SU1581741A1 - Method of microclonal reproduction of aspen hybrides - Google Patents

Abstract

The invention relates to biotechnology and may be used in the reproduction of forest woody plants. The aim of the invention is to increase the yield of propagated plants and accelerate the multiplication process. The method consists in introducing the meristems of the axillary buds into the culture and the cultivation is carried out in three stages on the Murashige-Skooga nutrient medium modified with various phytohormone additives. 8 tab.

Description

The invention relates to biotechnology and may be used in the reproduction of forest woody plants in forestry and agriculture.

The purpose of the invention is to increase the yield of propagated plants and accelerate the process of reproduction.

The method consists in using apical and axillary kidney meristems of non-multiplying grafting of aspen hybrids with poplar as initial explants; , 15-0.25 mg / l 6-BAP, 0.05-0.15 mg / l IAA (medium 1), followed by separation, subculturing and lengthening shoots are carried out on the host medium with additives Q, 05-0, 2 mg / l 6-BAP, 0.01 o, 03 mg / l PUU in the absence of adenine and inositol (medium 2) with pos eduyuschim rooting plants in a basic medium with a reduced concentration twice as mineral salts containing 0.3-0.6 mg / L INC (Medium 3). Cultures were harvested under illumination with fluorescent lamps (5000 lux) at a 16-hour light period at 25 ° C 5 70% relative

air humidity. Rooted plants - regenerants are planted in the soil in the greenhouse, followed by planting in open ground.

The method is carried out as follows. Young non-ligneous shoots 3-5 cm long are washed in a mild soap solution, then washed with tap water. Plant material is sterilized first for 1 min in 70% alcohol and 20 min in a 10% solution of chloramine followed by

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washing 3 times with distilled water. Under sterile conditions (in the laminar), the buds are separated from the shoots and the zone of the meristem of the hyperacine or axillary bud is separated. The initial explant is placed on the automated nutrient medium 1 in test tubes. Medium 1 for the growth of the kidneys contains, in addition to the Murashige-Skoog basic medium, 15-30 mg / l of adenine sulfate, 0.15-0.25 mg / l of 6-BAP, and 0.05-0.015 mg / l of NAA. On this medium for 4-5 weeks, swelling, disclosure of the kidney growth occurs, followed by the formation of a conglomerate of short shoots with 2-3 small leaves (up to 20-50 shoots). The resulting shoot conglomerate is divided under a microscope into individual shoots and placed on medium 2, in which shoots grow in length and the buds of axillary buds are planted. This medium except the main nutrient medium contains 0.05 -. 0.20 mg / l 6-BAP and 0.01-0.03 mg / l NAA in the absence of adenine and inositol. On medium 2, numerous axillary shoots are observed. Thus, at each initial shoot, 2-3 shoots are formed in the leaf axils. When the shoots originating from the axillary buds are transferred to fresh medium 2, the activation process of the axillary buds growth (i.e., multiplication) is observed. The multiplication process can be repeated many times, thus achieving the maximum yield of the number of shoots from one explant. Thus, the process of differentiation and growth of plants is 6–9 weeks. Hybrid shoots that have reached 3-4 cm in length are transplanted to nutrient medium 3 for rooting. This medium consists of mineral salts according to Murassige-Skoog, the concentration of which is halved, nicotinic acid — 0.1 mg / l, pyridoxy-x -NS1 0.1 mg / l, thiamine - HC1 1.0 mg / l and IMC 0.3-0.6 mg / l. The sucrose concentration is 2%. On this medium, the escapes form roots in 7–10 days. When the roots reach 3-5 cm in length, the plants are transplanted into the soil. Plants rooted in the vessel

transplanted into non-sterile soil and acclimatized to external conditions. Plant survival is 85-90%.

The method is illustrated by the following examples.

Example 1. Highly heterogeneous aspen hybrids with poplar P.tretmila xR are used as breeding material. alba, P.alba x P.Bolleana. Apical and axillary buds taken from trees, 26–32 years old, from several hybrid combinations, aspen 23 x white poplar 12, aspen 29 x white poplar 95, white poplar x Bolle 8. Trees from trees taken in February-March , placed in vessels with water to induce apical bud development. Young non-lignified shoots 3-5 cm long are cut off, washed in a mild soap solution, then washed with tap water. Plant material is sterilized first for 1 min in 70% alcohol and 20 min in a 10% solution of chloramine, then washed three times with sterilized distilled water. Thereafter, under sterile conditions (cabinet with laminar airflow of the WADW), the buds are separated from the shoots, and the covering scales are removed from them. The original Explant consists of the zone of the meristem of the apical or axillary kidney with a length of 2.0-3.0 mm, Tubes with medium 1 (Table 1) with the addition of a BAP of 0.2 mg / l and IAA 0.1 mg / l (optimal concentrations) autoclave at 1 atm for 30 min, water at 1.5 atm for 1 h. Explants are placed on nutrient medium 1 in biological tubes (21x200 ml), which are closed with sterile gauze plugs. The tubes are placed on the light platform under illumination with fluorescent lamps (5,000 lux) for a 16-hour photoperiod at 25 ° C and 70% relative humidity. Within 4-5 weeks on medium 1, the buds swell, open and grow, followed by the formation of a conglomerate of short shoots with 2-3 leaves (up to 20-50 shoots). Under a microscope (in a laminar), a conglomerate of short shoots is divided into individual shoots and placed on medium 2 (Table 1) with the addition of a BAP of 0.1 mg / l and NUK 0.02 mg / l (optimal concentrations), on which growth of shoots in length and laying of axillary buds for 2-4 weeks. At this stage, numerous axillary shoots develop. Conduct 2-fold multiplication of shoots, 10

20

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from the axillary buds, i.e. On each initial shoot, 2-3 shoots are formed in the leaf axils. When these shoots, arising from the axillary buds, are transferred to fresh nutrient medium 2, the process of activation of the axillary buds is repeated, which in turn gives new shoots for a new transplant. Duration of differentiation and shoot growth takes 6-9 weeks depending on the clone. Hybrid shoots that reach 3-4 cm in length are transplanted onto nutrient medium 3 with an additional 0.5 mg / l IMC (optimal concentration) (Table 1) for rooting. Root percentage is 84-90% depending on the clone. On this medium, the shoots form roots in 7-10 days. When the roots reach a length of 3-5 cm, the plants are transplanted into the soil.

The transfer of plants is carried out as follows. Plants with formed roots are carefully removed from the agar, the adherent agar is carefully separated, washed in water and directly transferred to the soil with sand (1: 1). The plants are abundantly watered. Plant survival is 85-90%. Normal growth and development of these plants is observed.

Example 2: Branches from trees, taken in February-March, are placed in vessels with water to induce the development of apical buds. From young green shoots, the buds are separated and cultivated on medium 1 with various concentrations of hormonal substances.

Tables 2–4 present the limiting values and optimal concentrations of BAP and IAA, respectively.

Example 3: Plant material is the same as in Example 1 and 2. After cultivation on medium 1, the obtained multiple micro-shoots are separated and subcultured individually on medium 2 with different concentrations of BAP and NAA with the aim of elongation. The experimental values and the optimal concentration of BAS and NUA are determined experimentally.

The results are presented in table.5 6.

PRI me R 4. After cultivation and on media 1 and 2, the grown micropogees are transplanted onto medium 3 to promote the formation of roots with different

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ItMK concentrations. Limit values and optimal concentrations of IMC are determined experimentally.

The results are shown in table.7. The plants of several hybrid combinations are introduced into the culture. Data on the comparatively effective regeneration, growth and rooting of shoots t are given in Table 8.

The advantages of the proposed method are: the possibility of industrial use in forestry of valuable high heterotic aspen hybrids with poplar, which previously could not be used in production due to the impossibility of their reproduction by grafting; the possibility of mass production of planting material with preservation of the genotype from elite trees; adjusting the rate of micro-multiplication depending on the production conditions; the possibility of multiple multiplication, a high percentage of rooted capacity and plant survival (85-90%); reducing the time of differentiation and plant growth to 6-9 weeks; preparation of rooted plants in greenhouse conditions for the beginning of spring vegetation in open ground; possibility of growing planting material regardless of the season.

Claims (1)

Invention Formula The method of microclonal propagation of aspen hybrids, including the cultivation of axillary bud meristems on a modified Murashige-Skoog medium containing, KN03. 2H20, MgS (V7II70, KGO ,,, Ka4EDTAh 2H20, FeS04-7H O, NEV03, Mp80f-411 0 2p804 7NgO, KI, NazMo04-2U20, CuS04, CaCl z 6H, jO, sucrose, nicotinic acid, pyridoxine HC1, thiamine HC1 and water to obtain regenerated plants, followed by planting them in open ground is different in that, in order to increase the yield of propagated plants and accelerate the propagation process, the cultivation is carried out in three stages, at the same time initiation and forming a shoot conglomerate — inositol, adenine sulfate, 6-benzylaminopurine, indolylacetic acid are added to the nutrient medium, in the following ratio of components, mg / l: NH4N03 KN03 CaClg 2H20 MgS04 7NgO KH2P04 Na1EDTA 2H20 FeSo4 711 0 Н3ВОЭ MnSOlfl 4H70 ZnSO 4 -7HgO 1650 1900 440 370 170 37.3 27.8 6.2 22.3 8.6 0.83 0.25 0.025 0.025 30000 100 KI NaMo04-21ljO CuSOf-5H20. CoC1a 6H70 Sucrose Inositol  Nicotine acid 0.5 Pyridoxine HC1 0.1 Thiamine HC10.1 Adenine sulfate 15-30 6-Benzylaminopurin0, 15-0.25 Indoliluk Susin acid 0.05-0.15 Water up to 1 l In the second stage of subculturing elongation of shoots, 6-benzylaminopurine, aphthylacetic acid is added to the nutrient medium, with the following ratio of components, mg / l: 1650 1900 NH4N03 QOL) b CaClg-2HgO MgS04-7II20 KNgR04 Na TA-2H20 FeS04-7HaO H3B03 MnSCV 4H20 ZnS04-7HtO KI NaMo04 2H10 CuS04 5NgO Sucrose Nicotine acid Pyridoxine HC1 Thiamine HC1 6-Benzylaminopurine Naphthylace acid Water 440 370 170 37.3 27.8 6.2 22.3 8.6 0.83 0.25 0.025 0.025 30000 0.5 0, .1 0.1 0.05-0.20 0.01-0.03 To 1 l in the third stage, rooting of plants is added to the nutrient medium. indolyl butyric acid, in the following ratio of components, mg / l: five 0 five NH4N03 Kn08 CaС12.21ЦО MgS047H20 KH2P04 NaiEDTA2H20 FeS047H20 NEV03 MnS04 4H20 ZnS04-7NO TCI NaMo04 2NaO CuS04 5H20 CoCl -6H20 Sucrose Nicotine acid Pyridoxine HC1 Thiamine HC1 Indolilmasl on acid 825 950 220 185 85 18.65 13.9 3.1 11.15 4.3 0.415 0.125 0.0125 0.0255 20000 0.1 0.1 0.1 0.3-0.6 Table 1  50 91581741 . Continuation of table 1 ten Table The composition of the main Environment - Concentration of IAA, Efficiency of the environment - Murashige-Skoog, -igm / l of kidney% mg / l 1235 1 0 055d Naphthylacetic x 0.1080 acid (NUK) - - Qj01 - °, 1563 0.63fo Indolilmasl on Table3 Acid (IMC) - - 0,3- ---- .. -. 0.6 BAL Concentration, Average Length, mg / l Shoot, cm T and blitz a 2. oh, 053.7 T - 0.104.2 BAL Concentration, Growth Efficiency 0.202.9 (mg / l) kidney,% 20 Tabl. 0.1564 About 2080 IAA Concentration, I Medium Length About 2571mg / lpobegov, cm .. 1-1 I-IL-SChCH1-. - ± g - - PT-IIU IT-Mil G1I-G1II - 250,013,4 0.02 4.1 Table3.0.03 2.7 r i Table Sul concentration - Efficiency - adenine veil, mg / l | kidney growth,% IttlK concentration, (Efficacy of feedg / l non-formation,% 1565 20820,375 30690,590 350,684 ITBLITSA 8 HybridsEfficiency Effective- Efficiency growth nzolicity of rooting, rotate nyu Ogobra-X check, Chzovan, Z Aspen 23 x white poplar 12 tree 1) 809190 Aspen 79 x white poplar 9569 7885 White poplar x x Poplar Bolle 8 74 6884 Note. The growth efficiency of isolated buds is the number of buds introduced into the culture, expressed in% of the initial quantity, the effectiveness of shoot formation, the number of shoots grown in tissue culture, expressed in 15 from the initial quantity; rooting efficiency is the number of rooted plants possessing a high survival rate in the soil and good growth of shoots and roots, expressed in Z from the initial amount.