CN108935099A - The method for tissue culture of Snakegourd Fruit - Google Patents
The method for tissue culture of Snakegourd Fruit Download PDFInfo
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- CN108935099A CN108935099A CN201810677701.7A CN201810677701A CN108935099A CN 108935099 A CN108935099 A CN 108935099A CN 201810677701 A CN201810677701 A CN 201810677701A CN 108935099 A CN108935099 A CN 108935099A
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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 method for tissue culture of Snakegourd Fruit, it include: 1) to carry out Snakegourd Fruit cutting shoots as explant to be heat-treated de- bacterium, then be placed in alcoholic solution and carry out two-stage sterilization, be subsequently placed in initial culture base carry out Fiber differentiation, in proliferated culture medium carry out Multiplying culture obtain differentiation seedling；2) differentiation seedling is subjected in root media culture of rootage, is then placed in 5 DEG C or less progress low-temperature treatments to obtain tissue culturing seedling；3) by tissue culturing seedling's hardening；Wherein, initial culture base is the MS culture medium containing methyl α-naphthyl acetate NAA, 6-benzyladenine 6-BA and inositol；Proliferated culture medium is the MS culture medium containing zeatin ZT, kanamycins, 2,4-D and tomato juice；Root media contains the 1/2MS culture medium of paclobutrazol, indolebutyric acid IBA and banana puree.The method for tissue culture of the Snakegourd Fruit has excellent survival rate and breeding potential.
Description
Technical field
The present invention relates to tissue cultures, and in particular, to a kind of method for tissue culture of Snakegourd Fruit.
Background technique
Snakegourd Fruit is Curcurbitaceae, also known as snakegourd, belongs to perennial climbing herb, long up to 10 meters.Rhizomes is plump, cylindric,
Crust yellow.Stem multi-branched, it is hairless；Leaf alternate, subcircular or heart, dioecism；Male flower number piece raceme, rare list
Raw, corolla lobe obovate, female flower Dan Sheng, ovary is oval, and fruit is subsphaeroidal, orange red when ripe, the flowering fruit bearing stage 7-11 month.Snakegourd Fruit point
It is distributed in LiaoNing, China, North China, East China, Central-South, Shaanxi, Gansu, Sichuan, Guizhou and Yunnan.It is born in 200-1800 meters of height above sea level of mountain
In slope hayashishita, shrubbery, meadow and the village side Pang Tian.Yin Ben kind be traditional Chinese medicine radices trichosanthis and snakegourd, therefore in its distributing region,
Outside, it widely cultivates, is distributed in Korea, Japan, Vietnam and Laos.Snakegourd Fruit have it is antipyretic quench the thirst, diuresis, kobadrin the effects of.Kind
Son is containing rouge flower fat oil；Fruit contains triterpenoid saponin, organic acid, resin, carbohydrate, pigment；Root contains protein, saponin(e, acids.
There are two types of the propagation methods of Snakegourd Fruit, wherein root division: it is fresh that mid or late March takes the raw section white of 3-5
The old root of healthy and strong female plant is divided into the segment of 7-10cm, and cave plants, and is watered with water, and the emergence of about more than 10 days is annual to combine intertillage spraying fertilizer 2-
3 times.Seminal propagation: 9-10 month picking fruit, it is slightly soft to pericarp, seed is taken out with plant ash seed dressing and wipes pulp, and dry hiding is passed the winter；
Also ventilation can be hung on carpopodium.Propagating seeding in cold frame carries out in early spring, and by seed tip insertion soil, often water spray keeps seedbed wet
Profit, when Seed sprouting, start to ventilate, bed temperature control after 22 DEG C or so, about 10 days be unearthed, see true leaf stretching can go up basin or
Cultivation is divided to educate.Live streaming carries out in April, selects fertile soil, the good ground of draining, opens cave and uses sufficient base manure, and 30x40 centimetres of hill spacing.
Program request seed after earthing, then thick 2 centimetres of mud are covered, about two weeks is unearthed, and when there is true leaf 2, every cave stays 1 plant of seedling, to climing length to 50
Centimetre when insert draw bar.But Snakegourd Fruit wild resource is limited and is seriously destroyed, although there are a small amount of introducing and planting in various regions, produces
Amount is lacked far from meeting market demands.
Summary of the invention
The object of the present invention is to provide a kind of method for tissue culture of Snakegourd Fruit, the method for tissue culture of the Snakegourd Fruit has excellent
Survival rate and breeding potential.
To achieve the goals above, the present invention provides a kind of method for tissue culture of Snakegourd Fruit, comprising:
1) Snakegourd Fruit cutting shoots are carried out being heat-treated de- bacterium as explant, is then placed in alcoholic solution and carries out two-stage sterilization, so
Be placed in initial culture base carry out Fiber differentiation, in proliferated culture medium carry out Multiplying culture obtain differentiation seedling；
2) differentiation seedling is subjected in root media culture of rootage, is then placed in 5 DEG C or less progress low-temperature treatments to obtain
Tissue culturing seedling；
3) by tissue culturing seedling's hardening；
Wherein, step 1) -2) condition of culture at least conform to the following conditions: it is sterile and ventilative；Initial culture base is to contain naphthalene
The MS culture medium of acetic acid NAA, 6-benzyladenine 6-BA and inositol；Proliferated culture medium be containing zeatin ZT, kanamycins, 2,
The MS culture medium of 4-D and tomato juice；Root media contains the 1/2MS culture medium of paclobutrazol, indolebutyric acid IBA and banana puree.
In the above-mentioned technical solutions, the present invention then in turn through Fiber differentiation, is increased using Snakegourd Fruit cutting shoots as explant
Culture, culture of rootage, hardening are grown to obtain mature Snakegourd Fruit seedling, are made in turn in each culture processes using specific culture medium
The Snakegourd Fruit seedling that must be obtained has excellent survival rate and high yield, and be capable of large area on this basis carries out cultivation Snakegourd Fruit, from
It and is that solid foundation has been established in the popularizations of the tissue cultures of Snakegourd Fruit.
Other features and advantages of the present invention will the following detailed description will be given in the detailed implementation section.
Specific embodiment
Detailed description of the preferred embodiments below.It should be understood that described herein specific
Embodiment is merely to illustrate and explain the present invention, and is not intended to restrict the invention.
The present invention provides a kind of method for tissue culture of Snakegourd Fruit, comprising:
1) Snakegourd Fruit cutting shoots are carried out being heat-treated de- bacterium as explant, is then placed in alcoholic solution and carries out two-stage sterilization, so
Be placed in initial culture base carry out Fiber differentiation, in proliferated culture medium carry out Multiplying culture obtain differentiation seedling；
2) differentiation seedling is subjected in root media culture of rootage, is then placed in 5 DEG C or less progress low-temperature treatments to obtain
Tissue culturing seedling；
3) by tissue culturing seedling's hardening；
Wherein, step 1) -2) condition of culture at least conform to the following conditions: it is sterile and ventilative；Initial culture base is to contain naphthalene
The MS culture medium of acetic acid NAA, 6-benzyladenine 6-BA and inositol；Proliferated culture medium be containing zeatin ZT, kanamycins, 2,
The MS culture medium of 4-D and tomato juice；Root media contains the 1/2MS culture medium of paclobutrazol, indolebutyric acid IBA and banana puree.
In above-mentioned method for tissue culture, the concentration of component in each culture medium can select in a wide range, still
In order to further increase the survival rate and reproductive efficiency of Snakegourd Fruit, it is preferable that in initial culture base, the concentration of NAA is 0.8-
The concentration of 1.3mg/L, 6-BA are 0.2-0.4mg/L, and the concentration of inositol is 0.8-1.3mg/L；In proliferated culture medium, ZT's is dense
Degree is 0.2-0.4mg/L, and the concentration of kanamycins is 0.8-1.3mg/L, and 2,4-D concentration is 0.4-0.6mg/L, tomato juice
Concentration is 2-5mg/L；In root media, the concentration of paclobutrazol is 0.05-0.1mg/L, and the concentration of IBA is 0.3-0.6mg/
L, the concentration of banana puree are 3-4.5g/L.
In various culture mediums of the invention, in order to preferably provide excellent nutrient and suitable training for each tissue
The condition of supporting, it is preferable that initial culture base, proliferated culture medium and root media agar and 14-18g/L containing 3-6g/L
Sucrose, and pH value be 6.0-7.0.
In the present invention, the condition for being heat-treated de- bacterium can select in a wide range, but go out to further increase
Bacterium effect, it is preferable that be heat-treated de- bacterium and meet the following conditions: de- bacterium temperature is 42-48 DEG C, and taking off the bacterium time is 3-6min.
In the present invention, the concentration of each component can also be in a wide range in the condition of two-stage sterilization and alcoholic solution
Selection, but in order to further increase sterilization effect, it is preferable that two-stage sterilization meets the following conditions: sterilization time 15-
25min, sterilising temp are 15-35 DEG C；The concentration of ethyl alcohol is 60-70 volume % in alcoholic solution.
In the present invention, the size of explant can select in a wide range, but in order to further increase Snakegourd Fruit
Survival rate and reproductive efficiency, it is preferable that the length of explant is 0.25-0.35cm.
In the present invention, the condition of Fiber differentiation can select in a wide range, but in order to further increase Snakegourd Fruit
Survival rate and reproductive efficiency, it is preferable that Fiber differentiation meets the following conditions: first dark culture 1-2 days is then transferred to alternation of light and darkness
Under conditions of cultivate 4-6 days；Wherein, alternation of light and darkness condition are as follows: light application time is 12-14h/ days, intensity of illumination 1400-
1600lux, cultivation temperature are 25-30 DEG C, relative humidity 50-60%.
In the present invention, the condition of Multiplying culture can select in a wide range, but in order to further increase Snakegourd Fruit
Survival rate and reproductive efficiency, it is preferable that Multiplying culture is cultivated 8-10 days under the conditions of alternation of light and darkness, alternation of light and darkness condition are as follows:
Light application time is 12-14h/ days, intensity of illumination 1700-1900lux, and cultivation temperature is 20-25 DEG C, relative humidity 50-
60%。
In the present invention, the condition of culture of rootage can select in a wide range, but in order to further increase Snakegourd Fruit
Survival rate and reproductive efficiency, it is preferable that culture of rootage is cultivated 10-15 days under the conditions of alternation of light and darkness, alternation of light and darkness condition are as follows:
Light application time is 12-14h/ days, intensity of illumination 2000-2200lux, and cultivation temperature is 20-25 DEG C, relative humidity 50-
60%。
In the present invention, the time of low-temperature treatment can select in a wide range, but in order to further increase Snakegourd Fruit
Survival rate and reproductive efficiency；Preferably, the time of the low-temperature treatment is 8-12h
In the present invention, sterile and ventilative condition acquisition can select in a wide range, but in order to further increase melon
The survival rate and reproductive efficiency of beach wormwood, it is preferable that step 1) -2) in sterile and ventilative condition pass through following operation obtain: culture
Environment is the tissue culture bottle with fungi-proofing ventilated membrane；Wherein, the actual conditions of ventilated membrane can also select in a wide range, but be
The survival rate and reproductive efficiency of succulent are further increased, it is highly preferred that ventilated membrane at least meets the following conditions: hole is
0.2-0.3um, percent opening 80-90 ﹪.
In the present invention, the condition of hardening can select in a wide range, but in order to further increase Snakegourd Fruit at
Motility rate and reproductive efficiency, it is preferable that the condition of hardening are as follows: light application time is 12-14h/ days, and interlunation is 10-12h/ days, light
It is 1800-2000lux according to intensity, temperature is 21-23 DEG C, relative humidity 75-85%, and the time is 5-7 days.
The present invention will be described in detail by way of examples below.In the examples below, MS culture medium and 1/2MS culture
The basic component of base is referring to table 1 and 2；It wherein, is the system of various concentration component in MS culture medium and 1/2MS culture medium in table 1
Table is counted, is the statistical form of same concentrations component in MS culture medium and 1/2MS culture medium in table 2.
Table 1
Table 2
Embodiment 1
1) carry out being heat-treated de- bacterium that (de- bacterium temperature is 46 DEG C, when taking off bacterium using Snakegourd Fruit cutting shoots that length is 0.3cm as explant
Between be 5min), being then placed in progress two-stage sterilization in alcoholic solution (concentration of ethyl alcohol be 60-70 volume %), (sterilization time is
20min, sterilising temp are 20 DEG C), being subsequently placed in progress Fiber differentiation in initial culture base, (first dark culture 1.5 days, is then transferred to
It is cultivated 5 days under conditions of alternation of light and darkness；Wherein, alternation of light and darkness condition are as follows: light application time is 13h/ days, and intensity of illumination is
1500lux, cultivation temperature be 28 DEG C, relative humidity 55%), in proliferated culture medium carry out Multiplying culture (in alternation of light and darkness item
It is cultivated 9 days under part, alternation of light and darkness condition are as follows: light application time is 13h/ days, intensity of illumination 1800lux, and cultivation temperature is 22 DEG C,
Relative humidity is 55%) to obtain differentiation seedling；
2) differentiation seedling culture of rootage is carried out (to cultivate 13 days under the conditions of alternation of light and darkness, alternation of light and darkness in root media
Condition are as follows: light application time be 13h/ days, intensity of illumination 2100lux, cultivation temperature be 23 DEG C, relative humidity 55%), then
5 DEG C or less progress low-temperature treatment 10h are placed in obtain tissue culturing seedling；
3) by tissue culturing seedling's hardening obtain Snakegourd Fruit seedling (light application time be 13h/ days, interlunation be 11h/ days, intensity of illumination
For 1900lux, temperature is 22 DEG C, and relative humidity 79%, the time is 6 days)；
Wherein, step 1) -2) condition of culture at least conform to the following conditions: it is sterile and ventilative, in the group with fungi-proofing ventilated membrane
It is carried out in training bottle, ventilated membrane meets the following conditions: hole 0.25um, 85 ﹪ of percent opening；Initial culture base is to contain 1mg/L
NAA, 0.3mg/L 6-BA, 1mg/L inositol MS culture medium；Proliferated culture medium is that is mould containing 0.3mg/L ZT, 1mg/L card
Element, 0.5mg/L 2,4-D concentration be, the MS culture medium of 3mg/L tomato juice；In root media, 0.08mg/L paclobutrazol,
The 1/2MS culture medium of the concentration of 0.5mg/L IBA, 3.5g/L banana puree；Initial culture base, proliferated culture medium and culture of rootage
The sucrose of the base agar containing 5g/L and 15g/L, and pH value is 6.5.
Embodiment 2
1) carry out being heat-treated de- bacterium that (de- bacterium temperature is 42 DEG C, takes off bacterium using Snakegourd Fruit cutting shoots that length is 0.25cm as explant
Time is 6min), it is then placed in alcoholic solution (concentration of ethyl alcohol be 60 volume %) and carries out two-stage sterilization (sterilization time is
15min, sterilising temp are 35 DEG C), being subsequently placed in progress Fiber differentiation in initial culture base, (first dark culture 1 day, is then transferred to light
It is cultivated 4 days under the conditions of dark alternate；Wherein, alternation of light and darkness condition are as follows: light application time is 12h/ days, intensity of illumination 1400lux,
Cultivation temperature be 25 DEG C, relative humidity 50%), in proliferated culture medium carry out Multiplying culture (cultivated under the conditions of alternation of light and darkness
8 days, alternation of light and darkness condition are as follows: light application time is 12h/ days, intensity of illumination 1700lux, and cultivation temperature is 20 DEG C, relative humidity
50%) to obtain differentiation seedling；
2) differentiation seedling culture of rootage is carried out (to cultivate 10 days under the conditions of alternation of light and darkness, alternation of light and darkness in root media
Condition are as follows: light application time be 12h/ days, intensity of illumination 2000lux, cultivation temperature be 20 DEG C, relative humidity 50%), then
5 DEG C or less progress low-temperature treatment 8h are placed in obtain tissue culturing seedling；
3) by tissue culturing seedling's hardening obtain Snakegourd Fruit seedling (light application time be 12h/ days, interlunation be 10h/ days, intensity of illumination
For 1800lux, temperature is 21 DEG C, and relative humidity 75%, the time is 5 days)；
Wherein, step 1) -2) condition of culture at least conform to the following conditions: it is sterile and ventilative, in the group with fungi-proofing ventilated membrane
It is carried out in training bottle, ventilated membrane meets the following conditions: hole 0.25um, 85 ﹪ of percent opening；Initial culture base is to contain 0.8mg/L
NAA, 0.2mg/L 6-BA, 0.8mg/L inositol MS culture medium；Proliferated culture medium be containing 0.2mg/L ZT, 0.8mg/L card that
Mycin, 0.4mg/L 2,4-D concentration be, the MS culture medium of 2mg/L tomato juice；In root media, 0.05mg/L multiple-effect
The 1/2MS culture medium of azoles, the concentration of 0.3mg/L IBA, 3g/L banana puree；Initial culture base, proliferated culture medium and training of taking root
The sucrose of feeding the base agar containing 3g/L and 14g/L, and pH value is 6.0.
Embodiment 3
1) carry out being heat-treated de- bacterium that (de- bacterium temperature is 48 DEG C, takes off bacterium using Snakegourd Fruit cutting shoots that length is 0.35cm as explant
Time is 3min), it is then placed in alcoholic solution (concentration of ethyl alcohol be 70 volume %) and carries out two-stage sterilization (sterilization time is
25min, sterilising temp are 35 DEG C), being subsequently placed in progress Fiber differentiation in initial culture base, (first dark culture 2 days, is then transferred to light
It is cultivated 6 days under the conditions of dark alternate；Wherein, alternation of light and darkness condition are as follows: light application time is 14h/ days, intensity of illumination 1600lux,
Cultivation temperature be 30 DEG C, relative humidity 60%), in proliferated culture medium carry out Multiplying culture (cultivated under the conditions of alternation of light and darkness
10 days, alternation of light and darkness condition are as follows: light application time is 14h/ days, intensity of illumination 1900lux, and cultivation temperature is 25 DEG C, relatively wet
Degree is 60%) to obtain differentiation seedling；
2) differentiation seedling culture of rootage is carried out (to cultivate 15 days under the conditions of alternation of light and darkness, alternation of light and darkness in root media
Condition are as follows: light application time be 14h/ days, intensity of illumination 2200lux, cultivation temperature be 25 DEG C, relative humidity 60%), then
5 DEG C or less progress low-temperature treatment 12h are placed in obtain tissue culturing seedling；
3) by tissue culturing seedling's hardening obtain Snakegourd Fruit seedling (light application time be 14h/ days, interlunation be 12h/ days, intensity of illumination
For 2000lux, temperature is 23 DEG C, and relative humidity 85%, the time is 7 days)；
Wherein, step 1) -2) condition of culture at least conform to the following conditions: it is sterile and ventilative, in the group with fungi-proofing ventilated membrane
It is carried out in training bottle, ventilated membrane meets the following conditions: hole 0.25um, 85 ﹪ of percent opening；Initial culture base is to contain 1.3mg/L
NAA, 0.4mg/L 6-BA, 1.3mg/L inositol MS culture medium；Proliferated culture medium be containing 0.4mg/L ZT, 1.3mg/L card that
Mycin, 0.6mg/L 2,4-D concentration be, the MS culture medium of 5mg/L tomato juice；In root media, 0.1mg/L multiple-effect
The 1/2MS culture medium of azoles, the concentration of 0.3-0.6mg/L IBA, 4.5g/L banana puree；Initial culture base, proliferated culture medium and
The sucrose of the root media agar containing 6g/L and 18g/L, and pH value is 7.0.
Application examples 1
The seedling in the various embodiments described above after 100 plants of hardening is taken to carry out plant strain growth of the culture up to 50% or more in matrix
Then 5 blades out count the survival rate (W) of seedling, while calculating the Snakegourd Fruit cutting shoots of 10 4cm a tissue cultures week
The quantity (N) for surviving Snakegourd Fruit that can be obtained after phase, concrete outcome is as shown in table 3.
Table 3
| W/% | N/ | |
| Embodiment 1 | 88 | 69 |
| Embodiment 2 | 79 | 59 |
| Embodiment 3 | 86 | 62 |
The preferred embodiment of the present invention has been described above in detail, still, the tool during present invention is not limited to the embodiments described above
Body details within the scope of the technical concept of the present invention can be with various simple variants of the technical solution of the present invention are made, these letters
Monotropic type all belongs to the scope of protection of the present invention.
It is further to note that specific technical features described in the above specific embodiments, in not lance
In the case where shield, can be combined in any appropriate way, in order to avoid unnecessary repetition, the present invention to it is various can
No further explanation will be given for the combination of energy.
In addition, various embodiments of the present invention can be combined randomly, as long as it is without prejudice to originally
The thought of invention, it should also be regarded as the disclosure of the present invention.
Claims (10)
1. a kind of method for tissue culture of Snakegourd Fruit characterized by comprising
1) Snakegourd Fruit cutting shoots are carried out being heat-treated de- bacterium as explant, is then placed in alcoholic solution and carries out two-stage sterilization, so
Be placed in initial culture base carry out Fiber differentiation, in proliferated culture medium carry out Multiplying culture obtain differentiation seedling；
2) by the differentiation seedling carried out in root media culture of rootage, then be placed in 5 DEG C or less progress low-temperature treatments with
Obtain tissue culturing seedling；
3) by tissue culturing seedling's hardening；
Wherein, step 1) -2) condition of culture at least conform to the following conditions: it is sterile and ventilative；The initial culture base be containing
There are the MS culture medium of methyl α-naphthyl acetate NAA, 6-benzyladenine 6-BA and inositol；The proliferated culture medium is containing zeatin ZT, card
The MS culture medium of that mycin, 2,4-D and tomato juice；The root media contains paclobutrazol, indolebutyric acid IBA and banana puree
1/2MS culture medium.
2. method for tissue culture according to claim 1, which is characterized in that in the initial culture base, the NAA's
Concentration is 0.8-1.3mg/L, and the concentration of the 6-BA is 0.2-0.4mg/L, and the concentration of the inositol is 0.8-1.3mg/L；
In the proliferated culture medium, the concentration of the ZT is 0.2-0.4mg/L, and the concentration of the kanamycins is 0.8-
The concentration of 1.3mg/L, 2, the 4-D are 0.4-0.6mg/L, and the concentration of the tomato juice is 2-5mg/L；
In the root media, the concentration of the paclobutrazol is 0.05-0.1mg/L, and the concentration of the IBA is 0.3-
0.6mg/L, the concentration of the banana puree are 3-4.5g/L.
3. method for tissue culture according to claim 1, which is characterized in that the initial culture base, proliferated culture medium with
And the sucrose of the root media agar containing 3-6g/L and 14-18g/L, and pH value is 6.0-7.0.
4. method for tissue culture according to claim 1, which is characterized in that the de- bacterium of heat treatment meets the following conditions:
De- bacterium temperature is 42-48 DEG C, and taking off the bacterium time is 3-6min.
5. method for tissue culture according to claim 1, which is characterized in that the two-stage sterilization meets the following conditions: going out
The bacterium time is 15-25min, and sterilising temp is 15-35 DEG C；
The concentration of ethyl alcohol is 60-70 volume % in the alcoholic solution.
6. method for tissue culture described in any one of -5 according to claim 1, which is characterized in that the length of the explant
For 0.25-0.35cm.
7. method for tissue culture described in any one of -5 according to claim 1, which is characterized in that the Fiber differentiation meets
The following conditions: first dark culture 1-2 days is then transferred under conditions of alternation of light and darkness and cultivates 4-6 days；Wherein, alternation of light and darkness condition are as follows:
Light application time is 12-14h/ days, intensity of illumination 1400-1600lux, and cultivation temperature is 25-30 DEG C, relative humidity 50-
60%。
8. method for tissue culture described in any one of -5 according to claim 1, which is characterized in that the Multiplying culture is in light
It is cultivated 8-10 days under dark alternation condition, alternation of light and darkness condition are as follows: light application time is 12-14h/ days, intensity of illumination 1700-
1900lux, cultivation temperature are 20-25 DEG C, relative humidity 50-60%.
9. method for tissue culture described in any one of -5 according to claim 1, which is characterized in that the culture of rootage is in light
It is cultivated 10-15 days under dark alternation condition, alternation of light and darkness condition are as follows: light application time is 12-14h/ days, intensity of illumination 2000-
2200lux, cultivation temperature are 20-25 DEG C, relative humidity 50-60%；
Preferably, the time of the low-temperature treatment is 8-12h.
10. method for tissue culture described in any one of -5 according to claim 1, which is characterized in that step 1) -2) in nothing
Bacterium and ventilative condition is obtained by following operation: culture environment is the tissue culture bottle with fungi-proofing ventilated membrane；
Preferably, ventilated membrane at least meets the following conditions: hole 0.2-0.3um, percent opening 80-90 ﹪；
It is highly preferred that the condition of the hardening are as follows: light application time is 12-14h/ days, and interlunation is 10-12h/ days, and illumination is strong
Degree is 1800-2000lux, and temperature is 21-23 DEG C, relative humidity 75-85%, and the time is 5-7 days.
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