WO2002001940A2

WO2002001940A2 - A method of generating fertile plants from isolated microspores

Info

Publication number: WO2002001940A2
Application number: PCT/DK2001/000456
Authority: WO
Inventors: Anni Jensen
Original assignee: Pajbjergfonden
Priority date: 2000-06-30
Filing date: 2001-06-29
Publication date: 2002-01-10
Also published as: AU2001268955A1; WO2002001940A3

Abstract

The present invention relates to the field of plant breeding. It concerns a method of generating fertile whole plants from microspores of wheat, that have been isolated from cold treated spikes. More specifically it concerns a method for the production of wheat plants from microspores co-cultured with ovaries. In other aspects, this invention relates to the regerated plants, and to seeds and progeny of the regenerated plants derived from cultured microspores/ovaries. The invention also relates to the use of a method for generating fertile whole plants from isolated microspores of wheat.

Description

A method of generating fertile plants from isolated microspores

Technical field of the invention

The present invention relates to the field of plant breeding. It concerns a method for the production of wheat plants from microspores co-cultured with ovaries. In other aspects, this invention relates to the regenerated plants, and to seeds and progeny of the regenerated plants derived from cultured microspores/ovaries.

Background of the invention

Crop and plant improvement is an area of great commercial interest. Wheat for example is a major world-wide cereal crop with an important commercial value. Un- fortunately current methods for improving breeding of wheat are time consuming and labour intensive. In traditional plant breeding seeds from those homozygous plants shown to possess the desired characteristics are selected for further breeding. They are crossed and the resulting offspring is inbred through generations of selfing (i.e. self fertilization) in order to obtain homozygous plants having new gene combinations. In each generation of selfing, the number of heterozygous plants is halved. Through generations of selfing, plants which are essentially homozygous may be obtained. A sufficient degree of homozygosity is generally believed to be obtained after 6-8 generation. This process is time consuming and generally takes from 3 to 6 years. Wheat plants displaying the desired characteristics are contin- uesly selected among the generations of selfing. For practical reasons the selection process takes place before the offspring has been inbred to 100 % homozygosity. This means that selection is carried out on the basis of both homozygous and heterozygous plants. The selection of heterozygous plants is disadvantageous since these plants are carrying recessive genes with unknown characteristics, which are passed on to the next generations. The process of developing inbred parents generally takes from 3-6 years. Another 3-4 years is required for field testing which gives a total of 7-10 years.

By the use of microspore in vitro cultures, the time period to produce a new homo- zygot wheat variety, may be reduced from 3-6 years to about 1-2 years. Further- more, selection becomes more efficient, as it is carried out exclusively with homo- zogous plants.

Various general methods, including anther and isolated microspore cultures, have been used in a variety of crops, for example tobacco and barley, to produce organisms with haploid genetic complements. Anthers are the structures that contain the male gametophytes, the haploid microspores or pollen. Microspores have a single haploid nucleus whereas mature pollen have 3 nuclei. "Haploids" contain only one- half of the chromosome number present in somatic cells. Somatic cells are those other than gametic cells, the latter being inherently haploid. Haploid complements may also be doubled to produce homozygous diploids. The production of doubled haploid plants is one way to produce truly homozygous lines. Douple haploid plants have both male and female sex organs and produce ears with good seed through self-pollination after chromosomes have been doubled.

As mentioned above, pollen grains develop from microspores, which are in turn produced by meiosis in pollen mother cells. The plants resulting from pollen cultures are also usually haploid and may be sterile. To remedy this the chromosome complement may be doubled by various agents, such as colchicine, a mitotic spindle inhibitor, the use of which results in chromosome duplication without cell division.

Occasionally, chromosome doubling occurs spontaneously. The plants resulting from induced or spontaneous chromosome doubling are diploid. Doubling of haploid complements allows homozygous lines to be produced from heterozygous parents in a single generation. Microspore culturing followed by chromosome doubling is one method of producing doubled haploids.

The use of doubled haploid plants in wheat breeding requires a successful haploid production system. Today the techniques for producing doubled haploids from culturing entire anthers are well established. Further, plant regeneration from isolated microspores has been reported for various cereal species, including corn (Coumans, et al., 1989; Pescitelli et al., 1989) barley, rice and wheat, wherein isolated microspores have been cultured.

Factors that affect the frequency of in vitro plant production include genotype, donor plant physiology, the stage of pollen development, pretreatment conditions and the concentrations and nature of ingredients in the media used for culture and regeneration. For example, sucrose levels, plant growth regulators and additives to culture media affect success.

The use of mannitol and of cold temperature pretreatment have been explored separately as means for increasing the frequency of embryo formation from isolated microspores. (Wei et al., 1986). Wei et al. applied mannitol pretreatment to isolated barley pollen, but had limited success, possibly because the methods were applied to binucleate pollen, whereas uninucleate pollen (microspores) are believed to be the most responsive in culture. Uninucleate cells most likely lost viability during isolation from the anthers. If mannitol pretreatment was applied to whole anthers containing microspores then results were said to be better.

There has been several reports on culturing isolated microspores from laboratory wheat. For example, Hu & Kasha (Hu, T. C, Kasha, K. J., 1999, Genome, Vol. 42,

Iss. 3, pp. 432-441) describe a cytological study of pretreatments used to improve isolated microspore cultures of laboratory wheat. Spikes were pretreated with 0.4 M mannitol solution under cold temperatures and an increase in the number of embryos and later green plants were observed.

Touraev et al. (Touraev, A., Indrianto, A., Wratschko, I., Vicente, O., 1996, Sex Plant Reprod., 9, pp. 209-215) report on the use of a combination of starvation and heat shock treatments to induce the formation of embryonic microspores in wheat. It has been observed that microspore cultures have not resulted in a similar amount of embryo-like structures as reported for the anther culture systems. Puolimatka et al. (Puolimatka, M., Laine, S., & Pauk, J., 1996, Cereal Research Communications, 24, 4, pp. 393-400.) present a study of the culture of directly isolated wheat microspores and examines the effect of wheat ovary co-cultivation. Ovary co-culturing was found to be beneficial to the development of embryo-like structures from micro- spores.

Further, Mejza et al. (Mejza, S. J., Morgant, V., DiBona, D. E., & Wong, J. R., 1993, 12, pp. 149-153) describe how co-culturing wheat microspores with barley ovaries were critical, in addition to how pretreatment with cold temperatures resulted in less embryos than pretreatment at high temperatures. Also, HU & Kasha (Hu, T. O, Kasha, K. J., 1997, Plant Cell Reports, 16, pp. 520- 525) report on the improvement of isolated microspore culture of laboratory wheat through ovary co-culture.

The present inventor has developed a new and improved culture technique for general use in commercial plant breeding, wherein the period of generation of fertile plants from haploid isolated microspore cultures is shortened by 3-4 years. The microspore cultures are co-cultured with ovaries. The present invention discloses a method which is successful for commercially desirable plant lines, such as wheat. The inventor has addressed the major problems in the culture of haploids, that is, low initial response frequency as determined by embryo-like growth, difficulties in plant regeneration, and difficulties in chromosome doubling to make diploids from haploids, the latter process leading to fertile plants. The inventor has combined environmental stress conditions which divert the microspores from microsporogenesis to embryogenesis, such as combining stress factors comprising culture medium components, such as mannitol with cold pretreatments and oxygen treatment during culturing, to recover surprisingly high yields of embryos and of regenerated fertile plants.

Summary of the invention

The present invention relates to plant breeding and discloses a method of generating fertile whole plants from isolated microspores of wheat, comprising the steps of:

- providing a first donor plant, exposing spikes of said donor plant to a cold treatment, isolating microspores from said spikes, obtaining said microspores,

- culturing said microspores in a culturing medium, - removing ovaries from a second donor plant,

- contacting said ovaries with said microspore cultures, obtaining microspore/ovary co-cultures, obtaining plant embryos, regenerating plantlets from said plant embryos, - growing said regenerating plantlets into whole fertile wheat plants, and - obtaining whole fertile wheat plants.

Detailed description of the invention

This invention relates to a method for the production of plants, and in particular fertile whole plants, from isolated microspores of wheat. Further, the present invention concerns the use of a method for generating fertile whole plants from isolated microspores of wheat to be employed in the commercial plant breeding industry.

The scope of the present invention is intended to further include the production of cereal plants, such as maize, sorghum, barley, rye, oat or rice plants.

In a general aspect, the invention presents a new and improved methodology for the production of plants which comprises subjecting plant compositions containing mi- crospores to a combination of stress factors. The microspores of the invention are co-cultured with ovaries, and the method comprises the steps of:

- providing a first donor plant,

- exposing spikes of said donor plant to a cold treatment, - isolating microspores from said spikes, obtaining said microspores,

- culturing said microspores in a culturing medium, removing ovaries from a second donor plant, contacting said ovaries with said microspore cultures, - obtaining microspore/ovary co-cultures,

- obtaining plant embryos,

- regenerating plantlets from said plant embryos,

- growing said regenerating plantlets into whole fertile plants, and

- obtaining whole fertile plants.

According to the invention a plant composition are obtained. The plant composition may be plant organs, such as spikes which include microspores. Microspores may be cultured to produce fertile plants. For plant breeding applications, elite breeding parent plants are preferred. The term "elite" is defined in a commercial context as the breeding parent plants having the highest yield, good resistance properties and/or good quality properties, such as wheat having excellent baking properties.

The following description is an illustration of one embodiment of the invention, wherein the first donor plant as well as the second donor plant are wheat. A preferred method for producing fertile wheat plants from microspore co-cultures will generally include the following steps:

According to the invention the microspores of the cultures are from spikes of a first donor plant. When spikes are employed as the plant donor, it is generally preferred to sterilize their surface. Following surface sterilization of the spikes, for example, with a solution of korsolin, the spikelets A are removed from spikes (small A portions of the spikes) and cut into 2-3 cm pieces and then placed in an isolation medium. In another embodiment the spikes are cut into cut into 2-3 cm pieces and then placed in an isolation medium. The amount of medium used may be dependent on the number of spikes or spikelets.

In one embodiment of the invention ovaries for the use in co-culturing, including wheat are from a second donor plant of the invention. The second donor plant may be selected from a group of plants different from the first donor plant. The second donor plant may be selected from wheat, barley, maize or rice. The ovaries may be selected from spikes when the second donor plant is wheat or barley, or the ovaries may be selected from tassels when the second donor plant is maize.

In another embodiment of the invention the second donor plant is selected from the same group of plants as the first donor plant. For example the ovaries and the microspores may all be selected from wheat. In one particular embodiment of the in- vention the second donor plant of wheat is identical to the first donor plant of wheat. The ovaries may be selected from the same individual donor plant as the microspores. In this embodiment the term "first" and "second" donor plant means the one and same donor plant. According to the invention the temperature under which the donor plant is growing is between 16-22 °C, preferably between 17-21 °C, more preferably between 18-20 °C. If the donor plant is grown in a greenhouse environment the temperature may be set to a temperature corresponding to the temperature of the season of the year. For example in the month of May a temperature of approximately 20-21 °C is preferred.

Further, the length of exposure to light under which the donor plant is growing is between 16-20 hours/24hours, such as between 16.5-19.5 hours/24 hours, for example between 17-19 hours/24 hours, such as 17.5-18.5 hours/24 hours, for exam- pie 17-18 hours/24 hours. By the term "light" is meant electromagnetic radiation in the wavelength range of 300-2000 nannometres. In one aspect mercury lamps may be used to mimic daylight and/or radiation having photosynthetic activity (400-700 nm).

The protocol of the present invention consists of pretreating a plant composition, for example, organs such as spikes containing microspores, under conditions which divert the microspores from gametophytic development to that of embryogenic development. The pretreatment includes incubation of the plant composition if it includes spikes, at a cold temperature which is a stress factor.

Although the microspore-containing plant organs such as spikes may generally be pretreated at any cold temperature below about 10 °C, a range between 1-10 °C is preferred, and a range between 2-7 °C is more preferred, particularly a temperature of between 3-6 °C is preferred. Other temperatures may yield embryos and regen- erated plants, however response rates may be less when pretreatment is outside the preferred temperature ranges. In the context of the invention "response rate" is defined as either the number of embryos or the number of regenerated plants per number of microspores initiated in culture.

The exposure of spikes to cold treatment may according to the invention be in a period of between 2-25 days, such as between 6-18 days, for example between 10- 14 days. Further, other pretreatment periods are envisioned within the scope of this invention, as long as the isolated microspores result in regenerated plants. After the pretreatment of whole spikes at cold temperatures, dissected spikes are further pre- treated in an environment that is capable of diverting microspores from their devel- opmental pathway. The function of the preculture medium is to switch the developmental program from one of pollen development to that of embryo development.

For microspore cultures originating from spikes, the spikes are preferably selected at a stage where the microspores are uninucleate, that is, include only one, rather than 2 or 3 nuclei. Methods to determine the correct developmental stage are well known to those skilled in the art. In a preferred embodiment of the invention microspores are separated from the donor spikes at the mid to late uni-nucleate developmental stage. The mid to late uni-nucleate microspore stage has been found to be the developmental stage most responsive to the disclosed methods of the invention. The mid to late uni-nucleate stage is the developmental stage of the microspore when the nucleus is situated in the center of the cell before the formation of a vacu- ole. In a particularly preferred embodiment of the invention the developmental stage is at the late uni-nucleate stage. During this stage the nucleus is migrating to a posi- tion next to the cell wall at the same time a vacuole is formed. Eventually the nucleus will have migrated to a position opposite the germination pit.

To isolate microspores, an isolation medium is preferred. An isolation medium may help in the separation of the microspores from spike walls, whilst the viability and embryogenic potential of the microspores are maintained. An illustrative embodiment of the isolation medium into which microspores are released from the disrupted spike includes mannitol, and optionally macronutrients.

According to the invention the term "medium" is defined as any combination of nutri- ents that permit the microspores/ovaries to develop into embryos or callus. The isolation medium allows for the blending and filtration of the microspores, and the culture medium is used for growing the microspores/ovaries. Many examples of suitable embryo/callus promoting media are well known to those skilled in the art. These media will typically comprise mineral salts, carbon sources, vitamins, growth regu- lators and water.

In those embodiments where microspores are obtained from anthers, microspores may be released from the anthers into isolation medium. One method of release is by disruption of the anthers, for example, by chopping the anthers into pieces with a sharp instrument, such as a razor blade, scalpel or Warring blender. The resulting mixture of released microspores, spike fragments and isolation medium are then passed through a filter in order to separate the microspores from the spike wall fragments. In the context of the present invention a filter is a mesh, having a mesh of about 150 μ pore size. The filter may consist of a nylon mesh, or of stainless steel. The filtrate which results from filtering the microspore-containing solution is preferably substantially free from spike fragments, cell walls and other debris after centrifugation.

Where appropriate and desired, the microspore filtrate may be washed several times in isolation medium prior to centrifugation. For best results, washing and subsequent centrifugation is repeated approximately two times. The purpose of the washing and centrifugation is to eliminate any toxic compounds which may be contained in the non-microspore part of the filtrate which are created by the release process. The centrifugation may be done at a spin speed of for example 800 rpm.

As an additional stress factor in the culture environment, spikes may be dissected into an isolation medium comprising a sugar alcohol, such as sorbitol, mannitol, in- ositol and/or carbohydrates, such as sucrose. Preferably, according to the invention the medium comprises mannitol. The mannitol concentration of the invention may be between 0.31-0.39 M, preferably between 0.32-0.37 M, more preferably between 0.33-0.36 M, such as 0.35 M. Further, the mannitol may be added to the plant organ during cold-treatment.

It is believed that the mannitol or other similar carbon structures or environmental stress factors induce starvation of the microspores and functions to force the microspores to focus their energies on entering developmental stages. The cells are unable to use, for example, mannitol as a carbon source at this stage of development. It is believed that these treatments confuse the cells causing them to develop into embryos and plants from microspores instead of developing into gamatophytes.

The isolated wheat microspores may then according to the invention be exposed to ovaries from a second donor plant such as wheat. The ovaries may be obtained by hand selecting the ovaries with tweezers transferring them to a dish containing culture medium. In a preferred embodiment of the invention the ovaries are selected for culturing on between day 5 and day 0 prior to culturing said microspore cultures. The microspores may be contacted with the ovaries to establish a co-culture on day 1 of culturing the microspores.

It is within the scope of the invention that the induced embryo/callus structures may be sub-cultured into a series of media which are capable of inducing tissue development, for example, roots and shoots. This medium may be designated maturation and regeneration medium.

In one aspect of the present invention the plant cultures are exposed to oxygen during culturing. It is believed that oxygen supplementation may increase the rate and success of cell growth. The cell cultures of the invention are exposed to oxygen by flushing a stream of oxygen through the cell culture container. This may be achieved by leading a stream of oxygen over the top of the culture medium by hand, or by having the exposure of oxygen automated, such as by placing the cell cultures in boxes or in a climate incubator and then centrally applying oxygen to the cell cultures through a valve in the lid or any other side of the box, or through a valve of any of the sides of the climate incubator. In one embodiment of the invention the individual culture containers in the box or climate incubator have their lids on. However, in yet another embodiment the individual culture containers in the box or climate incu- bator are without lids or sealing. Further, the lids or sealing may be oxygen permeable. By supplementing the cell cultures with oxygen a small over-pressure is created, which promotes the exchange of gases in the cultures. The concentration of oxygen in the plant culture medium is dependent on the surface to volume ratio of the medium in the container, such as a petri dish and the concentration in the im- mediate gas phase. In a preferred embodiment of the invention the cultures are exposed to oxygen approximately every 2-3 days. The duration of the oxygen exposure according to the invention is between 1-20 minutes, such as between 4-16 minutes, for example between 8-12 minutes. It is preferred that the oxygen is a 100 % oxygen having a pressure of 2 bar.

The culture medium of the invention may have a pH value of between 5.4-6.0. The components of the culture medium of the invention essentially comprises components capable of maintaining good microspore/ovary co-culture viability, and is described in detail below. In one embodiment the medium optionally comprises ascor- bic acid. The culture medium of the invention may be added freshly to the cultures. In one embodiment of the invention fresh medium is added to the cultures at least once a week. In a more preferred embodiment fresh medium is added to the cultures every 6 days, such as every 5 days, for example every 4 days, such as

3 days, for example every 2 days, or such as every day. The length of the intervals between the additions of fresh medium is dependent on the culture conditions, such as temperature, level of oxygen, density of the microspores, and growth rate of the microspores. In the present context "density" is measured as the number of cells per predefined culture area. For example the method of counting the cells in a counting chamber is known to the person skilled in the art. By the term "growth rate" is meant how fast the cells divide within a predefined time frame, i.e how "vital" the cells are. Other factors that are important for the intervals in between addition of fresh medium are the size and volume of the culture dish. The amount of fresh medium may be added considering the culture conditions, such as those mentioned above and the amount may differ from addition to addition. In one embodiment of the invention the fresh medium is added to the culture dishes in an amount of between 0.1 tol .O ml, such as between 0.2 to 0.9 ml, for example between 0.3 to 0.8, such as between 0.4 to 0.7 ml.

In one aspect of the invention the culture medium is a 2-layered medium. According to the invention it has proven beneficial to employ a bilayer plate support for the microspores/ovaries during culturing. This allows the cells to be near the surface of the medium. Several types of suitable supports are within the scope of the invention, one such support is gellan gum (Gel-Rite®). In one embodiment the bilayer support for development of the microspore/ovary cultures consist of a 2-layer medium, wherein the medium is on top of a solid base. Culturing isolated microspores/ovaries on a solid support prevents microspores/ovaries from sinking into the medium with oxygen tension and possibly death as a consequence.

In one aspect of the invention the solid support is a nylon mesh in the shape of a raft. A "raft" is defined as a support material which is capable of floating above the bottom of a tissue culture vessel, such as a petri dish. Further, in another embodiment the solid support is a stainless steal mesh. Also the solid medium allows the embryos to mature. The medium passes through the mesh while the microspores are retained at the medium-air interface. The surface tension of the liquid medium in the petri dish causes the raft to float. The medium is able to pass through the mesh and as a consequence the microspores/ovaries stay on top. The mesh raft will remain on top of the total volume of medium. An advantage of the mesh raft is to permit diffusion of nutrients to the microspores/ovaries. Optionally, the use of a raft also permits transfer of the microspores/ovaries from dish to dish during sub-culturing. In another embodiment the solid support is the basal part of the culture vessel (solid layer), where upon a liquid layer is situated.

In one aspect of the invention a raft cell support enables the transferral of the raft with its associated microspores/ovaries to various media containing desired ingredients, such as subculture media, without substantial loss, disruption or disturbance of the developing embryos.

After the petri dish has been incubated for an appropriate period of time, preferably 4 weeks, in the dark at a predefined temperature, such as 28°C all microspores/ovaries are transferred to a liquid or semi-solid based medium which promotes embryo maturation (regeneration medium). It is desirable to obtain high quality embryos, such as embryos exhibiting organized developmental structures, for example shoots and meristems. Embryos are aggregates of multi-cellular structures each generally arising during a period of approximately 3 to 4 weeks. The embryos are preferred for subsequent steps to regenerating plants.

If desired, intact fertile plants may then be regenerated in a regeneration medium. During the regeneration process individual embryos are induced to form plantlets. It is within the scope of the invention to use a sequence of regeneration media for whole plant formation from the embryos. The number of different media in the sequence may vary depending on the specific protocol used. Optionally as a final employment a rooting medium may be used prior to transplanting the plantlets into soil.

In one aspect of the invention a haploid embryonic cell culture is obtained and fur- ther the plant embryos obtained are haploid. To obtain whole fertile plants the number of chromosomes in haploid plant embryos must be doubled. One method of chromosome doubling is by using chromosome doubling agents. A widely applied chromosome doubling agent is colchicine, which disrupts the mitotic spindle during cell proliferation, and thus prevents the cell from dividing. In one embodiment the chromosome doubling agents may be added to the isolation medium, whereby di- haploid cell cultures are obtained. In another preferred embodiment of the invention the chromosome doubling agent may be added to plantlets. This may be performed by rinsing the soil off the roots and the placing the plantlets in a container, wherein the roots are covered with the chromosome doubling agent. In one embodiment of the invention the plantlets are placed in a container of approx. 0.05 % colchicine and 1 % DMSO, and left for approx. 13-20 hours.

In another aspect of the present invention when the plantlets reach a height of about 5-10 cm they are transferred to containers, i.e. pots for further growth into flowering plants for example in a greenhouse by methods well known to the skilled artisan.

According to the invention the ratio of fertile regenerated wheat plants per embryo is at least 7:10, such as 8:10, for example 9:10. The fertile regenerated whole plants are capable of producing seeds and progeny. These seeds and progeny are also within the scope of this invention. Thus, in yet a further aspect of the invention the seeds from the regenerated plants are prepared.

An object of the invention is to obtain a plant regenerated from the embryonic cell culture as described above. In one embodiment the plant may be haploid and in another embodiment the plant is dihaploid. Also the invention relates to producing progeny of the regenerated plants, and therefore encompass seeds obtained from the regenerated plants, either by crossing or selfing.

In yet another embodiment the plant of the invention is transgenic. In one aspect the method of the present invention may shorten the time required for improvement of crops by integrating new genetic traits. The method of regenerating plants from or microspore/ovary co-cultures may facilitate genetic manipulations directed at plant improvement. For example, nucleic acid segments may be introduced into microspores for integration in the chromosomes and expression on a whole plant level. Accordingly, it is within the scope of the present invention to transform plants, whereby transgenic plants are produced for example by the incorporation of large segments of DNA or artificial chromosomes using standard commercially available techniques. Experimentals

The following is an example of one embodiment of the invention, wherein microspores and ovaries from wheat are co-cultured.

Harvest of spikes:

Harvest at noon. The spike has just started to emerge. Leaves are cut off. The spikes are placed in container of 1.25 mM Hydroxyurea and then placed in a re- fridgerator. The spikes are moved to water the following morning (18 hours in hy- droxyurea).

Cold treatment: 14 days at 4 °C.

Sterilising spikes:

The spikes are rinsed of leaves and are collected in bundles of 5 spikes per bundle. 3 bundles are collected to sterilise 15 spikes at a time. They are rinsed for a few seconds in 70% ethanol with a drop of tween added. Thereafter the spikes are placed in a container with 2.5 % korsolin while stirring for 12 min. New fresh korsolin every day. Rinse in steril water 4 x and place in an empty steril container.

Dissection of spikelets and ovaries:

Spikelets from 5 spikes are removed from the spikes by twisting with tweezers so that the ovaries stay on the spikes. Spikelets are placed in a steril 100 ml beaker or 9 cm petri dishes, and the ovaries are transferred to 3.5 cm petri dishes with approx. 5 ml culture medium and 20 ovaries per dish.

Sterilising the blender:

The blender is a Warring with MC3 top. The stopper is put in a glas container with 96% ethanol for at least a couple of minuttes. The blending is with 96 % ethanol, 2 x 60 seconds. At high speed.

Blending:

Spikelets are blended for 2 x 3 sec. in 0.37 M mannitol. Mannitol is placed in the blender so that the bottom third of the top knives are covered. The blended sub- stance is lead through a coarse filter into a 100 ml beaker. The filtrate is transferred to 3 x 15 ml centrifuge tubes. The remains on the filter is reentered into the blender with a spoon. Mannitol is added to the blender so that the liquid reaches the top knives. Blending for 2 x 3 sec. The blended substance is lead through the same filter and the filtrate is transferred to 3 centrifuge tubes and an additional 2 centrifuge tubes. Accordingly, the filtrates from the 2 blendings are placed in 5 centrifuge tubes all together.

Centrifugation:

Centrifugation is carried out for 3 min at 800 rpm. The supernatant is discarded.

Collecting the pellets:

Pellets from the 5 centrifuge tubes are collected in a 15 ml centrifuge tubes with a pipette, and the centrifuge tube is filled with mannitol.

Centrifugation:

Adding medium:

Pellet is resuspended in 12 ml CHB-2 medium, which is transferred from the petri dishes containing the ovaries with a disposable pipette. The microspores are spread out evenly in the medium with the pipette. 12 ml microspore suspension are distributed with 4 ml in each petri dish with the ovaries. The dished are sealed with para- film. For the 2-layer medium the resuspension is in 6 ml CHB-2 medium divided by 2 ml per dish.

Induction:

The dishes are placed in plastic boxes with valves in darkness at 28 °C. 100 % oxygen (2 bar) is added every 3 day for 5 minutes.

Transferral to differentiation medium:

After approx. 4 weeks the embryos are transferred to the differentiation medium: The ovaries are removed with tweezers, and the medium is sucked up with a pipette and the embryos are transferred to containers with a spoon or spatula.

Mannitol 0.37 M: 134.8 g in 2 litres H₂O, 269.6 g in 4 litres H₂O, 20 ml/spike Maltose: 21 g in 100 ml Liquid 2-layer culture medium

(3 ml/spike liquid medium, 1 ml/spike 2-layer medium)

Claims

1. A method of generating fertile whole plants from isolated microspores of wheat, comprising the steps of:

- providing a first donor plant,

- exposing spikes of said donor plant to a cold treatment, isolating microspores from said spikes,

- obtaining said microspores, - culturing said microspores in a culturing medium,

- removing ovaries from a second donor plant,

- contacting said ovaries with said microspore cultures,

- obtaining microspore/ovarie co-cultures,

- obtaining plant embryos, - regenerating plantlets from said plant embryos,

- growing said regenerating plantlets into whole fertile wheat plants, and

- obtaining whole fertile wheat plants.

2. The method according to claim 1 , wherein the second donor plant is selected from a group of plants consisting of wheat, barley, maize or rice.

3. The method according to any of the preceding claims, wherein the length of exposure to light under which the donor plant is growing is between 16-20 hours/24hours, preferably between 16.5-19.5 hours/24 hours, more preferably between 17-19 hours/24 hours, such as 17.5-18.5 hours/24 hours, for example

17-18 hours/24 hours.

4. The method according to any of the preceding claims, wherein the temperature under which the donor plant is growing is between 16-22 °C, preferably between 17-21 °C, more preferably example between 18-20 °C.

5. The method according to any of the preceding claims, wherein the cold treatment comprises exposing said spikes to temperatures of between 1-10 °C, preferably between 2-7 °C, more preferably between 3-6 °C.

6. The method according to claim 5, wherein the exposure of the spikes to cold treatment is a period of between 2-25 days, such as between 6-18 days, for example between 10-14 days.

7. The method according to any of the preceding claims, wherein the microspores are separated from the donor spikes at the mid to late uni-nucleate developmental stage.

8. The method according to the claims 1-7, wherein the microspores are separated in a medium comprising a sugar alcohol.

9. The method according to any of the preceding claims, wherein the microspores are separated in a medium comprising mannitol.

10. The method according to claim 9, wherein the mannitol concentration is between

0.30-0.50 M, preferably between 0.32-0,39 M, more preferably between 0.33- 0.37 M.

11. The method according to any of the preceding claims, wherein the culture me- dium is a 2-layer medium.

12. The method according to claim 11 , wherein the medium has a pH value of between 5.4-6.0.

13. The method according to the claims 12-13, wherein the medium optionally comprises ascorbic acid.

14. The method according to any of the preceding claims, wherein the ovaries are contacted with the microspores to establish a co-culture on day 1 of culturing said microspore cultures.

15. The method according to any of the preceding claims, wherein the cultures are exposed to oxygen during culturing.

16. The method according claim 15, wherein the cultures are exposed to oxygen every 2-3 days.

17. The method according to the claims 15-16, wherein the duration of the oxygen exposure is between 1-20 minutes, such as between 4-16 minutes, for example between 8-12 minutes.

18. The method according to the claims 16-17, wherein the oxygen has a pressure of 2 bar.

19. The method according to any of the preceding claims, wherein the ratio of fertile regenerated wheat plants per embryo is at least 7:10, such as 8:10, for example 9:10.

20. The method according to any of the preceding claims, wherein the plant embryos are haploid.

21. The method according to any of the preceding claims, wherein whole plants are regenerated by chromosome doubling.

22. The method according to any of the preceding claims, further comprising preparing seeds from the regenerated plants.

23. The method according to any of the preceding claims, further comprising pro- ducing progeny of the regenerated plants.

24. A haploid embryonic cell culture obtained by the method as defined in the claims 1-23.

25. A dihaploid embryonic cell culture obtained by the method as defined in the claims 1-23.

26. A plant regenerated from the embryonic cell culture as defined in the claims 24- 25.

27. The plant according to claim 26, wherein the plant is haploid.

28. The plant according to claim 26, wherein the plant is dihaploid.

29. The plant according to claim 26, wherein the plant is transgenic.

30. A seed obtained from the plants as defined in the claims 26-29.

31. Use of a method for generating fertile whole plants from isolated microspores of wheat as defined in the claims 1-23.