SU1055437A1 - Method of preserving genofund of potato varieties and species - Google Patents

Abstract

1. A process for the gene conservation species and varieties of potato comprising preculture meristematic apexes on the medium, cooling the object in ampoules in the presence of a cryoprotectant, seeded crystallization subsequent freezing and storage in liquid nitrogen, characterized in that, in order to maintain a high level of viability meristems and simplify the cryopreservation method, meristematic tips for cooling are placed in the upper part of the ampoule divided by a permeable transverse partition And seeded crystallization of manufac out by placing the bottom of the vials in liquid nitrogen prior to crystal formation. (O 2. The method according to claim 1, wherein (L that the seed is produced for 1.0 + 0.3 s.

Description

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| The invention relates to agriculture, namely to methods for low-temperature storage of plant tissues and cells, and can be used in plant breeding and seed production for the long-term preservation of potato gene pools. There is a method of preserving the gene pool of potatoes, based on deep freezing of meristematic tips. The meristematic tips, after preliminary cultivation on a liquid nutrient medium for 3-5 days and then incubated on the medium with cryoprotectants for 1 hour, are quickly immersed at the tips of sterile needles or in plastic ampoules directly into liquid nitrogen. This method allows maintaining the viability of meristematic tops in the range from 27% to 76% for the cultivated and for wild species, respectively. 1 The disadvantage of this method is the large variation in viability between the frozen specimens after thawing. Also known is a method of cryopreservation of meristematic tops of potatoes after preliminary preparation by analogy with the indicated methods, the distinguishing feature of which is slow cooling of the apexes in the presence of a cryoprotectant at a rate of 0.3 ° C / min to -40 ° C with subsequent rapid immersion in liquid nitrogen. In this method, during freezing, a seed (artificial initiation of crystallization) of the cryoprotectant solution is made by introducing a piece of ice into the ampoule using a micropipette at a temperature of -5 ° C. Such a method allows maintaining the viability of the apexes at the level of 71% after cryopreservation 2. However, the method has several disadvantages. After preliminary preparation, the ampoules with meristematic tips are placed in an alcohol solution with a temperature of 5 ° C and after 10 minutes they crystallize. To do this, under sterile conditions, a piece of ice is individually placed in each vial using a microbooter, and after 10 minutes slowly (about 0.3 ° C / min) the temperature of the alcohol solution is reduced using a Dewar vial system cooled by liquid nitrogen. With a significant number of ampoules, this method complicates and lengthens the whole process of blinking. In addition, the possibility of infection of the meristematic tips in the seeding process is not excluded. Thus, the known method of cryopreservation is cumbersome and time consuming, as it requires sterile conditions during freezing and a number of additional operations. The aim of the invention is to maintain a high level of viability of meristematic potato tops during slow freezing and subsequent storage in liquid nitrogen, as well as to simplify the cryopreservation process. The goal is achieved by the fact that before freezing the meristematic tops are placed in the cryoprotectant solution in the upper part of the ampoule divided by a transverse partition with holes, which prevents direct damage of the meristematic tissue at the moment of priming, the cooling process is carried out in a programmatic freezer, and the seed of the cryoprotectant solution is found in the lower part of the ampoule under the septum, produced by simultaneous immersion of the bottom of all and the ampulla thallium. In this case, the seed was produced within 1.0 ± 0.3 s. The temperature in the chamber of the apparatus is kept constant until the formation of ice crystals in the entire volume of the cryoprotectant solution. This ensures slow dehydration of the meristematic tissue, which in turn prevents the crystallization of water inside the cells. Example. For freezing, the meristematic tips of a cultivated tetraploid potato variety of the variety “Domodedovo. Preliminary preparation of apexes consists in cultivating them in a liquid nutrient medium with 5% DMSO (dimethyl sulfoxide) for 24 hours. Apex size is 250-500 µm. Cultivation conditions: temperature 26 ° C, illumination 5,000 lux, constant light, humidity 70%. After preliminary preparation, the meristematic tops transferred B 1.0 ml of a 5% -doped DMSO solution in a nutrient medium into ampoules with a transverse partition tight to the walls and dividing the entire volume of DMSO solution into two equal parts communicating with each other with a diameter of 0.2 mm in the transverse partition. Thus, the object to be frozen is separated from the zone of formation and the initial rapid growth of ice crystals that occur at the moment of seeding. The meristematic upperings are cooled in a program freezer at a rate of 0.5 ° C min. At a temperature of -5 ° C, all ampoules are seeded at once. Located in a special stand, by immersing their bottom in liquid nitrogen. For 1.0 s. After that, the temperature in the freezing chamber is maintained at –5 ° C for 20 minutes, and then the cooling of the frozen object is continued at the initial rate to –40 ° C, after which it is immediately immersed in liquid nitrogen (–196 ° C). After storage in liquid nitrogen, ampoules are quickly thawed in water at 40 ° C, and meristematic tips are planted on an agar medium. The conditions for reclamation are the same as in the preliminary preparation of the material for freezing. The viability of the apexes after thawing is determined by counting the number of developing meristematic tips after recultivation on a nutrient medium for 7-10 days. The viability level of meristematic tops, depending on the method of cryoconservation, is shown in the table.

The proposed method allows an increase in the average viability of meristematic tips by 13% as compared with the known method, and is also less labor-consuming, since it significantly simplifies and shortens the freezing process by eliminating all the operations necessary for conducting the seed. a cryoprotectant solution under sterile conditions by introducing a piece of ice into it using a micropipette.

Seed method

The introduction of ice pieces into ampoules using a micropipette under sterile conditions

Immersion of the bottom of the ampoules with a transverse partition into liquid nitrogen for 1.0 s

Data taken from the work of Touilla 2.

The average level of viability of meristematic tops

after freezing (storage, thawing),%

96.0

71.0

95.0

84.0

Claims (2)

1. METHOD FOR PRESERVING THE VARIETY OF VARIETIES AND TYPES OF CARDS FEL, which includes the preliminary cultivation of meristematic tips on a nutrient medium, cooling of the object in ampoules in the presence of a cryoprotectant, crystallization seed, subsequent freezing and storage in liquid nitrogen, characterized in that, in order to maintain a high level of viability of the meristem and simplify the method of cryopreservation, meristematic tips for cooling is placed in the upper part of the ampoule, separated by a permeable transverse partition, and the crystallization seed is produced by placing the lower part of the ampoules into liquid nitrogen before the formation of crystals. 2. The method according to π. 1, characterized in that the seed is produced within 1.0 + 0.3 s. SU ,, „1055437