CN103181325B - Tissue culture rapid propagation system of European hornbeam - Google Patents
Tissue culture rapid propagation system of European hornbeam Download PDFInfo
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- CN103181325B CN103181325B CN201310110563.1A CN201310110563A CN103181325B CN 103181325 B CN103181325 B CN 103181325B CN 201310110563 A CN201310110563 A CN 201310110563A CN 103181325 B CN103181325 B CN 103181325B
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Abstract
The invention relates to a tissue culture rapid propagation system of European hornbeam, which is characterized by comprising the following process steps of: selecting a stem tip which fully grows and inoculating onto an induction medium after surface sterilization; culturing for 4 weeks and systemically calculating the induction rate; shearing a 1.5-2.0-cm stem segment, inoculating onto a proliferation medium and systemically calculating the proliferation times after 60 days; shearing a 3-4-cm long single seedling which grows strongly, inoculating onto a rooting medium and inducing rooting; culturing for 50 days and systemically calculating the rooting rate, the average root number and the average root length; and when the root length is 3-5 cm, hardening a seedling and transplanting, and investigating the transplanting survival rate of the seedling after 30 days. The tissue culture rapid propagation system has the advantages that a European hornbeam test tube seedling can be induced to quickly proliferate and generate a large amount of adventitious buds, the survival rate is up to 85 percent, and an effective way is provided for industrial seedling culture of the European hornbeam.
Description
Technical field
The present invention relates to a kind of tissue culture and rapid propagation method of Carpinus betulus, belong to Plant Tissue Breeding field.
Background technology
Carpinus betulus (
carpinus betulus) have another name called Western carpinus cardata, deciduous tree, belong to Betulaceae (Betulaceae) Carpinus (
carpinusl.), be mainly distributed in Europe, Iran, Turkey.It is thick with leaves, and plant type is compact, blade profile is beautiful, and Forming Autumn-color Leaf is golden yellow, and fruit ear is peculiar, rather attractive in appearance, is road, urban green space and the good seeds of turning the four sides green.In addition, its multi-branched, extremely resistance to pruning, can form various moulding by artificial shaping, in order to enrich plants landscape, as being often trimmed to hedgerow in UK and USA garden in early days.Because its cold resistance is strong, wind resistance power is strong again, wide adaptability, and few damage by disease and insect, is the desirable seeds of Deposits in Eastern Coastal China urban landscaping and afforestation.Carpinus betulus spire contains cancer-resisting substance and is just being subject to increasing research, and its material is the quite hard handle that can manufacture floor, musical instrument, turning and various tool etc. also, also can manufacture high-quality charcoal.Therefore accelerate Carpinus betulus, introduce a fine variety and the paces of applying, make it in industry, urban forestry, afforestation and the afforestation of China, to be applied widely, market prospects are very wide.
At present, Carpinus betulus is mainly by seminal propagation, but after seed maturity, outside meeting forms hard shell, affects the germination rate of seed, causes germination rate lower.By lamination, process the germination that can promote seed, but the time of lamination is longer, and still lower through the germination rate of lamination processing seed.Carpinus betulus cuttage is difficult to take root, and yellow and blanching, colligation are processed and can be improved its cuttage root-taking rate, but rooting rate is still lower.The tissue-culturing rapid propagation research of Carpinus betulus rarely has report in the past, only indivedual scholars utilize stem section and terminal bud, as explant, Carpinus betulus is carried out to Vitro Quick Reproduction and have obtained whole plant, but established certain theoretical foundation for the factorial seedling growth of Carpinus betulus, but also had a certain distance from factorial seedling growth.
Summary of the invention
Main purpose of the present invention is to overcome in Carpinus betulus tissue culture procedures, and indefinite bud is difficult to the deficiency that induction, survival rate and reproduction coefficient are low, and a kind of quick breeding method for tissue culture of Carpinus betulus is provided.
Technical solution of the present invention: a kind of tissue culture and rapid propagation method of Carpinus betulus, is characterized in that the method comprises following processing step:
1) select the full stem apex of growth, after surface sterilization, be seeded in containing on the WPM minimal medium of 0.5 ~ 10.0mg/L 6-benzylaminopurine and carry out adventitious bud induction culture, in medium, also comprise 6.5g/L agar powder and 30.0g/L sucrose;
2) after indefinite bud grows, clip 1.5 ~ 2.0cm stem apex is at the 6-benzylaminopurine of additional 0.1 ~ 2.0mg/L, and the kinetin of 0.1 ~ 2.0mg/L, breeds cultivation on the indolebutyric acid plant hormone MS minimal medium of 0 ~ 0.5mg/L;
3) single seedling of clip robust growth, long 3 ~ 4cm carries out culture of rootage, and minimal medium macroelement reduces 1/2, and other elements remain unchanged, and are called for short 1/2 minimal medium; Additional 0 ~ 1.0 mg/L indolebutyric acid, the methyl α-naphthyl acetate of 0 ~ 1.0mg/; Cultivation temperature is 25 ± 2 ℃, and pH 5.8 ~ 6.0, illumination 12h/ days, and intensity of illumination is 2000 lx;
4) when single shoot root grows to 3 ~ 5cm, the bottle mouth sealing film of rooting tube plantlet is opened under ， culturing room scattered light and carried out opening hardening, test-tube plantlet is contacted with the external world gradually, in 1 week, ambient humidity is remained on to 70% ~ 80%, progressively improve the ability that seedling adapts to external environment; Within 5 days, take out afterwards test-tube plantlet and clean the culture material of root, be transplanted in seedling medium and grow, matrix is that 2:2:1 forms by the weight ratio of perlite, peat soil, garden mould, after fully mixing, with carbendazim, sterilizes.
Beneficial effect of the present invention: solved sprouting in Carpinus betulus group training process and be difficult for the problem that startup, survival rate and reproduction coefficient are low, by the adjustment of nutrient media components, induced Carpinus betulus to produce a large amount of indefinite buds and higher rooting rate, after transplanting, survival rate is up to 85%, a kind of quick breeding method for tissue culture of Carpinus betulus is provided, significant for the factorial seedling growth of Carpinus betulus.
Embodiment
A tissue culture and rapid propagation method for Carpinus betulus, comprises following processing step:
1) select the full stem apex of growth, after surface sterilization, being placed on and inducing cultivation, minimal medium containing 0.5 ~ 10.0mg/L 6-benzylaminopurine (benzylaminopurine is called for short BA) is WPM(Woody Plant Medium, be called for short WPM), mainly comprise following composition: NH
4nO
3400mg/L, Ca (NO
3)
24H
2o 556mg/L, CaCl
22H
2o 96mg/L, MgSO
47H
2o 370mg/L, KH
2pO
4170mg/L, K
2sO
4990mg/L, H
3bO
36.2mg/L, MnSO
4h
2o 17.7mg/L, ZnSO
47H
2o 8.6mg/L, NaMoO
42H
2o 0.25mg/L, CuSO
45H
2o 0.25mg/L, FeSO
47H
2o 27.8mg/L, Na
2-EDTA 37.3mg/L, inositol 100mg/L, glycine 2mg/L, nicotinic acid 0.5mg/L, nicotinic acid thiamine 1.0mg/L, puridoxine hydrochloride 0.5mg/L; Minimal medium also comprises agar powder and the 30.0g/L sucrose of 6.5g/L.
2) after adventitious bud inducing grows, 1.5 ~ 2.0cm stem apex is bred cultivation, and minimal medium is MS(Murashige and Skoog, is called for short MS); 6-benzylaminopurine (the benzylaminopurine of additional 0.1 ~ 2.0mg/L, be called for short BA), the kinetin of 0.1 ~ 2.0mg/L (kinetin is called for short KT), the indolebutyric acid of 0 ~ 0.5mg/L (indole butyric acid is called for short IBA) plant hormone; Also comprise the agar powder of 6.0g/L and the sucrose of 30.0g/L; Mainly comprise following composition:: NH
4nO
31650mg/L, KNO
31900mg/L, MgSO
47H
2o 370mg/L, KH
2pO
4170mg/L, CaCl
22H
2o 440 mg/L, KI 0.83mg/L, H
3bO
36.2mg/L, MnSO
44H
2o 22.3 mg/L, ZnSO
47H
2o 8.6mg/L, NaMoO
42H
2o 0.25mg/L, CuSO
45H
2o 0.025mg/L, CoCl
26H
2o 0.025mg/L, FeSO
47H
2o 27.8mg/L, Na
2-EDTA 37.25mg/L, inositol 100mg/L, glycine 2mg/L, nicotinic acid 0.5mg/L, puridoxine hydrochloride 0.5mg/L, thiamine hydrochloride 0.1 mg/L.
3) single seedling of clip robust growth, long 3 ~ 4cm carries out culture of rootage, and minimal medium is that 1/2WPM(WPM medium macroelement reduces 1/2, and other elements remain unchanged, and are called for short 1/2WPM) medium, mainly comprise following composition: NH
4nO
3200mg/L, Ca (NO
3)
24H
2o 278mg/L, CaCl
22H
2o 48mg/L, MgSO
47H
2o 185mg/L, KH
2pO
485mg/L, K
2sO
4495mg/L, H
3bO
36.2mg/L, MnSO
4h
2o 17.7mg/L, ZnSO
47H
2o 8.6mg/L, NaMoO
42H
2o 0.25mg/L, CuSO
45H
2o 0.25mg/L, FeSO
47H
2o 27.8mg/L, Na
2-EDTA 37.3mg/L, inositol 100mg/L, glycine 2mg/L, nicotinic acid 0.5mg/L, nicotinic acid thiamine 1.0mg/L, puridoxine hydrochloride 0.5mg/L; Additional 0 ~ 1.0 mg/L indolebutyric acid (indole butyric acid is called for short IBA), the methyl α-naphthyl acetate of 0 ~ 1.0mg/ (naphthylene acetic acid is called for short NAA); The sucrose that also comprises 6.5g/L agar powder and 15.0g/L, pH 5.8 ~ 6.0, illumination 12h/d, intensity of illumination is 2000 lx.While being cultured to 60 days, add up its propagation multiple, average plant height, rooting rate, mean elements and average root long.When root grows to 3 ~ 5cm, the bottle mouth sealing film of rooting tube plantlet is opened under ， culturing room scattered light and carried out opening hardening, test-tube plantlet is contacted with the external world gradually, in 1 week, ambient humidity is remained on to 70% ~ 80%, progressively improve the ability that seedling adapts to external environment.After 5d, take out test-tube plantlet and clean the culture material of root, be transplanted in seedling medium and grow, matrix is comprised of perlite, peat soil, garden mould 2:2:1, after fully mixing, with carbendazim, sterilizes.
Choose the full stem apex of growth, after surface sterilization, be seeded on inducing culture; Cultivate 4 weeks statistics inductivities.Clip 1.5 ~ 2.0cm stem section is inoculated on proliferated culture medium, 60 days statistics propagation multiples.Single seedling of clip robust growth, long 3 ~ 4cm is inoculated in root induction on root media.Cultivate 50 days statistics rooting rates, mean elements and average root long.When root grows to 3 ~ 5cm, carry out acclimatization and transplants, after 30 days, investigate its transplanting survival rate.
Embodiment 1
Choose the full stem apex of growth, after surface sterilization, be seeded on the inducing culture of additional 0.5mg/L BA; Minimal medium is WPM, and minimal medium also comprises the sucrose of 6.5g/L agar powder and 30.0g/L.Treat that indefinite bud grows, clip 1.5 ~ 2.0cm stem section is bred, and minimal medium is MS; Additional plant hormone is that the BA of 0.1mg/L is, the IBA of the KT of 0.5mg/L and 0.01mg/L; Also comprise the agar powder of 6.0g/L and the sucrose of 30.0g/L.60 days statistics propagation multiples.Single seedling of clip robust growth, long 3 ~ 4cm carries out culture of rootage, and minimal medium is 1/2WPM; Additional plant hormone is the NAA of 0.01mg/L, the IBA of 0.5mg/L; Also comprise the agar powder of 6.5g/L and the sucrose of 15.0g/L, PH 5.8 ~ 6.0, illumination 12h/d, and intensity of illumination is 2000 lx.Within 60 days, statistics propagation multiple, average plant height, rooting rate, mean elements and average root are long afterwards.Result shows that each explant on average breaks up 2.86 indefinite buds, and average bud reaches 3.25cm; Rooting rate reaches 56.8%, and mean elements reaches 2.95, and average root reaches 4.48cm.The bottle mouth sealing film of rooting tube plantlet is opened under ， culturing room scattered light and carried out opening hardening, test-tube plantlet is contacted with the external world gradually, in 1 week, ambient humidity is remained on to 70% ~ 80%, progressively improve the ability that seedling adapts to external environment.After 5d, take out test-tube plantlet and clean the culture material of root, be transplanted in seedling medium and grow, matrix is comprised of perlite, peat soil, garden mould 2:2:1, and after fully mixing, with carbendazim sterilization, survival rate reaches 70%.
Embodiment 2
Choose the full stem apex of growth, after surface sterilization, be seeded on the inducing culture of additional 2.0mg/L BA; Minimal medium is WPM, and minimal medium also comprises the sucrose of 6.5g/L agar powder and 30.0g/L.Treat that indefinite bud grows, clip 1.5 ~ 2.0cm stem section is bred, and minimal medium is MS; Additional plant hormone is that the BA of 1.0mg/L is, the IBA of the KT of 2.0mg/L and 0.01mg/L; Also comprise the agar powder of 6.0g/L and the sucrose of 30.0g/L.60 days statistics propagation multiples.Single seedling of clip robust growth, long 3 ~ 4cm carries out culture of rootage, and minimal medium is 1/2WPM; Additional plant hormone is the NAA of 0.5mg/L, the IBA of 0.5mg/L; Also comprise the agar powder of 6.5g/L and the sucrose of 15.0g/L, PH 5.8 ~ 6.0, illumination 12h/d, and intensity of illumination is 2000 lx.Within 60 days, statistics propagation multiple, average plant height, rooting rate, mean elements and average root are long afterwards.Result shows that each explant on average breaks up 2.54 indefinite buds, and average bud reaches 3.89cm; Rooting rate reaches 65.8%, and mean elements reaches 2.35, and average root reaches 3.08cm.The bottle mouth sealing film of rooting tube plantlet is opened under ， culturing room scattered light and carried out opening hardening, test-tube plantlet is contacted with the external world gradually, in 1 week, ambient humidity is remained on to 70% ~ 80%, progressively improve the ability that seedling adapts to external environment.After 5d, take out test-tube plantlet and clean the culture material of root, be transplanted in seedling medium and grow, matrix is comprised of perlite, peat soil, garden mould 2:2:1, and after fully mixing, with carbendazim sterilization, survival rate reaches 65%.
Embodiment 3
Choose the full stem apex of growth, after surface sterilization, be seeded on the inducing culture of additional 1.0mg/L BA; Minimal medium is WPM, and minimal medium also comprises the sucrose of 6.5g/L agar powder and 30.0g/L.Treat that indefinite bud grows, clip 1.5 ~ 2.0cm stem section is bred, and minimal medium is MS; Additional plant hormone is that the BA of 1.0 mg/L is, the IBA of the KT of 0.5mg/L and 0.5mg/L; Also comprise the agar powder of 6.0g/L and the sucrose of 30.0g/L.60 days statistics propagation multiples.Single seedling of clip robust growth, long 3 ~ 4cm carries out culture of rootage, and minimal medium is 1/2WPM; Additional plant hormone is the NAA of 0.01mg/L; Also comprise the agar powder of 6.5g/L and the sucrose of 15.0g/L, PH 5.8 ~ 6.0, illumination 12h/d, and intensity of illumination is 2000 lx.Within 60 days, statistics propagation multiple, average plant height, rooting rate, mean elements and average root are long afterwards.Result shows that each explant on average breaks up 4.06 indefinite buds, and average bud reaches 3.87cm; Rooting rate reaches 76.8%, and mean elements reaches 3.65, and average root reaches 3.76cm.The bottle mouth sealing film of rooting tube plantlet is opened under ， culturing room scattered light and carried out opening hardening, test-tube plantlet is contacted with the external world gradually, in 1 week, ambient humidity is remained on to 70% ~ 80%, progressively improve the ability that seedling adapts to external environment.After 5d, take out test-tube plantlet and clean the culture material of root, be transplanted in seedling medium and grow, matrix is comprised of perlite, peat soil, garden mould 2:2:1, and after fully mixing, with carbendazim sterilization, transplanting survival rate is up to 85%.
Claims (1)
1. a tissue culture and rapid propagation method for Carpinus betulus, is characterized in that the method comprises following processing step:
1) choose the full stem apex of growth, after surface sterilization, be seeded on the inducing culture of additional 0.5mg/L BA; Minimal medium is WPM, and minimal medium also comprises the sucrose of 6.5g/L agar powder and 30.0g/L;
2) treat that indefinite bud grows, clip 1.5 ~ 2.0cm stem section is bred, and minimal medium is MS; Additional plant hormone is that the BA of 0.1mg/L is, the IBA of the KT of 0.5mg/L and 0.01mg/L; Also comprise the agar powder of 6.0g/L and the sucrose of 30.0g/L; 60 days statistics propagation multiples;
3) single seedling of clip robust growth, long 3 ~ 4cm carries out culture of rootage, and minimal medium is 1/2WPM; Additional plant hormone is the NAA of 0.01mg/L, the IBA of 0.5mg/L; Also comprise the agar powder of 6.5g/L and the sucrose of 15.0g/L, pH 5.8 ~ 6.0, illumination 12h/d, and intensity of illumination is 2000 lx;
4) within 60 days, statistics propagation multiple, average plant height, rooting rate, mean elements and average root are long afterwards; Result shows that each explant on average breaks up 2.86 indefinite buds, and average bud reaches 3.25cm; Rooting rate reaches 56.8%, and mean elements reaches 2.95, and average root reaches 4.48cm; The bottle mouth sealing film of rooting tube plantlet is opened under ， culturing room scattered light and carried out opening hardening, test-tube plantlet is contacted with the external world gradually, in 1 week, ambient humidity is remained on to 70% ~ 80%, progressively improve the ability that seedling adapts to external environment;
5) after 5d, take out the culture material that test-tube plantlet is cleaned root, be transplanted in seedling medium and grow, matrix is comprised of perlite, peat soil, garden mould 2:2:1, and after fully mixing, with carbendazim sterilization, survival rate reaches 70%.
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| CN104855258B (en) * | 2015-04-27 | 2017-04-12 | 南京林业大学 | Hormone treatment method for improving water culture rooting of carpinus betulus |
| CN108353785A (en) * | 2017-11-12 | 2018-08-03 | 柳州市天立农林科技有限公司 | A kind of orchid mating system |
| CN112567990A (en) * | 2020-11-28 | 2021-03-30 | 巢湖市年晟农业生态有限公司 | Cutting seedling method for improving survival rate of tea trees in alpine mountain areas |
| CN115804341A (en) * | 2022-11-18 | 2023-03-17 | 中国科学院武汉植物园 | Tissue culture method for rapidly propagating African white ginseng |
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