RU2538859C1 - Method of clonal micropropagation of grape in vitro - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention is a method of clonal micropropagation of grape in vitro, comprising micro-grafting of test tube plants, planting them on nutrient medium in the presence of the growth substances and cultivation, where planting the cultivation is carried out on a solid Murashige and Skoog nutrient medium containing the growth-regulating substance as the Melaphen preparation in the concentration of 10^-7-10^-11%.

EFFECT: method enables to increase the yield of the plants due to effective survival of micro-grafts, good regeneration, improvement of quality indicators of plants.

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Description

The invention relates to biotechnology, in particular, to methods of propagation of plants, and can be used in viticulture for accelerated propagation of promising grape varieties.

Various methods are known using mainly natural biologically active compounds, such as giberrellins, cytokinins, auxins, brassinosteroids. However, some of them are expensive, others have not been widely used due to lack of stability, insolubility in water or high toxicity (auxins). Therefore, the creation of methods for producing planting material using cheap, non-toxic, water-soluble chemicals that act like phytohormones in ultra-low concentrations is an urgent task.

A known method of clonal micropropagation of plants, including the introduction into the nutrient medium of cytokinins [Guidelines for clonal micropropagation of grapes. Yalta, 1986, pp. 25-29]. Its disadvantage is the high complexity of the processes associated with the frequent transplantation of explants, their weak rooting, and the possibility of violating the genetic stability of propagated plants.

A known method of clonal micropropagation of grapes in vitro, including microcentering of test plants and planting them on a nutrient medium Murashige and Skoog [Clonal micropropagation and improvement of planting material of grapes to create intensive varietal mother plants from it. Recommendations M., 1992, p. 12-13]. The disadvantage of this method is its limited effectiveness due to stressful conditions for microcranes in vitro, inhibition of the growth processes of explants with toxic substances secreted by them into the nutrient medium. Repeated subcultivation worsens their condition, reduces the yield of microcranes.

A known method for optimizing clonal micropropagation of grapes in vitro [RU 2264706 C2, publ. November 27, 2005, Bull. No. 33] is a prototype. The method includes microcrossing of test plants and planting them on a liquid nutrient medium with a reduced amount of microelements and vitamins with the addition of indolylacetic acid 0.1-0.3 mg / l and the drug Emistim at a concentration of 10 ^-7 -10 ^-10 %. The disadvantage of the prototype is the use of the expensive drug Emistim, which is a composition of growth substances of a cytokinin and gibberellin nature isolated from ginseng roots. The drug is used together with indolylacetic acid, which is also a growth-regulating drug.

The use of a liquid nutrient medium also complicates the process and reduces the rate of planting and the survival rate of micrograins. In addition, when a bacterial infection appears on a liquid nutrient medium, it is difficult to notice it until it develops to a large extent and leads to the death of a microchereon or even a plant.

To obtain a good, sustainable planting material for elite grape varieties, it is necessary to use highly effective and cheap plant growth regulators.

The objective of the invention is the expansion of the arsenal of known methods of clonal micropropagation of grapes in vitro, which can improve the survival rate of microcrops, plant regeneration and their quality indicators.

The technical result consists in the action of a synthetic growth regulating preparation Melafen in clonal micropropagation of grapes in vitro.

The technical result is achieved by the claimed method of clonal micropropagation of grapes in vitro, including microcentering of test plants, planting them on a nutrient medium in the presence of growth-regulating substances, which use the drug Melafen at a concentration of 10 ^-7 -10 ^-11 %.

Melafen is a melamine salt of bis (oxymethyl) phosphinic acid of the general formula:

The drug is soluble in water, low toxicity (its LD _{50 is} 2000 mg / kg for mice and 6000 mg / kg for rats). Melafen solutions are stable during storage for years.

The experimental data obtained on various crops (cereals, fodder and vegetables) made it possible to conclude that Melafen, like phytohormones, has a multifunctional effect. The orientation of physiological and biochemical changes in plants under the influence of the drug is similar to the action of cytokinins on the energy and metabolic metabolism of plants [Fattakhov SG, Loseva NL, Konovalov AI, etc. The effect of melafen on the growth and energy processes of plant cells // DAN. 2004, T.394, No. 1, S.127-129; Kostin V.I., Kostin O.V., Isaichev V.A. The state of research and the prospects for the use of the growth regulator Melafen in agriculture and biotechnology. Kazan, 2006, S.26-34].

Melafen, like cytokinins, is involved in the regulation of physiological processes throughout ontogenesis, in the regulation of metabolism of organs that have completed growth, increases the content of pigments, increases the intensity of photosynthesis, increases the rate of photophosphorylation, improves the energy metabolism of a plant [RU 2158735 C1, publ. November 10, 2000 Bull. No. 31].

Melafen acts in extremely small concentrations, which makes it environmentally friendly. Its synthesis is simple, which leads to its cheapness in comparison with drugs of this purpose.

It is known the influence of Melafen on the energy of germination and germination of seeds, increasing the yield and quality of grain products [RU 2158735 C1, publ. November 10, 2000 Bull. No. 31; RU 2390984 C1, publ. 06/10/2010, Bull. No. 16; RU 2354106 C2, publ. 05/10/2009, bull. No. 13].

It is known the use of Melafen in biotechnology, and in particular in methods for producing alkaloids when growing tissue culture, for example, Rauwolfia snake Rauwolfia Serpentina Benth [RU 2394100 C2, publ. 07/10/2010, Bull. No. 19; RU 2174555 C1, publ. 10/10/2001; RU 2323972 C1, publ. 05/10/2008, Bull. No. 13].

However, the possibility of using Melafen in the clonal micropropagation of grapes at the stage of microcherenking is unknown.

New in the claimed method is that microcranes are planted on a nutrient medium containing a synthetic growth regulating drug Melafen.

The inventive method is as follows.

Prepare a nutrient medium Murashige and Skoog of the following composition:

Macronutrients, mg / l:

NH ₄ NO ₃ ammonium nitrate 138.0 KNO ₃ potassium nitrate 950.0 MgSO ₄ · 7H ₂ O magnesium sulfate 7-water 185.0 KH ₂ PO ₄ potassium phosphate monosubstituted 68.0 CaCl ₂ · 2H ₂ O calcium chloride 2-aqueous 296.0

Trace elements, mg / l:

H ₃ BO ₃ boric acid 6.2 MnSO ₄ · 5H ₂ O 4-aqueous manganese sulfate 22.3 CuSO ₄ · 5H ₂ O copper sulfate 5-water 0.025 CoCl ₂ · 6H ₂ O cobalt chloride 6-water 0.025 ZnSO ₄ · 7H ₂ O zinc sulfate 4-aqueous 8.6 Na ₂ MoO ₄ · 2H ₂ O molybdenum sodium 2-0.025 0.3 KJ potassium iodide 0.9 FeSO ₄ · 7H ₂ O iron chelate: iron sulfate 7-water 27.8 Na ₂ EDTA Trilon B 37.3

Vitamins, mg / L

Mesoinositis 50.0 Thiamine HCl 0.2

as well as

Indolylacetic acid (IAA), mg / l 0.1 Sucrose (g / l) 30.0 Agar agar (g / l) 7.5

The composition of the medium add the drug Melafen in concentrations of 10 ^-7 -10 ^-11 %.

pH of the medium before autoclaving is 5.7-5.9.

Test tubes with a nutrient medium are covered with foil, placed in a tripod, which is closed with cellophane, tied with twine and autoclaved in an autoclave GK-100-3.

Vine plants are selected that are regenerated from apical meristems 0.1-0.2 mm in size and propagated in vitro. Microcutting is carried out in the operating room in the Fortran laminar box. Shoots having 8-10 internodes are placed on a sterile Petri dish, cut into micro stems having an eye with a leaf. The length of the microcranes is 10-12 mm, 2 mm above the eye, the rest is under the eye. The resulting microcranes are planted in test tubes on a solid nutrient medium.

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 25-27 ± 2 ° C, air humidity of 70-75%.

Observation of plant growth was carried out with the clonal micropropagation of the grape varieties Tsimladar, Pukhlyakovsky and Varyushkin using Melafen at concentrations of 10 ^-7 , 10 ^-9 , 10 ^-11 % for 3 months. Melafen concentrations were previously determined empirically for each propagated variety.

The composition of the nutrient medium, the conditions for planting, and the cultivation of micro-cuttings both in the control and in the experimental variants were the same, except that the drug Melafen was absent in the control in the nutrient medium.

Clear data were obtained on the positive effect of the drug Melafen on the clonal micropropagation of Tsimladar grapes (table 1). At concentrations of 10 ^-7 , 10 ^-9 , 10 ^-11 %, an improvement in all the quality indicators of plants was observed after 23 days of cultivation. The best results were obtained at a concentration of 10 ^-7 %. Slightly worse performance in the embodiment with a concentration of 10 ^-9 %. A further decrease in the positive effect was noted with a minimum concentration of the drug. Of particular note is a 1.6-fold increase in the growth rate compared to the control, as well as root formation, growth and foliage of plants, which increases the willingness of plants to adapt in non-sterile conditions, the efficiency of clonal micropropagation and the final yield of plants.

With further cultivation (48 days), the growth rate slows down, but exceeds the control option by 1.2 times; the advantages of the drug Melafen for all growth indicators at concentrations of 10 ^-7 , 10 ^-9 , 10 ^-11 % remain.

When studying the effect of the drug Melafen on varieties Pukhlyakovsky and Varyushkin (Tables 2, 3), such clear data were not obtained, but there is a tendency to improve the qualitative characteristics of healthy plants under the influence of the growth regulator Melafen.

The results presented in table 2, obtained during cultivation for 30 and 60 days of the Pukhlyakovsky variety, demonstrate an improvement in the survival rate of plants at all studied concentrations, an increase in root length at concentrations of 10 ^-9 , 10 ^-11 %, rhizogenic zone at a concentration of 10 ^-11 % , height and leafiness at concentrations of 10 ^-7 , 10 ^-11 % (30 days of cultivation), and with longer cultivation (60 days) - an increase in the height and length of roots at all concentrations, the rhizogenic zone at a concentration of 10 ^-11 %, leafyness at concent radios 10 ^-9 , 10 ^-11 %.

The data of table 3 - grade - Varyushkin demonstrate an increase in the length of the roots and the rhizogenic zone at all concentrations after 20 days of cultivation, after 54 days of cultivation, an increase in leafiness, the number of roots and rhizogenic zone at concentrations of 10 ^-7 , 10 ^-9 %, as well as an improvement in survival rate by compared with control at a concentration of 10 ^-9 %.

The results of the final counting, carried out 3 months after sowing on the Pukhlyakovsky variety, confirmed the positive effect of the drug Melafen on the growth processes of the obtained meriklon (table 4) at a concentration of 10 ^-11 %. The increase in length (47.0 cm versus 41.1 cm in the control) and the number (4.7 pcs. In the control 3.8 pcs.) Of roots, rhizogenic zone (220, 9 cm, in the control 156.2 cm), the length (15.9 cm, in the control 15.4 cm) and weight (0.06 mg, in the control 0.05 mg) of shoots, the number of leaves (15.9 pcs., in the control 12.1 pcs.) indicates the effectiveness of the indicated concentration. A high polarity coefficient indicates a predominant development of the root system at this concentration.

The results of tables 1-4 demonstrate that the use of Melafen as a growth regulator improves plant morphogenesis in an in vitro culture and increases the efficiency of clonal micropropagation of grapes by improving the growth rate at the first stage of cultivation and qualitative characteristics of plants.

Comparison of the effects of the drugs Emistim (prototype) and Melafen taken at a concentration of 10 ^-7 % on the efficiency of in vitro clonal micropropagation of grapes on the 50th day of cultivation using the example of Tsimladar cultivar are shown in table 5. From the results of the table it can be seen that the main qualitative indicators of plants in particular, the length of the roots, the size of the rhizogenic zone, the growth rate, the polarity coefficient, the height of the plants and the yield of plants when using Melafen is much better.

Thus, the use of the drug Melafen as a growth regulator made it possible to increase the survival rate and development of micrograins, to reduce, on average, 1.5-3 times the cycle of development of micrograins, their regeneration into plants; in regenerated plants, improve root formation and development of the rhizogenic zone, shoot growth and leafiness, plant readiness to adapt to non-sterile conditions, yield plants with improved quality indicators and, in general, increase the efficiency of clonal micropropagation of grapes.

It should be noted that Melafen exhibits an unexpected, non-obvious effect, expressed in improving the quality of plants during repeated subcultivation, that is, there is a "aftereffect" of Melafen. It is expressed in the fact that plants grown on a nutrient medium with the addition of Melafen, which were re-cut and planted on a nutrient medium without Melafen (a second subculture was carried out), also exhibit improved quality indicators. Verification of aftereffect Melafen gave an unexpected clearly defined result (table 6). In plants that were grown on a nutrient medium with Melafen at concentrations of 10 ^-7 , 10 ^-9 %, an improvement in root formation and growth processes was noted when replanting to fresh nutrient medium without Melafen. The length of the rhizogenic zone increased by 1.4–2.5 times, the height of plants by 1.3 times, and the growth rate increased. All this contributed to the fact that after 47 days the plants were ready for adaptation according to their parameters, that is, there was an acceleration of micropropagation and, in general, an increase in the efficiency of clonal micropropagation. This makes it possible to use Melafen not for every subculture, but every other time: the first subculture - nutrient medium (PS) + Melafen, the second subculture - PS without Melafen, etc. Improving the quality characteristics of plants contributes to the adaptation process (higher yield of adapted plants) with good plant conditions. Strong, erect, well-leafy shoots with dark green leaves contribute to the rapid adaptation of plants to open ground conditions when laying the base mother liquors.

An in vitro study of clonal micropropagation of grapes was also carried out using the Baklanovsky variety as an example using Murasig and Skoog medium containing Melafen in concentrations of 10 ^-5 , 10 ^-7 , 10 ^-8 , 10 ^-9 without indolylacetic acid (IAA), where the preparation Melafen in the above concentrations was used as the only growth-regulating substance. Indolylacetic acid at a concentration of 0.1 mg / L without the addition of Melafen in the nutrient medium Murasiga and Skoog was a control. The results showed that in the control the rhizogenic zone reaches 7.7 cm, and with Melafen as the only growth-regulating substance, 9.0 cm. Accounting was carried out after 40 days. After 70 days of accounting, the rhizogenic zone of plants was 12.1 cm in the presence of Melafen and 10.7 cm in the control. The height and foliage of plants in both cases were the same.

This indicates the possibility of using the drug Melafen for clonal micropropagation of grapes in vitro and without indolylacetic acid in a nutrient medium.

Thus, a new method of in vitro clonal micropropagation of grapes using a non-toxic, time-stable, water-soluble, synthetic growth-regulating preparation Melafen, effective in a concentration of 10 ^-7 -10 ^-11 %, is proposed.

Using the proposed method allows to increase the yield of plants due to more efficient survival of microcrops, regeneration of plants and improve their quality indicators. Given the low cost of the drug and the high effect, the application of the proposed method is economically feasible.

Table 1 The effect of Melafen on the indicators of clonal micropropagation of Tsimladar grapes, 2013 Melafen concentration,% Survival rate,% Roots Height cm Leaves, pcs. Growth rate, cm / day number length cm rhizogenic zone, cm 23 days cultivation The control 96.4 3.0 3.0 9.0 2.7 3.0 0.12 10 ^-5 78.6 3.3 2.6 8.4 2.9 3.0 0.13 10 ^-7 100.0 4.6 5.9 27.4 4.4 4.3 0.19 10 ^-9 100.0 4.3 3.6 15,2 4.3 3.8 0.19 ^10-11 100.0 3,1 3.6 11.1 4.3 3.8 0.19 48 days cultivation The control 92.9 4.9 4.2 20.3 8.9 7.2 0.19 10 ^-5 71,4 4.2 4.7 19.7 9.2 7.8 0.19 10 ^-7 100.0 5.5 5.8 31.9 10.6 8.9 0.22 10 ^-9 100.0 4.6 5.5 25.6 10.5 9.0 0.22 ^10-11 100.0 4.4 4.2 18.5 10.0 9.7 0.20

table 2 The influence of Melafen on the indicators of clonal micropropagation of grapes of the Pukhlyakovsky variety, 2013 Melafen concentration,% Survival rate,% Roots Height cm Leaves, pcs. Growth rate, cm / day Number length cm rhizogenic zone, cm 30 days cultivation The control 92.9 3,5 4,5 16,0 5.9 5,6 0.19 10 ^-5 100.0 2,3 4.7 10.8 5.9 5.8 0.20 10 ^-7 96.4 3,5 4.3 15.1 6.1 5.8 0.20 10 ^-9 100.0 2,5 5.5 13.8 5.7 5,6 0.20 ^10-11 96.4 3.3 5,4 17.8 6.1 5.8 0.20 60 days cultivation The control 92.9 4.2 5,4 22.7 12.0 12,4 0.20 10 ^-3 100.0 3,1 5,6 17.3 12,2 12.6 0.20 10 ^-7 92.9 3,7 5.5 20.3 12.7 12,2 0.21 10 ^-9 100.0 3.3 5.7 18.8 12.3 12.8 0.20 ^10-11 96.4 3.4 6.7 22.8 12.0 12.9 0.20

Table 3 The influence of Melafen on the indicators of clonal micropropagation of Varyushkin grapes, 2013 Melafen concentration,% Survival rate,% Roots Height cm Leaves, pcs. Growth rate, cm / day number length cm rhizogenic zone, cm 20 days of cultivation The control 100.0 4.0 2,3 9.5 3.2 2.9 0.17 10 ^-5 96.4 3.8 2.7 10,2 2.9 2,4 0.15 10 ^-7 100.0 4.0 2.7 10.8 2,8 2,8 0.15 10 ^-9 100.0 4.1 2,5 10,2 2,5 2.7 0.13 ^10-11 100.0 2.9 3.4 9.9 2.7 2,8 0.14 54 days cultivation The control 96.4 4.4 4.2 18.9 11,2 8.4 0.21 10 ^-5 96.4 4.0 4.2 17.0 9.6 8.9 0.18 10 ^-7 96.4 5.2 3.9 20.3 10.1 9.5 0.19 10 ^-9 100.0 4.7 4.1 19.2 9.9 8.6 0.18 ^10-11 92.9 3.6 5.1 18.3 8.9 8.1 0.16

Table 4 The condition of grape plants of the Pukhlyakovsky variety after 3 months of cultivation in a nutrient medium with Melafen, 2013 Melafen concentration,% Roots The escape Leaves Polarity ratio number length cm rhizogenic zone, cm length cm weight mg number area, cm weight mg The control 3.8 41.1 156.2 15.4 0.05 12.1 35,4 0.05 10.1 10 ^-5 2.9 32,4 93.9 15.4 0.04 12.8 38.6 0.04 6.1 10 ^-7 2.7 35.0 94.5 15.1 0.05 13,2 31.8 0.05 6.2 10 ^-9 2.7 32,2 86.9 16,2 0.05 14.5 29.8 0.04 5.3 ^10-11 4.7 47.0 220.9 15.9 0.06 15.9 32,7 0.05 13.8

Table 5 The influence of Melafen on the effectiveness of clonal micropropagation of healthy plants of Tsimladar grapes, 2013 Indicators The claimed technical solution, the concentration of Melafen 10 ^-7 % Prototype, Emistim concentration 10 ^-7 % 50 days cultivation The number of roots, pieces 5.5 8.3 Root length, cm 5.8 2.2 The size of the rhizogenic zone, cm 31.9 18.2 Growth rate cm / day 0.22 0.18 Polarity ratio 3.0 2.0 Plant height, cm 10.6 9.2 The number of leaves 8.9 6.9 The yield of plants,% 100.0 87.8

Table 6 The effect of the drug Melafen on the indicators of clonal micropropagation of grapes of the Pukhlyakovsky variety, 2013 Melafen concentration,% Roots Plant height, cm Growth rate, cm / day Polarity ratio number length cm rhizogenic zone, cm 47 days cultivation The control 4.6 3.3 15.0 6.0 0.12 2,5 10 ^-5 4.4 3,1 14.0 5.7 0.12 2,4 10 ^-7 5,0 5.3 26.5 7.6 0.16 3.4 10 ^-9 5,4 6.3 34.1 7.6 0.16 4,5

Claims (1)

The method of clonal micropropagation of grapes in vitro, including microcentering of test plants, planting them on a nutrient medium in the presence of growth substances and cultivation, characterized in that the planting and cultivation is carried out on solid nutrient medium Mursige and Skoog, containing Melafen at a concentration of 10 as a growth-regulating substance -10 ^{-7 -11%.}