CN109169277A - A kind of germ plasm resource in-vitro conservation method of dendrobium candidum - Google Patents
A kind of germ plasm resource in-vitro conservation method of dendrobium candidum Download PDFInfo
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- CN109169277A CN109169277A CN201811082917.5A CN201811082917A CN109169277A CN 109169277 A CN109169277 A CN 109169277A CN 201811082917 A CN201811082917 A CN 201811082917A CN 109169277 A CN109169277 A CN 109169277A
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- dendrobium candidum
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
Abstract
The present invention provides a kind of germ plasm resource in-vitro conservation method of dendrobium candidum, the techniqueflow of this method includes: the pretreatment of (1) dendrobium candidum plant；(2) acquisition of sterilizable material；(3) explant inoculation and Fiber differentiation；(4) germplasm grows preservation slowly；(5) kind germplasm rejuvenation proliferation；(6) water planting strengthening seedling and rooting；(7) hardening and transplanting.The present invention passes through the Effective Regulation to candidum tissue culturing condition, and replace subculture using two different culture mediums, the growth and breeding for saving material is effectively slowed down, subculture interval time is extended, reduce subculture number, to reduce possible hereditary variation while reducing and saving cost；Method of the invention is easy to operate, reliable and stable, can save germplasm up to long 4-5, and germplasm restoration ecosystem works well, and proliferation of propagation, the development and utilization for dendrobium candidum germ plasm resource can provide technical support within a short period of time.
Description
Technical field
The present invention relates to a kind of germ plasm resource in-vitro conservation methods of dendrobium candidum, belong to medicinal plant tissue cultures and kind
Technical field is deposited in quality guarantee.
Background technique
Dendrobium is the second largest category in orchid family, is herbaceos perennial.The platymiscium be mainly distributed on Tropical Asian,
Subtropical zone and Oceania etc. are regional, the whole world shared about more than 1500.There is 74 kind of 2 mutation in China at present, be distributed in more the Qinling Mountains with
The province in south, is distributed mainly on the ground such as Anhui, Jiangxi, Zhejiang, Fujian, Sichuan, Guangxi and Yunnan, wherein most with the yield in Yunnan
Greatly.Dendrobium Sw is most of to grow nonparasitically upon another plant as other orchids, and requires ecological environment very stringent；Happiness temperature
The environment of warm moist climate and half yin and half yang, can not resist cold, be suitable in nice and cool, wet, clear air ambient growth, suitable growth
Temperature is 15-28 degree, and suitable growth air humidity is 60% or more, not very stringent to clay fertilizer requirement, wild mostly loose and thick
It is grown on bark or trunk, some is also grown in crack of stone.Since Dendrobium Sw integrates medicinal and watches, in addition excessively
The features such as excavation utilizes, low reproduction rate and slow growth, all plants of the category are put into first-grade state protection plant list.
Dendrobium candidum gains the name also known as iron bracketplant, ribbed hedyotis herb etc. because its coat color is green in iron, is dendrobium nobile class plant
In the most rare, the highest kind of utility value, can be rated as the superfine product in dendrobium nobile.Dendrobium candidum is China's tradition rare Chinese medicine
Material, enjoys the good reputation of " gold in medicine ", and history tree is on the books.Dendrobium candidum is classified as " top grade ", road by Shennong's Herbal
Hiding is classified as first of " Chinese nine big mesonas ", civil to be called " help mesona ".Its is sweet in flavor, cold nature, returns stomach meridian, kidney channel,
With reinforcing stomach reg fluid, the effect of nourishing Yin and clearing heat, hurt for pyreticosis saliva, dry polydipsia, deficiency of stomach-Yin, deficiency of food is retched, after being ill abnormal heat
It does not move back, fire excess from yin deficiency, hectic fever due to yin labor heat, mesh is secretly unknown, muscles and bones impotence.Modern pharmacology research show the drug effect of dendrobium candidum at
Dividing mainly has dendrobium polysaccharide, amino acid, flavones, alkaloid and microelement etc., immune, antitumor, anti-oxidant, anti-ageing adjusting
Always, antifatigue, hypoglycemic, lowering blood pressure and blood fat, liver protection, shield stomach, anti-radiation, skin care, moisturizing, promotion hair growth etc.
Certain beneficial effect can be played, be modern metropolitan cities sub-health population enhancing immunity of organisms, relieve fatigue, promotes digest,
The preferred medicinal material for reducing blood glucose, has a vast market foreground and economic value.
Seeds of Dendrobium Candidum is minimum, and there are about 20,000 for each capsule, under field conditions (factors), Seeds of Dendrobium Candidum need according to
It could be sprouted by specific fungal induction, cause its natural propagation rate extremely low；In addition, dendrobium candidum wants the uniqueness of growing environment
It asks, dendrobium officinale slow growth is unable to satisfy medicinal demand.Further, since ecological disruption and exhaustive exploitation, wild iron sheet
Dendrobium Plants have been on the verge of exhaustion.In recent years, test tube seedling is bred with method for tissue culture rapid, high volume, it is short to improve breeding coefficient
A large amount of seedling is provided in period, is the effective way for solving iron sheet stone solution shortage of resources.Scale tissue culture produces iron sheet at present
Dendrobium nobile application is earliest and widest explant is aseptic seed, i.e., under isolated culture condition, seed forms protocorm after sprouting
Stem, protocorm can form seedling with direct development, protocorm can also be induced to generate a large amount of callus, divided again by callus
Change develops into seedling.But in order to realize the quick breeding of dendrobium candidum, manufacturing enterprise often uses the frequent subculture of protocorm, holds
Somaclonal variation is easily led to, so that the original germplasm saved be made to lose, it was reported that manufacturing enterprise average every 3 years will be more
Change provenance expand again it is numerous.Therefore, on the basis of tissue culture technique establishes vegetative propagation system, carry out germplasm Plantlet in vitro and grind
Study carefully, saves the elite germplasm of precious wild resource and artificial cultivation, it is very necessary.
Germplasm in-vitro conservation method can be divided into short-term, medium-term and long-term and three kinds of long-term preservation by the holding time.At present in iron
In terms of the short-term preservation of skin dendrobium nobile germ plasm resource, remarkable break-throughs are had been achieved for, it generally can be with using conventional organization cultural method
Save germplasm materials 1 year within, if the holding time it is too long may due to subculture number excessively cause make a variation and plant sexual involution；
In terms of long-term preservation, Liquid nitrogen method is generally used, but the method technical equipment is more demanding, at present still in examination
Test the stage.Therefore team of the present invention has made intensive studies in terms of the slow growth preserving seed of dendrobium candidum, by a series of
Technical measures, limitation germplasm materials growth, extend Subculture Time, reduce blastation, to be realization dendrobium candidum germplasm money
The medium-term and long-term preservation in source provides scientific theory and practical basis.
Summary of the invention
The present invention is dedicated to providing for defect or deficiency existing for current dendrobium candidum germ plasm resource Plantlet in vitro aspect
A kind of explant materials are convenient, subculture interval time is long, subculture number is few, preservation is at low cost, holding time 4-5, germplasm are extensive
Multiple growth result is good, can preferably distinct variety kind preserving seed method, so that meeting scientific development utilizes dendrobium candidum
The needs of germ plasm resource.
The purpose of the present invention is what is solved by the following technical programs:
A kind of germ plasm resource in-vitro conservation method of dendrobium candidum, which is characterized in that the techniqueflow of this method includes following step
It is rapid:
(1) dendrobium candidum plant pre-processes
The dendrobium candidum plant of robust growth, no disease and pests harm is selected, carries out root cleaning after materials, pre- place is then immersed into root
It manages in liquid, the preculture 5-7d under 8-10 DEG C of dark surrounds；
(2) acquisition of sterilizable material
The tender stem-segment with node of dendrobium candidum is selected, careful tripping is gently washed with suds or dish washing liquid, in tap water
Under rinse well；It being subsequently placed on superclean bench, first impregnates 1min with 70% isopropanol, sterile distilled water rinses 3-4 times, then
3 drop Tween 80s are added to sterilize 8-10min with 2.0% glutaraldehyde, during which needing constantly to shake makes stem section come into full contact with solution, nothing
Bacterium distilled water flushing 3-4 times, it is spare after blotting surface moisture with filter paper；
(3) explant inoculation and Fiber differentiation
Explant after above-mentioned disinfection is cut into the small stem section of long 1.0-1.5cm, every section includes an internode, then its level is inoculated with
In on bud inducement cultivation base, culture 38-44d induces the adventitious bud grown thickly；Condition of culture control is 25-27 DEG C of temperature, illumination
Intensity 1400-1600lx, photoperiod 10-12h light/12-14h are dark；
(4) germplasm grows preservation slowly
Adventitious bud wait induce it is long to 2.0-3.0cm high when, it is taken out from bottle, single plant is cut into and is transferred to subculture medium
1. or subculture medium 2. on, be first placed in the condition of 15-18 DEG C of temperature, intensity of illumination 800-1200lx, daily illumination 10-12h
Lower culture 15d, being subsequently placed under the condition of culture of low temperature, dim light grows it slowly, and every 14 months subcultures are primary, 2 kinds of subcultures
Culture medium is used alternatingly, and then proceeds by slow growth and saves；
(5) germplasm rejuvenation is proliferated
After the dendrobium candidum preserving seed phase, the adventitious bud that slow growth saves is transferred in rejuvenation culture medium, temperature is placed in
23-27 DEG C, intensity of illumination 1800-2200lx, restoration ecosystem under conditions of daily illumination 12h, every 28d or so subculture 1 time, subculture
The vigorous dendrobium candidum Multiple Buds of the growing way of 2-3 available proliferation；
(6) water planting strengthening seedling and rooting
Above-mentioned Multiple Buds are each individually cut and are transferred in strengthening seedling and rooting culture solution, 23-27 DEG C of temperature, intensity of illumination are placed in
The candidum tissue culturing seedling that 25-30d obtains robust growth is cultivated under conditions of 1800-2200lx, daily illumination 12-16h；
(7) hardening and transplanting
When tissue-cultured seedling root system length to 6-8 item, true leaf 5-6 piece, tissue-cultured seedling is moved in greenhouse, under room temperature, half dark conditions
Open bottleneck hardening 5-7d；Then tissue-cultured seedling is taken out out of bottle, root is soaked in 30% S-Ethyl ethylthio sulfonate missible oil, 500 times of dilutions
After steeping 15min, it is transplanted into the hole tray equipped with cultivation matrix, canopy temperature is controlled at 20-28 DEG C, humidity 65-85% at this time,
Obscurity 70-80% carries out conventional cultivation management.
The kind of the dendrobium candidum is " gloomy mountain 2 ".
The formula of pretreatment fluid in the step (1) are as follows: health polyphenol 3.5mg/L, 1.1 mg/L of furalane, dimension life
Plain 0.8 mg/L of C, 1.0 mg/L of methyl jasmonate spend No. 1 0.5g/L of treasured.
The formula of bud inducement cultivation base in the step (3) are as follows: 1/2MS+1.8mg/L6-BA+0.2mg/LNAA+
0.05mg/L triacontanol+cerous nitrate 0.5mg/L, pH value is 5.3 ~ 5.7.
Low temperature in the step (4), dim light condition of culture be 5-7 DEG C of temperature, intensity of illumination 500-800lx, illumination week
Phase is that 12h light/12h is dark.
The formula of subculture medium 1. in the step (4) are as follows: KNO3910 ~ 950mg/L, (NH4)2 SO4 158~
164mg/L, KH2PO4130 ~ 170mg/L, MgSO4·7H2O 263 ~ 273mg/L, CaCl2·2H2210 ~ 230mg/L of O,
MnSO4·4H2O 20 ~ 23mg/L, ZnSO4·7H2O 7 ~ 9mg/L, H3BO30.6 ~ 0.7mg/L of 4.0 ~ 4.4mg/L, KI,
CuSO4·5H2O 0.03 ~ 0.05mg/L, CoCl2·6H2O 0.022 ~ 0.026mg/L, Na2MoO4·2H2O 0.20~
0.30mg/L, FeSO4·7H2O 27 ~ 28mg/L, Na236 ~ 37mg/L of-EDTA, 11 ~ 15mg/L of altheine, proline-4 ~
6mg/L, 0.3 ~ 0.6mg/L of vitamin B1,0.4 ~ 0.7mg/L of vitamin B6,0.4 ~ 0.6mg/L of niacin, Titanium Citrate 2.0 ~
2.4g/L, 5.2 ~ 5.6mg/L of polyvinyl alcohol adjust 1.9 ~ 2.3mg/L of phosphine, 31 ~ 35g/L of sucrose, sweet potato 13 ~ 16g/L of mud, yellow rotten
3.4 ~ 3.8 mg/L of acid, ancymidol 0.07 ~ 0.09 mg/L, 2.5 ~ 3.0mg/L of sodium alginate, Prunella vulgaris leaching liquor 45 ~
51mL/L, PPM 2.5 ~ 2.9mg/L of antibacterial agent, agar 6 ~ 7g/L, pH are 5.3 ~ 5.7.
The formula of subculture medium 2. in the step (4) are as follows: KNO3 210 ~ 250mg/L, NH4H2PO4 770~810mg/
L, MgSO4·7H2O 184 ~ 190mg/L, Ca (NO3)2·2H2O 357 ~ 363mg/L, MnSO4·4H210 ~ 13mg/L of O,
ZnSO4·7H2O 7 ~ 9mg/L, H3BO3 4.0 ~ 4.4mg/L, KI 0.6 ~ 0.7mg/L, CuSO4·5H20.03 ~ 0.05mg/L of O,
CoCl2·6H2O 0.022 ~ 0.026mg/L, Na2MoO4·2H2O 0.20 ~ 0.30mg/L, FeSO4·7H227 ~ 28mg/L of O,
Na235 ~ 36mg/L of-EDTA, 85 ~ 100mg/L of inositol, 4 ~ 6mg/L of cysteine, 3 ~ 5mg/L of serine, vitamin B1 0.3 ~
0.6mg/L, 0.4 ~ 0.7mg/L of vitamin B6 adjust 1.5 ~ 1.7 mg/L of phosphine, taurine 0.6 ~ 0.8mg/L, PPM antibacterial agent
2.5 ~ 2.9mg/L, 4.3 ~ 5.3g/L of spirulina powder, 31 ~ 35g/L of sucrose, 4.1 ~ 4.5mg/L of trigonelline, Bitter Melon Juice 55 ~
60mL/L, 23 ~ 27mg/L of ferulic acid, 6.6 ~ 7.0 mg/L of putrescine, polyvinylpyrrolidone 0.5 ~ 0.6
G/L, agar 6 ~ 7g/L, pH are 5.3 ~ 5.7.
Subculture number is no more than 5 times in the step (4).
The formula of rejuvenation culture medium in the step (5) are as follows: MS+2-(3,4- dichlorophenoxy) triethylamine 2.1mg/L+5-
Base beta-lactam 32mg/L+ Prunella vulgaris leaching liquor 33mL/L+ citric acid 1.5g/L is urinated, pH is 5.6 ~ 6.0.
The formula of strengthening seedling and rooting culture solution in the step (6) are as follows: 1/2MS+NAA0.5mg/L+ fulvic acid 4.5g/L, pH
It is 5.6 ~ 6.0.
The present invention has the following advantages compared with prior art:
1, for the present invention using the stem-segment with node of dendrobium candidum " gloomy mountain 2 " as explant, materials are convenient, and power of regeneration is strong；By right
The pretreatment of dendrobium candidum plant improves the inductivity of adventitious bud, provides more preservation materials for the work of subsequent preserving seed
Material；Using the adventitious bud that induces as preserving seed material, after the preserving seed phase, can while restoration ecosystem into
The a large amount of proliferation of row, so that the development and utilization for dendrobium candidum provide a large amount of germ plasm resource.
2, the present invention is by changing external environmental condition, adjusting nutrient media components and using two different squamous subcultures
Base such as is used alternatingly at the means, has delayed the growth for saving material, has extended the subculture interval for saving material, avoid frequently after
The hereditary variation that generation may cause, while human and material resources are also saved, so that the preserving seed phase is extended to 4-5.
3, slow growth conditions are constantly in using the germplasm materials that method of the invention saves, germplasm can be kept excellent
Characteristic, and restoration ecosystem, formation intact plant can be used to expand numerous application and breeding research at any time within a short period of time.
Specific embodiment
With reference to embodiment, the embodiment of the present invention is furthur described in detail.
Embodiment 1
2013-2017, the dendrobium candidum cultivated using materials from the Jinhua, Zhejiang Province city mountain Xian Yuan dendrobium candidum planting base are " gloomy
Mountain 2 " is used as germplasm origin, and technology path according to the invention carries out the test of germplasm Plantlet in vitro, and detailed step is as follows:
(1) dendrobium candidum plant pre-processes
The dendrobium candidum plant of robust growth, no disease and pests harm is selected, root cleaning is carried out after materials, then by root in April, 2013
(the formula of pretreatment fluid are as follows: health polyphenol 3.5mg/L, 1.1 mg/L of furalane, vitamin C in pretreatment fluid is immersed in portion
0.8 mg/L, 1.0 mg/L of methyl jasmonate spend No. 1 0.5g/L of treasured), the preculture 5-7d under 8-10 DEG C of dark surrounds.
(2) acquisition of sterilizable material
The tender stem-segment with node of dendrobium candidum is selected, careful tripping is gently washed with suds or dish washing liquid, in tap water
Under rinse well；It being subsequently placed on superclean bench, first impregnates 1min with 70% isopropanol, sterile distilled water rinses 3-4 times, then
Add 2-3 drop Tween 80 to sterilize 8-10min with 2.0% glutaraldehyde, during which need constantly to shake that stem section is made to come into full contact with solution,
Sterile distilled water rinses 3-4 times, spare after blotting surface moisture with filter paper.
(3) explant inoculation and Fiber differentiation
Explant after above-mentioned disinfection is cut into the small stem section of long 1.0-1.5cm, every section includes an internode, then its level is inoculated with
In (culture medium prescription are as follows: 1/2MS+1.8mg/L6-BA+0.2mg/LNAA+0.05mg/L+ melissane on bud inducement cultivation base
Alcohol+cerous nitrate 0.5mg/L, pH value is that 5.5), culture 38-44d induces the adventitious bud grown thickly；Condition of culture control is temperature
26 DEG C, intensity of illumination 1500lx, photoperiod 10h light/14h it is dark.
(4) germplasm grows preservation slowly
Adventitious bud wait induce it is long to 2.0-3.0cm high when, it is taken out from bottle, single plant is cut into and is transferred to subculture medium
1. or subculture medium 2. on, be first placed under conditions of 16 DEG C of temperature, intensity of illumination 1000lx, daily illumination 12h and cultivate 15d,
Be subsequently placed in 6 DEG C of temperature, intensity of illumination 650lx, daily illumination 12h condition of culture under grow it slowly, every 14 months after
In generation, is primary, and 2 kinds of subculture mediums are used alternatingly, and then proceeds by slow growth and saves；Adventitious bud survival rate is counted after subculture 4 times
With average plant height increment；
The formula of subculture medium 1. are as follows: KNO3930mg/L, (NH4)2 SO4161mg/L, KH2PO4150mg/L, MgSO4·
7H2O 268mg/L, CaCl2·2H2O 220mg/L, MnSO4·4H2O 21.5mg/L, ZnSO4·7H2O 8mg/L, H3BO3
4.2mg/L, KI 0.65mg/L, CuSO4·5H2O 0.04mg/L, CoCl2·6H2O 0.024mg/L, Na2MoO4·2H2O
0.25mg/L, FeSO4·7H2O 27.5mg/L, Na2- EDTA 36.5mg/L, altheine 13mg/L, proline 5mg/L,
Vitamin B1 0.45mg/L, vitamin B6 0.55mg/L, niacin 0.5mg/L, Titanium Citrate 2.2g/L, polyvinyl alcohol 5.4mg/
L adjusts phosphine 2.1mg/L, sucrose 33g/L, sweet potato mud 14g/L, fulvic acid 3.6mg/L, ancymidol 0.08mg/L, alginic acid
Sodium 2.7mg/L, Prunella vulgaris leaching liquor 48mL/L, PPM antibacterial agent 2.7mg/L, agar 8g/L, pH 5.5；
The formula of subculture medium 2. are as follows: KNO3 230mg/L, NH4H2PO4 790mg/L, MgSO4·7H2O 187mg/L, Ca
(NO3)2·2H2O 360mg/L, MnSO4·4H2O 11.5mg/L, ZnSO4·7H2O 8mg/L, H3BO3 4.2mg/L, KI
0.65mg/L, CuSO4·5H2O 0.04mg/L, CoCl2·6H2O 0.024mg/L, Na2MoO4·2H2O 0.25mg/L,
FeSO4·7H2O 27.5mg/L, Na2- EDTA 36.5mg/L, inositol 92.5mg/L, cysteine 5mg/L, serine 4mg/L,
Vitamin B1 0.45mg/L, vitamin B6 0.55mg/L adjust phosphine 1.6mg/L, taurine 0.7mg/L, PPM antibacterial agent
2.7mg/L, spirulina powder 4.8g/L, sucrose 33g/L, trigonelline 4.3mg/L, Bitter Melon Juice 58mL/L, 3- methoxyl group -4- hydroxyl
Base cinnamic acid 25mg/L, putrescine 6.8 mg/L, polyvinylpyrrolidone 0.55g/L, agar 8g/L, pH 5.5.
(5) germplasm rejuvenation is proliferated
Dendrobium candidum preserving seed is transferred in rejuvenation culture medium (rejuvenation after 8 months 4 years, by the adventitious bud that slow growth saves
The formula of culture medium are as follows: MS+2- (3,4- dichlorophenoxy) triethylamine 2.1mg/L+5- urinates base beta-lactam 32mg/L+ Prunella vulgaris
Leaching liquor 33mL/L+ citric acid 1.5g/L, pH 5.8), it is placed in 25 DEG C of temperature, intensity of illumination 2000lx, daily illumination 12h
Under the conditions of restoration ecosystem, every 28d or so subculture 1 time observes adventitious bud growing way after subculture 2 times, counts adventitious bud proliferation coefficient.
(6) water planting strengthening seedling and rooting
Above-mentioned Multiple Buds are each individually cut and are transferred in strengthening seedling and rooting culture solution the (formula of culture solution are as follows: 1/2MS+
NAA0.5mg/L+ fulvic acid 4.5g/L, pH 5.8), it is placed in the item of 25 DEG C of temperature, intensity of illumination 2000lx, daily illumination 14h
After cultivating 25-30d under part, tissue-cultured seedling rooting rate is counted.
(7) hardening and transplanting
When tissue-cultured seedling root system length to 6-8 item, true leaf 5-6 piece, tissue-cultured seedling is moved in greenhouse, under room temperature, half dark conditions
Open bottleneck hardening 5-7d；Then tissue-cultured seedling is taken out out of bottle, root is soaked in 30% S-Ethyl ethylthio sulfonate missible oil, 500 times of dilutions
It after steeping 15min, is transplanted into the cultivation matrix in greenhouse, canopy temperature control at this time hides at 20-28 DEG C, humidity 65-85%
Luminosity 70-80% carries out conventional cultivation management；The transplant survival of dendrobium candidum " gloomy mountain 2 " tissue-cultured seedling is counted after transplanting 1 month
Rate.
Embodiment 2
Using germplasm origin " gloomy mountain 2 " same as Example 1, cancellation step (1) directly carries out adventitious bud inducing, culture
The adventitious bud induction frequency of Statistics Implementation example 1 and the present embodiment when 40d compares the influence pre-processed to adventitious bud inducing.
Embodiment 3
The preserving seed test on dendrobium candidum " gloomy mountain 2 " is carried out using germplasm origin same as Example 1 and techniqueflow,
The difference is that the condition of preserving seed in step (4) is changed to 4 DEG C of temperature, dark condition, the condition of culture derives from history forever
Loyalty etc. " the low temperature Plantlet in vitro of dendrobium candidum germ plasm resource ".
Embodiment 4
The preserving seed test on dendrobium candidum " gloomy mountain 2 " is carried out using germplasm origin same as Example 1 and techniqueflow,
Make the difference is that 1/2MS+0.5mg/L NAA+20mg/L sucrose+0.5g/L active carbon+7g/L agar is used alone in step (4)
For subculture medium, the formula is from " dendrobium candidum germplasm room temperature Plantlet in vitro " such as Shi Yongzhong.
Embodiment 5
The preserving seed test on dendrobium candidum " gloomy mountain 2 " is carried out using germplasm origin same as Example 1 and techniqueflow,
The difference is that 1/2MS+BA0.5mg/L+NAA0.1mg/L+ potato juice 50g/L+ bananas juice 50g/L is used alone in step (4)
+ active carbon 5g/L+ sucrose 25g/L is as subculture medium, and bravely " Dendrobium Sw germ plasm resource is in vitro from Huang for the formula
It saves ".
Embodiment 6
The preserving seed test on dendrobium candidum " gloomy mountain 2 " is carried out using germplasm origin same as Example 1 and techniqueflow,
The difference is that 1/2MS+1% sucrose is used alone in step (4) as subculture medium, the formula is from " iron sheets such as Luo Jifeng
The research of Rapid Propagation of Dendrobium and isolated lipid tissue ".
Embodiment 7
The preserving seed test on dendrobium candidum " gloomy mountain 2 " is carried out using germplasm origin same as Example 1 and techniqueflow,
The difference is that using 1/2MS+0.5mg/L BA as rejuvenation culture medium in step (6), the formula is from " iron sheets such as Shi Yongzhong
The low temperature Plantlet in vitro of dendrobium nobile germ plasm resource ".
As a result it counts
1, by " the gloomy mountain 2 " adventitious bud induction frequency of dendrobium candidum in embodiment 1,2 statistics such as the following table 1.
As can be seen from Table 1, the inductivity of dendrobium candidum adventitious bud can be improved in plant preprocess method of the invention, from
And more preservation materials are provided for the test of subsequent preserving seed.
2, " gloomy mountain 2 " in embodiment 1 and embodiment 3-7 adventitious bud survival rate and is averaged after preserving seed 8 months 4 years
Adventitious bud growing way after plant height increment, germplasm rejuvenation, adventitious bud proliferation coefficient, rooting rate, transplanting survival rate statistics such as the following table 2.
In conjunction with table 2, the data of comparing embodiment 1 and embodiment 3 find preserving seed condition of the invention more suitable for iron
The preserving seed of skin dendrobium nobile " gloomy mountain 2 "；The data of comparing embodiment 1 and embodiment 4-6, discovery are protected using method of the invention
" gloomy mountain 2 " is deposited after germplasm 8 months 4 years, the survival rate highest of adventitious bud, average plant height increment is also most, it is of the invention after
The growth of germplasm materials can effectively be delayed for the method that culture medium and 2 kinds of subculture mediums are used alternatingly, reduce nutrients
The consumption of matter, and it is remarkably improved the survival rate after its preservation；For germplasm materials after rejuvenation is proliferated, adventitious bud growing way is good
It is good, being proliferated, take root, be able to maintain stronger vigor in terms of transplanting, it is seen that subculture medium of the invention and preserving seed side
Method is smaller to the injury for saving material, and normal growth conditions can be restored within a short period of time by saving material；Comparing embodiment 1
With the data of embodiment 7, discovery is more advantageous to the restoration ecosystem of preserving seed material using rejuvenation culture medium of the invention.This hair
Bright dendrobium candidum germplasm in-vitro conservation method is steady for the plasm resource protection of dendrobium candidum, guarantee dendrobium candidum variety and quality
It is qualitative all to have great importance.
Above description is the detailed description for the present invention preferably possible embodiments, but embodiment is not limited to this hair
Bright patent claim, it is all the present invention suggested by technical spirit under completed same changes or modifications change, should all belong to
In the covered the scope of the patents of the present invention.
Claims (10)
1. a kind of germ plasm resource in-vitro conservation method of dendrobium candidum, which is characterized in that the techniqueflow of this method includes following
Step:
(1) dendrobium candidum plant pre-processes
The dendrobium candidum plant of robust growth, no disease and pests harm is selected, carries out root cleaning after materials, pre- place is then immersed into root
It manages in liquid, the preculture 5-7d under 8-10 DEG C of dark surrounds；
(2) acquisition of sterilizable material
The tender stem-segment with node of dendrobium candidum is selected, careful tripping is gently washed with suds or dish washing liquid, in tap water
Under rinse well；It being subsequently placed on superclean bench, first impregnates 1min with 70% isopropanol, sterile distilled water rinses 3-4 times, then
3 drop Tween 80s are added to sterilize 8-10min with 2.0% glutaraldehyde, during which needing constantly to shake makes stem section come into full contact with solution, nothing
Bacterium distilled water flushing 3-4 times, it is spare after blotting surface moisture with filter paper；
(3) explant inoculation and Fiber differentiation
Explant after above-mentioned disinfection is cut into the small stem section of long 1.0-1.5cm, every section includes an internode, then its level is inoculated with
In on bud inducement cultivation base, culture 38-44d induces the adventitious bud grown thickly；Condition of culture control is 25-27 DEG C of temperature, illumination
Intensity 1400-1600lx, photoperiod 10-12h light/12-14h are dark；
(4) germplasm grows preservation slowly
Adventitious bud wait induce it is long to 2.0-3.0cm high when, it is taken out from bottle, single plant is cut into and is transferred to subculture medium
1. or subculture medium 2. on, be first placed in the condition of 15-18 DEG C of temperature, intensity of illumination 800-1200lx, daily illumination 10-12h
Lower culture 15d, being subsequently placed under the condition of culture of low temperature, dim light grows it slowly, and every 14 months subcultures are primary, 2 kinds of subcultures
Culture medium is used alternatingly, and then proceeds by slow growth and saves；
(5) germplasm rejuvenation is proliferated
After the dendrobium candidum preserving seed phase, the adventitious bud that slow growth saves is transferred in rejuvenation culture medium, temperature is placed in
23-27 DEG C, intensity of illumination 1800-2200lx, restoration ecosystem under conditions of daily illumination 12h, every 28d or so subculture 1 time, subculture
The vigorous dendrobium candidum Multiple Buds of the growing way of 2-3 available proliferation；
(6) water planting strengthening seedling and rooting
Above-mentioned Multiple Buds are each individually cut and are transferred in strengthening seedling and rooting culture solution, 23-27 DEG C of temperature, intensity of illumination are placed in
The candidum tissue culturing seedling that 25-30d obtains robust growth is cultivated under conditions of 1800-2200lx, daily illumination 12-16h；
(7) hardening and transplanting
When tissue-cultured seedling root system length to 6-8 item, true leaf 5-6 piece, tissue-cultured seedling is moved in greenhouse, under room temperature, half dark conditions
Open bottleneck hardening 5-7d；Then tissue-cultured seedling is taken out out of bottle, root is soaked in 30% S-Ethyl ethylthio sulfonate missible oil, 500 times of dilutions
After steeping 15min, it is transplanted into the hole tray equipped with cultivation matrix, canopy temperature is controlled at 20-28 DEG C, humidity 65-85% at this time,
Obscurity 70-80% carries out conventional cultivation management.
2. a kind of germ plasm resource in-vitro conservation method of dendrobium candidum according to claim 1, which is characterized in that the iron
The kind of skin dendrobium nobile is " gloomy mountain 2 ".
3. a kind of germ plasm resource in-vitro conservation method of dendrobium candidum according to claim 1, which is characterized in that the step
Suddenly in (1) pretreatment fluid formula are as follows: health polyphenol 3.5mg/L, 1.1 mg/L of furalane, 0.8 mg/L of vitamin C,
1.0 mg/L of methyl jasmonate spends No. 1 0.5g/L of treasured.
4. a kind of germ plasm resource in-vitro conservation method of dendrobium candidum according to claim 1, which is characterized in that the step
Suddenly in (3) bud inducement cultivation base formula are as follows: 1/2MS+1.8mg/L6-BA+0.2mg/LNAA+0.05mg/L triacontanol+nitre
Sour cerium 0.5mg/L, pH value are 5.3 ~ 5.7.
5. a kind of germ plasm resource in-vitro conservation method of dendrobium candidum according to claim 1, which is characterized in that the step
Suddenly low temperature in (4), dim light condition of culture be 5-7 DEG C of temperature, intensity of illumination 500-800lx, periodicity of illumination are 12h light/12h
Secretly.
6. a kind of germ plasm resource in-vitro conservation method of dendrobium candidum according to claim 1, which is characterized in that the step
Suddenly the formula of subculture medium 1. in (4) are as follows: KNO3910 ~ 950mg/L, (NH4)2 SO4158 ~ 164mg/L, KH2PO4 130~
170mg/L, MgSO4·7H2O 263 ~ 273mg/L, CaCl2·2H2O 210 ~ 230mg/L, MnSO4·4H220 ~ 23mg/L of O,
ZnSO4·7H2O 7 ~ 9mg/L, H3BO34.0 ~ 4.4mg/L, KI 0.6 ~ 0.7mg/L, CuSO4·5H20.03 ~ 0.05mg/L of O,
CoCl2·6H2O 0.022 ~ 0.026mg/L, Na2MoO4·2H2O 0.20 ~ 0.30mg/L, FeSO4·7H227 ~ 28mg/L of O,
Na236 ~ 37mg/L of-EDTA, 11 ~ 15mg/L of altheine, proline-4 ~ 6mg/L, 0.3 ~ 0.6mg/L of vitamin B1, dimension
Raw element 0.4 ~ 0.7mg/L of B6,0.4 ~ 0.6mg/L of niacin, 2.0 ~ 2.4g/L of Titanium Citrate, 5.2 ~ 5.6mg/L of polyvinyl alcohol, are adjusted
Save 1.9 ~ 2.3mg/L of phosphine, 31 ~ 35g/L of sucrose, sweet potato 13 ~ 16g/L of mud, 3.4 ~ 3.8 mg/L of fulvic acid, ancymidol 0.07
~ 0.09 mg/L, 2.5 ~ 3.0mg/L of sodium alginate, Prunella vulgaris leaching liquor 45 ~ 51mL/L, PPM 2.5 ~ 2.9mg/L of antibacterial agent, fine jade
Rouge 6 ~ 7g/L, pH are 5.3 ~ 5.7.
7. a kind of germ plasm resource in-vitro conservation method of dendrobium candidum according to claim 1, which is characterized in that the step
Suddenly the formula of subculture medium 2. in (4) are as follows: KNO3 210 ~ 250mg/L, NH4H2PO4 770 ~ 810mg/L, MgSO4·7H2O
184 ~ 190mg/L, Ca (NO3)2·2H2O 357 ~ 363mg/L, MnSO4·4H2O 10 ~ 13mg/L, ZnSO4·7H2O 7~9mg/
L, H3BO3 4.0 ~ 4.4mg/L, KI 0.6 ~ 0.7mg/L, CuSO4·5H2O 0.03 ~ 0.05mg/L, CoCl2·6H2O 0.022~
0.026mg/L, Na2MoO4·2H2O 0.20 ~ 0.30mg/L, FeSO4·7H2O 27 ~ 28mg/L, Na235 ~ 36mg/L of-EDTA,
85 ~ 100mg/L of inositol, 4 ~ 6mg/L of cysteine, 3 ~ 5mg/L of serine, 0.3 ~ 0.6mg/L of vitamin B1, vitamin B6
0.4 ~ 0.7mg/L adjusts 1.5 ~ 1.7 mg/L of phosphine, taurine 0.6 ~ 0.8mg/L, PPM 2.5 ~ 2.9mg/L of antibacterial agent, spirulina
4.3 ~ 5.3g/L of powder, 31 ~ 35g/L of sucrose, 4.1 ~ 4.5mg/L of trigonelline, Bitter Melon Juice 55 ~ 60mL/L, 3- methoxyl group -4- hydroxyl
Base 23 ~ 27mg/L of cinnamic acid, 6.6 ~ 7.0 mg/L of putrescine, 0.5 ~ 0.6 g/L of polyvinylpyrrolidone, agar 6 ~ 7g/L, pH are
5.3~5.7。
8. a kind of germ plasm resource in-vitro conservation method of dendrobium candidum according to claim 1, which is characterized in that the step
Suddenly subculture number is no more than 5 times in (4).
9. a kind of germ plasm resource in-vitro conservation method of dendrobium candidum according to claim 1, which is characterized in that the step
Suddenly in (5) rejuvenation culture medium formula are as follows: MS+2-(3,4- dichlorophenoxy) triethylamine 2.1mg/L+5- urinate base beta-lactam
32mg/L+ Prunella vulgaris leaching liquor 33mL/L+ citric acid 1.5g/L, pH are 5.6 ~ 6.0.
10. a kind of germ plasm resource in-vitro conservation method of dendrobium candidum according to claim 1, which is characterized in that described
The formula of strengthening seedling and rooting culture solution in step (6) are as follows: 1/2MS+NAA0.5mg/L+ fulvic acid 4.5g/L, pH are 5.6 ~ 6.0.
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