SU1606045A1 - Method of storing unripe embrio of wheat - Google Patents

Abstract

The invention relates to biotechnology, in particular to breeding and genetic research, and can be used in agriculture to create new varieties. The aim of the invention is to increase the viability of wheat embryos. Embryos taken on days 16–20 after self-pollination are placed in a 0.30– 0.75% dimethyl sulfoxide solution for 20–24 h, after which they are dehydrated at 2–4 ° C, then a two-stage cryopreservation first in pairs and then directly in liquid nitrogen. 4 tab.

Description

The invention relates to biotechnology and can be used in agriculture, in particular for the rapid production of breeding material, as well as for the creation of new highly productive varieties of plants.

The need to use the culture of immature embryos in cell selection work is due to the fact that the genome of mature embryos is more stable and weakly subject to change under the action of various kinds of mutant factors. This makes it difficult to work in the field of genetic engineering to obtain new plant varieties. The main difficulty in working with em-f brions that have not completed their development is that they artificially lose their viability and cannot be stored for a long time. Since ten thousand embryos are needed to obtain new plant varieties, the question of their storage is urgent and requires an early decision. The production of immature embryos under field conditions in large quantities is limited by the growing season, and under artificial conditions large areas of greenhouse are required for raising and planting. In addition, during storage, the embryos lose the properties necessary for cell selection.

The purpose of the invention is to increase the fertility of wheat embryos.

The method of storage of immature wheat embryos consists of their preliminary preparation, dehydration and freezing: o

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In this case, wheat seeds 16-20 days after self-pollination are placed in a 0.3-0.75% solution of dimethyl sulfoxide (DMSO) for 20-24 hours, after which they are dehydrated in a vacuum desiccator with CaCl at a temperature of (+2) - (+ 4) ° C. When the moisture content of immature seeds is reduced to 20–25% of their cryoconcervirus, placed in pairs of liquid nitrogen, then immersed in liquid nitrogen. Reheating is carried out in a water bath at + 40 ° C. The viability of the embryos is determined after separation of the immature embryos from the seeds and their cultivation in Gamborg medium.

The choice of temperature parameters for dehydration in the range (+2) - (+4) C is due to the fact that it is within these temperature limits that functional damage to the tissues of immature embryos is prevented during dehydration.

Dehydration of immature embryos at +105 С sharply reduces the viability of immature wheat embryos due to damage resulting from disruption of growth processes caused by pathological changes in cell – cell interactions and disruption of the proliferation mechanisms of cell membrane structures.

The decrease in the viability of immature seeds when dehydrated at temperatures pax below 0 ° C indicates that part of the water bound to the macromolecules in the embryo freezes and forms 1 ice crystals that damage the cell membranes. When defrosting, this ice can recrystallize and cause even more cell damage. The dehydration of immature seeds of wheat to 20–25% moisture is due to the fact that at lower or higher levels of water content, vital processes are damaged, which determine the viability of embryos after cryopreservation under conditions of liquid nitrogen.

Conducting a two-stage cryopreservation ensures complete safety of the viability of the embryos. In the case of direct immersion of immature seeds in liquid nitrogen, embryo damage occurs. This is due to the fact that part of the water, which constitutes the movable compartment of the weak

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water can locally crystal and damage the internal organization of the cell.

As a result of the research, it was found that to maintain the viability of wheat embryos, the following conditions are not important: preparing immature wheat embryos for storage: processing time 16-20 days after self-pollination; the optimum concentration of cryoprotectant is in the range from 0.3 to 0.75%; incubation time immature. Cryoprotectant nuclei in solution is 20-24 hours.

Example 1. A study was conducted on prototypes of seeds of wheat 1 Kharkov 11. Plants are grown in field conditions. After 14,16,18, 20,22 days after self-pollination, immature seeds are placed at 0.1; 0.3, 0.5; 0.75; 1.0% DMSO solution for 22 hours. After that, the seeds are dehydrated at a temperature in a vacuum desiccator on CaCl. When a moisture content of 20% is reached, the seeds are frozen by placing liquid nitrogen in vapors and then directly placing it in liquid nitrogen. Heating was carried out at a temperature of +40 s in a water bath. The viability of embryos separated from immature seeds is determined by counting the number of calli on Gamborg B medium after 7 days of recultivation.

The results of studies of the viability of immature wheat embryos of Kharkov variety 11 during incubation in DMSO solutions,%, are presented in table. one.

Example 2. Wheat varieties from Kharkiv 11 variety, 18 days after self-injection, are examined experimentally. Immature seeds, separated from plants, are immersed in a 0.5% solution of cerebrospinal fluid at 18.2 :), 22.24.28 h, and then dehydrated over CaCl at + 3 ° C in a vacuum desiccator. Further, the experiment is carried out similarly to that indicated in Example 1. The results of the experiment are presented in Table. 2

Example 3. Wheat varieties from Kharkov variety 11, 18 days after self-pollination, research and comfort in the experiment. The immature seeds of the immersion seeds are separated from the plants: they are sown in a 0.5% solution of DMSO, and then in a vacuum desiccator and dehydrated over CaClg in a refrigerator at a temperature of -2, о, + 2, +3, +4,. The experiment is then carried out analogously to example 1. The results of the experiment are presented in Table. 3

As evidenced by the data given in table. 1-3, - the best results are obtained when working with immature embryos 18 days after self-pollination, which are incubated in 0.5% DMSO solution for 22 hours during dehydration in the temperature range from +2 to.

To confirm the effectiveness of the method, the viability of immature wheat embryos is determined according to the prototype method and the proposed method after cryopreservation.

Comparative data on the viability of immature embryos after cryo. conservation according to the method of the prototype and the proposed method are presented in table. four,

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valuable immature embryos necessary for cell selection, which contributes to the acceleration of work on the selection of new plant varieties. In addition, the method allows for the preservation of cryogenic embryos and their storage for a long period of time with a high level of viability, which is important for the preservation of hereditary characteristics of plants.

Claims (1)

Invention Formula 20 The method of storage of immature wheat embryos, including their preliminary preparation, dehydration and freezing, is distinguished by the fact that, in order to improve the viability of the embryos, the preliminary preparation is carried out by placing the embryos taken , 20 days after self-help, According to the data of Table 4, -25 0.30-0, 75% solution of dimethyl sulfoxide for 20-24 hours, dehydration of pro-. water at a temperature of +2 - 4 s and freezing is carried out by two-step cryopreservation, first in vapor, and then directly in the liquid. The proposed method is more effective. The yield of viable immature embryos processed by the proposed method exceeds the yield according to the method of the prototype by 56%. Thus, the proposed method allows to increase the yield of 30 com nitrogen. , 1606045 but valuable immature embryos necessary for cell selection, which helps accelerate the work on the selection of new plant varieties. In addition, the method allows for the preservation of cryogenic embryos and their storage for a long period of time with a high level of viability, which is important for the preservation of hereditary characteristics of plants. Invention Formula 20 oxide by 2 tons when freezing stage cree vapor, and after com nitrogen.   wheat embryos varieties XapbKOBCKa "l 1 e- 18 days after self-cultivation, incubation in a 0.5% DMSO solution, And in relation to the control Table 1  1606045 " Table3 Viability of immature wheat embryos of Kharkiv variety 11, 18 days after self-pollination, incubation in 0.5% DMSO solution,% 43.4 (100%) 67.8 (156.2%) P 0.01 Editor S. Lisin Compiled by V. Demkin Tehre, p; M.Didyk Proofreader N.Korol Order 3504 Circulation 416 VNIIPI State Committee for Inventions and Discoveries at the State Committee on Science and Technology of the USSR 113035, Moscow, Zh-35, 4/5 Raushsk nab. Production and publishing plant Patent, Uzhgorod, st. Gagarin, 101 A4 table Subscription