The method of reproduction buckwheat in vitro with obtaining selection and valuable somaclonal

 

(57) Abstract:

The invention relates to agriculture and can be used during the reproduction of buckwheat in vitro with obtaining selection and valuable somaclonal. Performed amputation of the explants, their cultivation accessing primary callus tissue, induction of organogenesis and obtaining regenerated plants. As explants use sections of hypocotyl week aseptic seedlings buckwheat. Regenerants were multiply and perpetuate the division of the stem on microscience on the same nutrient medium. Then translate regenerants were in non-sterile conditions and force propylaeum inside the clone. Get seeds somaclonal that multiply on spatially isolated areas and evaluate in the field. The method allows to obtain qualitatively new source material for further somaclonal breeding buckwheat aimed at her saltoluokta. 5 table.

The invention relates to agriculture, in particular to plant breeding and agricultural biotechnology.

A known method of reproduction buckwheat in vitro, including amputation of explants, their cultivation with recip is Roscoe's certificate of the USSR No. 1704715, IPC5A 01 H 4/00, 1992)

The disadvantage of this method of reproduction buckwheat in vitro is:

- the complexity of the isolation of immature embryos;

selection for culturing embryos of the desired size from 0, 5 up to 3 mm;

- no stage of micropropagation, which can lead to loss of regenerant when landing in non-sterile conditions in case of his death or in the absence of tying full of fruit;

- not provided selection and evaluation of regenerated plants in the field, which limits their use in applied aspects for selection of buckwheat.

Also known is a method of reproduction buckwheat in vitro, including amputation of explants, their cultivation accessing primary callus tissue, induction of organogenesis and obtaining regenerated plants (see, for example, USSR author's certificate No. 1658928, IPC5A 01 H 4/00, 1991)

The disadvantage of this method of reproduction buckwheat in vitro is:

- not planned stage of dedifferentiation explants - formation of callus tissue, it is well known that this is not conducive to the accumulation of somaclonal variation caused by long cultivated the microclonal reproduction may but ineffectively, as the environment with the preparation of 6-benzylaminopurine (BAP) contributes to the appearance of abnormal forms of shoots, reducing the efficiency of micropropagation.

The purpose of the invention to increase the genetic diversity of plants buckwheat, by creating a new source of material involved in saltoluokta way somaclonal selection.

This goal is achieved by the fact that the method of reproduction buckwheat in vitro with obtaining selection and valuable somaclones, including isolation of explants, their cultivation accessing primary callus tissue, induction of organogenesis and obtaining regenerated plants, the explants use sections of hypocotyl week aseptic seedlings, plants-regenerants multiply and perpetuate the division of the stem on microscience on the same nutrient medium, transferred regenerants were in non-sterile conditions and force propylaeum inside the clone, get seeds somaclonal that multiply on spatially isolated areas and evaluate in the field.

Compared with the prototype signs of inventive step of the proposed method of reproduction buckwheat in vitro is of hypocotyl week aseptic seedlings...", so you can:

to get significantly more callus cultures, as one of aseptic seedlings, you can use several sections of the hypocotyl, and from one embryo is only one;

to facilitate the process of introducing in vitro, as removed time-consuming operation of extracting the germ of a certain size;

- use lots of hypocotyl week aseptic seedlings in work all year round and requires no additional seeding to obtain immature embryos.

2. "...regenerants were multiply and perpetuate the division of the stem on microscience on the same nutrient medium...", so you can:

- more cost effective for one passage on the same nutrient medium composition to obtain rooted plants-regenerants buckwheat, suitable for translation in non-sterile conditions, and for further multiplication in vitro;

to avoid abnormal forms of shoots, because BAP is not used;

- reduce the time for obtaining regenerated plants and to accelerate the production of seeds somaclonal.

3. "...translate regenerants were in non-sterile conditions and force propylaeum ve cultivation and to promote more rapid identification and selection recessive inherited traits;

- to simplify and accelerate the breeding process, as it is carried out under controlled conditions.

4. "...propagation in spatially isolated areas and evaluate field conditions", so you can:

is to preserve the genetic purity of somaclonal, to increase the homozygosity of the material of interest selection and valuable features;

to obtain a sufficient amount of seed material for subsequent evaluation;

- select somaclone, different from the original varieties in breeding and valuable characteristics;

to accelerate and increase the efficiency of breeding work, due to the receipt of new original material with the required characteristics.

Signs specified in the characterizing part of the description achieve the goal of proving that the claimed method of reproduction buckwheat in vitro with obtaining selection and valuable somaclones, has novelty. The set of features shown in the comparison of the properties of the claimed and known solutions, gives grounds to conclude that the claimed method involves an inventive step.

The proposed method of reproduction buckwheat in vitro with obtaining selection and valuable somaclonal as follows. Three 10 minutes in 70% ethanol and 10 minutes in a 10% solution of bleach, remove the pericarp, is placed for 5 minutes in 70% ethanol for 5 minutes in a 10% solution of bleach and not less than three times washed with sterile distilled water, soaking for at least 5 minutes each wash. Placed thus treated seeds in test tubes with cotton-gauze plugs for culture on hormone-free medium Murashige and Skoog up to receive weekly aseptic seedlings, assuming a 16-hour photoperiod and a light intensity of 4 to 5 thousand Lux, temperature 232C. Hypocotyl week aseptic seedlings divided by the explants from 3 to 4 mm, Then the explants are placed in test tubes with the medium of Gamborg5supplemented in mg/l: ascorbic acid 10.0 g; casein hydrolysate 1000; 2,4-dichlorophenoxyacetic acid (2,4-D) 5,0; kinetin 0,1; sucrose 25000; agar 7000; when hydrogen exponent (pH) is from 5.5 to 5.6, and cultured for 5 to 10 days in an incubator without lighting and temperature, equal to 232With, before the formation of callus tissue. Then the tubes with callus tissue is placed in conditions of 16 hours http://www.fips.ru/chr/176.gif" ALIGN="ABSMIDDLE">With at least 25 days. Further, to support the growth of callus tissue produced on the environment Gamborg5supplemented in mg/l: hydrolyzed casein 1000; 2,4-dichlorophenoxy-acetic acid (2,4-D) 1,0; kinetin and 1.0; sucrose 20000; agar 7000; at pH from 5.5 to 5.6.

Table 1 presents callosobruchus the ability of the hypocotyl weekly sprouts buckwheat in comparison with the prototype.

From the data presented in table 1, we can conclude that the explants-hypocotyl weekly sprouts buckwheat have maximum callosobruchus capacity equal 90,6%.

For proliferation organogenic or embroidering structures callus tissue cultured on fresh medium of Gamborg5supplemented in mg/l: hydrolyzed casein 1000; indolyl-3-acetic acid (IAA) 0,2; BAP 2,0; agar 7000; at pH from 5.5 to 5.8. Obtained regenerants were propagated and rooted on the environment "And" including, the Murashige and Skoog medium, supplemented in mg/l: thiamine-Hcl 2,0; pyridoxine-Hcl and 1.0; a hydrolysate of casein 1000; 3-indolylmethane acid (IBA) 0,2; indolyl-3-acetic acid (IAA) 0,5; sucrose 20000; agar 6000; at pH from 5.3 to 6.0 paleoscience develops normally formed plant-regenerant buckwheat with the root system, who is ready for transplantation in non-sterile conditions or use for further breeding in vitro culture. Table 2 presents the results micropropagation of plants-regenerants buckwheat within 25 days on the used media.

From the data presented in table 2 show that use of the environment "And" most effective to obtain a regenerated plants with a root system and reaches 66,7%.

Before planting in sterile controlled culture conditions of the room with the tubes removed with a cotton-gauze tube and a tube with plants-regenerants in a horizontal position for 5 days with light 5 thousand Lux, then regenerants were planted in pots with substrate: soil-peat-sand (4:1:1). After landing create conditions of high humidity in the vessel using a plastic cover, which is removed after acclimatization of plants to normal humidity. Regenerants were developed, and in the flowering phase of their force propylaeum within a single clone. Obtained from the seeds of somaclonal plated on spatially isolated areas for breeding. Razmnozheny is logical; evaluation of germination and the plants prior to harvesting; evaluation of resistance to lodging; determine the main quantitative characteristics of buckwheat: plant height, the thickness of the first internode, length of the first internode, the number of nodes on main stem, number of branches of the first, second order, the number of inflorescences with fruits, number of inflorescences without fruit, the height of attachment of first inflorescence, the productivity of the plant, weight of 1000 grains, planchette, cereal yield, protein content, fat routine. Assess ecological plasticity index plants. The presence of somaclonal variability of signs installed after statistical processing of the data in comparison with the original grade. Table 3 shows data on the variability of quantitative traits in somaclonal buckwheat varieties "seaside local".

From the presented data in table 3 shows that somaclone on the characteristics exceed the values of the source forms.

The offspring of somaclonal has a different degree of ecological plasticity on the basis of productivity per plant.

the different somaclonal different and distinguishable from the original varieties.

There are manifestations of variability colors and shapes of the fruits of somaclonal buckwheat in comparison with the original grade.

In the breeding nursery, starting from the second generation conduct individual and individual-family selections with the purpose of securing the desired features.

As a result, the three best somaclonal lines buckwheat, which has experienced in the control kennel, table 5.

The application of the present invention will increase the genetic diversity of plants buckwheat, by creating a new source of material involved in saltoluokta way somaclonal breeding.

The method of reproduction buckwheat in vitro with obtaining selection and valuable somaclones, including isolation of explants, their cultivation accessing primary callus tissue, induction of organogenesis and obtaining regenerated plants, characterized in that as sexplanet use sections of hypocotyl week aseptic seedlings, plants-regenerants multiply and perpetuate the division of the stem on microscience on the same nutrient medium, transferred plants regenerant is oraut on spatially isolated areas and evaluate in the field.

 

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