RU2302106C1 - Method for preparation of in vitro propagated strawberry (fragraria l) plants to ex vitro cultivation - Google Patents

Abstract

FIELD: biotechnology, agriculture, horticulture.

SUBSTANCE: strawberry (Fragraria L.) plants are propagated in vitro, hold on nutrient medium, containing 4-10 % of sucrose, for 20-60 days, at dark and at temperature of 1-10°C. Then prepared plants are transferred into soil substrate.

EFFECT: strawberry plants of increased productivity.

2 dwg, 2 tbl, 1 ex

Description

The invention relates to biotechnology and agriculture, namely to horticulture, and can be used in nursery for the preparation of plants propagated in vitro to ex vitro cultivation conditions.

Biotechnological systems of large-scale accelerated propagation in vitro of healthy planting stock suggest the intensive use of biologically active substances of cytokinin action (benzylaminopurin - BAP, etc.), known as drugs that delay the aging of plant leaves (Kulaeva ON "Cytokinins and their physiological effect" // "Successes of modern biology", 1967, T.63, No. 1, P.28-53; Kulaeva ON "Cytokinins, their structure and function", M .: "Science", 1973).

The use of 6-benzylaminopurine (BAP) for cultivating plants on artificial in vitro nutrient media in order to intensify plant propagation can lead to reenewalization of plant material and delay flowering of strawberry plants after transplanting them into the ground (Mohamed F., Swartz HJ, Buta JG "The role of abscisic acid and plant growth regulators in tissue culture-induced rejuvenation of strawberry ex vitro "// Plant cell, Tissue and Organ Culture, 1991, V.25 No. 1, pp. 75-84).

It is known that in the generative organs of plants there is an increased content of Sugars (Milyaeva E.L., Komarova E.N. “Sugar content in stem apexes of two-colored rudbeckia during the flowering evocation” // Plant Physiology, 1988, vol. 35, issue 1 , p.187-189; E.L. Milyaeva, E.N. Komarova, V.G. Kochankov. "Evolution of flowering and carbohydrate content in the stem apexes of Rudbeckia bicolor upon transition to flowering in complete darkness" // Plant Physiology, 1996 , Vol. 43, No. 2, pp. 299-302; BMLamp, JH Connel, RADuncan, M. Viveros, VSPolito. "Almond Flower Development: Floral Initiation and Organogenesis // J. Amer. Soc. Hort. Sci. 2001, V.126, No. 6, pp. 689-696).

In addition, after cultivation on nutrient media supplemented with high concentrations of sugars, the resistance of plant material to deep stresses increases significantly (Vysotskaya ON, Popov AS "Method for cryopreservation of meristems isolated from plants of strawberry garden (Fragaria L.) in vitro "/ Patent No. 2220563, C1, 2004).

There is a method of stimulating flowering plants in vitro. In this method, callus was obtained from the stems of flowering tobacco plants, which formed buds on a nutrient medium if the glucose concentration in it reached 3.5-6.5% (Konstantinova T.N., Bavrina T.V., Aksenova N.P., Golyanovskaya SA "The effect of glucose on the morphogenesis of stem calli of photoperiodically neutral tobacco Trapezoid" // Plant Physiology, 1972, V.10, Issue 1, pp. 89-98).

The disadvantage of this method is that it proposes to use reducing sugar: glucose, which is easily oxidized and which is preferably introduced into the nutrient medium through special filters after the autoclaving step (cold sterilization method). This method can be considered a similar solution to our task.

The closest technical solution is a method of converting in vitro propagated plants to non-sterile conditions, namely, that strawberry plants with or without roots immediately after in vitro cultivation (light, 23 ° C) in a nutrient medium containing 6-benzylaminopurine (0 , 5 mg / l), indolylbutyric acid (0.1 mg / l) and a standard amount of sucrose (30 g / l) were transferred to non-sterile conditions where these plants took root and adapted to non-sterile conditions (Bozena Borkowska Morphological and physiological characteristics of micropropagated strawberry plants rooted in vitro or ex vitro // Scientia Horticulturae, 2001, V.89, P.195-206).

The disadvantage of this method, we consider the absence of the effect of stimulation of the formation of generative organs in strawberry plants ex vitro.

The problem to which the invention is directed is such preparation of plants propagated in vitro, which allows them not only to successfully adapt to non-sterile conditions, but also quickly transfer ex vitro to the generative state.

The problem is solved in that before transferring to the soil substrate propagated in vitro plants are placed on an agarized nutrient medium with 4-10% sucrose, incubated for 20-60 days in the dark at a temperature of from 1 to 10 ° C.

The essence of the present invention is that the proposed method for preparing in vitro propagated garden strawberry plants (Fragaria L.) for ex vitro conditions, which consists in placing these plants on an agarized nutrient medium with 4-10% sucrose and keeping them for 20-60 days in the dark at a temperature of 1-10 ° C, it allows not only to prepare plants for transplantation into non-sterile conditions, but also to stimulate the vegetative and generative productivity of these plants ex vitro (table 1; table 2, appendix: figure 1, figure 2).

The invention is illustrated by the following example:

Example 1

Plants of strawberry garden (Fragaria L.) cultivar Roxana (Roxana) propagated in vitro on agarized nutrient medium Murashige and Skoog (Murashige T., F.Skoog. A revised medium for rapid growth and bio-assays with tobacco tissue cultures // Physiol. Plantarum, 1962, 15, P.473-497) supplemented with 6-benzylaminopurine (1 mg / L) and indolylacetic acid (0.1 mg / L) and 30 g / l sucrose.

Plants obtained as a result of propagation of plants with or without roots in vitro on an agarized nutrient medium Murashige and Skoog without indolylacetic acid and benzylaminopurine, supplemented with 4-10% sucrose, are placed for 20-60 days in the dark at a temperature of 1-10 ° C.

After this, the strawberry plants are transplanted into the soil mixture in the greenhouse, and then transferred to the field.

Of the plants planted in the soil mixture, more than 95% remained alive and not only mustaches, but also flowers were formed in the greenhouse before being transferred to the field conditions (Appendix, figure 2).

After being transferred to field conditions, these strawberry plants formed 55-56% more generative organs, and 38-77% more vegetative organs than those that were cultivated on medium with 3% sucrose before being transferred to non-sterile conditions (Table 1, Table .2).

After transferring to field conditions, these strawberry plants formed 67-68% more generative organs, and vegetative organs 43-92% more than those that were cultivated on medium with 3% sucrose before transferring to non-sterile conditions (Table 1, Table .2).

Table 1.
The effect of the preparation method for planting in vitro plants in the soil on the ex vitro generative productivity of wild strawberries (Fragaria L.), Roxane variety Option The average number of peduncles per plant The average number of generative formations per plant Control ^* 18.0 ± 2.2 (100%) 78.9 ± 9.8 (100%) Method ^** 28.0 ± 2.2 (156%) 122.2 ± 9.7 (155%) ^* Preparation of plants in vitro in an environment with 3% sucrose at 20-28 ° C and a 16-hour day. ^** Preparation of plants in vitro for 20-60 days in the dark at 1-10 ° C in an environment with 4-10% sucrose.

Table 2.
The influence of the method of preparation for planting in vitro plants in the soil on the vegetative productivity of ex vitro strawberry garden (Fragaria L.) Roxane cultivar. Option The average number of leaves Average number of mustache Average number of outlets 2003 2004 2003 2004 2003 2004 Control ^* 22.9 ± 2.8 (100%) 50.6 ± 5.5 (100%) 10.0 ± 1.3 (100%) - 24.1 ± 3.8 (100%) 34.5 ± 4.8 (100%) Method ^** 35.9 ± 3.0 (157%) 70.0 ± 7.4 (138%) 17.7 ± 1.5 (177%) - 37.3 ± 4.4 (155%) 47.6 ± 5.6 (138%) ^* Preparation of plants in vitro in an environment with 3% sucrose at 20-28 ° C and a 16-hour day. ^** Preparation of plants in vitro for 20-60 days in the dark at 1-10 ° C in an environment with 4-10% sucrose.

The technical result of the claimed method is that an in vitro stay of 20-60 days in the dark at a temperature of from 1 to 10 ° C in a nutrient medium with 4-10% sucrose stimulates the generative and vegetative productivity of in vitro propagated strawberry plants (Fragaria L.) in the first years of cultivation ex vitro (table 1, table 2) and even accelerates their flowering (figure 1, figure 2). This effect can be illustrated by the fact that, per one plant, the following indicators increase: the average number of peduncles by 56%, the average number of generative formations by 55%, the average number of leaves by 38-57%, the average number of whiskers by 77%, and the average number sockets at 38-55%.

Claims (1)

A method of preparing in vitro propagated plants of garden strawberry (Fragaria L.) for ex vitro cultivation conditions, comprising preparing in vitro plants, characterized in that the plants are incubated on a nutrient medium with 4-10% sucrose for 20-60 days in the dark at a temperature of from 1 to 10 ° C, and then transferred to the soil substrate.

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