcribed in the plant cell. Therefore, the dsRNA molecule is not comprised in a nucleic acid expression construct such as a viral vector.

In accordance with some embodiments of the invention, the dsRNA molecule is not derived from a viral vector. According to some embodiments, dsRNA is not a product of a natural viral infection. In accordance with some embodiments, the naked dsRNA may comprise regulatory elements for in vitro transcription, such as the T7 promoter. In accordance with some embodiments of the invention, the naked dsRNA can

TMPI modify, for example, modify yourself:

FROM t TO INDUSTRIAL PROPERTY

<img file="MX360866B_D0016.tif" />

confer higher bioavailability, seed penetration and / or improved shelf life.

As used herein, the term dsRNA refers to two strands of antiparallel polyribonucleic acids linked through base pairing. The two strands can be of identical lengths or of different lengths, provided that there is a sufficient degree of homology between the two and a double strand structure is formed with a complementary length of at least 80%, 90%

95% or 100%.

According to an embodiment of the invention, the dsRNA molecule does not possess protruding strands. According to another embodiment of the invention, the dsRNA molecule comprises protruding strands. According to other embodiments, the strands are aligned in such a way that there are at least 1, 2 or 3 bases at the end of the strands that are not aligned (that is, they do not have complementary bases in the opposite strands), so that a protruding end of 1, 2 or 3 residues can be found on one or both ends of the double strand when the chains are paired.

As mentioned, any dsRNA molecule can be used in accordance with the present disclosure, as long as it can be amplified by an RNA-dependent RNA polymerase (RDRP).

The present disclosure relates to various lengths of dsRNA, whereby the short version is

<td>say x</td><td>has</td><td>a</td><td>length</td><td>minor or</td><td>same</td><td>at 50 bp (per</td>

<td>example,</td><td> 17-50),</td><td>is</td><td>denominate</td><td>siRNA or</td><td>miRNA.</td><td>Molecules more</td>

<td>long</td><td>dsRNA,</td><td>of</td><td>51-600 bp,</td><td colspan="2">what can be</td><td>further</td>

Processed to produce siRNA molecules, they are called dsRNAs herein.

Small inhibitory double-stranded RNA molecules (generally 17-30 base pairs, but also longer, eg 31-50 bp) that induce RNA interference pathways are called siRNAs. Typically, siRNAs are chemically synthesized as 21mers, with a 19 bp double-stranded region, and protruding symmetrical 2-base ends at the 3 'ends, although it has recently been reported that double-stranded molecules of

25-30 chemically synthesized bases in length can have a power increase of up to 100 times with respect to

21mers in the same location. It has been postulated that the increase in potency observed using longer RNA molecules in the RNAi trigger would result from providing the Dicer enzyme with a substrate (27mer) rather than a product (21mer), which increases the rate or efficiency input of double-stranded siRNA molecules to the RISC.

It has been observed that the position of 1 protrusion influences the potency of a siRNA and that asymmetric double-stranded molecules containing a 3 'protruding end in the antisense strand are generally more powerful than those possessing 3' protruding ends in the sense strand (Rose et al., 2005). This can be attributed to an asymmetric load on the RISC, while opposite efficiency patterns are observed when antisense transcripts are attacked.

Strands of a double-stranded interference RNA (eg, a siRNA) can join to form a hairpin or stem-loop structure (eg, shRNA). Thus, as mentioned above, the silencing agent of some embodiments of the invention may also be short hairpins (shRNA).

The term '' shRNA '', as used herein, refers to an RNA agent that possesses a stem-loop-like structure, comprising a first and a second region of a complementary sequence, the level of complementarity and orientation of the regions sufficient to allow the pairing of the regions bases, and the first and second regions being linked by a loop, this loop resulting from a mismatch of the nucleotide bases (or nucleotide analogs) of the loop region. Nucleotide number is between, and includes, 3 to 23, or 5 to 15, or 7 to 13, or 4 to

9, 9 to 11. Some of the nucleotides in the loop may participate in base pair interactions with other nucleotides in the loop. Examples of the oligonucleotide sequences that can be used to form the loop include the 5'-UUCAAGAGA-3 'sequences (Brummelkamp, ​​TR et al. (2002) Science 296: 550, DEQ ID NO: 22) and 5' -UUUGUGUAG3 '(Castanotto, D. et al. (2002) RNA 8: 1454, SEQ ID NO: 23).

One of skill in the art will recognize that the single strand of oligonucleotides forms a stem-loop type hairpin structure that includes a double stranded region capable of interacting with the RNAi machinery.

As used herein, the phrase mircro RNA (also interchangeably referred to herein as miRNA or miR) or a precursor refers to a micro RNA (miRNA) molecule that acts as a post-transcriptional regulator . Typically, miRNA molecules are RNA molecules of about 20 to 22 nucleotides in length that can be loaded into the RISC complex and direct the cleavage of another RNA molecule, where the other RNA molecule comprises an essentially complementary nucleotide sequence to the nucleotide sequence of the miRNA molecule.

Typically, a miRNA molecule is pn from a "pre-miRNA" or, as used herein, from a pre-miRNA molecule by proteins such as DCL proteins, present in any plant, and it is loaded into the RISC complex where it can guide the cleavage of the target RNA molecules.

Pre-microRNA molecules are typically processed from pri-microRNA (primary transcripts). The single-stranded RNA segments that flank the pre10 microRNAs are important for the processing of primiRNAs into pre-miRNAs. The cleavage site appears to be determined by the distance from the stem-ssRNA junction (Han et al. 2006, Cell 125, 887-901, 887-901).

As used herein, a pre-miRNA molecule is a molecule of about 100 to about 200 nucleotides, preferably about

100 nucleotides to around 130 nucleotides that can adopt a secondary structure comprising an imperfect double-stranded stem and a single-stranded RNA loop (also called '' hairpin ''), further comprising the nucleotide sequence of the miRNA (and its complementary sequence ) in the double-stranded RNA stem.

In accordance with this specific embodiment, the miRNA and its complement are located between around 10 and around

<img file="MX360866B_D0017.tif" />

20 nucleotides from the free ends of the

INDUSTRIAL strand of the miRNA. The length and sequence of the region of

<td>single strand</td><td>of the loop</td><td>they are not critical</td><td>and</td><td>may vary</td>

<td colspan="3">considerably, for example between 30 and</td><td> 50</td><td>nucleotides of</td>

<td>length.</td><td></td><td></td><td></td><td></td>

<td colspan="2">Complementarity</td><td>between miRNA and</td><td>its</td><td>complement no</td>

<td>needs to be</td><td>perfect and</td><td>about 1</td><td>to</td><td>3 bubbles</td>

Unpaired nucleotides can be tolerated. The secondary structure adopted by an RNA molecule can be predicted by conventional algorithms in the art such as mFOLD.

The particular strand of the double-stranded RNA stem of the pre-miRNA that is released by DCL activity and loaded in the RISC complex is determined by the degree of complementarity at the 5 'end, whereby the strand that at its 5 end 'It has fewer hydrogen bonds between the nucleotides of the different strands of the stem of the cleaved dsRNA is loaded in the RISC complex and will determine the sequence specificity of the degradation of the target RNA molecule. However, if the miRNA molecule of a given synthetic premiRNA is not empirically functional (because the wrong strand is loaded into the complex

RISC), it will be immediately apparent that the problem can be solved by exchanging the position of the miRNA molecule and its complement in the respective strands of the pre-miRNA molecule. As is known in the art, the bond between A and Ü through two hydrogen bonds, or between G and U through two hydrogen bonds is less strong than the bond between G and C through three hydrogen bonds . Examples of hairpin sequences are provided in Tables 1-8 below.

The naturally occurring miRNA molecules can be comprised within the naturally occurring pre10 miRNA molecules but can also be introduced into pre-existing scaffold pre-miRNA molecules,

<td>through</td><td>the</td><td>exchange of</td><td>sequences</td><td>of</td><td>nucleotides of the</td>

<td>molecule</td><td>of</td><td>miRNA they are</td><td>processed</td><td colspan="2">normally in bliss</td>

<td>molecule</td><td>of</td><td>pre-miRNA already</td><td>existing</td><td>by</td><td>the sequence of</td>

<td colspan="2">15 nucleotides</td><td>from another miRNA</td><td>of interest.</td><td>The</td><td>pre scaffold</td>

miRNA can also be completely synthetic. Likewise, synthetic miRNA molecules can be comprised, and processed, in pre-miRNA scaffold molecules or in synthetic premiRNA scaffold. Some pre-miRNA scaffolding may be preferred over others for their efficiency in being correctly processed into engineered microRNAs, particularly when expressed as chimeric genes in which other regions of DNA, such as leader or transcription termination sequences and regions of Polyadenylation are incorporated into the primaric pre-microRNA transcripts.

In accordance with the present disclosure, dsRNA molecules can be naturally occurring or synthetic.

The dsRNA can be a mixture of short and long dsRNA molecules such as dsRNA, siRNA, siRNA + dsRNA, siRNA + miRNA, or a combination thereof. According to a specific embodiment, the dsRNA is a siRNA (100%). According to a specific embodiment, dsRNA is a combination of siRNA + dsRNA in various proportions. For example, in a 1: 1 ratio: a dsRNA mixed with the same sequence after treatment with RNase III. According to a specific embodiment, the dsRNA constitutes purified dsRNA (100%). According to another embodiment, the ratio of dsRNA to siRNA is 2: 1, 1.5: 1, 1.3: 1, 1: 0.01, 1: 0.05, or

1: 0.1. According to another embodiment the ratio of dsRNA to siRNA is from 2: 1 to 1: 0.1.

The dsRNA molecule is designed to specifically target a target gene of interest. It will be appreciated that dsRNA can be used to down-regulate one or more target genes. If a number of target genes are targeted, a heterogeneous composition comprising a variety of dsRNA molecules is used to target a number

<img file="MX360866B_D0018.tif" />

<img file="MX360866B_D0019.tif" />

of genes. Alternatively, such diversity,_:

INDUSTRIAL dsRNA can be applied separately to seeds (but not as a single composition). According to a specific embodiment, a set of different dsRNA molecules were used for a single target, which can be applied either separately or together (i.e., co-formulation).

According to an embodiment of the invention, the target gene is endogenous to the plant. Downregulation of this gene is typically important to improve an agricultural, horticultural, or nutritional characteristic of the plant (improvement or increase is defined in more detail below). It should be appreciated that dsRNA treatment may lead to upregulation of the target gene (which follows a suggested mechanism disclosed below); however, such upward regulation may be transitory. The present inventors managed to confer resistance to biotic stress by modulating the expression of endogenous genes in cucumber and tomato and thus conferring resistance to infections as demonstrated in the

Example 35 and 41.

As used herein, endogenous refers to a gene whose expression (mRNA or protein) takes place in the plant. Typically, the gene is endogenous naturally in the plant or originates from the plant. Therefore, the plant can be a wild plant. However, it can also be a genetically modified (transgenic) plant.

Downregulation of the target gene may be important to confer improvement of one, or at least one (eg, two or more) characteristics such as biomass, vigor, yield, resistance to abiotic stress, or improvement of use efficiency of nitrogen.

Examples of target genes include, but are not limited to, an enzyme, a structural protein, a plant regulatory protein, a target gene for a miRNA, or a noncoding RNA such as a plant miRNA.

WO2011067745, WO 2009125401 and WO 2012056401 constitute examples of miRNA sequences or targets of miRNAs (eg mRNA167, miRNAl56, miR164 and their targets NFY, SPL17 and

NAC, respectively) whose expression can be silenced to improve a plant characteristic. Other examples of target genes that may be subject to modulation in accordance with the present disclosure are described in the section on

Examples below.

The target gene may comprise a nucleic acid sequence that is transcribed as an mRNA and encodes for a polypeptide.

Alternatively, the target gene can be a non-coding gene such as a miRNA or a siRNA.

For example, in order to silence expression of an mRNA of interest, synthesis of the appropriate dsRNA for use with some embodiments can be selected as follows. First, the mRNA sequence is analyzed including the 3 'UTR and 5' UTR regions.

The mRNA sequence is then compared to an appropriate genomic database using any sequence alignment software, such as the BLAST software available through the NCBI server (wwwpuntoncbipuntonlmpuntonihpuntogov / BLAST /). Putative mRNA regions that show significant homologies to other coding regions are filtered.

Appropriate target sequences are selected as templates for dsRNA synthesis. Those sequences that show low homology with other genes will be preferred in order to reduce the effect on genes other than the target gene.

It will be appreciated that the RNA silencing agent of some of the embodiments of the invention should not be limited to molecules containing only RNA, but also encompasses chemically modified nucleotides and other non-nucleotide components.

DsRNA can be synthesized using any method known in the art, including enzymatic or solid phase synthesis. These methods are particularly useful in the case of short polynucleotide sequences with or without modifications as explained above. Equipment and reagents for carrying out solid phase synthesis are commercially available from, for example, Applied Biosystems. Any other method can be used for such synthesis; current oligonucleotide synthesis is within the capabilities of one skilled in the art and can be accomplished through methodologies set forth in detail in, for example,

Sambrook, J. and Russell, DW (2001), Molecular Cloning: A

Laboratory Manual; Ausubel, RM et al., Eds. (1994, 1989),

Current Protocols in Molecular Biology, Volumes I-III,

John Wiley &amp; Sons, Baltimore, Maryland; Perbal, B. (1988), A

Practical Guide to Molecular Cloning, John Wiley &amp; Sons, New

York; and Gait, MJ, ed. (1984), Oligonucleot ide

Synthesis; using solid phase chemistry, eg, cyanoethyl phosphoramidite, followed by deprotection, salt removal, and purification, eg, through an automated trityl-on method or HPLC.

As mentioned, the naked dsRNA molecule is brought into direct contact with the seed.

The seed can belong to any plant, such as plants of the Viridiplantae superfamily, including mono and dicot plants. Other plants are listed below. In accordance with one embodiment of the invention, the plant cells possess RNA-dependent RNA polymerase activity and the dsRNA target RNA molecule to ensure amplification of the dsRNA.

The term plant as used herein, encompasses whole plants, ancestors and progeny of plants, parts of plants, including seeds, shoots, stems, roots (including tubers), and cells, tissues, and organs isolated from plants. . The plant can occur in any form, including suspension cultures, embryos, meristemic regions, callus tissue, leaves, gametophytes, sporophytes, pollen, and macrospores. It will be appreciated that the plant, or its seeds, may be transgenic.

As used herein, the phrase "plant cell" refers to plant cells that are derived from and isolated from disintegrated plant tissue or plant cell cultures.

As referred to herein, the phrase "plant cell culture" refers to any type of genetically modified, native (naturally occurring) plant cells, plant cell lines and cells that do not assemble to form a plant. complete, so that at least one biological structure of the plant is not present. Optionally, the cultivation of plant cells, in connection with this aspect of the present invention, may include a particular type of plant cell or a variety of different types of plant cell. It should be noted that optionally plant cultures possessing a particular type of plant cell may originally be derived from a variety of different types of these plant cells.

In accordance with some embodiments of the invention, any plant of commercial or scientific interest is contemplated.

Among the plants particularly useful for the methods of the invention are all the plants belonging to the Viridiplantae superfamily, in particulates, monocotyledonous and dicotyledonous plants including legumes for feed or forage, ornamental plants, food crops, trees or shrubs selected from the list including Acacia spp., Acer spp.,

Actinidia spp., Aesculus spp., Agathis australis, Albizia amara, Alsophila tricolor, Andropogon spp., Arachis spp,

Areca catechu, Astelia fragrans, Astragalus cicer, Baikiaea plurijuga, Betula spp., Brassica spp., Bruguiera gymnorrhiza, i ivi ri iiiys⁰

African Burkea, Butea frondosa, Cadaba farir

OF U igalplj PROPERTY ........

spp, Camellia sinensis, Canna indica, Capsicum spp., Cassia spp., Centroema pubescens, Chacoomeles spp., Cinnamomum cassia, Coffea arabica, Colophospermum mopane, Coronillia varia, Cotoneaster serótina, Crataegus spp., Cucumis spp.

Cupressus spp., Cyathea deaibata, Cydonia oblonga,

Japanese Cryptomeria, Cymbopogon spp., Cynthea deaibata,

Cydonia oblonga, Dalbergia monetaria, Davallia divaricata,

Desmodium spp., Dicksonia squarosa, Dibeteropogon amplectens,

Dioclea spp., Dolichos spp., Dorycnium rectum, Echinochloa pyramidalis, Ehraffia spp., Eleusine coracana, Eragrestis spp., Erythrina spp., Eucalyptus spp., Euclea schimperi,

Eulalia vi / losa, Pagopyrum spp., Feijoa sellowlana, Fragaria spp., Flemingia spp, Freycinetia banksli, Geranium thunbergii, GinAgo biloba, Glycine javanica, Gliricidia spp, Gossypium hirsutum, Grevillea spp., Guibourtia coleos

Hedysarum spp., Hemaffhia altissima, Heteropogon contoffus,

Hordeum vulgare, Hyparrhenia rufa, Hypericum erectum,

Hypeffhelia dissolute, Indigo incamata, Iris spp.,

Leptarrhena pyrolifolia, Lespediza spp., Lettuca spp.,

Leucaena leucocephala, Loudetia simplex, Lotonus bainesli, Lotus spp., Macrotyloma axillare, Malus spp., Manihot esculenta, Medicago saliva, Metasequoia glyptostroboides,

Musa sapientum, Nicotianum spp., Onobrychis spp., Ornithopus spp., Oryza spp., Peltophorum africanum, P

Persea gratissima, Petunia spp., Phaseolus spp., Phoenix canariensis, Phormium cookianum, Photinia spp., Picea glauca,

Pinus spp., Pisum sativam, Podocarpus totara, Pogonarthria fleckii, Pogonaffhria squarrosa, Populus spp., Prosopis cineraria, Pseudotsuga menziesii, Pterolobium stellatum,

Pyrus communis, Quercus spp., Rhaphiolepsis umbellata,

Rhopalostylis sapida, Rhus natalensis, Ribes grossularia,

Ribes spp., Robinia pseudoacacia, Rosa spp., Rubus spp.,

Salix spp., Schyzachyrium sanguineum, Sciadopitys vefficillata, Sequoia sempervirens, Sequoiadendron giganteum,

Sorghum bicolor, Spinacia spp., Sporobolus fimbriatus,

Stiburus alopecuroides, Stylosanthos humilis, Tadehagi spp,

Taxodium distichum, Themeda triandra, Trifolium spp.,

Triticum spp., Tsuga heterophylla, Vaccinium spp., Vicia spp., Vitis vinifera, Watsonia pyramidata, Zantedeschia aethiopica, Zea mays, amaranth, artichoke, asparagus, broccoli, brussels sprout, cabbage, cane, carrot, celery , flax, kale, lentil, oilseed rape, okra, onion, potato, rice, soy bean, raffia, sugar beet, sunflower, tomato, zucchini tea, corn, wheat, barley, rye, barley, peanut, lentil and alfalfa, cotton, rapeseed, cane, pepper, sunflower, tobacco, eggplant, eucalyptus, trees, ornamental plants, perennial herbs and forage crops. Alternately, algae and other plants not belonging to the group of

Viridiplantae.

According to some embodiments of the invention, the plants used by the method of the invention include, but are not limited to, cotton, vegetables of the Brassica genus, oilseed rape, sesame, olive, oil palm, banana, wheat, corn, barley, alfalfa, peanut, sunflower, rice, oats, sugarcane, soybeans, grass for grass, barley, rye, sorghum, sugarcane, chicory, lettuce, tomato, zucchini, pepper, eggplant, cucumber, melon, watermelon, beans, hibiscus, apple, roses, strawberries, chili, garlic, peas, lentils, canela, chrysanthemum, arabidopsis, broccoli, cabbage, beet, quinoa, spinach, zucchini, onion, leek, tobacco, potato, sugar beet, papaya, pineapple, mango,

Arabidopsis thaliana, as well as plants used in horticulture, floriculture, or forestry, such as, but not limited to, poplar, spruce, eucalyptus, pine, an ornamental plant, a perennial herb, a crop for forage, coniferous plants, mosses, algae , as well as other plants listed on the Web (dot) nationmaster (dot) com / encyclopedia / Plantae.

According to a specific embodiment, the plant is selected from the group comprising corn, tomato, cotton and sorghum.

According to a specific embodiment, the seed is an uncoated or fresh seed that has not been subjected to chemical / physical treatment.

The washing of the seeds is carried out for 30 min to 4 hours. Other exemplary wash ranges are from minute to 10 minutes, 10 minutes to 30 minutes. The washing solution can include a weak detergent such as Tween-20.

The detergent concentration can be 0.01-0.2% or 0.2-1 or, or ·

The seed may undergo conditioning or washing prior to contacting the dsRNA.

As used herein the term conditioning refers to the control of the level of hydration within the seeds so that the metabolic activity necessary for germination can occur but at the same time the appearance of the radicle is prevented. Different metabolic activities occur within the seed at different levels of wetting (Leopold and Vertucci, 1989;

Taylor, 1997). The last physiological activity in the germination process is the appearance of the radicle. The initiation of the appearance of the radicle requires a high water content of the seeds. By limiting the water content of the i ivi ri iiys ° seeds, all pas can occur

OF EA PROPERTY 1 | Β; || Οί ........

necessary for germination without the irreversible act of the appearance of the radicle. Prior to the appearance of the radicle, the seed is considered tolerant to drying, therefore the moisture content of the conditioned seed can be decreased during drying. After drying, the conditioned seeds can be stored until the moment of sowing.

Several different conditioning methods are used commercially. Among them, liquid or osmotic conditioning and solid matrix conditioning have had the greatest follow-up (Khan et al., 1991).

According to an embodiment of the invention, the conditioning is carried out in the presence of salt, a chelating agent, polyethylene glycol or a combination thereof (eg chelating agent and salt).

Alternatively the conditioning is carried out in the presence of water such as deionized water or doubly deionized water. According to a specific embodiment, the conditioning is carried out in the presence of 100% ddW.

Different types of seed conditioning are commonly used:

Osmoconditioning - is the conditioning technique. The seeds are incubated in well aerated solutions with low water potential, and then washed and dried. The low water potential of the solutions can be achieved by adding osmotic agents such as mannitol, polyethylene glycol (PEG), or salts such as KC1.

Hydroconditioning - is accomplished by continually or successively adding a limited amount of water to the seeds. A barrel is used for this purpose and the water can also be applied using humid airs. on-farm steeping is an economical and useful technique that is performed by incubating the seeds (cereals, legumes} for a limited time in warm water.

Matrix conditioning - is the incubation of the seeds in an insoluble solid matrix (vermiculite, diatomaceous earth, highly crosslinked hydroabsorbent polymers) with a limited amount of water. This method confers slow embedding.

Pre-germinated seeds - only possible with a few species. In contrast to normal conditioning, seeds are allowed to develop a protrusion of the radicle. This is followed by classification for specific stages, a treatment that reduces tolerance to drying and drying. The use of pregerminated seeds causes rapid development and seedlings.

Thus, in accordance with an exemplary embodiment, the seeds are conditioned seeds.

It should be noted that it is possible to treat the seeds with water (double distilled water, ddW), prior to contacting the dsRNA without producing effective seed conditioning. For example, a water treatment can be performed for a short period (for example,

30 seconds to 1 hour, 30 seconds to 0.5 hour, 30 seconds to min, 30 seconds to 5 min or 45 seconds to 5 min).

It is to be understood that the dsRNA may be comprised in water (eg, tap water, distilled water, or doubly distilled water), that is, free from any of the aforementioned effective conditioning concentrations of salts, chelating agent, popylethylene glycol, or combinations thereof (eg chelating agent and salt).

According to an exemplary embodiment, the seeds are unconditioned seeds.

A non-limiting exemplary method of introducing dsRNA into seed is provided in the

Example 1, which is considered an integral part of the specification.

The temperature during washing / conditioning and drying can be the same or different.

According to an exemplary embodiment, washing / conditioning is carried out at 4-28 ° C.

According to an exemplary embodiment, the conditioning / washing solution or the dsRNA-containing solution is devoid of a solid vehicle.

According to an exemplary embodiment, the conditioning / washing solution or the dsRNA-containing solution is devoid of a transfer agent such as a surfactant or a salt.

In accordance with a further embodiment of the invention, the seeds to be contacted with the dsRNA molecule are washed in order to remove the agents to which the seeds have been subjected, such as a pesticide, a fungicide, an insecticide, a fertilizer , a coating agent and a coloring agent.

Thus, in accordance with an exemplary embodiment, the seeds (prior to dsRNA treatment) are substantially free of (i.e., do not contain effective amounts) pesticide, fungicide, insecticide, fertilizer, coating agent, or coloring agent.

The seeds are then dried.

According to an exemplified embodiment it is carried out at 20-37 ° C, 20-30 ° C, 22-37 ° C, 15-22 ° C or

20-25 ° C for 10-20 hours, 10-16 hours or even 2-5 hours.

Several considerations need to be made to calculate the concentration of naked dsRNA in the contacting solution.

They depend on at least one seed size, seed weight, seed volume, seed surface area, seed density, and seed permeability.

For example, in relation to seed size, weight, volume and surface area, it is estimated that corn seeds will require a longer treatment than

Arabidopsis and tomato. Regarding permeability and density, it is estimated that wheat seeds will require longer treatments at higher concentrations than tomato seeds.

Exemplary concentrations of dsRNA in the treatment solution include, but are not limited to, 0.01-0.3 pg / μΐ, 0.01-0.15 gg / μΐ, 0.04-0.15 μς / μΐ, 0.1-100 gg / gl; 0.1-50 μρ / μΐ, 0.1-10 gg / μΐ, 0.1-5 μg / μl, 0.1-1 gg / μΐ, 0.1-0.5 gg / μΐ, 0.15-0.5 gg / μΐ, 0.1-0.3 gg / μΐ, 0.010 .1 gg / μΐ, 0.01-0.05 gg / μΐ, 0.02-0.04 gg / μΐ

0.001-0.02 pg / μΐ. According to an embodiment and concentration of the dsRNA in the treatment solution is

0.01-0.15 or 0.04-0.15 pg / μΐ.

According to a specific embodiment, contacting with the dsRNA is carried out in the presence of a chelating agent such as EDTA or another chelating agent such as

DTPA (0.01-0.1 mM).

The contacting solution may comprise a transfer agent such as a surfactant or a salt.

Examples of transfer agents include, but are not limited to, salts such as sodium or lithium salts of fatty acids (such as tallow or tallow amines or lipofectamine of phospholipids or lipofectin (1-20 nM, or 0.1 -1 nM)) and organosilicone surfactants.

Other surfactants include organosilicone surfactants including nonionic organosilicone surfactants, for example, trisiloxane ethoxylate surfactants or a silicone polyether copolymer such as a commercially available heptamethyltrisiloxane polyether copolymer of allyloxypolypropylene glycol. Available as SilwetTM L-77 Surfactant with CAS Number 27306-78-1 and

EPA number: CAL.REG.NO. 5905-50073-AA, currently available

<img file="MX360866B_D0020.tif" />

from Momentive Performance Materials, A

Useful physical agents include (a) abrasives such as carborundum, corundum, sand, calcite, pumice, garnet and the like (b) nanoparticles such as carbon nanotubes (c) physical force. Carbon nanotubes are disclosed in Kam et al. (2004) J. Am. Chem.

Soc., 126 (22): 6850-6851, Liu et al. (2009) Nano Lett.,

9 (3): 1007-1010, and in Khodakovskaya et al. (2009) ACS Nano,

3 (10): 3221-3227. Physical force agents include heating, cooling, applying positive pressure, or treating with ultrasound. Agents for laboratory conditioning of a plant to be permeable to polynucleotides include, for example, the application of a chemical agent, enzyme treatment, heating or cooling, treatment with positive or negative pressure, or ultrasound treatment. Plant conditioning agents in a field include chemical agents such as surfactants and salts.

For contacting the seeds with the dsRNA any method known in the art can be used as long as a suppressive amount of dsRNA penetrates the seeds. Possible examples include

<img file="MX360866B_D0021.tif" />

<img file="MX360866B_D0022.tif" />

non-limiting soaking, spraying or &lt;

INDUSTRIAL with powder, emulsion, suspension or solution; similarly, polynucleotide molecules are applied to the plant using any convenient method, for example, spraying or dabbing with a solution, emulsion, or suspension.

As used herein, a suppressive amount refers to an amount of dsRNA that is sufficient to downregulate the target gene by at least 20%, 30%, 40%, 50%, or more, for example 60 %, 70%, 80%, 90% or more, even 100%. The suppressive amount may be the result of the formation of an amplification in the plant.

According to a specific embodiment, the contacting can be carried out by soaking (that is, inoculation) in such a way that shaking the seeds with the treatment solution can improve penetration and soaking and therefore decrease the treatment time. .

Stirring is typically done at 50-150 RPM and depends on the volume of the treatment solution. Stirring can be carried out for 4-24 hours (1-4 hours, 10 minutes to 1 hour or 30 seconds to 10 minutes). The present disclosure also provides for a short incubation time such as up to 10 minutes. Some examples include, without limitation, 30 seconds to 7 minutes, 30 seconds to 5 minutes, 30 seconds to 3 minutes, 30 seconds to 2 minutes, 30 seconds to 1 minute or 1 minute to 5 minutes. Immersion is also considered within the scope of the present invention. Thus, the seeds are immersed in the dsRNA solution for seconds, for example, 1-10 seconds, 1-5 seconds, 1-3 seconds or 1-2 seconds. During this period, dsRNA can be absorbed from the seed surface. The absorbed dsRNA covering the seed can penetrate the seed or seedling during germination. Incubation takes place in the dark at 10 4-28 ° C or 15-22 ° C (eg 8-15 ° C, 4-8 ° C, 22-28 ° C).

According to a specific embodiment, contacting occurs prior to the rupture of the seed dormancy and the appearance of the embryo.

After contacting, preferably prior to the rupture of the seed dormancy and the appearance of the embryo, the seeds can be subjected to treatments (for example, coating) with the agents mentioned above (for example, pesticide, fungicide, etc. .).

The contacting is carried out in such a way that the dsRNA enters the embryo, endosperm, coating or combination of all three.

After contact with the treatment solution, the seeds can be dried for up to 30 hours at 25-37 ° C. For example, dry them for 16 hours at 30 ° C.

According to a specific embodiment, the seed (eg, an isolated seed) comprises the naked exogenous dsRNA and at least 10-20 dsRNA molecules are found in the endosperm of the isolated seed.

As used herein the term "isolated" refers to separation from the natural physiological environment. In the case of a seed, the isolated seed is separated from other parts of the plant. In the case of a nucleic acid molecule (eg dsRNA) separated from the cytoplasm.

According to a specific embodiment, dsRNA is not expressed from the plant genome, and thus does not constitute an integral part of the genome.

In accordance with a specific embodiment an isolated seed is disclosed comprising an exogenous dsRNA that is present at a similar concentration (for example, about 1: 1, 2: 1 or 1: 2) in an embryo and an endosperm of the seed. It is suggested that the direct introduction of naked dsRNA into the seed results in a higher concentration of dsRNA in the endosperm than that observed when dsRNA is expressed from a nucleic acid expression construct.

According to a specific embodiment ε an isolated seed comprising an exogenous dsRNA that is spatially distributed in an embryo and an endosperm of the plant seed in a spatial distribution that differs from a spatial distribution of exogenous dsRNA in a seed derived from a transgenic plant that recombinantly expresses said exogenous dsRNA.

Methods for measuring the location of RNA molecules in the seed are well known in the art. The use of been as described in the Examples section is an example of them.

According to an alternative or additional embodiment, an isolated seed comprising an exogenous dsRNA is disclosed, where a ratio of concentrations of said exogenous dsRNA to siRNA maturing therefrom is higher in the seed compared to a transgenic seed which recombinantly expresses said exogenous dsRNA.

As used herein the term major refers to at least about 3%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 50%, 60%, 70% , 80%, 90% or even several times higher.

According to an alternative or additional embodiment, an isolated seed comprising an exogenous dsRNA is disclosed, where the plant seed is devoid of a heterologous promoter .MPI to promote expression. · * Ήί

INDUSTJlSAL ^ϊ:·! Β '· *^ϊ: exogenous, where a special distribution of said exogenous dsRNA and / or siRNA maturing therefrom is altered in the seed compared to it in a transgenic seed that recombinantly expresses said exogenous dsRNA.

The term "recombinantly" refers to an expression from a nucleic acid construct.

Pursuant to a further embodiment a

<td>seed of</td><td>a</td><td>plant</td><td>obtainable (or obtained</td><td>) through</td>

<td>any of</td><td>the</td><td>methods</td><td>that are disclosed in</td><td>the present</td>

<td>memory.</td><td></td><td></td><td></td><td></td>

<td>Between</td><td>the</td><td>methods</td><td>to qualify as</td><td>successful the</td>

Introduction of dsRNA are found by way of non-limitation, RTPCR (eg, quantification of the level of the target gene or naked dsRNA), phenotypic analysis such as biomass, vigor, yield, stress tolerance, root architecture, dimensions of leaves, grain size and weight, oil content, cellulose, as well as cell biology techniques.

<td colspan="3">According to the realizations</td><td rowspan="2">of the of the</td><td rowspan="2">invention, target gene</td><td colspan="2">sometimes</td>

<td>it is noted</td><td>the</td><td>upward regulation</td><td>for</td><td>the</td>

<td>treatment</td><td>of</td><td>the seed, such</td><td>how</td><td>It is described</td><td>in</td><td>the</td>

present memory. This has been observed for principal genes that function as master regulators, such as targets for microRNAs (eg SPL and NAC) and other genes involved in regulating major processes (eg HY5). See for example Examples 23 and 32 of the Examples section below.

Without being limited to theory, it is suggested that this phenomenon could be related to a potential feedback loop in the regulation of the expression of these genes. These genes are probably tightly regulated and therefore plants could react to changes in their expression in one direction by overcompensating for a major change in gene expression in the opposite direction. Accordingly, it is possible for example that a gene initially exhibits down-regulation in the first hours or days after treatment, which can be altered to up-regulation later in the life cycle of the plant. See for example Example 32 in the Examples section below. Therefore, the present inventors observed upregulation of the NAC gene in corn five days after treatment and downregulation 10 and 12 days after treatment. This was later confirmed in lettuce for the Hy gene.

5.5 or 5.6

The seeds can be stored for several months before being planted (for example, at 4-10 ° C).

The resulting seed can be germinated in the dark to produce a plant.

Thus a plant or part of a plant is disclosed which comprises a naked exogenous dsRNA and which is devoid of a heterologous promoter to promote expression of the dsRNA in the plant.

As used herein devoid of a heterologous promoter to promote dsRNA expression means that the plant or plant cell does not contain a cis-acting (eg, heterologous) regulatory sequence that transcribes the dsRNA in the plant .

As used herein the term heterologous refers to exogenous, which is not naturally found within the cell of the virgin plant (such as according to the position of integration, or not naturally found within the plant cell). Therefore, the seed isolated in the absence of a heterologous promoter sequence to promote the expression of dsRNA in the plant comprises a homogeneous population (before amplification) or heterogeneous (secondary siRNAs, after amplification) of the non-transcriptional dsRNA of the plant.

The present methodology can be used for gene expression such as in a plant, the method comprising:

(a) contacting a plant seed with a bare dsRNA, under conditions that allow the dsRNA to penetrate the seed, thereby introducing the dsRNA into the seed; and optionally (b) generating a plant from seed.

When used for down-regulation of a plant gene, naked dsRNA is engineered for a desired specificity using bioinformatic tools that are well known in the art (eg BLAST).

This methodology can be used in various applications, ranging from basic research, such as for the purpose of evaluating the function of a gene, to the generation of plants with altered characteristics that have valuable commercial use.

Such plants may exhibit beneficial agricultural characteristics including altered morphology, altered flowering, altered stress tolerance (i.e., biotic viral and / or abiotic streaks), altered biomass vigor and / or altered yield, and the like.

The phrase abiotic stress as used herein refers to any adverse effect on

...... .a metabolism, growth, viab

...... DELA PROPERTY 1 | Β; || ΡΚ; ........

reproduction of a plant. Abiotic stress can be induced by any suboptimal environmental growth conditions such as, for example, a water deficit or drought, flooding, freezing, low or high temperature, strong winds, heavy metal toxicity, anaerobiosis, high and low levels of nutrients (for example nutrient deficiency), high or low levels of salts (for example salinity), air pollution, high or low light intensities (eg insufficient light) or ÜV radiation. Abiotic stress can be a short-term effect (for example an acute effect, for example lasting for about a week) or alternatively it can be persistent (for example a chronic effect, for example lasting for 10 days or more ). The present invention contemplates situations in which there is a single abiotic stress condition or alternatively situations in which there are two or more abiotic stresses.

According to an exemplary embodiment, abiotic stress refers to salinity.

According to another exemplary embodiment, abiotic stress refers to drought.

According to another exemplary embodiment, abiotic stress refers to heat stress.

As used herein, the phrase "abiotic stress tolerance" refers to the ability of a plant to tolerate abiotic stress without exhibiting substantial physiological or physical damage (eg, impaired metabolism, growth, viability, and / or reproducibility). of the plant).

As used herein, the phrase "Nitrogen Use Efficiency" (NUE) refers to a measure of crop production per unit nitrogen input fertilizer. Fertilizer efficiency (FUE) is a measure of NUE. Crop production can be measured by biomass, vigor, or yield. The efficiency in the use of nitrogen in a plant is typically the result of an alteration in at least one of the uptake, propagation, absorbance, accumulation, relocation (within the plant) and use of the nitrogen absorbed by the plant. An improved NUE is (sic) with respect to that of the non-transgenic plant (that is, it does not possess the transgene of the transgenic plant) of the same species and for the same stage of development and cultivated under the same conditions.

As used herein the phrase "nitrogen limiting conditions" refers to growth conditions including example, concentration) of nitrogen (eg, ammonium or nitrate) applied that is less than the level necessary for optimal metabolism, growth reproduction and / or viability of the plant.

As used herein the term / phrase biomass, biomass of a plant, or biomass of the plant refers to the amount (eg, measured in grams of air-dried tissue) of tissue produced from the plant in a growing season. An increase in the biomass of the plant can damage the whole plant or parts of it such as the aerial parts (for example, harvesters), the vegetative biomass, roots and / or seeds or the content of the same (for example, oil, starch, etc.).

As used herein the term / phrase vigor, vigor of a plant, or vigor of plant refers to the amount (eg, measured by weight) of tissue produced by the plant at a given time. Increased vigor could determine or affect plant yield or yield by time or area of ​​growth. Furthermore, early vigor (eg seed and / or seedling) results in improved field tolerance.

As used in the present term / phrase yield, yield of a plant or yield of plant refers to the quantity (for example, as determined by weight or size) or units (for example, numbers) of tissues or organs produced by plant or by growing season.

An increase in plant yield can affect the economic benefit that can be obtained from the plant in a certain growth area and / or growth time.

According to an exemplary embodiment the yield is measured according to the cellulose content, the oil content, the starch content and the like.

According to another exemplary embodiment the performance is measured according to the oil content.

According to another exemplary embodiment the yield is measured according to the protein content.

According to another exemplary embodiment, the yield is measured according to the number of seeds, the weight of the seed, the number of fruits or the weight of the fruit per plant or part of it (for example, pit, seed).

The performance of the plant can be affected by several parameters, among which are, without limitation, the biomass of the plant; the vigor of the plant;

the growth rate of the plant; performance in

<img file="MX360866B_D0023.tif" />

seeds; the amount of seeds or grains;

seeds or grains; oil yield; the content of oil, starch and / or protein in the harvested organs (for example, seeds or vegetative parts of the plant); number of flowers (for example buds) per panicle (for example expressed as the ratio of the number of filled seeds to the number of primary panicles); the harvest index; the number of plants cultivated per area; the number and size of organs harvested per plant and per area; the number of plants per cultivation area (for example density);

the number of organs harvested in a field; the total area of ​​the sheet; carbon assimilation and carbon partitioning (eg carbon distribution / allocation within the plant); shade resistance; the number of harvestable organs (for example seeds), seeds per pod, weight per seed; and modified architecture (such as increased diameter, thickness, or improved physical properties (eg, elasticity) of the stem].

An improved plant NUE results in the field in either harvesting similar amounts of yield by implementing fewer fertilizers, or higher yields with the same levels of fertilizers. Therefore, an improved NUE or FUE has a direct effect on plant performance in the field.

<img file="MX360866B_D0024.tif" />

As used in this mea

GAVE! THE ILLNESS ILMsIiSI ..... «.....

INDUSTRIAL ^::* !! Í | Ír''W^i: improve or increase refers to an increase of at least

<td rowspan="2"></td><td rowspan="2">around around</td><td rowspan="2">of of</td><td rowspan="2"> 2 4</td><td rowspan="2">OR, O r or O r</td><td rowspan="2">to the to the</td><td rowspan="2">less less</td><td rowspan="2">around around</td><td rowspan="2">of of</td><td colspan="2">Or 2- O Ό!</td><td rowspan="2">to the to the</td><td rowspan="2">less less</td>

<td> 5</td><td>Q, O z</td>

<td> 5</td><td>around</td><td>of</td><td> 10</td><td>or OZ</td><td>to the</td><td>less</td><td>around</td><td>of</td><td> 15</td><td>g. oz</td><td>to the</td><td>less</td>

<td></td><td>around</td><td>of</td><td> 20</td><td> %,</td><td>to the</td><td>less</td><td>around</td><td>of</td><td> 25</td><td>or. O r</td><td>to the</td><td>less</td>

<td></td><td>around</td><td>of</td><td> 30</td><td>g, 0 Z</td><td>to the</td><td>less</td><td>around</td><td>of</td><td> 35</td><td>g, or</td><td>to the</td><td>less</td>

<td></td><td>around</td><td>of</td><td> 40</td><td> %,</td><td>to the</td><td>less</td><td>around</td><td>of</td><td> 45</td><td> %,</td><td>to the</td><td>less</td>

<td></td><td>around</td><td>of</td><td> 50</td><td></td><td>to the</td><td>less</td><td>around</td><td>of</td><td> 60</td><td>OR, O t</td><td>to the</td><td>less</td>

<td> 10</td><td>around</td><td>of</td><td> 70</td><td> %,</td><td>to the</td><td>less</td><td>around</td><td>of</td><td> 80</td><td>OR_ ° Z</td><td>to the</td><td>less</td>

<td></td><td>around</td><td>of</td><td> 90</td><td>Q. Ό</td><td colspan="2">or greater in</td><td>the NUE,</td><td>in</td><td>the</td><td colspan="3">tolerance to</td>

stress, yield, biomass, or vigor of a plant, compared to virgin or wild plants [ie isogenic plants (not modified to understand dsRNA) of the invention].

As mentioned, the dsRNA target gene may not be an endogenous gene of the plant but an exogenous gene of the plant, such as one of a plant virus or bacterium that feeds on or depends on the plant for growth, replication and / or survival.

Therefore, according to an aspect of the invention, a method is disclosed for inhibiting the expression of a target gene in a plant virus, the method comprising providing the plant to the plant virus (contacting under conditions of infection) as present memory (at least part of it includes naked dsRNA), thereby inhibiting the expression of a target gene in the plant virus.

A number of virus genera are transmitted, both persistently and non-persistently, through soil-borne zoosporic protozoa. These protozoans are viral pathogens in themselves, but are parasitic organisms. The transmission of viruses takes place when they are associated with the roots of the plant. Examples include Polymyxa graminis, whose involvement in the transmission of viral diseases in cereal crops has been demonstrated, and Polymyxa betas that transmits the yellow vein necrotic virus in beets. Plasmidiophorids also produce wounds in the roots of the plant through which other viruses can enter.

Specific examples of viruses that can be treated under this disclosure include, but are not limited to:

(1) Tobacco mosaic virus (TMV, virus to RNA) that infects plants, especially tobacco and other members of the Solanaceae family.

(2) Tomato tanning virus (TSWV, virus a

RNA) that causes serious diseases of economic importance, representatives of 35 plant families, including monocotyledons and dicotyledons.

This broad spectrum of ornamental hosts, vegetables and field crops is unique to viruses that infect plants. It belongs to tospoviruses in the Mediterranean area and affects vegetable crops, especially tomato, peppers and lettuce (Turina et al.,

2012, Adv Virus Res 84; 403-437).

(3) Tomato spoon leaf virus (TYLCV), which is transmitted by the whitefly, mainly affects tomato plants. Geminivirus (virus to DNA) of the genus

Begomoviruses (including sweepoviruses and legumoviruses) most devastating pathogens affecting a variety of crops, including cassava, sweet potatoes, beans, tomato, cotton, and grain legumes (Rey et al 2012, Viruses

4; 1753-1791). Its members include the TYLCV mentioned above and tomato leaf curl virus (ToLCV).

(4) Cucumber Mosaic Virus (CMV) - CMV has a broad host spectrum and attacks a wide variety of vegetables, ornamentals, and other plants (up to 191 host species in 40 families). The most important vegetables that are affected by cucumber mosaic virus include peppers (Capsici cucurbits, tomatoes (Lycopersicon esculentum Mili.) And bananas (Musa L. spp.).

Other host vegetables include: cucumber, cantaloupe, zucchini, tomato, spinach, celery, bell pepper, watercress, beets, sweet potato, turnip, chayolet, pickles, watermelon, squash, French lemon, squash, white beans, beans, onion, physalis, eggplant, potato, rhubarb, carrot, dill, fennel, parsnip, parsley, loofah, and artichoke (Chabbouh and Cherif, 1990, FAO Plant Prot. Bull. 38: 52-53.).

Ornamental hosts include: Chinese asters, chrysanthemum, delphinium, sage, geranium, gilia, gladiolus, heliotrope, hyacinth, larkspur, lily, marigold, nasturtium, violet, petunia, flox, dragon's mouth, tulip, and ( Chupp and Sherf, 1960; Agrios, 1978).

(5) Potato Y virus (PVY) - one of the most important plant viruses, which affect potato production.

(6) Cauliflower mosaic virus (CaMV, virus to DNA (Rothnie et al., 1994)).

(7) African cassava mosaic virus (ACMV).

(8) Sharka virus (PPV) is the most devastating viral disease of stone fruits of the Prunus genus.

(9) Bromine mosaic virus (BMV) usually

Κ f [Ό I ^ ί ^ 5ΐ? · '' Βί »2 _ IVJL Jl JL itl'W * β^ο infects Bromus inermis and other herbs, pi. ·. ·. . «^ ¡Í -

INDUSTRIAL virtually everywhere wheat is grown.

(10) Potato virus X (PVX) There are no vector insects or fungi for this virus. This virus causes mild symptoms, or does not cause symptoms, in most varieties of potatoes, but when potato virus Y is present, the synergy between these two viruses causes severe symptoms in potatoes.

Additional viruses:

Citrus sadness virus (CTV) - causes the most economically damaging disease of citrus, including bitter orange (Citrus aurantium} and any other Citrus species grafted on a bitter orange, orange foot) C. sinensis), grapefruit (C. paradisi), lime and Seville orange (C. aurantifolia), and tangerine (C.

reticulata). CTV is also known to infect Aeglopsis chevalieri, Afraegle paniculata, Pamburus missionis and

Passiflora graoilis. CTV has a global distribution and can be found anywhere citrus trees grow.

Barley yellow dwarf virus (BYDV) viral disease of cereals with the widest distribution.

It affects the economically important species of barley, oats, wheat, corn, triticale and rice.

100

Leaf curl virus d &lt;

infects potatoes and other family members

Solanaceae.

The tomato bush dwarf virus (TBSV), virus to DNA, is a member of the genus of Tombusviruses and mostly affects tomatoes and aubergines.

Additional revisions:

Hamilton et al., 1981, J Gen Virol 54; 223-241 mentions TMV, PVX, PVY, CMV, and CaMV.

Additional scientific articles:

Makkouk et al., 2012, Adv Virus Res 84; 367-402 - Viruses that affect peas and beans with narrow host range (broad bean necrotic yellowing virus (FBNYN)) and broad host spectrum (mosaic virus alfalfa (AMV) and CMV).

The plant virus target gene encodes an essential product for the viability and / or infection capacity of the viral pathogen, therefore its down regulation (mediated by naked dsRNA) results in a reduced ability of the pathogen to survive and infecting host cells.

Therefore, such down regulation results in a detrimental effect for the maintenance of the viability and / or infection capacity of the viral pathogen, since it prevents or reduces the pathogen's ability to feed and survive.

101 from nutrients derived cells

By virtue of this decrease in the viability and / or capacity of infection of the viral pathogen, resistance is facilitated and / or favors tolerance to infection by a pathogen of the plant cells. Treatment can be directed against genes in the mature (adult), immature (juvenile), or embryonic stages.

As used herein, a plant virus resistance trait is a characteristic of a plant that makes the host plant resistant to attack by a viral pathogen that is usually capable of inflicting damage or loss on the plant. . Once the pathogen is supplied with plant material that includes the naked dsRNA, expression of the gene within the target virus is suppressed and suppression of gene expression in the target virus results in resistance of the plant to the virus.

<td>With</td><td>the</td><td>In order to consolidate antiviral activity, the</td>

<td>Present</td><td colspan="2">disclosure also contemplates the control of</td>

<td>reduction</td><td>of</td><td>the capacity of infection or replication and the</td>

<td>level of</td><td>the</td><td>host symptoms after said</td>

treatment.

In order to enhance antiviral activity, embodiments of the present invention provide a composition containing two or more different agents, each

102 one of which is toxic to the at least one of them comprising a dsRNA described herein. In some embodiments, the second agent may be an agent selected from the group consisting of metabolic enzyme inhibitors that participate in the synthesis of amino acids or carbohydrates; cell division inhibitors, cell wall synthesis inhibitors, DNA or RNA synthesis inhibitors, gyrase inhibitors, tubulin assembly inhibitors, ATP synthesis inhibitors, kinase inhibitors

MAP, inhibitors of lipid synthesis or oxidation, inhibitors of ester synthesis and inhibitors of melanin synthesis.

On the other hand, plants that are generated in accordance with the disclosures of the present invention or parts thereof may exhibit altered nutritional or therapeutic efficacy and as such may be used in the food or feed and drug industries. Similarly, plants that are generated in accordance with the disclosures of the present invention or parts thereof may exhibit altered oil or cellulose content and as such may be implemented in the construction or oil industry.

The seeds of the present invention can be packaged in a seed storage device comprising a

103 plurality of seeds (at least some example, 5%, 10% or more) contain a naked exogenous dsRNA, where the seed is devoid of a heterologous promoter to promote expression of the dsRNA.

The device for storing seeds can be a bag, a plastic bag, a paper bag, a container with a soft structure or a container with a hard structure.

The reagents of the present invention can be packaged in a kit comprising naked dsRNA, instructions for introducing dsRNA into the seeds, and optionally a conditioning solution.

The compositions of some embodiments of the invention may, if desired, be presented in a dispenser package or container, which may contain one or more dosage forms containing the active ingredient. The package may, for example, comprise a metal or plastic foil, such as a blister pack. The package or dispensing device may be accompanied by instructions for insertion into the seed.

According to an exemplary embodiment, the naked dsRNA and the co nditioning solution are contained in separate containers.

As used herein, the term

OR,

Around · around refers to ± 10

104

The terms comprise, comprising including / n, possessing / n and its conjugates mean including / n without being limited to.

The term consists of means including / n and is limited to.

The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the composition , claimed method or structure.

As used herein, the singular form un, una and el includes plural references unless the context clearly dictates otherwise. For example, the term a compound or at least one compound can include a plurality of compounds, including mixtures thereof.

Throughout this application, various embodiments of the present invention may be presented in the range format. It should be understood that the description in the range format is provided merely for convenience and brevity and is not to be understood as an inflexible limitation on the scope of the invention. Accordingly, it should be considered that the description of a range has disclosed

105 specifies all possible individual numeric sub-ranges within that range. For example, it should be considered that the description of a range such as between 1 and 6 has specifically disclosed sub-ranges such as between

one and 3, between 1 and 4, between 1 and 5, between 2 and 4, between 2 and 6, between 3 and 6, etc., as well as the individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. The above applies regardless of the width of the range.

Whenever a range is indicated herein, it is understood that it includes any number (rational or integral) within the indicated range. The phrases "ranging from a first indicated number to a second indicated number and ranging from a first indicated number to a second indicated number" are used interchangeably herein and mean that the first and second indicated numbers are included and all the rational and integral numbers between them.

As used herein the term method refers to ways, forms, techniques, and procedures for accomplishing a given task including, but not limited to, those ways, forms, techniques, and procedures that are either known or can be readily developed by practitioners of chemical, pharmacological, biological, biochemical disciplines

106 and medical.

It should be appreciated that certain features of the invention, which, for clarity, are described in the context of separate embodiments, may also be disclosed in combination in a single embodiment. Conversely, various features of the invention, which, for the sake of brevity, are described in the context of a single embodiment, may also be disclosed separately or in a suitable subcombination or in any other convenient manner. described embodiment.

Certain features that are described in the context of various embodiments are not considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Various embodiments and aspects of the present invention as outlined above and claimed in the claims section, below, are experimentally supported by the following examples.

EXAMPLES

Reference is now made to the following examples, which, in conjunction with the foregoing descriptions, illustrate the invention in a non-limiting manner.

In general, the nomenclature used in the

107 present memory and the techniques of laboi,

INDUSTRIAL used in the present invention include recombinant DNA, molecular, biochemical, microbiological, and molecular techniques. A detailed description of such techniques is found in the literature. See for example

Molecular Cloning: A laboratory Manual Sambrook et al., (1989); Current Protocols in Molecular Biology Volumes IIII Ausubel, RM, ed. (1994); Ausubel et al., Current

Protocols in Molecular Biology, John Wiley and Sons,

Baltimore, Maryland (1989); Perbal, A Practical Guide to

Molecular Cloning, John Wiley &amp; Sons, New York (1988);

Watson et al., Recombinant DNA, Scientific American Books,

New York; Birren et al. (eds) Genome Analysis: A Laboratory

Manual Series, Vols. 1-4, Coid Spring Harbor Laboratory

Press, New York (1998); methodologies as set forth in

US Patent Nos. 4,666,828; 4,683,202;

4,801,531; 5,192,659 and 5,272,057; Cell Biology: A Laboratory

Handbook, Volumes I-III Cellis, JE, ed. (1994); Culture of Animal Cells - A Manual of Basic Technique by Freshney,

Wiley-Liss, NY (1994), Third Edition; Current Protocols in Immunology Volumes I-III Coligan JE, ed. (1994);

Stites et al. (eds), Basic and Clinical Immunology (8th

Edition), Appleton &amp; Lange, Norwalk, CT (1994); Mishell and

Shiigi (eds)

Selected Methods in Cellular Immunology, W.

108

H. Freeman and Co., New York (1980); detailed description of the immunoassays available in the

<td>literature</td><td colspan="2">patent and scientific, see,</td><td>for example,</td>

<td>Patents</td><td>Americans Nos.</td><td> 3.791.932;</td><td> 3.839.153;</td>

<td> 3.850.752;</td><td> 3.850.578; 3.853.987;</td><td> 3.867.517</td><td> ; 3.879.262;</td>

<td> 3.901.654;</td><td> 3.935.074; 3.984.533;</td><td> 3.996.345</td><td> ; 4.034.074;</td>

4,098,876; 4,879,219; 5,011,771 and 5,281,521; Oligonucleotide

Synthesis Gait, MJ, ed. (1984); Nucleic Acid

Hybridization Hames, BD, and Higgins SJ, eds. (1985);

Transcription and Translation Hames, BD, and Higgins S.

J., eds. (1984); Animal Cell Culture Freshney, RI, ed.

(1986); Immobilized Cells and Enzymes IRL Press, (1986); TO

Practical Guide to Molecular Cloning Perbal, B., (1984) and

Methods in Enzymology Vol. 1-317, Academic Press; PCR

Protocols: A Guide To Methods and Applications, Academic

Press, San Diego, CA (1990); Marshak et al., Strategies for

Protein Purification and Characterization - A Laboratory

Course Manual CSHL Press (1996); all of which are incorporated by reference herein as if fully disclosed herein. Other general references are provided throughout this document. The techniques disclosed in the aforementioned publications are considered well known in the art and are provided for the convenience of the reader. All the

109 information contained in such publications by reference in this report.

EXAMPLE 1

PROTOCOLS FOR dsRNA PRODUCTION AND TREATMENT

OF THE SEED

Generation of dsRNA / siRNA sequences

The dsRNA sequences were specifically designed for each gene using in vitro transcription of the PCR products. Part of the mRNA, including either the ORF,

3 'UTR or 5' UTR for which the dsRNA to be produced was amplified by PCR using gene-specific primers, which contain the T7 promoter sequence on either side. This product was used as a template for dsRNA production using commercial kits such as the MaxiScript dsRNA kit (Life Technologies) or T7 High Yield

RNA Synthesis kit (NEB). The sample was then treated with DNase Turbo at 37 ° C for 15-30 min followed by phenol treatment and nucleic acid precipitation.

Next, one of two different reactions was carried out: (1) dsRNA is ready for use, (2) dsRNA was processed with Dicer (Shortcut RNase III (NEB)) to create small interference RNAs (siRNA).

Either dsRNA or a combination of dsRNA and siRNA is

110 used for the semil treatments described below. All of the dsRNA sequences disclosed herein are listed as DNA (a simple transformation is performed by converting T&gt; U).

General protocol for seed treatment for gene silencing using a dsRNA / siRNA mix

The uncoated organic corn seeds were of the popcorn variety, the uncoated organic whole grain rice and organic soybeans were purchased from Nitsat Haduvdevan (Israel). The wheat seeds were from AB Seeds (Israel). The lettuce seeds were of the Sun Valley variety. Fresh tomato seeds were obtained from M82 tomato fruits, which were propagated in the researchers' establishment. The uncoated or fresh seeds were washed with double distilled water (DDW) prior to treatment for hours. The seeds were then dried at 20-30 ° C for up to 24 hours. After the drying step, the seeds were treated with a solution containing the dsRNA formulation, which is prepared with dsRNA at a final concentration of 1-261 pg / ml in 0.lmM EDTA.

The treatment was carried out by gently shaking the seeds in the solution for up to 60 hours in a

111 dark growth at 15-25 ° C. Finally, 1 briefly washed up to three times and planted in the soil or germinated at 25 ° C in a dark growth chamber and planted in soil or dried for 0-30 hours and germinated at 2 5 ° C in a dark growth chamber and planted in soil or planted directly in soil. Control seeds were treated similarly, with a solution that did not contain dsRNA or with nonspecific dsRNA.

EXAMPLE 2

STABILITY OF THE dsRNA IN RICE, TOMATO AND

SORGHUM

As an example for an exogenous gene that is not present / not expressed in plants, ORFs encoding the replicase and the CGMMV covering protein (Cucumber green mottle mosaic virus, registration number) were used.

AF417242) as targets for dsRNA treatment of plant seeds using the protocol described in

Example 1. Rice, tomato and sorghum seeds were washed for 4 hours at 20 ° C, and tomato and sorghum seeds were dried at 30 ° C and rice seeds at 20 ° C overnight. The seeds were immediately treated at 15 ° C with 132.7 pg / ml dsRNA (final concentration) for 39 hours in the case of

112

<td>rice,</td><td>as shown</td><td>in</td><td>the</td><td>Figure</td><td> 1; 9:</td><td></td><td></td>

<td colspan="3">(final concentration) during</td><td> 48</td><td>hours</td><td>for him</td><td>tomato like</td><td>I know</td>

<td>shows</td><td>in the figure</td><td>2A and</td><td> 75</td><td>pg / ml</td><td>dsRNA</td><td colspan="2">(concentration</td>

<td>final)</td><td>for 40 hours</td><td>for</td><td>the</td><td>sorghum</td><td>how I know</td><td>shows in</td><td>the</td>

<td>5 Figure</td><td>2B. Briefly,</td><td>the</td><td colspan="3">Derived ORFs</td><td>virus</td><td>I know</td>

amplified by PCR with forward and reverse primers containing the T7 sequence (5'-TAATACGACTCACTATAGGG-3 ', SEQ ID

NO: 1) on your 5 '(see Table 1, below). The PCR products were purified from agarose gel and since they transport the T7 primers at both ends they were used as templates for in vitro transcription dependent on

T7, deriving in the dsRNA product of the CGMMV genes. The PCR of a constitutive gene, tubulin (direct primer 5'-GGTGCTCTGAACGTGGATG-3 '(SEQ) was used as a positive control

ID NO: 2), and reverse primer 5'-CATCATCGCCATCCTCATTCTC-3 '(SEQ

ID NO: 3)).

Table 1: The PCR primers served as templates for in vitro transcription and detection of CGMMV, and 20 CGMMV dsRNA products.

<td></td><td>N</td><td></td><td></td><td>Fattening</td><td>Zeb</td>

<td>Nom</td><td>ombre</td><td></td><td></td><td>dor</td><td>adore</td>

<td>bre of</td><td>of the</td><td>Sequence</td><td>of the</td><td>direct / S</td><td>reverse/</td>

<td>virus</td><td>produ</td><td>product / SEQ ID NO:</td><td></td><td>EQ ID NO:</td><td>SEQ ID</td>

113

..... or'"......._

<td></td><td>cto</td><td></td><td>INSTITUTE MEaíCAN MÍ, A PROFÍEDA</td><td>) ......................... llÉPto ) ...............</td>

<td></td><td></td><td>TAATACGACTCACTATAGGG</td><td></td><td>With</td>

<td></td><td></td><td>GGTAAGCGGCATTCTAAACCTCCAA</td><td></td><td>together 1:</td>

<td></td><td></td><td>ATCGGAGGTTGGACTCTGCTTCTGA</td><td>TAAT</td><td>TAA</td>

<td></td><td></td><td>AGAGTCCAGTTCTGTTTCTTTTGAA</td><td>ACGACTCAC</td><td>TACGACTC</td>

<td></td><td></td><td>GATGGCTTACAATCCGATCACACCT</td><td>TATAGGGGG</td><td>ACTATAGG</td>

<td></td><td></td><td>AGCAAACTTATTGCGTTTAGTGCTT</td><td>TAAGCGGCA</td><td>GGAAGACC</td>

<td></td><td></td><td>CTTATGTTCCCGTCAGGACTTTACT</td><td>TTCTAAACC</td><td>CTCGAAAC</td>

<td></td><td></td><td>TAATTTTCTAGTTGCTTCACAAGGT</td><td> /5</td><td>TAAGC / 4</td>

<td></td><td></td><td>ACCGCTTTCCAGACTCAAGCGGGAA</td><td></td><td></td>

<td></td><td></td><td>GAGATTCTTTCCGCGAGTCCCTGTC</td><td></td><td></td>

<td></td><td></td><td>TGCGTTACCCTCGTCTGTCGTAGAT</td><td></td><td></td>

<td></td><td></td><td>attaattctagattcccagatgcgg</td><td></td><td></td>

<td></td><td></td><td>_{GTTTTTACGCTTTCCTCAACGGTCC}</td><td></td><td></td>

<td></td><td></td><td>TGTGTTGAGGCCTATCTTCGTTTCG</td><td></td><td></td>

<td></td><td></td><td>CTTCTCAGCTCCACGGATACGCGTA</td><td></td><td></td>

<td> 1)</td><td></td><td>ATAGGGTCATTGAGGTTGTAGATCC</td><td></td><td></td>

<td>CGMMV</td><td></td><td>TAGCAATCCTACGACTGCTGAGTCG</td><td></td><td></td>

<td>(NCBI</td><td>P</td><td>CTTAACGCCGTAAAGCGTACTGATG</td><td></td><td>With</td>

<td>number</td><td>roduc</td><td>ACGCGTCTACGGCCGCTAGGGCTGA</td><td></td><td>together 2:</td>

<td>of</td><td>to</td><td>GATAGATAAT TTAATAGAGT CTAT T</td><td></td><td>ACT</td>

<td>registry</td><td>CGMMV</td><td>TCTAAGGGTTTTGATGTTTACGATA</td><td>CTTC</td><td>CAGCAGTC</td>

<td>AF417242</td><td>dsRNA</td><td>GGGCTTCATTTGAAGCCGCGTTTTC</td><td>TTATGTTCC</td><td>GTAGGATT</td>

<td> )</td><td> 1</td><td>GGTAGTCTGGTCAGAGGCTACCACC</td><td>CGTCAGG / 7</td><td>G / 6</td>

114 ϊ

<td></td><td></td><td>TCGAAAGCTTAGTTTCGAGGGTCTT</td><td colspan="2"></td>

<td></td><td></td><td>CCCCTATAGTGAGTCGTATTA / 8</td><td></td><td></td>

<td></td><td></td><td>TAATACGACTCACTATAGGG</td><td></td><td></td>

<td></td><td></td><td>GCTTTACCGCCACTAAGAACTCTGT</td><td></td><td></td>

<td></td><td></td><td>ACACTCCCTTGCGGGTGGTCTGAGG</td><td></td><td></td>

<td></td><td></td><td>CTTCTTGAATTGGAATATATGATGA</td><td></td><td></td>

<td></td><td></td><td>TGCAAGTGCCCTACGGCTCACCTTG</td><td></td><td></td>

<td></td><td></td><td>TTATGACATCGGCGGTAACTATACG</td><td></td><td></td>

<td></td><td></td><td>CAGCAC TT GTT CAAAGGTAGATCAT</td><td></td><td></td>

<td></td><td></td><td>ATGTGCATTGCTGCAATCCGTGCCT</td><td></td><td></td>

<td></td><td></td><td>AGATCTTAAAGATGTTGCGAGGAAT</td><td></td><td></td>

<td></td><td></td><td>GTGATGTACAACGATATGATCACGC</td><td></td><td></td>

<td></td><td></td><td>AACATGTACAGAGGCACAAGGGATC</td><td></td><td></td>

<td></td><td></td><td>TGGCGGGTGCAGACCTCTTCCAACT</td><td></td><td></td>

<td></td><td></td><td>TTCCAGATAGATGCATTCAGGAGGT</td><td></td><td></td>

<td></td><td></td><td>ACGATAGTTCTCCCTGTGCGGTCAC</td><td></td><td></td>

<td></td><td></td><td>CTGTTCAGACGTTTTCCAAGAGTGT</td><td></td><td>With</td>

<td></td><td></td><td>TCCTATGATTTTGGGAGTGGTAGGG</td><td></td><td>together 3:</td>

<td></td><td>Ρ</td><td>ATAATCATGCAGTCTCGTTGCATTC</td><td>TAAT</td><td>TAA</td>

<td></td><td>roduc</td><td>AATCTACGATATCCCTTATTCTTCG</td><td>ACGACTCAC</td><td>TACGACTC</td>

<td></td><td>to</td><td>ATCGGACCTGCTCTTCATAGGAAAA</td><td>TATAGGGGC</td><td>ACTATAGG</td>

<td></td><td>CGMMV</td><td>ATGTGCGAGTTTGTTATGCAGCCTT</td><td>TTTACCGCC</td><td>GCATCACC</td>

<td></td><td>dsRNA</td><td>TCATTTCTCGGAGGCATTGCTTTTA</td><td>ACTAAGAAC</td><td>ATCGACCC</td>

<td></td><td> 2</td><td>GGTTCGCCTGTAGGTAATTTAAATA</td><td> /10</td><td>TAAAC / 9</td>

115

GTATTGGGGCTCAGTTTAGGGTCGA

MPI

Trreτο Mexican DI! THE INDUSTRY ITEM :.

<img file="MX360866B_D0025.tif" />

TGGTGATGCCCTATAGTGAGTCGTA

TTA / 11

Exogenous dsRNA was found to be stable for at least three weeks in rice seedlings as can be seen in Figures 1A-C and at least 10 days in tomato seedlings and four weeks in sorghum plants as seen in Figures 2A-B.

EXAMPLE 3

THE dsRNA IS NOT INTEGRATED IN THE RICE GENOME

Rice seeds were treated with an exogenous dsRNA as in Example 2. Plants were germinated and grown for five weeks, DNA was extracted, and PCR reactions were performed to demonstrate that dsRNA did not integrate into the rice genome. (Figure 3). Two sets of primers were used that gave a positive reaction when verifying the RNA level, set 1 (see Table 1) of primers was the set of primers that was used to amplify the template (the entire dsRNA sequence). Set 2 (see Tablel) are the primers that are

116 they were used in PCR in Figure 1. It was constitutive of endogenous rice (tubulin) as a positive control for the PCR reaction (see Table 2).

Table 2: Tubulin primers used for PCR amplification.

<td>Name</td><td>and</td><td>Sequence of</td><td>Lon</td>

<td>direction</td><td>of the</td><td>primer / (SEQ ID NO :)</td><td>gitude</td>

<td>primer</td><td></td><td></td><td>of the</td>

<td></td><td></td><td></td><td>primer</td>

<td>TubAl Bear</td><td> _73</td><td>GGTGCTCTGAACGTGGATG /</td><td> 19</td>

<td>6F</td><td></td><td> 12</td><td></td>

<td>TubAl Bear</td><td> _13</td><td>CATCATCGCCATCCTCATTC</td><td> 22</td>

<td>42R</td><td></td><td>TC / 13</td><td></td>

EXAMPLE 4

EXOGENOUS dsRNA MOLECULES ARE HIGHLY STABLE AND

ARE NOT INCORPORATED IN THE GENOME OF TREATED PLANTS

Corn seeds were treated using the protocol

117 described in Example 1, the seeds were washed

20 ° c, dried at 30 ° C overnight and immediately treated with 40 pg / ml dsRNA (final concentration) directed against the β-glucuronidase (GUS) reporter gene for hours at 15 ° C, dried and they germinated. Leaves and roots were harvested from control and dsGUS-treated plants 7 and 15 days after germination. It was extracted

RNA was harvested from tissues and RT-PCR was run with GUS-specific primers (Table 3). Furthermore, an endogenous corn constitutive gene (ubiquitin) was used as a positive control for the PCR reaction. The molecules of

GUS dsRNA were extremely stable in treated seeds, and can be detected in corn plants 7 and 15 days after seed germination (Figures 4AC). Also, the GUS dsRNA molecules were not incorporated into the treated corn plants one week after germination (Figures 5A-B).

Table 3: Primers for PCR amplification of the GUS and Ubiquitin genes and the GUS dsRNA product.

<td>Name</td><td>Primer Sequence / SEQ ID NO:</td><td> 1</td>

<td>re del</td><td></td><td>ongi</td>

<td>primer</td><td></td><td>your D</td>

<td></td><td></td><td>of the</td>

118

<td></td><td></td><td>dor</td>

<td>GUS_</td><td>TAATACGACTCACTATAGGGAGATCGACGGCCTGT</td><td></td>

<td>T7 For</td><td>GGGCATTC / 15</td><td></td>

<td>GUS_</td><td>TAATACGACTCACTATAGGGAGCATTCCCGGCGGG</td><td>i</td>

<td>T7 Rev</td><td>ATAGTCTG / 16</td><td> 3</td>

<td>GUS2</td><td></td><td></td>

<td>08 For</td><td>CAGCGCGAAGTCTTTATACC / 17</td><td> 3</td>

<td>GUS2</td><td></td><td>TO</td>

<td>8 9Rev</td><td>CTTTGCCGTAATGAGTGACC / 18</td><td> 0</td>

<td>zmaU</td><td></td><td>F TO</td>

<td>BQ-947F</td><td>CCATAACCCTGGAGGTTGAG / 19</td><td> 0</td>

<td>zmaU</td><td></td><td>TO</td>

<td>BQ1043R</td><td>ATCAGACGCTGCTGGTCTGG / 20</td><td> 0</td>

<td>Prod</td><td>TAATACGACTCACTATAGGGAGATCGACGGCCTGT</td><td></td>

<td>GUS ucto</td><td>GGGCATTCAGTCTGGATCGCGAAAACTGTGGAATTGATCA</td><td></td>

<td>dsRNA</td><td>GCGTTGGTGGGAAAGCGCGTTACAAGAAAGCCGGGCAATT</td><td></td>

<td></td><td>GCTGTGCCAGGCAGTTTTAACGATCAGTTCGCCGATGCAG</td><td></td>

<td></td><td>ATATTCGTAATTATGCGGGCAACGTCTGGTATCAGCGCGA</td><td></td>

<td></td><td>AGTCTTTATACCGAAAGGTTGGGCAGGCCAGCGTATCGTG</td><td></td>

<td></td><td>CTGCGTTTCGATGCGGTCACTCATTACGGCAAAGTGTGGG</td><td></td>

<td></td><td>TCAATAATCAGGAAGTGATGGAGCATCAGGGCGGCTATAC</td><td></td>

<td></td><td>GCCATTTGAAGCCGATGTCACGCCGTATGTTATTGCCGGG</td><td></td>

<td></td><td>AAAAGTGTACGTATCACCGTTTGTGTGAACAACGAACTGA</td><td></td>

119

ACTGGCAGACTATCCCGCCGGGAATGCTCCCTATA

TCGTATTA / 21

EXAMPLE 5

FLUORESCENCE MOCROSCOPY OF SYRNA SEQUENCES

ON SEVERAL PLANT SEEDS

Plant seeds were treated following the protocol described in Example 1. Seeds were washed for 4 h at 20 ° C, dried at 25 ° C and immediately treated with a fluorescent siRNA (siGLO, 2μΜ final concentration, Thermo Scientific) at 15 ° C for 24 h.

The quality of the siGLO was verified by gel electrophoresis analysis as can be seen in Figure 6.

Fluorescent images were taken from the seeds 24-48 hours after treatment using a microscope.

Olympus at the lowest multiplication level of the target &lt;5X for larger seeds such as rice and tomato seeds, and 10X for smaller seeds such as arabidopsis seeds). In order to eliminate the possibility of non-specific fluorescence, each seed treated with dsRNA is shown next to a control seed that received no treatment (Figures 7-8).

In order to evaluate the efficiency in the distribution of

120 fluorescent siRNA inside the seed!

different seeds of sliced ​​plants and images of these were captured with a fluorescent binocular 48 hours after treatment. Images of each seed were captured along with a control seed that received no treatment. Light and fluorescence images were taken as appropriate for samples of rice, tomato, cucumber, bean, sorghum and wheat seeds (Figures 9-12). It is evident that the siRNA is distributed at various levels between the embryo and the endosperm. The above supports the following models:

The dsRNA molecules enter the embryo directly, carried by the water-based solution that is used for seed treatment.

DsRNA molecules enter the endosperm as part of the endosperm's water absorption process. These lu8ego molecules feed the embryo as it develops as part of the nutrient flow from the endosperm during germination and seed development.

The present findings suggest that the molecules of

RNA used to treat seeds both penetrate the embryo and function in the embryo as it develops as well as penetrate the endosperm and feed the embryo after germination.

121

EXAMPLE 6

EXPERIMENT OF THE TEMPORARY COURSE OF THE TREATMENT WITH SIGLO

A time course experiment was performed for rice seeds to monitor the kinetics of siGLO penetration into the seeds after seed treatment (Figure 13). The results indicate that siRNA efficiently penetrates plant seeds using the protocol described in

Example 1.

EXAMPLE 7

SILENCING THE PDS-1 GENE IN RICE USING A

DsRNA / siRNA MIX

The rice seeds were washed in a washing solution for 4 h at 20 ° C, dried at 25 ° C and immediately treated with a dsRNA / siRNA mixture at a total concentration of 60 pg / ml at 15 ° C for 40 hours. The seeds were germinated at room temperature for several days and the development of the seed was monitored. The seeds treated with PDS and the dsRNA / siRNA mixture exhibited a slow and delayed development, as can be seen from the smaller size of the seedlings and the limited rooting. In these experiments,

122 two products of the PDS-1 gene are combined (see Ί

Table 4: Two PDS-1 gene products to be silenced by mixing dsPU ^ / siPNR.

<td></td><td>Nom</td><td>Oh</td><td>Number</td><td>Sequence</td><td>Sequence</td>

<td>bre</td><td>of the</td><td>ganism</td><td>or of</td><td>nucleotide</td><td>nucleotide</td>

<td>gen</td><td></td><td></td><td>registry</td><td>of the product</td><td>of the product</td>

<td></td><td></td><td></td><td>NCBI</td><td>1 / SEQ ID NO:</td><td>2 / SEQ ID NO:</td>

<td></td><td>Phy</td><td>Ze</td><td>BT084</td><td>TAATACGACTC</td><td>TAATACGACTC</td>

<td colspan="2">toene</td><td>to mays</td><td> 155.1</td><td>ACTATAGGGAGAT T GG</td><td>ACTATAGGGTGATCGG</td>

<td colspan="2">Desaturate</td><td></td><td></td><td>CGAGCTTAGGATTGAG</td><td>GTGAACGATGAGGTTT</td>

<td>I know</td><td>(PDS-</td><td></td><td></td><td>GATCGTTTACAGTGGA</td><td>TTATTGCAATGTCCAA</td>

<td> 1)</td><td></td><td></td><td></td><td>AAGAACACTCTATGAT</td><td>GGCACTCAATTTCATA</td>

<td></td><td></td><td></td><td></td><td>ATTCGCCATGCCAAAC</td><td>AATCCTGATGAGCTAT</td>

<td></td><td></td><td></td><td></td><td>AAGCCAGGAGAATT CA</td><td>CTATGCAGTGCATTTT</td>

<td></td><td></td><td></td><td></td><td>GCCGGTTTGATTTCCC</td><td>GATTGCTTTGAACCGA</td>

<td></td><td></td><td></td><td></td><td>AGAAACTTTGCCAGCA</td><td>TTTCTTCAGGAGAAGC</td>

<td></td><td></td><td></td><td></td><td>CCTATAAATGGGATAT</td><td>ATGGTTCTAAAATGGC</td>

<td></td><td></td><td></td><td></td><td>GGGCCATATTGAGAAA</td><td>ATTCTTGGATGGTAAT</td>

<td></td><td></td><td></td><td></td><td>CAATGAAATGCTTACC</td><td>CCGCCTGAAAGGCTAT</td>

<td></td><td></td><td></td><td></td><td>TGGCCCGAGAAGGTGA</td><td>GCATGCCTATTGTTGA</td>

<td></td><td></td><td></td><td></td><td>AGTTTGCAATCGGACT</td><td>TCACATTCGGTCTAGG</td>

<td></td><td></td><td></td><td></td><td>TCTGCCAGCAATGGTT</td><td>GGTGGAGAGGTCCGCC</td>

123

GGTGGTCAACCTTATG

TTGAAGCTCAAGATGG

CTTAACCGTTTCAGAA

TGGATGAAAAAGCAGG

GTGTTCCTGATCGGGT

GAACGATGAGGTTTTT

ATTGCAATGTCCAAGG

CACTCAATTTCATAAA

TCCTGATGAGCTATCT

ATGCAGTGCATTTTGA

TTGCTTTGAACCGATT

T CT T CAGGAGAAGCAT

GGTTCTAAAATGGCAT

TCTTGGATGGTAATCC

GCCTGAAAGGCTATCT

CCCTATAGTGAGTCGT

ATTA / 44

TGJ

AAAGATAGAGCTGAAT

CCTGATGGAACTGTAA

AACACTTCGCACTTAG

TGATGGAACTCAGATA

ACTGGAGATGCTTATG

TTTGTGCAACACCAGT

CGATATCTTCAAGCTT

CTTGTACCTCAAGAGT

GGAGTGAAATTACTTA

TTTCAAGAAACTGGAG

AAGTTGGTGGGAGTTC

CTGTTATCAATGTTCA

TATATGGTTTGACAGA

AAACTGAACAACACAT

ATGACCACCTTCTTTT

CAGCAGGAGTTCACTT

TTAAGTGTCTATGCAG

ACATGTCAGTAACCTG

CAAGGAATACTATGAC

CCAAACCGTTCAATGC

TGGCCCTATAGTGAGT

CGTATTA / 45

124

The experiment was carried out

<img file="MX360866B_D0026.tif" />

biological repeats and the results are presented in the

Figures 14A-D.

EXAMPLE 8

CHLOROPHYLL WHITENING AND GROWTH INHIBITION

AFTER THE SILENCING OF PDS

The rice seeds were treated as described in Example 7 and their subsequent seedling growth and development was monitored. Thirty days after the PDS-1 silencing treatment, the general phenotype of the two groups of plants, those of control and those with PDS silencing, was recorded. Silencing PDS has been reported to cause chlorophyll bleaching and inhibition of growth (Peretz et al., 2007, Plant Physiol 145:

1251-1263), which correlates with the phenotype of the PDS-silencing plants of the invention, since they are smaller in size and paler in color, respectively, compared to control plants (see Figure 15).

EXAMPLE 9

DETECTION OF THE TWO PRODUCTS OF THE PDS-1 GENE BY PCR IN

REAL TIME

After treatment with the dsRNA / siRNA mixture

125 (1: 1 ratio) as described in determine expression levels of gene products

PDS-1 by real-time PCR using specifically designed primers (Direct: GATTGCTGGAGCAGGATTAG SEQ

ID NO: 46, Reverse: CCCTTGCCTCAAGCAATATG, SEQ ID NO: 47). For normalization purposes, the expression of UBQ5 was also determined using primers (direct

ACCACTTCGACCGCCACTACT, SEQ ID NO: 48, reverse

ACGCCTAAGCCTGCTGGTT, SEQ ID NO: 49). The results are shown in Figures 16A-C.

EXAMPLE 10

SILENCE OF GENE DIANA HAP2E

Rice seeds were treated using the protocol described in Example 1, The seeds were washed for 4 h at room temperature, dried overnight at 25 ° C and immediately treated with a concentration of Hap2e dsRNA of 152pg / ml, for 41 hours at 15 ° C (for the Hap2e dsRNA sequences see Table 11 below). Control rice seeds and those treated with Hap2e dsRNA that germinated 5 days after treatment did not show any difference in their root development (Figure 17). RNA was extracted from the sprouts of the germinated seeds, 5 and 7 days after germination, and RT-PCR was run. Then

126 to test 3 different sets of primer:

located in various regions of dsRNA molecules (Figure

18), the main primer set (primer set 3) was used to assess the expression levels of endogenous Hap2e in dsRNA treated plants versus control (untreated) plants. Down-regulation of Hap2e mRNA expression was achieved in the treated plants, at a level of about 50% or more silencing compared to control plants, with an efficiency of 25% (Figure 19).

Other rice seeds were treated under the same conditions as those in Figure 17 with a concentration of

Hap2e dsRNA of 145.7 pg / ml, for 42 hours. RT-PCR using random primers + Oligo dT on RNA extracted from seedlings 18 days after germination also exhibited down-regulation of Hap2e mRNA in plants treated with dsRNA (Figure 20), with 50% efficiency of reaching the down regulation of more than 25% compared to control.

Table 5: Primers used for RT-PCR of Hap2e dsRNA molecules

<td>Co</td><td>Ub</td><td>Name</td><td>Sequence of</td><td>L</td>

<td>together</td><td>ication</td><td>and direction</td><td>primer / SEQ ID NO:</td><td>ongitu</td>

<td>of</td><td>of the</td><td>primer</td><td></td><td>d of</td>

127

C ".......

<td>primer is</td><td>conjunct or of primer is</td><td></td><td>ϊΝΧΠΧϋΐΟ MEXiCAr M LA INDUSTrí</td><td>0L „-^: 11 «β&gt; Ι1ίβΐ | Ο D ..... iffi .......... í or r</td>

<td> 1</td><td>In</td><td>osaHAP</td><td>ACCGGCATCAGCTCAG</td><td> 2</td>

<td></td><td>dsRNA</td><td>2E501F3</td><td>TCTC / 50</td><td> 0</td>

<td></td><td></td><td>osaHAP</td><td>TGCTGTTCTCTGGGCA</td><td> 2</td>

<td></td><td></td><td>2E589R3</td><td>CAGG / 51</td><td> 0</td>

<td> 2</td><td>A</td><td>osaHAP</td><td>TCCCCTCAGATATTAA</td><td> 2</td>

<td></td><td>ion</td><td>2E11F5</td><td>CAAC / 52</td><td> 0</td>

<td></td><td></td><td>osaHAP</td><td>AGGAGGAAAGGCAGCT</td><td> 2</td>

<td></td><td></td><td>2E108R5</td><td>TCTGTG / 53</td><td> 2</td>

<td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td>

<td> 3</td><td>Af</td><td>osaHAP</td><td>GTGACTCGTCACCAAC</td><td> 2</td>

<td></td><td>out of</td><td>2E122F7</td><td>AAAG / 54</td><td> 0</td>

<td></td><td>dsRNA</td><td>osaHAP</td><td>TGTGTTGTCCGTTGAG</td><td> 2</td>

<td></td><td></td><td>2E202R7</td><td>ACTG / 55</td><td> 0</td>

EXAMPLE 11

SILENCING OF THE DIANA NFY GENE IN CORN SEEDS

Corn seeds were treated using the protocol described in Example 1, The seeds were washed for 4 h at room temperature, dried overnight at 30 ° C and

128 they were immediately treated with a concentration:

56pg / ml, for 40 hours at 15 ° C (for the sequence of NFY dsRNA see Table 11). RT-PCR was performed on the RNA extracted from control shoots and seeds treated with NFY dsRNA 10 days after germination to determine the level of expression of the NFY target gene (see Table 6). Downregulation of the gene was successfully achieved as shown in Figure 21.

Table 6: Primers used for RT-PCR of NFYA dsRNA molecules. in corn seeds 3 10 days after germination.

<td>Name</td><td>and</td><td>Sequence of</td><td>Lon</td>

<td>direction</td><td>of the</td><td>primer</td><td>gitude</td>

<td>primer</td><td></td><td></td><td>of the</td>

<td></td><td></td><td></td><td>primer</td>

<td>zma-NFYA3_</td><td> _345</td><td> /57</td><td> 20</td>

<td>F3</td><td></td><td>TCGGAAGCCGTACCTTC</td><td></td>

<td></td><td></td><td>GTG</td><td></td>

<td>zma-NFYA3</td><td></td><td>CCTGGAGCTGCTGCTTTG</td><td> 20</td>

<td>_442R3</td><td></td><td>TG / 58</td><td></td>

<td>zma-</td><td></td><td>TACCAGGCGTCGAGTGGT</td><td> 20</td>

<td>NFYA3_457F4</td><td></td><td>TC / 59</td><td></td>

<td colspan="2">zma-NFY-</td><td>GAAGAGGGCGTGCAAATG</td><td> 20</td>

129

<td>A3 542R4</td><td>GG / 60</td><td></td>

<td></td><td></td><td></td>

EXAMPLE 12

SILENCING OF THE DIANA NFY GENE IN TOMATO SEEDS

Tomato seeds were treated using the protocol described in Example 1. The unwashed seeds were treated with a concentration of NFY dsRNA of 200pg / ml, for 24 hours at 15 ° C, the seeds were briefly washed twice and immediately planted in soil without drying. It has been made

RT-PCR on the RNA extracted from control sprouts and tomato seeds treated with NFY dsRNA 3 weeks after germination to determine the level of expression of the NFY target gene (see Table 7). Downregulation of the gene was successfully achieved as shown in Figure

22.

Tomato plants 55 days after treatment with the NFY dsRNA molecules were compared with control plants of the same age. The comparison showed important evident phenotypic differences, most notably a change in height was observed, being that the treated plants were significantly shorter than the untreated control plants (Figures 23, 24).

Table 7: Primers used for RT-PCR of molecules

130 of NFYA dsRNA in tomato and product NFY dsRNA

<td rowspan="2">Name and direction primer</td><td rowspan="2">Primer / SEQ ID sequence NOT:</td><td colspan="2">L ongitu</td>

<td>d EC r</td><td>of the ; bado</td>

<td>slyNFY A125F3</td><td>CTATTGCGTGTGCTCCAAAC / 61</td><td> 0</td><td> 2</td>

<td>slyNFY A212R3</td><td>ACATGAGGAGGAACCAAAGG / 62</td><td> 0</td><td> 2</td>

<td>Produc</td><td>CTAATACGACTCACTATAGGGAGAGGCTCAA</td><td></td><td>P</td>

<td>to NFY</td><td>GAACCAGTTTATGTTAATGCTAAGCAGTATCGAAGG</td><td colspan="2">roduct</td>

<td>dsRNA 1</td><td>ATCCTGCAGCGAAGACAGTCACGTGCTAAAGCAGAA CTTGAAAAGAAGCAAATAAAGGGTAGAAAGCCATAT CT TC ACGAGTC TC GACAT C AGC AT GC AC T GAGGAGG GTAAGGGCCTCGGGTGGACGTTTTGCCAAAAAGACA GATGCTTCTAAGGGTACTGGTTCTGTGAGTTCATCG GGTTCTGAACCTTTGCAGTTCAATGCTGCTGATATT CAAAAGAGGAATGAAAATGGAAGGTTGGCCGAGCTT CAGCAGTCTTATTCAAATGGTAGCAGTTATGGCAAT CAAAGTAGCTTTCAAGAATCCAAGGATGAGTACCAG TTTGCTAAAAGCAGGGAAGGAGGTTTTTTTGTCAAG TAATTGGAGATACGTT CAT GT GTAAACTAGC T CT TG CCCTCTCCCTATAGTGAGTCGTATTAG / 63</td><td>or</td><td> 1</td>

131

<td></td><td colspan="2"></td>

<td>Produc to NFY dsRNA 2</td><td>CTAATACGACTCACTATAGGGAGAGCAGTTA TGGCAATCAAAGTAGCTTTCAAGAATCCAAGGATGA GTACCAGTTTGCTAAAAGCAGGGAAGGAGGTTTTTT TGTCAAGTAATTGGAGATACGTTCATGTGTAAACTA GCTCTTGCCCTGCAACGAGGGTAGAGTATGAGCAAG AGGAGTTTACAGGGATTGTTTCATTTCTTGGCTTTT CAAGATAGGCGGCAATTCATTCTTGGCTTTTTACTT TAGTGTTAAAGGGAGCAACAGAGGTGACGAGGGTAT CAGTGTTGCAGCATTTGCTTGGAGATTACATCTTCC CT TAT GTACAGAGAT GGAT GAAC TTAGAACTAGGAT TAGAAAGTTTTTCAGTAAGTTTATGTTTGGCCAGTT ACTGTAGTTTTAGTTTAGGAGACCATGTAAAAAGGT TGTTAGTTTTGCAAAAGGATCTTTTTTCTTTCCCTA ATTGGTGCATTCTCCCTATAGTGAGTCGTATTAG / 6 4</td><td>P roduct or 2</td>

EXAMPLE 13

SILENCING OF THE DIANA NAC GENE IN CORN SEEDS

Corn seeds were treated using the protocol described in Example 1, the seeds were washed for 4 h at room temperature, dried overnight at 30 ° C and immediately washed with a concentration of NAC dsRNA concentration of 90pg / ml, for 40 hours at 15 ° C e

132

<img file="MX360866B_D0027.tif" />

immediately germinated (due to the sequence

INDUSTRIAL see Table 11). RT-PCR was performed on the RNA extracted from control sprouts and corn seeds treated with NAC dsRNA days after germination to determine the expression level of the target NAC gene (see Table 8). Downregulation of the gene was successfully achieved as shown in Figure 25.

Table 8: Primers used for RT-PCR of NAC dsRNA molecules in corn.

<td>Name and</td><td>Sequence of</td><td>Lon</td>

<td>address of</td><td>primer</td><td>gitude</td>

<td>primer</td><td></td><td>of the primer</td>

<td>zmaNAC5 267F3</td><td>/ 65 CGAGTCGGGATACTGGA AGG</td><td> 20</td>

<td>zmaNAC5_342R3</td><td>CTTCTTCATGCCGACGAG GG / 66</td><td> 20</td>

<td>zmaNAC5 187F4</td><td>ACGATGGGCGAGAAGGAG TG / 67</td><td> 20</td>

<td>zmaNAC5_250R4</td><td>TCAGTCCCGTCGGGTACT TG / 68</td><td> 20</td>

133

EXAMPLE 14

SILENCING THE DIANA ARF-8 GENE IN RICE SEEDS

Rice seeds were treated using the protocol described in Example 1, the seeds were washed for 4 h, dried overnight at 20 ° C and immediately treated with an ARF-8 dsRNA concentration of 66.2pg / ml, for hours at 15 ° C. RT-PCR was performed on RNA extracted from control rice seeds and those treated with ARF-8 dsRNA 18 days after germination to determine the level of expression of the target gene ARF-8 (see Table 9). Downregulation of the gene was successfully achieved as shown in Figure 26.

Table 9: Primers used for RT-PCR of ARF-8 mRNA molecules in rice and in ARF-8 dsRNA product

<td>Name</td><td>Primer sequence</td><td> 1</td>

<td>and direction</td><td></td><td>ongi</td>

<td>primer</td><td></td><td>your D</td>

<td></td><td></td><td>of the</td>

<td></td><td></td><td>fat</td>

<td></td><td></td><td>dor</td>

<td>osaARF</td><td>AGGGTCACATCCCGAACTAC / 69</td><td></td>

134

<td>8_140F3</td><td colspan="2"></td>

<td>osaARF 8_233R3</td><td>ACCTCGTCAGTCTCCACATC / 70</td><td>z i. 0</td>

<td>osaARF 8_1674F4</td><td>GTTGGATTCGAGCTTCCTTC / 71</td><td>• VM- or</td>

<td>osaARF 8_1757R4</td><td>TGCTGCTGCTCACTAGCTAC / 72</td><td>'T'S- OR</td>

<td>Produc to ARF8 dsRNA</td><td>CTAATACGACTCACTATAGGGAGACAGTCCGTT GGCCTAGTTCCTATTGGAGATCTGTGAAGGTTGGTTGG GATGAATCAACTGCAGGGGAAAGACCACCAAGAGTTTC TTTATGGGAAATTGAACCATTGACAACCTTTCCAATGT ATCCATCTCTGTTCCCACTGAGAGTTAAGCATCCTTGG TATTCAGGAGTTGCTTCCCTGCATGATGACAGCAATGC TTTAATGTGGCTGAGAGGAGTTGCTGGTGAGGGAGGTT TTCAGTCTCTGAACTTTCAGTCACCTGGTATTGGCTCC TGGGGACAACAGAGGCTCCATCCATCCTTACTGAGCAG C GAT CAC GAT CAGTACCAAGCAGTAGT TGCT GCT GCT G CTGCTTCCCAATCTGGTGGTTACTTAAAACAGCAATTC TTGCACCTTCAGCAACCTATGCAGTCCCCTCAAGAACA CTGCAACCTCAACCCTCTCCCTATAGTGAGTCGTATTA G / 73</td><td></td>

EXAMPLE 15

SILENCING OF THE DIANA SPL17 GENE IN RICE SEEDS

135

Rice seeds were treated using

OF PROPERTY described in Example 1, the seeds were washed for 4 h, dried overnight at 20 ° C and immediately treated with a concentration of SPL17 dsRNA of 200pg / ml, for 41 hours at 15 ° C (for the sequence of SPL17 dsRNA see

Table 11). Control rice seeds and those treated with SPL17 dsRNA that germinated 5 days after treatment did not observe any visual difference (Figure 27). RNA from 5-day-old shoots was extracted from these sprouted seeds and RT-PCR was run to determine the expression levels of SPL17 in the control and treated plant groups. Two different sets of primers were tested (see Table 10), located in various regions of the dsRNA molecules (Figure 28). When RT-PCR was run on RNA extracted from 14-week-old plants, down-regulation of SPL17 mRNA expression was achieved in highly efficient treated plants compared to control plants (Figure 29).

Table 10: Primers used for RT-PCR of SPL17 dsRNA molecules in rice seeds 5 days after germination.

<td>Conju</td><td>Name</td><td>and</td><td>Sequence</td><td>of the</td><td>Lon</td>

<td>nto and</td><td>direction</td><td>of the</td><td>primer</td><td></td><td>gitude</td>

136

<td>Location of primers</td><td>primer</td><td></td><td>primer</td>

<td> 1</td><td>osaSPL17</td><td>CTCAGCCATGGGATACTAC</td><td> 20</td>

<td>in dsRNA</td><td>119F3</td><td>C / 74</td><td></td>

<td></td><td>osaSPL17__</td><td>GCTGGCCGTTGACGACATT</td><td> 20</td>

<td></td><td>189R3</td><td>G / 75</td><td></td>

<td> 2</td><td>osa spll7</td><td>TTCAGCCACTCCACCAATG</td><td> 19</td>

<td>outside of</td><td>454Fwd</td><td> /76</td><td></td>

<td>dsRNA</td><td>osa spll7</td><td>AAGAAGATGAT CAAT GGT C</td><td> 21</td>

<td></td><td>512Rev</td><td>TC / 77</td><td></td>

EXAMPLE 16

Silencing the MicroRNA Target Genes with Complementary dsRNA / siRNA

The high specificity and efficiency of post-transcriptional gene silencing by target gene-specific dsRNA has enabled a preferred method of generating eukaryotic organisms with the preferred phenotype, where expression of one or more genes is decreased or inactivated. DsRNA sequences have been designed to silence microRNA target genes of maize (Zea mays) and rice (Oryza sative). Specifically, microRNAs that have been shown to be associated with stress tolerance will be used.

137 abiotic. Table 11 below provides examples for target gene sequences that are produced using PCR amplification to test the gene silencing capabilities of their respective dsRNA / siRNA mixtures. These dsRNA molecules will then be used to override the endogenous level of the selected target genes.

Table 11: Sequences of the target gene and primers for PCR

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>L</td>

<td></td><td></td><td></td><td>N</td><td></td><td>C</td><td>C</td><td>ongit</td>

<td>D</td><td>C</td><td></td><td>number</td><td></td><td>stabber</td><td>stabber</td><td>You</td>

<td>iana</td><td>in</td><td> 0</td><td>of</td><td></td><td>direct</td><td>invers</td><td>of the</td>

<td>for</td><td>dian</td><td>rganis</td><td>register</td><td>Sequence</td><td>o / SEQ</td><td>o / SEQ</td><td>produ</td>

<td>Mir</td><td>to</td><td>mo</td><td>ro</td><td>target / SEQ ID NO:</td><td>ID NO:</td><td>ID NO:</td><td>cto</td>

<td>m</td><td></td><td>Z</td><td>N</td><td>GATGAAGATCATGG</td><td>T</td><td>T</td><td> 5</td>

<td>IR169</td><td>FY-</td><td>ea</td><td>M_0011</td><td>GAAGGATAATCAGGACACA</td><td>AATACG</td><td>AATACG</td><td>37 bp</td>

<td></td><td>A3</td><td>mays</td><td> 53839</td><td>TTGAAGCCAGTATTGTCCT</td><td>ACTCAC</td><td>ACTCAC</td><td></td>

<td></td><td></td><td></td><td></td><td>TGGGGAAGGAAGGGTCTGC</td><td>TATAGG</td><td>TATAGG</td><td></td>

<td></td><td></td><td></td><td></td><td>CTTTTTGGCCCCAAAAATA</td><td>GCCGCA</td><td>GTGCAT</td><td></td>

<td></td><td></td><td></td><td></td><td>GATTACAACCCGTCTTTTC</td><td>TGCCAT</td><td>GCCGTT</td><td></td>

<td></td><td></td><td></td><td></td><td>CTTATATTCCTTATACTGC</td><td>TGTCCA</td><td>CACGAC</td><td></td>

<td></td><td></td><td></td><td></td><td>TGACGCTTACTATGGTGGC</td><td>TCC / 84</td><td>CAG / 85</td><td></td>

<td></td><td></td><td></td><td></td><td>GTTGGGGTCTTGACAGGAT</td><td>T</td><td>T</td><td> 6</td>

138

<img file="MX360866B_D0028.tif" />

139

ΑΡ2Ε

O ryza sativa

TO

B28803

1.1

GTGAGCAAGGTGGAGGCGG

CTCGAAGCTGGTCGTGAAC

GGCATGCAGCAGCGTGTTT

C CAC CATAAGGT GAAGAGA

AGTGGGCACGACACCAT TC

CCAGGCGCGCACTGCCTGT

GGCAACTCATCCTTGGCTT

TT GAAAC TAT GAATAT GCA

ATGGACATGTAGCTTTGAG

TTCCTCAGAATAA / 78

TCAGTGTTTGTCCC

CT CAGATATTAACAACAAT

GATAGTTGTGGGGAGCGGG

ACCATGGCACTAAGTCGGT

ATTGTCTTTGGGGAACACA

GAAGCTGCCTTTCCTCCTT

CAAAGTTCGATTACAACCA

GCCTTTTGCATGTGTTTCT

TATCCATATGGTACTGATC

CATATTATGGTGGAGTATC

AACAGGATACACT TCACAT

GCATTTGTTCATCCTCAAA

TTACTGGTGCTGCAAACTC

TAGGATGCCATTGGCTGTT

T

AATACG

ACTCAC

TATAGG

GCTGCC

TTTCCT

CCTTCA

AAGTTC / 88

AT'AATACG

ACTCAC

TATAGG

GCATTG

T

AATACG

ACTCAC

TATAGG

GTGCTG

TTCTCT

GGGCAC

AGG / 89

T

AATACG

ACTCAC

TATAGG

GTTCGT bp bp

140

<img file="MX360866B_D0029.tif" />

360)866

141 m

IR156

PL17 ryza N19298 sativa 8.1

CACCGTGTCCCCGTCATGA

GGTGAAAACCTCGGGATCG

CGGGACACGGGCGGTTCTG

GTTTACCCTCACTGGCGCA

CTCCGGTGTGCCCGTGGCA

ATTCATCCTTGGCTTATGA

AGTATCTACCTGATAATAG

TCTGCTGTCAGTTTATATG

CAATGCAACCTCTGTCAGA

TAAACTCTTATAGTTTGTT

TTATTGTAAGCTATGACTG

AACGAACTGT / 79

<img file="MX360866B_D0030.tif" />

CATGGTGGCTCAGC

GGCTGGGGCACCAATGCTC

CACCACCCAGCCTTTGAGC

TCACCTCAGGTGGATGTCT

CGCGGGAGTCGCCACCGAC

TCCAGCTGTGCTCTCTCTC

TTCTGTCAACTCAGCCATG

GGATACTACCCAAAGCACC

AGCAGCCACAACCGGTCCC

CGCCAATGTCGTCAACGGC

CAGCGCCTTCGGAGGCGGC

AACAACCCGGTGTCGCCCT

<td>T</td><td>T</td><td> 5</td>

<td>AATACG</td><td>AATACG</td><td>00 bp</td>

<td>ACTCAC</td><td>ACTCAC</td><td></td>

<td>TATAGG</td><td>TATAGG</td><td></td>

<td>GTCACC</td><td>GCATTG</td><td></td>

<td>TCAGGT</td><td>GTGGAG</td><td></td>

<td>GGATGT</td><td>TGGCTG</td><td></td>

<td>CTC / 92</td><td>AAG / 93</td><td></td>

<td>T</td><td>T</td><td> 4</td>

<td>AATACG</td><td>AATACG</td><td>97 bp</td>

<td>ACTCAC</td><td>ACTCAC</td><td></td>

<td>TATAGG</td><td>TATAGG</td><td></td>

142

<img file="MX360866B_D0031.tif" />

143

- &gt; »

<img file="MX360866B_D0032.tif" />

144

CCCGCAGGGTCGTCCCCGG

CGGCGGCTGGTGCTCTCTC

TCTTCTGTCATCGGCCAGA

GGCAGCGTGGCGGGCGCCA

GCGGGCCCTGGCTGGTCAC

GGCGGCGCGGGAGGACATC

CCGGCGCGCTCCAGCGCGG

CGCTCGACGACCTTATCGC

CGAGAACCGCGCCGCCGCG

CTCCTCGCGCGGCAGTACT

TCGTCTCCGACCGCTCGCC

GGCGCCCAGACGGGATTTC

GTCGCCTCT / 81

<img file="MX360866B_D0033.tif" />

IR164

AC (TF homo logo

O ryza sativa

N

M_0010

64881.

ATGAGCGGGATGAA

TTCGCTGAGCATGGTGGAG

GCGAGGCTGCCGCCGGGGT

TCAGGTTCCACCCGCGAGA

CGACGAGCTCGTGCTGGAC

TACCTGGAAAGGAAGCTCC

TCGACGGCGGCGTGGGCGG

CGCCGCGGCGGCGGCGGCG

GCGGTCACCATCTACGGCT

GCCCGGTGATGGTCGACGT

CGATCTCAACAAGTGCGAG

<td>T</td><td>T</td>

<td>AATACG</td><td>AATACG</td>

<td>ACTCAC</td><td>ACTCAC</td>

<td>TATAGG</td><td>TATAGG</td>

<td>GTTCAG</td><td>GCCGTT</td>

<td>GTTCCA</td><td>GGCAGC</td>

<td>CCCGCG</td><td>TTGGCA</td>

<td>AGAC / 1</td><td>ATGG / 1</td>

<td> 00</td><td> 01</td>

T

AATACG

T

AATACG

5 bp bp

145 * Item

<img file="MX360866B_D0034.tif" />

146

IMPI

<td></td><td></td><td></td><td></td><td>GGTTATGAGCAAGTGCCCT</td><td> 1</td><td>: anus ....... i ...... ssill OLÁD ..... j ΓκϊΜ</td><td>Are »</td><td></td>

<td></td><td></td><td></td><td></td><td>GCTTCTCCAATAATCCCTC</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td>TCAACAGCCATCGTCGTCG</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td>ATGAATGTTCCGTTGACAT</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td>CGGCCATGGTTGATCAAGA</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td>GCAAAACAATATGGGTAGG</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td>GCGATCAAGGATGTGCTGA</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td>GCCAATT / 82</td><td></td><td></td><td></td><td></td>

<td></td><td> 1</td><td>Ζ</td><td>Ν</td><td>ATGGAGCACGACGT</td><td>Τ</td><td>τ</td><td></td><td> 5</td>

<td></td><td>AC5</td><td>ea</td><td>Μ_0011</td><td>GCACCACCAGCAGGCCATG</td><td>AATACG</td><td>AATACG</td><td> 65</td><td>bp</td>

<td></td><td></td><td>mays</td><td> 54298.</td><td>GAGCTGCCGCCGGGGTTCC</td><td>ACTCAC</td><td>ACTCAC</td><td></td><td></td>

<td></td><td></td><td></td><td> 1</td><td>GATTCCACCCCACCGACGA</td><td>TATAGG</td><td>TATAGG</td><td></td><td></td>

<td></td><td></td><td></td><td></td><td>GGAGCTCATCACGCACTAC</td><td>GCCACC</td><td>GCGACG</td><td></td><td></td>

<td></td><td></td><td></td><td></td><td>CTCGCCAGGAAGGCCGCCG</td><td>GACGAG</td><td>TCCTCC</td><td></td><td></td>

<td></td><td></td><td></td><td></td><td>ACGCCCGCTTCGCCCCGCG</td><td>GAGCTC</td><td>ACCAAC</td><td></td><td></td>

<td></td><td></td><td></td><td></td><td>CGCCGTCGGCGAGGCCGAC</td><td>ATC / 10</td><td>ATC / 10</td><td></td><td></td>

<td></td><td></td><td></td><td></td><td>CT CAACAAGTGCGAGCCAT</td><td> 4</td><td> 5</td><td></td><td></td>

<td></td><td></td><td></td><td></td><td>GGGACCTGCCATCCCGGGC</td><td>Τ</td><td>T</td><td></td><td> 6</td>

<td></td><td></td><td></td><td></td><td>GACGATGGGCGAGAAGGAG</td><td>AATACG</td><td>AATACG</td><td> 64</td><td>bp</td>

<td></td><td></td><td></td><td></td><td>TGGTACTTCTTCTGCGTCA</td><td>ACTCAC</td><td>ACTCAC</td><td></td><td></td>

<td></td><td></td><td></td><td></td><td>AGGACCGCAAGTACCCGAC</td><td>TATAGG</td><td>TATAGG</td><td></td><td></td>

<td></td><td></td><td></td><td></td><td>GGGACTGAGGACGAACCGG</td><td>GAGGCC</td><td>GTCAGG</td><td></td><td></td>

<td></td><td></td><td></td><td></td><td>GCCACCGAGTCGGGATACT</td><td>GACCTC</td><td>AAGAAC</td><td></td><td></td>

<td></td><td></td><td></td><td></td><td>GGAAGGCGACGGGCAAGGA</td><td>AACAAG</td><td>TGGCCC</td><td></td><td></td>

147

<img file="MX360866B_D0035.tif" />

148

<img file="MX360866B_D0036.tif" />

3, i

EXAMPLE 17

SILENCING THE ARF-8 GENE IN TOMATO SEEDS

Tomato seeds were treated using the protocol described in Example 1, the unwashed seeds were treated with an ARF-8 dsRNA concentration of 200pg / ml, for 24 hours at 15 ° C and immediately planted in soil. Gene expression levels were examined using RTPCR, 3 and 8 weeks after treatment (see Table 12). Changes in expression were observed in dsRNA-treated plants 3 weeks after treatment (Figure 30A-B).

Plants that were treated with the dsRNA molecules specific for the ARF8 gene showed a phenotypic difference compared to the control ones. This phenotypic difference was observed at different times (55, 62 and 72 days) and was demonstrated by an increase in height (Figures 31AC). While the height of the control plants was ~ 36cm, the dsRNA-treated plants reached an average height of ~ 30cm (Figure 31D). In addition to lower height (delayed vertical development), plants treated with dsRNA were more branched (increased development

149 horizontal) compared to both plants, the plants treated with the specific dsRNA for ARF8 were shorter and more branched in relation to their control counterparts 55 and 72 days after treatment, as can be seen in Figures 32A and 32B.

Table 12: Primers used for RT-PCR of ARF-8 mRNA molecules in Tomato and ARF-8 dsRNA product

<td>Name</td><td>Primer sequence</td><td>L</td>

<td>and direction</td><td></td><td>ongitu</td>

<td>primer</td><td></td><td>d of priming r</td>

<td>slyARF</td><td>CCTCAACAGTCCTGGATGTC / 108</td><td> 2</td>

<td>8_1816F4</td><td></td><td> 0</td>

<td>sly</td><td>CCCGTAAGTTGGAAGTGATG / 10 9</td><td> 2</td>

<td>ARF_8_1896R4</td><td></td><td> 0</td>

<td>Product</td><td>CTAATACGACTCACTATAGGGAGAGCTCTT</td><td>P</td>

<td>or ARF 8</td><td>CCTCCCTACAACTGTGTCTAACGTCGCTACTACAT</td><td>roduct</td>

<td>dsRNA 1</td><td>CAATTGATGCTGATATATCCTCTATGCCACTAGGG ACTTCTGGATTTCCGAATCCCTTGTATAGTTATGT GCAAGATTCTACTGACTTGTTGCATAATGTAGGGC AAGCTGATGCACAAACTGTGCCCCGTACATTTGTC AAGGTTTACAAATCAGCGTCCCTTGGGAGGTCATT</td><td>or 1</td>

150

GGACATCACTCGGTTCAACAGCTATCATGAGC

GACAGGAATTAGGGCAGATGTTCGGTATCGAAGGG

TTGCTTGAAGACCCTCAAAGATCAGGCTGGCAGCT

TGTATTTGTTGACAGGGAGAATGATGTCCTTCTCC

TTGGAGACGATCCGTGGGAGGAATTTGTCAATAAT

GTTTGGTACATCAAAATTCTTTCACCCGAGGATGT

GCAGAAACTGGGGAAAGAGGAGGTTGGATCCCTCT

CCCTATAGTGAGTCGTATTAG / 110

Product or ARF 8 dsRNA 2

CTAATACGACTCACTATAGGGAGATGGGAG

ATTGAGCCTTTGACTACTTTTCCGATGTATCCATC

TCTTTTTCCTCTAAGGCTAAAGAGGCCTTTCTATC

AAGGAACCTCATCTTATCAGGATAGTAACAATGAA

GCTATTAATCGAATGTCATGGTTAAGAGGGAATGC

TGGTGAGCTAGGACATCATTCAATGAATCTTCAGT

CTTTTGGCATGCTTCCTTGGATGCAACAGAGAGTC

GATTCAACAATTCTCCCAAATGATATTAATCAGCA

CTATCAAGCTATGCTGGCTACTGGC

TTGCAAAGTTTTGGGAGTGGAGATTTACTG

AAACAGCAATTAATGCAGTTTCAGCAGCCTGTCCA

ATATCTGCAACATGCAAGTACTGAGAATTCAATTT

TGCATCAGCAGCAGCAGCAGCAGCAGCAAATAATG

CAGCAAGCAGTTCATCAGCATATGCTGCCTGCTCA

AACCCAAATGCTGTCAGAGAACCTTCAAAGGCAAT

P roduct 2

151

CCCAGCATCAATCCATCTCCCTATAGTGAGTCi

TTAG / 111

EXAMPLE 18

SILENCING THE FW2.2 GENE IN TOMATO SEEDS

Tomato seeds were treated using the protocol described in Example 1, the unwashed seeds were treated with a concentration of FW2.2 dsRNA of 100pg / ml, for 24 hours at 15 ° C they were immediately planted in soil. Gene expression levels were examined using RTPCR, 9 weeks after germination (primers are listed in Table 13). A decrease in

<td>about halfway down the level of</td><td>expression</td><td>of</td><td>FW2.2</td>

<td colspan="2">in dsRNA-treated plants compared</td><td colspan="2">with the</td>

<td>control plants (Figure 33).</td><td></td><td></td><td></td>

<td>Even so, the plants were treated with</td><td>molecules</td><td>of</td><td>dsRNA</td>

specific for the FW2.2 gene did not show phenotypic differences compared to control plants, ruling out a toxic effect as an alternative explanation for the phenotypic effects seen in the previous examples. The plants presented a similar height and appearance 72 days after treatment (Figure

34) .

152

Table 13: Primers used for RT-PCR of the FW2.2 dsRNA molecules in tomato and FW2.2 dsRNA product

<td>Name hey</td><td>Primer sequence</td><td>L ongit</td>

<td>direction</td><td></td><td>You</td>

<td>of the</td><td></td><td>of the</td>

<td>primer</td><td></td><td>fat</td>

<td></td><td></td><td>or</td>

<td>slyFW</td><td>GAGGCACCTTGTGTTGATTG / 112</td><td> 2</td>

<td>2_316F2</td><td></td><td> 0</td>

<td>slyFW</td><td>CAAAGCCACGGTTCTTAAGC / 113</td><td> 2</td>

<td>2_406R2</td><td></td><td> 0</td>

<td></td><td>CTAATACGACTCACTATAGGGAGATCCAGGTCC</td><td></td>

<td>Product</td><td>AATGAAACAACCTTATGTTCCTCCTCACTATGTATCTG</td><td></td>

<td>FW2.2</td><td>CCCCCGGCACCACCACGGCGCGGTGGTCGACTGGTCTT</td><td></td>

<td>dsRNA</td><td>TGTCATTGTTTTGATGACCCTGCTAACTGTTTAGTTAC</td><td></td>

<td></td><td>TAGTGTTTGCCCTTGTATCACCTTTGGACAGATTTCTG</td><td></td>

<td></td><td>AAATACTAAACAAAGGAACAACTTCATGTGGGAGTAGA</td><td></td>

<td></td><td>GGTGCATTATATTGTTTGCTGGGATTGACAGGATTGCC</td><td></td>

<td></td><td>TAGCCTATATTCCTGCTTCTACAGGTCTAAAATGAGGG</td><td></td>

<td></td><td>GGCAATATGATCTGGAAGAGGCACCTTGTGTTGATTGT</td><td></td>

<td></td><td>CTTGTACATGTATTCTGTGAACCTTGTGCTCTTTGCCA</td><td></td>

<td></td><td>AGAATACAGAGAGCTTAAGAACCGTGGCTTTGATATGG</td><td></td>

153

<img file="MX360866B_D0037.tif" />

XICANO

PIETY

Striatum

GAATAGGGTGGCAAGCTAATATGGATAGACAAAG

OF THE IRO!

GGAGTTACCATGCCCCCTTATCATGCAGGCATGACCTC

TCCCTATAGTGAGTCGTATTAG / 114

EXAMPLE 19

THE DOWNWARD REGULATION OF THE GEN DELLA IN RICE DRIVES IN

GREATER ROOT DEVELOPMENT OF GERM SEEDS

Rice seeds were treated using the protocol described in Example 1, the seeds were washed for 4 h, dried for 24 h at room temperature and immediately treated with a DELLA dsRNA concentration of 66pg / ml, for 36 hours at 15 ° C . The 10 rice seeds were treated with dsRNA directed against the Della gene (see

Table 15 below), which is a known repressor of plant growth. Arabidopsis seedlings with the mutant Della gene are larger with a more extensive root system (Josse, EM, Gan, Y., Bou-Torrent, J.,

Stewart, KL, Gilday, AD, Jeffree, CE, Vaistij, FE,

Martinez-Garcia, JF, Nagy, F., Graham, IA, and Halliday,

KJ (2011). A DELLA in disguise: SPATULA restrains the growth of the developing Arabidopsis seedlings. Plant cell

23: 1337-1351.). Figure 35 shows a mimicry of the phenotypes of using the treatment of the seeds with dsRNA, being that the treated seedlings were of greater

154 size and with roots more extensive than the plants

EXAMPLE 20

THE DOWN REGULATION OF THE NRR GENE IN RICE DRIVES IN

INCREASED DEVELOPMENT OF SEED ROOTS AND SPROUTS

GERMINATED

Rice seeds were treated using the protocol described in Example 1, the seeds were washed for 4 h, dried for 24 h at room temperature and immediately treated with a NRR dsRNA concentration of approximately 13pg / ml, for 36 hours at 15 ° C . Rice seeds were treated with dsRNA directed against the NRR gene, which was found to regulate root growth in response to macronutrients in rice (Zhang et al., 2012, Mol Plant 5 (1): 63 -72). Transgenic rice seedlings, with reduced levels of NRR using

RNAi were shown to have more extensive roots when grown under nitrogen limiting conditions. Figure 36 shows a mimic of this phenotype using the treatment of the seeds with dsRNA, being that the resulting treated seedlings were larger and with more extensive roots than the control seedlings.

Table 14: NRR dsRNA molecule products in rice

155

Primer Name and Address

Primer ongitu d primer sequence

Produ

CTAATACGACTCACTATAGGGAGAAGCTCCTG cto

NRR

AACCCATCATTGAAGAACCAGTGCTTAGCCTTGATCC roduct dsRNA 1

AGTTGCAGCAGCCATTTCGATGATGTCTGGCAGTGAG

AACGTAAT GGAT GAAAC TATAGAGGTT GCAGATAT CA

GCGACATTCAGAATGACTCTCTTTTAAGCGAAGTATT

ATACGAGTGCGAGAAGGAACTCATGGAGAAGTCCGCA

ATCGAAGAGACTATTTCTGAACTGCTGGACGTCAAGA

TTCCTATGCTGCAAGTGGAAGAGTTCCCTAGGGAAAC

CCAAGTACAACTACCGGCCATGGAGAAGGAGAAGCCA

TCAGTTCCTGAATGTTGTTCACTCCAGAAAAGTGTCA

GTTCTGGGTGCCTCAACTCAGCTGATTGGATCAATGG

ACCAGCCAGGCCAAACTTCCTGGACTTCCAAGGATTG

GACTTTGAGACAGCGTTTGGGTTGAGGAGGGCATACA

GCGAAGGAGACATTCTCCCTATAGTGAGTCGTATTAG / 115

Produ

C TAATAC GAC TCACTATAGGGAGACAT GGAGA cto

NRR

AGTCCGCAATCGAAGAGACTATTTCTGAACTGCTGGA dsRNA 2

CGTCAAGATTCCTATGCTGCAAGTGGAAGAGTTCCCT roduct or 2

AGGGAAACCCAAGTACAACTACCGGCCATGGAGAAGG

156

AGAAGCCATCAGTTCCTGAATGTTGTTCACTCCA

AAGTGTCAGTTCTGGGTGCCTCAACTCAGCTGATTGG

ATCAATGGACCAGCCAGGCCAAACTTCCTGGACTTCC

AAGGATTGGACTTTGAGACAGCGTTTGGGTTGAGGAG

GGCATACAGCGAAGGAGACATTCAGAATCTTGGAGCT

AGCACCCCTCGACCCGGGAACTCAGGAAACGCTCAAT

TAGCATCTTGCGAGAGGCTTGTAACCATCAGTGACCT

GAAATCTGAAGAAAGGAAGCAGAAGCTATCTAGGTAC

AGAAAGAAGAAGGT GAAGAGAAACTT T GGCAGAAAGA

TCAAGTATGCTTGCAGGAAGGCTCTCTCCCTATAGTG

AGTCGTATTAG / 116

EXAMPLE 21

SIMULTANEOUS SILENCING OF THREE ENDOGENOUS GENES

In the present example, the effect of simultaneous silencing of three genes is tested. Rice seeds were treated using the protocol described in Example 1, the seeds were washed for 4 h, dried overnight at room temperature and immediately treated with a solution containing a mixture (152.3pg / ml final concentration) of dsRNA against three genes: Hap2e (59.9pg / ml, see

Table 11), Della (44pg / ml see Table 15 below) and SQS (48.4pg / ml see Table 16 below) for 42 h at 15 ° C.

RNA was extracted from sprouts of the germinated seeds,

157 days after germination, and each of the three genes was run (see Table 15 below).

As can be seen in Figure 37, the down-regulation of the three genes was highly effective, since the treated plants exhibited a decrease in the expression of each individual gene in varied amounts, which varied between a minimum of a decrease in the 10% and total gene silencing (equals 100% down regulation).

Table 15: Primers used for RT-PCR analysis for the level of expression of the Hap2e, Della and SQS genes and the dsRNA products.

<td>Name</td><td>Primer sequence</td><td colspan="2">L</td>

<td>and direction</td><td></td><td colspan="2">ongitu</td>

<td>primer</td><td></td><td>d</td><td>of the</td>

<td></td><td></td><td colspan="2">priming</td>

<td></td><td></td><td>r</td><td></td>

<td>osaHAP</td><td>GTGACTCGTCACCAACAAAG / 117</td><td></td><td> 2</td>

<td>2E122F7</td><td></td><td> 0</td><td></td>

<td>osaHAP</td><td>TGTGTTGTCCGTTGAGACTG / 118</td><td></td><td> 2</td>

<td>2E202R7</td><td></td><td> 0</td><td></td>

<td>osaDel</td><td>CAGTTCGCGCACACCATTCG / 119</td><td></td><td> 2</td>

<td>lal410F5</td><td></td><td> 0</td><td></td>

158

<td>osaDel lal494R5</td><td>GCAGCATGAACGGCTCCAAG / 120</td><td> 0</td>

<td>osaSQS 465F3</td><td>TCCGCAATGCCGTGTGCATC / 121 •</td><td> 2 0</td>

<td>osaSQS 543R3</td><td>GCGGCAGGAATGCTAGTGTC / 122</td><td> 2 0</td>

<td>Produc to Della dsRNA</td><td>CTAATACGACTCACTATAGGGAGAGCCCACT TCTACGAGTCCTGCCCCTACCTCAAGTTCGCCCACT TCACCGCAAATCAAGCCATCCTCGAGGCTTTCGCCG GCTGCCACCGCGTCCACGTCGTCGACTTCGGCATCA AGCAGGGGATGCAATGGCCAGCTCTCCTCCAGGCCC TCGCCCTTCGTCCCGGCGGCCCCCCATCGTTCCGCC TCACCGGCGTCGGCCCCCCGCAGCCGGACGAGACCG ACGCCTTGCAGCAGGTGGGTTGGAAGCTTGCCCAGT TCGCGCACACCATTCGCGTCGACTTCCAGTACCGGG GACTCGTCGCCGCCACTCTCGCGGACTTGGAGCCGT TCATGCTGCAGCCGGAGGGCGAGGCGGACGCGAACG AGGAGCCTGAGGTGATCGCCGTCAACTCGGTGTTCG AGCTGCACCGGCTGCTCGCGCAGCCCGGCGCGCTGG AGAAGGTCCTGGGCACGGTGCACGCGGTGCGGCCAA GGATCGTCACCGTGGTAGAGTCTCCCTATAGTGAGT CGTATTAG / 123</td><td></td>

<td>Produc to SQS</td><td>CTAATACGACTCACTATAGGGAGAATATCTA CAACCGCGACTGGCATTATTCATGTGGAACAAAAGA</td><td>P roduct</td>

159

<td>dsRNA</td><td> 1</td><td>CTACAAATTACTGATGGATAAGTTTCGCCTTl CACGGCTTTCTTGGAGCTTGGTCAAGGTTATCAAGA GGCAATTGAAGAAATCACTAGGCTAATGGGAGCAGG aatggcaaaatttatctgcaaggaggttgaaactgt T GATGAC TACAAT GAGT ACT GT C ACT AT GT AGC AGG GCTAGTGGGGTATGGGCTTTCCAGGCTCTTTCATGC TGGTGGGACGGAAGATCTGGCTTCAGATTCACTTTC AAATTCAATGGGCTTGTTTCTGCAGAAAATCAATAT AATTAGGGAT TAT T TGGAGGACATAAACGAGATAC C AAAGTCACGTATGTTCTGGCCTCGAGAAATATGGAG TAAATATGTCAATAAACTCGAGGATTTGAAATACGA GGAAAATTCAGAAAAGGCAGTTCAGTGTTTGAATGA TATGGTGACTAACGCTCTGTCTCATCTCCCTATAGT GAGTCGTATTAG / 56</td><td></td>

<td colspan="2">Produc</td><td>CTAATACGACTCACTATAGGGAGACGCTCTG</td><td>P</td>

<td>to</td><td>SQS</td><td>TCTCATGCTGAAGACTGCCTCCAATACATGTCAGCA</td><td>roduct</td>

<td>dsRNA</td><td> 2</td><td>TTGAAGGATCATGCCATTTTCCGTTTTTGTGCAATA CCTCAGATAATGGCAATTGGGACATGTGCTATTTGC TACAATAATGTGAATGTCTTTAGAGGAGTTGTTAAG ATGAGGCGTGGGCTCACTGCACGAGTAATTGATGAG ACAAACACAATGT CAGAT GT CTATACT GCT TTCTAT GAGTTCTCTTCGCTGATAGAATCGAAGATTGATAAT AATGATCCAAATGCTTCCCTAACGCGGAAACGTGTT</td><td>or 2</td>

160

GATGCGATAAAGAGAACCTGCAAGTCATCTT ·

CTAAAGAGAAGGGGATACGATTTGGAGAAGTCAAAG

TACAACTCCATGCTGATAATGGTTGTACTTCTGTTG

GTGGCTCTCCCTATAGTGAGTCGTATTAG / 14

EXAMPLE 22

FLUORESCENCE MICROSCOPY OF SYRNA FRAGMENTS IN

TOMATO

Tomato seeds were treated with a fluorescent siRNA (siGLO, final concentration ΙμΜ, Thermo

Scientific) at 15 ° C for 24 h.

The seeds were sectioned and fluorescent images were taken 24 hours after treatment using a Leica confocal myroscope. As shown in the Figures

38A-D, a treated seed (Figures 38A, C) is shown along with a control seed that was buffered (Figures 38B, D). It is clear that the siRNA is distributed at various levels in the embryo and in the endosperm.

EXAMPLES 23-29

SPL EXPRESSION IS AFFECTED BY DOSE AND

KINETICS OF SEED TREATMENT WITH dsRNA

EXAMPLE 23

ALTERED SPL EXPRESSION AFTER TREATMENT WITH 50 pg / ml dsRNA IN TOMATO

161

Tomato seeds were treated with u

NO: 12 6) derived from the SPL gene and with GUS dsRNA as a control according to the protocol described in Example 1. The treatment was carried out by gently shaking the seeds in the solution for up to 24 hours in a dark growth chamber at 15- 25 ° C followed by washing with water three times for one minute. After treatment, the seeds were planted in soil and grown at around 25 ° C with a 16 hour photoperiod. The plants were watered with running water as necessary. The primers used for the in vitro transcription of SPL dsRNA and the sequence of the dsRNA are listed in Table 16. The sequence of GUS dsRNA is shown in Table 3.

Table 16: Primers used for in vitro transcription of SPL dsRNA and the resulting dsRNA product.

<td></td><td></td><td>Sequence / SEQ ID NO:</td><td>L</td>

<td>iana</td><td></td><td></td><td>ongitu</td>

<td></td><td></td><td></td><td>d (nt)</td>

<td></td><td>Fattening</td><td>CTAATACGACTCACTATAGGGAGATGGCC</td><td> 4</td>

<td></td><td>dor</td><td>CAATAGGTTCTCCTCA / 12 4</td><td> 5</td>

<td></td><td>direct</td><td></td><td></td>

<td>PL</td><td>Fattening</td><td>CTAATACGACTCACTATAGGGAGAGCTCTC</td><td> 4</td>

<td></td><td>dor</td><td>CATTGATGCTGATGC / 125</td><td> 4</td>

<td></td><td>reverse</td><td></td><td></td>

162 dsRN

C TAATACGACT CACTATAGGGAGAT (

CAATAGGTTCTCCTCATATGGATGGAAACTAACA

AATGGGAAGGGAAGAGAAGCATTACTGAAGCTGA

AAAGGAAGAGGATGAACATGGAAGTGTTGAAGAG

GATAGCAAAAGAAAAAGGGTATTGACTCTCTCTG

GTAGGAAGCTAGTTGGTGAAGGGTCGGCACATCC

TTCTTGCCAGGTCGATCAGTGCACTGCAGATATG

GCAGATGCCAAGCCATACCATCGCCGCCACAAGG

TGTGTGAGTTCCATTCAAAGTCTCCAATAGTACT

TAT TAGT GGAC TC CAGAAGC GAT TCT GT CAGCAA

TGTAGCAGATTTCATCTGTTAGCAGAGTTTGATG

ATGCTAAGAGGAGTTGCCGAAGGCGTTTGGCAGG

TCACAATGAGCGCCGCCGTAAAATTACATATGAC

T CT CATGGAGAAAAT TT GGGC T GAAGAAGCAT CA

GCATCAATGGCAGCTCTCCCTATAGTGAGTCGTA

TTAG / 126

Before all treatment, the concentration and purity of the dsRNA were evaluated by absorbance (NanoDrop) and by HPLC. The dsRNA concentration according to the NanoDrop measurement was 1864 pg / ml for SPL and 1964 pg / ml for GUS. HPLC showed that the sample contained mainly dsRNA but that other molecules (including nucleotides and ssRNA) were also present in the solution. According to

163 HPLC analysis, the SPL solution contained

IMPL

M INDUSTRIAL LROUD

<img file="MX360866B_D0038.tif" />

dsRNA and 838 pg / ml ssRNA. The GUS solution contained 633 pg / ml dsRNA and 167 pg / ml ssRNA (see Figures 39A-B).

The seeds were treated with dsRNA at a concentration of 50 pg / ml (determined by HPLC measurement) for minutes, 2, 6 and 24 hours. Further HPLC analysis of the dsRNA solutions for SPL and GUS after incubation of the seeds revealed that the ssRNA peak has disappeared (see Figure 39).

Total RNA was extracted from sprouted seed leaves, 17 days (in plants treated for 24 hours) and 18 days (in plants treated for 10 minutes, 2 and 6 hours) after treatment. CDNA was prepared using oligodT primers and the level of SPL mRNA expression was determined in treated and control plants by real-time PCR with

SYBR Green (Quanta BioSciences). The primers used for real-time PCR of SPL were located outside the dsRNA region, in the 3 'UTR. The primer sequences are listed in Table 17 along with the constitutive genes (Expressed and CAC) that were used as nomalizers (in the case of SPL Expressed was used as a normalizer).

Table 17: Primers used to determine the level

164 expression of SPL mRNA by PCR at time

<td>iana</td><td>D</td><td>odo in time] real</td><td>Mét PCR 30</td><td colspan="2"></td><td>Sequence / SEQ ID NO:</td><td>ongi your D (nt)</td>

<td></td><td></td><td></td><td></td><td></td><td>Fat</td><td>CAATTCCCGGATTTCTAAGC</td><td></td>

<td></td><td></td><td></td><td>SYB</td><td>or</td><td>direct</td><td> /127</td><td>C</td>

<td></td><td></td><td colspan="2">R Green</td><td></td><td>Fat</td><td>CCCTTTACACAAGGGAAATG</td><td></td>

<td></td><td></td><td></td><td></td><td>or</td><td>reverse</td><td> /128</td><td> 0</td>

<td>PL</td><td>S</td><td></td><td></td><td>or</td><td>Fat direct</td><td>TTCTGAAGCAACATAAACAA GATGTG / 129</td><td> 6</td>

<td></td><td></td><td>man</td><td>Taq</td><td>or</td><td>Fat reverse</td><td>AATTTGCTTAGAAATCCGGG AAT / 130</td><td> 3</td>

<td></td><td></td><td></td><td></td><td colspan="2">Taqma n probe</td><td>6FAM- TTAAGCATGCTCTCTATCT- MGBNFQ / 131</td><td> 9</td>

<td colspan="2">AND xpress</td><td></td><td>SYB</td><td>or</td><td>Fat direct</td><td>GCTAAGAACGCTGGACCTAA TG / 132</td><td> 2</td>

<td>ed</td><td></td><td colspan="2">R Green</td><td>or</td><td>Fat reverse</td><td>AGAATAGCATCCGGTCTCAG / 133</td><td> 0</td>

<td>AC</td><td>C</td><td>man</td><td>Taq</td><td>or</td><td>Fat direct</td><td>GGTGGCGCCTTTGATGAA / 1 3. 4</td><td> 8</td>

<td></td><td></td><td></td><td></td><td colspan="2">Fat</td><td>TCCAATAGCTCGTAAATCAG</td><td></td>

165 or reverse

AACAA / 135

VICTaqma n probe

ATGCCATCCGCAATAAMGBNFQ / 136

This analysis showed a significant up-regulation (Wilcoxon ranksum test, p value &lt;0.05) upregulation of SPL mRNA at all incubation times. The median level of expression of SPL in plants treated with SPL dsRNA for 10 minutes, 2 hours, 6 hours and 24 hours was 2.54,

2.85, 2.69 and 2.73 times higher than in the control plants treated with GUS dsRNA, respectively (Figures 40A-B to 43AB).

To verify that the effect on SPL mRNA derives specifically from treatment with SPL dsRNA, the level of SPL mRNA was measured after treatment with both GUS dsRNA (a control sequence that does not have a relevant homology with silencing in the tomato genome) and FW2.2 dsRNA (which is an endogenous tomato sequence, see Table 13). Plants treated with 50 pg / ml dsRNA for 24 hours showed upregulation of SPL when treated with the SPL sequence but not when treated with the GUS or FW2.2 sequences (Figures 40A-B). Figures 41A-B a

43A-B show the effect of varying the treatment period

166

I IVI Γ 1 eiiW * '' * β^ο on the expression of SPL. All the times

OF EA PROPERTY l | gj || O) Í ........

Assayed, that is, 24h-10 minutes, showed an effect on the expression levels of SPL mRNA.

In another experiment, tomato seeds of the Oregon Spring variety were treated with a dsRNA independently prepared for the SPL gene at a concentration of 50 pg / ml for 24 hours. The seeds were then washed three times with water, and transferred to seed growth boxes (9 seeds per box) containing

12 my water. The seeds in the boxes were germinated in a growth chamber in the light at 25 ° C for 7 days, and then the shoot tissue was collected for analysis of

RNA. The RNA analysis was carried out by Taqman, using a CAC test for tomato for the normalization of the values ​​to correct the differences in the concentration of the sample (Table 17). Table 18 shows that in seedlings germinated from seeds treated with SPL dsRNA, mRNA levels of the SPL gene were increased by a factor of 7 compared to seedlings germinated from seeds treated with GUS dsRNA. Treatment with AFR8 dsRNA and 0.1 mM EDTA (buffer) had no significant effect on SPL mRNA levels compared to treatment with GUS dsRNA.

167

Table 18: mRNA concentrations for tomato seedling shoot tissues.

<td>Tr atamien to</td><td>N number</td><td>R Q Half</td><td>D is V. Its T.</td><td>Q. OR change resp. GUS control</td><td>C ambio (multiple iplos )</td><td>Goes lor p of Dunnett</td>

<td>Bu f fer</td><td> 1 2</td><td> 0 .298</td><td> 0 .551</td><td> 330%</td><td> 4 .3</td><td> 0. 4723</td>

<td>GU S</td><td> 1 5</td><td> 0 .069</td><td> 0 .059</td><td> 0%</td><td> 1 .0</td><td> 1</td>

<td>YES .ARF8-1</td><td> 1 1</td><td> 0 . 196</td><td> 0 .338</td><td> 183%</td><td> 2 .8</td><td> 0. 846</td>

<td>YES .SPL</td><td> 1 8</td><td> 0 . 463</td><td> 0 . 655</td><td> 568%</td><td> 6 . 7</td><td> 0. 0577</td>

EXAMPLE 24

ALTERED SPL EXPRESSION AFTER DIVING OF

TOMATO SEEDS IN dsRNA

Tomato seeds were treated with a dsRNA derived from the SPL gene as described in Examples 1 and 23.

Treatment with dsRNA at a concentration of 50 pg / ml was carried out by immersing the seeds in the dsRNA solution at

168 room temperature and immediately wash double distilled (DDW). Total RNA was extracted from sprouted seed leaves 13 days after treatment. CDNA was prepared using oligo-dT primers and the level of SPL mRNA expression was determined in treated and control plants by real-time PCR, as described in

Example 23.

This analysis showed a significant upregulation of SPL mRNA (Figures 44A-B). The median level of expression of SPL in plants treated with SPL dsRNA was 1.7 8 times higher than in control plants soaked with 50 pg / ml GUS dsRNA.

EXAMPLE 25

ALTERED SPL EXPRESSION AFTER TREATMENT WITH 25 pg / ml dsRNA IN TOMOATE

Tomato seeds were treated with a dsRNA derived from the SPL gene as described in Examples 1 and 23.

The seeds were treated with dsRNA at a concentration of 25 pg / ml for 10 minutes, 2 and 24 hours. Total RNA was extracted from sprouted seed leaves, 17 days (in plants treated for 24 hours) and 18 days (in plants treated for 10 minutes and 2 hours) after treatment. CDNA was prepared using oligo-dT primers and

169 SPL mRNA expression level stopped

DE LA ΡΕΟΠΕρΑΓ) treated and controlled by real-time PCR, as described in Example 23.

This analysis showed a significant upregulation of SPL mRNA for incubation times of 2 and 24 hours, and an upregulation trend for an incubation time of 10 minutes (Figures 45A-B a 47AB). The median level of SPL expression in the plants treated with SPL dsRNA for 2 and 24 hours was 2.23 and 2.4810 times higher than in control plants treated with GUS dsRNA, respectively.

EXAMPLE 26

ALTERED EXPRESSION OF SPL AFTER TREATMENT WITH 1 or 5 pg / ml dsRNA IN TOMATO

Tomato seeds were treated with a dsRNA derived from the SPL gene as described in Examples 1 and 23.

The seeds were treated with dsRNA at a concentration of 1 or 5 pg / ml for 10 minutes, 2 and 24 hours. 20 total RNA was extracted from sprouted seed leaves, 17 days (in plants treated for 24 hours) and 18 days (in plants treated for 10 minutes and 2 hours) after treatment. CDNA was prepared using oligo-dT primers and the level of SPL mRNA expression was determined in plants

170 treated and control by PCR in time described in Example 23.

An upregulation trend in SPL mRNA expression was detected for all incubation times with 1 pg / ml dsRNA (Figures 48A-B through 50A-B).

EXAMPLE 27

ALTERED SPL EXPRESSION AFTER TREATMENT WITH

SPL siRNA IN TOMATO dsRNA derived from the SPL gene (Table 16) was processed with

ShortCut® RNase III (NEB) as described in Example 1.

Tomato seeds were treated with the resulting siRNA at a concentration of 50 pg / ml for 2 hours at

25 ° C. Total RNA was extracted from sprouted seed leaves 13 days after treatment. CDNA was prepared using oligo-dT primers and the level of SPL mRNA expression was determined in treated and control plants by real-time PCR, as described in Example 23.

An upregulation trend in SPL mRNA expression was detected. The median level of expression of

SPL in plants treated with SPL siRNA was 1.89 higher than in control plants treated with GUS siRNA (Figures

51A-B).

171

EXAMPLE 28

ALTERED EXPRESSION OF FW2.2 AFTER TREATMENT WITH pg / ml dsRNA IN TOMATO

Tomato seeds were treated with a dsRNA derived from the FW2.2 gene as described in Examples 1 and

2. 3. As a control, the seeds were treated with GUS dsRNA at the same concentration. The sequence of FW2.2 dsRNA is shown in Table 13.

Before treatment, dsRNA concentration and purity were evaluated by absorbance (NanoDrop) and by

HPLC. The concentration of the dsRNA according to the NanoDrop measurement was 1791 pg / ml. HPLC showed that the sample contains mainly dsRNA but that other species (nucleotides and ssRNA) were also present in the solution. According to HPLC analysis, the FW2.2 solution contained 1410 pg / ml of dsRNA and 473 pg / ml of ssRNA (see Figures 52A-B).

The seeds were treated with dsRNA at a concentration of 50 pg / ml (determined by HPLC measurement) for 2, 5 and 24 hours. Total RNA was extracted from sprouted seed leaves, 17 days (in plants treated for hours) and 18 days (in plants treated for 2 and 6 hours) after treatment. CDNA was prepared using oligo-dT primers and the expression level of

FW2.2 mRNA in treated and control plants by PCR in

172 real time. The primers used for FW2.2 PCI were TCTCTGGGCTTGTATCATCC (Direct Primer, SEQ ID

NO: 137) and GCTGCTCAAGGTGTTTGTG (Reverse Primer, SEQ ID NO:

138). These primers are located outside the dsRNA region, at the 3 'UTR.

Down-regulation of FW2.2 mRNA was achieved after seed treatment at all incubation times (p ^ Ol value). The median level of FW2.2 expression in the plants treated with FW2.2 dsRNA for 2, 6, and 24 hours was 1.26, 1.09, and 1.41 times lower than in the control plants treated with GUS dsRNA, respectively (Figures 53A- B to 55A-B).

EXAMPLE 29

ALTERED DELLA EXPRESSION AFTER TREATMENT WITH

DELLA dsRNA IN RICE

The rice seeds were treated using the protocol described in Example 1. The seeds were washed for 4 h, dried overnight at room temperature and treated with DELLA dsRNA (see Table 15) at a concentration of 142 pg / ml, for 24 hours at 15 ° C. Total RNA was extracted from sprouted seed leaves 13 days after treatment. CDNA was prepared using oligo-dT primers and the level of DELLA mRNA expression was determined

173 in plants treated and controlled by PCI (see Table 15).

This analysis showed a significant downward regulation of DELLA mRNA. The median level of expression of DELLA in the plants treated with DELLA dsRNA was 3.73 times lower than in control plants treated with

GUS dsRNA (Figures 56A-B).

EXAMPLE 30

DELAYED DEVELOPMENT OF PLANTS AND EXPRESSION

ALTERATION OF PDS AFTER TREATMENT WITH PDS dsRNA IN

WHEAT

Wheat seeds were treated using the protocol described in Example 1. The seeds were treated with a mixture of two PDS dsRNA fragments (see Table 19) for 4 hours at 15 ° C and then germinated on a wet paper towel. The concentration of dsRNAs in the mixture was 136 pg / ml for fragment # 1 (SEQ ID NO: 141) and 125 pg / ml for fragment # 2 (SEQ ID NO: 144). Three days after treatment, the seeds were treated with the PDS dsRNA mixture showing a slowed down development, as can be seen from the smallest seedlings and the least rooted seedling compared to the control seeds treated with either 200 pg / ml GUS dsRNA or

O.lmM EDTA (Figures 57A-B).

174

Table 19: Primers used for in vitro transcription of PDS dsRNAs for wheat and the resulting dsRNA products.

<td>D iana</td><td></td><td>Sequence / SEQ ID NO:</td><td>L ongitu d (nt)</td>

<td rowspan="3">P DS</td><td>Primer direct # 1</td><td>CTCGTAATACGACTCACTATAGGGCGA ACAAGAATCTGCCGGACTAC / 139</td><td> 4 7</td>

<td>Primer inverse # 1</td><td>CTCGTAATACGACTCACTATAGGGCGA CATCCTTCCATGCAGCTAAC / 140</td><td> 4 7</td>

<td>dsRNA #one</td><td>CTCGTAATACGACTCACTATAGGGCGA ACAAGAATCTGCCGGACTACTTGCTTCAGTA TGGATACCAGCTGCCTATCATCTATGAACAT AGCTGGAGCGAAGCAAGTAAGATCTTTTGCT GGACAACTTCATACGCAGAGGTGTTTCACAA GTAGCAGCGTCCAGGCACTAAAAACTAGTCA TCGTACGACCTCCCTTGGCTTAAGGAATAAA GTAAAAGGATCACGTCATGGACTTCGTGCTC TGCAGGTTGTTTGCCAAGATTTTCCAAGGCC TCCACTAGAGAACACGATTAACTATTTGGAA GCTGGCCAGCTTTCTTCGTCGTTTAGAAGCA GTGAACGCCCCAGTAAACCATTACAGGTCGT GATTGCTGGTGCAGGACTGGCTGGTCTATCA</td><td> 5 17</td>

175

<td rowspan="4"></td><td></td><td>ACTGCAAAATACCTGGCAGACGCTGGCl AACCCATAGTGCTTGAGGCAAGAGATGTGTT GGGCGGAAAGTTAGCTGCATGGAAGGATGTC GCCCTATAGTGAGTCGTATTACGAG / 141</td><td></td>

<td>Primer direct # 2</td><td>CTCGTAATACGACTCACTATAGGGCGA CGGAACAGTGAAGCACTTTG / 14 2</td><td> 4 7</td>

<td>Primer inverse # 2</td><td>CTCGTAATACGACTCACTATAGGGCGA TTCGGGACGGTCTTGTAAAC / 14 3</td><td> 4 7</td>

<td>dsRNA #2</td><td>CTCGTAATACGACTCACTATAGGGCGA CGGAACAGT GAAGCAC TTT GCAC TTAC T GAT GGGACTCAAATAACTGGAGATGCATATGTTT TT GCAGCAC CAGT T GATAT CT TCAAGCT T CT T GTACCACAAGAGT GGAGAGAGATC TC T TAT TTCAAAAGGCTGGATAAGTTGGTGGGAGTTC CTGTCATCAATGTTCATATATGGTTTGACAG AAAACTGAAGAACACGTATGACCACCTTCTT TTCAGCAGGAGTTCACTTTTAAGCGTTTATG CAGACATGTCTTTAGCGTGCAAGGAGTACTA TGATCCAAACCGTTCGATGCTGGAGTTGGTT TTTGCTCCAGCAGAGGAATGGATCGGACGGA GT GACACCGAAAT CAT CGAAGC AAC TAT GCT AGAGCTAGCCAAGTTGTTTCCTGATGAAATC GCTGCTGACCAGAGTAAAGCAAAGATTCTTA AATACCATGTTGTGAAGACACCGAGGTCCGT</td><td> 5 37</td>

176

<img file="MX360866B_D0039.tif" />

TTACAAGACCGTCCCGAATCGCCCTAT

FROM «© PIEDAD

AGTCGTATTACGAG / 14 4

EXAMPLE 31

ALTERED TB1 EXPRESSION AFTER TREATMENT WITH

TBl dsRNA IN CORN

Corn seeds were treated using the protocol described in Example 1. The seeds were washed for 4 h, dried overnight at 30 ° C and treated with TBl dsRNA (see Table 20) at a concentration of 25 pg / ml , for hours at 15 ° C. As a control, the seeds were treated with

CGMMV dsRNA (Table 1, product 1, SEQ ID NO: 8) at the same concentration. Total RNA was extracted from germinated seed leaves 7.5 weeks after germination. CDNA was prepared using oligo-dT primers and the expression level of

TBl mRNA was determined in treated and control plants by real-time PCR, using GPM120 as a normalizer (see Table 20).

This analysis showed downregulation of TBl mRNA after dsRNA treatment. The median level of TBl expression in plants treated with TBl dsRNA was

9.88 times less than in control plants treated with CGMMV dsRNA (Figures 58A-B).

177

Table 20: maize TBl dsRNA and cebac for real-time PCR of TBl and GPM120 mRNAs.

<td>Gave Ana</td><td></td><td>Sequence / SEQ ID NOT:</td><td>The ngitud (nt)</td>

<td>TB one</td><td>dsRNA</td><td>CTAATACGACTCACTATAG GGAGGTGATCAACTCGCCGGACCT GCCGGTGCAGGCGCTGATGGACCA CGCGCCGGCGCCGGCTACAGAGCT GGGCGCCTGCGCCAGTGGTGCAGA AGGATCCGGCGCCAGCCTCGACAG GGCGGCTGCCGCGGCGAGGAAAGA CCGGCACAGCAAGATATGCACCGC CGGCGGGATGAGGGACCGCCGGAT GCGGCTCTCCCTTGACGTCGCGCG CAAATTCTTCGCGCTGCAGGACAT GCTTGGCTTCGACAAGGCAAGCAA GACGGTACAGTGGCTCCTCAACAC GTCCAAGTCCGCCATCCAGGAGAT CATGGCCGACGACGCGTCTTCGGA GTGCGTGGAGGACGGCTCCAGCAG CCTCTCCGTCGACGGCAAGCACAA CCCGGCAGAGCAGCTGGGAGGAGG AGGAGATCAGAAGCCCAAGGGTAA</td><td> 48 1</td>

178

<td></td><td></td><td>TTGCCGTCTCCCTATAGTGAGTCG</td><td></td>

<td></td><td></td><td>TATTAG / 145</td><td></td>

<td></td><td>Fat</td><td>AATCGGTGTCGTCGATTTG</td><td> 20</td>

<td></td><td>or direct</td><td>G / 146</td><td></td>

<td></td><td>Fat</td><td>GGCGGATACTGTTTGATCT</td><td> 20</td>

<td></td><td>or reverse</td><td>C / 147</td><td></td>

<td></td><td>Fat</td><td>GCTGCGTGTTGTGCGTTCT</td><td> 20</td>

<td>GP</td><td>or direct</td><td>G / 148</td><td></td>

<td>M120</td><td>Fat</td><td>TCGTCGCGTGCTGTCTGTT</td><td> 20</td>

<td></td><td>or reverse</td><td>C / 149</td><td></td>

EXAMPLE 32

ALTERED EXPRESSION OF NAC AFTER TREATMENT WITH

NAC dsRNA IN CORN

The two indicated dsRNA NAC sequences (Table 11) were synthesized in vitro using a T7 convergent method

RNA polymerase, and were diluted in 0.1 mM EDTA at a concentration of 50 pg / ml of total nucleic acids. Fifteen corn seeds (LH244) were incubated in 7.5 ml of this dsRNA solution in a 15 ml tube in the dark at 15 ° C, with slight oscillation, for 24 hours. A set of seeds was washed three times with water after treatment with dsRNAs, and then dried overnight at ° C. A second set were planted directly after

179 of soaking in the dsRNA solution.

After treatment, the seeds were either transferred to seed germination boxes (15 seeds per box) containing wet filter paper and germinated in a growth chamber set at 25 ° C in the dark, or planted in soil and they germinated in a greenhouse.

The bud tissues, including the mesocotyl base and coleoptilko, were harvested from the seeds that germinated in the germination boxes 5 days after the treatment for RNA analysis. Seeds that had been dried had only 4 days to germinate, while seeds that had germinated without the drying step had 5 days to germinate. However, drying resulted in improved germination synchronicity so that the 2 sets of plants had similar developmental stages. RNA analysis was carried out by Taqman, using the Zm.GPM120 gene for normalization of the values ​​in order to correct the differences in the concentration of the sample (Table 21). RQ values ​​were transformed into loglO for analysis. Data points that deviated from general behavior were defined as 3 standard deviations from the mean and were removed from the dataset prior to analysis.

180

Seed treatment with NAC c ** ήί

GAVE! INDUSTRIAL PROPERTY increased NAC expression such that NAC mRNA levels were 1.7 to 2.2 times the GUS control values ​​(Table 22 and Figures 59A and 59B).

In another experiment, the seeds were treated as described above and 7 were planted directly into the soil. 12 days after planting, a decrease in NAC mRNA expression in leaf VI was observed in the seeds that were not washed and dried before planting (Figure 59C and Table

2. 3) . These plants also showed a decrease in plant height 16 days after planting (Table 24).

Table 21: Primers used to determine the level of NAC mRNA expression by real-time PCR.

<td>Diana</td><td></td><td>Sequence / SEQ ID NO:</td><td>Lon gitude (nt)</td>

<td></td><td>Fat</td><td>CTGGATTGGAAACTGGGATTG</td><td> 22</td>

<td></td><td>or direct</td><td>T / 150</td><td></td>

<td></td><td>Fat</td><td>TTGCCCCATTTTGCATATAGC</td><td> 21</td>

<td>NAC</td><td>or reverse</td><td> /151</td><td></td>

<td></td><td></td><td>6FAM-</td><td> 17</td>

<td></td><td>Taqma</td><td>ATTGTGCCGTTGAATAT-</td><td></td>

<td></td><td>n probe</td><td>MGBNFQ / 152</td><td></td>

181

<td></td><td>Fat</td><td>AGGCTTTCGCTGCGTC</td><td></td>

<td></td><td>or direct</td><td> 3</td><td></td>

<td>GPM12</td><td>Fat</td><td>TGGCCCATCCAAACTCAGA / 1</td><td> 19</td>

<td> 0</td><td>or reverse</td><td> 54</td><td></td>

<td></td><td>Taqma</td><td>VIC-TGCGTTCTGCTTGAAT-</td><td> 16</td>

<td></td><td>n probe</td><td>MGBNFQ / 155</td><td></td>

Table 22: mRNA concentrations for the NAC gene in 5-day-old corn seedling shoot tissue.

<td>Treatment</td><td>Number</td><td>Mean RQ</td><td>Std Dev</td><td>Std Err Mean</td><td>Lower 95%</td><td>Upper95%</td><td>fold change</td><td>Dunnett's comparison te control on loglO transformed RQ va lúes (signficant if pVal &lt;0.05)</td>

<td>NAC</td><td> 22</td><td> 0.904</td><td> 1.225</td><td> 0.261</td><td> 0.361</td><td> 1.447</td><td> 1.7</td><td> 0.088</td>

<td>GUS</td><td> 22</td><td> 0.543</td><td> 0.623</td><td> 0.133</td><td> 0.267</td><td> 0.819</td><td></td><td> 1.000</td>

<td>NAC Dry</td><td> 21</td><td> 0.401</td><td> 0.267</td><td> 0.058</td><td> 0.279</td><td> 0.522</td><td> 2.2</td><td> 0.000</td>

<td>GUS Dry</td><td> 21</td><td> 0.185</td><td> 0.092</td><td> 0.020</td><td> 0.142</td><td> 0.227</td><td></td><td> 1.000</td>

The GUS_dry and NAC_dry seeds were washed and dried after treatment with dsRNAs. The other set was placed directly into the seed germination boxes after treatment with dsRNAs. Comparisons with a control were made using Dunnett's method, Group 10 control = GUS_dry or GUS, as applicable.

Table 23: mRNA concentrations for the NAC gene in 12-day-old corn leaf tissue.

182

<td>Tr</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>eatment</td><td>umbe</td><td></td><td>ean</td><td>td</td><td>td</td><td>ower</td><td>pper</td><td></td><td>hear</td><td>nnett's</td>

<td></td><td>r</td><td></td><td>RQ</td><td>Dev</td><td>Err</td><td> 95%</td><td> 95%</td><td>chang</td><td>chan</td><td>p value</td>

<td></td><td></td><td></td><td></td><td></td><td>Mea</td><td></td><td></td><td>and</td><td>ge</td><td></td>

<td></td><td></td><td></td><td></td><td></td><td>n</td><td></td><td></td><td>desde</td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>GUS</td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>contr</td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>ol</td><td></td><td></td>

<td>GU</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 1</td>

<td>S</td><td> 2</td><td></td><td> .56</td><td> .17</td><td> .25</td><td> .04</td><td> .08</td><td></td><td></td><td></td>

<td>NA</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> -</td><td> (</td><td> 0.</td>

<td>C</td><td> 1</td><td></td><td> . 94</td><td> .72</td><td> . 16</td><td> . 62</td><td> .27</td><td> 24.2%</td><td> .76</td><td> 0441</td>

Table 24: Height of 16-day-old corn plants.

<td></td><td></td><td>M</td><td></td><td></td><td> 1</td><td></td><td>g. OR</td><td></td>

<td></td><td></td><td>ean</td><td>c</td><td>td</td><td>pper</td><td>ower</td><td></td><td>Dunnett '</td>

<td>Tre</td><td></td><td>Height</td><td>td</td><td>Err</td><td> 95%</td><td> 95%</td><td>chang</td><td>s value</td>

<td>atment</td><td></td><td>(cm)</td><td>Dev</td><td>Mean</td><td>Mean</td><td>Mean</td><td>and</td><td>P</td>

<td></td><td></td><td></td><td></td><td></td><td> 49.9</td><td> 47.8</td><td></td><td></td>

<td>GUS</td><td> 3</td><td> 48.91</td><td> 3.37</td><td> 0.51</td><td> 4</td><td> 7</td><td></td><td> 1.0000</td>

<td></td><td></td><td></td><td></td><td></td><td> 46.3</td><td> 44.5</td><td> —</td><td></td>

<td>NAC</td><td> 3</td><td> 45.47</td><td> 2.86</td><td> 0.44</td><td> 4</td><td> 9</td><td> 7.04%</td><td> 0.0001</td>

183

EXAMPLE 33

ALTERED EXPRESSION OF HY5 mRNAs IN LETTUCE PLANTS

AFTER TREATMENT OF SEEDS WITH HY5 dsRNA

The LONG HYPOCOTYL 5 (HY5) gene codes for a key positive regulator of the plant's response to light (Oyama, Shimura et al. 1997). Lettuce contains 2 Arabidopsis HY5 related genes on chromosomes 5 and 6 which possess 7 9% DNA sequence identity with each other. A region of ~ 500 base pairs was selected from the cDNA sequence of each gene as a trigger (Table 25), and dsRNA was prepared in vitro using the convergent method of T7 RNA polymerase. The dsRNAs were solubilized in 0.1 mM EDTA at a concentration of 50 pg / ml. Thirty lettuce seeds of the Sun Valley variety were incubated in

1.5 ml of this dsRNA solution in a 2 ml eppendorf in the dark at 15 ° C, with slight agitation, for 24 hours.

The seeds were then washed three times with water, and then either transferred to seed germination boxes (9 seeds per box) containing 12 ml of water, or to soil. The seeds in the boxes were germinated in the light at 25 ° C for 7 days, and then the shoot tissue was collected for RNA analysis. RNA analysis was carried out using

184

Taqman, using an ubiquitin assay to normalize the values ​​to correct for differences in sample concentration (Table 25).

Table 25: dsRNA of HY5.5 and HY5. 6 and primers used for real-time PCR.

<td>D iana</td><td></td><td>Sequence / SEQ ID NO:</td><td>ongi your D (nt)</td>

<td>H Y5.5</td><td>dsRN TO</td><td>AGAGTTTCGGCTCAACAAGCAAGGGAGAG GAAGAAGGCATAC TT GAAT GAAT T GGAAGT GC GA GTAAAAGAAATTGAAAAGAAGAACTCCGAGCTTG AAGAGCGACTTTCAACTCTCCAAAATGAGAATCA AATGCTTAGACATATCTTGAAAAACACTACAGCC GGTATGCAAGAAAAGAAGTAGACATATGATTAGA AGAGGAAAAGCATTACAT GT GCAATCCGAAT CAT AGCTTGAAAATCGAAGGGTTTGGTTTAGGATCGA GACTTGTTATTGTGGTTATTTCTTTTCCTAGCAA ACATAATGAGAATCCAACCATCTTTACGTACGAT TCGATTAAAGATCTTTAAGTCATGTAGGTGGTAA TGGGCTGTGTTTCTAAATGACCAAAAAAGATGTA AAGTATT GCATATGATAT GGGT TT TAATTTGTAG CAC / 156</td><td> 40</td>

185

<td></td><td colspan="2">Fattening</td><td colspan="2">CATGTGCAATCCGAATCATAGC /</td>

<td></td><td>dor</td><td></td><td></td><td> 2</td>

<td></td><td colspan="2">direct</td><td></td><td></td>

<td></td><td></td><td>Fattening</td><td>ACCACAATAACAAGTCTCGATCCTAA / 15</td><td></td>

<td></td><td>dor</td><td></td><td> 8</td><td> 6</td>

<td></td><td colspan="2">reverse</td><td></td><td></td>

<td></td><td></td><td>Taqm</td><td>6FAM-TGAAAATCGAAGGGTTTG-</td><td></td>

<td></td><td colspan="2">an probe</td><td>MGBNFQ / 159</td><td> 8</td>

<td></td><td></td><td></td><td>ATGCAGGAGCAAGCAGCAACGAGTTCCAT</td><td></td>

<td></td><td></td><td></td><td>GGCGGCTAGTCTACCTTCAAGTAGCGAGAGATCT</td><td> 01</td>

<td></td><td></td><td></td><td>TCAAGCTCTGCTCTACAAATTGAAATTAAAGAAG</td><td></td>

<td></td><td></td><td></td><td>GAAT GGAAAGTGAT GACGAGATCAGAAGAGT GCC</td><td></td>

<td></td><td></td><td></td><td>GGATATGGGCGGAGAAGCCGCCGGAGCATCAAGA</td><td></td>

<td>Η</td><td>TO</td><td>dsRN</td><td>TCCGGCAGAGAAACCGGTTCAAATCAAAATAATC CAGACCGGGTTCAACACTCAGCTGAAGGAACAAA GAAAAGAGGGAAAACTCCTGCTGATAGAGAAAGC</td><td></td>

<td>Υ5.6</td><td></td><td></td><td>AAGCGATTAAAGAGATTGTTGAGGAATAGAGTAT</td><td></td>

<td></td><td></td><td></td><td>CG GCTCAACAAGCAAGAGAGAGAAAGAAGGC GTA</td><td></td>

<td></td><td></td><td></td><td>CATGACCGAGTTGGAGAGCCGAGTTAAAGAGTTG</td><td></td>

<td></td><td></td><td></td><td>GAGAAGAAGAACTCGGAGCTTGAAGAACGTTT / 1</td><td></td>

<td></td><td></td><td></td><td> 60</td><td></td>

<td></td><td></td><td>Fattening</td><td>CCACAATGCAAAATGAAAACCA / 161</td><td></td>

<td></td><td>dor</td><td></td><td></td><td> 2</td>

<td></td><td colspan="2">direct</td><td></td><td></td>

186

<td></td><td>Fattening dor reverse</td><td>GCATCCCAGACGTTGTGTTCT / l</td><td> 1</td>

<td></td><td>Taqm</td><td>6FAM-TGCTTAGACACATCTTG-</td><td></td>

<td></td><td>an probe</td><td>MGBNFQ / 163</td><td> 7</td>

<td></td><td>Fattening</td><td>TTGTCTTGAATTTTAGCTTTGACGTT / 16</td><td></td>

<td></td><td>dor</td><td> 4</td><td> 6</td>

<td></td><td>direct</td><td></td><td></td>

<td>or</td><td>Fattening</td><td>CCTTGACCGGAAAAACAATCA / 165</td><td></td>

<td>biquit</td><td>dor</td><td></td><td> 1</td>

<td>in</td><td>reverse</td><td></td><td></td>

<td></td><td></td><td>VIC-</td><td></td>

<td></td><td>Taqm</td><td>TCAATGGTGTCGGAGCTTTCCACTTCC-</td><td> 7</td>

<td></td><td>an probe</td><td>TAMRA / 166</td><td></td>

Table 26 and Figure 60A show that the mRNA levels of the HY5.5 gene were raised 2-3 times when the seeds were treated with dsRNAs of the HY5.5 or HY5.6 genes compared to the GUS dsRNA-treated seedlings. The mRNA levels of the HY5.6 gene were raised about 2-fold after treatment with HY5 dsRNAs compared to treatment with GUS dsRNA (Table 27 and Figure 60B).

Table 26: Concentration of HY5.5 mRNa in outbreaks of

187 One week old seedlings treated with dsRNAs.

<td colspan="3">HY5.5, Means for Oneway Anova, a = 0.05</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Treatment</td><td>Number</td><td>Mean RQ</td><td>Std Dev</td><td>Std Err Mean</td><td>Lower 95%</td><td>Upper95%</td><td>% change from GUS control</td><td>Dunnett p valued</td>

<td>buffer control</td><td> 28</td><td> 0.013</td><td> 0.005</td><td> 0.001</td><td> 0.011</td><td> 0.015</td><td> -8%</td><td>0.99É</td>

<td>GUS-1 dsRNA</td><td> 29</td><td> 0.014</td><td> 0.005</td><td> 0.001</td><td> 0.012</td><td> 0.016</td><td> 0%</td><td>1.00C</td>

<td>SI.Hy5-5 + SI.Hy5-6 dsRNA</td><td> 25</td><td> 0.033</td><td> 0.013</td><td> 0.003</td><td> 0.028</td><td> 0.038</td><td> 135%</td><td> &lt;.0001</td>

<td>SI.Hy5-5 dsRNA</td><td> 27</td><td> 0.035</td><td> 0.019</td><td> 0.004</td><td> 0.028</td><td> 0.042</td><td> 148%</td><td> &lt;.0001</td>

<td>SI.Hy5-6 dsRNA</td><td> 30</td><td> 0.046</td><td> 0.026</td><td> 0.005</td><td> 0.037</td><td> 0.056</td><td> 227%</td><td> &lt;.0001</td>

Comparisons with a control using the method of

Dunnett, Control group = GUS-1.

Table 27: Concentration of HY5.6 mRNa in seedling shoots of one week old treated with different dsRNAs.

<td colspan="9">HY5.6, Means for Oneway Anova, a = 0.05</td>

<td>Treatment</td><td>Number</td><td>Mean RQ</td><td>Std Dev</td><td>Std Err Mean</td><td>Lower 95%</td><td>Upper95%</td><td>% change from GUS control</td><td>Dunnett p valued</td>

<td>buffer control</td><td> 28</td><td> 0.002</td><td> 0.001</td><td> 0.000</td><td> 0.002</td><td> 0.003</td><td> -22%</td><td> 0.213</td>

<td>GUS-1 dsRNA</td><td> 29</td><td> 0.003</td><td> 0.001</td><td> 0.000</td><td> 0.003</td><td> 0.003</td><td> 0%</td><td>1.00C</td>

<td>YES .Hy5-5 + YES. Hy5-6 dsRNA</td><td> 25</td><td> 0.005</td><td> 0.002</td><td> 0.000</td><td> 0.004</td><td> 0.005</td><td> 53%</td><td>o.ooc</td>

<td>SI.Hy5-5 dsRNA</td><td> 27</td><td> 0.005</td><td> 0.002</td><td> 0.000</td><td> 0.004</td><td> 0.005</td><td> 48%</td><td>o.ooc</td>

<td>SI.Hy5-6 dsRNA</td><td> 29</td><td> 0.008</td><td> 0.002</td><td> 0.000</td><td> 0.007</td><td> 0.008</td><td> 142%</td><td>O.OOC</td>

Comparisons with a control using the method of

Dunnett, Control group = GUS-1.

In another experiment, the plants were treated as described above and cultivated for 2 weeks in the greenhouse. Then, a plume of leaves was harvested and the mRNA concentration was analyzed. In these older plants

188 age, a decrease in exf was observed

Table 28 and Figure 60C shows the mRNA levels of the gene

HY5.5 in plants when treated with dsRNAs of the HY5.5 or HY5.6 genes compared to plants grown from GUS dsRNA and lettuce DHFR dsRNA treated seeds (Table 30) as controls. Table 29 and Figure 60D shows the mRNA levels of the HY5.6 gene in plants when the seeds were treated with dsRNAs of the HY5.5 or HY5.6 genes compared to plants that grew from GUS dsRNA-treated seeds. and with

Lettuce dsRNA DHFR (Table 30) as controls.

Table 28: Concentration of HY5.5 mRNa in leaves of two-week-old plants treated with different dsRNAs.

<td>Tr atamien</td><td>ume</td><td>Q</td><td>is V</td><td>rro</td><td>I nferi</td><td>S uperi</td><td></td><td>AC mbio</td><td>Valo</td>

<td>to</td><td>ro</td><td></td><td> •</td><td>r</td><td>or</td><td>or</td><td>camb</td><td>(multiple</td><td>rp</td>

<td></td><td></td><td>edi</td><td>Its T</td><td>Its T</td><td> 95%</td><td> 95%</td><td>io</td><td>the)</td><td>of</td>

<td></td><td></td><td>to</td><td> -</td><td></td><td></td><td></td><td>resp</td><td></td><td>Dunn</td>

<td></td><td></td><td></td><td></td><td>Med</td><td></td><td></td><td> •</td><td></td><td>ett</td>

<td></td><td></td><td></td><td></td><td>ia</td><td></td><td></td><td>GUS</td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>cont</td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>role</td><td></td><td></td>

189

<td>Bu</td><td></td><td></td><td></td><td></td><td> 0</td><td> 0</td><td></td><td></td><td></td>

<td>ff er</td><td> 9</td><td> . 17</td><td> . 08</td><td> .01</td><td> . 146</td><td> .212</td><td> 5%</td><td> 4</td><td> .002</td>

<td></td><td></td><td> 9</td><td> 6</td><td> 6</td><td></td><td></td><td></td><td></td><td> 8</td>

<td>GU</td><td></td><td></td><td></td><td></td><td> 0</td><td> 0</td><td></td><td> 1.</td><td></td>

<td>Sl</td><td> 6</td><td> . 12</td><td> . 07</td><td> .01</td><td> .093</td><td> . 154</td><td>or. 0</td><td> 0</td><td></td>

<td></td><td></td><td> 4</td><td> 6</td><td> 5</td><td></td><td></td><td></td><td></td><td></td>

<td>Ls</td><td></td><td></td><td></td><td></td><td> 0</td><td> 0</td><td></td><td> 0.</td><td></td>

<td>. DHFR</td><td> 9</td><td> . 10</td><td> . 04</td><td> .00</td><td> .084</td><td> . 121</td><td> 17%</td><td> 8</td><td> . 533</td>

<td></td><td></td><td> 3</td><td> 8</td><td> 9</td><td></td><td></td><td></td><td></td><td></td>

<td>Ls</td><td></td><td></td><td></td><td></td><td> 0</td><td> 0</td><td></td><td> 0.</td><td></td>

<td>.Hy5-5</td><td> 8</td><td> . 05</td><td> . 03</td><td> . 00</td><td> .043</td><td> .073</td><td> 53%</td><td> 5</td><td> .000</td>

<td></td><td></td><td> 8</td><td> 8</td><td> 7</td><td></td><td></td><td></td><td></td><td> 3</td>

<td>Ls</td><td></td><td></td><td></td><td></td><td> 0</td><td> 0</td><td></td><td> 0.</td><td></td>

<td>.Hy5-5</td><td> 9</td><td> .06</td><td> . 03</td><td> . 00</td><td> . 054</td><td> .076</td><td> 47%</td><td> 5</td><td> .001</td>

<td> +</td><td></td><td> 5</td><td> 0</td><td> 6</td><td></td><td></td><td></td><td></td><td> 3</td>

<td>Ls.Hy5-</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> 6</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Ls</td><td></td><td></td><td></td><td></td><td> 0</td><td> 0</td><td></td><td> 0 .</td><td></td>

<td>.Hy5-6</td><td> 7</td><td> .06</td><td> . 05</td><td> . 01</td><td> . 040</td><td> .082</td><td> 51%</td><td> 5</td><td> . 000</td>

<td></td><td></td><td> 1</td><td> 4</td><td> 0</td><td></td><td></td><td></td><td></td><td> 7</td>

Table 29: Concentration of HY5.6 mRNa in leaves of two-week-old plants treated with different dsRNAs.

190

<td>Tr atamien to</td><td>ume ro</td><td>Q edi to</td><td>is V Its T</td><td>rro r Its T Med ia</td><td>I nferi or 95%</td><td>S uperi or 95%</td><td>camb io resp GUS cont role</td><td>AC mbio (multiple the)</td><td>Valo rp of Dunn ett</td>

<td>Bu</td><td></td><td> .00</td><td> . 00</td><td> .00</td><td> 0</td><td> 0</td><td></td><td> 1.</td><td> . 016</td>

<td>ff er</td><td> 9</td><td> 5</td><td> 3</td><td> 1</td><td> .004</td><td> .006</td><td> 1%</td><td> 4</td><td> 6</td>

<td>GU</td><td></td><td> . 00</td><td> . 00</td><td> .00</td><td> 0</td><td> 0</td><td></td><td> 1.</td><td></td>

<td>Sl</td><td> 6</td><td> 3</td><td> 2</td><td> 0</td><td> .003</td><td> .004</td><td>Q. Ό</td><td> 0</td><td>. ooo</td>

<td>Ls</td><td></td><td> .00</td><td> .00</td><td> .00</td><td> 0</td><td> 0</td><td></td><td> 1.</td><td> . 998</td>

<td>. DHFR</td><td> 8</td><td> 3</td><td> 1</td><td> 0</td><td> . 003</td><td> .004</td><td> 4%</td><td> 0</td><td> 4</td>

<td>Ls</td><td></td><td> .00</td><td> . 00</td><td> .00</td><td> 0</td><td> 0</td><td></td><td> 0.</td><td> . 445</td>

<td>.Hy5-5</td><td> 9</td><td> 3</td><td> 2</td><td> 0</td><td> .002</td><td> .003</td><td> 20%</td><td> 8</td><td> 8</td>

<td>Ls</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>.Hy5-5</td><td></td><td> . 00</td><td> .00</td><td> .00</td><td> 0</td><td> 0</td><td></td><td> 0.</td><td></td>

<td> +</td><td> 9</td><td> 2</td><td> 1</td><td> 0</td><td> .002</td><td> .002</td><td> 43%</td><td> 6</td><td> . 009</td>

191

<td>Ls.Hy5-</td><td></td><td></td><td colspan="7"></td>

<td> 6</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Ls</td><td></td><td> .00</td><td> .00</td><td> .00</td><td> 0</td><td> 0</td><td></td><td> 0.</td><td> .018</td>

<td>.Hy5-6</td><td> 6</td><td> 2</td><td> 1</td><td> 0</td><td> .002</td><td> . 002</td><td> 41%</td><td> 6</td><td> 8</td>

EXAMPLE 34

ALTERED EXPRESSION OF DIHYDROFLAVONOL 4-REDUCTASE (DHFR) mRNA IN LETTUCE PLANTS AFTER TREATMENT OF

SEEDS WITH DHFR dsRNA

Dihydroflavonol 4-Reductase (DHFR) is an enzyme in the anthocyanin biosynthetic pathway. A DHFR gene has been identified in lettuce and a region of 524 base pairs was selected from the cDNA sequence as a trigger (Table

30). DsRNA was prepared in vitro using the convergent method of T7 RNA polymerase for the DFR sequence and for the GUS sequence.

Table 30: DHFR dsRNA and primers used for real-time PCR.

<td>D</td><td></td><td>Sequence / SEQ ID NO:</td><td></td>

<td>iana</td><td></td><td></td><td>ongi</td>

<td></td><td></td><td></td><td>your D</td>

<td></td><td></td><td></td><td>(nt)</td>

192

<td></td><td></td><td>AGAGGATTCTCCGACCACCGTGTC CTGGAGCTGCCGGATTCATTGGTTCATGGCTCGT TATGAGACTTCTTGAACGTGGGTATAATGTTCAT GCCACTGTTCGTGACCCTGATGACATAAAGAAAG TGAAACATTTATTGGAACTACCAAAAGCAGCAAC AAACTTGACGTTATGGAAGGCAGATTTGACACAA GAAGGAAGCTTTGATGAAGCCATTGAAGGTTGTC</td><td> 24</td>

<td></td><td>dsRN</td><td>ATGGAGTCTTTCATGTGGCTACGCCTATGGACTT</td><td></td>

<td></td><td>TO</td><td>T CAGTC CAAGGAT CCT GAGAAT GAGATCAT AAAG</td><td></td>

<td></td><td></td><td>CCAACAATAGAAGGTGTATTAAGCATCGTAAGAT</td><td></td>

<td></td><td></td><td>CATGTGTGAAAGTCAAAACAGTCAAGAAATTGGT</td><td></td>

<td>D</td><td></td><td>GTTTACATCCTCTGCGGGGACAGTGAACGTGCAC</td><td></td>

<td>HFR</td><td></td><td>GGAAATGATCAACTTCCGGTCTATGACGAGTCTC</td><td></td>

<td></td><td></td><td>ATTGGAGCGATTTGGACTTCATCTACTCCAAGAA</td><td></td>

<td></td><td></td><td>AATGACTGCATGGATGTATTTCGTATCAAAAACA</td><td></td>

<td></td><td></td><td>TTGGCAGAAAAAGCAGCAT / 167</td><td></td>

<td></td><td>Fattening</td><td>GGAGATGTTCAAAGGAGCAAT TG / 168</td><td></td>

<td></td><td>dor</td><td></td><td> 3</td>

<td></td><td>direct</td><td></td><td></td>

<td></td><td>Fattening</td><td>TTGATTGTGGAATATGGAAGCATT / 169</td><td></td>

<td></td><td>dor</td><td></td><td> 4</td>

<td></td><td>reverse</td><td></td><td></td>

<td></td><td>Taqm</td><td>6FAM-TAGTTGCAGAGAGAAAG-</td><td></td>

<td></td><td>an probe</td><td>MGBNFQ / 170</td><td> 7</td>

193

The dsRNAs were stabilized in 0.1 mM EDTA at a concentration of 50 pg / ml. The lettuce variety 8N LLF 652713141 Batavia is a highly pigmented red variety that was used in these studies. Thirty lettuces were incubated in 1.5 ml of the dsRNA solution in a 1 ml eppendorf in the dark at 15 ° C, with slight shaking, for 24 hours. The seeds were then washed three times with water, and then either transferred to the seed germination boxes (10 seeds per box) containing 12 ml of water, or to soil. The seeds in the boxes were germinated at light at ° C for 7 days, and then the shoot tissue was collected for RNA analysis. RNA analysis was carried out by Taqman, using a lettuce ubiquitin assay to normalize the values ​​to correct for differences in the concentration of the samples (Tables 25 and 30).

DHFR mRNA levels decreased significantly when seeds were treated with dsRNA for DHFR compared to seeds treated with GUS dsRNA or the control buffer (Table 31 and Figure 61).

Table 31: DHFR mRNa concentration in one-week-old seedling sprouts treated with dsRNA.

194

<td>Treatment</td><td>Number</td><td>Mean RQ</td><td>Std Dev</td><td>Std Err Mean</td><td>Lower 95%</td><td>1Ό MEXICAN 1 LA IIOLÍLDAD Upper95%</td><td>........ ........ change</td><td>o: innett's: ompariso to control</td>

<td>buffer control</td><td> 25</td><td> 7.800</td><td> 3.555</td><td> 0.711</td><td> 6.333</td><td> 9.268</td><td> 1.1</td><td>0.73Í</td>

<td>GUS-2 dsRNA</td><td> 28</td><td> 7.256</td><td> 2.792</td><td> 0.528</td><td> 6.173</td><td> 8.338</td><td> 1.0</td><td></td>

<td>Ls.DHFRdsRNA</td><td> 23</td><td> 4.966</td><td> 2.548</td><td> 0.531</td><td> 3.864</td><td> 6.068</td><td> 0.7</td><td>o.oi;</td>

Comparisons with a control using the method of

Dunnett, Control group = GUS-2.

EXAMPLE 35

ALTERED DND1 mRNA EXPRESSION AND REDUCED IRRITATION OF

THE RKN ROOT IN CUCUMBER AFTER SEED TREATMENT

WITH DND1 dsRNAs

DNDl, defense no death, is a negative regulator of plant defenses. DND1 mutations lead to constitutive systemic resistance and elevated levels of salicylic acid (Clough et al. 2000). The seeds of the cucumber variety Straight 8 were placed in a monolayer in a plastic box and covered with 5-12 volumes of water at room temperature (~ 20C). The seeds were washed for 4 hours in water with slight agitation. After washing, the seeds were air dried on filter paper at ~ 30C for 12-24hr. The cucumber DNDl dsRNAs (Table 32) was resuspended in 0.lmM EDTA (diluted from 0.5M pH8.0 stock solution) at 100Dg / ml. A ratio of

1: 5 (w / v) of the dsRNA solution to seed (for example for lgr of seed a 5mL solution of dsRNa in EDTA). The

195 seeds were placed in a conical tube incubated in the dark at 15C with slight shaking for

24hr. After incubation, the seeds were washed three times with gentle shaking for 1 minute each in one volume of water to completely fill the plastic container, and dried on filter paper before planting.

Table 32: Cucumber DND1 and GFP dsRNAs and primers used for real-time PCR.

<td>iana</td><td>D</td><td></td><td>Sequence / SEQ ID NO:</td><td>ongi your D (nt)</td>

<td></td><td></td><td></td><td>GTCTTGGAATGCTACGCCTGTACCCAAGT</td><td></td>

<td></td><td></td><td></td><td>GGGCGTTCCAGCCTTCCACTCCACCAGCTGCGAC</td><td> 98</td>

<td></td><td></td><td></td><td>CACGCCCACCAACAACCCGAATGGGAAGCCTCCG</td><td></td>

<td></td><td></td><td>dsRN</td><td>CGGGCTCTTCCCTGGTTCCAATCCAACCCACAAA</td><td></td>

<td></td><td>D</td><td>A # 1</td><td>ATCCTCACCAGCGCCCCGACATTCTTCGGCGGGT</td><td></td>

<td>ND1</td><td></td><td>(T33</td><td>TGCTTCGGGACGGTTCTGGACCCAAGAAAGAAAC</td><td></td>

<td></td><td></td><td> 787)</td><td>CGGTTCAGAGATGGAACCGGGTTCTGTTATTGGC</td><td></td>

<td></td><td></td><td></td><td>CCGGGGAATGTCTCTTGCGGTTGATCCGCTTTAC</td><td></td>

<td></td><td></td><td></td><td>TTCTATGCTCTGTCTATTGGAAGAGGAGGATGGC</td><td></td>

<td></td><td></td><td></td><td>CTTGCCTGTACATGGATGGTGGGTTGGCTGCCGG</td><td></td>

196

<td rowspan="3"></td><td></td><td>AGTTACGGTGGTTCGAACGTGTCTTGAT. CACTTGTGGCACGTGTGGCTTCAGTTCAGGCTTG CTTACGTGTCGAAAGAGAGTATGGTGATTGGGTG TGGGAAACTGGTGTGGGATGCACGTGATATTGCT TCTCACTATGTTCGTTCTTTCAAAGGC / 171</td><td></td>

<td>dsRN A # 2 (T33788)</td><td>GTACGGTGCTTAGTGGATTGTTGCTTTTC ACTCTTTTGATTGGTAATATTCAGGTACTTTTGC ACGCTGTCATGGCAAGGAGGCGAAAAATGCAGCT GAGATGT CGAGAT Τ T GGAGT GGT GGAT GAGGAGA CGACAATTGCCATCTCGTTTGAAACATCGAGTTC GACACTATGAGCACCAGAGATGGGCAGCTATGGG AGGAGAAGATGAGATGGAACTAATCAATGATTTG CCAGAAGGTCT TAGAAGAGATATCAAACGT CATC TTTGTGTTGACCTAATCAGAAAGGTGCCTCTCTT TCAAAACCTGGAGGAGCTGATTCTAGACAACATA TGTGACAAAGTCAAGCCACTTGTATTCTCCAAAG ATGAAAAGATAAT CAGAGAAGGAGATCCTGTTCC AAGGATGTTATTCATAGTGTGTGGACGAGTAAAA CGTAGCCAAAGCCTGAGCAAGGGCATGACAGCGA CAAGTTTTATTGAACCGGGAGGATTTCTTGGTGA C / 172</td><td> 06</td>

<td>Fattening dor direct</td><td>CAGCGAGTTGCTTCTTGTATCCA / 173</td><td> 3</td>

197

<td colspan="2" rowspan="2"></td><td colspan="2" rowspan="2">Fattening dor reverse</td><td colspan="2">TCCTCAGAGCAAGACAAAGATAA (</td>

<td> 74</td><td> 7</td>

<td></td><td></td><td></td><td>Taqm</td><td>6FAM-ACATTGTGAGAGAAACAAGT-</td><td></td>

<td></td><td></td><td>an</td><td>probe</td><td>MGBNFQ / 175</td><td> 0</td>

<td></td><td></td><td></td><td></td><td>GGTGATGCAACATACGGAAAACTTACCCT</td><td></td>

<td></td><td></td><td></td><td></td><td>TAAATTTATTTGCACTACTGGAAAACTACCTGTT</td><td> 99</td>

<td></td><td></td><td></td><td></td><td>CCATGGCCAACACTTGTCACTACTTTCTCTTATG</td><td></td>

<td></td><td></td><td></td><td></td><td>GTGTTCAATGCTTTTCAAGATACCCAGATCATAT</td><td></td>

<td></td><td></td><td></td><td></td><td>GAAGCGGCACGACTTCTTCAAGAGCGCCATGCCT</td><td></td>

<td></td><td></td><td></td><td></td><td>GAGGGATACGTGCAGGAGAGGACCATCTTCTTCA</td><td></td>

<td>FP</td><td>G</td><td>TO</td><td>dsRN</td><td>AGGACGACGGGAACTACAAGACACGTGCTGAAGT CAAGTTTGAGGGAGACACCCTCGTCAACAGGATC GAGCTTAAGGGAATCGATTTCAAGGAGGACGGAA ACATCCTCGGCCACAAGTTGGAATACAACTACAA CTCCCACAACGTATACATCATGGCCGACAAGCAA AAGAACGGCATCAAAGCCAACTTCAAGACCCGCC ACAACATCGAAGACGGCGGCGTGCAACTCGCTGA TCATTATCAACAAAATACTCCAATTGGCGATGGC CCTGTCCTTTTACCAGACAACCATTACC / 176</td><td></td>

<td></td><td></td><td></td><td>Fattening</td><td>ATGGCTTGCTGCCTGATGTATC / 177</td><td></td>

<td></td><td>AND</td><td>dor</td><td></td><td></td><td> 2</td>

<td>LFla</td><td></td><td colspan="2">direct</td><td></td><td></td>

<td></td><td></td><td colspan="2">Fattening</td><td>GGTGGCAACAGCAGCATTCA / 178</td><td></td>

198

<td></td><td>dor</td><td></td><td></td>

<td></td><td>reverse</td><td></td><td></td>

<td></td><td>Taqm</td><td>VIC-ATGTTGTGCCTAAGGAC-</td><td></td>

<td></td><td>an probe</td><td>MGBNFQ / 179</td><td> 7 -</td>

RNA analysis was performed by Taqman real-time PCR using a cucumber ELFla assay for normalization of values ​​to correct for differences in sample concentration (see Table 32 for primer sequences and Taqman probes). Analysis of samples of fifteen-day-old cucumber leaves germinated from DNDl dsRNAs-treated seeds demonstrated altered expression of the DNDl gene compared to control seeds treated with GFP dsRNA (see Table for GFP dsRNA sequence and Table 33 and Figure 62A by real-time PCR analysis).

Table 33: Concentration of DNDl mRNa in 15-day-old cucumber leaves treated with DNDl dsRNAs.

<td colspan="3">Means and Std Deviations, Cs.DNOl</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Treatment</td><td>Number</td><td>Mean RQ</td><td>Std Dev</td><td>Std Err Mea</td><td>Lower 95%</td><td>Upper95%</td><td>% change from GFP control</td><td>fold change</td><td>Dune p-vali LOGH</td>

<td>Formulation.NI</td><td> 19</td><td> 0.806</td><td> 0.157</td><td> 0.036</td><td> 0.730</td><td> 0.881</td><td> 3%</td><td> 1.0</td><td> (</td>

<td>T33776 GFP NI</td><td> 20</td><td> 0.782</td><td> 0.209</td><td> 0.047</td><td> 0.684</td><td> 0.879</td><td> 0%</td><td> 1.0</td><td> 1</td>

<td>T33787 DND NI</td><td> 20</td><td> 0.934</td><td> 0.150</td><td> 0.033</td><td> 0.864</td><td> 1.004</td><td> 19%</td><td> 1.2</td><td> (</td>

<td>T33788 DNDJMI</td><td> 20</td><td> 1.060</td><td> 0.204</td><td> 0.046</td><td> 0.965</td><td> 1.156</td><td> 36%</td><td> 1.4</td><td> •</td>

Comparisons with a control using the method of

199

Dunnett, Control group = GFP-1.

In another experiment, one hundred cucumber seeds were treated with dsRNA containing the cucumber DND1 transcribed sequences (Table 32, dsRNA # 2 T33788) as described above. The treated seeds were treated in 1 / 4MS plates for 3 days. 10 ml of dry sand was added to each glass container and the seedlings were planted by tilting the container and placing the seedling in the correct orientation such that the cotyledons were just above the sand and then tilting it again to cover the radicles with sand . 3.3 ml of water was added to each container and the containers were placed on shelves under banks of fluorescent light. 250 vermiform eggs or 300 J2 RKN were inoculated in each tube in 50 ul of deionized or spring water. Plants were watered as necessary. Harvest of the cucumber plants was carried out at the sil days after inoculation, washing to remove the sand that covers the roots. A percentage value of root irritation was taken for each sample, showing a decrease of 26% and 21% in the irritation of

RKN compared to control treatments with 0.1 mM

EDTA and GFP dsRNA, respectively (Figure 62B).

200

EXAMPLE 36

ALTERED EXPRESSION OF PMR5 mRNA IN CUCUMBER AFTER

TREATMENT OF SEEDS WITH PMR5 dsRNAs

PMR5 belongs to the family of plant-specific genes of unknown function. Mutations in the Arabidopsis PMR5 gene, powdery mildew resistance, possess pectin-enriched cell walls and confer resistance to powdery mildew (Vogel et al. 2004).

Cucumber seeds were treated with PMR5 dsRNAs (Table 34) as described in Example 35. Leaves germinated from treated seeds were analyzed as described in Example 35. Alteration in PMR5 gene expression is evident in seeds treated with PMR5 compared to seeds treated with GFP (Table 35 and Figure 63).

Table 34: Cucumber PMR5 dsRNAs and primers used for real-time PCR.

<td>D iana</td><td></td><td>Sequence / SEQ ID NO:</td><td>ongi your D (nt)</td>

<td>P</td><td>dsRN A # 1</td><td>GATCCTGAGTTCAACTGCCAAGCTTACGG CAGACCCGATTCAAATTACCTCAAGTACCGTTGG</td><td> 09</td>

<td>MR5</td><td>(T33</td><td>CAGCCGCTCGATTGTGAGCTCCCAAGGTTCGATG</td><td></td>

201

<td rowspan="2"></td><td> 789)</td><td>gggctgagtttttgatgagaatgagagg; TGTGATGTTTGTTGGTGATTCATTGGGGAGAAAC CAATGGGAGTCATTGATTTGTTTGATCGTGTCAT CTTCTCCTCAAACTCCTACTCAAATGACTAGAGG AGAACCTCTTTCAACCTTCAGATTCCTGGAATAT GAGTTAACTGTGTCCTATTACAAAGCCCCGTATC TTGTGGACATAGAGATAGAGAATGGGAAGAGAGT GTTGAAGCTGGAGGAGATATCAATGAATGGAAAT GCTTGGGTTGGAGCTGATGTTATTTCCTTCAACA CTGGACATTGGTGGAGCCACACTGGCTCTCTACA AGGGT GGGATTACAT GGAATCAGGAGGATCATAC TATCAAGACATGGATCGGTTGGGTGCAATGGAAA AGGC / 180</td><td></td>

<td>dsRN A # 2 (T33790)</td><td>GGCTCTCTACAAGGGTGGGATTACATGGA ATCAGGAGGATCATACTATCAAGACATGGATCGG TTGGGTGCAATGGAAAAGGCTTTAAGAACATGGG CTGATTGGGTTGAGAAGAACATTGATGTCTCTAG AACAAGGGTTTTCTTCCAAGCTATCTCCCCCACA CAT TACAATCCAT CT GAATGGAACACGGGGACAG CAT CGAT GAT GACAT CAACGAAAAAT T GT TAT GG GGAAACGGCACCAATGGGGGGGACGACGTACCCG GGAGGGTACCCTATTCAAATGAGGGTTGTGGATG AAGTGATAAGGGAGATGAGGAAGCCAGTATACTT ATTGGACATAACAATGTTATCTGAGCTAAGAAAA</td><td> 00</td>

202

<td></td><td></td><td>GATGGACACCCTTCCATTTATAGTGGTG.</td><td></td>

<td></td><td></td><td>ATCCTCAACAAAGGGCTAACCCAGATAGATCAGC</td><td></td>

<td></td><td></td><td>GGATTGTAGCCATTGGTGTCTTCCTGGCTTACCA</td><td></td>

<td></td><td></td><td>GATACTTGGAACCAATTGTTTTATACTGC / 181</td><td></td>

<td></td><td>Fattening</td><td>AGCTTCCTCAGCTTTGATTCTCAGT / 18 2</td><td></td>

<td></td><td>dor</td><td></td><td> 5</td>

<td></td><td>direct</td><td></td><td></td>

<td></td><td>Fattening</td><td>GCGATTATGGTGGTCGCTGTT / 183</td><td></td>

<td></td><td>dor</td><td></td><td> 1</td>

<td></td><td>reverse</td><td></td><td></td>

<td></td><td>Taqm</td><td>6FAM-TGAAGCACCATTACCG-</td><td></td>

<td></td><td>an probe</td><td>MGBNFQ / 184</td><td> 6</td>

Table 35: Concentration of PMR5 mRNa in 15-day-old cucumber leaves germinated from seeds treated with PMR5 dsRNAs.

<td colspan="3">Means and Std Deviations, Cs.PMRS</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Treatment</td><td>Number</td><td>Mean RQ</td><td>Std Dev</td><td>Std Err Mea</td><td>Lower 95%</td><td>Upper95%</td><td>% change from GFP control</td><td>fold change</td><td>Dunn p-vali LOG1</td>

<td>Formulation NI</td><td> 20</td><td> 0.304</td><td> 0.118</td><td> 0.026</td><td> 0.249</td><td> 0.359</td><td> 13%</td><td> 1.1</td><td> (</td>

<td>T33776 GFP NI</td><td> 20</td><td> 0.268</td><td> 0.119</td><td> 0.027</td><td> 0.212</td><td> 0.323</td><td> 0%</td><td> 1.0</td><td> 1</td>

<td>T33789 PMR5 NI</td><td> 18</td><td> 0.604</td><td> 0.233</td><td> 0.055</td><td> 0.488</td><td> 0.721</td><td> 126%</td><td> 2.3</td><td> -</td>

<td>T33790 PMR5 NI</td><td> 19</td><td> 0.536</td><td> 0.279</td><td> 0.064</td><td> 0.401</td><td> 0.670</td><td> 100%</td><td> 2.0</td><td> «</td>

EXAMPLE 37

ALTERED EXPRESSION OF TubG mRNA IN CUCUMBER AFTER

SEED TREATMENT WITH TubG dsRNAs

TubG encodes a γ-tubulin protein (Snustad

203

DP, et al. 1992). Cucumber seeds

TubG dsRNAs (Table 36) as described in Example 35. The leaves germinated from the treated seeds were analyzed as described in Example 35. The alteration in the expression of the TubG gene is evident in the leaves of seeds treated with compared to GFP-treated seeds (Table 37 and Figure 64).

Table 36: Cucumber TubG dsRNAs and primers used for real-time PCR.

<td>D iana</td><td></td><td>Sequence / SEQ ID NO:</td><td>ongi your D (nt)</td>

<td>T ubG</td><td>dsRN A # 1 (T33 791)</td><td>GTGGGAACCAGATCGGAATGGAGTTCTGG AAGCAGCTTTGCCTCGAGCATGGAATCAGCAAAG ACGGCATTCTTGAAGATTTTGCTACTCAGGGAGG TGACCGGAAAGATGTATTCTTCTATCAAGCCGAT GATCAGCACTACATACCAAGAGCTTTACTTATTG ACCTGGAGCCCAGGGTCATTAATGGTATCCAGAA CAGTGAATATCGAAATCTCTACAACCACGAGAAC ATCTTTGTTTCAGATCATGGAGGTGGTGCTGGAA ATAACTGGGCCAGTGGATATCATCAGGGAAAGGG C GT T GAAGAGGATAT CAT GGACAT GAT T GACAGA GAAGCAGATGGAAGCGATAGCCTTGAGGGTTTTG</td><td> 12</td>

204

<td rowspan="3"></td><td></td><td>TTCTATGCCACTCAATTGCTGGAGGGAC GGGCATGGGTTCATATCTTCTGGAGACTCTGAAT GATCGCTACAGCAAAAAACTGGTTCAGACGTACA GTGT T TT TCCTAAT CAGAT GGAAACAAGTGAT GT TGTAGTC / 185</td><td></td>

<td>dsRN A # 2 (T33792)</td><td>GCCTTACAACTCACTTTTGACTTTAAAGC GACTAACACTCAATGCTGATTGTGTTGTTGTTCT TGATAATACTGCCCTAAATAGAATAGCTGTAGAA CGCCTTCATCTATCAáATCCAACCTTTGCACAAA CAAACTCCTTAGTGTCGACTGTAATGTCAGCTAG CACAACCACTTTGAGATACCCAGGATATATGAAC AATGACTTGGTTGGACTCTTGGCCTCTCTAATTC CAACACCAAGATGCCATTTTCTAATGACAGGATA CACACCACTCACGGTTGAGCGCCAGGCTAATGTG ATAAGGAAAACCACTGTTCTTGATGTCATGAGAA GACTTCTCCAGACAAAAAATATTATGGTCTCCTC GTAT GC TC GAACAAAAGAAGCTAGTCAAGCAAAA TACATATCAATATTGAATATCATACAGGGAGAAG TGGACCCTACACAGGTTCATGAAAGTTTGCAGAG AATACGTGAAAGAAAGCTGGTGAATTTTATTGAG TGGGGGC / 186</td><td> 12</td>

<td>Fattening dor direct</td><td>GGGTCAGTGGTCTTATGTTAGC / 187</td><td> 2</td>

205

<img file="MX360866B_D0040.tif" />

Table 37: Concentration of TubG mRNa in 15-day-old cucumber leaves germinated from seeds treated with TubG dsRNAs.

<td colspan="3">Means and Std Deuiations, Cs.TubS</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Treatment</td><td>Number</td><td>Mean RQ</td><td>Std Deu</td><td>Std Err Mea</td><td>Lower95%</td><td>Upper95%</td><td>% change from GFP control</td><td>fold change</td><td>Dunn ' p-vali LOGll</td>

<td>NI formulation</td><td> 20</td><td> 0.562</td><td> 0.215</td><td> 0.048</td><td> 0.462</td><td> 0.663</td><td> 18%</td><td> 1.2</td><td> (</td>

<td>T33776 GFP NI</td><td> 20</td><td> 0.478</td><td> 0.177</td><td> 0.040</td><td> 0.395</td><td> 0.561</td><td> 0%</td><td> 1.0</td><td> 1</td>

<td>T33791 TubG NI</td><td> 20</td><td> 1.252</td><td> 0.493</td><td> 0.110</td><td> 1.021</td><td> 1.483</td><td> 162%</td><td> 2.6</td><td> &lt;</td>

<td>T33792 TubG NI</td><td> 18</td><td> 1.117</td><td> 0.360</td><td> 0.085</td><td> 0.938</td><td> 1.297</td><td> 134%</td><td> 2.3</td><td> &lt;</td>

EXAMPLE 38

ALTERED EXPRESSION OF DND1 mRNA IN TOMATO AFTER

TREATMENT OF SEEDS WITH DNDl dsRNAs

Tomato seeds of the Microtom variety were treated with DNDl dsRNAs (Table 38) as described in

Example 35 (except that a 2ml eppendorf was used instead of a 50ml conical tube in the dsRNA incubation step). RNA analysis was performed by Taqman real-time PCR using an assay for tomato TIP41

206 for the normalization of the values ​​in order to differences in the concentration of the sample (see Table 38 by sequences of primers and Taqman probes). The alteration in the expression of the DNDl gene is evident in the leaves of seeds treated with compared to the seeds treated with GFP (Table 39 and Figure 65).

Table 38: tomato DNDl dsRNAs and primers used for real-time PCR.

<td>D iana</td><td></td><td>Sequence / SEQ ID NO:</td><td>ongi your D (nt)</td>

<td>D ND1</td><td>dsRN A # 1 (T33 781)</td><td>G AT G AC G AC AT CAAT C CAAT CT CAAAT TC CATTGAATGTTATGCATGTACTCAAGTTGGCGTC CCTGTTTTCCACTCCACCAGTTGCGATGGAGCTA ACCAACCGGAGTGGGAAGCTTCAGCCGGTTCTTC TCTAGTTCCAATTCAAAACCGGACGGATTCAAAA ACCGGAAAATCCCGGTCCAGTCGCAGCCGGCACA CATCGGGGCCGTTCGGGCGTGTATTAGACCCTCG AAGCAAGCGCGTGCAGAGATGGAACCGAATGATT TTATTGGCACGTGGCATGGCTTTAGCCGTTGATC CTCTATTCTTTTACGCCTTATCCATCGGCCGCGG TGGATCGCCGTGTTTGTACATGGACGGCAGCCTG</td><td> 00</td>

207

.....0 .

<td rowspan="4"></td><td></td><td>GCGGCTATCGTCACCGTGATTCGGACTAi GAVE! THE J'ROPtn INDUSTi ACGCCGTGCACCTCTTCCATTTGTGGTTGCAGTT TCGTTTGGCTTACGTGTCGAGAGAATCGCTGGTG GTTGGTTGTGGGAAACTCGTGTGGGATGC / 24</td><td>NO IBI! .................. AD lll EAL</td>

<td>dsRN A # 2 (T33782)</td><td>GATTTTGGTTCGATGCTTTTGTCATCCTT CCCGTTCCACAGGCTGTATTCTGGCTGGTGGTTC CAAAACTAATAAGAGAAGAGCAGATAAAGCTTAT AATGACGATCCTTTTATTAATGTTCTTGTTCCAG TTCCTTCCCAAAGTTTATCACTGTATAAGCTTAA TGAGAAGGATGCAAAAGGTTACAGGATATATTTT TGGTACCATCTGGTGGGGATTTGGACTTAATCTC ATTGCTTATTTTATTGCTTCTCATGTTGCTGGGG GATGCTGGTATGTTCTTGCAATACAAAGAGTGGC TTCATGTCTAAGGCAGCAGTGTGAGCGCAACCCT TCGTGTAATCTATCTTTGTCTTGCTCAGAGGAGG TGTGTTATCAGTTTCTGTTGCCAACAGGAACTGT GGGAAATCCATGTGCTGGGAACTCAACAACAGTG ACCAGGAAGCCAATGTGTTTGGATGTCAATGGAC CATTTCCATATGGGATATACCAATGGGCAC / 25</td><td> 01</td>

<td>Fattening dor direct</td><td>CTCACCAAGACGTCCGCTTCT / 26</td><td> 1</td>

<td>Fattening dor</td><td>GGTTGAACTGATCTTCGTCGGAAT / 27</td><td> 4</td>

208

<td></td><td>reverse</td><td colspan="2"></td>

<td></td><td>Taqm</td><td>6FAM-CTCTCAAAGTGGTTTGGC-</td><td></td>

<td></td><td>an probe</td><td>MGBNFQ / 28</td><td> 8</td>

<td></td><td>Fattening</td><td>AACAGGTGGTGCTCGACTATGACT / 29</td><td></td>

<td></td><td>dor</td><td></td><td> 4</td>

<td></td><td>direct</td><td></td><td></td>

<td>T</td><td>Fattening</td><td>TGCTTTCGACAGTTTCACTTCCA / 3 0</td><td></td>

<td>IP41</td><td>dor</td><td></td><td> 3</td>

<td></td><td>reverse</td><td></td><td></td>

<td></td><td>Taqm</td><td>VIC-ACCTTCACAACACCTTACT-</td><td></td>

<td></td><td>an probe</td><td>MGBNFQ / 31</td><td> 9</td>

Table 39: Concentration of DND1 mRNa in 15-day-old tomato leaves germinated from seeds treated with DND1 dsRNAs.

<td colspan="3">Means and Std Deuiatlons, SI.DND1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Treatment</td><td>Number</td><td>Mean RQ</td><td>Std Oev</td><td>Std Err Mea</td><td>Lower 95%</td><td>Upper95%</td><td>% change fromGFP control</td><td>fold change</td><td>Dunnetl p-value LOGIOF</td>

<td>Formulation NI</td><td> 19</td><td> 1.078</td><td> 0.265</td><td> 0.061</td><td> 0.950</td><td> 1.205</td><td> 23%</td><td> 1.2</td><td> 0.083</td>

<td>T3377G GFP NI</td><td> 19</td><td> 0.879</td><td> 0.303</td><td> 0.070</td><td> 0.733</td><td> 1.025</td><td> 0%</td><td> 1.0</td><td> 1</td>

<td>T33781 DND1 NI</td><td> 17</td><td> 0.599</td><td> 0.213</td><td> 0.052</td><td> 0.489</td><td> 0.709</td><td> -32%</td><td> 0.7</td><td> 0.001</td>

<td>T33782 DND1 NI</td><td> 17</td><td> 0.501</td><td> 0.164</td><td> 0.040</td><td> 0.417</td><td> 0.586</td><td> -43%</td><td> 0.6</td><td> &lt;.000</td>

EXAMPLE 39

ALTERED EXPRESSION OF PMR5 mRNA IN TOMATO AFTER

SEED TREATMENT WITH PMR5 dsRNAs

Tomato seeds were treated with PMR5 dsRNAs (Table 40) as described in Example 35 and 39. The leaves

209 sprouted from the treated seeds;

as described in Example 38. The alteration in the expression of the PMR5 gene is evident in the leaves of seeds treated with compared to the seeds treated with GFP (Table 41 and Figure 66).

Table 40: tomato PMR5 dsRNAs and primers used for real-time PCR.

<td>D iana</td><td></td><td>Sequence / SEQ ID NO:</td><td>ongi your D (nt)</td>

<td>P MR5</td><td>dsRN A # 1 (T33 783)</td><td>GTAGCTTTATCTGTTATATTATTAAGGAA TCACCATAATAATAACAATAATTATAATAACCCA AATCACAGAAAC CCAAT TCTT CAAGGAAAT CAAA CTTCATGTTCTCTCTTTATAGGTAGTTGGGTTTA CGATGAAACTTACCCATTTTACCAATCAGCTTCT TGCCCCGCCGTCGATCCACAGTTCAACTGTCAAC TCTACGGCCGACCCGATACGGAATACCTAAAGTA TCGATGGAAACCGGCGAACTGTGAGCTACCCAGG TTTAATGGGCTTGAGTTTCTGTTGAAAATGAAAG GGAAAACAGTGATGTTTGTGGGTGATTCATTAGG CCGGGATCAGTGGGAGTCGTTGATTTGTATGATT TCAGCTGATGTACCTAAAGCTCAAACGCAGATGT CGAGGCTTTACCCTATTTCAACTTTCAAGTTCCT</td><td> 99</td>

210

<td></td><td></td><td colspan="2">GGATTACGGAGTTGCTATTTCATATTAC</td>

<td></td><td></td><td>C CATATC TAGT GGACATAGACACT GTAC / 32</td><td></td>

<td></td><td></td><td>GATGTATTATCTTTTAATACTGGTCATTG</td><td></td>

<td></td><td></td><td>GTGGACTCACAAAGGTCCTCTTCAAGGGTGGGAC</td><td> 01</td>

<td></td><td></td><td>AAC GTAGAAGCAGGAGGGACAATGTATGAAGACA</td><td></td>

<td></td><td></td><td>TGGATCCACTAATTGCAATGGAAAAAGGGCTAAG</td><td></td>

<td></td><td></td><td>AAC GT GGGCAAGAT GGGT T GATACCAATAT T GAC</td><td></td>

<td></td><td></td><td>AGAAGTAGAACCAGACTCTTCTTTCAGGGCATTT</td><td></td>

<td></td><td>dsRN</td><td>CACCTACGCACTACAATCCGAGTGAATGGAACGC</td><td></td>

<td></td><td>A # 2</td><td>GGGTGCATCAACAGGGAGTTGTTACGGGGAGACA</td><td></td>

<td></td><td>(T33784)</td><td>ATCCCCGTAACAACCACCCCTATGACGAGCACGT</td><td></td>

<td></td><td></td><td>ACCCGGGTCCCGATTTGGATCAATCAAATGTGAT</td><td></td>

<td></td><td></td><td>CCAAAAAGTTATAAGAGAAATGGACAATCCACCT</td><td></td>

<td></td><td></td><td>TTCTTGCTAGACATAACATTGTTATCAACAATGA</td><td></td>

<td></td><td></td><td>GGAAAGATGCACATCCATCTATTTACAGTGGTGA</td><td></td>

<td></td><td></td><td>TCTCAATTCTCAACAAAGAATTAACCCTAACAAA</td><td></td>

<td></td><td></td><td>CCTGATTGTAGCCATTGGTGTCTGCCTGGC / 33</td><td></td>

<td></td><td>Fattening</td><td>CTCTTTCCTTAACCCTTTTTTAAATTTCT</td><td></td>

<td></td><td>dor</td><td>C / 34</td><td> 0</td>

<td></td><td>direct</td><td></td><td></td>

<td></td><td>Fattening</td><td>AGAAGAAGACATAAT GTAGT T GAAGAACA</td><td></td>

<td></td><td>dor</td><td>AG / 35</td><td> 1</td>

<td></td><td>reverse</td><td></td><td></td>

<td></td><td>Taqm</td><td>SFAM-CAAATGGAGCTTCTCTC-</td><td></td>

211 an probe

MGBNFQ / 36

Ϊ í

Ϊ

Ϊ

Table 41: PMR5 mRNa concentration in 15-day-old tomato leaves germinated from seeds treated with PMR5 dsRNAs.

<td colspan="3">Means and Std Devlations, SI.PMR5</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Treatment</td><td>Number</td><td>Mean RQ</td><td>Std Dev</td><td>Std Err Mea</td><td>Lower 95%</td><td>Upper 95%</td><td>% change from GFP control</td><td>fold change</td><td>Dunneti p-value LOGIOF</td>

<td>NI formulation</td><td> 19</td><td> 0.295</td><td> 0.223</td><td> 0.051</td><td> 0.188</td><td> 0.402</td><td> 54%</td><td> 1.5</td><td> 0.3</td>

<td>T33776 GFP NI</td><td> 19</td><td> 0.192</td><td> 0.060</td><td> 0.014</td><td> 0.163</td><td> 0.221</td><td> 0%</td><td> 1.0</td><td> 1.0</td>

<td>T33783 PMR5 NI</td><td> 10</td><td> 0.512</td><td> 0.427</td><td> 0.135</td><td> 0.206</td><td> 0.817</td><td> 167%</td><td> 2.7</td><td> 0.0</td>

<td>T33784 PMR5 N1</td><td> 11</td><td> 0.479</td><td> 0.368</td><td> 0.111</td><td> 0.232</td><td> 0.727</td><td> 150%</td><td> 2.5</td><td> 0.0</td>

EXAMPLE 40

ALTERED EXPRESSION OF MLO mRNA IN TOMATO AFTER

TREATMENT OF SEEDS WITH MLO dsRNAs

MLO, the powdery mildew resistance locus O protein encodes a plant-specific membrane protein and has been shown to play a role in powdery mildew resistance in Arabidopsis, barley and tomato (Bai et. Al 2007).

Tomato seeds were treated with MLO dsRNAs (Table 42) as described in Examples 35 and 38. Leaves germinated from the treated seeds were analyzed as described in Example 38. Alteration in MLO gene expression gene is evident in the leaves of seeds treated with compared to the seeds treated with GFP (Table

43 and Figure 67).

212

Table 42: dsRNAs of tomato and cebat MLO for real-time PCR.

<td>D iana</td><td></td><td>Sequence / SEQ ID NO:</td><td>ongi your D (nt)</td>

<td rowspan="2">M THE</td><td>dsRN A # 1 (T33 779)</td><td>GCACT TGAAAAGAT CAAAGCT GAAC T TAT GCTGTTGGGATTCTTATCACTGTTGTTGACAGTG TTGCAAGATCCAGTTTCTAACTTATGTGTCCCCA AGAGTGTTGGTTATTCATGGCATCCTTGTATGGC AAAGGAAGATGCCAAGTCTGAGTATGATGACCCT TGTCTACCAAAGGGAAAAGTGCAATTTGCATCTT CATATGCAATACACCAGCTCCATATCTTCATCTT TGTATTGGCAGTTGCTCATGTATTGTACTGTATA GCAACTTTTGCTTTGGGCAGGCTAAAGATGAGAA AATGGAGGGCATGGGAGGATGAAACAAAAACAAT GGAGTACCAATTCTACAACGACCCTGAGAGATTC AGATTTGCAAGGGAGACCTCGTTTGGACGTAGGC ATTTGCATTTCTGGAGCAAGTCCCCCGTGTTGCT CTCGATAGTTTGTTTCTTTCGGCAATTCTTCTCA TCAGTTGCAAAAGTTGACTATTTAACCCTTAGAC / 37</td><td> 05</td>

<td>dsRN A # 2</td><td>GGCACATTTAACTCCACAAAATCAAAATA ATTTTGATTTTCAATTATACATTAACAGAGCAGT</td><td> 00</td>

213

<img file="MX360866B_D0041.tif" />

INDUST 1.

(Τ33780)

Fattening

TGACAAAGACTTCAAAGTTGTTGTTGGAj

CCTGCATTATGGCTCTTCACGGTGCTATATTTTC

TGACTACTACCGATCGATTGTACTCGTATCTTTG

GGTGCCATTTATCCCACTTGTAATAATATTGCTA

GTTGGCACAAAACTTCAAATGATCATAACAGAAA

TGGGAGTAAGGATTTCAGAAAGGGGAGACATAGT

AAAAGGTGTACCTGTGGTGGAGACTGGTGACCAT

CTTTTCTGGTTTAATCGCCCTGCCCTTGTCCTAT

TCTTGATTAACTTTGTACTCTTTCAGAATGCGTT

TCAAGTTGCTTTCTTTTTTTGGAGTTGGTGGAAA tttggtttcccatcttgctttcataagaatgctg

CAGACCTAGCCATAAGGCTAACCATGGGGGTGAT

CATACAGGTCCATTGCAGCTATGTGACTC / 3 8

GCAATTGCTGTGGTTTGCTTCA / 39 direct dor

Fattening

TTTCCAGTAACCACTCTCCAATGTG / 40 reverse dor

Taqm an probe

6FAM-CTTGCTCGCTATTTCTAMGBNFQ / 41

Table 43: MLO mRNa concentration in 15-day-old tomato leaves germinated from treated seeds

214 ¿.'Tfc with MLO dsRNAs.

<td colspan="3">Means and Std Deuiations, SI.MLO</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Treatment</td><td>Number</td><td>Mean RQ</td><td>Std Dev</td><td>Std ErrMea</td><td>Lower95%</td><td>Upper9S%</td><td>% change fromGFP control</td><td>fold change</td><td>Dunnetl p-value LOGIOF</td>

<td>Formulation NI</td><td> 19</td><td> 2.098</td><td> 0.441</td><td> 0.101</td><td> 1.885</td><td> 2.311</td><td> -2%</td><td> 1.0</td><td> 0.9</td>

<td>T33776 GFP NI</td><td> 19</td><td> 2.151</td><td> 0.327</td><td> 0.075</td><td> 1.994</td><td> 2.309</td><td> 0%</td><td> 1.0</td><td> 1.0</td>

<td>T33779 MLO NI</td><td> 17</td><td> 0.863</td><td>0.42S</td><td> 0.103</td><td>at 645</td><td> 1.082</td><td> -60%</td><td> 0.4</td><td> &lt;.0</td>

<td>T33780 MLO NI</td><td> 17</td><td> 0.895</td><td> 0.434</td><td> 0.105</td><td> 0.672</td><td> 1.118</td><td> -58%</td><td> 0.4</td><td> &lt;.0</td>

EXAMPLE 41

DECREASE IN powdery mildew in tomato and modulation of

MRNA EXPRESSION AFTER SEED TREATMENT WITH dsRNAs

Bil is a negative regulator of programmed cell death. Microtom tomato seeds were treated with tomato Bil containing dsRNAs (Table 44, dsRNA # 2) and PMR5 (Table 40, dsRNA # 1) dsNAs as described in Examples and 38.

Table 44: dsRNAs for Tomato bil.

<td>D iana</td><td></td><td>Sequence / SEQ ID NO:</td><td>ongi your D (nt)</td>

<td>B</td><td>dsRN</td><td>GTGCTTTAGTGGCATCGGCTGCTGGGGCT</td><td></td>

<td>il</td><td>A # 1</td><td>TACCTTCACATTCTATGGAATATCGGTGGCCTCC</td><td> 01</td>

215

<td></td><td>(Τ33</td><td colspan="2">T CACAACAATGGCT T GCAT GGGAAGCAT</td>

<td></td><td> 777)</td><td>GCTTCTCTCAGCTCCTCCTTATCAAGAGCAAAAA AGGGTGGCTCTTCTGATGGCAGCTGCACTTTTTG AAGGCGCCTCTATTGGTCCTCTGATTGAGCTGGG CATTAACTTCGATCCAAGCATTGTGTTTGGCGCT TTTGTAGGTTGTGCTGTGGTTTTTGGTTGCTTCT CAGCTGCTGCCATGTTGGCAAGGCGCAGGGAGTA CTTGTACCTCGGGGGCCTTCTTTCATCTGGCGTC TCCCTTCTCTTCTGGTTGCACTTTGCATCCTCCA TTTTTGGTGGTTCCATGGCTGTTTTCAAGTTTGA GTTGTATTTTGGACTCTTGGTGTTTGTGGGCTAC ATCGTCTTTGACACCCAAGAAATTATTGAGAAGG CTCACTTGGGTGATATGGATTACGTTAAGC / 42</td><td></td>

<td></td><td>dsRN A # 2 (T33778)</td><td>GCCAGATCTCACCTCTCGTTCAAACTCAT CTCAAGCAGGTGTACCTTACGCTATGCTGTGCTT TAGTGGCATCGGCTGCTGGGGCTTACCTTCACAT TCTATGGAATATCGGTGGCCTCCTCACAACAATG GCTTGCATGGGAAGCATGGTGTGGCTTCTCTCAG CTCCTCCTTATCAAGAGCAAAAAAGGGTGGCTCT TCTGATGGCAGCTGCACTTTTTGAAGGCGCCTCT ATTGGTCCTCTGATTGAGCTGGGCATTAACTTCG ATCCAAGCATTGTGTTTGGCGCTTTTGTAGGTTG TGCTGTGGTTTTTGGTTGCTTCTCAGCTGCTGCC ATGTTGGCAAGGCGCAGGGAGTACTTGTACCTCG</td><td> 00</td>

216

GGGGCCTTCTTTCATCTGGCGTCTCCCT '

CTGGTTGCACTTTGCATCCTCCATTTTTGGTGGT

TCCATGGCTGTTTTCAAGTTTGAGTTGTATTTTG

GACTCTTGGTGTTTGTGGGCTACATCGTC / 43

Seeds treated with dsRNA were planted in soil

M200 with fertilizer (Hummerts) and placed in a growth chamber. About one tomato seed per pot was planted in the soil and the soil surface was soaked with water. The growth chamber configuration was 26 / 24C, 16hr light cycle, 50% humidity, light intensity 3, and 4x / week irrigation by automated sub-irrigation. Fourteen days after planting the pots were arranged according to similar sizes. The replication number was 5 plants / treatment. On day 15 the plants were randomly mixed and changed to the following growth chamber conditions: 22 / 20C, 16 hr light cycle, 70% humidity, light intensity 3 and M, W, F irrigation. Infection with powdery mildew (Oidium neolycopersici) was as follows:

one. 10 μΐ Tween-20 were mixed in 200 ml of deionized H2O in a 250ml flask.

2. About 20-25 leaves of highly infected tomato plants were sectioned and placed in a

217

Α ιΜ_ Jl A ΐ | ΐίΐΐι *^:the second flask. Approximately added

...... ”'Say LA J« LffiAt »l | ¡g || pllt ..........

Tween 20 solution to the leaves until the powdery mildew is gone.

This was accomplished by shaking the flask. The solution was then transferred to a bottle with a spray nozzle, the nozzle was inserted and sprayed on the plants. The trays were rotated 4X during spraying.

Eight days after infection, samples were taken for qPCR. Plants treated with PMR5 (Table 40, dsRNA # 1) showed significant upregulation of PMR5 expression compared to plants treated with

GFP and 0.lmM EDTA (Formulation) as controls (Table 45).

Table 45: Concentration of PMR5 mRNa in 21-day-old tomato leaves germinated from seeds treated with PMR5 dsRNA and infected with powdery mildew.

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>or</td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>c</td><td></td><td></td><td></td><td></td><td>change</td><td></td>

<td></td><td></td><td></td><td></td><td>N</td><td></td><td>c</td><td>td</td><td></td><td></td><td> 1</td><td></td><td>t</td><td>desde</td><td></td>

<td></td><td></td><td>N</td><td>ean</td><td></td><td>td</td><td></td><td>Err</td><td></td><td>ower</td><td></td><td>pper</td><td></td><td>GFP</td><td>hear</td>

<td>Treatment</td><td colspan="2">umber</td><td>RQ</td><td></td><td>Dev</td><td></td><td>Mean</td><td></td><td> 95%</td><td></td><td> 95%</td><td></td><td>control</td><td>chane</td>

<td>Formulate</td><td></td><td> 1</td><td></td><td>C</td><td></td><td> 0</td><td></td><td> 0</td><td></td><td> 0</td><td></td><td>C</td><td></td><td></td>

<td>on_PM</td><td> 8</td><td></td><td> .392</td><td></td><td> . 077</td><td></td><td> .018</td><td></td><td> .354</td><td></td><td> .430</td><td></td><td> 7%</td><td> . 9</td>

<td>T33 776_GF</td><td></td><td> 1</td><td></td><td>C</td><td></td><td>c</td><td></td><td>c</td><td></td><td>C</td><td></td><td>or</td><td></td><td></td>

<td>P_PM</td><td> 8</td><td></td><td> . 421</td><td></td><td> .105</td><td></td><td> . 025</td><td></td><td> .369</td><td></td><td> .474</td><td></td><td> 0%</td><td> . 0</td>

R5 ΡΜ

Τ33783 ΡΜ

<td></td><td> 1</td><td>C</td><td>218 C</td><td>C</td><td>n</td>

<td> 6</td><td></td><td> .734</td><td> .426</td><td> . 106</td><td> . 507</td>

<img file="MX360866B_D0042.tif" />

Fourteen days after infection with the disease, development was measured according to the percentage of the leaf area that was covered with powdery mildew. Scores were found between 0.10.25 and 50% infection. The data was analyzed using Anova Single Factor Analysis (o¡ = .l). LSD was calculated and adequate error bars were created for the graphs. Treatment averages are used to plot a percent decrease in disease compared to GFP control. This analysis showed a 23% decrease in disease compared to tomato seeds treated with GFP (p value &lt;0.1, Figure 68).

EXAMPLE 42

MODULATION OF THE EXPRESSION OF PHYA mRNA IN SOYA AFTER

OF TREATMENT OF SEEDS WITH dsRNAs

The soy bean phytochrome A (PHYA) genes E3 and E4 were used as targets by treating the seeds with dsRNAs containing sequences from those genes. The PHYAE3 and PHYAE4 dsRNA sequences (Table 46) were synthesized in vitro using a convergent method of T7 RNA polymerase. The dsRNAs for the two genes were combined and brought to a

219 final concentration of 50 pg / ml of nud acids

0.1 mM EDTA. Fifteen soybean seeds were incubated (Williams

82) in 7.5 ml of these dsRNA solution or in dsRNA from a GUS control in a 15 ml tube in the dark at 15 ° C, with slight oscillation, for 24 hours. After treatment, the seeds were transferred to seed germination boxes (15 seeds per box) containing wet filter paper and germinated in a growth chamber set at 25 ° C in the dark.

Sprout tissues, excluding cotyledons, were harvested from the seeds that germinated in the seed germination boxes 5 days after treatment for RNA analysis. The RNA analysis was carried out by Taqman, using the Gm.refl6 gene for the normalization of the values ​​in order to correct the differences in the concentration of the sample (Table 46). The values ​​of

RQ were transformed to loglO for analysis after removal of scattered data.

Treatment of the seeds with PHYAE3 and PHYAE4 dsRNAs led to an increase in the expression of PHYAE3 in seedling sprouts so that the levels of PHYAE3 mRNA were 1.7 times the values ​​of the GUS control (Table 47 and

Figure 69).

220

Table 46: dsRNAs for PHYAE3 and PHYj primers used for real-time PCR.

<td>D iana</td><td colspan="2"></td><td>Sequence / SEQ ID NO:</td><td>ongi your D (nt)</td>

<td>P HYAE3</td><td>TO</td><td>dsRN</td><td>TCAAGAAGATGTTGGACATGGCATTGCAG GGTGAGGAAGAGAGAAATGTCCAATTTGAGATCC AAACACATCATATGAAGATTGATTCTGGTCCCAT CAGCTTGGTAGTTAATGCTTGTGCAAGCAGGGAT CTTCAAGATAATGTTGTGGGAGTTTGTTTTCTGG CACAAGATATAACT GCTCAGAAAACAATGAT GGA CAAATTCACCCGAATTGAAGGTGACTACAAGGCA ATTGTACAGAACCCAAACCCATTGATCCCTCCAA TAT T TGGCACAGAT GAATTTGGTTGGTGTTGTGA ATGGAATTCAGCTATGGCAAAATTAACTGGATGG AAGCGAGAGGAGGTAATGGATAAAATGCTTTTAG GAGAGGTTTTCGGGACCCAAATAGCTTGTTGTCG CCTAAGGAATCATGAAGCTGTTGTTAACTTTAGC ATTGTACTTAATACAGCCATGGCTGGTTTGGAAA CAGAGAAGGTTCCTTTTGGTTTCTTTGCTCGTGA TGGAAAGC / 190</td><td> 13</td>

<td></td><td>dor</td><td>Fattening</td><td>TCCCTCTTAGGTATGCTTGTCAATT / 191</td><td> 5</td>

221

<td rowspan="3"></td><td>direct</td><td colspan="2"></td>

<td>Fattening dor reverse</td><td>TCTCTAGCTCTTTGCTCACATGAAC / 192</td><td> 5</td>

<td>Taqm an probe</td><td>6FAM-CTGGCTCAAGTATTTG- MGBNFQ / 193</td><td> 6</td>

<td>Ρ ΗΥΑΕ4</td><td>dsRN TO</td><td>TCAAGAAGATGCTTAACTTAGCACTGCTA GGT GAAGAAGAGAAGAAT GT CCAAT TT GAGAT CA AAACACATGGGTCTAAGATGGATTCTGGTCCTAT TAGTTTAGTAGTAAATGCTTGCGCAAGCAGGGAT CTTCGAGATAATGTTGTTGGGGTTTGTTTTGTGG CCCATGATATAACTGCTCAGAAGAATGTCATGGA CAAATTTACGCGTATTGAAGGTGATTACAAGGCA ATTGTACAGAACCGCAATCCATTAATCCCTCCTA TATTTGGCACAGATGAATTTGGCTGGTGTTGTGA GTGGAATCCAGCTATGACGAAGTTAACTGGATGG AAGCGAGAGGAGGTGATGGATAAAATGCTTTTGG GAGAGCTTTTTGGCACCCATATGGCTGCTTGTCG CCTAAAGAATCAAGAAGCTTTTGTTAATTTGGGT GTTGTACTTAATAAAGCCATGACTGGTTTGGAAA CAGAGAAGGTTCCTTTTGGTTTCTTTGCTCGGAA TGGCAAGTATGTGGAATGCC / 19 4</td><td> 25</td>

<td>G m. ref1</td><td></td><td>CAAGGTTATGAAAATTATGGGTATGC / 19 5</td><td> 6</td>

222

<td colspan="2"> 6</td><td>CCCGGATAACTGCCATACATG / 1</td><td> 1</td>

<td></td><td></td><td>VIC-CTGCTGCTGGACAGGATCCCA-</td><td></td>

<td></td><td></td><td>TAMRA / 197</td><td> 1</td>

Table 47: mRNA concentrations for PHYA genes in one-week-old seedling sprout tissue.

<td>Tr eatment</td><td>7 ssay</td><td>i umbe r</td><td>ean RQ</td><td>td Dev</td><td>td Err Mean</td><td>ower 95%</td><td>pper 95%</td><td>fold chan ge</td><td>Du nnett's p value during RQ log 10 analysi s</td>

<td>GU S</td><td>I HYAE 3</td><td> 5</td><td> . 045</td><td> . 012</td><td> .003</td><td> .038</td><td> .052</td><td></td><td> 1</td>

<td>PH YAE3E4</td><td>I HYAE 3</td><td> 5</td><td> . 076</td><td> . 047</td><td> . 012</td><td> .050</td><td> .101</td><td>to . 7</td><td> 0. 0141</td>

Although the invention has been described

223 in conjunction with specific embodiments it is apparent that one skilled in the art will be able to identify many alternatives, modifications and variations. Accordingly, it is intended to encompass all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

All publications, patents and patent applications mentioned in this specification are incorporated by reference herein in their entirety, such as if each publication, patent or patent application specifically and individually indicated that it was to be incorporated by reference in the present memory. Furthermore, the citation or identification of any reference in this application is not to be understood as an assumption that such reference is available in the prior art to the present invention. Insofar as section headings are used, they are not necessarily to be construed as limiting.

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