RU2039427C1 - Method for reproduction of currants in vitro - Google Patents

Abstract

FIELD: agriculture. SUBSTANCE: currants are reproduced in the tissue culture by singling out the explant and its subsequent cultivation at the stages of introduction into the culture, reproduction and rooting on nutrient medium whose qualitative composition corresponds to the Murasige-Skuga medium. The relation of ions of NO $\genfrac{}{}{0ex}{}{\text{-}}{\text{3}}$ :SO $\genfrac{}{}{0ex}{}{\text{2}}{\text{4}}$ ^-:PO $\genfrac{}{}{0ex}{}{\text{3}}{\text{4}}$ ^-:K⁺:Ca²⁺:NH $\genfrac{}{}{0ex}{}{\text{+}}{\text{4}}$ at the stage of introduction into the culture is 30:8:18:20:12:12, at the reproduction stage 36: 8:6:19:13:18 and at the rooting stage 24:7:15:21:14:19. In each cultivation stage the total content of these ions is 100 m equiv. EFFECT: higher efficiency. 1 dwg, 7 tbl

Description

 The invention relates to the field of agriculture, namely to clonal micropropagation of currants.

A known method of mathematical design of the experiment in the selection of the optimal nutrient medium for growing a culture of tomato plant cells in vitro [1]
The disadvantage of this method is that it requires a large number of explants (the experimental design contains 25 variants of 20 replications per variant) plus the availability of a computer, R-Multi program and an experienced programmer. In addition, the scheme of experiment variants contains a reduced number of variants (25) and calculations that raise doubts among specialists in this field.

The closest technical solution known is the method for determining the optimal ratio of macroions in a nutrient solution [2] according to which the ratio between NO ₃ ^- SO ₄ ^2- PO ₄ ^3- : K ⁺ Ca ²⁺ Mg ²⁺ is determined according to just eight experimental options. The method is based on varying the ratios between the six elements of mineral nutrition with their constant total dose in the nutrient solution, since in each variant the sum of NO ₃ ^- + SO ₄ ^2- + PO ₄ ^3- + K ⁺ + + Ca ²⁺ + Mg ^{2 + is the} same. In each variant, the relative amount (fraction) of one element prevails with constant (but smaller) relative amounts of all other elements. Based on the experimentally obtained data (wheat yield), the optimal ratios of the studied elements of mineral nutrition are calculated according to the Omes formulas. After that, tests are carried out on the obtained optimized ratios on plants of the same species and variety in comparison with conventional fertilizers.

 This method does not allow the selection of the optimal composition of the nutrient medium for plants grown using clonal micropropagation.

 The purpose of the invention is to increase the yield of planting material by optimizing the ratio of ions of the nutrient medium, providing increased regeneration and growth of additional shoots.

This goal is achieved by the fact that in the known method of propagation of currants in vitro, including isolating the explant and subsequent cultivation at the stages of introduction into the culture, reproduction and rooting on a nutrient medium, the qualitative composition of the corresponding medium Murashige-Skoog (MS), characterized in that at the stage of introduction into the culture, the ratio of NO ₃ ^- ions: SO ₄ ^2- PO ₄ ^3- K ⁺ Ca ²⁺ NH ₄ ⁺ is 30 8 18 20 12 12, at the propagation stage 36 8 6 19 13 18, at the rooting stage 24 7 15 21 14 19, respectively, with the total content of these ions in the nutrient medium on each of tapov culturing equal to 10 m eq.

PRI me R implementation. The method of propagation of currants in vitro is as follows. According to the scheme presented by Omes [2], the total level of the tested macroions NO ₃ ^- + SO ₄ ^2- + PO ₄ ^3- + K ⁺ + Ca ²⁺ + + NH ₄ ⁺ in the nutrient medium at each stage of in vitro cultivation was initially determined. Since the common medium for the propagation of currant in vitro is the nutrient medium MS, the effect of the dose rate of macro salts in the medium MS was tested on the regeneration and growth of currant explants in the range 0.50.75 1 1.25 1.5 2 the total content of salts of the medium MS.

 The drawing shows the effect of the multiplicity of the total dose (DM) of the macronutrients of the MS medium on the number of kidneys per 1 explant of the currant of the Riddle variety.

 The zone of optimum content of macroions in the nutrient medium of MS at the stage of reproduction was between 1 and 1.25 of the total dose. At this limit, the biometric difference between the explants was not significant. At the same time, a decrease and a 2-fold increase in the multiplicity of salts in the MS medium led to a decrease in the growth rate of shoots in length, a decrease in the number of buds by 2.5 times. At the other stages of cultivation (introduction to culture and rooting), the optimum for this indicator was within the same limits. For the convenience of calculating the options for further experiments and for reasons of economy of chemical reagents, we determined the sum of ions equal to 100 mEq for all stages of in vitro cultivation, which is 1.13 of the total dose of salts of MS medium.

The next step was to determine the optimal ratios between the three anions and the three cations of the nutrient medium (NO ₃ ^- SO ₄ ^2- PO ₄ ^3- : K ⁺ Ca ²⁺ NH ₄ ⁺ ). The scheme of experiment options is presented in table. 1. Instead of a magnesium ion, an NH ₄ ⁺ ion was used.

 In salt terms, these components of the environment are presented in table. 2.

The remaining components of the nutrient medium did not change according to the variants and corresponded to the well-known method developed in NIZISNP for clonal micropropagation of black and red currants and contained trace elements, mg / l: KJ 0.83, H ₃ BO ₃ 6.2, MnSO ₄ ˙ 7H ₂ O 22.3, ZnSO ₄ ˙ 7H ₂ O 8.6, NaMoO ₄ ˙ 2H ₂ O 0.25, CuSO ₄ ˙ 5H ₂ O 0.025, CoCl ₂ ˙ 6H ₂ O 0.025, Na ˙ EDTA 37.3 FeSO ₄ ˙ 7H ₂ O 27.8, vitamins: thiamine 0.5, pyridoxine 0.5, riboflavin 0.5, glycine 0.5, ascorbic acid 50, mesoinositol 100, sucrose 30, agar 7.

 As growth regulators, we used 6-benzylaminopurin (6-BAP) at the injection stage at a concentration of 0.5 mg / L, at the 6-BAP propagation stage, 1.5 mg / L, at the rooting stage indolylbutyric acid (IMA) at a concentration of 0, 5 mg / l.

 The experience is set in five repetitions. At the stage of introduction, the variant included 15 test tubes, at the stage of reproduction and rooting, 10 jars in the variant (3 explants per jar). The determination of optimal macroion ratios was carried out using the Riddle variety as an example. The found optimums were tested on other varieties of black and red currants.

 After planting explants of the Riddle variety on nutrient media, biometric measurements were performed weekly (number of buds, shoot length, number of shoots, length and number of roots). The optimal media ratios were calculated on the basis of experimentally obtained data (by the number of formed kidneys) using the Omes method.

(1) где Yⁱ урожай в варианте с преобладанием элемента i, Σ Y сумма количества почек, полученных в трех вариантах анионной серии.PRI me R calculation. Based on the data presented in table. 3, initially calculate the "rough optimum" OM (optimum brutto) in fractions of a unit for each element of the anion series according to the formula:
OB

(1) where Y ^{i is the} crop in the variant with the predominance of the element i, Σ Y is the sum of the number of buds obtained in the three variants of the anion series.

=

0,599
OB

=

0,212
OB

=

0,189 (min) Затем находим безразмерный поправочный коэффициент, фактор коррекции F_o(correction factor) для перехода от грубого к точному оптимуму:
F_o 0,88 ˙ n ˙ (OB_min + V) 0,6, (2) где n число элементов в соотношении (в анионной серии 3),
ОВ_min минимальный из грубых оптимумов серии,
V (value уровень) принятая в опыте доля минорного элемента в долях единицы.OB

=

0.599
OB

=

0.212
OB

=

0.189 (min) Then we find the dimensionless correction factor, the correction factor F _o (correction factor) to go from the rough to the exact optimum:
F _o 0.88 ˙ n ˙ (OB _min + V) 0.6, (2) where n is the number of elements in the ratio (in the anion series 3),
OB _{min is the} smallest of the crude optima in the series,
V (value level) accepted in the experiment, the share of the minor element in fractions of a unit.

(3)
OP

=

0,725
OP

=

0,154
OP

=

0,121 Таким образом, соотношения между NO₃ ^- SO₄ ^2- PO₄ ^3- равны 0,725 0,154 0,121 (в долях единицы) или 72,5 15,4 12,1 (в м.экв. ). Следующий этап нахождение оптимальных соотношений в катионной серии опыта:
OB_K

0,355
OB_Ca=

0,305
OB

=

= 0,628
F 0,88 ˙ 3 (0,305 + 0,16) 0,6 0,628
OP_K=

0,392
OP_Ca=

0,258
OP

=

0,352
Оптимальные соотношения между К⁺Ca²⁺ H₄ ⁺ равны 0,392 0,258 0,352. Аналогично находим оптимум между парными соотношениями N K в смешанной серии опыта (7 и 8 варианты).F 0.88 ˙ 3 ˙ (0.189 + 0.16) 0.6 0.321. Finally, with the help of the corrections, we pass from the gross optimum of OM to the exact or possible optimum of OP (optimum possible) for each of the elements of the series:
OP

(3)
OP

=

0.725
OP

=

0.154
OP

=

0.121 Thus, the ratios between NO ₃ ^- SO ₄ ^2- PO ₄ ^3- are 0.725 0.154 0.121 (in fractions of a unit) or 72.5 15.4 12.1 (in m.eq.). The next step is finding the optimal ratios in the cationic series of experiments:
OB _K

0.355
OB _Ca =

0,305
OB

=

= 0.628
F 0.88 ˙ 3 (0.305 + 0.16) 0.6 0.628
OP _K =

0.392
OP _Ca =

0.258
OP

=

0.352
The optimal ratios between K ⁺ Ca ²⁺ H ₄ ⁺ are 0.392 0.258 0.352. Similarly, we find the optimum between the pair NK ratios in the mixed series of experiments (options 7 and 8).

0,59
OB_K=

0,409
F 0,88 ˙ 2 ˙ (0,409 + 0,147) 0,6 0,379
OP_N

0,650
OP_K=

0,350 Оптимальные соотношения между NO₃ ^- K⁺ равны 0,65 0,35. Далее рассуждаем следующим образом: на 65 частей NO₃ ^- приходится 35 частей К⁺. Сколько должно приходиться К⁺ на 72,5 частей NO₃ ^-? Строим пропорцию.OB _N

0.59
OB _K =

0.409
F 0.88 ˙ 2 ˙ (0.409 + 0.147) 0.6 0.379
OP _N

0.650
OP _K =

0.350 The optimal ratios between NO ₃ ^- K ⁺ are 0.65 0.35. Next, we reason as follows: for 65 parts of NO ₃ ^- accounts for 35 parts of K ⁺ . How much should K ⁺ account for 72.5 parts of NO ₃ ^- ? We build a proportion.

65 NO ₃ 35 K
x 39 K ⁺ .

72.5 x From the cationic ratio it is known that 39.8 parts of K ⁺ account for 25.8 parts of Ca ²⁺ and 35.2 parts of NH ₄ ⁺ . And how much Ca ²⁺ and NH ₄ ⁺ should be in 39 parts of K ⁺ ?
39.2 K ⁺ 25.8 Ca ²⁺ 39.2 K ⁺ 35.2 NH ₄ ⁺
39.0 x 39.0 y
x 25.7 y 35.0 Thus, the ratio between NO ₃ ^- SO ₄ ^2- : PO ₄ ^3- K ⁺ Ca ²⁺ NH ₄ ⁺ is 72.515.4 12.1 39.0 25.7 35.0 ( in m ˙ eq).

The sum of ions must be equal to 100 mEq, and in this ratio it is equal to 199.7 mEq. Therefore, each member of the ratio of ions must be reduced by 1.1997 times or 2 times. Then we get the optimal ratio of ions NO ₃ ^- SO ₄ ^2- PO ₄ ^3- K ⁺ Ca ²⁺ NH ₄ ⁺ , equal to 36.25 7.7 6.0 19.5 12.917.5, which after rounding to the nearest whole will look like like 36: 8 6 19 13 18.

As a result of experiments at all stages of cultivation of currant in vitro, optimal ratios were obtained between the 6 most important macroions of the nutrient medium, which were:
I at the stage of introducing NO ₃ ^- SO ₄ ^2- PO ₄ ^3- K ⁺ Ca ²⁺ into the culture: NH ₄ ⁺ 30 8 1820 12 12 (we called PCl a nutrient medium with such a ratio of macroions);
II at the stage of propagation of NO ₃ ^- SO ₄ ^2- PO ₄ ^3- K ⁺ Ca ²⁺ NH ₄ ⁺ = 36 8 6 19 1318 (we called PC4 a nutrient medium with this ratio of ions);
III at the stage of rooting NO ₃ ^- SO ₄ ^2- PO ₄ ^3- K ⁺ Ca ²⁺ NH ₄ ⁺ = 24 7 15 21 1419 (we called PC7 a nutrient medium with this ratio of ions).

 In salt expression, the resulting ratios are presented in table. 4.

 After finding the optimal ratios of macroions of nutrient media for each stage of in vitro cultivation, the obtained media were tested for propagation and rooting of explants of two varieties of currant.

 I stage of introduction to the culture. A comparative study of the effect of the obtained PC1 medium for the stage of introduction into the culture with the MS medium (control) showed the advantage of the proposed PC1 medium (Table 5). Explants of blackcurrant cultivars Zagadka, Belorusskaya Sweet, Shiryaevskaya nutrient medium RS 1 contributed to an increase of 2-2.6 and 4.8 times, respectively, the growth of shoots and the extension of the growth activity of the explant during passage. In the control, two weeks after the start of the passage, the growth stopped due to the formation of a black wound callus of a dense consistency, which hindered the flow of nutrients into the depth of the explant. In redcurrant varieties on PC1 medium, shoot growth increased by 50-70% compared with the control.

 II stage of reproduction. As experience has shown, all tested currant varieties developed more actively on PC4 than on MS (Table 6).

 As a result of the planting of explants of the Early Sweet variety on PC4 medium, the breeding level increased by 7.5 times, in the Shiryaevskaya variety by 6.9 times, in the more plastic variety Chulkovskaya, which multiplies well on standard medium, the breeding level increased by 1.5 times. The period of active shoot growth on PC4 medium was 1 week longer than on MS medium.

 III stage of rooting shoots. As a result of the application of the PC7 nutrient medium, it was possible to achieve 100% rooting in almost all tested currant varieties (Table 7).

 The obtained ratio of macroions of the PC7 nutrient medium ensured a more active growth of the explant root system. The root length of the first order of branching was 78% on average higher than the control (MS medium). The appearance of roots of the 2nd order of branching in these plants was noted 5 days earlier than the control. According to the number of first-order roots formed, explants on PC7 medium exceeded those on MS medium by 68.8%. In the Medveditsa variety, the number of roots of this order exceeded the control by 2.27 times. All these advantages ensured the production of better quality currant microplants, which transferred adaptation much more easily and practically without loss after transferring them from culture vessels to non-sterile conditions.

All these advantages of optimized ratios in comparison with the conventional MS medium have contributed to increasing the efficiency of the clonal micropropagation method of this valuable crop and to increasing the yield of planting material per unit area. This allowed to reduce labor costs and equipment and chemical costs. reagents for obtaining the I-th plant by 20.8% and make clonal micropropagation of currants profitable with a profitability level of 30%
Using the proposed method for optimizing the ratios between NO ₃ ^- SO ₄ ^2- PO ₄ ^3- K ⁺ Ca ²⁺ NH ₄ ⁺ does not require a large number of explants, since the scheme of the experimental variants includes only 8 variants, the help of computers and complex mathematical calculations are not required.

Claims (1)

METHOD FOR REPRODUCING CURRANT IN VITRO, including isolating the explant and subsequent cultivation at the stages of introduction into the culture, reproduction and rooting on a nutrient medium, in terms of quality composition corresponding to the Murashige-Skoog medium, characterized in that at the stage of introducing into the culture the ratio of NO ions $\genfrac{}{}{0ex}{}{\text{-}}{\text{3}}$ : SO $\genfrac{}{}{0ex}{}{\text{2}}{\text{4}}$ ^- : PO $\genfrac{}{}{0ex}{}{\text{3}}{\text{4}}$ ^- : K ⁺ : Ca ²⁺ : NH $\genfrac{}{}{0ex}{}{\text{+}}{\text{4}}$ equal to 30: 8: 18: 20: 12: 12 at the breeding stage, the ratio of NO $\genfrac{}{}{0ex}{}{\text{-}}{\text{3}}$ : SO $\genfrac{}{}{0ex}{}{\text{2}}{\text{4}}$ ^- : PO $\genfrac{}{}{0ex}{}{\text{3}}{\text{4}}$ ^- : K ⁺ : Ca ²⁺ : NH $\genfrac{}{}{0ex}{}{\text{+}}{\text{4}}$ equal to 36: 8: 6: 19: 13: 18, at the rooting stage, the ratio of NO $\genfrac{}{}{0ex}{}{\text{-}}{\text{3}}$ : SO $\genfrac{}{}{0ex}{}{\text{2}}{\text{4}}$ ^- : PO $\genfrac{}{}{0ex}{}{\text{3}}{\text{4}}$ ^- : K ⁺ : Ca ²⁺ : NH $\genfrac{}{}{0ex}{}{\text{+}}{\text{4}}$ equal to 24: 7: 15: 21: 14: 19, respectively, while the total content of these ions in the medium at each of the stages of cultivation is 100 M · equiv.

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