RU2827225C1 - Method for growing lingonberry (vaccinium vitis-idaea linnaeus) - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention represents a method for cultivation of lingonberry. Method includes planting an explant on a growing medium, introducing the explant into a culture in vitro to obtain regenerated plants, their micro-cutting and rooting of micro-cuttings in a growing medium containing carbohydrates, subsequent adaptation of plants to non-sterile conditions ex vitro, characterized by that the explants are sterilized in a solution of silver nitrate 0.2% for 10 minutes, as a source of carbohydrates in the growing medium, glucose is used in amount of 20.0 g/l of the growing medium, at the stage of micropropagation proper, Cytodef is added to the growing medium in concentration of 0.1 mg/l; at the stage of microshoots rooting in the culture in vitro, IBA in concentration of 1.0 mg/l and birch charcoal in amount of 5.0 g/l are added to the growing medium, plants are adapted in vitro to soil conditions ex vitro, using a substrate of a mixture of transitional peat and sphagnum tirr in ratio of 3:1.

EFFECT: invention ensures increase of lingonberry plants reproduction factor at reduction of labour costs for production of seedlings.

1 cl, 5 tbl

Description

The invention relates to the field of agriculture, forestry and biotechnology and can be used for accelerated biological production of improved planting material by microclonal propagation for the purpose of subsequent establishment of berry plantations.

There are known classical methods of obtaining planting material for lingonberries: it can be propagated both by seeds and by vegetative-rhizome cuttings, lignified and green stem cuttings, partial bushes with part of the rhizomes, and rarely by pieces of rhizomes. The disadvantages of these methods are: a small yield of planting material, seasonality in the work (limited time for harvesting material), the need to maintain high humidity for 4 weeks for the adaptation of cuttings, which leads to an increase in the costs of obtaining a large volume of planting material and an increase in the cost of the technology (Holloway P. Rooting of Lingonberry, Vaccinium vitis-idaea, Stem Cuttings // Plant. Propagator. 1985. Vol. 31. No. 4. P. 7-9; Sidorovich E.A., Ruban H.N., Sherstenikina A.V. Introduction and experience of growing large-fruited cranberry, highbush blueberry and lingonberry // Review information. Minsk: BelNIINTI, 1991. P. 1-53; Tyak G.V., Cherkasov A.F., Altukhova S.A. Experience of growing lingonberry in the conditions of the Kostroma region // Issues of use and restoration of wood and non-wood resources of the southern taiga forest. Moscow, 1998. pp. 50-63; Morozov O.V. Culture of common lingonberry (Vaccinium vitis-idea L): problems and prospects: monograph. Minsk: Belarusian science, 2008. 150 p; Tyak G.V., Kurlovich L.E., Makarov S.S. et al. Reproduction of promising hybrid forms of common lingonberry (Vaccinium vitis-idaea L.) // Bulletin of the Belarusian State Agricultural Academy named after V.R. Filippov. 2022. No. 1 (66). pp. 113-118).

However, to obtain purebred and healthy planting material free from viral and fungal infections, the method of clonal micropropagation of plants in vitro culture is used, which allows in the shortest possible time to obtain a large number of viable plants intended both for gardeners and for industrial cultivation (Kalinin F.L., Kushnir G.P., Sarnatskaya V.V. Technology of microclonal propagation of plants. Kyiv: Naukova Dumka, 1992. 232 p.; Butenko R.G. Biology of cells of higher plants in vitro and biotechnology based on them. Moscow: FBK-Press, 1999. 160 p.; Shevelukha B.S. et al. Agricultural biotechnology and bioengineering: textbook. Moscow: URSS, 2015.715 p.).

A method of clonal micropropagation of lingonberry is known (patent BY 991 C1, A01 H 4/00, 15.12.1995. Sidorovich E.A., Kutas N.N. Method of propagation of Vaccinium vitis-idaea L. in vitro) using a modified Anderson nutrient medium with the addition of 2-isopentenyladenine and indyl acetic acid, but this method is aimed at obtaining a callus culture. In this case, there is a specific reaction of the plant to the introduction of certain growth regulators into the medium, depending on the variety, which leads to a positive effect only on a particular crop or variety. In turn, this contributes to a decrease in the value of the development and leads to the loss of its universality for other varieties (Sidorovich E.A., Kutas E.N. Clonal micropropagation of introduced varieties of highbush blueberry and common lingonberry in vitro culture in connection with genotypes // Bulletin of the National Academy of Sciences of Belarus Series of Belarusian Science. 1998. No. 3. pp. 5-9; Bozhiday T.N., Kukharchik N.V. Influence of genotype and auxin on the process of ex vitro rhizogenesis of common lingonberry (Vaccinium vitis-idaea L.) varieties // Biotechnology in fruit growing: materials of the International Scientific Conf. (Samokhvalovichi, June 13-17, 2016). Samokhvalovichi, 2016. pp. 99-101).

The closest to the invention in terms of the set of essential features are the methods of clonal micropropagation of lingonberry using a nutrient medium according to Anderson's prescription (1975) with the addition of growth regulators of cytokinin, auxin and phenolic nature (Gajdosova A., Ostrolucka M.G., Libiakova G., Ondruskova E. Protocol for Micropropagation of Vaccinium vitis-idaea L. // Protocols for Micropropagation of Woody Trees and Fruits / M. Jain, H. Haggman (eds.). 2007. P. 457-464). However, the disadvantages of this method include an insufficiently high reproduction coefficient at the stage of shoot proliferation and an insufficient level of rooting and development of the root and aboveground systems of plants at the stage of rooting in vitro culture. This leads to a decrease in the yield of planting material and an increase in the chain of the technological cycle of its production.

The claimed invention is aimed at solving the problem of: increasing the multiplication coefficient and rooting percentage of micro shoots; reducing the costs of obtaining standard planting material; increasing crop yield.

The technical result of the proposed invention consists in increasing the reproduction rate of lingonberry plants while reducing labor costs for growing seedlings.

To solve the specified problem and achieve the stated result in the method of growing lingonberries, including planting an explant on a nutrient medium, introducing it into an in vitro culture to obtain regenerated plants, their microcutting and rooting the microcuttings in an in vitro culture on a nutrient medium containing carbohydrates, and subsequent adaptation of the plants to non-sterile ex vitro conditions, in accordance with the proposed technical solution:

- explants are sterilized in a solution of _AgNO3 0.2% for 10 minutes;

- glucose is used as a source of carbohydrates in the nutrient medium in an amount of 20.0 g/l of the nutrient medium;

- at the stage of micropropagation itself, Cytodef is added to Anderson’s nutrient medium at a concentration of 0.1 mg/l;

- at the stage of rooting of microshoots in vitro culture, indolebutyric acid 1.0 mg/l and birch charcoal 5.0 g/l are added to the nutrient medium;

- at the stage of in vitro plant adaptation to ex vitro soil conditions, a substrate made of a mixture of transitional peat and sphagnum moss in a ratio of 3:1 is used.

The advantages of the proposed method, explaining the essence of the invention, are presented in Tables 1-5.

Information confirming the possibility of implementing the method

Examples of specific implementation

Sterilization of plant material. To sterilize stems, buds, and other plant fragments intended for explant isolation, use aqueous solutions of 5% hydrogen peroxide, 0.2% silver nitrate, 5% disinfectant Lysoformin 3000, 0.01% disinfectant Nika-2, disinfectant Domestos in a 1:3 dilution. Before sterilization, plant stems are thoroughly washed with a brush and soap in warm running water, rinsed with bidistilled water and placed in absolute alcohol for 5 seconds, and then in the main sterilizing solution for 5-15 minutes.

Stage of introduction into in vitro culture. Virus-free planting material for clonal micropropagation of plants can be obtained by the method of culture of isolated apical meristems, which are the healthiest, virus-free part of the plant and represent a cone of actively dividing cells 0.1 mm high (100 μm) and 0.25 mm wide. At the stage of introduction into culture, the explant is placed on a modified Anderson nutrient medium (Table 1) with the addition of the growth regulator Cytodef at a concentration of 0.1 mg/l. Regenerated plants obtained from apical meristems are propagated by microcuttings.

As a result of the research, it was revealed that at the stage of introduction into the in vitro culture of lingonberry, the most effective main sterilizing agent was a 0.2% solution of silver nitrate with an exposure of 10 minutes, where the maximum viability of explants was revealed (90%) (Table 2).

Clonal micropropagation by cuttings. When micro plants are cultivated on a nutrient medium with the addition of cytokinin 6-BAP, apical dominance is removed, which leads to the activation of the development of axillary meristems, which subsequently develop into microshoots. The resulting adventitious microshoots are separated from the mother explant and independently cultivated on a new nutrient medium. To increase the multiplication coefficient, microcutting is used - dividing shoots into cuttings containing one or two axillary buds.

In a laminar flow hood, remove the original plants from the culture vessel and place them on a sterile mattress. Using a scalpel and tweezers, divide the lingonberry plants into micro-cuttings with 2-3 internodes and transplant to a 1/2 WPM nutrient medium with the addition of a growth regulator Cytodef 0.1 mg/l. The pH of the medium should be 4.0...4.5. After this, the neck of the vessel must be burned over the flame of an alcohol lamp, covered with cling film and placed in a light room with illumination of 2500-3000 lux, a 16-hour photoperiod, a temperature of +25°C and air humidity of 70%. Cultivation is carried out for 25-30 days. The biomaterial obtained at the end of the passage is again used for further reproduction in vitro.

It was established that at the stage of micropropagation itself, the highest values of the total length of lingonberry microshoots (on average 6.3 cm) were detected on Anderson's nutrient medium with the addition of the growth regulator Cytodef at a concentration of 0.1 mg/l (Table 3).

The process of in vitro root formation. At this stage, it is necessary to create the most favorable composition of the nutrient medium, ensuring the receipt of a high percentage of rooted microshoots. For this purpose, at the last stage of clonal micropropagation, the concentration of mineral salts, sucrose is reduced, and cytokinins are excluded from the nutrient medium. The main regulatory factor of root formation is the presence of auxins in the nutrient medium. IMC or IAA in concentrations from 1.0 to 5.0 mg / l are most often used for this. The choice of hormone and its concentration depends on the species and varietal characteristics of the plants under study. Rooted microshoots are planted in soil conditions for adaptation.

In a laminar flow cabinet, remove the original plants from the culture vessel and place them on a sterile mattress. Using a scalpel and tweezers, divide the plants into micro-cuttings 1.0-1.5 cm long with two internodes, removing the lower leaves and transplant them to a nutrient medium containing auxin IMC at a concentration of 0.5-1.0 mg/l. After this, the neck of the vessel must be burned over the flame of an alcohol lamp, covered with cling film and placed in a light room with illumination of 2500-3000 lux, a photoperiod of 16/8 h, a temperature of +25°C and a relative humidity of 80%. Cultivation is carried out for 30-50 days. The biomaterial obtained at the end of the passage is again used for further reproduction in vitro.

It was revealed that at the stage of rooting of in vitro lingonberry microshoots, the highest values of the total root length (14.2 cm) were noted on Anderson's nutrient medium with the addition of IMC at a concentration of 1.0 mg/l and birch charcoal in an amount of 5.0 g/l (Table 4).

Adaptation of in vitro plants to non-sterile ex vitro conditions. The most favorable time of year for adapting test-tube plants to soil is considered to be the period from the 2nd decade of April. Then plants with a well-developed root system with 2-3 leaves are able to adapt to ex vitro conditions. The soil substrate is pre-watered with a 5% solution of potassium permanganate and left for 14 days in a dark place. For forest berry crops, high-moor or transitional peat and sand (3:1) are used as a substrate and mulched on top with sphagnum moss, since moss is a good antiseptic and moisture retainer. The prepared substrate is laid out in cassettes or microgreenhouses, into which the in vitro plants are transplanted. The temperature should be +25°C, humidity - 80 ... 90%. For successful and 100% survival rate, it is advisable to use a fogging unit (usually used on an industrial scale). For small batches of plants, it is advisable to use micro greenhouses or use individual plant closures in polyethylene cups for 14-20 days, which are then removed. As the plants grow, they are transplanted into larger containers with fresh substrate. Their further cultivation is carried out according to the accepted agricultural technology for this type of plant.

In vitro plants with a well-developed root system are removed from the test tube with tweezers. The roots are washed in a 1% solution of potassium permanganate (faint pink color) to prevent the development of pathogenic microflora. The plants are transplanted into cassettes with soil, pressed down on the soil and watered. After that, the cassettes are placed in lighting conditions (5000 lux). Every day, it is necessary to spray the plants with water for 1 week. After 10 days, conduct the first revision of the plants.

It was noted that the composition of the substrate had different effects on the survival rate, shoot length and number of lingonberry leaves. The best survival rate (100%) of plants was achieved in the variant using a mixture of transitional peat and sphagnum toxin in a ratio of 3:1, while on transitional peat this indicator was 80%, on a mixture of peat with sand 3:1 - 75%, on a mixture of peat with perlite 3:1 - 65% (Table 5).

Technical and economic advantages or other effectiveness of the invention compared to the prototype

This technology for obtaining lingonberry (Vaccinium vitis-idaea L.) planting material can be used for mass production of improved planting material with subsequent establishment of mother plants, nurseries and variety plots. The proposed method involves the use of plant meristems as explants, which are transplanted to a modified nutrient medium with the addition of glucose, birch charcoal, as well as cytokinins (Cytodef) and auxins (IMK). This method allows increasing the reproduction rate of lingonberry plants by 5 or more times, its adaptation to non-sterile conditions using a substrate made of a mixture of transitional peat and sphagnum toxin (3:1) up to 100%, and also reducing labor costs in growing lingonberry seedlings by 2 times.

Claims (1)

A method for growing common lingonberry, including planting an explant on a nutrient medium, rooting in vitro culture to obtain regenerated plants, their microcutting and rooting the microcuttings in vitro culture on a nutrient medium containing carbohydrates, subsequent adaptation of the plants to non-sterile conditions ex vitro, characterized in that the explants are sterilized in a 0.2% silver nitrate solution for 10 minutes, glucose is used as a source of carbohydrates in the nutrient medium in an amount of 20.0 g / l of the nutrient medium, at the stage of actual micropropagation, Cytodef is added to the nutrient medium at a concentration of 0.1 mg / l, at the stage of rooting microshoots in vitro culture, IMC is added to the nutrient medium at a concentration of 1.0 mg / l and birch charcoal in an amount of 5.0 g / l, the plants are adapted in vitro to the soil conditions ex vitro, using a substrate from a mixture of transitional peat and sphagnum moss in a ratio of 3:1.