RU2440414C1 - Nutrient medium for microreproduction of chinese magnolia (schisandra chinensis (turcz) baill) in conditions in vitro - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: nutrient medium includes regulators of growth 6- "БАП", "ИМК", vitamins by Murashige-Skoog (1962), agar. Mineral base contains macro- and micro elements by Quoirin-Lepoivre in modification of A.Standardi - F.Catalano (1984), casein hydrolysate, glucose and water in specified ratio of medium components.

EFFECT: increase of reproduction coefficient and increase of length of forming sprouts of Chinese magnolia length in culture in vitro.

6 dwg, 3 ex

Description

Usage: biotechnology and agriculture.

The inventive medium containing (mg / l): ammonium nitrate 400.0 ± 12.0; potassium nitrate 1800.0 ± 54.0; magnesium sulfate 360.0 ± 10.8; potassium phosphate 270.0 ± 8.1; calcium nitrate 1200.0 ± 36.0; iron sulfate 27.8 ± 0.83; sodium ethylenediaminetetraacetate 37.3 ± 1.1; manganese sulfate 1.0 ± 0.03; boric acid 6.2 ± 0.19; zinc sulfate 8.6 ± 0.26; sodium molybdenum acid 0.25 ± 0.008; copper sulfate 0.025 ± 0.001; cobalt chloride 0.025 ± 0.001; potassium iodide 0.08 ± 0.002; thiamine 0.4 ± 0.01; pyridoxine 0.5 ± 0.02; nicotinic acid 0.5 ± 0.02; glycine 2.0 ± 0.06; inositol 100.0 ± 3.0; casein hydrolyzate 250.0 ± 7.5; glucose 15000.0 ± 450.0; agar 7000.0 ± 210.0; 6-benzylaminopurine 1.0 ± 0.03; indolylbutyric acid 0.1 ± 0.003 and water up to 1 liter. The invention relates to biotechnology of plants and can be used to multiply crops that are difficult to propagate vegetatively.

Schizandra chinensis (Schisandra chinensis (Turcz.) Baill.) Is a valuable medicinal plant rich in biologically active substances and microelements, and refers to hard-rooted crops whose vegetative propagation by the green cuttings method is difficult and is characterized by low efficiency. At present, the first varieties of this non-traditional culture have been obtained, therefore, the development of effective methods of reproduction, which allow preserving varietal characters in vegetative offspring, is especially relevant.

An alternative to green cuttings is the method of clonal micropropagation through the culture of meristems, the high efficiency of which has already been proven on the example of many agricultural, ornamental and woody plants. However, to date, the composition of the nutrient medium that ensures the intensive formation of micro-shoots of lemongrass from isolated meristems has not yet been determined. This explains the fact that attempts to cultivate lemongrass in vitro are so few and so far have not brought significant positive results.

Known nutrient medium containing a mineral base according to Mura-shige-Skoog [1], zeatin 0.1 mg / l or 2-iP 1 mg / l and 1-3 ml / l tincture of Schisandra seeds [3].

Also known is a nutrient medium containing macronutrients according to Mura-shige-Skoog [1] and microelements according to Harada [4], mesoinositol 100 mg / l, nicotinic acid 0.5 mg / l, thiamine 0.5 mg / l, pyridoxine 0, 5 mg / l, ascorbic acid 1 mg / l, casein hydrolyzate 500 mg / l, sucrose 30 g / l, agar 7 g / l, as well as 2-iP 5 mg / l or 6-BAP 0.25-0, 5 mg / l [5].

Common disadvantages of these media are the almost complete absence of a multiplication factor, tissue necrosis and a high level of explant death, reaching 65-70%. As a result, only single regenerants were obtained for the entire cultivation period, the height of which did not exceed 8-10 mm.

Known nutrient medium according to the recipe Lloyd-McCown [6] containing 6-BAP 1 mg / l, sucrose 30 g / l and agar 7 g / l [7].

Despite the high reproduction rate, this medium is effective only when seeds with a germ and not a bud are used as primary explants, which does not allow the genetic characteristics of the mother plant to be preserved in the vegetative offspring, and therefore is not suitable for micropropagation of varieties and valuable magnotypes of magnolia vine.

The closest in essence to the claimed invention is a nutrient medium comprising macrocells according to Murashige-Skoog [1], microelements according to Harada [4], mesoinositol 100 mg / l, nicotinic acid 0.5 mg / l, thia min 0.5 mg / l, pyridoxine 0.5 mg / l, ascorbic acid 1 mg / l, casein hydrolyzate 500 mg / l, sucrose 30 g / l, agar 7 g / l, as well as 6-BAP 1 mg / l, IMC 0.3 mg / l [8].

The disadvantages of the prototype are the long lag period (for several months) at the stage of introduction into the culture and the gradual death of the meristems in the process of cultivation. This recipe of the nutrient medium does not allow to fully realize the regenerative potential of lemongrass and to obtain shoots longer than 1.5 cm long, suitable for rooting in vitro.

The aim of the invention is to increase the reproduction rate and increase the length of the forming shoots of Schisandra chinensis in vitro culture.

The goal is achieved due to the fact that the nutrient medium contains (mg / l): ammonium nitrate 400.0 ± 12.0; potassium nitrate 1800.0 ± 54.0; magnesium sulfate 360.0 ± 10.8; potassium phosphate 270.0 ± 8.1; calcium nitrate 1200.0 ± 36.0; iron sulfate 27.8 ± 0.83; sodium ethylenediaminetetraacetate 37.3 ± 1.1; manganese sulfate 1.0 ± 0.03; boric acid 6.2 ± 0.19; zinc sulfate 8.6 ± 0.26; sodium molybdenum acid 0.25 ± 0.008; copper sulfate 0.025 ± 0.001; cobalt chloride 0.025 ± 0.001; potassium iodide 0.08 ± 0.002; thiamine 0.4 ± 0.01; pyridoxine 0.5 ± 0.02; nicotinic acid 0.5 ± 0.02; glycine 2.0 ± 0.06; inositol 100.0 ± 3.0; casein hydrolyzate 250.0 ± 7.5; glucose 15000.0 ± 450.0; agar 7000.0 ± 210.0; 6-benzylaminopurine 1.0 ± 0.03; indolylbutyric acid 0.1 ± 0.003 and water up to 1 liter.

Example 1. Schizandra explants obtained from sterile apical and lateral buds were placed on a nutrient medium containing Quoirin-Lepoivre mineral base in the modification of Standardi-Catalano [2], vitamins according to Murashige-Skoog [1], growth regulators 6-BAP and IMA, as well as various concentrations of casein hydrolyzate according to options.

As follows from the data presented in Fig.1-2, casein hydrolyzate at a concentration of 250 mg / l significantly increased the shoot formation of Schisandra in vitro. In this case, microcranes in this variant of the experiment already on the 12th day of cultivation and then during the passage exceeded the control in terms of the multiplication coefficient. The differences are significant at a five percent level of significance of the null hypothesis (α = 0.05).

Example 2. Schizandra explants obtained from sterile apical and lateral buds were placed in control on Murashige-Skoog prescription medium [1], and in experiment, on Quoirin-Lepoivre nutrient medium in modification of Standardi-Catalano [2]. The media included the same set of vitamins according to Murashige-Skoog and growth regulators (6-BAP, IMC). Each option was further divided into two, containing 250 mg / l casein hydrolyzate and without it (figure 3). Explants cultivated on a modified QL medium, both with casein hydrolyzate and without it, significantly exceeded the corresponding control variants in the Murasige-Skoog medium at a five percent significance level of the null hypothesis (α = 0.05), which confirms the unsuitability of the mineral base of the Murasige medium - Skoog for micro-breeding schisandra and demonstrates a clear advantage of the proposed modified prescription over existing analogues (figure 4).

Example 3. Schizandra explants obtained from sterile apical and lateral kidneys were placed on a nutrient medium of the claimed composition containing 6-BAP, IMA, casein hydrolyzate 250 mg / l and various types and concentrations of sugars according to the options (Fig. 5). Substitution of sucrose for glucose in a concentration of 15 g / l in the medium made it possible to significantly increase the average shoot length of lemongrass and to obtain in sufficient quantity shoots longer than 1.5 cm long suitable for rooting in vitro (Fig. 6). The differences are significant at a five percent level of significance of the null hypothesis (α = 0.05).

The presented research results prove that the proposed nutrient medium for micro propagation of Schisandra chinensis for the first time allows to achieve a multiplication factor equal to an average of 2.2-2.4 new shoots per explant with an average shoot length of 25.7 mm, using apical and lateral buds as explants , which could not be achieved on well-known media-analogues.

Information sources

1. Murashige T., Skoog F. A revised medium for rapid growth and bio assays with tobacco tissue cultures // Physiol. Plant. - 1962. - vol. 5., No. 95 - P.473-497.

2. Standardi A., Catalano F. Tissue culture propagation of kiwifruit // Comb. proc. Intern, plant propagators' soc. - 1984. - vol. 34. - P.236-243.

3. Tut EA Acceleration of vegetative propagation of healthy planting material of actinidia and lemongrass. Abstract. dis. ... cand. S.-kh. sciences. - M., 2007 .-- 22 p.

4. Harada H. A new method for obtaining protoplasts from mesophyll cells // Z. Plantzenphysiol. - 1973. - vol. 69. - No. 1. - P.77-80.

5. Tut EA, Upadyshev MT Features of micropropagation of actinidia and Schisandra chinensis // Agricultural Biology. - 2008. - No. 3. - S.96-101.

6. Lloyd G., McCown B. Commercially feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot-tip culture // Comb. Proc. Intl. Prop. Soc. - 1980. - vol. 30. - P.421-427.

7. Hong M.H., Kim O.T., Park J.I., Hwang B. Micropropagation of Schisandra chinensis Baillon using glucose from cotyledonary nodes // J. of Plant Biology. - 2004 .-- vol. 47, No. 3. - P.270-274.

8. Upadyshev M.T., Tut E.A. Features of propagation of actinidia and Schisandra chinensis in vitro and green cuttings // Fruit growing and berry growing in Russia: Sat. scientific works. - M .: VSTISP, 2004. - T.XI. - S.200-209.

Claims (1)

Nutrient medium for the clonal micropropagation of Schisandra chinensis (Turcz.) Baill.), Containing ammonium nitrate, potassium nitrate, magnesium sulfate, potassium phosphate, calcium nitrate, ferric sulfate, ethylene diamino tetrate, sodium sulfate, sodium sulfate, ethylene diaminoacetate, sodium sulfate, molybdenum acid, copper sulfate, cobalt chloride, potassium iodide, thiamine, pyridoxine, nicotinic acid, glycine, inositol, casein hydrolyzate, glucose, agar, 6-benzylaminopurine, indolylbutyric acid and ode in the following ratio, mg / l:
NH ₄ NO ₃ 400.0 ± 12.0 Kno ₃ 1800.0 ± 54.0 MgSO ₄ · H ₂ O 360.0 ± 10.8 KH ₂ PO ₄ 270.0 ± 8.1 Ca (NO ₃ ) ₂ · 4H ₂ O 1200.0 ± 36.0 FeSO ₄ · 7H ₂ O 27.8 ± 0.83 Na ₂ EDTA · 2H ₂ O 37.3 ± 1.1 MnSO ₄ · 4H ₂ O 1.0 ± 0.03 H ₃ IN ₃ 6.2 ± 0.19 ZnSO ₄ · 7H ₂ O 8.6 ± 0.26 Na ₂ MoO ₄ · 2H ₂ O 0.25 ± 0.008 CuSO ₄ · 5H ₂ O 0.025 ± 0.001 CoCl ₂ · 6H ₂ O 0.025 ± 0.001 Ki 0.08 ± 0.002 Thiamine (B ₁ ) 0.4 ± 0.01 Pyridoxine (B ₆ ) 0.5 ± 0.02 A nicotinic acid 0.5 ± 0.02 Glycine 2.0 ± 0.06 Inositol 100.0 ± 3.0 Casein Hydrolyzate 250.0 ± 7.5 Glucose 15000.0 ± 450.0 Agar 7000.0 ± 210.0 6-BAP 1.0 ± 0.03 IMC 0.1 ± 0.003 Water up to 1 l pH 5.5-5.8

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