Regenerating graminea plants belonging to subfamily pooideae from protoplasts

The present invention relates to grasses of the Pooideae subfamily (Subfamilia) regenerated from protoplasts, protoplasts with regenerated cell walls (plant cells) or callus derived from protoplasts, and to a generally applicable method for regenerating these plants. Another object of the present invention relates to embryogenic cell cultures (suspension culture or callus culture) as well as calli which serve as starting material for protoplasts which can be regenerated in themselves into whole plants. The present invention also encompasses methods for producing the aforementioned embryogenic cell cultures, cold preserving said embryogenic cell cultures and embryogenic calli, and transgenic plants from the Pooideae subfamily, which may regenerate from genetically modified protoplasts.

Most of the plants on which human nutrition is primarily dependent belong to the group of plants known under the generic term grassland plants. Gramineae (Poaceae) plants are commercially the most important family in the class of monocots. Lawns include the following subfamilies and genera

<td>subfamily</td><td>A genus within the subfamily</td>

<td>Bambusoiade</td><td>Bamboo</td>

<td>Andropogonoideae</td><td>Saccharum (sugarcane) Sorghum Zea (corn)</td>

<td>Arundineae</td><td>Phragmites</td>

<td>Oryzoideae</td><td>Oryza (rice)</td>

<td>Panicoideae</td><td>Panicum (* *) Pennisetum (*) Setaria (*)</td>

<td>Pooideae (Festuciadeae)</td><td>Poa (**) Festuca (**) Lolium (**) Bromus (**) Trisetum (**) Agrostis (**) Phleum (**) Dactylis (**) Alopecurus (**) Avena (zab λ) Triticum (wheat λ) Secale (rose λ) Hordeum (barley a)</td>

* millets * grasses λ small grains

Within the subfamilies of grasses, the Pooideae subfamily is a very important group of plants in economic terms. This includes, for example, closely related subgroups of grasses and small-grain cereals.

Interestingly, it is the plants of the Pooideae subfamily that are the most difficult to manipulate with scientific methods. To date, no suitable general procedure is known which would allow the regeneration of plants, productive plants or transgenic plants with stably incorporated exogenous DNA from protoplasts, although such regeneration from cultured protoplasts is a prerequisite for, for example, somatic hybridization and production of transgenic plants using . The current state of knowledge in the field of grain transformation is described briefly by Cocking and Davey (1987) in their review paper.

A process for the isolation of protoplasts as starting material for cereal cultures and the properties of these protoplasts are described, for example, in Cereal Tissue and Cell Culture, edited by Bright SWJ and Jones MGK (1985); Publishers: Nijhoff M. and Junk W. Dordrecht]

Stable transformation of grasses has been achieved by chemical or electrically stimulated uptake of crude DNA into protoplasts ("Direkter gentransfer", Potrykus et al., 1985; Lörz et al., 1985; Fromm et al., 1986), however, starting from the cell line produced in the study was not possible.

So far, only plants that do not belong to the Pooideae subfamily have been successfully regenerated from the group of lawns. For example, Abdullah et al. (1986) report an effective plant regeneration through somatic embryogenesis from rice protoplasts (Oryzoideae subfamily). Yamada et al. Also describe plant regeneration in rice from calli, which originate from protoplasts. Rhodes et al. (1988) describe the regeneration of non-productive maize plants. Cocking and Davey (1987) discuss the state of the art to date in the field of gene transfer to cereals.

Regeneration of lawn plants in the Pooideae subfamily is known from tissue cultures. Hanning et al. (1982) describe embryo and germ development from Dactylis glomerata L., a callus tissue that developed from leaf segments.

The following is an example of additional plants from the Pooideae subfamily, the regeneration of which is described in the following publications:

Lolium rigidum (Skene et al., 1983); Lolium perenne, Lolium multiflorum (Ahloowalia, 1975); Lolium multiflorum, Festuca arundinacea (Kasperbauer et al., 1979); Alopecurus arundinaceus, Agropyron crytatum, Stipa viridula, Bromus inermis, Agropyron smithiii (Lo et al., 1980); and Agrostis palustris (Krans et al., 1982).

For a further overview of tissue cultures in fodder grasses see Ahloowalia (1984).

In the above cases, the regeneration of the Pooideae does not start from the same starting material as the present invention but

EN 220 186 Β from other cell culture types. In none of the above examples is it demonstrated that regeneration is accomplished by de novo somatic embryogenesis. In addition, the references cited above do not include data on the isolation and cultivation of protoplasts or the regeneration of plants from protoplasts.

Consequently, despite the significant interest in the genetic transformation and regeneration of lawns from the Pooideae subfamily, to date there is no in vitro method that allows the successful regeneration of plants or productive plants from protoplasts that may be transformed (Cocking and Davey, 1987). ).

So far, all research and efforts in this direction have failed, that is, they have either led to non-viable embryos or to non-viable plant blue, which have been destroyed at an early stage of development and therefore failed to be transplanted into the ground (Ahloowalia, 1984).

As a result, no available method has been disclosed so far to allow the production of protoplasts from the Pooideae subfamily, which would lead to differentiation into whole plant, especially productive plant, even less, to protoplasts derived from protoplasts or callus, stem.

These and other objects have been accomplished within the scope of the present invention, which describes the preparation of protoplasts that can be made into cellular or callus colonies. Protoplasts can be transformed, if desired, and the resulting callus can be regenerated into whole plants (Pooideae). This method of producing protoplasts, which are capable of dividing and developing into callus, which can then grow into a whole plant, requires new embryogenic cell cultures (suspension or callus cultures) or embryos as starting material.

These embryogenic cell cultures and embryos, as well as methods for their preparation and identification, are described in this application and are part of the present invention.

In addition, embryogenic callus from which suspensions can be derived can be used as starting material for protoplasts. This callus, as well as suspensions, embryos and methods for its preparation and identification, are described in the present application and form part of the present invention.

These embryogenic cultures are the source of protoplasts, which can be transformed with exogenous DNA and from which cell division and callus formation can be achieved. This callus can then be regenerated into a whole plant, including a whole plant, which can be grown in foil.

Until this invention was made, it could not be deduced from the state of the art that it is possible to regenerate lawns from the Pooideae subfamily, in particular productive lawns, or from cells or callus derived from protoplasts. It was even less predictable that protoplasts of plants from the Pooideae subfamily could also be regenerated into transgenic plants, especially productive transgenic plants, which contain exogenous DNA stably incorporated into their genome.

The invention thus relates in particular to embryogenic cell cultures (suspension or callus cultures) derived from lawns of the Pooideae subfamily, which can be isolated from protoplasts, wherein said protoplasts first regenerate, proliferate, proliferate, proliferate, including, I can regenerate.

The present invention also relates to protoplasts of plants of the Pooideae subfamily and to plant cells formed therefrom (after regeneration of the cell wall) from which whole plants, preferably whole crops, derived from cell cultures or embryogenic cell suspension can be regenerated.

Another object of the present invention are plant cells, calli, embryogenic cell cultures, embryos, young plants and plants derived from said protoplasts.

The present invention also encompasses regenerated lawn plants of the Pooideae subfamily, and their propagation material, in particular plants derived from protoplasts or plant cells that incorporate exogenous DNA into their genome, preferably exogenous DNA that can be expressed in the plant. . In the context of the present invention, propagation material refers to plant materials that are capable of reproducing sexually or asexually or in vitro or in vivo, with protoplasts, cells, calli, tissues, organs, zygotes, embryos, pollen or seeds that are transgenic from the Pooideae subfamily. can be obtained from plants. The present invention further provides progeny of said plants of the Pooideae subfamily, as well as mutants and variants thereof, including those derived from plants obtained by somatic cell fusion, genetic modification, or mutant selection.

The present invention further relates to a process for the production of protoplasts and plant cells from plants of the Pooideae subfamily capable of regenerating whole plants, preferably whole crop plants, and to a process for making callus derived from said protoplasts or plant cells and they are able to regenerate into whole plants, especially whole plants. The present invention further relates to a process for regenerating plants of the Pooideae subfamily from said callus. These procedures will be described in detail later.

These and other objects of the present invention will be apparent from the following detailed description.

HU 220 186 Β

We now briefly describe the figures.

First figure. The image shows colonies of Dactylis glomerata L., which are derived from protoplasts and grow on agarose medium in which a liquid medium is suspended.

Second figure. The image shows a young plant developed from a protoplast-derived callus of Dactylis glomerata L., where the callus was grown on SH-O medium.

Third figure. The image shows a container with a rooted young plant grown on SH-0 medium from Dactylis glomerata L. callus derived from protoplasts.

4th figure. The image shows Dactylis glomerata L., regenerated from protoplasts (left), along with a wild-type Dactylis glomerata L. (right).

5th figure. The figure shows plasmid pCIB709, which can be used to transform Dactylis glomerata L. protoplasts to introduce resistance to hygromycin. Plasmid pCIB709 was deposited with the American Type Culture Collection (Rockville, Maryland, United States of America) under accession number ATCC 40428 under the terms of the Budapest Treaty. The date of deposit is February 12, 1988 (The nucleotide sequence of the plasmid is shown in Figure 7).

explanations:

35S Prom: 35S Promoter Area

Hygro gene: structural gene for hygromycin phosphotransferase (APH TypIV)

35Sterm .: CaMV region with 3'-polyadenylation site of CaMV 35S transcript

6th figure. The figure is a "Southem blot" analysis of the DNA of various calli of Dactylis glomerata L., which are obtained after transformation of protoplasts with plasmid pCIB709. The assay is performed using the Xba I-Sst I fragments which serve as molecular probe.

7th figure. The figure shows the nucleotide sequence of plasmid pCIB709.

1-2. lanes 10 and 2 ng, respectively, of pCIB709 cleaved with BamHI restriction endonuclease.

4-8. Lane 4: BamHI-cleaved DNA from calli cultures of Dactylis glomerata, wherein calli culture is derived from protoplasts previously transformed with pCIB709.

9th Lanes 17 and 17: BamHI-cleaved DNA from Dactylis glomerata L. callus derived from untransformed protoplasts.

10-13. Rows: BamHI digested DNA from callus cultures of Dactylis glomerata L., wherein callus cultures are derived from protoplasts previously transformed with pCIB709.

14th Lane 4: BamHI-cleaved DNA from calli cultures of Dactylis glomerata L., wherein calli culture is derived from protoplasts previously transformed with pCIB709.

Lanes 15, 16: BamHI-cleaved DNA from calli cultures of Dactylis glomerata L., wherein calli culture is derived from protoplasts previously transformed with pCIB709.

4th line: blind

Lanes 6, 10, 12 and 15 show the presence of foreign DNA integrated into the genome of Dactylis glomerata L. cells. The 1063 bp fragment expected for digestion of the integrated hygromycin gene in plasmid pCIB709 with BamHI (nucleotides 583-1646 of pCIB709) is indicated by an arrow.

definitions

In order to provide a clear and unambiguous understanding of the specification and the claims and the terms used therein, the following definitions are provided:

Plant Cell: A structural and physiological unit of a plant consisting of a protoplast and a cell wall.

Plant Tissue: A group of plant cells organized in structural and functional units.

Plant organ: A defined and noticeably differentiated area of ​​a plant, such as roots, stems, leaves, flower buds or embryos. A plant organ can be made up of different cell and tissue types.

Protoplast: isolated plant cell without cell wall.

Cell culture: proliferating cell mass in an undifferentiated or partially differentiated state.

Embryo: a very small and early stage of development of a plant that has evolved either from a zygote (sexual embryo) or from an embryogenic somatic cell (somatic embryo) and is already at a recognizable morphology, structure, and cellular organization; this includes the cellular, globular, and cotyledon stages. (For example, the embryonic development of maize was described by Randolp in 1936 and the embryonic development of grass by Brown in 1960.)

Cell Node: A group of connected cells that are adhered to each other; cell nodules are usually formed by one or more progenitor cells or protoplasts by cell division.

Young plant: a multicellular structure consisting of a shoot and a root and appearing as a small plant in appearance.

Dicamba: 3,6-dichloro-2-methoxybenzoic acid.

MES: 2- (N-morpholine) ethanesulfonic acid.

2,4-D: 2,4-dichlorophenoxyacetic acid.

Picloram: 4-amino-3,6,6-trichloro-picolinic acid.

Tris-HCl: α, α, α-tris (hydroxymethyl) methylamine hydrochloride.

EDTA: 1-ethylenediamine-N, N, N ', N'-tetraacetic acid.

PEG: polyethylene glycol.

HU 220 186 Β

Agarose: The preparation and purification of agarose are described, for example, by Guieseley and Renn (1975). Agarose is a component of agar. Commercially available agar generally consists of a mixture of neutral agarose and ionic agaropectin with a plurality of substitutes. Commercial agarose can generally be obtained from known agar. Usually, a number of substitutes are still retained, which then determine the physicochemical properties of the agarose, such as the temperature of the peduncle or the melting point. Low melting agarose, especially Sea Plaque Agarose, is particularly preferred as a solidifying agent in the processes of the present invention.

SH-0 medium: Hormone-free medium according to Schenk and Hildebrandt (1972). [The SH medium may be liquid or may be solidified with 0.8% (w / v) agar or 0.5% (w / v) GelRite.] The medium may be heated or autoclaved at 128 ° C and 1 ° C by standard procedures, 2 kg / cm² (-1,2-10⁵ Pa) is sterilized by treatment for 15-20 minutes under pressure.

GelRite: GelRite Gellan Gum, Scott Laboratories, Inc., Fiskerville, Rhode Island, # 02823.

SH-30 medium: SH-0 medium at 30 pmol / L dicamba.

SH-45 medium: SH-0 medium at 45 pmol / L dicamba.

KM-8p medium: 8p medium according to Kao et al. (1975). This medium may be in liquid form or may be solidified with agar, agarose or GelRite and may be prepared and used in the absence of ascorbic acid, vitamin A or vitamin D. Components of the medium, except for the solidifying agents, can normally be sterilized by filtration through a 0.2 µm filter.

RY-2 Medium: Medium according to Yamada et al. (1986).

OMS Medium: Medium according to Murashige and Skoog (1962). For example, the medium may be solidified with 0.8% (w / v) agar or agarose or 0.5% (w / v) GelRite. For the purpose described in the present claims, this medium may be prepared by including vitamin supplementation of B5 media according to Gambory et al. (1968).

Cellulase RS: Cellulase RS, Yakult Hansha Co., Ltd., 1.1.19 Higashi-Shinbasi, Minato-ku, Tokyo, 105, Japan.

Pectolyase Y-23: Seishin Pharmaceutical Co., Ltd., 4-13 Koami-cho, Nihanbashi, Tokyo, Japan.

Parafilm: Parafilm laboratory film - American Can Co., Greenwich, Connecticut, 06830, USA.

Nalgene Filter: Nalge Co., Division of Sybron Corp., Rochester, New York, 14602, United States.

BglII: BglII restriction enzyme; New England Biolabs, 32 Tozer Road, Beverly, Massachussets, 01915, United States; or other manufacturers of your choice.

BamHI: BamHI restriction enzyme; New England

Biolabs, 32 Tozer Road, Beverly, Massachussets,

01915, United States of America; or other manufacturers of your choice.

Casein hydrolyzate: Casein hydrolyzate - enzymatic hydrolyzate from cow's milk; Type 1; Sigma Co., PO Box 14508, St. Louis, Missouri, 63178, United States.

Hygromycin B: Cytotoxin, Hygromycin B, purified; Calbiochem Behring Diagnostica, Catalog Number 400050, La Jolla, California, 92037, United States of America, Lot 702296.

Gene Screen Plus: NEN Research Products, NEF 976, 549 Albany St., Boston, Massachussets 02118, United States.

TBE Buffer: Trisborate Buffer - A standard buffer for electrophoresis, see Maniatis et al., 1982.

Spin Saul: Sephadex G25 Ready-Packed Column, Boehringer Mannheim Biochemicals, Cat. No. 100402, Piscataway, New Jersey, USA.

SDS: Sodium dodecyl sulfate.

SSC: 1.54 mmol / l NaCl, 0.154 mmol / l sodium citrate, as described by Maniatis et al. (1982).

CET AB: Hexadecyltrimethylammonium bromide.

IBI Random Primer Kit: Primary Time random primer set, International Biotechnologies Inc., PO Box 1565, New Haven, Connecticut 07606, United States (Cat. No. 77800; Serial No. F630-01).

The present invention surprisingly demonstrates that lawn grass plants of the Pooideae subfamily, in particular fruit lawn grass plants, can be regenerated from special types of protoplasts, as well as cells and callus derived from these protoplasts.

This regeneration of plants, especially productive plants, is also possible when protoplasts contain exogenous DNA. It is advantageous here if the DNA of the protoplast is stably incorporated into the genome of the protoplast, which can be expressed in plants.

Any lawn herb of the Pooideae subfamily may be used within the scope of this invention. However, plants of the Pooideae subfamily belonging to the herbaceous species, such as, for example, all genera belonging to the group Poa, Festuca, Lolium, Bromus, Trisetum, Agrostis, Phleum, Alopecurus and Dactylis, are preferred. Dactylis plants are particularly preferred.

Likewise, plants of the Pooideae subfamily of the genus Avena (oat), Triticum (wheat), Secale (rye) and Hordeum (barley) are also preferred within the scope of this invention.

A special type of Pooideae protoplast, which divides and forms cell cultures that are capable of regenerating to a whole plant, is derived from cell cultures, especially embryogenic cell cultures. Of the embryogenic cell cultures, embryogenic suspension and callus cultures are preferred.

HU 220 186 Β

Cell cultures may be prepared from appropriate parts of plants of the Pooideae subfamily. Suitable plant parts within the scope of the present invention include, but are not limited to, basic sections of young inoculated leaves, immature sexual embryos, immature inflorescences, mature seeds or germ tissues derived from plants of the Pooideae subfamily.

Process step A: Preparation of an embryogenic suspension from plant tissue

For embryogenic callus production, we start from a suitable section of a plant of the Pooideae subfamily, usually from the base section of a young leaf. Particularly preferred are the young, indoors leaves of plants of the Pooideae subfamily. This process step can be carried out analogously to the procedure of Hannig et al. (1982) for Dactylis glomerata L. The procedure described therein is not limited to a particular species, but can be applied to all other plants of the Pooideae subfamily. This notice is incorporated by reference in the present invention.

For example, the leaves can be cut into small sections or segments of 1-5 mm in length or diameter. The shape and size of this piece of letter are not critical. These segments are plated on medium suitable for callus maintenance and cultured there until callus and / or embryogenic structures are formed. A suitable medium for this purpose is, for example, SH medium (Schenk and Hildebrandt, 1972) with 30 pmol / L dicamba and 0.8% (w / v) agar or agarose as a gelling agent. Further suitable media are described by George et al., 1987. Callus and / or embryogenic structures usually appear after 2 to 6 weeks after plating. The initiation and maintenance of the callus may be carried out in the light or preferably in the dark at a temperature of 0-50 ° C, preferably 20-32 ° C and most preferably 25-28 ° C. The embryogenic callus can also be produced by other known methods, such as the method of Lührs and Lörz (1987), as well as the methods of references given in this publication, which describe methods for barley. These methods can also be applied to other Pooideae plants and are hereby incorporated by reference in their entirety.

Initiation of the suspension culture begins by transferring fresh pieces of embryogenic callus into a suitable liquid medium, for example, 0.5 g of callus in 50 ml of liquid medium (Gray et al. (1985)) containing 45 pmol / L dicambat and 4 g / L casein hydrolyzate. . The alternation between the light and dark phases during the growing period may be advantageous. The suspension is cultivated with a light period of 5 to 20 hours, preferably 16 hours, followed by a dark period of 5 to 20 hours, preferably 8 hours. The luminous intensity is typically 0.1 pE / m²s and 100 pE / m²s (E = Einstein; m = meter; s = second), preferably 30 pE / m²s and 80 pE / m²s. It is also advantageous to move the suspension during the culture phase. The suspension can be moved, for example, in a Delong flask which is sealed with a light and gas-permeable plastic film or any other suitable closure by moving the flasks at 100-150 rpm on a rotary shaker. After about 3 to 5 weeks, the larger cells are pelleted for about 30 seconds, the larger pieces are removed to retain only the smaller cells, and they are transferred to fresh medium to start new cultures.

These process steps can be repeated periodically, preferably every 3-4 weeks, always employing only promising cultures, estimating the smaller size and quality of the cell nodules. After 4-20, usually 6-8 transfers, the suspension is substantially free of non-embryogenic cells and most embryogenic cell nodules are typically in the range 150-2000 µm.

Within the scope of this invention, a process leading to the production of embryogenic suspensions, which consist primarily of low pre-embryogenic cell mass, is particularly preferred. The ratio of preembryonic cell mass can be significantly increased by first allowing the larger material to settle and then cultivating only the above portion of the suspension.

Thus, the present invention includes an embryogenic cell culture, suspension culture, or callus culture derived from lawn grass plants of the Pooideae subfamily, wherein said protoplasts are capable of regenerating, dividing and forming callus. The so-called callus itself can then be regenerated to a complete plant, and in particular to a full-grown plant.

Cell cultures derived from grasses (suspension cultures and callus cultures) are particularly preferred within the scope of the present invention, especially grasses of the genera Poa, Festuca, Lolium, Bromus, Trisetum, Agrostis, Phleum, Alopecurus and Dactylis. Embryogenic cell cultures of Dactylis glomerata L. are particularly preferred.

Other preferred objects within the scope of the present invention include embryogenic cell cultures (suspension and callus cultures) of small-grain cereals, particularly those of the genera Avena, Triticum, Secale and Hordeum.

A further object of the present invention is a callus derived from lawn grass plants of the Pooideae subfamily, which can be isolated from protoplasts, wherein said protoplasts are capable of regenerating, dividing and forming a cell wall, which can then call itself a full plant and, in particular, a full crop. .

Protoplasts can be isolated in which said protoplasts are capable of regenerating, dividing and forming a cell wall, which can then be regenerated again into a complete plant and, in particular, to a full-grown plant.

Embryogenic callus of grasses, especially those of the genus Poa, Lolium, Bromus, Trisetum, Agrostis, Phleum, Alopecurus or Dactylis, are particularly preferred in the context of the present invention.

EN 220 186 Β Β............ Embryogenic callus from Dactylis glomerata L. is particularly preferred.

A further preferred object of the present invention is a callus which is derived from a small-grain cereal, in particular from the genus Avena, Triticum, Secale or Hordeum.

Also included within the scope of the present invention are lawn plants of the Pooideae subfamily, in particular, productive lawn plants and propagating material thereof, which are regenerated from protoplasts or plant cells derived from embryogenic cell culture.

Procedure for Cryopreservation of Cell Cultures (Suspension and Callus Cultures) on Lawns

Some plant tissues can be preserved in the cold by known methods, such as those described by Withers (1986) and found in the references cited therein. However, these methods are not yet generally applicable, and are particularly applicable to cold preservation of embryogenic cell cultures (suspension and callus cultures) of grasses.

The present invention has now surprisingly shown that it is possible to preserve embryogenic cell cultures, including suspensions and callus cultures, in suspension form from all plants of the Graminae family by low temperature cryopreservation.

The procedure for cold preservation of embryogenic cell cultures, including suspension and callus cultures, derived from plants of the Graminae family, includes the following process steps:

(a) dispersion of active, growing suspension culture cells or callus in a suitable liquid medium;

(b) cooling said culture to near freezing point (0-5 ° C);

(c) mixing said chilled culture with a suitable freezing aid at about the same temperature;

(d) cooling the resulting mixture to a temperature of about 0.01-20 ° C / min, preferably 0.1-5 ° C / min, most preferably 0.2-2 ° C / min, most preferably 0.5- 1 ° C / min to (-20) - (-60) ° C, preferably (-35) to (-50) ° C, and more particularly (-38) to (- 44) ° C;

(e) blast freezing the cooled mixture in liquid air or liquid nitrogen; and (f) storing the frozen mixture below -100 ° C, preferably at the temperature of liquid nitrogen or liquid air.

This procedure for cold preservation of embryogenic cell cultures (suspension and callus cultures) is generally applicable to all plants of the Graminae family. It is understood that, in the context of the term Graminae, these plants include both Bambusoidee (e.g. Bamboo), Andropogonideae (e.g. Saccharum, Sorghum and Zea), Arundineae (e.g. Phragmites), Oryzoideae (e.g. Oryza), Panicoideae (e.g. Panicoideae) and Pooideae (e.g., herbs including Poa, Festuca, Lolium, Bromus, Trisetum, Agrostis, Phleum, But not limited to members of the genera Alopecurus and Dactylis or small-grain cereals, including the genera Avena, Triticum, Secale and Hordeum).

Within the Graminae, a particularly preferred target group is the characteristic groups of the Pooideae listed directly above, which may be summed up in terms of grasses and small-grains.

In a typical embodiment of the method of the present invention, an appropriate amount of active growing growing callus or active growing suspension culture cells (normally 1-40 days, preferably 2-10 days from the start of batch culture) is first dissolved in an appropriate liquid medium. Suitable liquid media include, for example, SH-O, SH-30 or SH-45, OMS, KM-8p, RY-2, mannitol, sucrose or other sugar and sugar alcohol solutions, and amino acids (e.g. such as proline) or even simply water, without limiting the invention to these. A medium which is also suitable for cell growth or an aqueous solution of sugars or sugar alcohols is preferred. Typically, 0.01-0.1 g of callus is dispersed in 1 ml of liquid medium and then cooled on ice.

Suitable freezing aids are primarily a mixture of osmotically active components and DMSO (dimethylsulfoxide) in water. They are also normally cooled on ice prior to addition to the pre-cooled dispersion described in process step (b), although in this case higher temperatures up to room temperature are acceptable. Thus, the temperature of the cooling aid solution is not critical.

Suitable in this connection are, for example, aqueous solutions of 0.5 to 2 mol / l glycerol, 0.5 to 2 mol / l proline and 0.5 to 4 mol / l dimethylsulfoxide (pH 5.6) or 0.5 to 2 mol / l. but not limited to aqueous glycerol, 0.5-2 mol / L sucrose and 0.5-4 mol / L dimethyl sulfoxide (pH 5-7). Other suitable components for use in freezing aid solutions include sugars, sugar alcohols, amino acids and polymers such as PEG (polyethylene glycol). Solutions containing DMSO as an ingredient are preferably freshly prepared or frozen before each use. While other freezing aid solutions may be prepared under certain conditions prior to use, fresh or frozen solutions are preferred in this case.

The freezing aid solution is typically from 1 second to 4 weeks, preferably from 1 second to 1 day, and most preferably from 1 second to 1 hour.

Add to the solution containing the cells of the dispersed suspension culture or the dispersed callus for a period of 186 Β. The cells are exposed to this freezing aid solution on ice for a suitable period of time, preferably between 1 minute and 2 days, particularly preferably between 5 minutes and 6 hours, and most preferably between 30 minutes and 2 hours. During or after this time, aliquots for cold preservation are transferred to a suitable sterile vessel (tube) or other suitable container and orally stored thereon on ice.

The container may be submerged in a liquid bath at a temperature of 0 ° C to 4 ° C prior to the introduction of said aliquots. This process step is not necessarily necessary, but may be beneficial in some cases. The bath may consist of ethanol or any other suitable coolant. The bath is normally provided with a stirring device which provides for constant mixing of the refrigerant and is connected to a device which allows the refrigerant to be cooled at a defined, controllable rate.

As soon as the vessel is in the refrigerant, the temperature is reduced at a specified, appropriate rate. Suitable cooling rates are in the range of 0.01 to 20 ° C / min, preferably in the range of 0.1 to 5 ° C / min, particularly preferably in the range of 0.2 to 2 ° C / min, and very particularly preferably in the range of 0.5-1 ° C / min. When the temperature reaches a very low temperature which is between 30 (-20) and (-60) ° C, preferably between -35 ° C and -50 ° C, and most preferably between -38 ° C and -44 ° C range, the container is subjected to a freezing "shock" if, for example, it is immersed in liquid nitrogen or liquid air. The optimum start temperature for immersion in liquid nitrogen or liquid air may vary depending on the culture used, but is generally between -20 ° C and -50 ° C, and for those skilled in the art. 40 chess, very easy to determine. The vessels can then be stored in liquid nitrogen or liquid air, either in the liquid itself or in the vapor phase above it, where the temperature does not exceed -100 ° C. 45

For some cultures, it may be advantageous to keep the temperature of the vessel at a low level for a period of time rather than immersing the vessel in liquid nitrogen or liquid air immediately upon reaching the optimum temperature. 50

In order to regain viable cell cultures, the vessels are removed from the liquid nitrogen with the callus material and preferably immersed in a warm water bath with vigorous shaking until all are thawed; the bath temperature is 10 ° C to 50 ° C, preferably 55 ° C to 40 ° C. Surprisingly, within the scope of this invention, it is shown that dishes containing cold-conserved cells of plants of the Pooideae subfamily can simply be melted by standing in air at room temperature until all 60 ice have melted. Finally, the vessels may be left on ice for a period of 1 second to 60 minutes, preferably 1 to 10 minutes, before transferring the callus material contained therein to a suitable culture medium.

The contents of the dishes are plated on a suitable solid medium. Typically, 0.5 ml of thawed culture is added to a 10 cm diameter Petri dish containing 30-50 ml of culture medium. The solid medium is either poured out as an oblique culture or cavernous at its perimeter to allow any remaining freezing aids to flow from the cells. The cells may be washed once or several times with liquid medium or any other suitable solution, such as a sugar or sugar alcohol solution or an amino acid solution, prior to plating on the appropriate medium.

Petri dishes, as described previously for embryogenic callus, were incubated at 27 ° C in the dark. The callus, like the normal embryogenic callus, is then cultured as described herein.

Step B). Isolation and purification of protoplasts that can be regenerated into whole plants, including whole crops

Starting from embryogenic suspension cultures obtained in Step A, protoplasts are obtained. The isolation and purification of the protoplasts can be accomplished by first isolating embryogenic cell nodules from the suspension medium by, for example, filtering the suspension culture obtained in Process A) on a Nalgene filter unit (0.2 µm) and finally incubating with the appropriate enzyme solution. it can remove the cell wall without damaging the protoplasts. The enzyme is used as a filter-sterilized solution. All manipulations involving cell or other cultures are performed under sterile conditions using sterile materials. A suitable enzyme solution is, for example, a solution of the following composition:

Enzyme solution:

Cellulase RS CaCl₂.h₂O NaH₂PO₄.h₂O MES (pH 5-7)

Glucose (final concentration)

2% (w / v) 7 mmol / l 0.7 mmol / l 3.0 mmol / 1550 mOs / kg H₂SHE

Normally, this mixture is shaken at 50 rpm and low light (5 pE / m).²s) with gentle movement without being critical. Digestion is carried out at 0 ° C to 50 ° C, preferably at 10 ° C to 35 ° C, and most preferably at 26 ° C to 32 ° C until the protoplasts are released. The digestion time is typically from a few seconds to 2 days, preferably from 1 hour to 1 day, and most preferably from 3 to 5 hours.

The released protoplasts are harvested and purified using standard techniques such as filtration, centrifugation and washing. In some cases, an additional cleaning step may be included. Protoplasts are applied to the surface of a suitable medium, such as, for example, KM-8p culture medium.

HU 220 186 Β

700 mOs / kg H₂It is set to an osmotic value or other suitable medium, such as that described by George et al., 1987.

After centrifugation at 10 g for 10 minutes, the protoplasts found in the interface range were collected. Finally, the protoplasts can be resuspended in the same culture medium and screened, for example, on a steel sieve (20 µm hole width).

Without this additional purification step, the contamination of the protoplast preparation material with total, undigested cells would result in a total cell count ranging from 0.001 to 0.01%. This value cannot be further reduced by the purification step described above. However, this additional purification step results in a significant loss of protoplasts having very dense cytoplasm. This results in the spreading efficiency being up to ten times lower when the process includes the said purification step. Thus, the protoplasts are purified by other known methods, such as by flotation in sucrose solution or other high-density buffer solution, such as Percoll's solution, as described above.

The protoplast yield and, consequently, the spreading efficiency are optimally achieved when serially culturing the suspension culture used for protoplast isolation 1-30 days, preferably 5-10 days prior to protoplast isolation.

The enzyme solution described above is a modification of the enzyme mixture described by Lu et al. (1981), which is more effective than any other enzyme solution tested, and⁶-70 · 10⁶ yields protoplast / gram fresh weight. Alternatively, 2% (w / v) Cellulase RS may be used in KM-8p or any other medium, such as that described by George et al. (1987), to achieve remarkable protoplast production. The use of glucose as an osmotic agent has been shown to be equivalent to sucrose protoplastisolation, and to some extent it exceeds yield in mannitol and subsequently affects spreading efficiency. Other known and suitable enzyme mixtures may be used in the process of the invention.

Protoplasts obtained after filtration, e.g., through a 20 µm screen, having an average aperture width of 12 µm to 15 µm, exhibit optically dense cytoplasm.

The present invention thus also relates to a process for the production of protoplasts which are capable of regenerating to a complete plant, in particular a full-grown plant, derived from the Pooideae subfamily of lawn grasses. This procedure is characterized by the following procedural steps:

(a) isolating tissue from a suitable portion of the lawn-grass plant of the Pooideae subfamily, in particular, a basic section of young indoors leaves, immature sexual embryos, immature inflorescences, mature seeds or germ tissues, most preferably the youngest and innermost leaves;

(b) cultivating these tissues to induce the formation of embryogenic callus and embryos;

(c) from time to time transferring the embryogenic callus and the secondary culture of the embryos into fresh medium allowing continuous growth;

(d) isolating embryogenic cell nodules after 0-500, preferably 0-100, and most preferably 3-50; and (e) removing the cell walls with an appropriate mixture of enzymes and isolating and purifying the resulting protoplasts.

Further objects of the present invention are protoplasts (including plant cells obtained after regeneration of the cell wall) of grass plants of the Pooideae subfamily, which are capable of regenerating to a complete plant, in particular a full-grown plant. Preferably, protoplasts or cells are obtained from either cell cultures or embryogenic cell suspensions.

Also included are lawn herbs of the Pooideae subfamily, in particular, productive lawn plants and their propagation material, which have been regenerated from said protoplasts or said plant cells.

Step C). Development of protoplast cultures and use of callus that can regenerate to whole plant and whole crop

The purified protoplasts obtained from step B) are transferred to a suitable liquid medium or plated on a suitable solid medium, whether or not said protoplast has been treated with exogenous DNA. (Treatment with exogenous DNA will be described in detail in a later section.) Suitable media for the purposes of this invention are those media which are KM-8p, RY-2 (Potrykus et al., 1979), SH-30 or SH-45 media. and contain sufficient concentrations of sugar and plant growth regulators. Preferred media are KM-8p and SH-45 medium containing a suitable solidifying medium. The preferred solidifying agent is agarose, especially Sea Plaque Agarose (Marin Colloids Division, FMC Corp., PO Box, Rockland, ME 04841, USA). When used with Sea Plaque Agarose, it may be present in a concentration of 0.1% to 2.5% (w / v), preferably 0.6% to 1.5% (w / v).

The protoplast plating on agarose-containing media can be performed in an analogous manner to the methods described in Shillito et al., 1983, European Patent Application No. 0,129,688 to Shillito et al., Or Adams et al., 1983. These publications are hereby incorporated by reference in their entirety.

The media in which the protoplasts are cultured may contain further suitable ingredients that facilitate colonization and division of the protoplasts. These substances include, for example, 2,4-D, Dicamba, Picloram or other plant growth regulators. Appropriate plant growth9

EN 220,186 are known to those of skill in the art.

The concentration of these substances is usually in the range of 0.01 mg / L to 100 mg / L.

Salicylic acid and its derivatives are capable of promoting cell division and colonization of Pooideae protoplasts. Salicylic acid derivatives include, but are not limited to, O-acyl and O-aryl derivatives. O-acyl derivatives include, but are not limited to, low-carbon acyl groups such as C 1-7, preferably C 1-4, most particularly C 2-3. The term "O-aryl derivatives" as used herein refers to 5 or 6 membered rings which may or may not be fused to one another (without limiting our process thereto). The rings may be unsubstituted or substituted with one or more groups including C 1-5 alkyl, C 1-4 alkoxy, nitro and amino, where the amino group itself may be substituted with C 1-4 alkyl and halogen atoms, especially chlorine and bromine.

Salicylic acid derivatives include carboxylic acid esters. Preferred carboxylic acid esters are aryl and alkyl esters wherein the alkyl group has 1 to 4 carbon atoms.

Salicylic acid derivatives also include compounds in which the ring of the salicylic acid is further substituted with, for example, one or more groups, such as C 1 -C 4 alkyl, halogen (especially chloro and bromo), nitro and amino, and the like. amino groups which are themselves substituted by C 1 -C 4 alkyl.

Preferred compounds that promote the proliferation of Pooideae protoplasts and cells include:

O-acetoxybenzoic acid (aspirin, acetylsalicylic acid);

O-hydroxybenzoic acid (salicylic acid);

O-methoxybenzoic acid (methyl salicylic acid);

O-carbamoyl-dimethoxy-benzoic acid.

The concentration of salicylic acid or a derivative thereof in the medium is preferably from 0.1 mg / l to 3000 mg / l, preferably from 10 mg / l to 300 mg / l, and most preferably from 100 mg / l.

The medium in which the protoplasts are cultured may additionally comprise medium previously conditioned by growth of appropriate cells, such as cells from Zea mays, Dactylis glomerata or other grasses. Preferably, the medium in which a suspension of a lawn grass embryogen is cultured. The medium in which the embryogenic suspension of Dactylis glomerata is cultivated is particularly preferred. The conditioned medium as described above may comprise from 0 to 100% (v / v), preferably from 5 to 50% (v / v) and most preferably from 30 to 40% (v / v) of the total medium.

The protoplasts are cultured in solid or liquid media for a period of 12 weeks, preferably 6 weeks, and most preferably 1 to 3 weeks, without periodically inoculating cultures. In a particularly preferred embodiment of the present invention, the solid medium may be introduced into a liquid medium as described in EP-0,129,688 (Shillito et al.), Or may be treated in some other suitable manner to effect cell division and / or colonization of protoplasts. promote.

The protoplasts are cultured in the light or preferably in the dark at temperatures between 0 ° C and 50 ° C, preferably 20 ° C and 32 ° C, and most preferably between 25 ° C and 28 ° C. The luminous intensities are typically 0.1 pE / rrPs and 200 pE / m²s, preferably 30 pE / m²s and 90 pE / m²s.

The spreading efficiency of KM-8p media varies between 0.5% and 10%, depending on the quality of protoplast preparation. Addition of 30-40% (v / v) conditioned suspension culture medium (suspension medium conditioned by growth of cells in it and adjusted to an osmotic value of 550 mOsm / kg) does not result in significant growth in protoplast culture medium. , but accelerates the process of proliferation of young colonies from protoplasts.

In a preferred embodiment of the present invention, the protoplasts are plated on agarose solidified medium. The first cell division can be observed approximately 2 days after plating the protoplasts. Further divisions occur every 2-3. on the sun. This proliferation process is not synchronized, which can lead to the fact that the first cell division occurs only after 7 days. 5-20 days, preferably 10-14 days after plating, the agarose-solidified medium is cut into segments and the segments containing the cell colonies are transferred to liquid medium. This process is commonly known as the bead culture technique and is fully described in Shillito et al., 1983, and in European Patent Application EP-0,129,688 (Shillito et al.).

Instead of splitting the solid medium, it can be liquefied and introduced into the liquid medium in this liquid form. This modification was described by Adams et al. (1983) and can be performed in an analogous manner. In both cases (slicing or liquefaction), KM-8p containing glucose and sucrose may be used, with good colon growth. However, the optimal liquid component is SH-45 medium containing 4 g / l casein hydrolyzate for colonization. Within 2-3 weeks of the introduction of the bead culture, new suspension cultures are recognized within the plates. Microscopic observation of agarose discs shows that colonies farthest from the surface normally grow and smaller amounts of cells are released in the medium,

The other colonies remain firmly fixed in the agarose. The new suspension multiplies very rapidly and after a further two weeks they are transferred in the same manner as the suspension cultures or plated in SH-30 to develop callus. In another embodiment, the agarose is dispersed in a Petri dish containing agarose-solidified SH-45 medium and further cultured.

The present invention thus also relates to a process for the production of cell cultures (suspension and callus cultures) from protoplasts of lawn-bearing plants of the Pooideae subfamily capable of regenerating to a complete plant, and in particular to a complete crop. This procedure is characterized by the following procedural steps:

(a) cultivating protoplasts derived from lawns of the Pooideae subfamily capable of regenerating whole plants in appropriate culture media until cell colonies are formed;

(b) culturing said cell colonies or portions thereof in a suitable medium for the production of cell cultures; and (c) isolating the resulting cell cultures.

Step (b) may not be necessary. It is likewise possible to leave the protoplasts in the culture medium defined in step (a) until cell cultures or embryos are formed.

Also included are lawn herbs of the Pooideae subfamily, in particular fruit lawn plants and their propagation material, which are regenerated from said cell cultures.

Preferred objects of the present invention include spreading protoplasts on agarose-solidified media, liquefying or disrupting agarose-solidified media, transferring the fluidized or disintegrated media into liquid media and culturing to cell colonies.

Preferably, the method wherein portions of the cell colonies mentioned in step (b) are derived from cells or cell masses released from the liquid medium.

The callus and suspension cultures obtained in these and other process steps may be cold-preserved in analogy to the procedure described in Process A).

Step D). Regeneration of young plants from callus

The callus obtained from the protoplasts (process step C), especially if it is a crumbly, granular callus, is cultured once or more, preferably every other week, in a suitable fresh medium to induce embryo formation in this manner. Suitable inducing media include, for example, SH medium with appropriate concentrations of sugar and plant growth regulator.

Each embryo produced in this manner is then transferred to a medium that provides appropriate conditions for the induction of maturation and germination. Suitable media include, for example, SH-30 or OMS media modified to contain sugars and plant growth regulators at appropriate concentrations. Plates were cultured under light [10 pE / m²s and 200 pE / m²s; cold white light from a fluorescent lamp or a mixture of daylight and Gro-Lux (Sylvania) fluorescent lamp]. It will be understood that other types of fluorescent light may be used as desired. The first embryos can be observed 2-5 weeks after the inoculation. In some cases, one or more transfer to fresh medium for maturation of embryos may be beneficial. Embryos develop further and, over a reasonable period of time, typically between 1 week and 6 months, and in particular between 1 and 3 months, produce young plant blue.

In another embodiment, the callus obtained from the protoplasts (process step C), particularly when it is a friable, granular callus, is cultivated once or several times, preferably every other week, in an appropriate fresh medium to induce embryo formation and maturation. An example of a suitable medium for this purpose is, but is not limited to, OMS medium, which contains sufficient concentrations of sugars but does not contain plant growth regulators. Plates were cultured under light [10 pE / m²s and 200 pE / m²s range; cold white light from a fluorescent lamp or a mixture of daylight with Gro-Lux (Sylvania) fluorescent lamp or any other suitable fluorescent lamp of choice]. Embryos develop further and, over a reasonable period of time, typically between 1 week and 6 months, in particular between 1 and 3 months, produce young plant-blue.

Step E). Preparation of plants, preferably fruit plants, from young plant blue

The young plant blue obtained in the previous step D) is transferred to a suitable medium, for example SH-O medium or OMS medium which does not contain growth regulators. Alternatively, a growth regulator may be added to stimulate the growth of roots or stems. Appropriate growth regulators are known to those of skill in the art. Young plants are grown on this medium until roots are formed. It is important to remove all calli from the young plant blue as it has been shown that these calli have a negative effect on the growth of the plants. To counteract this, the plants are rinsed with distilled water, for example, during transfers. Calliums that subsequently form must be removed at regular intervals, preferably every 3-30 days, particularly preferably every 1-2 weeks. The time required for root formation is typically 1-4 weeks, preferably 2 weeks. Plants that have a well-developed root system can be transplanted into the ground and taken to a greenhouse where it will slowly harden. In this context, a root length of between 1 and 10 cm, in particular between 2 and 5 cm, may be considered sufficient11.

EN 220 186 Β range. Alternatively, the seedlings are further cultured for an indefinite period after transferring the seedlings to each of the inoculated cultures for an indefinite period of time, separating the shoot shoots and transferring the seedlings to fresh medium, such as SH-0 or OMS,

Thus, the process of the present invention is characterized by the following process steps for the regeneration of whole lawn plants of the Pooideae subfamily, in particular of full-grown lawn plants:

(a) callus, derived from lawn grass plants of the Pooideae subfamily, obtained from protoplasts and capable of regenerating to a complete plant, cultivating a culture medium capable of inducing embryo formation until embryo formation;

(b) culturing the embryos in a medium suitable for inducing the maturation and germination of said embryos; and (c) cultivating the resulting blue flowers to a stage at which they can be transplanted into the soil and matured.

Flower formation can be induced either by the method described by Heide (1987) or by any other suitable method, which always meets the needs of the species or variant used. A method for inducing flowering in the Pooideae subfamily is known.

Seeds from these plants may be germinated and / or potted or sterilized by appropriate treatment, followed by Murashige and Skoog medium, which does not contain growth regulators (OMS medium) and 0.8% strain, agarose, GelRite or any other suitable, gelatinized, spread. Seeds can also be seeded in media containing between 10 pg / ml and 1000 pg / ml of hygromycin B, thereby demonstrating the transmission of hygromycin resistance.

Thus, the process of the present invention for the regeneration of fertile lawns of the Pooideae subfamily from callus comprises the following process steps:

(a) callus, derived from lawn grass plants of the Pooideae subfamily, obtained from protoplasts and capable of regenerating to a complete plant, cultivating a culture medium capable of inducing embryo formation until embryo formation;

(b) culturing the embryos in a medium suitable for inducing the maturation and germination of said embryos;

(c) cultivating the resulting blue flowers to a stage where they can be transplanted to the ground and matured; and (d) obtaining seeds as a result of controlled or free pollination.

Further objects of the present invention include lawn herbs of the Pooideae subfamily, in particular productive lawn plants and propagating material, which are regenerated from said callus.

Step F). Treatment of protoplasts with exogenous DNA

Pooideae protoplasts can be treated with exogenous DNA to form cells that contain all or part of the exogenous DNA stably integrated in their genome. Exogenous DNA in the context of the present invention is understood to be any DNA that can be fitted to protoplasts. Said DNA may be either homologous or heterologous to the transformed plant. The exogenous DNA may comprise a promoter that is active in plants of the Graminae family, particularly plants of the Pooideae subfamily, or a promoter that is present in the plant genome. In addition, the exogenous DNA may contain one or more genes that alter the genotype, especially the phenotype, of the resulting cell or plant regenerated therefrom. However, it is desirable that the gene sequence encoding one or more desired proteinaceous products be expressed and produce one or more functional enzymes or polypeptides in the resulting cell or plant. Exogenous DNA may be a chimeric gene or a portion thereof.

Exogenous DNA within the meaning of the present invention is also by definition a non-coding DNA that has a regulatory role, for example, that plays a role in the regulation of transcription.

Treatment of protoplasts with exogenous DNA may be accomplished by one of the methods described in Paszkovski et al., 1984; European Patent Application EP-0,164,575 (Paszkovski et al.); Shillito et al. (1985); Potrykus et al. (1985); Loerz et al. (1985); Fromm et al. (1986); British Patent Application GB-2,140,822 (Mettler); and Negrutiu et al. (1987). These publications are incorporated herein by reference.

The exogenous DNA may also be incorporated into other protoplasts of any desired form, such as purely linear or circular DNA, encapsulated in liposomes, spheroplasts, constituents of other protoplasts, salts, etc. The uptake of foreign DNA can be stimulated by any suitable, suitable and known method, in particular those described in the publications listed above.

Preferred chimeric genes for the present invention are those conferring beneficial properties to the transformed protoplasts and to the tissues developed, and particularly to plants, such as increased resistance to pathogens (e.g., phytopathogenic fungi, bacteria, viruses); resistance to chemicals (e.g. herbicides (e.g. triazines, sulfonylureas, imidazolines, triazole pyrimidines, Bialaphos, Glyphosate, etc.), insecticides and other biocides); resistance to harmful (endaphirous or atmospheric) environmental influences (such as heat, cold, wind, unfavorable soil conditions, humidity, drought, etc.); or the backup and storage materials are elevated 12

EN 220 186 Β in leaves, seeds, tubers, roots, stems, etc. Desired materials that the transgenic plant can produce include proteins, starches, sugars, amino acids, alkaloids, fragrances, colorants, fats, and the like.

Resistance to cytotoxins can be achieved, for example, by transferring a gene; this provides an enzyme that detoxifies cytotoxin for expression in plant cells; such as neomycin phosphotransferase II. type IV or aminoglycoside phosphotransferase IV. types that aid in the detoxification of kanamycin, hygromycin, and other aminoglycoside antibiotics, or glutathione-S-transferase, cytochrome P-450, or other catabolically active enzymes known to detoxify triazine, sulfonylurea, and other herbicides. Resistance to cytotoxins can also be mediated by a gene that expresses in a plant a suitable form of a "target enzyme" (the target of cytotoxin activity) that is resistant to cytotoxin activity; such as, for example, a variant of acetohydroxyacid synthase which is insensitive to the inhibitory action of sulfonylureas, imidazolinones or other herbicides which interact with this particular metabolic step; or a variant of EPSP synthase which is insensitive to the inhibitory effect of Glyphosate. It may be advantageous to express these altered target enzymes in such a way that they can be transported to the correct cell compartment, such as the chloroplasts in the above case.

In certain cases, it may be advantageous to direct the gene products to the mitochondria, vacuoles, endoplasmic reticulum, or other cell areas, preferably to the intercellular space (apoplasts).

For example, resistance to certain fungal species can be achieved by "gating" a gene that expresses chitinase in plant tissue. Many phytopathogenic fungi contain chitin as integral constituents in the bud and spore structure, such as Basidiomycetes (fungi and rust fungi), Ascomycetes and Fungi imperfecti (including Altemaria, Bipolar, Exerophilum turcicum, Collectotrica, Gleocercos). Chitinase is able to inhibit mycelial growth of certain pathogens in vitro. A plant leaf or root that expresses chitinase constitutively or in response to the penetration of a pathogen is protected against attack by a large number of different fungi. In some situations, constitutive expression may be advantageous over inducible expression, which occurs in many plants as a natural reaction to pathogenic diseases, since chitinase is directly present in high concentrations without the need to wait for the log phase for the new synthesis.

For example, resistance to insects can be transmitted by a gene encoding a polypeptide which is toxic to insects and their larvae; such as the crystalline protein of Bacillus thuringiensis (Barton et al., 1987); Valch et al., 1987]. Another class of proteins that mediate insect resistance is protease inhibitors. Protease inhibitors are a common component of plant storage structures (Ryan, 1973). It was demonstrated that a Bowman-Birk protease inhibitor isolated and purified from soybean inhibits the enteric protease of Tenebrio larvae (Birk et al., 1963). The gene encoding the trypsin inhibitor from cow pea was described by Hilder et al. (1987).

A gene encoding a protease inhibitor may be operable in a suitable vector under the control of a plant promoter, in particular a constitutive promoter such as the CaMV 35S promoter (described by Odell et al. (1985)). Certain genes, such as the soybean-derived Bowman-Birk protease inhibitor coding sequence, may be obtained by the cDNA cloning method described by Hammond et al. (1984). A further possibility for the production of a protease inhibitor consists of its synthetic production if it contains less than 100 amino acids, such as the trypsin inhibitor of Lima bean. The coding sequence can be predicted by reversing the amino acid sequence. Subsequently, at each end, a restriction site is inserted which is suitable for the desired vector. The synthetic gene is prepared by synthesizing 30-60 base pairs of overlapping oligonucleotide fragments, which are subsequently subjected to a kinase reaction and linked (Maniatis et al., 1982) and finally cloned into a suitable vector. DNA sequence analysis can then identify the clone that contains the insert in the correct orientation. Isolated plasmid DNA is used for insertion into the protoplast (Abel et al., 1986).

Also included within the scope of the present invention are genes encoding pharmaceutically active ingredients, such as alkaloids, steroids, hormones and other physiologically active substances, as well as flavins, vitamins and coloring agents. Genes which may be used in the context of the present invention include, but are not limited to, plant-specific genes, such as the zein gene (Wienand et al., 1981), mammalian-specific genes, such as the insulin gene, the somatostatin gene, the interleukin gene, the t-PA gene (Pennica et al., 1983) and so on. or genes of microbial origin, such as the NPTII gene, and synthetic genes, such as the insulin gene (Itakura et al., 1975).

In addition to the coding DNA itself, non-coding DNA may be used to transform protoplasts, which generally has a regulatory function and may be of interest, for example, in regulating the transcription process.

There are some plant genes known to be induced by various internal and external factors, such as plant hormones, heroes, chemicals, pathogens, oxygen deficiency and light.

For example, gene regulation with plant hormone may include abscisic acid (ABS),

220 that it induces an excess of mRNA in cotton during the late embryonic phase.

Another example is gibberellic acid (GA3), which induces malate synthase transcription in castor seeds and induces the α-amylase isoenzyme in the barley gluten layer.

The regulation of soybean heat-sensitive protein genes has been studied in detail. Treatment of the plant for several hours at 40 ° C results in de novo synthesis of the so-called heat shock protein. Many of these genes have been isolated in the meanwhile and individually analyzed for their regulation. The expression of this gene is primarily regulated at the transcriptional level (Shoffl et al., Cited by Willmitzer (1988)). The promoter of the hps70 gene is fused to the neomycin phosphotransferase II (NPTII) gene. This may have demonstrated that the chimeric gene is inducible by heroes (Spona et al., 1985).

Another group of inducible genes in plants is the nuclear codon of the light-regulated genes, particularly the small subunit of ribulose-1,5-bisphosphate carboxylase (RUBISCO). Morelli et al. (1985) and Herrera-Estrella et al. (1984) have demonstrated that the 5'-flanking sequence of the RUBISCO gene from peas is capable of transferring light inducibility to a messenger gene when linked to this gene in chimeric form. This observation can be extended to other light-inducible genes, such as the a / b binding protein of chlorophyll.

The maize alcohol dehydrogenase gene (adh gene) has been the subject of intensive research. The adh 1 gene has been isolated from maize and has been shown to be capable of expressing a portion of the 5'flanking DNA (e.g., chloramphenicol acetyltransferase; CAT) when the transiently transformed tissue is exposed to anaerobic conditions (Howard and et al., 1987).

In a preferred embodiment of the present invention, protoplasts obtained from plants of the Pooideae subfamily are transformed by a combination of electroporation and polyethylene glycol treatment. Immediately after purification of process step B, the protoplasts obtained are subjected to electroporation (see, e.g., Shillito et al. (1985) and EP-0,164,575 (Paszkovski et al.)). After the final rinse, the protoplasts were resuspended in electroporation buffer. For example, a suitable solution of mannitol in the context of this invention is MgCl₂concentration. An aqueous DNA solution can then be added to the protoplast suspension. In a particular embodiment of the invention, plasmid pCIB709, previously linearized by treatment with appropriate restriction endonuclease, is used. The resulting mixture is gently shaken. In one particular embodiment of the present invention, half volume is 24% (w / v), 0.5 mol / l mannitol and 30 mmol / l MgCl₂is added in PEG solution. After thorough mixing, the protoplasts are transferred to a chamber of a Dialog electroporator (DIA-LÓG GmbH Haffstrasse 34, D-4000 Düsseldorf 13, NSW), with 2 to 10 strokes, preferably 3 strokes, at an output voltage of about 2000 V / cm and 5000 V / cm. and 10 ps at an exponential damping constant at 30 second intervals. The sample is then transferred to a Petri dish supplemented with 1-25% (w / v) agarose as a gelling agent. The protoplasts are evenly distributed on the medium before the agarose solidifies. From this culture of transformed protoplasts, transgenic plants, including productive transgenic plants, of the Pooideae subfamily are regenerated as described in Chapters C-F.

In another preferred embodiment of the present invention, the Pooideae protoplasts are transformed according to the method described by Negrutiu et al. (1987). In this case, the purified protoplasts, after the final rinse, are suspended in a 0.5 M mannitol solution containing between 15 mM and 45 mM MgCl2.₂contains. The DNA was added in the form of an aqueous solution followed by the addition of an equal volume of a 36% (w / v) PEG solution (Negrutiu et al., 1987). The resulting solution is mixed well and incubated for 5 to 60 minutes, preferably 30 minutes at 10 to 32 ° C, preferably at room temperature (about 25 ° C). The solution is stirred occasionally during the incubation phase. After the incubation is complete, the protoplasts are washed and plated on appropriate culture medium. Suitable culture media include, but are not limited to, KM-8p medium containing 0.3 to 2.5% (w / v) agarose, preferably 0.6 to 2% (w / v), as a solidifying agent. The protoplasts are uniformly distributed on the medium before solidification of the agarose. From this culture of transformed protoplasts, transgenic plants, including productive transgenic plants, into the Pooideae subfamily are regenerated as described in Chapters C-F.

Preferred exogenous DNAs within the present invention are a

7th The plasmid pCIB709 shown in FIG.

Step G). Selection of transformed colonies

The agarose solidified medium from step F containing the transformed protoplasts is exposed to light or preferably dark for a period of 5-30 days, preferably 8-15 days, and most preferably 10 days at 0-50 ° C, preferably 20-32 ° C. At a temperature of between 25 ° C and 28 ° C. The solid medium is then cut into 5 slices, for example, and as described by Shillito et al., 1983; European Patent Application EP 0.129,688 (Shillito et al); Shillito et al. (1985); or EP-0,164,575 (Paszkovski et al.), selected in a bead type culture system. The number and size of media segments are not critical. In a specific embodiment of the present invention, four of these slices are each individually transferred to a suitable medium, for example, SH-45 culture medium containing 4 g / l of casein hydrolyzate and 20-100 pg / ml.

HU 220 186 Β contains hygromycin B. Slice 5 was transferred to the same medium but without hygromycin (control).

About 4-5. After one week, the putative transformed cell colonies were excised from agarose and cultured in a suitable culture medium, such as SH-45 containing 20-100 pg / ml hygromycin, at 50-80 rpm on a rotary shaker. After a further 4-5 weeks, all colonies that could provide a new suspension culture were transferred to fresh medium containing 10 20 pg / ml hygromycin. At least two additional inoculated cultures are prepared from the new suspension in the presence of 20 pg / ml hygromycin B. They are cultured under the same conditions as described above until callus formation begins. 15

The callus, the suspension cultures, and the cultures obtained from the materials prepared in this process step may be preserved cold as described in process step A).

Step H). Regeneration of transferred plants into the Pooideae subfamily from callus

From the transformed callus [process step G], transformed plants of the Pooideae subfamily can be regenerated in accordance with the procedures described in Chapters D and E. 25

Thus, the process of the present invention enables the regeneration of protoplasts derived from lawn grass plants of the Pooideae subfamily into a whole plant, in particular a whole crop. In particular, it allows the exogenous DNA to be stably incorporated into the genome of these plants, thereby altering their genotype or phenotype. In addition, protoplasts may be fused intraspecally or interspecifically with other protoplasts to form new combinations of core DNAs. These protoplasts can now be used as cloning agents in mutation and selection steps to produce the desired phenotype.

Desired phenotypic examples include resistance to toxic concentrations of natural or synthetic chemicals, including insecticides, herbicides, fungicides, bactericides, heavy metals, salts, pathotoxins, metabolic inhibitors, structural and functional analogues of cellular metabolites, to limit our procedure to these. Further examples of the desired phenotype to be selected for include resistance to adverse environmental influences such as cold or warm temperatures or biological effects such as pathogens. 50

The following embodiments are provided for purposes of further illustration of the present invention and are not intended to limit the scope of the invention in any way.

EXAMPLES

First example

Preparation of embryogenic suspension cultures from tissues of Dactylis glomerata L. 60

The embryogenic callus is based on the basic section of the youngest leaves of the knot-blossom eider (Dactylis glomerata L.) grown in the greenhouse, as described by Hanning et al. (1982). The leaves were sterilized by immersing the leaves in immersion in Clorox diluted 1:10 for 10 minutes [Clorox 5.25% (w / v) sodium hypochlorite, manufactured by The Clorox Company, Oakland, California 94623, USA ] and then cut under sterile conditions into pieces of 1-5 mm in length or diameter. These pieces were plated on sterile SH-30 medium containing 0.8% (w / v) agarose as a solidifying agent. At a culture temperature of about 25 ° C, callus and / or embryogenic cultures appear within 2 to 6 weeks after plating.

The starting material for the preparation of embryogenic suspension cultures is embryogenic callus, which is added in an amount of about 0.5 g (fresh weight) to 50 ml of liquid medium described by Gray et al., Containing 45 pmol / l Dicambat and 4 g / l casein hydrolyzate. The suspension culture was placed in a 125 ml Delong flask, sealed with a metal cap and parafilm at 27 ° C and 16 h light (40 pE / m).^{1 2}s) and incubated for 8 hours at about 130 rpm on a circular shaker at a light / dark rhythm. After about 4 weeks, the large set of nodules are allowed to settle for about 30 seconds and then 10 ml aliquots are taken from the upper portion containing the small nodules and transferred to 50 ml of fresh medium. This procedure is performed every 3-4. week, where the best-promising cell nodes are always used, according to the smaller nodule size and better quality resulting from the presence of small cells rich in cytoplasm. After 5 to 8 inoculations, the suspensions are substantially free of non-embryogenic cells and the vast majority of embryogenic cell nodules are relatively small (150-2000 pm).

Second example

Isolation and purification of Dactylis glomerata L. protoplasts

Protoplasts were obtained from the embryogenic suspension culture (Example 1) by first isolating the cells by sterile filtration on a 0.2 µm Nalgene filter unit and then adding 0.5 g (fresh weight) of protoplast in a 12.5 ml Petri dish. is added to the enzyme mixture. An enzyme solution consisting of:

<td>Cellulase RS</td><td>2% (w / v)</td>

<td>CaCl₂.h₂SHE</td><td>7 mmol / l</td>

<td>NaH₂SHE₄.h₂SHE</td><td>0.7 mmol / L</td>

<td>MES (pH 5.6)</td><td>3.0 mmol / l</td>

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

<td>(final concentration)</td><td>550 mOs / kg H₂O (pH 5</td>

then sterilized by filtration. The mixture was shaken at 50 rpm in twilight (&lt;5 pE / m).²s) Shake for 4-5 hours. The digested material is then filtered through a stainless steel sieve (100 µm mesh width) and transferred to 12 ml centrifuge tubes

HU 220 186 Β, centrifuged at 60-100 g for 5 minutes. The protoplast-containing pellet is then washed three times with KM-8p protoplast culture medium, which is fixed at 550 mOs / kg H with glucose.₂Set to O. Here, a flotation step can be incorporated to further purify the protoplasts. In this case, the washed protoplasts with sucrose are 700 mOs / kg H₂It is placed on the surface of KM-8p medium (10 ml) adjusted to an osmotic value. After centrifugation for 10 minutes at 60-100 g, protoplasts concentrated at the interface were collected using a fine pipette. Finally, the protoplasts were resuspended in 1-2 ml of KM-8p culture medium and filtered through a stainless steel grid (20 µm). The remaining protoplasts are harvested, washed, and in KM-8p medium or other osmotic suitable medium as shown in Figure 6. and resuspended for culture in Example 1B.

Third example

Protoplast culture and callus growth of Dactylis glomerata L. (a) About 5 · 10⁵ protoplast / ml was plated on KM-8p culture medium containing 1.3% (w / v) SeaPlaque Agarose (FMC Corp. Marine Colloids Division, Rockland, Maine, USA) and 30-40% (v / v) ) contains conditioned medium. The conditioned medium was obtained from a 3-4 week embryogenic cell culture of Dactylis glomerata L., filtered through a sterile Nalgene filter (0.2 µm) with the addition of glucose at 550 mOsm / kg H₂Adjust to an osmotic value and finally sterilize by filtration. The plates are then sterilized in the dark at a constant temperature of 28 ° C. After 10-14 days, the agarose was cut into wedge-shaped pieces and transferred to a bead culture system (Shillito et al., 1983), 20 ml of SH-45 suspension culture medium containing 3% (w / v) sucrose. medium in 3 ml of original agarose solidified culture. Plates were placed on a shaker at 50 rpm and 8 pE / m²s at light intensity. The release of cells from the agarose into the surrounding liquid medium leads to the formation of a new suspension culture. The resulting suspension-cultured cells are plated on agar-solidified SH-30 medium and cultured in the dark at 25 ° C until callus formation.

(b) The protoplasts are cultured in the same manner as described in Example 3 (a) above, except that the culture medium also contains 100 mg / L Oacetylsalicylic acid.

(c) The protoplasts are cultured in the same manner as described in Example 3 (a) above, except that the culture medium still contains 30 mg / L Oacetylsalicylic acid.

(d) The protoplasts are cultured in the same manner as described in Examples 3 (a) to (c) above, except that the medium does not contain conditioned media.

4th example

Regeneration of Dactylis glomerata L. plants from callus itself derived from protoplasts (a) Dactylis glomerata L. callus (as in Example 3) obtained from protoplasts was cultured in solidified SH-30 medium. Transfer to a secondary culture every other week. All embryos that develop during this time are harvested, plated on germination medium (SH-O) and exposed to light (45 pE / m).²s and 55 pE / m²s) incubate. Germination of these embryos takes 1-4 weeks. The resulting young plant blossoms are transferred to SH-O medium and incubated in light to form a root system. After reaching the 6-12 leaf stage, they are transferred to a greenhouse and slowly gain strength there.

(b) Callus (as in Example 3) obtained from protoplasts in 0.24% (w / v) GelRite-solidified SH-O medium (45 pE / m).²s and 55 pE / m²s). Transfer to a secondary culture every other week. The resulting young plant blossoms are applied to a 1: 1 mixture of SH-O and OMS media, solidified with a combination of 0.12% (w / v) GelRite and 0.4% (w / v) agar, and exposed to light. cultured. After reaching the 6-12 leaf stage, they are transferred to a greenhouse and slowly gain strength there.

(c) The small young plant blue obtained as described in Examples 4 (a) and 4 (b) is plated on OMS medium containing 0.8% (w / v) agar and cultured under light to form a root system. . After reaching the 6-12 leaf stage, they are transferred to a greenhouse and slowly gain strength there.

(d) Small young plant blues obtained as described in Example 4 (a) for a 1: 1 mixture of SH-O and OMS medium containing 0.12% (w / v) GelRite and 0 for rooting. , Solidified with 4% (w / v) agar, applied and cultured under light. After reaching the 6-12 leaf stage, they are transferred to a greenhouse and slowly gain strength there.

5th Example 1 Construction of plasmid pCIB709 with the hygromycin resistance gene expressed in an E. coli replicon plant (35S / Hyg>)

The coding sequence for the structural genes for hygromycin resistance was isolated from plasmid pLG90 (Gritz and Davies, 1983) in the form of a BamHI fragment containing about 1150 bases. Plasmid pLG9 is available from Linda Gritz, Applied Biotechnology, 80 Rogers Street, Cambridge, Massachussets 02141, USA. To construct plasmid pCIB709, the isolated BamHI fragment was inserted at the BamHI site of pCIB710 (Rothstein et al., 1987). Plasmid pCIB710 contains the regulatory region of the 35S transcript of CaMV (cauliflower mosaic virus) where

EN 220 186 Β promoter and terminator sites are separated by a unique BamHI site.

The resulting plasmid pCIB709 was deposited with the American Type Culture Collection (Rockville, Maryland, USA) under ATCC 40428.

Prior to use for transformation, plasmid pCIB709 can be linearized by treatment with PvuII. This construct then contains a hygromycin resistance gene (aminoglycoside phosphotransferase type IV) along with the 5 'and 3' expression signals of the CaMV 35S transcript from the cauliflower mosaic virus in a plasmid pCU. The sequence of pCIB709 is shown in Figure 7.

6th example

Transformation of Protoplasts of Dactylis glomerata L. by Electroporation (a) Directly coupled to purification of the protoplasts, electroporation was performed according to Shillito et al. (1985) using the linearized plasmid pCIB709 shown in Figure 7. Protoplasts after last flushing 7 · 10⁶ protoplast / ml in electroporation buffer (0.4 M mannitol, 6 mM MgCl₂) resuspended. The protoplasts were then transferred into 0.7 mL aliquots into plastic centrifuge tubes (10 mL). Plasmid DNA [62 μΐ water in PvuII-digested plasmid pCIB709 and sonicated calf DNA (Sigma) at a final concentration of 10 pg / ml (pCIB709) and 50 pg / ml (calf DNA) was then added to the tubes. added. Then 0.38 ml of polyethylene glycol solution (PEG solution) [24% (w / v) PEG 6000, 0.4 mol / l mannitol, 30 mmol / l MgCl₂and 0.1% (w / v) MES (pH 5.6)] and the solution is gently mixed. The protoplast suspension is then transferred to a Dialog electroporator chamber and treated with 10 strokes having an output voltage of 3250 V / cm and an exponential damping constant of 10 ps at 30-second intervals. The sample is then removed from the chamber and transferred to a 10 cm diameter Petri dish. 10 After addition of ml of KM-8p medium containing 1.2% SeaPlaque agarose, the protoplasts were dispersed evenly throughout the medium before the agarose solidified.

(b) Example 6 (a) is repeated, except that the output voltage in this case is 3500 V / cm.

(c) Example 6 (a) is repeated, except that the output voltage in this case is 4000 V / cm.

(d) Example 6 (a) is repeated, except that the output voltage in this case is 5000 V / cm.

(e) Example 6 (a) is repeated, except that the output voltage in this case is 3000 V / cm.

(f) Example 6 (a) is repeated, except that the output voltage in this case is 2500 V / cm.

(g) Any one of Examples 6 (a) to (f) is repeated except that PEG having a molecular weight of 4000 is used.

(h) Any of Examples 6 (a) to (f) is repeated except that PEG having a molecular weight of 8000 is used.

(i) Any of Examples 6 (a) to (h) is repeated except that the final concentration of PEG is in the range of 10% to 30% (w / v).

(j) Any one of Examples 6 (a) to (i) is repeated except that heat shock treatment is additionally performed as described by Shillito et al. (1985) and Potrykus et al. (1985).

7th example

Transformation of Dactylis glomerata L. by polyethylene glycol treatment (a) PEG-mediated direct gene transfer was performed as described by Negrutiu et al., 1987. The DNA used is the linearized form of pCIB709.

The protoplasts were rinsed with 15 mmol / L MgCl after the final rinse₂in a 0.5 mol / l mannitol solution containing 2 · 16⁶/ ml. The protoplast suspensions were aliquoted into 1 ml aliquots into plastic centrifuge tubes (10 ml). Prior to the addition of the PEG solution [40% (w / v)], DNA was administered first as detailed in Example 6. The solutions were mixed gently and incubated at room temperature for 30 minutes with occasional shaking. 1.4 ml of wash solution is then added and the individual components of the tubes are gently mixed. The wash solution was 87 mM mannitol, 15 mM CaCl₂of 27 mM MgCl₂It consists of 39 mM KCl, 7 mM tris HCl and 1.76 g / l m-inositol (pH 9.0). After standing for 4 minutes, an additional 1.4 ml aliquot of the wash solution is added and all layers are thoroughly mixed. The tubes are then centrifuged at about 60 g for 10 minutes and the supernatant discarded. The protoplasts forming the pellet were taken up in 1 ml of KM-8p and transferred to a 10 cm Petri dish. 10 After addition of ml of medium containing 1.2% (w / v) SeaPlaque Agarose, before the agarose solidified, the protoplasts were distributed evenly throughout the medium.

(b) The transformation was carried out as described in Example 7 (a) except that the pH of the wash solution was adjusted to 5.6.

(c) Transformation was performed as described in Example 7 (a) except that the pH of the wash solution was adjusted to 7.0.

(d) Transformation was performed as described in any of Examples 7 (a) to (c) except that 6,000 molecular weight PEG was used as PEG.

(e) The transformation is carried out as described in any one of Examples 7 (a) to (c) except that the PEG used is PEG of 2000 molecular weight.

ί

(F) The transformation is carried out in the same manner as described in any of Examples 7 (a) to (c) except that the PEG used is a PEG of 8000 molecular weight.

(g) Transformation was performed as described in any of Examples 7 (a) to (f) except that heat shock treatment was first applied as described by Shillito et al. (1985) and Potrykus et al. (1985).

(h) Transformation was carried out as described in any of Examples 7 (a) to (g) except that the wash solution was made from 154 mM NaCl, 125 mM CaCl₂and 5 mM glucose and adjusted to pH 6.0 with KOH.

(i) Transformation was performed as described in any of Examples 7 (a) to (g) except that the wash solution was 0.2 M CaCl2.₂and 0.1% (w / v) MES and adjusted to pH 6.0 with KOH.

(j) Transformation was performed as described in any of Examples 7 (a) to (g) except that the wash solution was 0.2 M CaCl2.₂and 7M Tris-HCl and adjusted to pH 9.0 with KOH.

8th example

Transformation of Dactylis glomerata L. Protoplasts by Electroporation or Polyethylene Glycol Treatment (a) Transformation was performed as described in Example 6 or 7 except that plasmid pCIB709 was cleaved with BglII before being used for transformation.

(b) Transformation was performed as described in Example 6 or Example 7 except that plasmid pCIB709 was cleaved with restriction enzyme HindIII before being used for transformation.

9th example

Transformation of Dactylis glomerata L. protoplasts by electroporation or polyethylene glycol treatment

The transformation is carried out in the same manner as in

6th or Example 7, except that the protoplasts were treated at 45 ° C for 5 minutes before aliquots were dispensed into individual centrifuge tubes for subsequent transformation or after aliquots were dispensed and PEG was added.

10th example

Selection of transformed colonies (a) The culture plates (Petri dishes) were incubated with the protoplasts for 10 days at 25 ° C in the dark and subsequently cultured [bead culture; Shillito et al. (1983)], cut into 5 equal pieces. 4 aliquots were added to 20-20 ml of SH-45 culture medium containing 4 g / l casein hydrolyzate and 20 pg / ml hygromycin B. 5. aliquots are also transferred to 20 ml of almost identical medium, which, however, does not contain hygromycin B, thereby serving as a non-selective control. After 4-5 weeks, the putative colonies from the protoplasts, which were washed in the presence of hygromycin B, were excised from the agarose and transferred to a 19 mm diameter petri dish containing 2 ml of liquid SH-45 containing 20 pg / ml hygromycin B . The Petri dishes are shaken on a shaker at about 50 rpm. After a further 4-5 weeks, all colonies showing growth and resulting in a new suspension were transferred to a 125 ml Erlenmeyer flask and cultured, except for hygromycin (20 pg / ml), in the same manner as the parent culture.

Each of the new slurries is 1-3. weekly inoculated culture using SH-45 medium containing 4 g / l casein hydrolyzate and 20 pg / ml hygromycin B. Cells from this suspension were plated on SH-30 solid medium containing 20 pg / ml hygromycin B and incubated at 25 ° C in the dark. From the callus that develops from these plated cells, every 2-3. weekly inoculated culture in fresh medium. It follows that all cells that grow in the presence of hygromycin B are transformants.

(b) The selection is carried out as in this

10th However, in this case, colonies of protoplasts growing on hygromycin-containing medium were transferred to an agar plate containing SH-30 containing 20 pg / ml hygromycin B and incubated at 25 ° C in the dark.

11th example

Regeneration of transformed Dactylis glomerata L. plants

Plants were regenerated in the same manner as described in Example 4 for the untransformed material.

12th example

Extraction of DNA from callus and leaf tissue

DNA is extracted from the callus and leaves of regenerated plants using a modified CETAB method (Roger and Bendich, 1985). This procedure is exemplified here as Dactylis glomerata L. but can be applied with the same efficiency to tissues of any desired plant of the Pooideae subfamily. However, any other conventional method of DNA extraction using the material described above can be used to prepare the DNA.

The callus grown on SH-0 and SH-30 medium is deep-frozen on ice and then rubbed to a fine powder under liquid nitrogen temperature. The powder thus obtained is then transferred to polypropylene centrifuge tubes (5 ml) pre-cooled at liquid nitrogen temperature. Care should be taken to ensure that the powder never releases during this process step. The powder was then freeze-dried overnight and finally distributed into 2.2 ml Eppendorf tubes, where each tube was

&lt;220 ml of powder was added to the 186 Β Vecse. One ml of CETAB extraction buffer was added to each tube and the tubes were incubated at 60 ° C for 30-45 minutes. After cooling to room temperature, a 24: 1 mixture of chloroform-isoamyl alcohol (1 mL) is added and the whole is thoroughly mixed. This is followed by centrifugation for 30 seconds at 3000 rpm in an Eppendorf centrifuge. After transferring the aqueous phase into fresh tubes, 1/10 volume of 10% (w / v) CETAB solution is added and the chloroform extraction is repeated. The aqueous phase is again transferred to fresh tubes and mixed with an equal volume of precipitation buffer. This leads to the precipitation of DNA and RNA at room temperature. This is then allowed to proceed for 30 minutes to 1 hour before the tubes are centrifuged again. The supernatant was discarded and the pellet was resuspended in high salt TE buffer (see below) at 65 ° C for 30 minutes.

Buffers and Solutions Used CETAB Extraction Buffer 1% (w / v)

<td>10% CETAB</td><td>CETAB tris (pH 8.0; 50 mmol / L) EDTA (10 mmol / L) NaCl (0.7 mol / liter) 0.5% (w / v) PVP, 360,000 molar weight [PVP = polyvinylpyrrolidone] 10% (w / v) CETAB</td>

<td>Kicsapópuffer</td><td>NaCl (0.7 mol / liter) 1% (w / v) CETAB</td>

<td>High salt concentration</td><td>tris (pH 8.0; 50 mmol / l) EDTA (1 mmol / l) tris (pH 8.0; 10 mmol / l)</td>

<td>TE solution</td><td>EDTA (1 mmol / liter)</td>

<td>TE buffer</td><td>NaCl (1 mol / liter) tris (pH 8.0; 10 mmol / l)</td>

<td>1/10 TE</td><td>EDTA (1 mmol / L) Tris pH 8.0; 1 mmol / L</td>

<td>Example 13 Purification of DNA</td><td>EDTA (0.1 mmol / liter)</td>

DNA prepared by the method described in Example 12 or by any other suitable method may be purified using any known method known in the art. Examples of suitable methods include, but are not limited to, gradient centrifugation of ethidium bromide-CsCl, treatment with phenol / chloroform, and purification on a step gradient without ethidium bromide. These procedures are described by Maniatis et al. (1982).

(a) Purification by phenol / chloroform treatment

The nucleic acids obtained in Example 12 were precipitated with 2 volumes of cold (-20 ° C) ethanol and centrifuged at 5000 g for 2-3 minutes. The supernatant was removed and the precipitate was washed with 70% and 100% ethanol. The nucleic acids are then partially dried with a sterile air stream. The DNA was dissolved in 200 μΐ of 1/10 TE buffer overnight. The DNA solution was transferred to Eppendorf centrifuge tubes and 10 μΐ of RNase solution (2 mg / ml) pre-heated to deactivate any DNase present before the tubes were incubated at 37 ° C for 1 hour. Then 0.25 volumes of 5 mol / L NaCl solution are added and the DNA is 0.4 volumes of 30% PEG solution (molecular weight 6000 to 8000) containing 1.5 mol / L NaCl and precipitated by addition and incubation for 1 hour at -20 ° C. The tubes were then centrifuged for 5 minutes, the supernatant was discarded, and the precipitate was washed with cold absolute ethanol. After drying briefly with a sterile air stream, the pellet is resuspended in 0.3 ml of TE buffer. The solution was extracted with phenol / chloroform / isoamyl alcohol (25: 24: 1) equilibrated in TE buffer and centrifuged for 30 seconds in an Eppendorf centrifuge. The aqueous phase is then transferred to fresh tubes. The solution was extracted with chloroform / isoamyl alcohol (24: 1) and centrifuged for 30 minutes, and the aqueous phase was finally transferred to fresh tubes. The chloroform extraction is repeated. Then, 1/10 volumes of 3 M sodium acetate were added to precipitate the DNA, followed by the addition of 70% and 100% ethanol. The precipitate was collected by centrifugation, washed with 70% and 100% ethanol, dried briefly in a sterile air stream and dissolved in a sufficient amount of TE buffer to give an appropriate solution for Southem blot analysis. Its DNA concentration is between 0.25 pg / L and 1 pg / L.

(b) Step gradient purification without ethidium bromide

The nucleic acids were purified with a stepwise gradient of CsCl consisting of a bottom layer of 5.7 M CsCl (in TE buffer) and 1.0 M CsCl (in TE buffer) above. The nucleic acids are incorporated into the top layer. Tubes containing a stepped gradient were centrifuged overnight with an eccentric rotary rotor (e.g. Beckman SW 50.1; 45,000 rpm). DNA is isolated from the interface, while RNA is obtained from the bottom of the tubes. The DNA was diluted with two volumes of water and precipitated with 2 volumes of ice-cold ethanol as described in Example 13 (a). The precipitate is then resuspended in TE buffer, re-precipitated with ethanol and used for Southem spot analysis.

14th example

Detection of foreign DNA sequence in the genome of transformed Dactylis glomerata L. plants by Southern blot analysis

Southem hybridization is performed essentially as described by Maniatis et al. (1982). DNA from Dactylis glomerata L. (purified according to the procedure described in Examples 13 (a) and 13 (b)) was digested with BamHI. 5 µg of this are plated on a 1% agarose gel and separated according to fragment size. The gel was added to 0.25 M HCl for about 20 minutes, followed by H₂Rinse with O and place in 0.4 M NaOH for an additional 30 min.

HU 220 186 Β

The DNA was then applied overnight to GeneScreen Plus (NEN Slot Product., NEF 976, Item No. 3300GP62, Item No. 3300GP62) (as described in the instruction manual supplied with the product) in the usual manner, where 0.4 mol NaOH is used as the transfer buffer. After overnight transfer, the filter was removed, washed with 2xSSC (0.3 M NaCl, 0.03 M sodium citrate) for 5 minutes and air dried. The blot was pre-hybridized for 4 hours at 65 ° C with buffer containing 10 g / L bovine serum albumin (non-fat, Sigma, Cat. No. A-4503), 7% SDS, 1 mM Na-EDTA and Contains 0.52 M sodium phosphate buffer, pH 7.0. The "Random Primer" method is then used to generate a radiolabeled probe sample which is freed of excess nucleotides on a "Spin" column using the IBI "Prime Time" labeling kit or any suitable method. The DNA probe consists of a fragment of pCIB709 which contains the 35S promoter region and the IV region. contains the structural gene for aminoglycoside phosphotransferase. The hybridization reaction takes place overnight at 65 ° C. The stain was then washed four times with SW wash buffer, where both final rinses were performed at 65 ° C. The stain was then subjected to additional rinses for 2 hours at 65 ° C in 0.2 χ SSC containing 1% SDS and 5 mM Na-EDTA. The wet spot is wrapped in light-permeable household foil (Saranwrap) or any other suitable foil and exposed to an X-ray film (Kodak X-Omat AR film, Eastman Kodak, Rochester, New York 14650, 165 1454). Evaluation of the developed X-ray film allows for a clear detection of hybridization between the test sample and the DNA obtained from pCIB709-transformed callus or from a pCIB709-transformed plant. Thus, the pCIB709 DNA is clearly incorporated into the large molecule DNA of the transformant.

Solutions and buffers

SW hybridization buffer 1% (w / v) bovine serum albumin (fat free); &lt;

> 0.52 M sodium phosphate, pH 7.0;

7% (w / v) SDS;

mmol / L EDTA

Wash Solution 0.04 M Sodium Phosphate, pH 7.0;

mmol / L Na-EDTA 1% (w / v) SDS; 0.125 mol / liter SDS.

15th example

Cold preservation of Dactylis glomerata L. callus cultures (1) The actively growing callus from Dactylis glomerata L. plants is transferred to liquid SH-0 medium. The amount of callus is customarily between about 0.5 and 1 g of callus per 20 ml of culture medium. The flasks are carefully moved with the callus and mixed to disperse and disperse the callus lumps. The culture and the freezing excipient are then separated by chilling on ice.

(2) An equal volume of freeze P is then added over a 5 minute period and the mixture is left on ice for 1 hour. During this time, 1.0 mL aliquots are dispensed into labeled and pre-cooled 18 mL plastic vials suitable for cold preservation (e.g., Vangard Cryos Cryogenic Vials, Sumimoto-Bakelite Co., Ltd., Japan, Cat. No. M S4502). and incubating on ice. The P freezing aid consists of 1 mol / L glycerol, 1 mol / L L-proline and mol / L dimethylsulfoxide (DMSO) (Sigma, catalog number 02650, entry 57F-8816) in water, pH 5.6. This solution is freshly prepared for each use (glycerol / proline / water mixture can be frozen frozen).

(3) After incubating the cells and freezing aid for one hour, the dish is placed on a surface of a liquid bath at 0 ° C. The bath may consist of ethanol or other suitable, known and suitable refrigerants. The bath is provided with a mixing device which ensures constant mixing of the refrigerant and is connected to a device which enables the cooling of the refrigerant at a controlled rate.

(4) As soon as the vessel is in the refrigerant, reduce the temperature to 0.5 ° C / min. When the temperature reaches -40 ° C, the vessel is placed in liquid nitrogen and stored either in the liquid itself or in a gas atmosphere above it where the temperature should not exceed -100 ° C.

16th example

Cold Preservation of Embryogenic Cell Cultures of Dactylis glomerata L. (A) 1. A suspension culture of Dactylis glomerata L. cells is placed on ice 2 to 10 days after inoculation of a secondary culture and cooled. The freezing aid solution is also cooled in the usual manner on ice. The freezing excipient consists of 1 M glycerol, 1 M L-proline, 2 M dimethylsulfoxide (DMSO) in water, pH 5.6. The freezing aid solution is freshly prepared before each use (the glycerol / proline / water mixture may be stored frozen).

Second The freezing aid is added over a period of 5 minutes. The cells then remain under constant ice cooling for 1 hour in the refrigerant. During or after this time, aliquots are taken out, divided into pots and kept on ice. The vessels are then treated in the same manner as described for Example 15 for callus.

(B) Cold preservation is performed as described in Example 16 (a), except that the refrigerant

The process step (1) consists of 1 mol / l glycerol, 1 mol / l sucrose and 2 mol / l DMSO in water (pH 5.6).

17th example

Recovery of Growth Cultures from Cold Canned Dactylis glomerata L. (A) 1. A vessel treated with Example 15 was removed from liquid nitrogen.

Second Melt the vessel while allowing it to stand at room temperature until all the ice melts.

Third The contents of the wells are dispensed into SH-0 culture medium solidified with GelRite or agar. Typically, 0.5 ml of the thawed material is then distributed on a 10 cm diameter Petri dish containing 30-50 ml of culture medium. The solid medium is either distributed in an oblique shape or cavity at its edges to ensure that any remaining freezing aid is run from the cells.

4th The material was incubated in the medium at 27 ° C in the dark. Growth begins in 1-4 weeks. From these calliums, as described previously for a normal embryogenic callus, a secondary culture is prepared.

(B) Recovery of growth cultures from cold-preserved Dactylis glomerata L. as described in Example 17 (A). In this case, the vessels are thawed very quickly in each case by placing the vessel in a water bath at about 40 ° C and keeping it there until all the ice melts.

18th example

Cold canning of Zea mays callus

Cold preservation of actively growing callus Zea mays is carried out in the same manner as described in Example 15 for Dactylis glomerata L.

19th example

Cold preservation of embryogenic suspension cell culture of Zea mays

Cold preservation of Zea mays embryogenic suspension cell culture is performed as described for Examples 16 (A) and 16 (B) for Dactylis glomerata L.

20th example

Recovery of growth-capable cultures from cold-preserved Zea mays

Recovery of growth cultures from cold-preserved Zea mays was carried out in the same manner as described for Dactylis glomerata L. in Examples 17 (A) and 17 (B).

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