RU2743967C1 - Method of creating planting material for chrysanthemums in vitro - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology. The invention is a method of growing regenerative plants on the basis of in vitro ray flowers of chrysanthemums . The method was devised for in vitro cultivation of ray flowers of chrysanthemums and can be used for mass production of genetically homogeneous planting material of valuable varieties and hybrids of chrysanthemums. Ray flowers are used as the primary explant and are cultivated on the basis of nutrient medium which contains all the mineral salts of Murashige and Skoog medium and also such hormones as 6-benzylaminopurine (BAP) at a concentration of 1 mg/l and indoleacetic acid (IAA) at a concentration of 0.5 mg/l and Aminoven 15% at a concentration of 3 ml/l.

EFFECT: invention increases yields of chrysanthemums.

1 cl, 1 ex

Description

The invention relates to the field of agricultural biotechnology, and can be used for the mass production of genetically homogeneous planting material of valuable varieties and hybrids of chrysanthemums.

A number of methods for producing regenerant plants in vitro are known: activation of the development of meristems existing in the plant, induction of the formation of adventive buds directly on the primary explant, somatic embryogenesis, induction of the formation of adventive buds in the primary and transplant callus tissue (Patent RU No. 2560592, Patent RU No. 2619052) ...

However, all of these methods have both their advantages and disadvantages.

In vitro cloning of chrysanthemums is being carried out in many countries, including Russia. However, the proposed technologies are ineffective, since they depend on the quantity and quality of the starting material. As a rule, cuttings with one or two axillary buds, as well as leaf blades, are used as the primary explant for in vitro propagation of chrysanthemum plants. Seeds are less commonly used.

For example, there is a known method of reproduction of chrysanthemums by activating the development of meristems existing in the plant. The method provides for the cultivation on a nutrient medium of cuttings 10-15 mm long with one axillary bud, which are cut from the donor plant. For mass production of genetically homogeneous planting material of valuable varieties and hybrids of plants, it is necessary to cut such cuttings from an intact plant in an amount of at least 50-100 pieces, since when introduced into an in vitro culture after sterilization, only 50% of explants can survive due to incorrect selection of a sterilizing agent and its temporary exposure (see the abstract of thesis ... Ph.D. / Grand Jaramillo Roberto Carlos. - Moscow Agricultural Academy named after K.A. Timiryazev. - Moscow, 2009. - 19 p.). It should be noted that the disadvantage of this method is that the isolation of such a number of cuttings from one plant can lead to the complete death of the plant and the loss of valuable varietal material. In addition, the disadvantage of this method is also a low multiplication factor. So, from one axillary bud, only one microshot develops at the first passage. When it is subsequently micro-cut, the multiplication factor is, as a rule, 5-7. Hence it follows that during the next passage (1 month of cultivation) only 5-7 plants can be obtained. If the multiplication factor at each passage is 5-7, then within 6 months of cultivation, 16807 regenerant plants can be obtained.

The known method provides for the propagation of chrysanthemum plants in vitro on a nutrient medium containing electrochemically activated aqueous solutions: neutral cathode-treated anolyte (ANK anolyte) and catholyte. Cuttings with one or two axillary buds are cut from intact plants and sterilized with anolyte ANK solution at a concentration of 0.03% for 10 minutes. This allowed the authors to obtain a yield of 90-100% of sterile and viable explants. At the stage of micropropagation proper, the authors propose to use catholyte as a stimulator of plant growth and development, which avoids rapid aging of tissues, as well as vitrification (hyperhydration) of shoots, which occurs during micropropagation (Patent RU No. 2560592).

The disadvantage of this method is that, as in the first analogue, the isolation of cuttings is carried out from one plant, which can lead to its complete death and even to the loss of valuable varietal material. In addition, the disadvantage of this method is also a low multiplication factor.

There is also known a method of in vitro propagation of chrysanthemum plants - obtaining regenerant plants in the primary and transplant callus tissue. As a primary explant, the authors propose to use seeds (Patent RU No. 2619052) - a prototype.

The method provides for the cultivation of seeds on the nutrient medium of Murasig and Skoog (MS) with the addition of 0.7% agar-agar, 1 mg / l BAP, 0.1-1 mg / l IAA for 1 passage (26 days) until callus appears. Then the calli are transplanted onto the MC nutrient medium with a half concentration of all components and with the addition of 0.2-1 mg / l BAP. and cultivated for 2-4 passages until the appearance of regenerant plants.

The disadvantage of this method is that regenerant plants obtained from dedifferentiated cells of callus tissue, as a rule, are genetically heterogeneous among themselves (somaclonal variability is observed, which manifests itself both at the morphological and genetic levels) and differ from the donor plant. Such plants are of interest only to breeders and not to producers of high quality planting material.

From the analysis of known similar technical solutions, it was revealed that a technical problem in this area is the need to expand the arsenal of tools for the mass production of genetically homogeneous planting material of valuable varieties and hybrids of chrysanthemums.

The technical result of the invention is to accelerate the process of mass production of genetically homogeneous, high-quality planting material of chrysanthemums for industrial floriculture of valuable varieties and hybrids of chrysanthemums.

To solve this problem and achieve the claimed technical result, reed flowers are used as the primary explant, which are isolated from the opened flower buds and cultivated on a nutrient medium containing mineral salts according to the recipe of Murasig and Skoog. The hormones 6-benzylaminopurine (BAP) at a concentration of 1 mg / l and indoleacetic acid (IAA) 0.5 mg / l), as well as the drug Aminoven 15% at a concentration of 3 ml / l, serve as an inducer of the formation of adventive kidneys. By the end of the passage, adventive buds developed into shoots are separated from the maternal explant and independently cultivated on MS medium containing only 15% Aminoven, taken at the same concentration.

A specific example of implementation of the proposed method

For propagation of chrysanthemums, you can use one cut flowering shoot, which has one or more flowers. Divide each chrysanthemum flower into separate petals (reed flowers). Up to 50 reed flowers can be isolated from one flower, which are placed in gauze bags and under the conditions of a laminar box, they are surface sterilized with 0.1% mercuric chloride solution for 5 minutes, followed by keeping in sterile distilled water. The reed petals are removed from the gauze bag and evenly spread on the surface of the nutrient medium in Petri dishes. The cultivation of reed petals is carried out on a nutrient medium containing mineral salts according to the recipe of Murasig and Skoog. Hormones (6-benzylaminopurine (BAP) at a concentration of 1 mg / l and indoleacetic acid (IAA) 0.5 mg / l), as well as the drug Aminoven 15% at a concentration of 3 ml / l, serve as an inducer of the formation of adventive kidneys. Petri dishes with explants are transferred to a light room, where the temperature is maintained at 230C, a 16-hour photoperiod, illumination with white fluorescent lamps, illumination intensity 3,000 lux. After 15 days, adventitious buds appear in the basal part of the reed petals, the average number of which is 12-15 pcs. By the end of the passage (30 days), adventive buds develop into shoots, which are separated from the maternal explant and independently cultivated on MS medium without hormones, but containing 15% Aminoven at a concentration of 3 ml / l. With an average multiplication factor of 5-7 and isolation from one flower bud of only 10 reed petals, then within 6 months of cultivation, 312130 regenerant plants can be obtained.

The proposed method for obtaining genetically homogeneous planting material for chrysanthemums combines a number of positive properties that allow it to be used in practical work:

1. The technology assumes the use of readily available material (flowers) for cloning in small quantities.

2. The presence in the composition of the nutrient medium of a strong inducer of morphogenesis (15% Aminoven 3 ml / l) (cheap technology).

3. The proposed method is easy to implement in comparison with the previously proposed inventions of the authors.

4. The proposed technology makes it possible to obtain 18 times more planting material of chrysanthemums in comparison with the previously proposed inventions of the authors.

The claimed invention is aimed at eliminating the disadvantages that are inherent in the most common methods for obtaining high-quality planting material for chrysanthemum plants. No methods with similar technology known in the scientific, technical and patent literature have been found. The result obtained with this solution and due to the use of reed flowers as a primary explant was not achieved in the known solutions.

The use of the invention will increase the yield of genetically stable, high-quality planting material of chrysanthemums by 18 times, which makes it possible to obtain them in an amount sufficient for industrial floriculture of valuable varieties and hybrids of chrysanthemums.

Claims (1)

A method for obtaining planting material of chrysanthemum in vitro, including the cultivation of explants on a nutrient medium containing mineral salts according to the recipe of Murashige and Skoog with the hormones 6-benzylaminopurine and indoleacetic acid, characterized in that reed chrysanthemum flowers are used as the primary explant and during their cultivation additionally add the preparation Aminoven 15% at a concentration of 3 ml / l to the nutrient medium with hormones, then the adventitious buds developed in shoots by the end of the passage are separated from the maternal explant and independently cultivated on MS medium containing only the preparation of 15% Aminoven taken in the same concentration.