Ovule sterilization method

FIELD: agriculture, in particular, fruit and ovules sterilization processes.

SUBSTANCE: method involves providing two-staged sterilization procedure with the use of 0.8%-silver nitrate solution: initially treating fruits for 8-10 min, followed by treatment for 5-6 min of ovules released from fruits.

EFFECT: reduced infecting of ovules upon introducing thereof into in vitro culture and, correspondingly, increased efficiency of selection works.

4 tbl

 

The invention relates to agricultural biotechnology, in particular to methods of producing sterile plant material in the culture of isolated organs, tissues and cells. The invention contributes to the protection of the environment.

There is a method of sterilization of plant tissues by the action of active chlorine (calcium hypochlorite and sodium, bleach, bleach). (Regbank. "The culture of isolated tissues and physiology of plants morphogenesis", 1964, p.24-28).

The disadvantages of the methods of application of active chlorine should include the need to prepare every time fresh solutions of high concentration sterilizers, a long period of sterilization, the need for repeated washing material after sterilization and low efficiency of sterilization.

A method of obtaining sterile germ and other generative organs (Ahistorically-Richter. "The culture of isolated embryos and some other methods of growing plants in vitro", 1974, p.34-39), where as the sterilizing agent used 90-95° alcohol, in which is immersed the fruit or seed and then fired over the flame of an alcohol burner.

The disadvantage of this method is that it is not always achieved positive effect sterilization.

Known sterilization method ovules, on the expectation by the Department of the fruit, the washing, sterilization (Npiskunova, Aviko, Vmin. "Biotechnology research garden and other valuable perennial crops", 1997, p.50-51).

The disadvantage of this method is the lack of a positive effect of sterilization, which affects the low efficiency in a separate crossing combinations peach (the number of infected embryos and 17.2-27.8 per cent), apricot (an 11.7-12.5%) and plums (sort of Idyll).

The purpose of sterilization of the ovules of grapes is a disinfectant, reducing infection ovules when their in vitro culture.

This objective is achieved in that the sterilization method of the ovules, including the Department of the fruit, washing, sterilization, sterilization is carried out in 2 stages of 0.8% solution of silver nitrate will first process the fruit for 8-10 minutes, and then separated from them ovule within 5-6 minutes.

New in the proposed method is that for the first time, silver nitrate is used for disinfection in the tissue and organ culture of grapes.

It is essential that the method applies the two-time processing of the same disinfectant as berries and ovules. Thus at the beginning of berries treated with 0.8 per cent solution of silver nitrate for 8-10 minutes, then separated from them ovule also handle ,8% solution of silver nitrate for 5-6 minutes. Due to the proposed method the infection of ovules obtained by self-fertilization of seedless varieties, when you enter in the culture decreased to 6.2-8.5%, and resulting from crossing of seedless varieties of grapes seedless up to 0-2,4%.

The method is as follows.

Bunch of grapes obtained from selfing or crossing seedless grapes, bring in the laboratory with parchment insulator, in which it is in the vineyard. Berries is separated, washed with warm soapy water, then running and distilled water and placed in a glass tube with a glass lid. Prepare a 0.8% solution AgO3(nitrate of silver) and sterilized in two stages. First pour berries this solution for 8-10 minutes. Then the solution is drained, the berries are dried with blotting paper and allocate ovule, which at the optimum time - 5 minutes also pour this solution. The solution is drained and the tube with the ovules are transferred into the box ("operating"). In Boxing ovule washed with three portions of sterile distilled water. After that, the ovules are planted each in a separate tube on a solid nutrient medium white. Culturing ovules is carried out in a culture room at a temperature of 25-27°C, 16 hour daylight and the intensity of light is Oia 4-5 thousand Lux. Regularly monitor the status of ovules and remove infected.

The effectiveness of various methods of step sterilisation of ovules obtained by self-pollination seedless grapes are illustrated in table 1.

As follows from the data processing berries 10,0% solution of calcium hypochlorite and sodium with further processing of the extracted ovules in a solution of calcium hypochlorite or roasting them in the flame of a torch after lowering to 1 second in solution 96,0% alcohol does not give a positive result. Infection of ovules is 78,6-100%. Not marked reduction of infection in the processing of berries with a solution of 0.8% AgNO3subsequent firing of the ovules in the flame of the burner. Only under the direct processing of ovules with a solution of 0.8% AgNO3there is a significant improvement in sterilization. The death of ovules from infection decreased to 17.8% during preview processing of berries 10.0% solution of calcium hypochlorite and to 6.5-8.5% during preview processing of berries also 0,8% solution of AgNO3.

Thus, the processing of berries and isolated from them ovules of 0.8% solution of AgNO3provides improved sterilization, which is expressed in the reduction of infection of ovules to a minimum size.

Table 2 presents the results of various sterilization hybrid samepage is, resulting from crossing seedless grapes with grapes. Also, how and when different processing methods ovules obtained from selfing, proved ineffective sterilization berries and ovules solution of calcium hypochlorite. Observed 100% mortality from infection. Somewhat better than the results obtained when processing the berries with a solution of calcium hypochlorite and ovules solution of AgNO3or at roasting them in the flame of the burner, but the death of ovules from infection remained very high. More effective was the use for processing berries sodium hypochlorite solution with the subsequent burning of the ovules in the flame of the burner, as well as of calcium hypochlorite with a subsequent more prolonged treatment with a solution of silver nitrate. The death of ovules from infection decreased, but still remained very high - 32,1-39,3%. And only two-time processing of berries and ovules solution of AgNO3practically nullified the death of ovules from the infection.

Sterilization of ovules obtained from selfing seedless grapes, and ovules, resulting from crossing of seedless varieties of grapes seedless varieties carried out in two stages, significantly reduces their infection, resulting in increased efficiency of selection.

Table 3 presents the results to the assessments of the impact of disinfectant for the infection of ovules after landing them on a nutrient medium.

Table 4 presents the results concerning the effect of sterilization method on the infection of ovules grapes after landing them on a nutrient medium.

The sterilization method of the ovules, including the Department of the fruit, washing, sterilization, wherein the sterilization is carried out in two stages of 0.8% solution of nitric acid silver will first process the fruit for 8-10 min, and then separated from them ovule within 5-6 minutes



 

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