CN103598101B - A kind of method of dendrobium aphyllum tissue-culture quick propagation - Google Patents
A kind of method of dendrobium aphyllum tissue-culture quick propagation Download PDFInfo
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Abstract
The invention provides a kind of method of dendrobium aphyllum tissue-culture quick propagation, sprouted by the mature seed that Dendrobium aphyllum (Roxb.) C. E. Fisch. selfing is ftractureed, the formation of protocorm and differentiation, and obtain whole plant after culture of rootage and transplant survival.Intercept with traditional high-order bud or plant division propagation method compared with, the present invention adopts tissue culture technology to carry out Dendrobium aphyllum (Roxb.) C. E. Fisch. breeding, and carried out the group training of Dendrobium aphyllum (Roxb.) C. E. Fisch. by the approach of protocorm, its reproduction speed is fast, a large amount of seedling can be obtained, seedling robust growth, can not only cultivate the Dendrobium aphyllum (Roxb.) C. E. Fisch. seedling that a large amount of proterties is consistent in a short time, transplanting survival rate is high, meet the need of market, and it is low to have cost, the cycle is short, and output height waits many advantages.
Description
Technical field
The present invention relates to a kind of method of dendrobium aphyllum tissue-culture quick propagation, belong to field of plant tissue culture technique.
Background technology
Dendrobium aphyllum (Roxb.) C. E. Fisch. (Den.aphyllum (Roxb.) C.E.Fischer) has another name called the sky bow stem of noble dendrobium, fall to hang down the spring stem of noble dendrobium, for the perennial herbaceous plant that grows nonparasitically upon another plant of the orchid family Dendrobium, be born in height above sea level 400 ~ 1500 meters, in sun-drenched evergreen broad-leaved forest on trunk or under sparse woods on rock, be mainly distributed in the country such as Yunnan Province of China and India, Burma, Thailand, Laos, Vietnam, Malaysia.It is important ornamental plants, and be again one of important former plant of the commodity traditional Chinese medicine stem of noble dendrobium, be used as medicine with fresh stem or dry stem, being that China is among the people commonly uses one of medicinal dendrobium kind simultaneously.Dendrobium aphyllum (Roxb.) C. E. Fisch. clearing heat and relieving fidgetness, clearing liver-fire for calming endogenous wind, diuresis is detoxified.Control the polydipsia of pyreticosis Tianjin wound, syndrome of upper hyperactivity of liver yang, food poisoning.Cure mainly cough, sore-throat, mouth parched and tongue scorched, empyrosis.
Along with people are to the increase of the medicinal demand of Dendrobium aphyllum (Roxb.) C. E. Fisch.; domestic wild resource reduces increasingly; Wild plant can not meet the daily demand of people far away; in order to wild resource can be protected again while meeting people's demand; walk sustainable development path, just in the urgent need to carrying out artificial cultivation by way of supplying raw materials.At present, the method for domestic breeding Dendrobium aphyllum (Roxb.) C. E. Fisch. mainly contains the intercepting of high-order bud, division propagation etc., and these methods require high to female parent material, and consumption is large, is also subject to the restriction in season, is difficult to carry out the spread of scale, standardized production and improved seeds.
Summary of the invention
Be difficult to carry out scale, standardized production to Dendrobium aphyllum (Roxb.) C. E. Fisch. for solving prior art; and the problems such as cultivation period length; the invention provides a kind of method of dendrobium aphyllum tissue-culture quick propagation; tissue culture technology is adopted to carry out Dendrobium aphyllum (Roxb.) C. E. Fisch. breeding; the tissue cultures being carried out Dendrobium aphyllum (Roxb.) C. E. Fisch. by the approach of protocorm can obtain a large amount of seedling; the Dendrobium aphyllum (Roxb.) C. E. Fisch. seedling that a large amount of proterties is consistent can not only be cultivated in a short time, meet the need of market, the protection of Dendrobium aphyllum (Roxb.) C. E. Fisch. resource also tool is of great significance.
The present invention is realized by following technical proposal: a kind of method of dendrobium aphyllum tissue-culture quick propagation, it is characterized in that through the following step:
(1) mature seed Dendrobium aphyllum (Roxb.) C. E. Fisch. selfing ftractureed, with after the hydrogen peroxide sterilization 10 ~ 15min of 3%, blots surface liquid, then is inoculated in by seed in following protocorm induction medium:
MS culture fluid
6-benzyl aminopurine 6-BA0.01 ~ 0.5mg/L
Methyl α-naphthyl acetate NAA0.05 ~ 0.5mg/L
Sucrose 30000mg/L
Agar 4500mg/L
Banana 50000mg/L
Potato 25000mg/L
pH5.8～6.0，
Be 1500 ~ 2000lx in intensity of illumination, under temperature is 25 ± 2 DEG C, light application time is the condition of 10 ~ 12h/d, cultivates 20 ~ 30 days, obtain the protocorm expanded, continue cultivation 50 ~ 60 days, make protocorm differentiation become seedling;
(2) step (1) gained seedling is transferred in following proliferated culture medium:
1/2MS culture fluid
6-benzyl aminopurine 6-BA0.01 ~ 0.5mg/L
Methyl α-naphthyl acetate NAA0.05 ~ 0.5mg/L
Sucrose 30000mg/L
Agar 4500mg/L
Banana 50000mg/L
Potato 25000mg/L
pH5.8～6.0，
Under the condition of culture identical with step (1), carry out squamous subculture 7 ~ 10 times, each cultivation cycle is 45 ~ 60d, obtains Multiple Buds;
(3) when the propagation radix of step (2) reaches production aequum, the Multiple Buds of step (2) below gained 2cm is returned step (2) and continue squamous subculture, the healthy and strong Multiple Buds of more than 2cm is inoculated in following media:
1/2MS culture fluid
Methyl α-naphthyl acetate NAA0.1 ~ 0.5mg/L
Indolebutyric acid IBA0.1 ~ 0.5mg/L
Sucrose 30000mg/L
Agar 5000mg/L
Banana 50000mg/L
Potato 25000mg/L
Active carbon 500 ~ 1000mg/L
pH5.8～6.0，
Intensity of illumination be 1500 ~ 2000lx, temperature is 25 ± 2 DEG C, light application time carries out culture of rootage 45 ~ 60d under being the condition of 10 ~ 12h/d, obtains seedling of taking root;
(4) after seedling of being taken root by step (3) gained carries out conventional hardening 6 ~ 8d under outdoor scattered light, seedling of taking root cleans its medium through routine, be placed in mass concentration be 5 ~ 10% carbendazim solution to sterilize 30min, transplanting with jig saw wood after drying moisture again plants in ventilation greenhouse, maintenance humidity is 70 ~ 80%, temperature is 15 ~ 28 DEG C, namely completes Dendrobium aphyllum (Roxb.) C. E. Fisch. tissue-culturing quick-propagation.
1/2MS medium in described step (2) and (3) is the culture fluid that in conventional MS culture fluid, full dose concentration of element reduces by half.
The present invention has following advantages and effect:
(1) in culturing room, realize whole year production by tissue culture propagation technology, both saved land resources, turn improved economic benefit, overcome the difficult point that seedling cannot carry out producing in the anniversary;
(2) carry out group training with the seed of physical maturity selfing cracking, adopt aseptic culture, improve the survival rate of seed tissue culture propagation; Replace traditional mercuric chloride to carry out sterilizing with hydrogen peroxide, decrease the pollution to environment;
(3) reproduction speed is fast, and 45 ~ 60 days is one-period, and breeding rate reaches 3 ~ 5, seedling robust growth;
(4) integration is optimized to the key link in whole Dendrobium aphyllum (Roxb.) C. E. Fisch. seeling industry techniqueflow, medium is improved, adopt the 1/2MS culture fluid that full dose element reduces by half respectively, reduce drug dosage, cost-saving, synchronously carry out bud inducement and Multiplying culture, simplify the cultivation program of tissue culture technology, only need the medium of two kinds of formulas, without calli induction, directly grow up to sprouting, root of hair is many, reduces the probability that indefinite bud morphs, the generation of effective control variant, transplanting survival rate is high, reaches 90%, is conducive to the plan of arranging production.Have cost low, the cycle is short, and output height waits many advantages.
Embodiment
In order to better the present invention is described, provide embodiments of the invention below, but content of the present invention is not limited in this.
Embodiment 1
(1) mature seed Dendrobium aphyllum (Roxb.) C. E. Fisch. selfing ftractureed, with after the hydrogen peroxide sterilization 10min of 3%, blots surface liquid, then is inoculated in by seed in following protocorm induction medium:
MS culture fluid
6-benzyl aminopurine 6-BA0.2mg/L
Methyl α-naphthyl acetate NAA0.1mg/L
Sucrose 30000mg/L
Agar 4500mg/L
Banana 50000mg/L
Potato 25000mg/L
pH5.8，
Be 1500lx in intensity of illumination, under temperature is 27 DEG C, light application time is the condition of 10h/d, cultivates 20 days, obtain the protocorm expanded, continue cultivation 55 days, make protocorm differentiation become seedling;
(2) step (1) gained seedling is transferred in following proliferated culture medium:
1/2MS culture fluid
6-benzyl aminopurine 6-BA0.05mg/L
Methyl α-naphthyl acetate NAA0.2mg/L
Sucrose 30000mg/L
Agar 4500mg/L
Banana 50000mg/L
Potato 25000mg/L
pH6.0，
Under the condition of culture identical with step (1), carry out squamous subculture 7 times, each cultivation cycle is 60d, obtains Multiple Buds;
(3) when the propagation radix of step (2) reaches production aequum, the Multiple Buds of step (2) below gained 2cm is returned step (2) and continue squamous subculture, the healthy and strong Multiple Buds of more than 2cm is inoculated in following media:
1/2MS culture fluid
Methyl α-naphthyl acetate NAA0.3mg/L
Indolebutyric acid IBA0.2mg/L
Sucrose 30000mg/L
Agar 5000mg/L
Banana 50000mg/L
Potato 25000mg/L
Active carbon 500mg/L
pH5.8，
Intensity of illumination be 2000lx, temperature is 27 DEG C, light application time carries out culture of rootage 50d under being the condition of 11h/d, obtains seedling of taking root;
(4) after seedling of being taken root by step (3) gained carries out conventional hardening 8d under outdoor scattered light, seedling of taking root cleans its medium through routine, be placed in mass concentration be 8% carbendazim solution to sterilize 30min, transplanting with jig saw wood after drying moisture again plants in ventilation greenhouse, maintenance humidity is 80%, temperature is 25 DEG C, namely completes Dendrobium aphyllum (Roxb.) C. E. Fisch. tissue-culturing quick-propagation.Transplanting survival rate can reach 90%.
Embodiment 2
(1) mature seed Dendrobium aphyllum (Roxb.) C. E. Fisch. selfing ftractureed, with after the hydrogen peroxide sterilization 13min of 3%, blots surface liquid, then is inoculated in by seed in following protocorm induction medium:
MS culture fluid
6-benzyl aminopurine 6-BA0.5mg/L
Methyl α-naphthyl acetate NAA0.05 ~ 0.5mg/L
Sucrose 30000mg/L
Agar 4500mg/L
Banana 50000mg/L
Potato 25000mg/L
pH5.9
Be 1800lx in intensity of illumination, under temperature is 25 DEG C, light application time is the condition of 11h/d, cultivates 25 days, obtain the protocorm expanded, continue cultivation 50 days, make protocorm differentiation become seedling;
(2) step (1) gained seedling is transferred in following proliferated culture medium:
1/2MS culture fluid
6-benzyl aminopurine 6-BA0.01mg/L
Methyl α-naphthyl acetate NAA0.5mg/L
Sucrose 30000mg/L
Agar 4500mg/L
Banana 50000mg/L
Potato 25000mg/L
pH5.9，
Under the condition of culture identical with step (1), carry out squamous subculture 9 times, each cultivation cycle is 50d, obtains Multiple Buds;
(3) when the propagation radix of step (2) reaches production aequum, the Multiple Buds of step (2) below gained 2cm is returned step (2) and continue squamous subculture, the healthy and strong Multiple Buds of more than 2cm is inoculated in following media:
1/2MS culture fluid
Methyl α-naphthyl acetate NAA0.5mg/L
Indolebutyric acid IBA0.5mg/L
Sucrose 30000mg/L
Agar 5000mg/L
Banana 50000mg/L
Potato 25000mg/L
Active carbon 750mg/L
pH6.0，
Intensity of illumination be 1500lx, temperature is 23 DEG C, light application time carries out culture of rootage 60d under being the condition of 12h/d, obtains seedling of taking root;
(4) after seedling of being taken root by step (3) gained carries out conventional hardening 7d under outdoor scattered light, seedling of taking root cleans its medium through routine, be placed in mass concentration be 5% carbendazim solution to sterilize 30min, transplanting with jig saw wood after drying moisture again plants in ventilation greenhouse, maintenance humidity is 75%, temperature is 15 DEG C, namely completes Dendrobium aphyllum (Roxb.) C. E. Fisch. tissue-culturing quick-propagation.Transplanting survival rate can reach 90%.
Embodiment 3
(1) mature seed Dendrobium aphyllum (Roxb.) C. E. Fisch. selfing ftractureed, with after the hydrogen peroxide sterilization 15min of 3%, blots surface liquid, then is inoculated in by seed in following protocorm induction medium:
MS culture fluid
6-benzyl aminopurine 6-BA0.01mg/L
Methyl α-naphthyl acetate NAA0.05mg/L
Sucrose 30000mg/L
Agar 4500mg/L
Banana 50000mg/L
Potato 25000mg/L
pH6.0，
Be 2000lx in intensity of illumination, under temperature is 27 DEG C, light application time is the condition of 12h/d, cultivates 30 days, obtain the protocorm expanded, continue cultivation 60 days, make protocorm differentiation become seedling;
(2) step (1) gained seedling is transferred in following proliferated culture medium:
1/2MS culture fluid
6-benzyl aminopurine 6-BA0.5mg/L
Methyl α-naphthyl acetate NAA0.05mg/L
Sucrose 30000mg/L
Agar 4500mg/L
Banana 50000mg/L
Potato 25000mg/L
pH5.8，
Under the condition of culture identical with step (1), carry out squamous subculture 10 times, each cultivation cycle is 45d, obtains Multiple Buds;
(3) when the propagation radix of step (2) reaches production aequum, the Multiple Buds of step (2) below gained 2cm is returned step (2) and continue squamous subculture, the healthy and strong Multiple Buds of more than 2cm is inoculated in following media:
1/2MS culture fluid
Methyl α-naphthyl acetate NAA0.1mg/L
Indolebutyric acid IBA0.1mg/L
Sucrose 30000mg/L
Agar 5000mg/L
Banana 50000mg/L
Potato 25000mg/L
Active carbon 1000mg/L
pH5.9，
Intensity of illumination be 1800lx, temperature is 25 DEG C, light application time carries out culture of rootage 45d under being the condition of 10h/d, obtains seedling of taking root;
(4) after seedling of being taken root by step (3) gained carries out conventional hardening 6d under outdoor scattered light, seedling of taking root cleans its medium through routine, be placed in mass concentration be 10% carbendazim solution to sterilize 30min, transplanting with jig saw wood after drying moisture again plants in ventilation greenhouse, maintenance humidity is 70%, temperature is 28 DEG C, namely completes Dendrobium aphyllum (Roxb.) C. E. Fisch. tissue-culturing quick-propagation.Transplanting survival rate can reach 90%.
Claims (1)
1. a method for dendrobium aphyllum tissue-culture quick propagation, is characterized in that through the following step:
(1) mature seed Dendrobium aphyllum (Roxb.) C. E. Fisch. selfing ftractureed, with after the hydrogen peroxide sterilization 10 ~ 15min of 3%, blots surface liquid, then is inoculated in by seed in following protocorm induction medium:
MS culture fluid
6-benzyl aminopurine 6-BA0.01 ~ 0.5mg/L
Methyl α-naphthyl acetate NAA0.05 ~ 0.5mg/L
Sucrose 30000mg/L
Agar 4500mg/L
Banana 50000mg/L
Potato 25000mg/L
pH5.8～6.0，
Be 1500 ~ 2000lx in intensity of illumination, under temperature is 25 ± 2 DEG C, light application time is the condition of 10 ~ 12h/d, cultivates 20 ~ 30 days, obtain the protocorm expanded, continue cultivation 50 ~ 60 days, make protocorm differentiation become seedling;
(2) step (1) gained seedling is transferred in following proliferated culture medium:
1/2MS culture fluid
6-benzyl aminopurine 6-BA0.01 ~ 0.5mg/L
Methyl α-naphthyl acetate NAA0.05 ~ 0.5mg/L
Sucrose 30000mg/L
Agar 4500mg/L
Banana 50000mg/L
Potato 25000mg/L
pH5.8～6.0，
Under the condition of culture identical with step (1), carry out squamous subculture 7 ~ 10 times, each cultivation cycle is 45 ~ 60d, obtains Multiple Buds; Described 1/2MS culture fluid is the culture fluid that in conventional MS culture fluid, full dose concentration of element reduces by half;
(3) when the propagation radix of step (2) reaches production aequum, the Multiple Buds of step (2) below gained 2cm is returned step (2) and continue squamous subculture, the healthy and strong Multiple Buds of more than 2cm is inoculated in following media:
1/2MS culture fluid
Methyl α-naphthyl acetate NAA0.1 ~ 0.5mg/L
Indolebutyric acid IBA0.1 ~ 0.5mg/L
Sucrose 30000mg/L
Agar 5000mg/L
Banana 50000mg/L
Potato 25000mg/L
Active carbon 500 ~ 1000mg/L
pH5.8～6.0，
Intensity of illumination be 1500 ~ 2000lx, temperature is 25 ± 2 DEG C, light application time carries out culture of rootage 45 ~ 60d under being the condition of 10 ~ 12h/d, obtains seedling of taking root; Described 1/2MS culture fluid is the culture fluid that in conventional MS culture fluid, full dose concentration of element reduces by half;
(4) after seedling of being taken root by step (3) gained carries out conventional hardening 6 ~ 8d under outdoor scattered light, seedling of taking root cleans its medium through routine, be placed in mass concentration be 5 ~ 10% carbendazim solution to sterilize 30min, transplanting with jig saw wood after drying moisture again plants in ventilation greenhouse, maintenance humidity is 70 ~ 80%, temperature is 15 ~ 28 DEG C, namely completes Dendrobium aphyllum (Roxb.) C. E. Fisch. tissue-culturing quick-propagation.
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| CN107926707A (en) * | 2017-12-18 | 2018-04-20 | 容县明曦铁皮石斛种植场 | A kind of Dendrobium crepidatum lindl et paxt. method for tissue culture |
| CN107836350A (en) * | 2017-12-18 | 2018-03-27 | 容县明曦铁皮石斛种植场 | One kind tool groove stem of noble dendrobium method for tissue culture |
| CN109392710A (en) * | 2018-11-14 | 2019-03-01 | 上海摩天农业科技有限公司 | A kind of method for culturing seedlings of dendrobium nobile |
| CN112237140A (en) * | 2020-10-16 | 2021-01-19 | 云南省农业科学院花卉研究所 | Method for blocking dendrobium officinale tissue culture seedling from being polluted by bacteria |
| CN112237139A (en) * | 2020-10-16 | 2021-01-19 | 云南省农业科学院花卉研究所 | Mutagenesis method for improving mutation rate of dendrobium officinale seeds |
| CN113331053B (en) * | 2021-05-26 | 2022-05-24 | 海南大学 | Separation breeding method of dendrobium pure-color flowers |
| CN113598045A (en) * | 2021-07-16 | 2021-11-05 | 上海植物园 | Culture medium for dendrobium yunnanense and rapid propagation method |
| CN116171855A (en) * | 2023-02-09 | 2023-05-30 | 云南山里红生物科技有限公司 | Dendrobium officinale tissue culture method and culture medium formula thereof |
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| CN101180950B (en) * | 2007-12-26 | 2010-12-08 | 浙江森禾种业股份有限公司 | Tissue cultivation rapid breeding method of spring dendrobium stem |
| TWI369179B (en) * | 2008-01-08 | 2012-08-01 | Nat Univ Kaohsiung | A method for producing polyploid plants of orchids |
| CN100586275C (en) * | 2008-01-18 | 2010-02-03 | 中国科学院昆明植物研究所 | Fast replication method for dendrobium |
| CN103371100B (en) * | 2012-04-17 | 2014-09-17 | 上海市农业科学院 | Tissue culture and rapid propagation method of nobile-type dendrobium seedlings |
| CN103155871B (en) * | 2013-03-07 | 2014-06-04 | 华中科技大学 | Dendrobium officinale sprout rapid propagation method with high efficiency |
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