RU2329639C2 - Method of potato microcloning - Google Patents

Abstract

FIELD: agriculture.

SUBSTANCE: invention agricultural biotechnologies. Healthy potato plants are cultivated in vitro by micrografting into nutrient medium containing macro- and microelements according to Murashige&Skoog formula, Fe-chelate, agar, vitamins according to White subscription, and sucrose. The regenerated plants obtained are replanted out. Additionally 3 mg/l of glycine, 1000-2000 mg/l of activated carbon and 1-2 mg/l of ascorbic acid are introduced into the nutrient medium. Original plants undergo micrografting at the apical, middle and basal parts. Apical and middle parts are vegetated in nutrient medium with sucrose content of 20 g/l at the temperature of 23-25°C in daytime and 17-18°C at night. Further regenerated potato plants are bedded. Basal part is vegetated in nutrient medium with sucrose content of 80 g/l in darkness at the temperature of 8-10°C, with further growing of potato microtubers in vitro.

EFFECT: allows improving survival rate and accelerate explants growth, increasing reproduction rate of regenerated plants and quantity of in vitro microtubers, as well as improving survival rate for regenerated plants after replanting them out.

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Description

The invention relates to agricultural biotechnology, potato growing, seed production of potatoes, and in particular to accelerated propagation of seed potatoes on a virus-free basis.

A known method of microclonal propagation of potatoes, including cultivating healthy potato plants in vitro. Cuttings are carried out on a nutrient medium containing macro- and microelements according to Murasige-Skoog, sucrose, Fe-chelate, glycerin, IAA (heteroauxin), agar-agar, White vitamins and cinnamic acid. The resulting plants are planted in the ground (RU No. 2181942, IPC ⁷ A01H 4/00, C12N 5/02, publ. 05/10/2002).

The disadvantages of this method are the low growth rate of plants in vitro, the small yield of regenerated plants and micro-tubers from one source plant in vitro, in addition, the survival rate of plants grown on a nutrient medium in vitro when transplanted into the soil is only 70-80%.

The technical result consists in increasing the viability and accelerating the growth of explants, increasing the reproduction rate of regenerated plants and the number of micro-tubers in vitro, as well as improving the survival rate of regenerated plants during transplantation into the ground.

The essence of the invention lies in the fact that in the method of microclonal propagation of potatoes, including cultivating healthy potato plants in vitro by microcrossing on a nutrient medium containing macro- and micronutrients according to the prescription Murashige-Skoog, Fe-chelate, agar-agar, White vitamins and sucrose , obtaining regenerated plants and planting regenerated plants in the soil, 3 mg / l of glycine, 1000-2000 mg / l of activated carbon and 1-2 mg / l of ascorbic acid are additionally introduced into the nutrient medium. Microcutting of the initial plants is carried out on the apical, middle and basal parts. The apical and middle parts are grown on a nutrient medium with sucrose in an amount of 20 g / l at a temperature of 23-25 ° C during the day and 17-18 ° C at night, followed by planting potato regenerated plants in the ground, and the basal part is grown on a nutrient medium containing sucrose in an amount of 80 g / l in the dark at a temperature of 8-10 ° C, followed by obtaining potato micro-tubers in vitro.

An increase or decrease in the temperature of growing regenerated plants from the proposed one (23-25 ° C during the day and 17-18 ° C at night) and the concentration of sucrose in the medium (20 g / l) leads to a decrease in the growth of regenerated plants and a decrease in their reproduction rate.

Changing the temperature limits of obtaining micro-tubers, proposed by this method (8-10 ° C), and the concentration of sucrose in the medium (80 g / l) leads to a decrease in the yield of micro-tubers and their mass.

When 1000-2000 mg / L of activated carbon and 1-2 mg / L of ascorbic acid are added to the nutrient medium, the stem grows faster, a well-developed root system is formed and a large number of internodes are formed, which allows repeated micro-cutting in 3-4 weeks. As a result, there is an increase in the reproduction rate, in connection with which the yield of test plants increases 3 months after planting one source to 1.5-2 thousand units, depending on the variety. The root system of in vitro potato regenerated plants cultivated on this medium is more powerful, and such plants are better rooted in the soil (survival rate 90-98%).

The experiments were carried out in the cell engineering laboratory of the Department of Botany and Plant Physiology of GOUVPO “Mordovian State University named after NP Ogarev” with collection of virus-free potato varieties. The nutrient medium in the control version (Table 1) was prepared according to the prototype, in the experimental one with the addition of 1-2 mg / l ascorbic acid and 1000-2000 mg / l activated carbon.

Table 1. The composition of the nutrient medium, mg / l Control (prototype) Proposed solution Ammonium nitrate 1650 1650 Potassium nitrate 1900 1900 Two-water calcium chloride 440 440 Magnesium sulfate heptahydrate 370 370 Potassium phosphate monosubstituted 170 170 Boric acid 6.2 6.2 Four-manganese sulfate 27.7 27.7 Zinc sulfate four-water 8.6 8.6 Potassium iodide 0.83 0.83 Two-water sodium molybdenum acid 0.25 0.25 Copper sulfate pentahydrate 0,025 0,025 Cobalt chloride hexahydrate 0,025 0,025 Trilon B 373 373 Iron sulphate heptahydrate 27.8 27.8 Glycine 3 3 Cinnamic acid 0.5-1.0 - Heteroauxin one - Thiamine 1,0 0.5 Pyridoxine 0.2 0.5 Calcium pantothenate 0.2 - Activated carbon - 1000-2000 Vitamin C - 1-2 Sucrose 30000 20000-80000 Agar agar 7000.0 7000 Water up to 1 l up to 1 l

The method is as follows.

In washed and dried tubes (19 × 210 mm), 7-10 ml of agarized medium is poured, closed with cotton-gauze plugs, made up in medical bottles, autoclaved for 20 minutes at a pressure of 1 atm and a temperature of 120 ° C. Use 2 options of nutrient media, which are distinguished by the content of sucrose. For the growth of regenerated plants, a nutrient medium is used that contains macro- and microelements according to the prescription Murashige-Skoog, Fe-chelate, sucrose, glycine, agar-agar, White vitamins, 20 g / l of sucrose and an additional 1000-2000 mg / l of activated coal and 1-2 mg / l of ascorbic acid, and to obtain micro-tubers a similar nutrient medium containing 80 mg / l of sucrose.

Microcutting of the initial plants is carried out in a laminar box. Test plants with 12-15 leaves are removed from the test tube and cut in a petri dish. 2 single-kidney cuttings from the apical (apical) and middle parts of the plant are planted in tweezers using tweezers on a nutrient medium containing 20 g / l of sucrose. Test tubes with cuttings are signed and placed in racks on racks with illumination of 4-5 thousand lux with fluorescent lamps, a photoperiod of 16 hours and maintain the temperature at night 17-18 ° C, daytime - 23-25 ° C for further obtaining regenerated plants. Test tubes with cuttings of the basal part (2-3 cuttings of the basal zone), planted on a nutrient medium containing 80 g / l of sucrose, are placed in the dark at a temperature of 8-10 ° C for further tuberization.

Table 2 presents the dynamics of the development of Nevsky potato plant regenerate plants in vitro depending on the composition of the nutrient medium according to the proposed method (containing 20 g / l sucrose).

Table 2. Culture medium Age of the regenerant plant, days 10 twenty thirty Root length mm Prototype 10.1 ± 0.5 21.7 ± 0.6 36.4 ± 0.8 Wednesday 1 11.7 ± 0.4 24.6 ± 0.5 40.1 ± 0.7 Wednesday 2 12.3 ± 0.3 25.1 ± 0.7 49.7 ± 0.8 Wednesday 3 14.3 ± 0.4 25.7 ± 0.5 48.3 ± 0.7 Wednesday 4 12.5 ± 0.8 25.8 ± 0.4 47.1 ± 0.5 Shoot length, cm Prototype 3.1 ± 0.5 5.7 ± 0.6 8.4 ± 0.8 Wednesday 1 3.7 ± 0.3 6.6 ± 0.2 9.1 ± 0.7 Wednesday 2 3.9 ± 0.3 7.1 ± 0.7 10.7 ± 0.8 Wednesday 3 4.3 ± 0.7 7.7 ± 0.3 11.3 ± 0.7 Wednesday 4 4.9 ± 0.8 8.8 ± 0.4 12.2 ± 0.5 Number of nodes / leaves Prototype 3.0 ± 0.1 5.7 ± 0.6 8.1 ± 1.7 Wednesday 1 3.2 ± 0.2 6.3 ± 0.4 9.1 ± 0.7 Wednesday 2 3.4 ± 0.3 6.6 ± 0.8 9.7 ± 0.6 Wednesday 3 3.3 ± 0.7 6.8 ± 0.7 10.3 ± 0.7 Wednesday 4 4.1 ± 0.8 8.8 ± 0.4 11.2 ± 0.5 Notes: Wednesday 1 - a nutrient medium containing 1 mg / l of ascorbic acid Wednesday 2 - a nutrient medium containing 2 mg / l of ascorbic acid Wednesday 3 - a nutrient medium containing 1 mg / l of ascorbic acid + 2 g / l of activated carbon Wednesday 4 - a nutrient medium containing 2 mg / l of ascorbic acid + 2 g / l of activated carbon

By the end of the first week, regenerated plants from microcherens in the variant with activated carbon and ascorbic acid were 0.5-1.5 cm higher than the plants of the control variant and reached 3.5-4.9 cm. Under experimental conditions, plants earlier and faster formed the root system: by the end of the second week, the length of the roots of regenerated plants reached 1.1-1.5 cm, and by the end of the third - 2.5-2.6 cm, while in the control it was only 0.5 -1.0 cm and 2.0-2.2 cm. Intensive root formation in the future ensured faster stem growth in reg nerantov. During the third week, the maximum daily increase in plants by the proposed method was 5-8 mm, in plants of the control group 3-6 mm. Plants of the control variant by the end of the third week of cultivation lagged behind in growth by 2.0-3.5 cm. Plants were ready for repeated cuttings in the experimental variants by the end of the 3rd - beginning of the 4th week. The number of cuttings by this time in the experimental plants was 8–11 against 6–8 in the control ones (Table 3).

Table 3 Grade Control (prototype) Wednesday 1 Wednesday 2 Wednesday 3 Wednesday 4 Zhukovsky early 10.3 ± 0.4 12.3 ± 0.4 12.2 ± 0.1 13.5 ± 0.2 15.2 ± 0.1 Luck 11.3 ± 0.4 12.3 ± 0.4 12.2 ± 0.1 13.5 ± 0.2 15.2 ± 0.1 Nevsky 9 ± 0.7 9.1 ± 0.2 9.7 ± 0.6 10.3 ± 0.7 11.2 ± 0.5 Nikulinsky 7.2 ± 0.7 7.5 ± 0.2 8.7 ± 0.6 9.3 ± 0.3 9.7 ± 0.1 Notes: Wednesday 1 - a nutrient medium containing 1 mg / l of ascorbic acid Wednesday 2 - a nutrient medium containing 2 mg / l of ascorbic acid Wednesday 3 - a nutrient medium containing 1 mg / l of ascorbic acid + 2 g / l of activated carbon Wednesday 4 - a nutrient medium containing 2 mg / l of ascorbic acid + 2 g / l of activated carbon

Table 4 presents the results of studies on the dynamics of the development of regenerated plants obtained from various types of Nevsky potato cuttings in vitro according to the proposed method on medium No. 4 containing 20 g / l sucrose and an additional 2000 mg / l activated carbon, 2 mg / l ascorbic acid.

Table 4 Explant Type Age of the regenerant plant, days 10 twenty thirty Root length mm apical 14.3 ± 0.1 25.7 ± 0.5 49.7 ± 0.8 average 12.3 ± 0.3 25.1 ± 0.7 48.3 ± 0.7 basal 11.7 ± 0.2 24.6 ± 0.5 40.1 ± 0.7 Shoot length, cm apical 4.9 ± 0.8 8.8 ± 0.4 16.2 ± 0.5 average 3.9 ± 0.3 7.1 ± 0.7 12.7 ± 0.8 basal 3.1 ± 0.5 5.7 ± 0.6 10.4 ± 0.8 Number of nodes / leaves apical 4.1 ± 0.8 8.8 ± 0.4 15.2 ± 0.5 average 3.4 ± 0.3 6.6 ± 0.8 12.7 ± 0.6 basal 3.3 ± 0.4 6.8 ± 0.7 10.3 ± 0.7

By the end of the first week, regenerant plants obtained from microcranes of the apical (apical) and middle parts of the plant were 0.5-1.0 cm higher than plants obtained from the basal part. They formed the root system earlier and faster. Intensive root formation in the future ensured faster stem growth in regenerated plants. Plants obtained from cuttings of the basal part (2-3 cuttings of the basal zone) were 1.5-3 cm behind in growth by the end of the third week of cultivation. Cuttings obtained from the apical and middle parts of plants were ready for repeated cuttings in the experimental version by the end of the 3rd week, and the basal part - by the 4th-5th week.

The reproduction rate of potato regenerant plants 4 weeks after the cuttings is presented in Table 5.

Table 5 Grade Apical Medium Basal Zhukovsky early 15.2 ± 0.1 13.5 ± 0.2 12.3 ± 0.4 Luck 15.3 ± 0.2 12.2 ± 0.1 11.3 ± 0.4 Nevsky 11.2 ± 0.5 9.7 ± 0.6 9.1 ± 0.2 Nikulinsky 9.7 ± 0.1 9.3 ± 0.3 7.5 ± 0.2

At the same time, the ability to tuberize in cuttings of different tiers of the plant is different. Cuttings of the apical part of the plant form an insignificant number of small tubers, and much later than cuttings from the basal part of the plant, which form large micro-tubers. The experiments revealed that the mass of microclubs obtained on single-kidney potato micrograins in vitro is higher when using cuttings of the basal part (Table 6).

The micro-tubers obtained by the proposed method were physiologically mature, were well stored, and during subsequent cultivation in vivo formed healthy plants

Table 6 Grade The mass of micro-tubers, mg Apical part middle part Basal part Zhukovsky early 12.8 ± 1.5 36.8 ± 2.1 81.6 ± 6.7 Luck 19.8 ± 1.4 46.8 ± 5.8 95.8 ± 5.7 Nevsky 17.2 ± 2.7 25.2 ± 2.6 90.3 ± 3.4 Nikulinsky 25.2 ± 2.6 46.3 ± 3.5 100.8 ± 3.5

The obtained potato regenerant plants with well-developed mechanical and conductive stem tissues, an extensive root system, and well-developed leaves when transplanted into the soil easily adapted to in vivo conditions, which ensured their high survival rate (90-98%) compared to the prototype (70- 80%).

It follows that the proposed method allows to increase the reproduction rate of potato regenerated plants in vitro for 3 months of sequential cuttings by 0.2-1.5 thousand pieces from one source by reducing the period between repeated cuttings, increasing the yield of micrograins and test plants and their best survival in the ground, as well as additional micro-tubers.

Claims (1)

A method of microclonal propagation of potatoes, including cultivating healthy potato plants in vitro by microcrossing onto a nutrient medium containing macro- and microelements according to the prescription Murashige-Skoog, Fe-chelate, agar-agar, White and sucrose vitamins, obtaining regenerated plants and planting plants regenerants into the soil, characterized in that 3 mg / l of glycine, 1000-2000 mg / l of activated carbon and 1-2 mg / l of ascorbic acid are additionally introduced into the nutrient medium; s, middle and basal parts, and the growing of the apical and middle parts is carried out on a nutrient medium with sucrose in an amount of 20 g / l at a temperature of 23-25 ° C during the day and 17-18 ° C at night, followed by planting potato regenerated plants in the ground and the cultivation of the basal part is carried out on a nutrient medium with a sucrose content of 80 g / l in the dark at a temperature of 8-10 ° C, followed by in vitro potato micro-tubers.