SU1701744A1 - Method for regeneration of wheat plants in tissue culture - Google Patents

Abstract

The invention relates to biotechnology, namely to biotechnological studies of plants by the method of cell and tissue culture, and can be used to create new forms of plants, in particular varieties and initial forms of wheat, possessing valuable traits. The aim of the invention is to increase the yield of regenerants and their viability. The method involves the production of a callus from the base of the leaf, followed by regeneration, while in the process of cultivation the phytohormone concentrations and the light intensity are gradually changed. 8 tab.

Description

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The invention relates to biotechnology, namely to biological studies of agricultural plants by the method of cell and tissue culture, and can be used in the work on the creation of new plant forms, in particular varieties and initial forms of wheat, possessing valuable characteristics.

The aim of the invention is to increase the yield of regenerants and their viability.

The method involves obtaining callus from the explant, induction of morphogenic callus formation, plant regeneration and rooting using nutrient media based on Murashige-Skoog medium, using the leaf base as the explant, and the phytohormone concentration and light intensity are changed stepwise: medium calluses induced at high

(2-4 mg / l) concentrations of 2,4-D, gradually adapt to ever lower concentrations of this hormone (from 2 to 0.1 mg / l) and lighting conditions (darkness - 500-1000 lux), then, in the regeneration medium, the content of zeatin is increased in steps (from 0.1 to 2 mg / l) at a constant concentration of IAA (0.01 mg / l) and the light intensity is increased from 1000 to 2000 lux; to root and increase the viability of the plants, In rooting medium, IAA content from 0.1 to 0.5 mg / l, increase the illumination to 4000 lux. Before planting in the soil, the plants are kept in a medium with half the salt content according to Murashige-Skoogu (MS) and the concentration of IAA is elevated compared to the regeneration medium - 0.1-0.5 mg / l. This is achieved by stimulating the development of the root system, increasing the viability of regenerants, allowing to prevent

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or reduce transplant shock due to the transfer of plants from in vitro conditions (tubes) to in vivo (soil). The developed method makes it possible to more finely regulate the morphogenetic reactions and adaptive potencies of the cell culture and thereby regenerate the plants from the considered low-potency wheat leaf tissues.

The method is carried out as follows.

Winter soft wheat grains (Tr, aestlvum Progress variety) are surface sterilized successively in solutions of diacid, ethanol, washed three times with sterile distilled water, and placed on solid salt medium according to MS and germinated in light of 2000hOO lx at 25 + 1 ° С, through For 7–8 days, the basal part of the leaf is sterilely isolated from the seedlings. Segments from the base of a sheet with a length of 3-5 mm are placed on solid nutrient medium for callusogenesis, containing salts according to MS, 100 mg / l of meso-inosite, 2 mg / l of glycine, 0.5 mg / l of nicotinic acid, 0.5 mg / l of pyridoxine , 1 mg / l thiamine, 3% sucrose, 0.7% agar, 2,4-D at pH 5.7. The explants are cultivated at 2515,5 ° C and relative humidity of 60-70%. The formed calli are transferred to fresh nutrient medium of the same composition and cultured for 2-3 weeks until colonies with an average diameter of 8-10 mm are formed. Next, the following steps are carried out sequentially:

a) transfer of calli to the medium of the same composition, but with a content of 1 mg / l 2,4-D, cultivation 12-14 days in the dark at 25tO, 5 ° C;

b) transplanting calluses to the same medium from 0.7 mg / l 2.4-D, cultivation 12-14 days at illumination of 500 ± 100 lx and 25iO, 5 ° C;

c) transplantation of calli on the medium with 0.5 mg / l 2.4-D, cultivation 12-14 days at illumination of 500 + 100 lux and 25 + 0.5 ° С;

d) transplantation of calluses on the medium with 0.3 mg / l 2.4-D, cultivation 12-14 days at 10001100 lux and 25 ± 0.5 ° С;

e) transplanting calluses to the medium with 0.1 mg / l 2.4-D, cultivation 12-14 days at 1000 + 100 lux and 25 ± 0.5 ° С.

The stage of cultivation of calluses at a concentration of 0.1 mg / l 2,4-D does not significantly affect the morphogenetic activity of the tissue and, in order to accelerate regeneration, it can be omitted.

The above procedure for the sequential transfer of callus to media with ever-decreasing concentrations of 2,4-D avoids callus necrosis,

observed during its direct transfer from the environment for callus genesis to regeneration, as well as significantly increase the yield of embryogenic callus (examples).

Further, embryogenic calluses, characterized by a dense, dusted consistency, are yellowish-green in color with clearly distinguished areas from weakly polymerized meristemoid cells.

0 is transferred to a regeneration medium. The following operations are performed successively:

a) transfer of embryogenic calli to the medium containing salts according to MS, 100 mg / l

5 mesoinositol, 2 mg / l glycine, 0.5 mg / l nicotinic acid, 0.5 mg / l pyridoxine, 1 mg / l thiamine, 3% sucrose, 0.7% agar. The concentration of phytohormones is 0.5 mg / l zeatin, 0.01 mg / l IAA, cultivated for 7-8 days

0 at 1000 lx, 27i1 ° C;

b) transplantation to medium of the same composition, but the concentration of zeatin is increased to 1 mg / l. The intensity of the light is increased to 2000 lux, cultivated for 7-8 days;

5c) transplantation to the same medium and cultivation under the same conditions, but the concentration of zeatin is increased to 2 mg / l.

Use of culture steps with zeatin concentrations below 0.5

0 mg / l does not significantly affect the regenerative activity, and these steps may not be performed.

Formed on regeneration medium shoots are transferred to medium for

5 rooting, containing half the salt concentration according to MS and various concentrations of IAA. Under these conditions, the regenerant goes gradually to the phototrophic nutritional method (since the medium is not

0 contains organic additives). The following steps are carried out sequentially:

a) transfer of shoots to half-mineral medium MS with a content of 0.3 mg / l IAA, illumination 2000 lx at a 16-h5 owl photoperiod, temperature 27 ± 1 ° C, cultivation 5-6 days;

b) cultivation of shoots under the same conditions, but the concentration of IAA is increased to 0.5 mg / l;

0c) transfer of plants to the same mineral medium, increase the concentration of IAA to 1 mg / l and light intensity to 4000 lx.

Cultivation of shoots on media with IAA concentrations below 0.3 mg / l does not significantly affect the viability of regenerants, and at concentrations above 1.0 mg / l, the percentage of rooted plants decreases.

Plant plants with a well-developed root system are planted in the soil

and carry out a full vegetation stage before seed production.

Obtained from leaf tissue of plant regenerants. Selection and genetic testing under field conditions. Among them are i and somaclonal variants with valuable traits.

PRI me R 1. The effects of different concentrations of 2,4-D on the morphogenetic activity of callus tissue are studied. Calluses induce 2,4-D on a medium with 2-4 mg / l. The yield of morphogenic calluses on this medium is 1-2%. The data are given in table 1,

41 r and m e r 2. The effects of various stages of subculturing are studied at successively decreasing concentrations of 2,4-D. The data are given in table 2.

As can be seen from a comparative analysis of the data of Tables 1 and 2, with a stepwise decrease in the concentration of 2.4-D in the culture medium, the percentage of morphogenic calli increases significantly (to 64%). At concentrations of 2,4-D below 0.3 mg / l, no further enhancement of the effect of a step change in the effect gradient is observed.

Example3 The effect of the conditions of pre-subculturing calli at various concentrations of 2,4-D on the appearance of necrotic areas in the calli when they are transplanted to a regeneration medium is studied. In the same experiment, the frequency of regeneration is estimated. The data are given in table 3.

Example 4, The effect of various concentrations of phytohormones in a regeneration medium on the frequency of occurrence of shoots is studied. Morphogenic calli are obtained by stepwise decreasing the concentration of 2,4-D from 4 to 0.3 mg / l. The data are given in table 4.

As can be seen from the above data, the optimal concentration for plant regeneration during direct transplantation is 1.0-2.0 mg / l. A further increase in concentration reduces the effect.

Example 5: The effect of stepwise changes in the concentration of zeatin on the regenerative capacity of calli is studied. Morphogenic calli are obtained as in Example 4. The concentration of IAA is constant and equal to 0.01 mg / l. The data are given in table.5.

From a comparative analysis of the data of Tables 4 and 5, the following follows: with a stepwise increase in the concentration of ze / in, the frequency of regeneration increases as compared with direct transplantation. In addition, the number of shoots from one callus also increases 1.5 times.

For example, the effect of various concentrations of IAA in the medium for rooting on the viability of regenerated plants, as measured by the number of plants rooted in the soil, is studied. The data are given in table.6.

As can be seen from the above data, the optimal IAA concentrations for rooting plants are in the range of 0.3-1.0 mg / l. A stepwise change in the concentration of IAA gives a 100% survival rate, while the root system of the plants is more powerful than with direct transplantation into the medium with 0.5 mg / l IAA.

Example. The effect of light on the frequency of morphogenic calli is studied. Conditions for the induction of callusogenesis, as in example 1. The data are given in table. 7.

It follows from the data that illumination of calluses increases the frequency of morphogenic calli appearance by 1.5 times.

The list of environmental components used in the implementation of the proposed method

The composition of the nutrient medium, mg / l MN4MOZ1650

KMOz1900

CaCl2 2H20440

MgS04 7H20370

KNAP04170

N32EDTA 2Н2037.3

FeS04 7H2027,8

NZOZ6.2

MnSCM 4H2022,3

ZnS04 7H208,6

KI0.83

Na2Mob4 2H200.25

CuSCM 5Н2О0.025

CoCI2 6H200.025

Saccharose30000

Glycine2,0

Mesoinositol 100,0

Nicotinic acid 0,5

Pyridoxine HCI1,0

Thiamine HCI1.0

Agar7000

PH5.7

Claims (1)

Hormones (Table 8) Formula of the Invention A method of regenerating wheat plants in tissue culture, including obtaining primary callus from an explant, induction of the formation and cultivation of embryogenic callus, obtaining regenerants and their rooting using nutrient media based on Murase-Skuga in the presence of phytohormones. tl and h and yusch and with the fact that in order to increase the yield of regenerants and their viability, the leaf base is used as an explant, when cultured embryogenic callus in a nutrient medium the concentration of 2,4-D is changed in steps from 2 to 0.1 mg / l and the illumination from 500 to 1000 lx; upon receipt of regenerants in the medium, the concentration of zeatin is changed in steps from 0.1 to Effect of different concentrations of 2.4 - D on morphogenesis Research of various step changes in the content of 2.4 - D The effect of phytohormone changes on regeneration 2 mg / l, the concentration of indolyl-3-acetic acid is set to 0.01 mg / l, and the illumination is increased from 1000 to 2000 lx, while rooting of the regenerants in the medium changes the concentration of indolyl-3-acetic acid from 0.1 to 0.5 mg / l and illumination from 2,000 to 4,000 lux. Table 1 table 2 Table 3 Effect of phytohormone content on spore formation Effect of changes in zeatin content on regenerative capacity The effect of the content of PUNK on rooting Table 4 Table 5 Table b The effect of light on morphogenesis The content of phytohormones in various environments Table 7 Table 8