CN109329049A - A kind of method of bletilla tissue culture seedlings Mycorrhizal - Google Patents
A kind of method of bletilla tissue culture seedlings Mycorrhizal Download PDFInfo
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- CN109329049A CN109329049A CN201811145716.5A CN201811145716A CN109329049A CN 109329049 A CN109329049 A CN 109329049A CN 201811145716 A CN201811145716 A CN 201811145716A CN 109329049 A CN109329049 A CN 109329049A
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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
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Abstract
The invention discloses a kind of methods of bletilla tissue culture seedlings Mycorrhizal, include the following steps: step 1, prepare liquid bacterial agent using the mycorrhizal fungi JSNLBJ-001 of the bletilla striata；Step 2, the liquid bacterial agent of suitable concentration is added in the culture medium in the white silk seedling stage before bletilla tissue culture seedlings transplanting toward bletilla tissue culture seedlings, carries out Mycorrhizal culture；Step 3, after the basal part of stem of tissue-cultured seedling cover with mycelia and it is observed that its root have mycelia attachment after, bletilla striata Mycorrhizal tissue-cultured seedling is taken out in tissue culture bottle, and removes the culture medium of its root and basal part of stem, then bletilla striata Mycorrhizal tissue-cultured seedling is transplanted in bletilla striata seedling medium and is tamed.The method of the present invention can provide good domesticated seedlings for bletilla striata field production, this method passes through before bletilla tissue culture seedlings are transplanted, liquid bacterial agent made from bletilla striata mycorrhizal fungi, which is added, makes its tissue-cultured seedling root system Mycorrhizal, after the tissue culture transplantation of seedlings of Mycorrhizal, survival rate is up to 99% or more, increment is big, and sprouting tiller is more and growing way is vigorous, and resistance is strong.
Description
Technical field
The present invention relates to a kind of methods of bletilla tissue culture seedlings Mycorrhizal, belong to seedling fostering technical field.
Background technique
Under field conditions (factors), orchid must establish symbiosis with mycorrhizal fungi, after forming mycorhiza, could complete it
Normal growth and development, the formation of mycorhiza be for orchid it is essential, mycorrhizal fungi to orchid provide carbon aquation
Object and other possible secondary metabolites, such as Metabolism, Vitamins and Hormones substance are closed, therefore, in a sense, orchid
It colonizes on fungi.The bletilla striata be perennial herb orchid, root structure be typical mycorhiza structure, seed without
Endosperm, the mycosymbiosis for needing to be adapted therewith under field conditions (factors) could sprout, and due to the rise of group culturation rapid propagating technology, solve
The problem that seed is sprouted, existing bletilla striata seedling tissue-culturing rapid propagation have become one of the main path of its cultivation seedling, but tissue-cultured seedling by
There is no the symbiosis therewith of corresponding mycorrhizal fungi in its root system, cause it during rooting culture, plants shoot survival percent, germination percentage
Low, resistance is poor, therefore, a kind of tissue culture method for the bletilla tissue culture seedlings that high-quality domesticated seedlings can be provided for bletilla striata field production
It develops necessary.
Summary of the invention
Goal of the invention: technical problem to be solved by the invention is to provide a kind of methods of bletilla tissue culture seedlings Mycorrhizal, should
Method is by being prepared into liquid bacterial agent using one plant of bletilla striata mycorrhizal fungi, being applied to bletilla striata tissue culture before bletilla tissue culture seedlings are transplanted
In seedling culture medium, make bletilla tissue culture seedlings Mycorrhizal, bletilla tissue culture seedlings Mycorrhizal carries out transplanting domestication again, and the tissue-cultured seedling of Mycorrhizal exists
During rooting culture, have the advantages that seedling high survival rate, germination percentage height and good stress resistance.
In order to solve the above technical problems, the technology used in the present invention means are as follows:
A kind of method of bletilla tissue culture seedlings Mycorrhizal, includes the following steps:
Step 1, liquid bacterial agent is prepared using the mycorrhizal fungi JSNLBJ-001 of the bletilla striata；
Step 2, suitable concentration is added in the culture medium in the white silk seedling stage before bletilla tissue culture seedlings transplanting toward bletilla tissue culture seedlings
Liquid bacterial agent, carry out Mycorrhizal culture；
Step 3, after the basal part of stem of tissue-cultured seedling cover with mycelia and it is observed that its root have mycelia attachment after, by bletilla striata mycorhiza
Change tissue-cultured seedling to take out in tissue culture bottle, and remove the culture medium of its root and basal part of stem, then moves bletilla striata Mycorrhizal tissue-cultured seedling
It plants and is tamed in bletilla striata seedling medium.
Wherein, in step 1, mycorrhizal fungi is pale purple purple spore bacterium (Purpureocilliumlilacinum), is preserved in
State's Microbiological Culture Collection administration committee common micro-organisms, deposit number are CGMCC NO:13690.
Wherein, in step 1, the production method of liquid bacterial agent are as follows: by mycorrhizal fungi JSNLBJ-001 bacterial strain from low-temperature preservation
Slant tube actication of culture after, be inoculated in PDA culture dish, in 24~30 DEG C of 5~10d of constant temperature incubation, then on bacterium colony side
Edge punching accesses bacteria cake in fluid nutrient medium, liquid 7~10d of shake culture, 26~28 DEG C of cultivation temperature, revolving speed at bacteria cake
120~160rpm；After spherical mycelia covers with fluid nutrient medium, puts it into blender and be broken into the liquid bacteria of suspension
Agent.
Wherein, the bletilla tissue culture seedlings of Mycorrhizal are used in the method for the present invention, for the finished product tissue-cultured seedling that can go out tissue culture bottle transplanting.
Wherein, the composition of the fluid nutrient medium are as follows: oat 20~30g/L+ glucose 25~30g/L+ calcium chloride dihydrate
2.5g/L+ 80~120g/L of potato, Medium's PH Value are 5.8~6.5；Wherein, potato is its leachate.Fluid nutrient medium
It is dispensed into after preparation in fungi triangular flask or culture bottle, dispensed loading amount is 80~100mL/ bottles, is then placed in high-pressure sterilizing pot
High pressure sterilization accesses bacteria cake after cooling.
Wherein, in step 2, the liquid bacterial agent additive amount is 10~15mL/ bottles.
Wherein, in step 2, after liquid bacterial agent accesses bletilla tissue culture seedlings, bottle cap is opened wide in greenhouse and places culture 10
~15d covers with tissue-cultured seedling basal part of stem to mycelia, after observing that there is mycelia attachment in bletilla tissue culture seedlings root, carries out out transplantation of seedlings.
Wherein, in step 3, bletilla striata seedling medium composition are as follows: 20~30 parts of perlite, 30~40 parts of rice husk, peat soil 100
~150 parts, 50~80 parts of fertile soil, 15~30 parts of calcium phosphate；After above-mentioned matrix components are mixed by formula volume ratio, carry out white
The transplanting of splendid achnatherum Mycorrhizal tissue-cultured seedling.
Wherein, in step 2, before bletilla tissue culture seedlings remove tissue culture bottle, tissue-cultured seedling is moved in greenhouse from culturing room,
When tissue-cultured seedling is moved in greenhouse, shading net need to being placed above greenhouse and being shaded, shading rate is 50~80%, is kept away
Exempt from illumination to burn by force very much tissue-cultured seedling, canopy temperature is controlled at 25~30 DEG C.
The utility model has the advantages that the method for the present invention can provide good domesticated seedlings for bletilla striata field production, this method passes through white
Before splendid achnatherum tissue culture transplantation of seedlings, liquid bacterial agent made from bletilla striata mycorrhizal fungi, which is added, makes its tissue-cultured seedling root system Mycorrhizal, the group of Mycorrhizal
After training transplantation of seedlings, for survival rate up to 99% or more, increment is big, and sprouting tiller is more and growing way is vigorous, and resistance is strong.With do not connect bacterium
The bletilla tissue culture seedlings of mycorrhiza fungi are compared, its germination percentage, survival rate and resistance are all significantly increased after bletilla tissue culture seedlings Mycorrhizal.
Specific embodiment
Technical scheme of the present invention is further explained combined with specific embodiments below.
Embodiment 1
The separation and identification of bacterial strain
Bacterial strain JSNLBJ-001 is in May, 2015 in Maoshan, Jiangsu Province genunie medicinal materials Co., Ltd of Jurong City of Jiangsu Province bletilla seed
Separation acquisition on the preferable plant of upgrowth situation in growing area.
Separation method are as follows: take wild fresh bletilla striata root, the tap water that the matrix soil on root segment surface is flowed rinses
After clean, the moisture on surface is blotted with filter paper on superclean bench.It is rinsed well again with the deionized water of sterilizing, then by several times
Immersion mass concentration is 75% alcohol 45s, and mass concentration is 2% liquor natrii hypochloritis's soaking disinfection 5min.After the completion of disinfection, use
The deionized water of sterilizing rinses 5~6 times to completely remove sodium hypochlorite, then the residual water of root surface is blotted with the filter paper of sterilizing
Then root segment is cut into the tissue segments of 0.5mm or so by drop with the scalpel of sterilizing.Tissue segments are transferred to added with streptomycin sulphate
On the PDA culture medium plate of (100 μ g/mL), 5 tissue segments are inoculated on each plate, every kind of culture medium connects 15 plates.It will divide
Endogenetic fungus from acquisition carries out tieback test, obtains bacterial strain uses therefor of the present invention, which is named as JSNLBJ-001, the bacterium
Strain can be obviously promoted the growth of bletilla striata seedling, improve the survival rate of seedling.
The colony characteristics of bacterial strain JSNLBJ-001 are as follows:
Morphological observation shows: cultivating 7d, colony diameter 3.5cm in PDA culture medium, grows slower.Bacterium colony ink
Green, felt shape, keratin, neat in edge have the transparent outer ring 1mm or so.
The amplification area ITS complete sequence is simultaneously sequenced, and the 16sRNA complete sequence that PCR amplification obtains is as follows.
The complete sequence (SEQ ID NO.1) that PCR amplification obtains:
CTAAGAAAGTTGGGCGTTTTACGGCGTGACCGCCTCCGCGCTCCGGTGCGAGGTGTGTGCTACTACGCA
GGGGAGGCTGCGGCGGGGTCGCCACTGCATTTCGGGGGCGGCTGGTGTGCCGTCCCCCAACACCGAGGCCCCCGGGG
GGGCTCGAGGGTTGAAATGACGCTCGAACAGGCATGCCCGCCAGAATGCTGGCGGGCGCAATGTGCGTTCAAAGATT
CGATGATTCACTGAATTCTGCAATTCACATTACTTATCGCATTTCGCTGCGTTCTTCATCGATGCCAGAACCAAGAG
ATCCGTTGTTGAAAGTTTTGATTCATTTGTTTTTGCTTGTGCAACTCAGAGAAGAAATTCCGCCCGCTGGGCGTAAT
GCAAGAGAGTTTGGGGTCCCTGCGGCGGGCGCCTGGGTCCGGCGCCGGCGCGGGGGCAGGCGGCCGGGGCGTTCCCG
CCGAGGCAACTGAGGTAAGGTTCACAGTGGGTTTGGGAGTTGTATAACTCGGTAATGATCCCTCCGCAGGTCACCCC
TACGGAA
Show that the bacterial strain is pale purple purple spore bacterium through morphology and molecular biology identification
(Purpureocilliumlilacinum), classification position is the snakelike cordyceps sinensis section purple spore of Ascomycota excrement shell Gammaproteobacteria Hypocreales
Belong to, bacterial strain number is JSNLBJ-001, has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, is protected
Hiding number is CGMCC NO:13690, and the deposit date is on March 10th, 2017, and preservation address is BeiChen West Road, Chaoyang District, BeiJing City
No. 3 Institute of Microorganism, Academia Sinica of No. 1 institute, postcode 100101.
Bletilla striata mycorrhizal fungi JSNLBJ-001 used in the method for the present invention is in 107629966 A of Publication No. CN
Patent in be documented.
Embodiment 2
The method of bletilla tissue culture seedlings Mycorrhizal of the present invention, includes the following steps:
Step 1, pale purple purple spore bacterium JSNLBJ-001 bacterial strain is inoculated in from after the slant tube actication of culture of low-temperature preservation
In PDA culture dish, in 28 DEG C of 6~8d of constant temperature incubation, then punch in colony edge into bacteria cake, it is spare；Match according to following components
Fluid nutrient medium processed: oat 30g/L+ glucose 30g/L+ calcium chloride dihydrate 2.5g/L+ potato 120g/L, Medium's PH Value
6.0；Wherein, potato uses its leachate；Fluid nutrient medium is dispensed into fungi triangular flask or culture bottle after preparing, point
Loading amount is 100mL/ bottles, is then placed in high pressure sterilization in high-pressure sterilizing pot, accesses bacteria cake after cooling；Fluid nutrient medium after sterilizing
It is put into isothermal vibration incubator after access mycorrhizal fungi bacteria cake and cultivates 10d, condition of culture are as follows: 28 DEG C of cultivation temperature, revolving speed
130rpm；After spherical mycelia covers with fluid nutrient medium, puts it into blender and be broken into the liquid bacterial agent of suspension；
Step 2, before bletilla tissue culture seedlings remove tissue culture bottle, tissue-cultured seedling is moved into the greenhouse that shading rate is 60% from culturing room
In greenhouse and bottle cap culture is opened, liquid bacterial agent made from step 1 is added in tissue-cultured seedling, carries out bletilla tissue culture seedlings Mycorrhizal
Culture, liquid bacterial agent additive amount are 15mL/ bottles；
Step 3, after liquid bacterial agent access bletilla tissue culture seedlings, bottle cap is opened wide in greenhouse and places culture 15d, to mycelia
Cover with tissue-cultured seedling basal part of stem, and it is observed that there is mycelia attachment in bletilla tissue culture seedlings root after, by bletilla striata Mycorrhizal tissue-cultured seedling from group
It is taken out in training bottle, and removes the culture medium of its root and basal part of stem, carry out out transplantation of seedlings, i.e., by bletilla striata Mycorrhizal tissue culture transplantation of seedlings
It is tamed into bletilla striata seedling medium；According to following component preparation bletilla striata seedling medium: 30 parts of perlite, 40 parts of rice husk, mud
100 parts, 80 parts of fertile soil, 30 parts of calcium phosphate of charcoal soil；Up to bletilla striata nursery after above-mentioned matrix component is mixed by volume
Matrix.
Embodiment 3
The method of bletilla tissue culture seedlings Mycorrhizal of the present invention, includes the following steps:
Step 1, pale purple purple spore bacterium JSNLBJ-001 bacterial strain is inoculated in from after the slant tube actication of culture of low-temperature preservation
In PDA culture dish, in 28 DEG C of 6~8d of constant temperature incubation, then punch in colony edge into bacteria cake, it is spare；Match according to following components
Fluid nutrient medium processed: oat 20g/L+ glucose 15g/L+ calcium chloride dihydrate 2.5g/L+ potato 80g/L, Medium's PH Value
6.0；Wherein, potato uses its leachate；Fluid nutrient medium is dispensed into fungi triangular flask or culture bottle after preparing, point
Loading amount is 100mL/ bottles, is then placed in high pressure sterilization in high-pressure sterilizing pot, accesses bacteria cake after cooling；Fluid nutrient medium after sterilizing
It is put into isothermal vibration incubator after access mycorrhizal fungi bacteria cake and cultivates 10d, condition of culture are as follows: 28 DEG C of cultivation temperature, revolving speed
130rpm: it after spherical mycelia covers with fluid nutrient medium, puts it into blender and is broken into the liquid bacterial agent of suspension；
Step 2, before bletilla tissue culture seedlings remove tissue culture bottle, tissue-cultured seedling is moved into the greenhouse that shading rate is 60% from culturing room
In greenhouse and bottle cap culture is opened, liquid bacterial agent made from step 1 is added in tissue-cultured seedling, carries out bletilla tissue culture seedlings Mycorrhizal
Culture, liquid bacterial agent additive amount are 10mL/ bottles；
Step 3, after liquid bacterial agent access bletilla tissue culture seedlings, bottle cap is opened wide in greenhouse and places culture 15d, to mycelia
Cover with tissue-cultured seedling basal part of stem, and it is observed that there is mycelia attachment in bletilla tissue culture seedlings root after, by bletilla striata Mycorrhizal tissue-cultured seedling from group
It is taken out in training bottle, and removes the culture medium of its root and basal part of stem, carry out out transplantation of seedlings, i.e., by bletilla striata Mycorrhizal tissue culture transplantation of seedlings
It is tamed into bletilla striata seedling medium；According to following component preparation bletilla striata seedling medium: 30 parts of perlite, 40 parts of rice husk, mud
100 parts, 80 parts of fertile soil, 30 parts of calcium phosphate of charcoal soil；Up to bletilla striata nursery after above-mentioned matrix component is mixed by volume
Matrix.
Comparative example 1
(1) before bletilla tissue culture seedlings bottle outlet, tissue-cultured seedling is moved in the greenhouse that shading rate is 60% simultaneously from culturing room
Bottle cap is opened, carries out practicing seedling 10d；
(2) according to following component preparation bletilla striata seedling medium: 30 parts of perlite, 40 parts of rice husk, 100 parts of peat soil, humic
80 parts, 30 parts of calcium phosphate of soil；Up to bletilla striata seedling medium after above-mentioned matrix component is mixed by volume；
(3) culture medium on bletilla tissue culture seedlings basal part of stem and root is cleaned, is planted in above-mentioned bletilla striata seedling medium.
Bletilla tissue culture seedlings in embodiment 2~3 and comparative example 1 are planted into the Mei Geshi in bletilla striata seedling medium
Applying example is 200 plants/group, and line space 10*10cm is inoculated in the seedling of plantation, after 90d, observes the bletilla striata that each embodiment is cultivated
Survival rate, germination percentage, plant height, the bulb size of tissue-cultured seedling, as shown in table 1.
Table 1
。
As shown in Table 1, the bletilla tissue culture seedlings obtained using the method for the present invention can make the survival rate of transplanting domestication, germination percentage all
It greatly improves, compared with the bletilla tissue culture seedlings for not connecing mycorrhizal fungi, its germination percentage after bletilla tissue culture seedlings Mycorrhizal of the present invention is survived
Rate, resistance all significantly improve.
Claims (8)
1. a kind of method of bletilla tissue culture seedlings Mycorrhizal, which comprises the steps of:
Step 1, liquid bacterial agent is prepared using the mycorrhizal fungi JSNLBJ-001 of the bletilla striata；
Step 2, the liquid of suitable concentration is added in the culture medium in the white silk seedling stage before bletilla tissue culture seedlings transplanting toward bletilla tissue culture seedlings
Body microbial inoculum carries out Mycorrhizal culture；
Step 3, after the basal part of stem of tissue-cultured seedling cover with mycelia and it is observed that its root have mycelia attachment after, by bletilla striata Mycorrhizal group
Training seedling takes out in tissue culture bottle, and removes the culture medium of its root and basal part of stem, is then transplanted to bletilla striata Mycorrhizal tissue-cultured seedling
It is tamed in bletilla striata seedling medium.
2. the method for bletilla tissue culture seedlings Mycorrhizal according to claim 1, it is characterised in that: in step 1, mycorrhizal fungi is
Pale purple purple spore bacterium (Purpureocilliumlilacinum), it is commonly micro- to be preserved in China Committee for Culture Collection of Microorganisms
Biology, deposit number are CGMCC NO:13690.
3. the method for bletilla tissue culture seedlings Mycorrhizal according to claim 1, it is characterised in that: in step 1, liquid bacterial agent
Production method are as follows: by mycorrhizal fungi JSNLBJ-001 bacterial strain from after the slant tube actication of culture of low-temperature preservation, be inoculated in PDA training
It supports in ware, in 24~30 DEG C of 5~10d of constant temperature incubation, is then punched in colony edge and access Liquid Culture at bacteria cake, and by bacteria cake
In base, liquid 7~10d of shake culture, 26~28 DEG C of cultivation temperature, 120~160rpm of revolving speed；Liquid training is covered with to spherical mycelia
After supporting base, puts it into blender and be broken into the liquid bacterial agent of suspension.
4. the method for bletilla tissue culture seedlings Mycorrhizal according to claim 3, it is characterised in that: the group of the fluid nutrient medium
Become: oat 20~30g/L+ glucose 25~30g/L+ calcium chloride dihydrate 2.5g/L+ 80~120g/L of potato, medium pH
Value is 5.8~6.5；Wherein, potato is its leachate.
5. the method for bletilla tissue culture seedlings Mycorrhizal according to claim 1, it is characterised in that: in step 2, the liquid bacteria
Agent additive amount is 10~15mL/ bottles.
6. the method for bletilla tissue culture seedlings Mycorrhizal according to claim 1, it is characterised in that: in step 2, in bletilla striata tissue culture
Before seedling removes tissue culture bottle, tissue-cultured seedling is moved in greenhouse from culturing room, greenhouse shading rate is 50~80%, in greenhouse
Temperature is at 25~30 DEG C.
7. the method for bletilla tissue culture seedlings Mycorrhizal according to claim 1, it is characterised in that: in step 2, liquid bacterial agent is connect
After entering bletilla tissue culture seedlings, bottle cap is opened wide in greenhouse and places 10~15d of culture, tissue-cultured seedling basal part of stem is covered with to mycelia, sees
Observe bletilla tissue culture seedlings root have mycelia attachment after, carry out out transplantation of seedlings.
8. the method for bletilla tissue culture seedlings Mycorrhizal according to claim 1, it is characterised in that: in step 3, bletilla striata nursery base
Matter composition are as follows: 20~30 parts of perlite, 30~40 parts of rice husk, 100~150 parts of peat soil, 50~80 parts of fertile soil, calcium phosphate 15
~30 parts；After above-mentioned matrix components are mixed by formula volume ratio, the transplanting of bletilla striata Mycorrhizal tissue-cultured seedling is carried out.
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| CN114080962A (en) * | 2021-11-17 | 2022-02-25 | 杭州市农业科学研究院 | Bletilla striata seedling growth promoting method based on Piriformospora indica |
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