SU1524479A1 - Method of cryogenic preservation of genetic collection of potato varieties and species - Google Patents

Abstract

The invention relates to biotechnology, namely, to cryogenic methods of storing living cells and plant tissues, in particular meristem, and can be used in plant breeding and horticulture to preserve the gene pool of potatoes for a long time. The aim of the invention is to increase the reliability of cryopreservation, increase the viability of the apexes and ensure the mentality of regenerated plants to the original. Cnoccf6 consists in taking as a starting material for isolating apexes r obviously healthy sterile plants grown in strictly controlled nifpyewbix conditions: adenine is added to the cryo-trotector solution additionally sucrose at a concentration of 5-10Z at a concentration of 10–40 mg / l, the vitamins are added according to the Staba formulation, most commonly ns .--; soil is auxins and gelsroliatin casein. 3 tab. g WITH

Description

The invention relates to biotechnology, in particular, to cryogenic methods of snoring living cells and plant tissues | in particular, a pex (meristematic feathers; shoots) and can be used in plant breeding and seed production for the long-term preservation of the potato gene pool.

The aim of the invention is to increase the reliability of cryopreservation, increase the viability of apexes and ensure the genetic identity of the regenerated plants to the original.

The method consists in that deliberately healthy sterile plants, grown under strictly controlled conditions, are taken as starting material for isolating apexes; carbohydrates, in particular sucrose, in concentration 5-IOZ, are additionally added to the cryoprotectant solution; adenine concentrations of 10–40 mg / l, vitamins are added according to the Staba formulation, at the same time excluding auxins and casein hydrolytic from the medium.

The essence of the invention is to replace the starting material for the isolation of the apexes with the highest quality in modifying the nutrient medium for their preliminary cultivation and recultivation.

after thawing and the introduction of additional substances, in addition to the cryoprotector, as which carbohydrates are used, in particular sucrose at a concentration of 5-10%. The most high-quality material is represented by collections of completely healthy sterile plants in test tubes, available in all breeding centers and world centers of the potato gene pool and numbering hundreds and thousands of different forms. Such collections are an indispensable stage of modern intensive seed farming of potatoes and are supported by clonal microcrania under strictly controlled laboratory conditions, i.e. they are as standardized as possible. When they are used, sterilization of the seedlings before the removal of their apexes is excluded. Modification of the medium for apexes, on the one hand, weakens the growth of their cells by stretching during the pre-culture period, which reduces vacuolization and cell oxygenation and is favorable for ck growth. NIN during the extreme procedure of deep freezing and falling defrosting. On the other hand, the proposed modification completely prevents callus formation during the reclamation period after thawing, providing only direct regeneration. The introduction of carbohydrates into the cryoprotectant solution, in particular sucrose, each molecule of which contains four water molecules, is not. It only reduces the toxicity of the CCNO and increases the viscosity of the solution, reducing the volume of ice formation, the rate of its spread and the likelihood of cell damage. At the same time, penetrating after DMSO and glue, carbohydrates bind water and 1HHX, preventing excessive compression of the protoplast during slow and freezing. Supplying a package with apexes with a wick and its location allows you to minimize the volume of the solution in the ampoule, significantly ci; o-. P drainage, which reduces the level of damage at this stage by increasing the crystallization centers that appear during siirioj / a iH-.

The method is illustrated by a primer, Dt freeze, uses aneh sy 300-450 µm, g; i. IR / .K: Sinus leaves of the middle part /: - 8

days of sterilization (plants of a cultivated tetraploid type of potato varieties Domodedovo. Preliminary preparation of apexes consists in cultivating them on a liquid nutrient medium, modified, as indicated by the plate, for 24-26 h, and the last 16 h of this period in the presence of cryoprotein added to the medium tectoron: 5% DMSO and 5% sucrose Condition of cultivation of plants: temperature 22-23 C, humidity 60-70%, illumination - 5-8 thousand lx, photoperiod 16 hours / day; apex: 26 C, humidity - 60-75 %, illumination 5-8 thousand lk, photoperiod - 24 hours. After Cooking apexes in a packet of filter paper with a wick are transferred to the vial under the cork, positioning the sachet along the side wall so that the wick and the sachet fit close to it and the wick is lowered to the bottom. 0.3 ml of the above cryoprotectant solution, cool the ampoule in an ice bath, and then in a PROGRAMMER freezer at a rate of U, 5 ° f / MHH. At a temperature of -A, all of the ampoules iia running from h in this tripod, by touching their bottom to the surface of the ground pzot during 0.3. 11c is also possible for priming: pilgling a special device. A software freezer was singing equipped with a large device that helps avoid short-term manual operation in the freezing chamber. In both cases, the apexes are distant from the zone of formation and the initial rapid growth of ice crystals in iron. the seed and cannot be damaged. This moment. After that, the temperature in the freezing chamber is held at 5 ° C for 10-20 minutes, and then the cooling is continued at an initial speed up to, after which it is immediately immersed in liquid nitrogen (-19b C). After storage in an ampoule, the ampoules are quickly thawed, chstnh.na in water, then ahui / .nbi in lg; d no1 | a bath quickly resist and sterile conditions and apexes on a simple environment TOI-0 same state: tavl. Conditions of recultivation and 11 hrn: e knots are the same as for the / C Guli m ary of apexes, but ocuf iienrtonTi is from 1000 to, 6000 lx. 2-ih, on s;: unitu1; cis day and

Further, every 5–7 days, the apekbel is divided into non-droplets, which are transplanted onto fresh agar, transferred to a tube, i.e., tubes for the rooting medium, continue to be reclaimed.

under the conditions indicated above for Table II. I shows the results, plants. After the resumption of growth, obtained by the proposed method, in the development and formation of a strong degree of dependence on the composition of the solution of the cryolayer on the stem of the stem, this protector. T a b i c i

The effect of cryoprotectants on the viability of apexes and the registration of plants

Variant — Final Cryo Concentration Number of Apexes, Z Ant Protectors, Z

DMSO glycerin sucrose vyzhnvshnich obrazhereggereiv tech. rovavpih I mon. callus plants

1502 HEO

250-582О82

35010 50О35

450209О9

555200О

655541О24

755106О6

table 2

The influence of the age of the original sterile plants on the yield of plants - regeneratsy after deep freezing and thawing of apexes

The age of the original apexes, plants., The days of generating

plants,%

. 2682

2833

3214

3610

414

Table 3

Effect of adenine-D nutrient content on the growth rate of regenerants from potato meristem

Option (co-length regenerants (mm / month c)

hold I

adenine to the port of Domodedovo variety Slapnsky grade Druzhny

medium mg / l)

mm% to control mm% to control mm I Z to control I role

control: without adenine

0.5

5.0

10.0

20.0

1,210 2,700 3,103 6,725 6,723

223.1 256.4 555.8 555.6

232.9 315.9 475.8 582.0

1.356 2.563 3.005 5, G06 6.440

189.0 221.6 376.5 474.9

20

Thus, from table. J; E: SCHNO that the proposed method is searingly distinguished by the number of plant regenerants and completely eliminates the overgrowth of surviving apexes with a calpe cloth — all survivors show direct, regenerated plants in a plant: for the Domodedovo variety, the number of surviving apex is 5% and the number of plants -1.genervi-25 tov also 82%. The nature of the split-up apexes leaves no doubt about the hf reggeeration, which eliminates the need for a complicated and time-consuming procedure for identifying the genotype JQ of the plants obtained. Consequently, the proposed method, which provides a high yield of plants — regenerants and eRono of labor and funds spent on maintaining laborigor), gx and field collections of varieties, forms and types of potatoes, PS is much less stringent than its counterparts. since it does not require ampoules of complex construction, which you have to manufacture .in mount the root and then sterilize the ampoules, allows / exclude operations of chemical sterilization and subsequent triple washing of the seedlings of tubers and always get Omo is a completely healthy breeding material for breeding, eliminating seasonal interruptions in work and the danger of infection of the samples by the superficial microflora of the club

CFIOCO6 cryopreservation of varieties and types of potatoes, pre-cultivation of plant sleep on nutrient-containing micro and macro programs Murashige-Skoog, p, ampoules, software packaged in the presence of a cryoprotectant with coreing of this solution and by stabilizing the temperature of spreading crystals of the solution volume raienie

temperature below -140 C,

exe

35

40

u and. and with that, with ce. Nor does the need to protect the viability of genetic supply. These regenerated raw materials of the source material are 26-28 days sterile grown under strictly control conditions, an additional 10-10 mg / l sphar ce ceration is added to the cryop solution, and an additional 5 to 10-40 mg / l is added to the body, Vitamite is manufactured according to Stubs, and in order to freeze an anchors, they use a bag of mage filters with a wick equal to the height of the ampoule.

Neva sprouts.

Editor N. Kozlov

50

Compiled by, Demkin

Tehred L. Sardyukova Proofreader Fr. Kravtsov

eight

Continuation of table. 3

Formula iinobject neither

CFIOCO6 cryopreservation of the gene pool of varieties and types of potatoes, including preliminary cultivation of plant apexon on a nutrient medium, containing micro and macronutrients according to the Murashige-Skoog progis, sizing them, ampoules, program freezing in packaging in the presence of a cryoprotectant solution with priming crystallization of the em. the solution and the subsequent stabilization of the temperature until the moment the crystals are distributed throughout the entire volume of the solution,:; radiation at a temperature below -140 ° C,

distinguish

u and. and with the fact that, with the Cp.... of increasing the cryogenic preservation, increasing the viability of the apexes and ensuring the genetic identity of the regenerated plants, 26-28 days old sterile plants, grown under strictly controlled conditions, are used as the starting material. cryoprotectant additionally introduced spharose in a concentration of 5–10%, additionally adeichn in the amount of 10–40 mg / l was added to the pi1l, additional vitamins were added via vitamine, and as a package for freezing apexes, pack of iltrovalnoy paper provided with a wick, the length equal to the height of the ampoule.

Claims (1)

The method of cryopreservation of the gene pool of varieties and wads of potatoes, including the preliminary cultivation of plant apexes on a nutrient medium containing micro- and macroelements according to the Murashige-Skoog recipe, placing them in ampoules, programmed freezing in the package in the presence of a cryoprotectant solution with crystallization of this solution and subsequent stabilization temperature until crystals spread throughout the volume of the solution, storage at temperatures below -140 C. _b featuring a dQ I that, with the aim of improving nadezhnos and protection, increasing the viability of apexes and ensuring the genetic identity of regenerated plants, use 26-28 days sterile plants grown under strictly controlled conditions as starting material, sucrose is additionally added to the cryoprotectant solution at a concentration of 5-10% in nutrient C (> food is additionally injected with adenine in an amount of 10–40 mg / l, vitamins are added according to Staba’s prescription, and as a package for filtering freezing of apexes, a filter paper bag equipped with a wick is used , length equal to the height of the ampoule.