RU2229219C2 - Method for buckwheat reproduction in vitro at obtaining selectionally valuable somaclones - Google Patents

Abstract

FIELD: agriculture, plant science, plant selection. SUBSTANCE: one should isolate explants, cultivate them at obtaining primary callus tissue, induce organogenesis to obtain plants-regenerants. As explants one should apply hypocotyl sections in week-aged aseptic buckwheat seedlings. Plants-regenerants should be reproduced and rooted due to stalk's division into microcuttings upon one nutritive medium. Then plants-regenerants are transferred into nonsterile conditions to be obligatory repollinated inside the clone. Somaclonal seeds are obtained to be reproduced at spatially isolated areas and evaluated under field conditions. The method enables to obtain qualitatively new initial material to conduct further somaclonal selection of buckwheat oriented to obtain its improved varieties. EFFECT: higher efficiency. 5 tbl

Description

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

A known method of propagation of buckwheat in vitro, including isolating explants, cultivating them with primary callus tissue, inducing organogenesis and obtaining regenerated plants (see, for example, USSR author's certificate No. 1704715, IPC ⁵ A 01 H 4/00, 1992 .)

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

- the complexity of isolating unripe embryos;

- selection for the cultivation of embryos of the required size from 0.5 to 3 mm;

- the absence of a stage of micropropagation, which can lead to the loss of regenerant when planting in non-sterile conditions in the event of its death or in the absence of tying of full fruits;

- no selection assessment of the obtained regenerated plants in the field is foreseen, which limits their use in applied aspects for the selection of buckwheat.

There is also known a method of propagation of buckwheat in vitro, including isolating explants, cultivating them to obtain primary callus tissue, inducing organogenesis and obtaining regenerated plants (see, for example, USSR author's certificate No. 1658928, IPC ⁵ A 01 H 4/00, 1991 g.)

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

- the explant dedifferentiation stage is not provided for - the formation of callus tissue, it is well known that this does not contribute to the accumulation of somaclonal variation caused by prolonged cultivation of callus cells on media with hormonal additives;

- the use of this method as a microclonal propagation is possible, but inefficient, since the medium with the preparation of 6-benzylaminopurin (BAP) contributes to the appearance of abnormal shoots that reduce the efficiency of micropropagation.

The purpose of the invention is to increase the genetic diversity of buckwheat plants by creating a qualitatively new source material involved in variety improvement by the method of somaclonal selection.

This goal is achieved by the fact that the method of propagation of buckwheat in vitro to produce selection-valuable somaclones, including isolating explants, cultivating them to obtain primary callus tissue, inducing organogenesis and obtaining regenerated plants, using hypocotyl sites of weekly aseptic seedlings as explants, regenerated plants are propagated and rooted by dividing the stem into micrograins on a single nutrient medium, regenerated plants are transferred to non-sterile conditions and forced eopylyayut inside clone somaclones get seeds that are propagated on spatially isolated sections and evaluated in the field.

Compared with the prototype, the features of the inventive step of the proposed method of propagation of buckwheat in vitro to obtain selection-valuable somaclones is:

1. "hypocotyl sites of weekly aseptic seedlings are used as explants ...", which allows:

- to obtain a significantly larger number of callus cultures, since from one aseptic seedling several plots of hypocotyl can be used, and from one germ only one;

to simplify the in vitro administration process, since the laborious operation of isolating embryos of a certain size is removed;

- use sections of the hypocotyl of weekly aseptic seedlings in work all year round and does not require additional sowing of seeds to obtain immature embryos.

2. "... regenerated plants propagate and root by dividing the stem into micro-stalks on one nutrient medium ...", which allows:

- it is economically more efficient to obtain rooted buckwheat regenerated plants suitable for translation into non-sterile conditions and for further propagation in vitro during one passage on the same composition of the nutrient medium;

- avoid the appearance of abnormal forms of shoots, since BAP is not used;

- reduce the time of receipt of regenerated plants and accelerate the production of somaclone seeds.

3. "... they transfer regenerated plants to non-sterile conditions and are forcedly pollinated inside the clone, receive somaclone seeds ...", which allows:

- consolidate the mutations that have arisen in the process of cultivation and contribute to a more rapid identification and selection of recessively inherited traits;

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

4. "... propagated in spatially isolated areas and evaluated in the field", which allows:

- to maintain the genetic purity of somaclones, to increase the homozygosity of the material according to interesting selection-valuable traits;

Obtain sufficient seed for subsequent evaluation

- select somaclones that differ from the original variety by breeding and valuable traits;

- accelerate and improve the efficiency of breeding, by obtaining new source material with the necessary characteristics.

The signs indicated in the distinctive part of the description of the achievement of the goal prove that the inventive method of propagation of buckwheat in vitro with obtaining selection-valuable somaclones has novelty. The combination of features shown in the comparison of the properties of the claimed and known solutions, gives reason to conclude that the claimed method has an inventive step.

The proposed method of propagation of buckwheat in vitro to obtain selection-valuable somaclones is as follows. Before sterilization, mature seeds are soaked for 12 hours in distilled water, then they are treated for 10 minutes in a 70% ethanol solution and for 10 minutes in a 10% chloramine solution, pericarp is removed, placed for 5 minutes in 70% - solution of ethanol and for 5 minutes in a 10% solution of chloramine and washed with sterile distilled water at least three times, keeping at least 5 minutes in each wash. The seeds thus treated are placed in test tubes with cotton-gauze plugs for cultivation on the hormone-free medium Murashige and Skoog until weekly aseptic seedlings are obtained, under the condition of a 16-hour photoperiod and light intensity from 4 to 5 thousand lux, at a temperature of 23 ± 2 ° С. Hypocotyls of weekly aseptic seedlings are divided into explants ranging in size from 3 to 4 mm. Then the explants are placed in test tubes with Hamburg B ₅ medium supplemented in mg / l: ascorbic acid 10.0; casein hydrolyzate 1000; 2,4-Dichlorophenoxyacetic acid (2,4-D) 5.0; kinetin 0.1; sucrose 25,000; agar 7000; at a hydrogen index (pH) equal to 5.5 to 5.6, and cultured for 5 to 10 days in an incubator without lighting and a temperature of 23 ± 2 ° C, until the formation of callus tissue. Then test tubes with callus tissue are placed under conditions of a 16-hour photoperiod and illumination intensity from 4 to 5 thousand lux, at a temperature of 23 ± 2 ° C for a period of at least 25 days. Further, in order to maintain the growth of callus tissue, it is cultivated on Hamburg B ₅ medium supplemented with mg / l: casein 1000 hydrolyzate; 2,4-Dichlorophenoxy-acetic acid (2,4-D) 1.0; kinetin 1.0; sucrose 20,000; agar 7000; at a pH of from 5.5 to 5.6.

Table 1 presents the callus-forming ability of the hypocotyl of weekly buckwheat seedlings in comparison with the prototype.

From the data presented in table 1, we can conclude that the hypocotyl explants of weekly buckwheat seedlings have a maximum callus-forming ability equal to 90.6%.

For the proliferation of organogenic or embryoidogenic structures, callus tissue is cultured on fresh Hamburg B ₅ medium supplemented with mg / l: casein hydrolyzate 1000; indolyl-3-acetic acid (IAA) 0.2; BAP 2.0; agar 7000; at a pH of from 5.5 to 5.8. The resulting regenerant plants are propagated and rooted in medium "A", including the medium Murasige and Skoog, supplemented in mg / l: thiamine-Hcl 2.0; pyridoxine-Hcl 1.0; casein hydrolyzate 1000; 3-indolylbutyric acid (IMA) 0.2; indolyl-3-acetic acid (IAA) 0.5; sucrose 20,000; agar 6000; at pH from 5.3 to 6.0 by dividing the stem into micro stalks with a length of 7 to 15 mm. Within 25 days of cultivation in this medium, a normally formed buckwheat regenerant plant with a root system develops from a microcranium, which is ready for transplantation into non-sterile conditions or use for further propagation in vitro culture. Table 2 presents the results of micropropagation of buckwheat regenerated plants for 25 days on used media.

From the data presented in table 2 it can be seen that the use of medium "A" is most effective for obtaining regenerated plants with a root system and reaches 66.7%.

Before planting in non-sterile controlled conditions of the culture room, the cotton-gauze plugs are removed from the tubes and the tubes with regenerated plants are placed in a horizontal position for 5 days under illumination of 5 thousand lux, then regenerated plants are planted in vessels with a substrate: soil-peat- sand (4: 1: 1). After planting, create conditions of increased humidity in the vessel using polyethylene shelters, which are removed after acclimatization of plants to normal air humidity. Regenerant plants develop, and in the flowering phase they are forcibly pollinated within one clone. The resulting somaclone seeds are sown in spatially isolated areas for propagation. Propagated somaclone seeds are sown further in breeding nurseries and make the following counts and observations: phenological; seedling assessment and condition of plants before harvesting; assessment of resistance to lodging; determine the main quantitative signs of buckwheat: plant height, thickness of the first internode, length of the first internode, number of nodes on the main stem, number of branches of the first, second order, number of inflorescences with fruits, number of inflorescences without fruits, attachment height of the first inflorescence, plant productivity, mass 1000 grains, filminess, cereal yield, protein, fat, and routine content. Ecological plasticity is assessed in terms of plant productivity. The presence of somaclonal variability of characters is established after statistical processing of data in comparison with the original variety. Table 3 shows the data on the variability of quantitative characters in buckwheat somaclones of the local Primorskaya variety.

From the data presented in table 3 it is seen that somaclones by the above signs exceed the values of the indicators of the initial forms.

The progeny of somaclones has a varying degree of ecological plasticity based on the productivity of one plant.

From the data presented in table 4. it can be seen that the value of the indicator of environmental plasticity in different somaclones is different and distinguishable from the original variety.

The manifestations of variability in color and shape of the fruit of buckwheat somaclones in comparison with the original variety are noted.

In the breeding nursery, starting from the second generation, individual and individual-family selections are carried out in order to consolidate the desired traits.

As a result, the three best somaclonal buckwheat lines that were tested in the control nursery were identified, table 5.

The application of the invention will increase the genetic diversity of buckwheat plants by creating a qualitatively new source material involved in variety improvement by the method of somaclonal selection.

Claims (1)

The method of propagation of buckwheat in vitro to obtain selection-valuable somaclones, including isolating explants, cultivating them with primary callus tissue, inducing organogenesis and obtaining regenerated plants, characterized in that hypocotyl plots of weekly aseptic seedlings are used as excipients, regenerated plants are propagated and rooted by dividing the stem into microcranes on the same nutrient medium, regenerate plants are transferred to non-sterile conditions and forcedly pollinated inside the clone, get seeds of somaclones, which are propagated in spatially isolated areas and evaluated in the field.