RU2515385C1 - Method of microclonal reproduction of black alder in vitro - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: in the method they cultivate callus cultures from sterile explants of stem segments, leaves, leaf stakes. Basic nutrient media are used, such as Murasige-Skuga and Woody Plant Medium with content and ratio of growth regulators: 6-benzylaminopurine - 0.2-1.5 mg/l, α-naphthyl acetic acid - 0.2-0.5 mg/l, indolebutyric acid - 2 mg/l. Further adaptation is carried out with light of 2000 lux and nutrient mode of regenerants in a greenhouse with production of planting material for forest cultures.

EFFECT: method makes it possible to produce planting material with high inherited properties.

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Description

The invention relates to the field of plant biotechnology.

Black alder (Alnus glutinosa (L.) Gaertn.) Is promising for the country's forestry, it can withstand prolonged flooding and grow on waterlogged ecotopes where other tree species cannot grow, and can grow on poor and barren dry soils (gravel, sand ) with close-lying groundwater, it is able to enrich the soil with nitrogen through litter and root excretions (up to 780 kg / ha for 5 years). Due to this quality, black alder in joint planting with more valuable breeds has a positive effect on them. The biosphere role of black alder in the bog ecosystem is unique, which makes it attractive for ecologists, foresters, breeders. It is irreplaceable in the development of models of sustainable and accelerated increase in the productivity of plantations, in plantation forestry for accelerated production of wood.

Due to the importance of growing breeding forms and varieties of black alder, there is a need to obtain planting material with high hereditary properties. In the prior art, no way to solve the problem.

The present invention solves the problem of creating the most effective method of propagation of black alder - microclonal propagation in vitro.

The invention consists in the following.

The method of microclonal propagation of black alder in vitro involves the cultivation of callus cultures from sterile explants of stem segments, leaves, leaf petioles using the base nutrient media Murashige-Skoog (MS), Woody Plant Medium (WPM) with the content and ratio of growth regulators: 6-benzylaminopurine ( 6-BAP) - 0.2 ÷ 1.5 mg / l, α-naphthylacetic acid (NAA) - 0.2 ÷ 0.5 mg / l, indolylbutyric acid (IMA) - 2 mg / l, and further adaptation at illumination of 2000 lux and the nutrient regime of regenerants in the greenhouse, and obtaining planting material for forest crops.

The method is carried out as follows.

For callus cultures, explants are renal stem segments, leaves, leaf petioles.

MS or WPM and their modifications with different contents and ratios of growth regulators (6-BAP, IAA, NAA, IMC) are used as base media. For direct regeneration, 6-BAP (0.2-1.5 mg / L) is used, and the induction of many buds and shoots is 6-BAP in combination with NAA (0.2-0.5 mg / L). Callus cultures are obtained using IMC (2 mg / L) in combination with 6-BAP (0.2-0.5 mg / L). The exclusion of IMC in the future and an increase in the concentration of 6-BAP to 1 mg / l are used to obtain morphogenic callus cultures. To proliferate callus of black alder, BCA (2 mg / L) in combination with 6-BAP (0.2-0.5 mg / L) is added to the nutrient medium. The development and elongation of shoots is achieved according to the following methods: the salt composition of the nutrient medium is diluted 2-3 times, supplemented with activated carbon (10-20 g / l), the basal ends of the shoots are treated with a solution of IMC (25 mg / l for 10-15 minutes )

The adaptation stage of development of the regenerated material provides for the following options:

1 - laboratory (open tubes for 3-5 days, then planted glasses in the ground, grow in a micro-greenhouse, followed by planting in open ground).

2 - direct landing in open ground using temporary shelter (film micro-greenhouses).

In the first version, environmental conditions are controlled (t 25-26 ° C, illumination 2000 lux with a 16-hour photoperiod); in the second, natural lighting and temperature conditions. High humidity is achieved by watering and film shelter.

The effectiveness of callusogenesis of different alder organs on the nutrient medium MS + IMC (2 mg / l) is shown in table 1.

Table 1 Explants Callusogenesis Efficiency Number of crops The rate of callusogenesis (day) The volume of tissue (cm ³ ) the general With callus,% Black alder stem segments fifteen one hundred 21 1,5-2 petioles 10 40,0 thirty traces leaves eleven 9.9 thirty traces

Table 2 shows the effectiveness of morphogenesis in cultures of various origins (callus, stem segments) on WPM medium depending on the content of BAP mg / mm:

table 2 BAP mg / mm Black alder callus culture the kidneys total amount total amount 0.1 5 - 10 - 0.2 5 - 10 - 0.5 6 1 / 16.6 10 4 / 40.0 0.6 5 2 / 40,0 10 7 / 70.0 1,0 5 - 10 2 / 20.0 1,5 5 - 10 - 2.0 - - 10 -

The life expectancy of morphogenic alder crops compared with other deciduous (birch) is much lower and limited to 2-3 weeks.

To preserve them, re-restoration is necessary. Otherwise, regardless of the origin, in cultures, intensive growth of callus tissue is noted, as a rule, inhibiting the development of shoots.

Claims (1)

The method of microclonal propagation of black alder in vitro, including the cultivation of callus cultures from sterile explants of stem segments, leaves, petioles, using the base nutrient medium Murashige-Skoog, Woody Plant Medium with the content and ratio of growth regulators: 6-benzylaminopurine - 0.2-1 , 5 mg / l, α-naphthylacetic acid - 0.2-0.5 mg / l, indolylbutyric acid - 2 mg / l, and further adaptation at illumination of 2000 lux and the nutrient regime of regenerants in the greenhouse, and obtaining planting material for forest cultures.