RU2711781C1 - Method of selecting in vitro acid-tolerant forms of red clover (trifolium pratense l.) - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to the field of biotechnology. Invention is a method for selection in vitro of acid-tolerant forms of red clover, including cultivation on nutrient medium of Gamborg Bmorphogenic culture of red clover, produced by sprouting of seeds and cultivating seedlings on Gamborg Bnutritive agarized medium with 2 mg/l of 6-benzylaminopurine and 100 mg/l of Al, where morphogenic tissue is obtained by subculturing epicotyls of seedlings without roots and with roots of less than 4–5 mm from selective nutritive agarized medium of Gamborg Bwith 2 mg/l of 6-benzylaminopurine and 100 mg/l of Alon medium of same composition, but without Al, and assessment of acid endurance of morphogenic tissue is carried out by growth index (not lower than 5.0) in 3–4 weeks of repeated cultivation of explants (pieces of morphogenic tissue) on selective medium with 100 mg/l Aland the ability of regenerating plants formed from morphogenic tissue to survive after 3–4 mowing in a soil greenhouse for at least 3 years of vegetation.EFFECT: invention allows selecting regenerating plants with higher acidity.1 cl, 3 tbl, 3 ex

Description

The invention relates to the field of agriculture and can be used in plant breeding to obtain acid-tolerant forms of meadow clover, in studies of the physiology and genetics of plants.

A known method of selection of acid-resistant forms of clover meadow by the method of roll culture [1].

The disadvantages of this method include the following. The method is limited to the selection of acid-resistant forms, but does not contain information about the vegetative propagation of the obtained forms and does not provide long-term preservation of valuable acid-resistant forms, which reduces the reproducibility of controlled crosses and genetic studies of the sign of acid resistance in such a strict cross-pollinated plant as meadow clover.

There is information in the literature on the method for the selection of acid-resistant forms of meadow clover by the pH of cell juice [2].

The disadvantages of this method include the fact that it does not contain information about the micropropagation of valuable acid-resistant genotypes of clover meadow. This does not allow long-term storage of selected forms.

A known method of selection in vitro acid-resistant forms of clover meadow [3]. The disadvantages of this method include the following. The number of acid resistant forms in the populations of variety samples selected by this method (sample 30 VIC 7) varies and may not exceed 7.4%. This significantly impoverishes the population according to other equally valuable breeding traits (high seed and feed productivity, early ripening, disease resistance), which do not correlate with the sign of acid resistance.

The purpose of the invention is the development of a method for in vitro selection of acid-tolerant forms of meadow clover.

In the proposed method, the goal is achieved by obtaining morphogenic tissue by subculturing epicotyls of seedlings without roots and with roots less than 4-5 mm from selective nutrient agar medium Hamburg ₅ with 2 mg / l 6-benzylaminopurine and 100 mg / l Al ³⁺ on Wednesday same composition, but without Al ³⁺ . The acid endurance of morphogenic tissue is assessed by the growth index (not lower than 5.0) after 3-4 weeks of re-cultivation of explants (pieces of morphogenic tissue) on selective medium with 100 mg / L Al ³⁺ and the ability of surviving regenerated plants from morphogenic tissue to survive 3-4 mowing in a greenhouse for at least 3 years of vegetation.

The method is as follows: from varieties of clover meadow with low resistance to soil acidity, in vitro selection of genotypes that withstand the concentration of selective factor Al ³⁺ 100 mg / l in the Gamborg B ₅ nutrient medium was selected. The medium and aluminum salt were sterilized separately by autoclaving for 30 minutes at 1.25 atm., And then mixed under sterile conditions until the medium solidified.

Morphogenic tissue was obtained by subculturing epicotyls of seedlings without roots and with roots less than 4-5 mm from a selective nutrient agarized Hamburg medium B ₅ with 2 mg / l 6-benzylaminopurine and 100 mg / l Al ³⁺ on a medium of the same composition, but without Al ³⁺ . The growth index was determined 3-4 weeks after re-culturing the explants (pieces of morphogenic tissue) on Hamburg B ₅ nutrient agar medium with 100 mg / L Al ³⁺ . We studied the morphobiological parameters of meadow clover regenerated plants formed in morphogenic tissue with a different growth index on a selective Hamburg B ₅ medium with 100 mg / L Al ³⁺ and surviving after 3-4 mowing for 3 years of vegetation in a ground greenhouse.

Example 1

Study of the toxic effect of Al ³⁺ on the root-forming ability of meadow clover seedlings in an in vitro culture.

Studies were carried out on F ₂ offspring of meadow clover plants with low acid resistance (Table 1). The number of genotypes with roots ≥4 mm in selective agarized Hamburg B ₅ medium with 100 mg / L Al ³ was insignificant in F ₂ I 0.9; F ₂ II 18.3 and F ₂ III 7.4, which was 2.1; 59.2 and 23.9 percent of the control (medium without Al ³⁺ ), respectively.

The greatest number of seedlings in populations of F ₂ offspring when germinated on a medium with 100 mg / L Al ³⁺ was without roots and with roots less than 4.0 mm (31.1 and 7.1 in F ₂ I; 20.7 and 11, 0 for F ₂ II and 32.0 and 10.6 for F ₂ III), i.e. 444.3% and 788.9% for F ₂ I; 279.7% 94.0% for F ₂ II; 400.0% and 96.4% in F ₂ III with respect to the control (medium without Al ³⁺ ).

Due to the low number of acid-resistant meadow clover genotypes selected by a previously developed method, there is a need for further studies to increase the number of meadow clover genotypes for selection for acid tolerance.

Example 2

A comparative assessment of acid resistance in vitro of three populations of F ₂ meadow clover genotypes (F ₂ I, F ₂ II, F ₂ III) was carried out. For this reason, aseptically grown meadow clover explants, after weighing, were cultured for 3-4 weeks on Hamburg ₅ agar medium with 2 mg / L 6-benzylaminopurine with 0 (control), 50 and 100 mg / L Al ³⁺ until morphogenic tissue with shoots formed. When determining the growth index (IR), the following results were obtained (Table 2). The highest IR in a selective medium with 100 mg / L Al ³⁺ was observed in morphogenic tissue F ₂ I (on average 7.3 at 41.0% of the control (medium without Al ³⁺ ), the smallest - in F ₂ III (average 2.9 at 37.1% of the control).

Shoots with roots formed in the morphogenic tissue of meadow clover with a growth index of at least 5.0 were planted from Petri dishes in cassettes for growing plants with soil (pH 4.1), and then in soil with normal acidity in an underground greenhouse when individually standing.

Example 3

A comparative study of morphobiological parameters in the growing experiment of the F ₂ generation of meadow clover regenerated plants obtained from morphogenic tissue with a different growth index on a selective medium with 100 mg / l Al ^{3+ was carried out} . The number of surviving meadow clover regenerated plants obtained from morphogenic tissue with a growth index of at least 5 after three mowing for three years of vegetation in a vegetation experiment on soil with normal acidity is F ₂ I 84.1%; F ₂ II - 89.1%; F ₂ III - 69.3%, which significantly exceeds this indicator for regenerated plants obtained from morphogenic tissue with IR below 5 (F ₂ I - 4.1 times; F ₂ II - 6.2 times; F ₂ III - 3.8 times) (table. 3). Moreover, these plants are superior in height 2.5-5.9 times, 2.0-4.3 times the number of stems and 2.5-2.6 times the number of inflorescences.

All this indicates that meadow clover regenerated plants obtained from morphogenic tissue with IR of at least 5.0 have greater acid endurance than those obtained from morphogenic tissue with IR of less than 5.0.

Thus, the developed method allows the selection in vitro of acid-tolerant forms of meadow clover by the morphogenic tissue growth index, which significantly increases the number of meadow clover genotypes that can overcome the toxic effects of Al ³⁺ and increased acidity of the medium and form plants with high feed and seed productivity.

SOURCES OF INFORMATION

1. Onuchina O.L., Tumasova M.I. Methods and results of selection of meadow clover for increased acid resistance // Feed production. - 2007.-№4. - from. 27-28.

2. Pat. 2138154 RF, IPC A01H 1/04. The method of selection of acid-resistant forms of meadow clover / Novoselov M.Yu., Payvin S.G. (RF) - No. 9810620/13; claimed January 15, 1998; publ. 09/27/1999. - Bull. Number 27. - 5 sec.

3. Pat. 2583304 of the Russian Federation. IPC A01H 1/04. In vitro selection method of acid-resistant forms of meadow clover / L. Solodkaya, A. N. Agafodorova, L. Lapotyshkina (RF) - No. 2014151553/10; stated 12/19/2014; publ. 05/10/2016. - Bull. No. 13.

Claims (1)

A method for in vitro selection of acid-tolerant forms of meadow clover, including cultivating on Hamburg B nutrient medium ₅ morphogenic culture of meadow clover obtained by germinating seeds and cultivating seedlings on Hamburg B ₅ nutrient agar medium with 2 mg / l 6-benzylaminopurine and 100 mg / l Al ³⁺ , characterized in that the morphogenic tissue is obtained by subculturing epicotyls of seedlings without roots and with roots less than 4-5 mm from a selective nutrient agar medium Gamborg B ₅ with 2 mg / l 6-benzylaminopurine and 100 mg / l Al ³⁺ on medium of the same composition, but without Al ³⁺ , and the acid endurance of morphogenic tissue is assessed by the growth index (not lower than 5.0) after 3-4 weeks of re-cultivation of explants (pieces of morphogenic tissue) on selective medium with 100 mg / l Al ³⁺ and the ability of regenerated plants formed from morphogenic tissue to survive after 3-4 mowing in a soil greenhouse for at least 3 years of vegetation.