RU2019960C1 - Method for production of salt-resistant alfalfa-regenerant plants - Google Patents

Abstract

FIELD: biotechnology, plant-growing, selection. SUBSTANCE: salt-resistant alfalfa-regenerant plants are cultivated by growing them from somatic embryoids which are produced by adding crushed soil with natural type of salination into nutrient medium. EFFECT: higher efficiency. 2 cl, 1 tbl

Description

 The invention relates to agriculture, agricultural biotechnology of plants, can be used in breeding and seed production.

The method of regeneration of alfalfa plants in vitro [1] (prototype) includes the selection of mother plants, the explantation of sterilized segments, their leaves on the Hamburg medium, modified by the addition of ammonium nitrogen and an increased content of iron chelate, cultivation to obtain callus, passaging on a nutrient medium to obtain somatic embryoids, transfer of somatic embryoids to a hormone-free medium with half the content of macro and micro salts for regeneration, transfer of the obtained regenerants to vu and their adaptation to open ground conditions, whereby previously obtained regenerant plants are used as maternal plants, somatic embryoids are obtained by adding 0.1-0.5 mg / l kinetin, 0.1-0.5 mg / l IAA and 5-15 mg / l of glycine and before transfer (passaging) on regeneration medium of cultured for 2-4 days at a temperature of 10-15 ^{° C} in the light. In this case, isolated embryoids are transferred to a half-agarized Hamburg agar medium without hormones. Transplanting seedlings into the soil is carried out during the formation of 2-3 leaves and the root system, pre-grown for acclimatization, for example, for 2-3 weeks, under optimal conditions of humidity under glass glasses.

 The disadvantage of the prototype method is that its use does not allow to obtain salt tolerant plants - regenerants that are resistant to a specific natural type of salinization of the soil.

 The aim of the invention is to obtain salt tolerant embryoids and regenerated plants during passage of camus by adding saline soil (CH) to the embryoid nutrient medium as a selective agent.

 This is achieved by the fact that in the method of regenerating alfalfa plants in vitro during the transfer of calluses to the medium for receiving somatic embryoids, crushed soil with a natural type of salinity is additionally added to it, and against this selective background, embryoids and regenerated plants are selected for resistance to the natural type salinization.

 Evidence of significant differences.

 In the well-known scientific, technical and patent literature, no totality of features similar to the claimed one was found.

 The use of saline soil in a nutrient medium as a selective additive and selective background is not obvious.

 The selection of embryoids and regenerated plants resistant to a particular natural type of salinization in vitro is also obvious.

 The use of an embryoidogenic medium for screening for salt tolerance is not obvious. The selection of resistant regenerant plants precisely on the medium forming the embryoids is caused by the fact that callus cells develop unstably on the callusogenic medium, and for their stabilization two or three cultivation passages are necessary.

 Even in the case of selection of resistant cells on a callusogenic medium when transplanting them onto the embryoid-forming medium, new cells appear on it, giving rise to embryoids that are not resistant to the selective factor.

 It is also advisable to carry out selection at the stage of regenerated plants, since in this case mass death of regenerated plants occurs and selection is carried out at the organism level for quick modification adaptation of plants to stressful conditions.

 Thus, the claimed combination of features meets the criteria of "novelty" and "significant differences".

 The method is as follows.

Maternal regenerant plants with valuable traits are selected. Parts of them, for example, leaflets, are isolated, sterilized in a 5-10% aqueous solution of chloramine "B" after washing three times in sterile distilled water. Ternary leaves are divided into separate leaflets and pieces are cut out of them, a series of partial injuries (injuries) are made on each of them over the entire surface of the leaf piece, placed on Hamburg's agarized callusogenic nutrient medium modified by the addition of ammonia nitrogen (200-300 mg / l) and increase in iron chelate (up to 15 mg / l). Material cultured to give callus, e.g. 3-4 weeks under continuous light fluorescent lamps (0.5-4.0 tys.lyuks), a temperature of 26 ± 1 ^° C leaves developed from the explant, callus divide for 3-5 h, and transferred to an agar medium, i.e. nutrient medium of the same composition as callusogenic, but in which hormones are replaced by kinetin 0.1-0.5 mg / l, IAA 0.1-0.5 mg / l and glycine 5-15 mg / l and In addition, finely chopped saline soil is additionally added. To obtain embryoids, calli are cultured for 2-4 weeks under the same conditions and a 16-hour photoperiod. Appeared after 2-3 weeks calli isolated embryoids was transferred to 2 days in a chamber with a temperature of 10-15 ^{° C} around the clock light. The resulting embryoids are transferred to an agarized nutrient medium, for example, Gamborg, twice diluted with water and without hormones. The regenerated seedlings are transplanted into the soil during the formation of 2-3 leaves and the root system and pre-grown for 2 weeks under optimal conditions of air humidity, for example, under glass glasses for better acclimatization of regenerated seedlings.

 PRI me R. To obtain and select salt tolerant embryoids, regenerate plants from the Krasnokutskaya 4009 variety and line 451 obtained in vitro were used as maternal, uterine regenerative plants.

 Well-formed ternary leaves were selected and cut on plants, they were sterilized in a 10% aqueous solution of chloramine "B". The concentration of the sterilizer depends on the infectious background of the grown plants. After sterilization, the leaves were washed three times with sterile distilled water. They cut into separate leaves and cut out pieces of 5 x 5 mm in size and did a series of injuries (injuries) on each of them over the entire leaf surface.

They were placed on a Hamburg agarized callus culture medium with a high content of iron chelate (15 mg / L), the addition of ammonium nitrogen (220-300 mg / L) and phytohormones: 2.4D 8 mg.L, kinetin 8 mg / L, NAA 0 5 mg / l; sucrose 3% and adjusting the pH of the medium to 5.8-6.0. Explants cultured 4 weeks under continuous light fluorescent lamps (3.0 th. Suite) and a temperature of 26 ± 1 ° ^C.

 Four weeks later, the calluses that developed from explant leaflets were divided by 3-5 hours and transferred to the same agar medium, the hormones of which were replaced by kinetin and IAA, at 0.1 mg / l each, and where the lycine was 7 mg / l. In addition, unlike the main invention, saline solonetzic) soil with a natural type of salinity was additionally used as a selective background agent) here.

The studies used soil samples of solonetzes containing a complex of salts with a maximum amount of Na, Cl ^- , SO- ⁴ (table).

 Soil solonetz samples were taken in the Ershov district, Saratov region, on the collective farm named after the XXII Party Congress. On these soils, alfalfa crops were inhibited and fell for 2-4 years of life.

 The residual plants and worms, as well as impurities of atypical soil (inclusions of clay, gravel, etc.) were removed from the dried samples. The soil samples were crushed to a dusty state in a porcelain cup, pestled and sieved through a sieve with a hole diameter of 1 mm.

 100, 200 g of crushed air-dry soil was weighed on a technical balance, poured into a liter flask, and 500 ml of distilled water was added. The contents of the flask were agitated for 5–10 min [2], then the prepared pre-melted, agarized organogenic nutrient medium of Hamburg was poured and the volume of the medium was adjusted to 1000 ml with distilled water.

 When spilling the nutrient medium, it was ensured that particles of salted soil were in suspension. This is necessary to evenly distribute the soil and create the same osmotic pressure in test tubes.

 The sterilization of the culture medium was carried out in the usual mode of autoclaving the culture medium. After sterilization, the tubes with agarized nutrient medium were shaken until a homogeneous mass was obtained and placed in cold water for quick solidification of the agar.

Cooked medium were placed for 2-3 days in an incubator at a temperature of 26 ^{° C} to provoke the development of fungal and bacterial infection.

Pieces of callus planted on this medium formed somatic embryoids after two weeks of cultivation. They were transferred to three days in a chamber at 10-15 ^{° C} with non-stop lighting. Then the embryoids were transferred to the Hamburg medium, diluted twice with water and without hormones. The transplantation of regenerated seedlings into the soil was carried out during the formation of 2-3 leaves and the root system and grown for two weeks under optimal conditions of humidity, for example, under glass glasses, for better acclimatization of regenerated seedlings.

 For the use of saline (solonetz) soil as a selective background in cell selection in vitro, various variants of agarized nutrient media were used. Variants of media from 1 to 4 had a mineral base according to Hamburg, and in versions 5 and 6 the mineral base of salts was reduced to 1/2 and 1/4 parts, i.e. 2 and 4 times. In the seventh variant of the selective agar medium, the mineral base of the Hamburg medium was absent, but the kinetin hormones 0.1 mg / L, IAA 0.1 mg / L, glycine 7 mg / L, and vitamins and sucrose were added according to the Hamburg medium prescription.

 In the variants from the third to the seventh, saline (solonetz) soil was added in an amount of 100 or 200 g / l, and in the second embodiment, a 1% NaCl solution (artificial salinization) was added to the Gamborg mineral base.

The following options for selective culture media were studied:
1 option Wednesday Hamburg - (prototype);
Option 2 - "- + 1% NaCl (analog);
3 option - "- +200 g / l (20%) CH (the claimed method);
4 option - "- +100 g / l (10%) CH -"-;
5 option - "- 1/2 Gamborg +200 g / l (20%) CH -"-;
6 option - "- 1/4 Gamborg +200 g / l (20%) CH -"-;
7 variant without mineral base +200 g / l (20%) CH - "-.

Hamburg environment
The first signs of inhibition of callus cells in an embryoidogenic agarized selective medium were observed in the seventh embodiment after seven to eight days. Callus was an unhealthy, black-brown, rough-type color. With further cultivation on this selective medium, complete necrosis of callus cells was observed due to a lack of mineral nutrition elements.

 In the first version (prototype) of an agarized embryoidogenic medium without adding soil, from callus cells, 733 embryoids were obtained in three passages, the type of callus remained globular, healthy, yellow-green in color.

 In the second version of the artificial selective nutrient medium (analog), with the addition of 1% NaCl, necrosis of callus cells was observed by the end of the second passage of cultivation. The color and type of callus varied from healthy, yellow-green, globular to gray, gray-brown flooded globular. This type of callus did not form embryoids.

 In the third embodiment (the claimed method), when saline soil was added to the agarized nutrient selective medium at a concentration of 20% (200 g / l), callus cells formed 82 embryoids, the type of callus did not change (i.e. it was globular), and the color changed from yellow green to brownish yellow.

 A decrease in the concentration of saline soil (the fourth option) to 10% (100 g / l) led to a rapid increase in callus mass, which has both a globular and a rough yellow-green type of callus. In this embodiment of a nutrient agarized selective medium, 112 embryoids were obtained.

 A decrease in the content of mineral salts of the medium in half and at a 20% content of CH (the fifth option) led to a change in the color of the callus from yellow-green to gray-brown with the formation of single embryoidogenic zones and only 3 embryoids were obtained. A low yield of embryoids was obtained, probably, due to the lack of elements of mineral nutrition and an increase in the toxicity of soil salts against this background.

 On a nutrient medium containing 1/4 of the composition of mineral salts and 20% (200 g / l) CH (sixth option), callus formed single (12) embryoidogenic zones. Callus had a rough and globular type, yellow-gray in color. Transplanted embryogenic zones onto a hormone-free medium formed ugly plants and died.

 Of the variants of the claimed method, the best are the third and fifth.

 The use of the third version of the nutrient medium allows one to obtain a large number of embryoids, from which, as a result of a further comprehensive assessment of plants, it is possible to choose salt tolerant regenerant plants with other valuable traits. An increase in the concentration of solonetz soil makes it possible to decrease the yield of embryoids and increase the probability of selection of salt tolerant embryoids and regenerated plants in the future.

 Using the fifth option allows you to save mineral salts of a selective medium and create tough, unfavorable conditions for the formation of embryoids in mineral nutrition. In this variant, the selection of salt tolerant plants is highly likely, because the medium contains an insufficient amount of mineral nutrition elements, which brings the development conditions of embryoids closer to the natural conditions for the development of plants on saline (solonetz) soil.

 Using the claimed method increases the yield of embryoids and the viability of calli compared to the analogue (adding 1% NaCl), which is currently widely used in breeding and genetic work on salt tolerance [1], but forms fewer embryoids when compared with the prototype.

 Thus, the claimed method in comparison with the prototype allows for in vitro selection against a natural background of salinization and to obtain salt tolerant embryoids. Obtained by the claimed method, regenerated plants when planting them on similar solonetzic soils were less depressed than those of the prototype, bloomed and gave seeds.

Claims (2)

 1. METHOD FOR PRODUCING SALT-RESISTANT ALFERANA REGENERANT PLANTS, including selection of donor plants, explantation of leaf segments on a nutrient medium to obtain callus, transfer of the obtained callus to a nutrient medium for the formation of somatic embryoids, transfer of embryoids to a nutrient medium for regeneration and adaptation of open ground conditions, characterized in that crushed soil is additionally introduced into the nutrient medium for the formation of somatic embryoids venous type of salinization.  2. The method according to claim 1, characterized in that the crushed soil with a natural type of salinization is introduced into the nutrient medium in an amount of 100-200 g / l.

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