RU2557389C2 - Method of producing dihaploid barley plants from cultured mycospors in vitro - Google Patents

Abstract

FIELD: agriculture.

SUBSTANCE: invention relates to the field of agricultural biotechnology. Method of producing dihaploid plants of barley from cultured microspores in vitro, comprising: growing donor plants at reduced air temperature of 15-20°C, light regime: 16 h day/8 h night, relative humidity of 60-70%, lighting intensity of 10000-15000 lux, with carrying out phytosanitary treatment, at that growing donor plants is carried out to the stage from opening of the sheath, when the inflorescence is in the flag leaf, - stress treatment of ears at 4°C, in tubes with a poor environment containing KCl - 1.5 g/l, MgSO₄×7H₂O - 0.25 g/l, CaCl₂×2H₂O - 0.1 g/l, mannitol - 60 g/l, potassium phosphate buffer - 1 ml/l, pH 7.0, for 7 days for shifting from gametophytic development path of microspore to sporophytic path - microspore isolation from spikelets in sterile conditions, - culturing the isolated microspores in a modified environment for embryogenesis induction, consisting of 6% maltose, 10 sets per 1.5 ml of the culture, and plant growth regulators in amount of 1 mg/l 2,4-D, 0.2 mg/l zeatin, and adding the aforementioned components is carried out before the addition of microspores, - regenerating plants from embryoids by culturing on solid nutrient Murashige and Skoog medium without the adding of plant growth regulators, - processing the barley haploid plants with antimitotic agent N-diacetyl-N-(β,γ-epoxypropyl) aminocolchicine for doubling chromosomes and obtaining dihaploid plants.

EFFECT: invention enables to obtain dihaploid homozygous lines of barley for breeding new varieties and hybrids with improved properties.

1 dwg, 2 tbl, 6 ex

Description

The field of technology. The invention relates to the field of agricultural biotechnology, in particular applied research related to the creation of new varieties and hybrids, and can be claimed by educational and scientific institutions of biotechnological and agricultural profile, seed companies and breeding stations.

The relevance of the invention. Barley is one of the most important crops, the selection of which shows the promise of using doubled haploids. The use of doubled barley haploids can significantly reduce the time of the breeding process, as well as increase its effectiveness (Maluszynski M. et al. 2003).

The most promising method for producing haploid plants is the method of inducing embryogenesis in a culture of isolated microspores (Touraev A. et al. 2009). The microspore is a haploid cell of higher plants, from which a mature pollen grain is formed during normal gametophyte development, ready for pollination. Using special technologies (stress treatment, the special composition of the nutrient medium, etc.), it is possible to change the pathway for the development of microspores from the gametophytic to sporophytic (formation of totipotent cells) and to induce the regeneration of the haploid plant (Touraev A. et al. 2009). The resulting haploid plant will have a single set of chromosomes, i.e. the genome will be represented by one allele of each gene. In case of artificial (use of antimitotic substances) or spontaneous (spontaneous) doubling of the genome, alleles also double and the haploid plant goes into a diploid (digaploid) state, i.e. it turns out a homozygous (inbred) plant. In contrast, with classical propagation in the genome of a plant, both male and female chromosomes combine, i.e. there is a combination of different alleles that can mask each other. The presence of only one double allele (in digaploids) allows the trait to manifest itself in the phenotype even if it is always recessive in the population initially. Genetic cleavage using digaploids is less complicated, which allows the use of relatively small populations to isolate a specific combination of genes (Wedzony M. et al. 2009).

The level of technology. The problem of creating dihaploid plants has been the subject of many reviews. There are publications and patents for inventions in which it is proposed to use remote hybridization (Patent WO 2005084420 (2005-09-15), Kasha KJ and Kao KN, 1970, Mujeeb-Kazi A. and Riera-Lizarazu O., 1996), female gametophyte (Patent JPH 0698648 (1994-04-12), Keller E. and Korzun L. 1996)), anther culture (Patents US 5770788 A (1995-06-06), US 6362393 B1 (1999-08-26)) and microspore culture (Patents KR 100952103 (2009-07-01), WO 2002052926 A3 (2002-01-02), SUS 6764854 B2 (2004-07-20). Due to the complexity of obtaining haploid plants, all works describe a solution to any particular problem applicable to a particular variety or plant species.

The method of obtaining haploid barley plants through the elimination of chromosomes

This method is based on the phenomenon of elimination of half of the chromosomes in hybrid barley cells in the early stages of embryogenesis (Kasha K.J. and Kao K.N., 1970). This process can be observed with interspecific crossing of barley (N.vulgare) with onion barley (N.bulbosum). The result of this process is the elimination of H.bulbosum chromosomes.

The method of obtaining haploid plants through interspecific hybridization has been improved over several years, which made it quite effective (Hayes P. et al. 2003, Devaux P. 2003). To date, the method of elimination of chromosomes has allowed to obtain a large number of haploid and dihaploid lines and this technology is used in breeding programs of a number of companies (Devaux R. 2003).

However, in this method, a number of significant drawbacks can be noted: the difficulty of selecting the obtained hybrids based on haploidy, the duration of the experimental process itself, and its low efficiency. The positive aspects of this technique include the fact that its implementation does not require special expensive equipment, and it can be carried out under the usual conditions for the breeding process and is almost independent of the genotype (Maluszynski M. et al. 2003).

Method for producing haploid plants from female gametophyte (parthenogenesis)

For several years, attempts have been made to cultivate in vitro isolated eggs, embryo sacs, dustless ovaries or ovules to produce haploids by parthenogenesis (Keller E. and Korzun L. 1996). This method has proven most effective on crops such as potatoes, sugar beets, onions and cucumbers (Metwally et al. 1998). Barley haploids were also obtained using the culture of the ovule, however, the method turned out to be ineffective (0.2-1.4%) and laborious, which did not allow it to be widely used for applied purposes (Yean-San L.H. 1987).

Method for producing haploid barley plants anther culture

Obtaining haploid plants from isolated anthers can take place in two ways: direct regeneration of somatic embryos (embryoidogenesis) and through callusogenesis. The efficiency of embryoid formation is a genotype-dependent process and can vary significantly, varying from 0 to 95% (Germana M.A. 2011).

The essence of the technique is that anthers are isolated from plants grown under controlled temperature and light conditions. Before the anthers are isolated, ears are cultivated for 14-28 days at low temperatures of 4-8 ° C, then the anthers are isolated and transferred to poor environments with mannitol (0.3-0.7 M) for 3-4 days. After stress treatment, the anthers are transferred to artificial nutrient media and cultured in the dark at 23-28 ° C for 3-5 weeks until embryoids are formed, which, in turn, are transferred to the regeneration medium. The composition of the media varies depending on the genotype used (Szarejko I., Kasha K.J. 1991, Cistué L. et al. 1994, Hoekstra S. et al. 1997, Jacquard C. 2006).

The main and significant drawback in addition to the high genotypic dependence of this technique is that the resulting plants can have a different degree of ploidy (due to the possibility of regeneration of plants from anther somatic tissues): from haploid to di-, poly- and aneuploid. Moreover, using this technology, a large number of albinos are formed among regenerated plants (Castillo A.M. et al. 2000, Li H, Devaux P. 2005, Jacquard C. 2006, Germana M.A. 2011).

The method of obtaining haploid plants through a culture of isolated microspores

Another approach for producing haploid plants is based on in vitro cultivation of isolated microspores. Externally, this method is similar to the method of isolated anthers, but at its core they are very different, as they are based on the use of various types of tissues and cells. Microspore culture is a process of cultivating in a liquid nutrient medium generative cells freed from anther somatic tissues. This method is technologically different from the anther culture. The main difference between the culture of isolated microspores and the anther culture is the process and time of isolation of microspore cells. The basic design of the microspore isolation experiment is as follows: isolated anthers or spike fragments are macerated in a nutrient medium. To effectively isolate a pure population of microspores, the resulting pulp is filtered and centrifuged several times. Further cultivation of microspores is carried out in liquid nutrient media, the composition of which varies depending on the genotypes or objectives of the experiment.

Currently, there are several patents for this method of producing digaploid plants. However, the methods proposed therein are developed and are suitable only for crops such as pepper (Capsicum annuum L.) (Patent KR 100952103 (2009-07-01)) and corn (Zéa máys L.) (WO 2002052926 A3 (2002-01-02 )), rice (SUS 6764854 B2 (2004-07-20)). Our proposed method allows to obtain haploid barley plants (H.vulgare).

Currently, two methods for producing haploid barley plants are described, which are closest to our work (Kasha K.J. et al. 2003, Devies P.A. 2003). However, they differ significantly from what we offer in such parameters as the cultivation conditions of donor plants, the age of the donor plant used, the presence and duration of stress treatment of plants, the method of isolation of microspores, the density of microspores in the induction nutrient medium and the composition of the induction nutrient medium and composition nutrient medium for plant regeneration.

The proposed methods also have several significant disadvantages: the need to determine the stage of development of microspores by cytological methods, the high frequency of obtaining albino plants, and the high dependence of the efficiency of obtaining digaploids on the plant genotype. We propose an efficient genotype-independent method for producing dihaploid barley plants with a high level of spontaneous doubling of chromosomes and a low yield of albino plants.

Duplication of chromosomes in haploids

In order for healthy offspring to be obtained from haploid plants, one of the developmental stages requires chromosome doubling in plant cells, which in most cases is induced by the use of an antimitotic agent, colchicine. This substance has the ability to bind to special cell structures responsible for division, to block the divergence of daughter cells, which leads to the formation of cells with a double (dihaploid) set of chromosomes. A significant drawback of colchicine is its high toxicity both to cells and to personnel (Ade R. and Rai M.K. 2010).

The method of treating plants with colchicine has not undergone significant changes since 1938, when it was first described (Levan A. 1938): roots, apical meristems, etc. are treated with colchicine. (Wong C.K. 1989, Swanson E.V. 1990, Mathias R. and Robbelen G. 1991). Plant treatment procedures due to the need to use high concentrations of the drug are potentially dangerous and expensive (De Raere R. et al. 1981, Hansen FL et al. 1988, Barnabas B. et al. 1991, Hassawi DS and Liang GH 1991, Zhao JP and Davidson D. 1984).

At the same time, the procedure for treating plants with colchicine does not always achieve the desired result, since after such an impact a large number of chimeric plants, aneuploids, etc. (Zhao J.P. and Davidson D. 1984, Barnabas B. et al. 1991, Mathias R. and Robbelen G. 1991).

The structural formula of colchicine was determined in 1955 (Corrodi and Hardegger, 1955). According to a certain structure, colchicine is a complex of trimethoxyphenyl, a seven-membered ring bearing acetamide at position 7 and a tropolone ring.

To double the chromosomes and convert haploid barley plants to dihaploid ones, we suggest using the amino derivative of colchicine, N-diacetyl-N- (β, γ-epoxypropyl) aminocolchicine (Figure). This analogue showed lower toxicity to plant cells and greater ability to bind tubulin (T. Resh and A. Touraev unpublished data).

Disclosure of invention

The developed technique is a method for producing haploid and dihaploid barley plants from microspores isolated from anthers and consists of the following key steps:

- cultivation of donor plants under certain conditions (vernalization of seeds and seedlings, low air temperature of 15-20 ° C, light mode: 16 hours a day / 8 hours a night, humidity 60-70%, light intensity 10,000-15,000 lux, phytosanitary treatment );

- the use of donor plants at the stage of opening the vagina of the flag leaf when the ear is inside the flag leaf;

- low-temperature treatment of freshly harvested ears of donor plants in a medium with mannitol (at 4 ° C, poor medium with 60 g / l mannitol, 7 days);

- the presence in the medium for the induction of embryogenesis of 6% maltose, ovaries (10 plants per 1.5 ml of culture) and plant growth regulators (1 mg / l 2,4-D, 0.2 mg / l zeatin).

Adding all components of the medium before adding microspores;

- regeneration of plants from embryoids on the medium without the addition of plant growth regulators,

- treatment of young haploid plants to obtain doubled haploids (soaking greenhouse plants in 0.05% N-diacetyl-N- (β, γ-epoxypropyl) aminocolchicine for 5 hours).

This technique was used on 6 varieties of domestic selection (3 winter and 3 spring). The efficiency of plant regeneration turned out to be high for all studied varieties, regardless of genotype, and ranged from 16 to 20 green plants per spike. Moreover, spontaneous doubling of chromosomes occurred in 78% of the regenerated plants obtained. As a result of the use of N-diacetyl-N- (β, γ-epoxypropyl) aminocolchicine, chromosome doubling occurred in 80% of the treated haploid plants, and thus the total efficiency of the method was 16-19 digaploid plants per one spike used. This indicator indicates the high efficiency of the proposed method.

Brief Description of the Drawings

The drawing shows the structural formula N-diacetyl-N- (β, γ-epoxypropyl) aminocolchicine

The implementation of the invention

Example 1. Growing donor plants

The cultivation of high-quality donor plants is a prerequisite for obtaining embryogenic microspores for subsequent embryogenesis and regeneration of haploid and dihaploid plants. In addition to the mandatory protection of plants from diseases such as powdery mildew, dwarf rust, root rot and insects (spider mites, aphids, thrips, etc.), optimal vegetation conditions must be created - high-quality lighting, water supply, soil substrates, fertilizers, moisture and air quality.

Preparation and germination of seeds. To increase germination, the seeds were kept at 4 ° C for at least 2 weeks, and then flaked to remove a dense seed skin. For disinfection, the seeds were treated with a 2% KMnO ₄ solution for 20 minutes. Seed treatment was carried out at room temperature, followed by washing with tap water. To prepare a 2% solution, 2 g of KMnO _{4 was} dissolved in 100 ml of water. Next, the seeds were soaked in water for 12-24 hours, and then transferred to wet filter paper and cultivated in the dark for 2-3 days until seedlings appeared, at a temperature of 15-20 ° C.

Sprouted barley seeds 3-5 pcs. sown in pots with sterile soil substrate (pH 6.5-7). Depth of sowing is 3-4 cm. Seeds were germinated in an artificial climate at a temperature of 15/12 ° C (day / night), with light conditions: 16 hours a day / 8 hours a night, air humidity 60-70% and light intensity 10000-15000 Suite After emergence, the temperature of cultivation increased to 15-20 ° C. Seeds of winter varieties were sown 1.5-2 months earlier than spring for the vernalization stage.

The vernalization of barley. Sprouted seedlings of winter barley varieties passed the vernalization stage for 1.5-2 months at 4 ° C and air humidity of 70-80%. After vernalization, the seedlings were transferred to conditions similar to the conditions of germination.

Breakthrough and rejection of atypical plants. After the formation of 2-3 leaves, before the beginning of the tillering stage, breakthrough and rejection of atypical plants was carried out. The first plant was left in the pots and the soil was sprinkled with soil. Watering with a mixture of water-soluble fertilizers was carried out once every 8-12 days.

Growing plants to obtain microspores.

Plants were grown by irrigation with tap water as the soil substrate dried at 60-70% air humidity. To improve aeration of the root system, the top soil layer was loosened at least once every 10 days. The room temperature was maintained within the range of 15-20 ° C, illumination of 10-15 thousand lux with a 16-hour photoperiod. Every 2 weeks was carried out radical top dressing of plants with organic fertilizers and trace elements.

Phytosanitary treatment. When growing donor plants, it is very important to prevent plant diseases and pests, as these factors greatly reduce plant fertility and the effectiveness of embryogenesis in microspore culture. Before planting and during the cultivation of plants, preventive treatment of plants with a mixture of fungicides and bactericides was carried out, and upon detection of affected plants, they were immediately isolated.

The treatment of plants with a mixture of fungicides and bactericides was stopped 2-3 weeks before the collection of microspores, because these drugs reduced the viability of the latter. After the flag leaf appeared in plants, weekly dressing with NPK fertilizer was performed weekly (20:20:20).

Example 2. Collection and low-temperature processing of spikelets

Spikelets were collected from donor plants at the stage when the vagina of the flag leaf was opened, but the inflorescence was still inside the flag leaf. Most of the microspores in such plants were in the late mononuclear phase of development (70-80% of microspores).

The stem was cut 8-10 cm below the spike (the flag leaf was removed), placed in a glass of water and transferred to the laboratory. Prior to sterilization, the vagina of the flag leaf was not removed.

Under sterile conditions (laminar boxing), the vagina of the flag leaf was removed from the ear and the ear was placed for 1 min in 70% ethanol and then thoroughly washed in sterile distilled water.

Then the ears were placed in test tubes with a poor environment and mannitol (KCl 1.5 g / l, MgSO ₄ × 7H ₂ O 0.25 g / l, CaCl ₂ × 2H ₂ O 0.1 g / l, mannitol 60 g / l potassium phosphate buffer (pH 7) 1 ml / l, pH 7.0) so that only the lower part (spine) of the ear is in the medium. The tubes were hermetically sealed and placed in a refrigerator (4 ° C, darkness) for 7 days.

Example 3. The allocation of microspores

Under sterile conditions (laminar box) using a sterile (autoclaved) tool, the upper and lower spikelets (1-2 cm) were removed from the ears. Spikes were cut into 2-3 cm segments and placed in a sterile blender bowl (Waring Blender, USA) with a volume of 50 to 200 ml (depending on the number of spikelets) so that the spikelets did not occupy more than half the volume. Cooled poor medium with mannitol (2-4 ° C) was immediately added to the spikelets in such a volume that it covered the spikelets (from 25 to 100 ml). It was crushed at low speeds for about 5 s, then the resulting suspension was passed through a filter (100 μm) into sterile flasks. Then, the suspension was separated into centrifugation tubes with a volume of 15 ml and centrifuged at 900 rpm for 5 minutes. The supernatant was removed, and the precipitated microspores were washed by double centrifugation in a cooled medium with mannitol (15 ml each) for 5 minutes at 600 rpm. The supernatant was removed and added to the precipitated microspores in a modified medium No. 1 (table. 1), to an approximate density of 2 × 10 ⁴ . To calculate the number of microspores per ml of medium, a Goryaev chamber was used. A suspension of microspores was transferred to Petri dishes (35 × 10 mm) of 1.5 ml per cup and freshly isolated ovaries (10 pcs. Per cup) were added to them. Each spike received 3-4 cups with microspore culture, which were tightly sealed with parafilm (Parafilm, USA) and cultured in the dark at a temperature of 25 ° C.

The ovaries were isolated under sterile conditions under a binocular light microscope from ears freshly cut and sterilized according to the method described above.

Table 1 The composition of the used nutrient media for producing barley haploids in microspore culture * Components (mg / L) Nutrient medium induction of embryogenesis (Wednesday No. 1) Culture medium for embryoid cultivation (Medium No. 2) Nutrient medium for plant cultivation (Environment No. 3) Kno ₃ 3000 3000 2000 (NH ₄ ) 2SO ₄ 450 450 - NH ₄ NO ₃ - - 1700 KH ₂ PO ₄ 400 400 180 CaCl ₂ · 2H ₂ O 200 200 450 MgSO ₄ · 7H ₂ O 200 200 400 Na ₂ EDTA 40 40 40 FeSO ₄ · 7H ₂ O thirty thirty thirty MnSO ₄ H ₂ O 10 10 - MnSO ₄ · 4H ₂ O - - 25 Н3ВО ₃ 3 3 6 ZnSO ₄ · 7H ₂ O 2 2 - ZnSO ₄ · 4H ₂ O - - 9 NaMoO ₄ · 2H ₂ O 0.25 0.25 0.25 CuSO ₄ 0,025 0,025 - CuSO ₄ · 5H ₂ O - - 0,025 CaCl ₂ · 6H ₂ O 0,025 0,025 0,025 Ki 0.75 0.75 0.8 Glycine - - 2 Myo-inositol one hundred one hundred one hundred Pyridoxine hcl one one 0.5 A nicotinic acid one one 0.5 Thiamine hcl 10 10 0.5 2,4-D one - - Zeatin 0.2 - - MES 2000 - - Glutamine 500 - - Maltose 60,000 20000 - Sucrose - - 30000 Fitagel - 2500 - Agar - - 7000 pH 6.2 5.8 5.8 * - Filters (0.22 microns) were used to sterilize liquid media; autoclaving was used to sterilize solid media. An antibiotic cefotaxime (200 mg / L) was added to all media (after autoclaving) to prevent culture contamination.

Example 4. Induction of embryoidogenesis

Microspore plates were cultured in the dark at 25 ° C. The content of live microspores after low-temperature processing and cultivation on the medium for the induction of embryogenesis (with maltose, phytohormones and ovaries) for 5 days ranged from 69 to 91% depending on the genotype (Table 2). Embryoids formed within 20-30 days. The average number of embryoids formed from microspores ranged from 188 to 214 pcs. on the used spike (table. 2).

table 2 The effectiveness of embryoidogenesis of barley microspores in vitro culture Varieties of barley The content of live microspores after 5 days of cultivation,% The average number of embryoids per spike The number of green plants per ear, pcs. The number of haploid plants before and after treatment with N-diacetyl-N- (β, γ-epoxypropyl) aminocolchicine, pcs. The number of digaploid plants, pcs. before after Pride oz 85% 205 19 four one eighteen MASTER oz 67% 192 19 6 2 17 ABROAD oz 88% 214 twenty 5 one 19 PRAIRIE yar 91% 250 19 2 0 19 ZEUS yar 81% 188 17 four one 16 DINA yar 69% 195 16 3 0 16

Example 5. Regeneration and cultivation of plants

After the embryoids reached 1.5–2 mm, they were transferred to Petri dishes (100 × 15 mm) with solid medium No. 2 for embryoid cultivation (Table 1), 20–25 pcs. per cup and cultured for 4-5 days in the dark. Then the plates were transferred to light at 25 ° C and cultured until seedlings formed (about 2 weeks). Approximately 8-10% of the formed embryoids were regenerated into full-fledged green plants, and about 2% of the formed plants were albinos (Table 2).

After the formation of 2-3 leaves from the obtained plants, green ones were selected and transferred to medium No. 3 by Murasige and Skoog (Murashige T. and Skoog F. 1962) for plant cultivation (Table 1). Cultivation was carried out in test tubes or containers with a light regime: 16 hours a day, 8 hours a night at 25 ° C until the formation of well-developed roots (2-4 weeks). Then the plants were kept at 4 ° C for 4 weeks in a climatic chamber under low-intensity light conditions: 8 hours a day, 16 hours a night (vernalization stage). Then, the temperature and light intensity were increased to 15 ° C and 12/12 h day / night, respectively. Under this regimen, another 1 week was cultivated.

After vernalization, plants were transferred from test tubes to pots with a soil mixture for growing in a greenhouse. During the first week after the plants were transferred to the greenhouse, plastic containers were left on them, which were slightly opened, for soft adaptation.

The effectiveness of the method was 16-20 green plants per spike used. Moreover, spontaneous duplication of chromosomes occurred in 78% of the obtained plants, which indicates the high efficiency of the developed protocol. The number of digaploids obtained was from 13 to 17 pcs. for one used ear. Ploidy of plants was determined using standard methods (Maluszynski M. et al.).

Example 6. The treatment of haploid barley plants with an antimitotic drug to obtain doubled haploids

Plants that have passed the stage of vernalization and adaptation to greenhouse conditions have grown to form at least two shoots. Then the plants were removed from the soil, the roots were washed and cut by 1-2 cm. The roots and the lower part of the shoots were lowered by 5-8 cm in a 0.05% solution of the antimitotic agent (500 mg / L N-diacetyl-N- ( β, γ-epoxypropyl) aminocolchicine + 20 ml / l DMSO + 100 mg / l gibberellic acid + 20 drops / l TWEEN 20) and left for 5 hours at a temperature of 15-20 ° C and intense light (10-15 thousand lux ) Then the roots were washed with running water and placed in soil. Next, the plants were cultivated according to the method described above for growing donor plants to obtain mature kernels.

As a result of the use of N-diacetyl-N- (β, γ-epoxypropyl) aminocolchicine, chromosome doubling occurred in 80% of the treated haploid plants and, thus, the total efficiency of the method ranged from 16-19 digaploid plants per one spike used (Table 2) .

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Claims (1)

1. A method of producing digaploid barley plants from cultured microspores in vitro, including:
- cultivation of donor plants at a low air temperature of 15-20 ° C, light mode: 16 hours a day / 8 hours a night, humidity 60-70%, lighting intensity 10,000-15,000 lux, with phytosanitary treatment, while growing plants - donors carry out to the stage from the opening of the vagina of the flag leaf, when the inflorescence is inside the flag leaf,
- stress treatment of ears at 4 ° C, in test tubes with a poor environment containing KCl - 1.5 g / l, MgSO ₄ × 7H ₂ O - 0.25 g / l, CaCl ₂ × 2Н ₂ О - 0.1 g / l, mannitol - 60 g / l, potassium phosphate buffer - 1 ml / l, pH 7.0, for 7 days to switch from the gametophytic pathway of microspore development to the sporophytic pathway,
- the selection of microspores from spikelets under sterile conditions,
- cultivation of the selected microspores on a modified medium for the induction of embryogenesis comprising 6% maltose, 10 ovaries per 1.5 ml of culture and plant growth regulators in an amount of 1 mg / l 2,4-D, 0.2 mg / l zeatin, and the addition of the above components before adding microspores,
- regeneration of plants from embryoids by cultivation on a solid nutrient medium Murashiga and Skoog without adding plant growth regulators,
- treatment of haploid barley plants with the antimitotic drug N-diacetyl-N- (β, γ-epoxypropyl) aminocolchicine to double the chromosomes and obtain dihaploid plants.

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