RU2118486C1 - Method for multiplying wood planting material - Google Patents

Abstract

FIELD: agriculture. SUBSTANCE: method involves in vitro growing of propagules; testing by immunoferment analysis; grafting plants, with propagules of proliferous crops which had passed test for presence of virus and other infection being used as graft. Mentioned plants are grafted on healthy grafts or seedlings adapted for unsterile conditions. Cut surfaces are treated with solutions of antioxidants and growth regulators promoting callusing. EFFECT: increased yield of healthy plants by removing limiting factors, eliminating labor consuming operations, reduced test and multiplication time. 5 cl, 6 tbl, 1 ex

Description

 The invention relates to the field of agriculture, in particular to biotechnology, and can be used in nursery for mass production of healthy planting material for woody plants.

 Biotechnical methods for obtaining and propagating virus-free planting stock are widely used to solve the most diverse problems of modern crop production. Their use is based on unequal concentrations of viral particles in various organs and tissues of plants. The minimum amount of pathogen is contained in apexes, which makes it possible by grafting them onto healthy specimens or rooting to obtain healthy planting material of the desired varieties (see CAMBELL AJApple virus inactivation by heat therapy and tip progation. Nature, 1962, v. 195, N 4840, p.520).

 The disadvantage of this technique is the need to use fairly large parts of the shoots, which reduces the effectiveness of recovery and limits the amount of source material.

 Therefore, it is more promising to obtain from relatively small initial explants, which use isolated apexes of 0.2–2.0 mm in size of proliferating shoot cultures and their further rooting.

 However, if it is necessary to recover relatively difficult root forms, which are most varieties of apple, pear and some stone fruits, these methods are also not effective enough.

 The closest technical solution known is the method of obtaining healthy citrus plants (see BITTERS W.P., MURASHIGET., RANGAN T. S. NAUER E.H., ROISTAHER C.N., HOLLIDAY P.B. Healhty trees from the test-tube.

 Citrograph, 1972, v 57, N 3, p.85-86-prototype), in which meristematic shoot apexes are grafted onto seedlings grown under sterile conditions, and then the resulting seedlings are grown to the required size.

 However, this method has the following disadvantages. The process of isolating apexes with a size of less than 1 mm and the procedure for grafting it onto seedlings is a laborious operation, the productivity of which is low, as a result of which the final yield of plants is low. The survival rate of such small structures is also unsatisfactory. In addition, all plants obtained require mandatory testing, since before this procedure their phytosanitary status is unknown.

 The problem to which the invention is directed is to increase the efficiency of healing and increase the final yield of plants by reducing the limiting factors and eliminating the most labor-intensive stages in plant production, including preventing the oxidation of phenolic compounds and stimulating the fusion of scion-rootstock components.

 This object is achieved by the fact that in the method of propagation of planting material of woody plants, including the cultivation of shoots in vitro on an artificial nutrient medium, testing by enzyme immunoassay, the plants are grafted on adapted to non-sterile, healthy test tube rootstock plants when they resume growth and reach 18- 25 cm

 In addition, the task is solved by the fact that in the proposed method of propagation of planting material of woody plants, vaccination is carried out on virus-free seedlings of 15-25 cm in size.

 In addition, the task is solved by the fact that in the proposed method of propagation of planting material of woody plants, the tops of shoots of proliferating cultures that have been tested for the presence of viruses and other pathogens by enzyme-linked immunosorbent assay, at least 15 mm long and at least 2 in diameter, are used as graft material. 5 mm.

 In addition, the task is solved by the fact that in the proposed method of propagating planting material of woody plants, the surface of the slices is treated with a solution of ascorbic acid at a concentration of 25-50 mg / l in conjunction with drugs that stimulate the development of wound callus at a concentration of 20-30 mg / l, which provides better intergrowth of scion-rootstock components.

 The problem is also solved by the fact that in the proposed method of propagation of planting material of woody plants, the surface of the slices is treated with a solution of sodium diethyl carbamate at a concentration of 1-2 g / l, together with drugs that stimulate the development of wound callus at a concentration of 20-30 mg / l.

 Due to the considerable size and the absence of the need for an inoculation operation under sterile conditions, labor productivity is significantly increased and the yield of plants increases. In addition, the use of already tested material makes further testing of all plants optional, which significantly reduces the entire process of seedling production. The use of drugs with antioxidant effects, as well as growth regulators that stimulate the formation of callus tissues at the site of contact of the stock-scion components, allows an even greater increase in the yield of finished products.

 The claimed technical solution differs from the prototype in that the developed shoots obtained in a proliferating culture of at least 15 mm in size, which have already been tested for the presence of viral and / or other infection, are used as graft components, and virus-free plant specimens adapted to non-sterile conditions.

 These differences allow us to conclude that the claimed technical solution meets the criterion of "novelty."

 The claimed technical solution has an inventive step, since it is not obvious to specialists of nursery breeders and biotechnologists, but is a product of the creative activity of the inventors.

 The claimed technical solution meets another required criterion of the invention - confirmation by experimental data obtained during the implementation of the method.

 The method includes growing in vitro shoots on an artificial nutrient medium, testing them by enzyme-linked immunosorbent assay, and plant grafting. Vaccination is carried out on healthy test tubes of rootstock plants adapted to non-sterile conditions when they resume growth and reach a size of 18-25 cm. Vaccination is carried out on virus-free seedlings of 15-25 cm in size. Tops of shoots of proliferating cultures tested for viruses and other pathogens by enzyme immunoassay, at least 15 mm long and at least 2.5 mm in diameter. Sliced surfaces of the grafted components are treated with a solution of ascorbic acid at a concentration of 25-50 mg / l in conjunction with drugs that stimulate the development of wound callus at a concentration of 20-30 mg / l. Sliced surfaces of the grafted components are treated with a solution of sodium diethylthiocarbomatate at a concentration of 1-2 g / l, together with drugs that stimulate the development of wound callus at a concentration of 20-30 mg / l.

 An example implementation of the method.

 Isolated apexes 0.2–2.0 mm in size are selected from the specimens to be improved and propagated, and proliferating shoot cultures are obtained on artificial nutrient media. Then, by the method of enzyme-linked immunosorbent assay, the cultures obtained are tested, for which 1-2 g of buds, leaves and shoots are taken from each meriklone and analyzed for the presence of viral and other infections. The cultures that showed negative results continue to propagate, and the shoots obtained, at least 15 mm in size, are used as micrograins for grafting on virus-free root forms adapted to non-sterile conditions or healthy seedlings. Moreover, to improve the conditions of fusion of the scion-rootstock components, treatment with drugs with antioxidant properties and growth regulators stimulating the formation of wound callus is used.

 The tables show the results of specific experiments on the implementation of the claimed method.

Using the proposed method of propagation of healthy planting material of woody plants provides the following advantages in comparison with the existing technology:
vaccines increase to 90% or more;
vaccination technique is simplified, since there is no need to observe sterile conditions;
almost 100% yield of healthy material is achieved;
the amount of work in clonal micropropagation systems is reduced due to the elimination of the rooting phase;
there is an opportunity to get healthy seedlings of hard-rooted forms.

Claims (5)

 1. The method of propagation of planting material of woody plants, including the cultivation of shoots in vitro on an artificial nutrient medium, testing by enzyme-linked immunosorbent assay, vaccination of plants, characterized in that the vaccination is carried out on adapted to non-sterile healthy test tube rootstock plants when they resume growth and achievement size 18-25 cm.  2. The method according to claim 1, characterized in that the vaccine is carried out on bisvirus seedlings of 15-25 cm in size.  3. The method according to claims 1 and 2, characterized in that the tops of shoots of proliferating cultures that have been tested for the presence of viruses and other pathogens by enzyme-linked immunosorbent assay, at least 15 mm long and at least 2.5 mm in diameter, are used as graft material.  4. The method according to claims 1 to 3, characterized in that the surface of the sections of the grafted components is treated with a solution of ascorbic acid at a concentration of 25-50 mg / l in conjunction with drugs that stimulate the development of wound callus at a concentration of 20-30 mg / l.  5. The method according to claims 1 to 4, characterized in that the surface of the sections is treated with a solution of sodium diethylthiocarbomatum at a concentration of 1-2 g / l together with preparations stimulating the development of wound callus at a concentration of 20-30 mg / l.

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