RU2834363C1 - Method for obtaining aseptic culture in vitro of hardwood plants of middle and senior classes of age - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology and can be used for propagation of hardwood plants of middle and senior age classes in conditions of tissue culture. Method of clonal micropropagation involves harvesting branches from mother woody plants of middle and senior age classes, forcing etiolated shoots, selection of primary explants, their sterilization, cultivation on a nutrient medium based on MS macro- and microsalts, sterilization of primary explants in 1% working solution of a disinfectant based on sodium hypochlorite (with active chlorine content of 5-15%) and 5% solution of Lysoformin 3000 and culturing explants on a sterile nutrient medium for 15-20 days.

EFFECT: method for in vitro introduction of hardwood plants of middle and senior age classes enables to obtain regenerated plants for their subsequent reproduction in order to preserve the gene pool of the initial parent forms and expand the collection fund.

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Description

The invention relates to the field of biotechnology and can be used for mass propagation of woody plants under tissue culture conditions.

The method of clonal micropropagation allows preserving the characteristics of the plant's mother organism.

For introduction into aseptic culture of deciduous trees, tissues at the juvenile stage of development with relatively easy regeneration are used. Introduction into culture of cells and tissues of plants belonging to the middle and senior age class of trees that have reached physiological maturity is limited by low regeneration and the ability to cell division and is accompanied by a large loss of the original material. The obtained regenerants of plants of the middle and senior age classes can be cultivated in an aseptic environment to preserve the gene pool and for further multiplication.

A method for growing poplar planting material is known (patent RU 2485755, IPC A01G 1/00, published 27.06.2013), including harvesting shoots, sterilizing them, inducing the development of the main axillary shoot on primary explants, rooting isolated shoots, multiplying them in vitro and transferring them to non-sterile conditions, characterized in that micro-cuttings are carried out on a WPM nutrient medium - Woody Plant Medium, with a reduced content of BAP - benzylaminopurine 0.2 mg / l and the addition of GC - gibberellic acid 0.2 mg / l, and planting test-tube microplants in non-sterile conditions is carried out without their preliminary adaptation to the conditions of the climatic chamber.

The disadvantage of this method is the complexity and labor intensity of the process; the method is only applicable to fast-growing crops with high shoot-forming capacity.

A method for forming a collection and long-term deposit of grapes in vitro is known (patent RU 2764104, IPC A01H 4/00, published on 13.01.2022), including introducing apical meristems into the culture and forming a collection based on them, with preliminary cultivation on a solid Murashige-Skoog (MS) nutrient medium, planting for storage, characterized in that apical meristems of 0.1-0.2 mm in size, isolated from test-tube plants previously introduced into the culture, are maintained on a nutrient medium with the addition of the Melafen growth regulator at a dilution of 10 ^-5 -10 ^-11 , and the resulting shoots are maintained on a Murashige-Skoog nutrient medium containing Cefotaxime at a concentration of 50-450 mg / l, and microcuttings of healthy plants isolated from the upper parts of shoots.

The disadvantage of this method is the introduction of meristems of test-tube plants from a laboratory collection into the in vitro culture, which increases the risk of mutation of the regenerants.

The closest to the claimed solution, accepted as a prototype, is the method of clonal micropropagation of walnut (USSR patent No. 1706478 A1, published on 23.01.1992), including the isolation of an explant from the mother plant, its planting on Murashige-Skoog (MS) nutrient medium and cultivation until a shoot is obtained, characterized in that, in order to increase the yield of regenerated plants, seedlings or grafted saplings are used as the mother plant, while the explants are isolated from etiolated herbaceous shoots of the mother plant, previously grown in the dark.

The disadvantage of this method is the use of seed-based source material, which is not a clone and does not ensure the preservation of the characteristics of the mother plant, and the complexity and labor-intensive nature of the preliminary grafting process.

The objective of the invention is to develop a method for obtaining an aseptic in vitro culture of explants regardless of the season, obtaining genetically identical regenerating clones from woody plants of middle and senior age classes. The technical result of the proposed invention coincides with the objective.

To solve the problem, a method is proposed for obtaining an aseptic in vitro culture of woody plants of deciduous species of middle and older age classes during the period of vegetation or dormancy of plants, including the isolation of an etiolated shoot of the cloned plant, planting on an MS nutrient medium in a vertical position and culturing with passaging of the explant at intervals of 14 days until a viable regenerant is obtained. During the growing season, the induction of etiolated shoots is carried out in complete darkness from dormant buds from trunk segments or skeletal branches of woody plants with subsequent step-by-step sterilization in a 1% working solution of a disinfectant based on sodium hypochlorite (with an active chlorine content of 5-15%) for 30 minutes and a 5% solution of Lysoformin 3000 for 10 minutes, and cultivated on an MS nutrient medium with a half composition of macro- and microsalts with the addition of gibberellic acid 0.4 mg / l, and during the dormant period, the induction of etiolated shoots is carried out in the dark from vegetative buds, sterilized and cultivated on an MS nutrient medium with a full composition of macro- and microsalts with the addition of ferulic acid 1 mg / l, 6-benzylaminopurine 1 mg / l, indoleacetic acid 0.5 mg/l, kinetin 0.5 mg/l.

The method is implemented as follows:

Example 1. The harvesting of trunk segments or large skeletal branches of middle-aged or older trees of nine deciduous species from the Arboretum of the Northern (Arctic) Federal University named after M.V. Lomonosov ( Crataegus Russanwvii Cin. (72 years old), C. douglasii Lindl (39 years old), C. dahurica Koehne ex Schneid (73 years old), C. chlorosarca Maxim. (71 years old), Acer ukurunduense Trautv. & CAMey. (43 years old), A. tegmentosum Maxim. (44 years old), Juglans mandshurica Maxim. (36 years old), Prunus maackii Rupr. (68 years old), Syringa amurensis Rupr. (40 years old)) was carried out during the growing season and placed in a dark room. Within 7-15 days, etiolated shoots developed from dormant buds at a temperature of 21-23°C, which were used as primary explants.

Sterilization of primary explants was performed in two stages. The first stage was performed in non-sterile conditions, placing etiolated shoots in a 1% working solution of a disinfectant based on sodium hypochlorite with an active chlorine content of 5-15% for 30 minutes. In the second stage, under aseptic conditions of a laminar flow hood, the explants were moved into a 5% solution of Lysoformin 3000 for 10-15 minutes, after which they were washed three times with sterile distilled water.

Sterilized explants were divided into segments with 1-2 axillary buds under aseptic conditions and placed in a vertical or slightly inclined position in culture vessels with a half composition of macro- and microsalts (analytical grade - CHDA) of the MS nutrient medium with the addition of gibberellic acid (GA) 0.4 mg/l. Addition of GA to the nutrient medium promotes elongation (further growing) of induced shoots. After 15-20 days, the shoots were transplanted to a medium with a full concentration of micro- and macrosalts for cultivation.

Example 2. During the dormant period, large branches with a diameter of 1-4 cm were harvested from middle-aged and older trees and placed in a dark room at a temperature of 21-23°C. Over the course of 7-15 days, etiolated shoots developed from vegetative buds and were used as primary explants.

Sterilization of primary explants was performed in two stages. The first stage of sterilization was performed under non-sterile conditions in a 1% disinfectant solution of sodium hypochlorite for 30 minutes. At the second stage, under aseptic conditions of a laminar flow hood, the explants were moved into a 5% solution of Lysoformin 3000 for 10-15 minutes, after which they were washed three times with sterile distilled water.

Sterilized explants were divided under aseptic conditions into segments with 1-2 axillary buds, placed in a vertical or slightly inclined position in culture vessels with MS nutrient medium with a complete composition of macro- and microsalts (analytical grade) with the addition of ferulic acid 1 mg/l, 6-benzylaminopurine 1 mg/l, indoleacetic acid 0.5 mg/l, kinetin 0.5 mg/l.

Within 15-20 days from the start of cultivation, shoots were formed in the culture vessel, characterized by dynamic growth in height with normally developed leaf blades, and no signs of somaclonal variability. Further passaging under sterile conditions was carried out after 30 days to reduce the likelihood of infection of the original explants with fungal diseases. During the observations, the presence of infection by pathogens was periodically visually monitored.

Only viable explants were considered in the experiment. During the morphogenesis of primary explants, the ability to regenerate (form callus) and the development of active microshoots were determined. The results of the experiments are given in Table 1.

Table 1 - Results of introducing etiolated shoots into aseptic culture

Species name Etiolated shoots from dormant buds, % Etiolated shoots from vegetative buds, % regenerating micro shoots regenerating micro shoots Crataegus Russanwvii Cin. 100 100 100 90 C. douglasii Lindl 100 85 100 80 C. dahurica Koehne ex Schneid 100 95 100 90 C. chlorosarca Maxim. 100 80 100 80 Acer ukurunduense Trautv. & C.A.Mey. 100 40 100 42 A. tegmentosum Maxim. 100 100 100 67 Juglans mandshurica Maxim. 100 33 100 26 Prunus maackii Rupr. 100 95 100 90 Syringa amurensis Rupr. 100 45 100 55

The results of the experiment presented in the table show that all viable explants are capable of regeneration and form callus tissue. 33-100% of explants obtained from dormant buds and 26-90% of explants obtained from vegetative buds are capable of morphogenesis and form microshoots suitable for further cultivation.

The efficiency of introducing etiolated shoots of woody plants belonging to the middle and senior age classes into aseptic culture is associated with the content of a large number of growth regulators in the tissues and a low content of phenolic compounds, which ensures a high capacity for regeneration.

The use of etiolated shoots of middle-aged and older deciduous woody plants as primary explants and sterilization of explants with non-aggressive sterilizing agents ensures high preservation of plant tissues and eliminates the need for daily multiple passages of explants on a nutrient medium. The resulting regenerant shoots continue to grow, unlike standard methods of introduction, where the bud only opens the covering scales, but the shoot does not develop further.

The proposed method of introducing explants of middle-aged and older woody plants into in vitro culture allows obtaining regenerated plants for their subsequent propagation in order to preserve the gene pool of the original maternal forms and expand the collection fund.

Claims (1)

A method for obtaining an aseptic in vitro culture of woody plants of deciduous species of middle and senior age classes during the growing season or dormancy of plants, including the isolation of an etiolated shoot of the cloned plant, planting it on an MS nutrient medium in a vertical position and culturing with passaging of the explant at intervals of 14 days until a viable regenerant is obtained, characterized in that the induction of etiolated shoots is carried out in complete darkness during the growing season from dormant buds from segments of the trunk or skeletal branches of woody plants with subsequent step-by-step sterilization in a 1% working solution of a disinfectant based on sodium hypochlorite with an active chlorine content of 5-15% for 30 minutes and a 5% solution of Lysoformin 3000 for 10 minutes and culturing on an MS nutrient medium with a half composition of macro- and microsalts with the addition of gibberellic acid 0.4 mg / l, and during the period dormancy induction of etiolated shoots is carried out in the dark from vegetative buds, sterilized and cultivated on MS nutrient medium with a complete composition of macro- and microsalts with the addition of ferulic acid 1 mg/l, 6-benzylaminopurine 1 mg/l, indoleacetic acid 0.5 mg/l, kinetin 0.5 mg/l.