RU2092036C1 - Method of micromultiplication of stevia, stevia rebaudiona l - Google Patents

Abstract

FIELD: agriculture, biotechnology. SUBSTANCE: method involves the use of terminal and/or axillary buds with lower stem tissues (size is 1-3 mm) as explant followed by sterilization with 0.1% dioxide solution for 3 min and 0.1% mercury dichloride solution for 2.5 min, culturing for 4-5 weeks on nutrient medium Murasige-Skoog containing phytohormones, indolylacetic acid 0.1-0.3 mg/l, benzyladenine 0.5-2.0 mg/l, gibberellic acid 0.5-2.0 mg/l up to formation of the rooted plant-regenerants, nonrooted regenerants and callus tissue. Then the rooted plants were replanted in soil and the nonrooted regenerants were micrografted and transferred for rooting on the nutrient medium Murasige-Skoog containing 2-6 mg/l indolylacetic acid and 1-2 mg/l gibberellic acid. Callus tissue is placed on the parent nutrient medium for the further culturing up to regenerants obtaining. EFFECT: improved method of micromultiplication. 2 tbl, 7 dwg

Description

 The invention relates to the field of biotechnology, and in particular to methods of propagation of Stevia (Stevia rebaudiana L.) using tissue culture in vitro, which can be used for the needs of the food and medical industries.

 A known method of micropropagation of stevia. In this method of micropropagation, stevia isolates the explant, sterilizes it, cultivates it on a nutrient medium containing macro- and micronutrients, vitamins, sucrose, agar and phytohormones, obtain regenerants, micro-cut them, grow, root and transplant regenerated plants into the ground.

 Micropropagation by this method is based either on an increase in the central shoot and its further cuttings for rooting, or on the formation of callus tissue from buds and leaf fragments. However, prolonged sterilization and a high concentration of the sterilizer in this method negatively affect the processes of shoot formation and plant growth. A significant part of the explants was only capable of forming callus. In addition, leaf fragments are used in this method, of which the possibility of obtaining plants free of viruses and fungi is small. The value of 5 mm declared in this method is insufficient to obtain a high yield of virus-free plants.

 The purpose of the invention is to increase the reproduction rate of virus-free plants and simplify the method of micropropagation.

 This goal is achieved by a new method of micropropagation of Stevia (Stevia rebaudiana L.), including isolating the explant, sterilizing it, cultivating in a nutrient medium Murashige-Skoog (MS) containing macro- and micronutrients, vitamins, sucrose, agar and phytohormones, obtaining regenerants, microcutting , growing, rooting and transplanting regenerated plants into the soil, moreover, according to the invention, terminal and / or axillary buds with underlying stem tissues of 1 to 3 mm in size are singled out as an explant, sterilization is carried out 0.1% with a solution of diocide for 3 min and a 0.1% mercuric chloride solution for 2.5 min, cultivation is carried out on nutrient medium Murashige-Skoog containing phytohormones indolylacetic acid (IAA) 0.1 0.3 mg / l, benzyladenine (BA) 0.5 2.0 mg / l and gibberellic acid (HA) 0.5 2.0 mg / l, the explant is cultivated for 4 to 5 weeks until the formation of rooted regenerated plants, unrooted regenerants and callus tissue, after which the plants are separated regenerants and plant them in the ground, and unrooted regenerants are micro-cut and transferred for rooting Murashige-Skoog medium containing 2.0-6.0 mg / L IAA and HA 1.0-2.0 mg / L, and callus tissue is placed on the initial medium for further cultivation until regenerants are obtained.

 The present invention is new, since no solution with the claimed combination of distinctive features has been found in the patent and scientific literature, that is, it is not known from the prior art, which means it meets the criterion of "novelty."

 The present invention has an inventive step, as it clearly does not follow from the prior art.

Example 1. Take the apical and lateral cuttings from Stevia donor plants measuring 10 15 mm and sterilize them with 0.1% diocide 3 min and 0.1% mercuric chloride 2.5 min, and washed in 3 portions of sterile water. Then, the apical kidneys with small underlying tissues of 0.2-0.3 mm in size are isolated from them and placed in MS medium containing thiamine 0.5, pyridoxine 0.5, nicotinic acid 1, sucrose 20,000, agar 8000, which additionally contains phytohormones IAA 0.1 mg / L, BA 0.5 mg / L and HA 0.5 mg / L. The explants are kept at a relative humidity of 70% photoperiod for 16 hours, illuminated with LB-80 fluorescent lamps at a temperature of 25 26 ^o C. After 2 weeks, an shoot with callus tissue on the basal part grows from the explanted kidney (Fig. 1).

 Then after 1 week, callus tissue increases and a large number of kidneys and regenerants arise (Fig. 2). After 1 week, there is a strong growth of regenerants with root formation in some of them (approximately 20%), suitable for planting directly in the ground (Fig. 3).

 The remaining regenerants are cut and transplanted into the MS medium (Fig. 4) containing phytohormones, mg / L: IAA 2, GK 1. After 2 weeks, the shoots are rooted and ready for planting in the ground (Fig. 5). This process can be repeated repeatedly (5–6 times) using the viability of the callus tissue formed after the first kidney implantation (Fig. 6). 2 weeks after the transplantation of this callus tissue into fresh medium, new buds and regenerants are born (Fig. 7).

 The results of example 1 are shown in table. 12.

Example 2. Take the apical and lateral cuttings from Stevia donor plants with a size of 10 15 mm and sterilize them with 0.1% diocide 3 min and 0.1% mercuric chloride 2.5 min, and washed in 3 portions of sterile water. Then, the apical kidneys with small underlying tissues of 0.2-0.3 mm in size are isolated from them and placed in MS medium containing thiamine 0.5, pyridoxine 0.5, nicotinic acid 1, sucrose 20,000, agar 8000, which additionally contains phytohormones IAA 0.5 mg / L, BA 1.0 mg / L and HA 2.0 mg / L. The explants are kept at a relative humidity of 70% photoperiod for 16 hours, illuminated with LB-80 fluorescent lamps at a temperature of 25-26 ^o C. After 2 weeks, an shoot with callus tissue on the basal part grows from the explanted kidney (Fig. 1).

 Then after 1 week, callus tissue increases and a large number of kidneys and regenerants arise (Fig. 2). After 1 week there is a strong growth of regenerants with root formation in some of them (approximately 20%), suitable for planting immediately in the ground (Fig. 3).

 The remaining regenerants are cut and transplanted into the MS medium (Fig. 4) with the content of phytohormones mg / L: IAA 4, HA 1.5. After 2 weeks, the shoots take root and are ready for planting in the ground (Fig. 5). This process can be repeated repeatedly (5–6 times), using the viability of the callus tissue formed after the first kidney implantation (Fig. 5). 2 weeks after the transplantation of this callus tissue into fresh medium, new buds and regenerants are born (Fig. 7).

 The results of example 2 are shown in table. 12.

Example 3. Take the apical and lateral cuttings from Stevia donor plants measuring 10 15 mm and sterilize them with 0.1% diocide 3 min and 0.1% mercuric chloride 2.5 min, and washed in 3 portions of sterile water. Then, the apical kidneys with small underlying tissues of 0.2-0.3 mm in size are isolated from them and placed in MS medium containing thiamine 0.5, pyridoxine 0.5, nicotinic acid 1, sucrose 20,000, agar 8000, which additionally contains phytohormones IAA 2.0 mg / L, BA 2.0 mg / L and HA 2.0 mg / L. The explants are kept at a relative humidity of 70% photoperiod for 16 hours, illuminated with LB-80 fluorescent lamps at a temperature of 25 26 ^o C. After 2 weeks, an shoot with callus tissue on the basal part grows from the explanted kidney (Fig. 1).

 Then after 1 week, callus tissue increases and a large number of kidneys and regenerants arise (Fig. 2). After 1 week there is a strong growth of regenerants with root formation in some of them (approximately 20%), suitable for planting immediately in the ground (Fig. 3).

 The remaining regenerants are cut and transplanted into the MS medium (Fig. 4) with the content of phytohormones mg / L: IAA 5, GK 2. After 2 weeks, the shoots are rooted and ready for planting in the ground (Fig. 5). This process can be repeated repeatedly (5–6 times), using the viability of the callus tissue formed after the first kidney implantation (Fig. 5). 2 weeks after the transplantation of this callus tissue into fresh medium, new buds and regenerants are born (Fig. 7).

 The results of example 3 are shown in table. 12.

 The effect of the complex of phytohormones that make up the main MS medium for micropropagation of stevia is shown in Table. one.

 The concentration and ratio of phytohormones introduced into the basic MS medium for rooting regenerated plants are given in Table. 2.

 From the results of the table. 1, 2 it follows that the inventive method of micropropagation of stevia allows for up to 250 regenerated plants from one explant within 4-6 weeks, i.e. the multiplication factor is 1: 250, and from one plant to an average of 10,000, including regenerant plants, both rooted after the first cultivation and in need of subsequent rooting. The remaining callus has the ability to regenerate new plants when passivated up to 5 6 times.

 The simplification of the proposed method consists in using a milder sterilization, after which there is no need to remove the negative effect of sterilizing substances, which consists in transplanting explants on fresh medium every 12 to 15 days.

 Repeated use of callus tissue greatly simplifies and accelerates the method, since sterilization and isolation of the meristemic kidneys is very time-consuming.

 The use of explants with a size of 1 3 mm can increase the yield of virus-free planting material.

Claims (1)

 A method of micropropagation of Stevia rebaudiana L. stevia, including isolating an explant, sterilizing it, culturing in a nutrient medium Murashige Skoog, containing macro- and micronutrients, vitamins, sucrose, agar and phytohormones, obtaining regenerants, microcrossing, growing, rooting and replanting plants characterized in that as an explant, terminal and / or axillary buds with underlying stem tissues of 1 to 3 mm in size are isolated, sterilization is carried out with a 0.1% solution of diocide for 3 min and a 0.1% solution ohm sublimate for 2.5 min, cultivation is carried out on a nutrient medium Murashige and Skoog containing phytohormones IAA 0.1 0.3 mg / l, benzyladenine 0.5 2.0 mg / l and gibberellic acid 0.5 2.0 mg / l, explants are cultured for 4–5 weeks until the formation of rooted regenerated plants, unrooted regenerants and callus tissue, after which the regenerated plants are separated and planted in the ground, and unrooted regenerants are microcraniated and transferred to the Muraga medium for rooting containing 2 6 mg / l indolylacetic acids and gibberellic acid 1 2 mg / l, and callus tissue is placed on the original nutrient medium for further cultivation to obtain regenerants.

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