RU2608638C2 - Method of producing planting material of bobovnik anagyroid - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology and reproductive biology. Invention represents a method of regeneration of plants of Bobovnik anagyroid in vitro, including preliminary processing of dry seeds, surface sterilization and cultivation in nutrient medium for sprouting, characterised by that as the preliminary treatment of seeds of Bobovnik anagyroid filled with hot water at a temperature of 90–100 °C and left for 20–30 minutes before the cooling water, surface sterilization is carried out, by placing seeds in 1 % aqueous solution of synthetic detergent for 15 minutes under continuous stirring conditions, and then washing with running water for 15–20 minutes, culturing is carried out for 3–4 weeks, then evolved seedings are planted in cups with soil substrate and placed into garden frame for 4–6 weeks for adaptation to non-sterile conditions, where nutrient medium comprises mineral salts and vitamins at MS, 20 g/l sucrose, 7 g/l agar, additionally contains 2.2 μM BAP, as feed medium selected medium WPM with addition of 2.2 μM BAP.

EFFECT: invention allows to reduce labor intensity and duration of the process of planting material, as well as simplify the process of planting material.

4 cl, 2 tbl, 2 dwg, 1 ex

Description

The invention relates to the field of biotechnology and reproductive biology of plants, namely for the mass production of planting material, and can be used in biotechnology, decorative gardening, introduction, as well as in order to preserve genetic resources and biological diversity of species. The method is intended for the regeneration of plants of the legume family, whose seed reproduction is difficult due to the phenomenon of hard seed (state of physical dormancy).

A known method of plant regeneration to obtain bean planting material using an in vitro culture (Timofeeva SN, Elkonin LA, Tyrnov VS Micropropagation of Laburnum anagyroides Medic. Through axillary shoot regeneration // In Vitro Cell.Dev. Biol. — Plant, 2014, v. 50, pp. 561–567), which consists in activating the growth and development of shoots from already existing meristems of the vegetative buds, which includes several successive stages. To initiate in vitro culture, surface-sterilized isolated kidneys were placed on a nutrient medium containing mineral salts and vitamins according to the prescription of the medium Murashige and Skoog (MS), sucrose (20 g / l) and agar (7 g / l); with the addition of phytoharmon 6-BAP (2.2 μM). After 2 weeks of cultivation, actively growing explants were transferred to proliferation medium, which differs from the initial medium by a concentration of mineral salts reduced to Ѕ by MS. After 6-8 weeks of cultivation on this medium, a bundle of several shoots 3-7 mm long developed from each bud from this kidney. Shoots ≥ 5 mm long were separated from the bunch and subcultured on freshly prepared medium until a new shoot bundle was obtained. This micropropagation cycle was repeated several times with regular transplants every 6-8 weeks. After obtaining a sufficient amount of experimental material, shoots ≥ 10 mm long, consisting of 3-4 internodes, were selected, transferred to a rooting medium, which differs from the medium for proliferation by a reduced concentration of mineral salts (¼ MS) and sucrose (10 g / l). To stimulate rhizogenesis, auxin of NAA (2.7 μM) was added to the medium. The obtained root plants were planted in glasses with soil substrate and placed in a greenhouse. After 1-2 months, after adaptation to non-sterile conditions, the plants were planted in the field.

However, the disadvantage of this method is that the success of in vitro culture is influenced by the explantation season. In addition, although the initiation frequency reached 70-75%, in the course of further cultivation, not all initiated explants produced intensively growing cultures with high regeneration potential. The application of this method also limits the duration of the process of obtaining stably proliferating cultures (about 6 months) and a low reproduction rate (each explant produced no more than 1-2 shoots with a length of ≥ 10 mm suitable for subsequent rooting). In addition, during long-term cultivation, the reproduction rate decreased, morphologically altered shoots appeared, and callusogenesis intensified.

There is also a method of producing seedlings of Bobovnik angovidnogo by treating seeds with concentrated sulfuric acid H ₂ SO ₄ for 0.5-1 hours.

However, working with sulfuric acid requires caution, since it is unsafe for the contractor.

Closest to the proposed invention is a method of regeneration of a woody plant from this. Legumes of Albizia odoratissima in vitro culture (Rajeswari V., Paliwal K. In vitro adventitious shoot organogenesis and plant regeneration from seedling explants of Albizia odoratissima Lf (Benth.) // In Vitro Cell. Dev. Biol. Plant, 2008, v. 44, pp. 78-83), according to which the albia seeds were preliminarily incubated for 15 min in 1N H ₂ SO ₄ , after which they were surface sterilized and placed on a germination medium containing Ѕ mineral salts by MS, sucrose (30 g / l ) and agar (8 g / l), without the addition of phytohormones. Epicotyls were isolated from the developed seedlings, which were cultured on MS medium with the addition of BAP (5-10 μM) for the induction of organogenic callus and the subsequent development of shoots. The resulting shoots were individually transferred to MS medium with the addition of NAA (25 μM), where they were kept for 24 hours to stimulate rhizogenesis. After this, the shoots were cultivated on the medium of the same composition, but without NAA. The resulting root plants were acclimatized in sterile vermiculite, and then planted in the soil.

The disadvantages of this method are the multi-stage production of root plants, which requires a large number of reagents and increases the duration of the process of obtaining regenerated plants. In this method, there is a stage of callus formation, which increases the likelihood of autoclonal variability in the resulting offspring. In addition, working with 1N H ₂ SO ₄ requires caution, as it is unsafe for the contractor.

The objective of the present invention is to develop a method for producing root plants (seedlings / planting material) Anovirus bobovnik.

The technical result consists in simplifying, reducing the complexity and duration of the process of obtaining planting material.

This object is achieved by the fact that in the method of regeneration of plants of the legume family in an in vitro culture, including pre-treatment of dry seeds, surface sterilization and cultivation in a nutrient medium for germination, according to the solution, an anovirus-like Bobovnik is selected, the preliminary treatment is carried out with hot water at a temperature from 90-100 ^° C, cultivation is carried out for 3-4 weeks, after which the developed seedlings are planted in glasses with soil substrate and placed in kroparnik for adaptation to non-sterile conditions.

The culture medium may contain mineral salts and vitamins according to MS, 20 g / l sucrose, 7 g / l agar, and an additional 2.2 μM BAP. As a nutrient medium, WPM can be selected with the addition of 2.2 μM BAP.

In a micro-greenhouse, moisture is gradually reduced, after the appearance of new leaves (after 4-6 weeks), the seedlings are suitable for transplantation and cultivation in the field. The novelty of the proposed solution is that the seeds are treated with hot water, and not H ₂ SO ₄ to obtain seedlings, the multi-stage inherent in the prototype is excluded, the duration of seedlings is reduced to 7-10 weeks.

The invention is illustrated by drawings: figure 1 - presents Bobovnik seedlings that developed during cultivation in vitro; figure 2 - adapted to non-sterile conditions bean seedlings,

where: 1 - Bobovnik seedlings, developed after 3 weeks of cultivation on MS medium with the addition of 2.2 µM BAP (on the left - pre-treatment of dry seeds with boiling water, on the right - control, without pre-treatment);

2 - Bobovnik seedlings adapted to non-sterile conditions,

on the left - 2 weeks after planting in the soil, on the right - 2 months after planting in the soil and growing in a greenhouse.

The method of obtaining seedlings (planting material) anovirus-shaped beanbill is as follows:

Dry bean seeds are placed in a measuring cup (200 ml), filled with boiling water (100 ml) and left for 20-30 minutes until the water cools. After that, the seeds are laid out in gauze bags of 30 pieces, placed in a 1% SMS solution (synthetic detergent, such as Biolan), where they are kept for 15 minutes with constant stirring, and then washed from traces of SMS with running water (15-20 minutes) . Surface sterilization is carried out according to the generally accepted method: bags with plant material are immersed for 1 min in 70% alcohol, then placed for 15 min in a 0.1% mercuric chloride solution, and then washed three times with sterile autoclaved water. Under sterile conditions of laminar, sterilized seeds are placed on the surface of a germination medium containing mineral salts and vitamins according to MS, sucrose (20 g / l) and agar (7 g / l), with the addition of BAP (2.2 μM) and grown in the light ( photoperiod 16 hours / 8 hours). After 3-4 weeks of cultivation, after the appearance of the first true leaf, the developed seedlings are planted in glasses with soil substrate and placed in a microparison to adapt to non-sterile conditions. After 4-6 weeks, the seedlings are suitable for transplanting into the soil and growing in the field.

An example of a practical implementation of the method of obtaining seedlings Bobovnik anagirovidny.

For the implementation of the proposed method as a donor plant used an instance of Laburnum anagyroides, grown in the field of the arboretum of the Botanical Garden of SSU (Saratov). The seeds used were collected in September 2010 and 2011.

Overcoming physical dormancy of seeds during germination. Bean seeds in the last phases of ripening and during storage gradually lose both their own moisture and the ability to absorb water vapor from the air. As a result, the complete waterproofness of the seed peel is formed, the so-called “hard seed” (or state of physical dormancy), which significantly complicates the germination of seeds. Various methods for overcoming the physical rest of seeds are known from the literature: scarification, soaking seeds in boiling water, treatment with concentrated H ₂ SO _4, or freezing. Modern methods of stimulating the germination of dormant seeds - treatment of seeds with physiologically active substances or germination on nutrient media under sterile conditions (Nikolaeva M.G., Razumova M.V., Gladkova V.N. Directory of germination of dormant seeds // Leningrad: publishing house "SCIENCE", 1985 - 245 p.).

Three variants of temperature seed pretreatment were investigated:

• Freezing when dry seeds were placed in a freezer at a temperature of -18 ^° C for 1 month, after which they were introduced into a sterile culture;

• Treatment with burning water, mainly boiling water; introduction to sterile culture.

• Frozen seeds were treated with hot water and introduced into a sterile culture. The obtained experimental results are shown in Table 1.

After identifying the factors influencing the overcoming of “hard seed”, the conditions of seed germination in vivo (soil substrate) and in vitro (MS medium + 0.5 mg / L 6-BAP) were comparatively studied. The obtained experimental results are shown in Table 2.

Table 1. The effect of temperature pre-treatment of seeds on their survival in a sterile culture (%) after 4 weeks of primary cultivation on media of various compositions

Note: 3 replicates of 15-20 pieces were studied; * p ≤ 0.05; ns - no significant difference; variants accompanied by identical Latin letters differ insignificantly according to the Duncan criterion.

Table 2. The effect of pre-treatment and seed germination conditions on the number of anovirus-shaped Bobovnik seedlings (%) after 1 month of cultivation

Note: 3 replicates of 10-15 each were studied; * p ≤ 0.05; variants accompanied by identical Latin letters differ insignificantly according to the Duncan criterion.

It was statistically significant that the most effective seed pre-treatment was hot water treatment 80.8. Thus, according to the proposed method, the treatment of dry seeds with hot water must be carried out precisely with boiling water (90-100 ^° C), followed by exposure to cooling the water to room temperature (20-30 minutes). With a short-term scalding of seeds in boiling water (2-3 min), the frequency of seed germination was lower compared to keeping the seeds in hot water before cooling. If the exposure time of seeds in cooling water was reduced, the number of germinated seeds also decreased. If soaking with boiling water was accompanied by a subsequent exposure of 20-30 min at a constant high temperature (50-60 ^° C), a significant deterioration in the quality of seedlings was observed.

In this regard, when using this method, the heat treatment process must be carried out in the specified temperature and time range.

The simplification of the method is achieved by eliminating the stages of animation and rooting of microprobe.

Claims (4)

1. The method of regeneration of Bobovnik anagovirus plants in vitro, including pre-treatment of dry seeds, surface sterilization and cultivation on a nutrient medium for germination, characterized in that, as a preliminary treatment, Bobovnik anagovirus seeds are poured with hot water at a temperature of 90-100 ° C and left 20-30 minutes before cooling water, surface sterilization is carried out by placing the seeds in a 1% aqueous solution of synthetic detergent for 15 minutes with constant stirring, and then washing with running water for 15-20 minutes, cultivation was carried out for 3-4 weeks, after which the fully developed seedlings were planted into pots with soil and placed in a substrate mikroparnik for 4-6 weeks to adapt to non-sterile conditions. 2. The method according to p. 1, characterized in that the nutrient medium contains mineral salts and vitamins according to MS, 20 g / l sucrose, 7 g / l agar. 3. The method according to p. 2, characterized in that the nutrient medium additionally contains 2.2 μM BAP. 4. The method according to p. 1, characterized in that the medium selected is WPM with the addition of 2.2 μM BAP.

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