RU2702765C2 - Microclonal propagation method of potatoes - Google Patents

Abstract

FIELD: agriculture.

SUBSTANCE: invention relates to the agriculture. For microclonal propagation of potatoes, propagation of tubed potato plants is performed in vitro using a hormone-free nutrient medium with pH 5.7–5.8, containing micro salts and macro salts according to Murashige and Skoog, Fe₂SO₄, CaCl₂, meso-inositol, thiamine, pyridoxine, nicotinic acid, saccharose, glucose, agar, biologically active substances of mycelial fungus Trichoderma lixii, with the following ratio of components: Macrosalt – 50 mg/l, Microsalt – 1 mg/l, Fe₂SO₄⋅7H₂O – 5.0 mg/l, CaCl₂⋅2H₂O – 440 mg/l, meso-inositol – 100 mg/l, thiamine – 1.0 mg/l, pyridoxine – 1.0 mg/l, nicotinic acid – 0.5 mg/l, acarose – 25 g/l, glucose – 25 g/l, agar – 6 g/l, biologically active substances of mycelial fungus Trichoderma lixii – 0.5 mg/l.

EFFECT: invention makes it possible to increase efficiency of reproduction of healthy pottyral potato plants.

1 cl, 2 tbl

Description

The present invention relates to agricultural biotechnology, and in particular to the technology of accelerated propagation of test tube potato plants in vitro. The invention can be used to improve the test tube potato plants in the production of seed-free virus-free planting material from meristem lines.

Potatoes (Solanum tuberosum var. Tuberosum) are grown everywhere in Russia, in almost all climatic zones. Relatively high productivity with a certain ease of cultivation allows the potato to consistently take the second place after cereal crops in domestic agricultural production.

An important feature of potatoes is that due to vegetative propagation, most of the diseases affecting it are transmitted through seed tubers, which are the primary source of infection for subsequent infection of plantings. Potatoes can be affected by diseases at all stages of the life cycle: before emergence, during the growing season and during storage. Many pathogens can also accumulate and persist for a long time in the soil.

Growing infection-free potatoes is a promising area of research today. Microclonal propagation of potatoes from meristemic lines allows you to get healed from viral, fungal and bacterial infections seed material.

Potato viral diseases are most often transmitted through seed material. During prolonged cultivation of sterile plants from meristem lines in vitro, contamination of the material with bacterial and fungal microflora often occurs. The reason for this is most often infected explants, less often the components of nutrient media; intra-laboratory transmission of the infection from air, from personnel, and from one cell culture to another is possible.

There is a method that reduces the infection of berry crops during prolonged cultivation from the apical meristems, which consists in including antibiotics, in particular tetracycline, in the nutrient medium, which reduces the contamination of individual microorganisms that accidentally hit the surface of the nutrient medium during transplants (Ivanov A.I., Prikhodko Yu.N., Prikhodko DP, Bogomolov AA “Improving the methodology for producing strawberry plants using the apical meristem culture method” / In the collection “Selection and agricultural technology of fruit growing x and fruit crops in the Middle Volga, Kuibyshevskoe book publishing house, 1989, p. 63-75) [1].

The disadvantage of this method is the restriction of the use of tetracycline for the production of a healthy test tube plant due to the fact that the antibiotic is active against only a few gram-negative bacteria. Microorganisms of most fungi are not resistant to tetracycline, which most often cause contamination of the nutrient medium during prolonged cultivation of sterile plants in vitro.

There is a method of introducing the nystatin antibiotic into the explant culture of strawberries and apple trees, the positive effect of its use with the zero effect of antibiotics macropen, cefipim, etc. (Buntsevich L.L., Paletskaya E.N., Kostyuk MA, Medvedeva N .I. “A study of the effectiveness of new generation antibiotics and sterilizers for suppressing bacterial and fungal contamination of the environment and explants” / Thematic online journal SKZNIISiV, 2012, No. 16 (4). - P. 10) [2].

The disadvantage of this method is the restriction of the use of the polyene antibiotic nystatin for the production of a healthy test tube due to the fact that the antibiotic is not active against bacteria. Nystatin is an antifungal, fungistatic agent.

It has been established that the morphological types of bacteria isolated from the culture medium of cherry and cherry plants are a mixture of various types of microorganisms. Studied 32 clones of contaminate and revealed that the sources of infection are rhizospheric, epiphytic and endophytic microflora of plants. Proceeding from this, antibiotics from the group of aminoglycosides with a wide antibacterial spectrum of action were used for decotamination of the nutrient medium (Bourgutin A.B., Feaktistov N.V., Punina N.V., Ignatov A.I. “Determination of the species affiliation of wood contaminating cultures plants in vitro I Zvenigorod, 2008. S. 80) [3].

The disadvantage of this method is the ability of microorganisms to have resistance to antibiotic substances of this series.

A known method of microclonal propagation of grapes in vitro, including microcentering of test plants and planting them on a nutrient medium Murashige and Skoog without growth substances and with a reduced content of macrocells, while planting and cultivation was carried out with the addition of indolylacetic acid 0.1-0.3 mg / l and emistima in a concentration of 10 ^-7 -10 ^-10 ml / l (RF patent No. 2264706 "Method for optimizing clonal micropropagation of grapes in vitro", A01H 4/00, A01N 25/00, C12N 5/00, 2005 ) [four]. Emistim is a bioregulator of plant growth and root formation, promotes the induction of rhizogenesis and elongation of cells of all tissues. As a result of its use, metabolic processes are optimized, and immunity in plants increases.

The disadvantage of this method is the limitation of the use of emistim for the production of a healthy test tube plant due to the fact that this bioregulator does not destroy bacterial infections that most often inhibit plants and lead to their death. Although at the same time its use for healing microcrops of grapes is not in doubt, since grapes are less likely to be infected with bacteria.

The closest in technical essence to the proposed is a method of preparing a nutrient medium for the propagation of test tube potato plants (patent KZ No. 25514 "Method for preparing a nutrient medium for the propagation of test tube potato plants", A01G 9/00 C05D 9/00, 2010) [5], including the rapid propagation of test tube potato plants in vitro using the optimized hormone-free nutrient medium Murashige and Skoog containing macrosalts 50 mg / l, microsols 1 mg / l, Fe ₂ SO ₄ 7H ₂ O 5.0 mg / l, CaCl ₂ ⋅ 2H ₂ O 440 mg / l, meso-inositol 100 mg / l, thiamine 1.0 mg / l, pyridoxine 1.0 mg / l, nicotinic acid 0.5 mg / l, sucrose 25 g / l, glucose 25 g / l, agar 6 g / l, pH 5.7-5.8 . The propagation of test tube potato plants from meristem lines was carried out by in vitro microcherenking. Test tube potato plants were removed from the test tube with tweezers and laid on a sterile mattress. The plant was cut with a scalpel into cuttings consisting of a piece of stem with a leaf and axillary bud. Cutting of test plants was carried out with an interval of 14 days. The grown plants with 5-6 cuttings were periodically cut and transplanted into test tubes with fresh nutrient medium Murasige and Skoog. Test plants were grown in a factorostat room at a temperature of 23-25 ° C and illumination of 7000 lux.

The disadvantage of this method is the lack of a component of the nutrient medium, which allows to reduce the contamination of fungal and bacterial microflora during prolonged cultivation of sterile potato plants from meristem lines in vitro.

The problem to which the invention is directed, is to increase the efficiency of reproduction of healthy test tube potato plants obtained from meristem lines in vitro using the optimized nutrient medium Murashige and Skoog.

The technical result is achieved by effective stimulation of root formation in vitro potato plants grown from meristem lines, and by complete decontamination of fungal and bacterial microflora.

The specified task and technical result is achieved by the fact that in the method of microclonal propagation of potatoes, including the propagation of test tube potato plants in vitro using the optimized hormone-free nutrient medium Murashige and Skoog containing macrosalts, microsols, Fe ₂ SO ₄ , CaCl ₂ , meso-inositol, thiamine , pyridoxine, nicotinic acid, sucrose, glucose, agar, pH 5.7-5.8, according to the invention, biologically active substances of the mycelial fungus Trichoderma lixii (GenBank: JQ350879.1) are additionally introduced into the nutrient medium in the following component ratio m: makrosoli 50 mg / l, mikrosoli 1 mg / L, Fe ₂ SO ₄ ⋅7H ₂ O 5.0 mg / l, CaCl ₂ ⋅2H ₂ O 440 mg / L, meso-inositol 100 mg / l, thiamine 1.0 mg / l, pyridoxine 1.0 mg / l, nicotinic acid 0.5 mg / l, sucrose 25 g / l, glucose 25 g / l, agar 6 g / l,

biologically active substances of the mycelial fungus Trichoderma lixii (GenBank: JQ350879.1) 0.5 mg / l, pH 5.7-5.8.

A distinctive feature of the proposed method is the introduction of a nutrient medium for the propagation of test tube plants of potato biologically active substances of the mycelial fungus Trichoderma lixii (GenBank: JQ350879.1) at a concentration of 0.5 mg / l, which provide decontamination from undesirable fungal and bacterial microflora of cultivated plant material and enrichment with growth factors.

It is proved that the biologically active substances of Trichoderma mycelium fungi are compounds of a non-peptide nature, without amide bonds, with immunomodulatory, growth-promoting properties (Pavlovskaya N.E. Influence of secondary metabolites of Trichoderma fungi on the sowing quality of pea seeds / N.E. Pavlovskaya, I.A. Gneusheva, I.Yu. Solokhina, I.V. Yakovleva // Agricultural Biology. 2012. No. 3. S.114-117) [6], bactericidal and fungicidal action against a number of phytopathogens widely distributed in vegetable crops , at number of potatoes (bacterial - Clavibacter michiganensis subsp. Michiganensis (causative agent of bacterial tomato cancer), Pseudomonas syringae pv. tomato (causative agent of bacterial spotting of tomato fruits), Xanthomonas euvesicatoria (causative agent of tomato bacterial spotting); cucumber), Didymella lycopersici (anamorph: Phoma lycopersici) (causative agent of stem rot of tomato), Phoma destructiva (causative agent of fomose rot of tomato), Fusarium oxysporum (causative agent of fusarium rot of root neck and tomarium rhizomeumata (tomato root), causative agent of fusarium rot of cucumber roots) (Gneusheva I.A. Antagonistic potential of Trichoderma spp. Strains against pathogens of fungal and bacterial plant diseases / I.A. Gneusheva, I.Yu. Solokhina, N.E. Pavlovskaya, A.V. Lushnikov // Materials of the All-Russian Scientific and Practical Conference "Food Security from Dependence to Independence", Orel. - November 29, 2017 in print) [7].

The method of microclonal propagation of potatoes is as follows.

Potato plants (explants) are selected, regenerated from apical meristems 0.1-0.2 mm in size, not infected with viral, bacterial and fungal infections. Plants should have 8-10 internodes.

Then prepare a solid optimized hormone-free nutrient medium Murashige and Skoog, containing 50 mg / l macro salts, 1 mg / l micro salts, Fe ₂ SO ₄ ⋅ 7H ₂ O 5.0 mg / l, CaCl ₂ ⋅ 2H ₂ O 440 mg / l, meso-inositol 100 mg / l, thiamine 1.0 mg / l, pyridoxine 1.0 mg / l, nicotinic acid 0.5 mg / l, sucrose 25 g / l, glucose 25 g / l, agar 6 g / l . pH of the medium before autoclaving 5.7-5.8. The medium is autoclaved in an autoclave under a pressure of 1.0 atm, a temperature of 110 ° C for 10 minutes.

The biologically active substances of the mycelial fungus Trichoderma lixii (GenBank: JQ350879.1) at a concentration of 0.5 mg / l are added under laminar box conditions to the nutrient medium cooled to + 55 ° C after autoclaving, which ensures their maximum effect. Then the medium is poured into test tubes.

Potato plants (explants) regenerated from the apical meristems are removed from the test tube with tweezers and placed on a sterile Petri dish. The plant is cut with a scalpel into cuttings consisting of a piece of stem with a leaf and axillary bud. The length of the microcranium is 10-12 mm, 2 mm above the eye, the rest under the eye.

The resulting micro-stems are planted in test tubes on a solid optimized hormone-free nutrient medium Murashige and Skoog. Cultivation is carried out in a culture room with illumination of 2.0-3.0 thousand lux, a photoperiod of 16 hours, a temperature of 23-25 ° C and an air humidity of 70-75%.

Evaluation of morphometric indicators and the presence of contaminants in grown plants with 5-6 cuttings is carried out with an interval of 14 days. Then transplanted into test tubes with fresh nutrient medium.

In the course of experimental studies, it was found that the claimed method promotes the propagation of healthy test tube plants of the Golubizna potato variety obtained from meristem lines in vitro using the optimized nutrient medium Murashige and Skoog with biologically active substances of the mycelial fungus Trichoderma lixii (GenBank: JQ350879 .1) at a concentration of 0.5 mg / L. The results of the study are shown in tables 1, 2.

As a result of microclonal propagation, 614 samples of healthy test tube potato plants from meristem lines free of viral, fungal, and bacterial infections were obtained.

Thus, it is the hallmark of the proposed method convincingly shows decontamination from undesirable fungal and bacterial microflora of cultivated plant material and stimulation of their growth and development.

Claims (2)

The method of microclonal propagation of potatoes, including the propagation of test tube potato plants in vitro using a hormone-free nutrient medium with a pH of 5.7-5.8 containing microsols and macrosalt according to Murasiga and Skoog, Fe ₂ SO ₄ , CaCl ₂ , meso-inositol, thiamine , pyridoxine, nicotinic acid, sucrose, glucose, agar, characterized in that the biologically active substances of the mycelial fungus Trichoderma lixii are additionally introduced into the nutrient medium in the following ratio of components: macrosalts 50 mg / l microsalts 1 mg / l Fe ₂ SO ₄ ⋅ 7H ₂ O 5.0 mg / L CaCl ₂ ⋅H ₂ O 440 mg / l meso-inositol 100 mg / l thiamine 1.0 mg / L pyridoxine 1.0 mg / L a nicotinic acid 0.5 mg / l sucrose 25 g / l glucose 25 g / l agar 6 g / l biologically active substances mycelial fungus Trichoderma lixii 0.5 mg / l