RU2565806C2 - Method of in vitro preparation of microshoots of ash, aspen, willow for subsequent rooting under ex vitro conditions - Google Patents

Abstract

FIELD: agriculture.

SUBSTANCE: invention relates to the field of plant biotechnology and forestry. The invention is a method of in vitro preparing microshoots of ash, aspen, willow for subsequent rooting under ex vitro conditions, comprising the transfer of plants after the step of replication on a nutrient medium WPM for elongation, with the addition of sucrose 30 g/l, agar-agar 9 g/l, inositol 100 mg/l, pyridoxine 0.1 mg/l, thiamine 0.1 mg/l and nicotinic acid 0.5 mg/l, where glutamine is added to the medium for elongation at a concentration of 0.5 or 1.0 mmol/l, while culturing the plants is carried out with increased illumination intensity - from 5 to 8 thousand lux.

EFFECT: invention enables to obtain physiologically and morphologically aligned shoots of aspen, ash and willow, which provides significant increase in the efficiency of their rooting ex vitro.

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Description

The invention relates to the field of plant biotechnology and forestry and can be used for the preparation of microprobe transgenic clones of ash, aspen, willow for subsequent rooting in ex vitro conditions.

Currently, forestry is already quite actively applying modern achievements in the field of plant biotechnology to produce healthy fast-growing planting stock. To do this, use the method of clonal micropropagation of plants.

Aspen (Populus tremula) and its hybrids are actively used in biotechnological research as a model culture for studying various aspects of the genetics of forest tree plants for several reasons: the relatively small genome size, the relative simplicity of its clonal micropropagation and genetic transformation, rapid growth. In forestry, aspen begins to take priority for use in the pulp and paper industry and obtain building materials.

Common ash is one of the most valuable hardwoods in the European part of Russia. This is the main breed in protective forest belts, in forest crops. This culture deserves widespread adoption in forest cultures during the reconstruction of low-value young growth in green areas around settlements. In addition, the wood of ordinary ash is widely used in agricultural engineering, in carriage building, in furniture and carpentry.

Due to their biological characteristics, species and hybrids of the genus Salix have wide distribution ranges. Growing willow allows you to get raw materials for creating wicker products, extracting tannins and other biologically active substances, producing pulp and paper, as well as willow wood biomass is an excellent raw material for bioenergy. However, for successful creation of targeted willow plantations, both productive genotypes and effective technologies for the production of high-quality clonal planting material are necessary.

An analysis of the literature did not provide information on the methods of rooting ex vitro common ash, aspen and willow, despite the availability of information on other crops.

A known method of rooting ash in ex vitro "In vitro and ex vitro rooting of micropropagated shoots using three green ash (Fraxinus pennsylvanica) clones" (Kim, MS, Klopfenstein, NB, Cregg, BM // New Forests, 1998, 16:43 -57), which is the closest technical solution. In it, plants of Pencilvan ash were cultivated on an artificial nutrient medium for the animation of MS (Murashige, T. and Skoog, F. 1962. // A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473-497) , with the addition of sucrose 30 g / l, 6-benzylaminopurine (BAP) 5 μmol, thidiazuron 5 μmol and indolylbutyric acid 1 μmol, for 4 weeks in the light. At the end of the passage, the apical parts of the shoots 50 mm long were cut off and the base of the shoot 10 mm long was dipped for 15 seconds into a solution containing a high concentration of various auxins. The prepared shoots were placed in a special Polyterra® soil, after which the plants were covered with a film, where a high level of humidity was maintained. Its disadvantages are: the explants obtained at the stage of animation are very fragile (brittle), and when transplanted into special soil, part of the shoots are damaged and die; the plant preparation scheme is difficult to implement, since each plant is immersed in auxin solution for 15 seconds, during which the plant withers, a strong genotypic effect is observed.

The aim of the invention is to simplify the production scheme, improve the quality of shoots, eliminate the strong influence of the genotype to obtain a aligned planting material.

This goal is achieved due to the fact that after the completion of the animation stage, the plants of ash, aspen and willow are transferred to an artificial nutrient medium for elongation, including mineral salts of WPM medium (Lloyd, G. and B.N. McCown. 1980. // Commercially feasible micropropagation of mountain laurel, (Kalmia latifolia) by use of shoot tip culture. Int. Plant Prop. Soc, Comb. Proc, 30: 421-427), 30 g / l sucrose, 9 g / l agar-agar, 100 mg / l inositol, 0.1 mg / l pyridoxine, 0.1 mg / l thiamine, 0.5 mg / l nicotinic acid and 0.5 mmol / l glutamine. Cultivation at the elongation stage is carried out at an increased level of illumination - 5 thousand lux.

The essence of the invention lies in the fact that the proposed method, which consists in transferring plants to a growth medium for elongation, including mineral salts of WPM medium, 30 g / l sucrose, 9 g / l agar-agar, 100 mg / l inositol, 0.1 mg / l of pyridoxine, 0.1 mg / l of thiamine, 0.5 mg / l of nicotinic acid and 0.5 mmol / l of glutamine, and cultivation at an increased level of illumination (5 thousand lux), provides physiologically and morphologically aligned shoots of ash , aspen and willow, which, in turn, provides a significant increase in the effectiveness of their rooting I'm in ex vitro.

Analysis of known methods of preparing plants for rooting in ex vitro conditions, carried out according to scientific, technical and patent documentation, showed that the set of essential features of the proposed method is not known from the prior art, therefore, it meets the condition of patentability of the invention - "novelty".

The proposed method is implemented as follows.

1. In non-sterile conditions, a nutrient medium is prepared, including mineral salts of WPM, 30 g / l of sucrose and 9 g / l of agar-agar. The medium is bottled, corked with foil and paper, tied with a rubber band. Autoclaving is carried out at 1 atm. (= 1 atm.) For 20 minutes. In a laminar box that cooled down to 55 ° C, 100 mg / L of inositol, 0.1 mg / L of pyridoxine, 0.1 mg / L of thiamine, 0.5 mg / L of nicotinic acid and 0.5 mmol / L of glutamine are added. . The nutrient medium is poured into sterile culture vessels. All manipulations with plant material are performed under sterile conditions in a laminar box. At all stages of cultivation, the number of explants in the vessels is 15-25 pieces.

2. The elongation step is carried out on a nutrient medium, including WPM mineral salts, 30 g / l sucrose, 9 g / l agar-agar, 100 mg / l inositol, 0.1 mg / l pyridoxine, 0.1 mg / l thiamine 0.5 mg / l nicotinic acid and 0.5 mmol / l glutamine. Containers with plants are placed on light culture racks with a light level of 5 thousand lux.

Tables 1-4 present the results of studies on the effect of the method of preparing plants of ash, aspen and willow in vitro for subsequent rooting in ex vitro.

Rooting under ex vitro conditions accelerates and simplifies the process of clonal micropropagation. However, the rooting of microprobe of some cultures obtained in vitro is often associated with problems such as low adaptation efficiency, fragility of microprobe, physiological heterogeneity of adapted plants, etc. To increase the physiological maturity of plants, an increased level of illumination of cultivated plants is used. In the case of ash, the results of the study showed that an increase in the level of illumination to 5 thousand lux contributed to a significant increase in the survival rate and a slight increase in the height of plants of both ash genotypes. A further increase in illumination (up to 8 thousand lux) did not increase the frequency of survival, and the height of plants slightly decreased compared to illumination of 5 thousand lux. The level of illumination did not affect the number of roots (table 1). The level of illumination similarly affected the survival rate and morphological characteristics of aspen and willow.

Various studies have shown that glutamine, which is one of the key amino acids, when added to the culture medium has a beneficial effect on many cultures. According to our data, the addition of glutamine to the growth medium for elongation at a concentration of more than 0.5 mmol contributed to a significant increase in plant height and an increase in the number of roots. Plant survival in all cases was very high (table 2).

In an experiment on the effect of glutamine on the effectiveness of subsequent willow adaptation, it was shown that, with a glutamine content of 0.5-1.0 mmol, the survival rate increased to 100%, while the height of the plants increased significantly (table 3).

The advantage of the proposed method for the preparation of microprobe is to increase their quality at the stage of elongation, which is manifested in a higher frequency of survival of plants and improved growth characteristics. In addition, the method allows to obtain more aligned shoots and, as a result, a greater number of plants of a commercial appearance (table 4).

The method is applied in experimental production conditions. It allows you to increase the efficiency of operation of greenhouse areas by 15-20%. Received and planted more than 10 thousand microplants.

List of tables

1. Table 1. The influence of the level of illumination at the stage of elongation in vitro on the efficiency of adaptation of ash in ex vitro.

2. Table 2. The effect of glutamine in the elongation medium on the subsequent survival of aspen in ex vitro.

3. Table 3. The effect of glutamine in the elongation medium on the subsequent survival of willow in ex vitro conditions.

4. Table 4. Average results demonstrating the effectiveness of the introduction of the elongation stage in combination with increased illumination, in comparison with the control (without the elongation stage).

Claims (1)

A method of preparing in vitro microprobe shoots of ash, aspen, willow for subsequent rooting in ex vitro conditions, comprising transferring plants after the stage of multiplication to WPM elongation medium with the addition of sucrose 30 g / l, agar-agar 9 g / l, inositol 100 mg / l, pyridoxine 0.1 mg / l, thiamine 0.1 mg / l and nicotinic acid 0.5 mg / l, characterized in that glutamine is added to the elongation medium at a concentration of 0.5 or 1.0 mmol / l At the same time, the cultivation of plants is carried out at high illumination - from 5 to 8 thousand lux.