RU2264706C2 - Method for optimization of clonal in vitro micro multiplication of grape - Google Patents

Abstract

FIELD: agricultural biotechnology, in particular, in vitro grape multiplication processes.

SUBSTANCE: method involves providing micro cutting of testing plants and planting thereof in liquid nutritive medium with reduced amount of macroelements and vitamins, with indole-acetic acid being added in an amount of 0.1-0.3 mg/l and emistim preparation with concentration of 10^-7-10^-10% being added into composition.

EFFECT: increased efficiency in multiplication of perspective sorts of grape sanitated from virus infection.

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Description

The invention relates to agricultural biotechnology, in particular to methods of propagation of plants, and can be used in viticulture for accelerated propagation of promising grape varieties recovered from viral infection, in studies on the physiology of grapes, serves to protect the environment.

A known method of clonal micropropagation of plants, including the introduction of cytokinins into the nutrient medium, which leads to the formation of shoots with relatively shortened internodes, and axillary buds and meristematic tubercles give rise to new shoots [Methodical recommendations for clonal micropropagation of grapes. Yalta, 1986, pp. 25-29]. A method for implementing this direction is associated with the proliferation of axillary buds and shoots.

The disadvantage of this method of clonal micropropagation is the high complexity of the process, since frequent explant transplantation is necessary; their weak rooting; the possibility of the formation of callus on the basal part of the aggregate of buds and shoots, which can lead to a violation of the genetic stability of propagated plants.

A known method of microclonal propagation of grapes in vitro, including microcentering of test plants with 8-10 internodes into fragments 10-12 mm long with a knot and leaf and planting them on a nutrient medium Murashige and Skoog [Clonal micropropagation and improvement of planting material of grapes to create varietal varieties from it mother cells of intensive type. Recommendations M., 1992, p.12-13] - the prototype.

The disadvantage of this method is that with clonal micropropagation at the initial stage, after microcrops, plants grow slowly, which limits the effectiveness of the method. One of the reasons for the slow growth is the stressful conditions in which microcherens appear in vitro. Inhibition of growth processes of explants by toxic substances secreted by them into the nutrient medium is observed. Fungal pathogens also negatively affect the development of plants and may contribute to their death. With repeated repeated subcultures, their condition worsens, the yield of micrograins decreases, which limits the effectiveness of the method. A way is needed to optimize this process. A new class of phytoregulators - adaptogens contributes to the activation and multipurpose stimulation of plant defense reactions. These include the preparation emistim, which is a composition of growth substances of a cytokinin and gibberellin nature, metabolic products of the symbiotic fungus Acremonium lichenicola isolated from ginseng roots. It positively affects the processes of plant growth and development, reduces the influence of adverse and stressful environmental factors.

All this creates the prerequisites for its use in in vitro technology to overcome the difficulties we have expressed earlier.

The aim of the invention is to increase the efficiency of clonal micropropagation of grape plants, recovered from viral infection by the method of apical meristems, at the stage of microcherenking shoots.

This goal is achieved by the fact that in the method of microclonal propagation of grapes in vitro, including microcutting of test plants with 8-10 internodes into fragments 10-12 mm long with an eye and a leaf and planting them in test tubes on a nutrient medium Murashige Skoog, they are planted and cultivated on a liquid nutrient medium Murashige and Skoog with a reduced amount of macronutrients and vitamins: NH ₄ NO ₃ - 130-140 mg / l, KNO _s - 920-950 mg / l, MgSO ₄ · 7H ₂ O - 180-191 mg / l, CaCl ₂ · 2H ₂ O - 290-300 mg / l, KH ₂ PO ₄ - 65-70 mg / l; mesoinositol 48-50 mg / l, thiamine - 0.20-0.22 mg / l, indolylacetic acid 0.1-0.3 mg / l with the addition of emistim in its composition at a concentration of 10 ^-7 -10 ^-10 % .

New in the proposed method is that microcirculation of test plants is planted on a liquid modified nutrient medium Murashige and Skoog with a reduced content of macrosalts, vitamins and indolylacetic acid. ·

It is significant that the content of macrosalts is reduced as follows: NH ₄ NO ₃ from 1650 to 138 mg / L, KNO ₃ from 1900 to 950 mg / L, MgSO ₄ · 7H ₂ O from 370 to 185 mg / L, CaCl ₂ · 2H ₂ O from 440 to 296 mg / L; KH ₂ PO ₄ from 444 to 68 mg / L; the content of mesoinositol from 100 mg / l to 50 mg / l, thiamine from 0.5 to 0.2 mg / l, nicotinic acid, indolylacetic acid in the amount of 0.1-0.3 mg / l with the addition of the composition of the nutrient medium of the drug emistim at a concentration of 10 ^-7 -10 ^-10 %.

Emistim has a twofold effect on microcranes: it promotes the induction of rhizogenesis and, in addition, affects the cone of growth of the kidneys of the eye, promoting cell division and elongation of the cells of all tissues. That is, in this case, the effect of emistim is aimed at microcranes in order to regenerate plants from it and is carried out simultaneously at the tissue and organism level.

As a result of its use, metabolic processes are optimized, immunity functions in plants are enhanced, and plant defense mechanisms against many pathogens are activated. Gibberellin and cytokinin activity of the drug positively affects the processes of plant growth. Thus, the drug emistim is a broad-spectrum drug.

Replacing a solid nutrient medium (agarized) with a liquid nutrient medium, reducing the amount of macrosalts, vitamins, and indolylacetic acid in its composition improves the survival rate of micrograins, the ratio between the development of the rhizogenic zone and the aerial part in plants, the formation of a larger number of leaves, and also the better development of plants after subcultivation, that is, the inhibition of growth processes that is observed at high concentrations is removed. In addition, by replacing agar with a liquid nutrient medium and reducing the number of reagents, the process of clonal micropropagation of grape plants is cheaper.

Thus, the ambiguity of the proposed method is obvious.

The method is as follows.

Vine plants are selected that are regenerated from apical meristems 0.1-0.2 mm in size and propagated in vitro. Plants should have 8-10 internodes. Then they begin to prepare the liquid nutrient medium Murashige and Skoog of the following composition, mg / l: macrocells - ammonium nitrate - NH ₄ NO ₃ - 138, potassium nitrate - KNO ₃ - 950, magnesium sulfate 7-water - MgSO ₄ · 7Н ₂ O - 185, monosubstituted potassium phosphate - KH ₂ PO ₄ - 68, 2-aqueous calcium chloride - CaCl ₂ · 2H ₂ O - 296; trace elements - boric acid - H ₃ BO ₃ - 6.2, 4-aqueous manganese sulfate - MnSO ₄ · 4H ₂ O - 22.3; 5-hydrous sulfate copper — CuSO ₄ · 5H ₂ O — 0.025, 6-aqueous cobalt chloride — CoCl ₂ · 6H ₂ O — 0.025; 4-aqueous zinc sulfate — ZnSO ₄ · 4H ₂ O — 8.6, 2-aqueous molybdenum sodium — Na ₂ MoO ₄ · 2H ₂ O — 0.25, potassium iodide — KJ — 0.86; iron chelate: iron sulfate 7-aqueous - FeSO ₄ · 7H ₂ O - 27.8, trilon-B - Na ₂ EDTA - 37.3; vitamins - mesoinositol - 50, thiamine HCl - 0.2; IAA - 0.1-3 mg / l; pH of the medium before autoclaving 5.7-5.9. Emistim is added to the composition of the nutrient medium at a concentration of 10 ^-7 -10 ^-11 %, which was previously determined empirically for each propagated variety.

Bridges are made of filter paper, placed in test tubes, the prepared nutrient medium is poured into them, covered with foil, the rack with the tubes is closed with cellophane, tied with twine and autoclaved in the GK-100-3 autoclave.

Microcutting is carried out in the operating room in the Fotran laminar box. Shoots having 8-10 internodes are removed with tweezers from the test tube, placed on a sterile Petri dish, cut into micro stems having a peephole with a leaf. The length of the microcranium is 10-12 mm, 2 mm above the eye, the rest under the eye. The obtained micro-stems are planted in test tubes on filter paper bridges that are immersed in a liquid 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%.

Observations of plant growth are carried out after 15 days.

The results of the development of test tube grape plants on the 50th day of cultivation on nutrient media with different contents of IAA and emistima are presented in Table 1.

Table 1
The state of test-tube plants of grapes of the Rupestris du Lo variety on nutrient media of various compositions Options The roots Shoots Polarity ratio The yield of plants,% culture medium IAA, mg / l emistim,% number, pcs length mm rhizogenic zone, mm length mm number of leaves Murasige and Skoog (prototype) 0 - 0.6 12,2 7.3 21.0 3.0 0.9 53.5 0.1 - 1,0 18.2 18.2 24.9 4.2 1.3 67.8 0.3 - 0.9 24.1 21.6 29.3 4.0 0.7 78.6 Murasige and Skoog (technical solution without emistim) 0 - 1.3 47.6 61.8 52.0 5,4 1.8 89.2 0.1 - 3.2 40,2 128.6 74.7 4.8 1.7 96.4 0.3 - 5.3 28,4 150.5 75.1 4.8 2.0 96.2 Murasige and Skoog (claimed techn. Resolved. With emistim) 0.3 0 3,1 54.0 168.0 85.7 5.2 2.0 89.4 0.3 ^10-10 3.4 71.8 246.4 110.0 5.7 2.2 100.0 0.3 10 ^-9 4.6 55.9 258.4 91.1 5.5 2,8 92.8 0.3 10 ^-8 4.7 50.1 237.8 85.3 4.8 2,8 89.8 0.3 10 ^-6 5,4 49.0 266.5 63.7 5,0 4.2 82.1

As follows from the above data, on the nutrient medium Murashige and Skoog (prototype), the formation of roots and their growth, the formation of shoots and leaves occurred much weaker than on the modified nutrient medium (technical solution without emistim).

IAA at a concentration of 0.3 mg / L on this nutrient medium until the 50th day of cultivation did not adversely affect the plants. Later (on the 64th day of cultivation), inhibition of root formation and growth was noted and, as a result, a decrease in the size of the rhizogenic zone, as well as a decrease in the number of formed leaves and the polarity coefficient, that is, the optimal ratio between the development of roots and shoots was disrupted.

table 2
The growth rate of test tube grape plants of the Rupestris du Lo variety on nutrient media of various compositions Options The growth rate mm / day on the day of cultivation culture medium IAA, mg / l emistim,% rhizogenic zone plants 17 31 49 64 17 31 49 64 Murasige and Skoog (prototype) 0 - - 0.05 0.1 0.5 0.2 0.3 0.4 0.6 0.1 - 0,03 0.01 0.1 0.9 0.4 0.4 0.5 0.7 0.3 - 0,03 0.06 0 0.7 0.3 0.4 0.6 0.7 Murasige and Skoog (techn. Resolved. Without emistima) 0 - 0.7 1.3 1,2 2.0 0.3 0.5 1,0 1,1 0.1 - 0.02 1,6 2.6 2.9 0.4 0.8 1,5 1.7 0.3 - 0.05 2.1 3.0 2,8 0.1 0.6 1,5 1.8 Murasige and Skoog (claimed techn. Resolved. With emistim) 0.3 0 0.1 3.3 3.4 - 0.4 1,6 1.7 - 0.3 ^10-10 2,3 4.0 5,0 - 0.4 1.8 2.2 - 0.3 10 ^-9 1,0 4.0 5.3 - 0.5 1.4 1.9 - 0.3 10 ^-8 1,5 3.3 4.8 - 0.4 1,2 1.7 - 0.3 10 ^-6 2.1 2.1 5,4 - 0.5 1,2 1.3 -

On the nutrient medium Murashige and Skoog (technical solution without emistim), all indicators of plant development improved: the number of roots increased 2.1-5.8 times, their length 1.1-3.9 times, the size of the rhizogenic zone 7.0 -8.4 times, the height of plants increased by 2.2-3.0 and the formation of leaves increased slightly.

When using IAA at a concentration of 0.1-0.3 mg / l, there was no significant difference in plant development. The length of the roots decreased, but their number increased, which did not adversely affect the size of the rhizogenic zone and the process of stem formation. Thus, on a modified medium Murashige and Skoog with a reduced amount of macrocells and vitamins, it is possible to use IAA at a concentration of 0.1-0.3 mg / l.

With the use of emistim, the improvement of all indicators of plant development continued, the optimal ratio between the development of roots and shoots (polarity coefficient = 2.2-2.8) was established, which ensured the yield of plants at optimal drug concentrations (10 ^-9 -10 ^-10 %) 92 , 8-100.0%.

In addition, emistim influenced the growth rate of the rhizogenic zone and shoots (Table 2). The growth rate with the addition of emistim to the nutrient medium already by the 49th day of cultivation exceeded almost 2 times the growth rates of test plants on nutrient media without emistim on the 64th day of cultivation. That is, there is an acceleration of the process of clonal micropropagation by more than two weeks and, as a result, an increase in its effectiveness.

Stimulation of the development of roots and shoots was noted in all varieties and stocks, but the concentrations providing stimulation were different for each variety (Table 3). So, in the Druzhba cultivar, Kober 5BB rootstocks, 1615-2, the best development of plants occurred when diluting emistim 10 ^-7 -10 ^-8 ml / l, in the Variety Tsvetochny when breeding 10 ^-10 .

When emistim is added to the nutrient medium of the Rupestris du Lo variety (Table 3), the number of roots increases, more significantly, the less dilution of the preparation. The effect of emistim on the length of the roots is less significant. An increase in the number of roots and their length affected the size of the rhizogenic zone in all variants of the experiment. With a dilution of 10 ^-10, it amounted to 246.4 mm, and with a dilution of 10 ^-7 - 266.5 mm.

Under the influence of emistima, changes also occurred in the aerial part of test plants. An increase in plant height was noted at drug concentrations of 10 ^-10 and 10 ^-9 %. At a concentration of emistim of 10 ^-7 %, inhibition of plant growth occurred, which is associated with excessive development of the root system. Similar changes occurred with the formation of leaves, a greater number were noted when diluting emistima 10 ^-10 and 10 ^-9 .

Thus, according to the length of the roots, the height of the plants and the formation of leaves, a variant was distinguished with the addition of the drug Emistim at a dilution of 10 ^-10 to the nutrient medium.

Table 3
The development of test plants of various grape varieties at various concentrations of emistim Options The output of plants, pcs. The roots Shoots number, pieces length mm rhizogenic zone, mm length mm number of leaves Cober 5BB The control 89.2 9.8 18,4 180.3 73.5 6.6 Emistim 10 ^-9 75.0 8.9 18.8 167.3 81.9 7.1 Amistim 10 ^-8 78.6 8.7 23.5 204.4 87.8 8.1 Amistim 10 ^-7 71,4 7.9 19.1 150.8 59.4 6.2 Amistim ^10-6 64,2 5,6 19.3 108,0 55.3 5.8 1615-2 The control 82.1 3.8 11.7 44,4 65.1 1.4 Emistim 10 ^-9 96.4 2,8 21.8 61.0 58.6 1,1 Amistim 10 ^-8 85.7 2,3 17.0 55.3 60.9 1.7 Amistim 10 ^-7 85.7 4.0 14,4 57.6 65.8 1.9 Amistim ^10-6 100.0 1.8 18,4 33.1 48.6 0.9 friendship The control 50,0 2.2 27.3 60.0 13,2 1.4 Emistim 10 ^-9 60.7 2.2 35,4 77.8 - - Emistim 10- ⁸ 60.7 3.2 23,4 74.9 23.9 1.7 Amistim 10 ^-7 67.8 2,8 26,4 73.9 18.5 1.9 Amistim ^10-6 75.0 0.9 8.6 7.7 2,5 0.9 Floral The control 92.8 4.0 21.0 84.0 29.5 3,1 Emistim 10 ^-10 100.0 3.6 25.1 90.3 33.8 3.4 Amistim 10 ^-8 85.7 4.85 14.5 69.6 23.3 2,5 Amistim ^10-6 92.8 2,8 12.8 35.8 15.0 1.8 Rupestris du Lo The control 89.4 3,1 54.0 168.0 85.7 5.2 Emistim 10 ^-10 100.0 3.4 71.8 246.4 110.0 5.7 Emistim 10 ^-9 92.8 4.6 55.9 258.4 91.1 5.5 Amistim 10 ^-8 89.2 4.7 50.1 237.8 85.3 4.8 Amistim ^10-6 82.1 5,4 49.0 266.5 63.3 5,0 Cabernet North The control 25.0 3,7 7.6 37,2 26.0 3.3 Emistim 10 ^-10 67.8 7.6 14.9 113.2 44.7 3,1 Emistim 10 ^-9 67.8 6.4 15.8 100,2 33,4 3.0 Amistim 10 ^-8 67.8 5.2 16.8 87.3 36.3 3.0 Amistim 10 ^-7 75.0 3.8 17.9 68.0 32,2 2.7

Similar results were obtained when cultured in a nutrient medium with an emistim of the Cabernet variety North. An increase in the number of roots, their length, and the size of the rhizogenic zone was noted in all variants with emistim, but the most significant increase in these indicators occurred with a dilution of 10 ^-10 %. This option also noted higher rates of plant growth and leaf formation.

It should be noted that in both grape varieties at an emistim concentration of 10 ^-7 %, inhibition of plant growth and leaf formation was observed, which negatively affects the multiplication rate of these varieties.

Observation of the course of development of plant varieties test tube rupestris du Lo (Table 4) showed that in the embodiment with emistimom 10 ^-10% was the smallest in the experiment, and with the initiation of culture, retarded growth of roots and shoots. In the control and other variants with emistim, both the absence of root growth and the absence of shoot growth until the 49th day of cultivation were observed. In this regard, the yield of plants in these variants was 82.1-92.8%, and in the variant with emistim 10 ^-10 -100%.

Table 4
The development of test tube grape plants with the addition of emistim in a nutrient medium Options Lack of growth The yield of plants,% roots, pieces per day shoots, pieces per day fifteen 34 49 63 fifteen 34 49 - Rupestris du Lo The control 5 3 2 - fifteen 34 49 - 89.4 Emistim 10 ^-10 3 1 - - 5 5 - - 100.0 Emistim 10 ^-9 6 2 2 - 6 2 2 - 92.8 Amistim 10 ^-8 10 3 2 - nine 2 2 - 89.2 Amistim 10 ^-7 thirteen 6 3 - 8 3 3 - 82.1 Cabernet North The control 25 22 22 21 24 23 21 25.0 Emistim 10 ^-10 12 5 8 7 27 fifteen eleven nine 67.8 Emistim 10 ^-9 fifteen 8 10 7 26 fifteen thirteen 8 67.8 Amistim 10 ^-8 thirteen 8 8 6 28 fifteen 10 7 75.0 Amistim 10 ^-7 14 fifteen eleven 12 26 17 thirteen 14 50,0

Under the influence of emistim, the Cabernet variety North also improved the formation and growth of roots and shoots, and the yield of plants increased by a factor of 2–3 compared with the control.

An analysis of the use of emistim for microcrossing of grape varieties showed that the preparation emistim contributes to an increase in the efficiency of clonal micropropagation of healthy plants. This is due to improved survival of microcrops, faster formation and growth of roots, shoots and leaves, which ensures, as we noted above, the acceleration of the process of clonal micropropagation by more than two weeks and, as a result, increase its efficiency.

The addition of the emistim preparation to the nutrient medium at the microcherenking stage, in addition to the foregoing, helps to improve the process of plant adaptation to non-sterile conditions, due to which, when the test plants are planted in peat pots, their death is practically absent, and the plant yield is 99.0-100%.

A correctly selected concentration of the drug is of great importance, since in addition to the stimulating effect, inhibition, excessive development of the rhizogenic zone, inhibiting the development of shoots and leaf formation can be observed, which leads to a decrease in the reproduction rate.

Table 5
The effect of emistim on the effectiveness of clonal micropropagation of healthy grape plants Indicators The claimed technical solution Prototype Rupestris du Lo The number of roots, pieces 3.4 0.3 The length of the roots, mm 71.8 2,3 The size of the rhizogenic zone, mm 246.4 20.8 Plant height, mm 110.0 49.6 The number of leaves, pieces 5.7 4.0 The yield of plants,% 100.0 78.6 Cabernet North The number of roots, pieces 8.3 4.6 The length of the roots, mm 22.0 16.6 The size of the rhizogenic zone, mm 182.6 76.3 Plant height, mm 92.0 51.0 The number of leaves, pcs 6.9 5,0 The yield of plants,% 67.8 25.0

Feasibility Study

The claimed technical solution helps to strengthen the immunity functions of propagated plants, activate their protective mechanisms against many pathogens, and improve the survival rate of microcrops.

With a decrease in the concentrations of macrosalts and vitamins in the composition of the nutrient medium, the indicators of plant development improve, which helps to improve the yield (up to 99-100%) and the quality of adapted plants and, ultimately, accelerate the process of laying the base mother liquors for transferring viticulture to a certified basis.

Claims (1)

The method of microclonal propagation of grapes in vitro, including microcrossing of test plants, planting them on a nutrient medium Murashige and Skoog without growth substances and with a reduced number of macrocells, characterized in that the planting and cultivation is carried out on a liquid nutrient medium with the addition of indolylacetic acid 0.1-0 , 3 mg / l and emistim in a concentration of 10 ^-7 -10 ^-10 %.