CN109006485A - A kind of germplasm in-vitro conservation method of peppermint - Google Patents
A kind of germplasm in-vitro conservation method of peppermint Download PDFInfo
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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 germplasm in-vitro conservation methods of peppermint, including following sport technique segment: (1) pretreatment of mint plants；(2) explant disinfection and inoculation；(3) adventitious bud induction culture；(4) preserving seed culture；(5) germplasm rejuvenation；(6) seedling culture；(7) acclimatization and transplants.One retention cycle of method provided by the invention can be reserved for peppermint germ plasm resource and reach 3-4, greatly extend the holding time, and the blastogenesis character saved is stablized, rejuvenation activity recovery can be passed through when needing, seedling is provided on a large scale for production at any time, is a kind of germplasm in-vitro conservation method that high practical value.
Description
Technical field
The invention belongs to biotechnologys and technical field of agriculture science, are related to a kind of germplasm in-vitro conservation method of peppermint,
More particularly to numerous method is expanded in rejuvenation after a kind of long-term preservation and preservation for realizing peppermint germplasm using tissue culture technique.
Background technique
Peppermint (Mentha haplocalyx Briq) be Labiatae Mentha herbaceos perennial, complete stool have it is dense
Strong cooling fragrance is a kind of adaptable, widely distributed plant resources with special aromatic odor.Peppermint is widely distributed
In the Temperate Region in China in the Northern Hemisphere, be distributed all over China, family's kind, it is wild all have, wherein Jiangsu, two province's cultivation yield of Anhui be most
Greatly.Peppermint has medicinal and edible dual value, and dry aerial parts can be used as medicine, and has effects that dispelling wind, heat dissipation, removing toxic substances,
It is that China is common for treating anemopyretic cold, headache, hot eyes, abscess of throat, toothache, aphtha, rubeola, morbilli, Breast bilges frowsty etc.
One of traditional Chinese medicine；Its cauline leaf can make vegetable edible, also can be used as flavoring agent, can also match wine, make tea.Peppermint main component is waved
Hair oil, flavonoids, organic acid, amino acid etc., wherein volatile oil is the most wide ingredient of purposes, and menthol can be obtained after processed
And peppermint oil dementholized, it is widely used in medicine, daily-use chemical industry and food industry.
China is universally acknowledged peppermint main production country, and mentholated product is with aroma of pure, peculiar smell is few, high-quality and enjoy great prestige generation
Boundary is known as " perfume (or spice) in Asia ", there is very important status in the international market.However in the fast development of peppermint industry,
Also there are some new problems.Peppermint is above mainly carried out by the way of rhizome, plant division and cuttage asexual numerous in production first
It grows.During long-term long-term vegetative propagation, virus can be accumulated by generation, serious virosis be broken out, to cause peppermint excellent
Breeding sexual involution is serious, and then influences the quality and yield of peppermint.Followed by order to meet the market demand, it is excellent to widely popularize high yield
The kind of matter, to cause the unification increasingly of peppermint kind, this will will lead to the diminution of peppermint and the platymiscium genetic resources
And failure.In addition there are scientific management is lacked when peppermint cultivation, leading to variety hybriding degradation also causes peppermint yield and quality to decline
Major reason.Therefore, it should reinforce collection and preservation to peppermint germ plasm resource, the breeding, prominent including local varieties, breeding
Mutation and rare species, wild species and nearly source wild plant, this undoubtedly can be to the utilization and breed improvement of China's peppermint germ plasm resource
Development play an important role.
At present China peppermint research be concentrated mainly on pharmacological action, volatile oil and involatile constituent extraction, Propogation and culture,
Cultivation management etc., and it is less to the research of its preserving seed.Preserving seed refers to is suitable for using natural or artificial creation
Environment makes inhereditary material contained in individual keep its genetic integrity so as to preservation germ plasm resource, has high vigor, and
Its heredity can be handed on by breeding.The method of preserving seed is broadly divided into original place preservation and strange land saves 2 major class,
Middle Plantlet in vitro is the strange land store method that germ plasm resource is realized by plant tissue culture technique.Shared by germplasm Plantlet in vitro
Space is small, can be reserved for a large amount of genetic resources in a smaller space, and overcoming field and saving needs that land occupation area is big, management
Costly disadvantage；Plantlet in vitro technology usually aseptically carries out simultaneously, easily controllable, while can be in Plantlet in vitro
Any stage carries out detoxification treatment, avoids disease and natural calamity, reduces during field saves and natural variation easily occurs
The phenomenon that degenerating with germplasm, to ensure that the Optimality and genetic stability of germplasm.
Team of the present invention finally has developed a kind of guarantor by the further investigation to the multi-party factor of peppermint preserving seed is influenced
The germplasm in-vitro conservation method for depositing of long duration, stabilization characteristics of genetics, the peppermint that germplasm survival rate is high, effect of rejuvenation is good, to peppermint
Industry development be of great significance.
Summary of the invention
Technical problem to be solved by the invention is to provide a kind of germplasm in-vitro conservation method of peppermint, this method both can be with
For saving the Wild ornamental resources of peppermint, is conducive to the continuity of peppermint genetic diversity, can also be used to save rejuvenation peppermint
Cultivation germplasm, the material as peppermint Propogation and culture.
The technical scheme of the invention to solve the technical problem is:
A kind of germplasm in-vitro conservation method of peppermint, which is characterized in that method includes the following steps:
(1) pretreatment of mint plants
Outdoor cropping, healthy and strong disease-free 1 year raw plant of peppermint are selected, entire plant is rinsed well with tap water, especially to be washed
Net root soil, then immerses plant root in the nutrient solution containing 0.1-0.3mg/L TDZ, 4 DEG C of Cold pretreatment 5-8
It；
(2) explant disinfection and inoculation
Pretreated mint plants are dipped in into a little detergent with hairbrush, rinse 30-40min under flowing water；Then it is tender to cut children
Stem section, remove blade, retain axillary bud, be transferred in superclean bench, first with 70% alcohol treatment 30s, then with sterile washing
It washs 3-4 times, then handles 4min with 2.5% sodium hypochlorite, finally with sterile water washing 3-4 times, filter paper sucks excessive moisture；By stem
The segment that section is cut into 1 cm left and right belt axillary bud is inoculated on adventitious bud induction culture base as explant；
(3) adventitious bud induction culture
Under conditions of above-mentioned culture medium is placed in 24-26 DEG C of temperature, intensity of illumination 1300-1500Lx, illumination 12h/d, 28- is cultivated
36d induces adventitious bud；
(4) preserving seed culture
It when adventitious bud wait induce grows to 1.0-1.5cm, is forwarded on preserving seed culture medium, seals up bottleneck and be placed on temperature
4-7 DEG C, intensity of illumination 500-800Lx, daily illumination 10-12h condition of culture under slowly grow, every 12 months subinoculations one
It is secondary, then proceed by preserving seed culture；
(5) germplasm rejuvenation
After the peppermint preserving seed phase, by the adventitious bud slowly grown in above-mentioned steps be transferred in rejuvenation culture medium cultivate with
Activity recovery, condition of culture control are 25-27 DEG C of temperature, intensity of illumination 2000-2200Lx, daily illumination 12-14h, and every 30d is left
Right subculture 1 time, the adventitious bud of subculture 2-3 times available proliferation；
(6) seedling culture
Above-mentioned adventitious bud is cut into strengthening seedling and rooting culture medium of individually transferring, 24-28 DEG C of temperature, intensity of illumination are placed in
25-30d is cultivated under conditions of 2600-3000Lx, daily illumination 14-16h, adventitious bud can grow up to plant height 5-8cm, have 7-10 item
The peppermint tissue-cultured seedling of root system；
(7) acclimatization and transplants
Peppermint tissue-cultured seedling is taken out together with tissue culture bottle from culturing room, is placed in 22-30 DEG C of greenhouse, is refined under natural lighting
Seedling 7d, then open bottleneck hardening 3-5 d；Then tissue-cultured seedling is carefully taken out from bottle, the culture medium residual of root is washed away, by root
Portion's immersion mass fraction is cultivated 3-5 hours for 0.1% spend in precious No. 1 solution, then is transplanted into crop field, carries out conventional cultivation management.
The formula of nutrient solution is Ca (H in the step (1)2PO4)2·H2O 0.4g/L、Ca(NO3)2 0.5g/L、KNO3
0.9g/L, urea 0.3g/L, MgSO4·7H2O 0.28g/L FeNa2-EDTA 0.12g/L、H3BO3 0.06mg/L、MnSO4·
4H2O 0.03mg/L、ZnSO4·7H2O 0.06mg/L、CuSO4·5H2O 0.06mg/L, (NH4)2MoO4 0.06mg/L, sub- essence
Amine 3.4mg/L.
The formula of adventitious bud induction culture base in the step (2) are as follows: the poly- second of MS+2,4-D1.0mg/L+IBA0.2mg/L+
Enol 12mg/L+ dithiothreitol (DTT) 0.8mg/L+yeast extract 5.3g/L, pH value is 5.8.
The formula of preserving seed culture medium in the step (4) are as follows: KNO3980 ~ 1060mg/L, NH4NO3 855~900mg/
L, KH2PO4110 ~ 140mg/L, MgSO4·7H2O 165 ~ 184mg/L, CaCl2·2H2O 203 ~ 213mg/L, MnSO4·4H2O
134 ~ 140mg/L, ZnSO4·7H2O 7.0 ~ 8.0mg/L, H3BO35.9 ~ 6.3mg/L, KI 0.7 ~ 0.8mg/L, CuSO4·
5H2O 0.024 ~ 0.026mg/L, CoCl2·6H2O 0.024 ~ 0.026mg/L, Na2MoO4·2H20.13 ~ 0.19mg/L of O,
FeSO4·7H2O 23 ~ 25mg/L, Na234 ~ 36mg/L of-EDTA, 47 ~ 55mg/L of inositol, proline-4 ~ 6mg/L, serine 2 ~
4mg/L, 0.2 ~ 0.4mg/L of vitamin B1,0.3 ~ 0.5mg/L of vitamin B6, vitamin B5 0.3 ~ 0.5mg/L, 3-(α-pyrrole
Piperidinyl) propyl alcohol 0.7 ~ 1.0 mg/L, 6.1 ~ 6.5mg/L of tea polyphenols, 0.5 ~ 0.7mg/L of silver thiosulfate, 0.5 ~ 0.8g/ of diatomite
L, 1.3 ~ 1.7mg/L of paclobutrazol, 55 ~ 60g/L of sucrose, 17 ~ 21g/L of glycine betaine, 2.2 ~ 2.6mg/L of calcium propionate, peppermint leaching liquor
22 ~ 28mL/L, coconut palm 30 ~ 36mL/L of cream, plant gel 2 ~ 4g/L, pH are 5.6 ~ 6.0.
The method that bottleneck is sealed up in the step (4) is to be sealed with the masking foil bilayer with a thickness of 0.01mm plus sealed membrane
Mouthful.
Subinoculation is no more than 4 times in the step (4).
The formula of rejuvenation culture medium in the step (5) are as follows: MS(is free of sucrose, agar)+11 ~ 15mg/L+ of glutathione
Humic acid 3.6 ~ 4.0mg/L+ sodium carboxymethylcellulose, 2.7 ~ 3.3g/L+ is to bromobenzene fluoroacetic acid 1.3 ~ 1.7mg/L+2-(3,4- bis-
Chlorophenoxy) triethylamine 0.8 ~ 1.0mg/L+ maltose 23 ~ 25g/L+ fructose 13 ~ 16g/L+ plant gel 2 ~ 4g/L, pH be
5.8。
The formula of root media in the step (5) are as follows: 1/2MS+NAA0.5mg/L+ active carbon 0.6g/L, pH are
5.8。
In the preserving seed culture medium, peppermint leaching liquor the preparation method comprises the following steps: taking fresh mint blade to smash to pieces, by quality
It is put into heat preservation extraction 1h in 60-70 DEG C of hot water than the material-water ratio for 1:3, obtains leaching liquor after the filtering of 200 mesh filter screens.
Beneficial effects of the present invention:
1, the present invention is furtherd investigate by the key link to entire peppermint germplasm Plantlet in vitro, finds out suitable peppermint kind
The optimum condition that quality guarantee is deposited effectively has delayed the growth of peppermint germplasm materials, has extended subculture cycle, to greatly extend
Preserving seed period of peppermint.
2, method germplasm survival rate of the invention is high, and genetic stability is good, and germplasm can easily be restored by rejuvenation
Growth, transplanting survival rate are up to 95% or more.
3, compared with conventional field planting store method, operation of the present invention is easy, saves soil, human and material resources, financial resources,
And do not limited by the seasonality of plant growth, the threat of natural calamity and pest and disease damage can also be avoided, is a kind of effective
Peppermint germplasm in-vitro conservation method, the plantation of peppermint excellent variety is promoted and the protection of wild resource all has important meaning
Justice.
Specific embodiment
With reference to embodiment, the embodiment of the present invention is furthur described in detail.Following embodiment is used for
Illustrate the present invention, but is not intended to limit the scope of the invention.
Embodiment 1
2012-2015, technology path according to the invention carry out the germplasm Plantlet in vitro test of peppermint, and detailed step is as follows:
(1) pretreatment of mint plants
In August, 2012 selects outdoor cropping, healthy and strong disease-free 1 year raw plant of peppermint, rinses entire plant with tap water and do
Only, root soil is especially cleaned, (nutrient solution prescription in the nutrient solution containing 0.2mg/L TDZ is then immersed into plant root
For Ca (H2PO4)2·H2O 0.4g/L、Ca(NO3)2 0.5g/L、KNO30.9g/L, urea 0.3g/L, MgSO4·7H2O
0.28g/L FeNa2-EDTA 0.12g/L、H3BO3 0.06mg/L、MnSO4·4H2O 0.03mg/L、ZnSO4·7H2O
0.06mg/L、CuSO4·5H2O 0.06mg/L, (NH4)2MoO4 0.06mg/L, spermidine 3.4mg/L), 4 DEG C of Cold pretreatments
5-8 days；
(2) explant disinfection and inoculation
Pretreated mint plants are dipped in into a little detergent with hairbrush, rinse 30-40min under flowing water；Then it is tender to cut children
Stem section, remove blade, retain axillary bud, be transferred in superclean bench, first with 70% alcohol treatment 30s, then use sterile water
Washing 3-4 times, then 4min is handled with 2.5% sodium hypochlorite, finally with sterile water washing 3-4 times, filter paper sucks excessive moisture；It will
Stem section is cut into the segment of 0.5cm left and right belt axillary bud as explant, is inoculated on adventitious bud induction culture base the (culture medium prescription
For MS+2,4-D1.0mg/L+IBA0.2mg/L+ polyvinyl alcohol 12mg/L+ dithiothreitol (DTT) 0.8mg/L+yeast extract 5.3g/
5.8) L, pH value are；
(3) adventitious bud induction culture
Culture 28-36d is induced under conditions of above-mentioned culture medium is placed in 25 DEG C of temperature, intensity of illumination 1400Lx, illumination 12h/d
Adventitious bud；
(4) preserving seed culture
It when adventitious bud wait induce grows to 1.0-1.5cm, is forwarded on preserving seed culture medium, with the tin with a thickness of 0.01mm
Foil paper bilayer plus sealed membrane seal up bottleneck be placed on 5 DEG C of temperature, intensity of illumination 700Lx, daily illumination 11h condition of culture under
Slowly growth, every 12 months subinoculations are primary, are statistics adventitious bud survival rate and average after preserving seed 3 years after subculture 3 times
Plant height increment；
The formula of preserving seed culture medium are as follows: KNO31020mg/L, NH4NO3877mg/L, KH2PO4125mg/L, MgSO4·
7H2O 174mg/L, CaCl2·2H2O 208mg/L, MnSO4·4H2O 137mg/L, ZnSO4·7H2O 7.5mg/L, H3BO3
6.1 mg/L, KI 0.75mg/L, CuSO4·5H2O 0.025mg/L, CoCl2·6H2O 0.025mg/L, Na2MoO4·2H2O
0.16mg/L, FeSO4·7H2O 24mg/L, Na2- EDTA 35mg/L, inositol 51mg/L, proline 5mg/L, serine 3mg/
L, vitamin B1 0.3mg/L, vitamin B6 0.4mg/L, vitamin B5 0.4mg/L, 3-(α-pyridyl group) propyl alcohol 0.85mg/
L, tea polyphenols 6.3mg/L, silver thiosulfate 0.6mg/L, diatomite 0.6g/L, paclobutrazol 1.5mg/L, sucrose 58g/L, glycine betaine
19g/L, calcium propionate 2.4mg/L, coconut palm cream 33mL/L, peppermint 22 ~ 28mL/L of leaching liquor, plant gel 3g/L, pH 5.8；
Wherein peppermint leaching liquor the preparation method comprises the following steps: fresh mint blade is taken to smash to pieces, be put into mass ratio for the material-water ratio of 1:3
Heat preservation extraction 1h in 60-70 DEG C of hot water, obtains leaching liquor after the filtering of 200 mesh filter screens.
Embodiment 2
Preserving seed is carried out using germplasm origin same as Example 1 and technology path, wherein by preserving seed in step (4)
Culture medium replaces with MS+ sucrose 40g/L+ mannitol 10g/L(formula from graceful " the in vitro guarantor of peppermint test tube seedling of kind favour
Deposit "), adventitious bud survival rate and average plant height increment are counted after preserving seed 3 years.
Embodiment 3
Preserving seed is carried out using germplasm origin same as Example 1 and technology path, wherein by preserving seed in step (4)
Culture medium replaces with MS culture medium, and adventitious bud survival rate and average plant height increment are counted after preserving seed 3 years.
Embodiment 4
The peppermint adventitious bud survived after in embodiment 1 subculture 3 times is continued into culture 7 months, then technology road according to the invention
Line carries out germplasm rejuvenation, and detailed step is as follows:
(1) germplasm rejuvenation
The adventitious bud of survival is transferred to rejuvenation culture medium (culture medium prescription is MS(without sucrose, agar)+glutathione
13mg/L+ humic acid 3.8mg/L+ sodium carboxymethylcellulose 3.0g/L+ is to bromobenzene fluoroacetic acid 1.5mg/L+2-(3,4- Dichlorophenoxy
Base) triethylamine 0.9mg/L+ maltose 24g/L+ fructose 14.5g/L+ plant gel 3g/L, pH 5.8) in culture to restore living
Property, condition of culture control be 26 DEG C of temperature, intensity of illumination 2100Lx, daily illumination 13h, every 30d or so subculture 1 time, subculture 2 times
Adventitious bud proliferation coefficient is counted afterwards；
(2) seedling culture
Above-mentioned adventitious bud is cut into strengthening seedling and rooting culture medium of individually transferring, 24-28 DEG C of temperature, intensity of illumination are placed in
25-30d is cultivated under conditions of 2600-3000Lx, daily illumination 14-16h, counts the rooting rate of adventitious bud；
(3) acclimatization and transplants
Peppermint tissue-cultured seedling is taken out together with tissue culture bottle from culturing room, is placed in 22-30 DEG C of greenhouse, is refined under natural lighting
Seedling 7d, then open bottleneck hardening 3-5 d.Then tissue-cultured seedling is carefully taken out from bottle, the culture medium residual of root is washed away, by root
Portion's immersion mass fraction is cultivated 3-5 hours for 0.1% spend in precious No. 1 solution, then is transplanted into crop field, carries out conventional cultivation management；
The transplanting survival rate of peppermint tissue-cultured seedling is counted after transplanting 1 month.
Embodiment 5
Germplasm rejuvenation is carried out using germplasm origin same as Example 4 and technology path, wherein by 4 step of embodiment (1)
Rejuvenation culture medium replace with MS+5.5g/L agar+30g/L sucrose (formula from kind favour it is graceful " peppermint test tube seedling it is in vitro
Save ").
Embodiment 6
Germplasm rejuvenation is carried out using germplasm origin same as Example 4 and technology path, wherein by 4 step of embodiment (1)
Rejuvenation culture medium replaces with+0.2 mg/LNAA(of the MS+4.4mg/L6-BA formula, and from Liu Ming, " Tissue Culture for Mint is ground
Study carefully ").
As a result it counts
1, by adventitious bud survival rate behind preserving seed 3 years in embodiment 1-3 and average plant height increment statistics such as the following table 1.
2, by adventitious bud value-added coefficient, rooting rate and transplanting survival rate statistics such as the following table 2 in embodiment 4-6.
As can be seen from Table 1, after being saved germplasm 3 years using method of the invention, the survival rate of adventitious bud still be can reach
62.7%, plant height increment average out to 1.7cm are significantly higher than other embodiments, illustrate that preserving seed culture medium of the invention can be with
Effectively delay the growth of peppermint seedling, while by factors such as the temperature of organization of regulation control culture, illumination, greatly extending thin
The holding time of lotus germplasm compensates for the shortcoming of current peppermint preserving seed；As can be seen from Table 2, embodiment 4 uses
Rejuvenation culture medium of the invention is best to the germplasm restoration ecosystem effect of long-term preservation, and the final field of Germplasms survives
Rate is up to 95% or more；Tissue-cultured seedling after transplanting is investigated and is counted by a series of biological characters, and tissue-cultured seedling is shown and the previous generation
The great consistency of maternal plant, still keeps the obvious characteristic of original strain；In conclusion the present invention is a kind of preserving seed time
Length, stabilization characteristics of genetics, the peppermint germplasm in-vitro conservation method that germplasm survival rate is high, effect of rejuvenation is good.
The specific implementation of the invention is not to be limited to these illustrations for the above content, and technology belonging to the present invention is led
For the those of ordinary skill in domain, without departing from the inventive concept of the premise, a number of simple deductions or replacements can also be made,
It all shall be regarded as belonging to present invention scope of patent protection determined by the appended claims.
Claims (9)
1. a kind of germplasm in-vitro conservation method of peppermint, which is characterized in that method includes the following steps:
(1) pretreatment of mint plants
Outdoor cropping, healthy and strong disease-free 1 year raw plant of peppermint are selected, entire plant is rinsed well with tap water, especially to be washed
Net root soil, then immerses plant root in the nutrient solution containing 0.1-0.3mg/L TDZ, 4 DEG C of Cold pretreatment 5-8
It；
(2) explant disinfection and inoculation
Pretreated mint plants are dipped in into a little detergent with hairbrush, rinse 30-40min under flowing water；Then it is tender to cut children
Stem section, remove blade, retain axillary bud, be transferred in superclean bench, first with 70% alcohol treatment 30s, then with sterile washing
It washs 3-4 times, then handles 4min with 2.5% sodium hypochlorite, finally with sterile water washing 3-4 times, filter paper sucks excessive moisture；By stem
The segment that section is cut into 1 cm left and right belt axillary bud is inoculated on adventitious bud induction culture base as explant；
(3) adventitious bud induction culture
Under conditions of above-mentioned culture medium is placed in 24-26 DEG C of temperature, intensity of illumination 1300-1500Lx, illumination 12h/d, 28- is cultivated
36d induces adventitious bud；
(4) preserving seed culture
It when adventitious bud wait induce grows to 1.0-1.5cm, is forwarded on preserving seed culture medium, seals up bottleneck and be placed on temperature
4-7 DEG C, intensity of illumination 500-800Lx, daily illumination 10-12h condition of culture under slowly grow, every 12 months subinoculations one
It is secondary, then proceed by preserving seed culture；
(5) germplasm rejuvenation
After the peppermint preserving seed phase, by the adventitious bud slowly grown in above-mentioned steps be transferred in rejuvenation culture medium cultivate with
Activity recovery, condition of culture control are 25-27 DEG C of temperature, intensity of illumination 2000-2200Lx, daily illumination 12-14h, and every 30d is left
Right subculture 1 time, the adventitious bud of subculture 2-3 times available proliferation；
(6) seedling culture
Above-mentioned adventitious bud is cut into strengthening seedling and rooting culture medium of individually transferring, 24-28 DEG C of temperature, intensity of illumination are placed in
25-30d is cultivated under conditions of 2600-3000Lx, daily illumination 14-16h, adventitious bud can grow up to plant height 5-8cm, have 7-10 item
The peppermint tissue-cultured seedling of root system；
(7) acclimatization and transplants
Peppermint tissue-cultured seedling is taken out together with tissue culture bottle from culturing room, is placed in 22-30 DEG C of greenhouse, is refined under natural lighting
Seedling 7d, then open bottleneck hardening 3-5 d；Then tissue-cultured seedling is carefully taken out from bottle, the culture medium residual of root is washed away, by root
Portion's immersion mass fraction is cultivated 3-5 hours for 0.1% spend in precious No. 1 solution, then is transplanted into crop field, carries out conventional cultivation management.
2. a kind of germplasm in-vitro conservation method of peppermint according to claim 1, which is characterized in that in the step (1)
The formula of nutrient solution is Ca (H2PO4)2·H2O 0.4g/L、Ca(NO3)2 0.5g/L、KNO30.9g/L, urea 0.3g/L,
MgSO4·7H2O 0.28g/L FeNa2-EDTA 0.12g/L、H3BO3 0.06mg/L、MnSO4·4H2O 0.03mg/L、
ZnSO4·7H2O 0.06mg/L、CuSO4·5H2O 0.06mg/L, (NH4)2MoO4 0.06mg/L, spermidine 3.4mg/L.
3. a kind of germplasm in-vitro conservation method of peppermint according to claim 1, which is characterized in that in the step (2)
The formula of adventitious bud induction culture base are as follows: MS+2,4-D1.0mg/L+IBA0.2mg/L+ polyvinyl alcohol 12mg/L+ dithiothreitol (DTT)
0.8mg/L+yeast extract 5.3g/L, pH value is 5.8.
4. a kind of germplasm in-vitro conservation method of peppermint according to claim 1, which is characterized in that in the step (4)
The formula of preserving seed culture medium are as follows: KNO3980 ~ 1060mg/L, NH4NO3855 ~ 900mg/L, KH2PO4110 ~ 140mg/L,
MgSO4·7H2O 165 ~ 184mg/L, CaCl2·2H2O 203 ~ 213mg/L, MnSO4·4H2O 134 ~ 140mg/L, ZnSO4·
7H2O 7.0 ~ 8.0mg/L, H3BO35.9 ~ 6.3mg/L, KI 0.7 ~ 0.8mg/L, CuSO4·5H20.024 ~ 0.026mg/L of O,
CoCl2·6H2O 0.024 ~ 0.026mg/L, Na2MoO4·2H2O 0.13 ~ 0.19mg/L, FeSO4·7H223 ~ 25mg/L of O,
Na234 ~ 36mg/L of-EDTA, 47 ~ 55mg/L of inositol, proline-4 ~ 6mg/L, 2 ~ 4mg/L of serine, vitamin B1 0.2 ~
0.4mg/L, 0.3 ~ 0.5mg/L of vitamin B6, vitamin B5 0.3 ~ 0.5mg/L, 3-(α-pyridyl group) propyl alcohol 0.7 ~ 1.0
Mg/L, 6.1 ~ 6.5mg/L of tea polyphenols, 0.5 ~ 0.7mg/L of silver thiosulfate, 0.5 ~ 0.8g/L of diatomite, paclobutrazol 1.3 ~
1.7mg/L, 55 ~ 60g/L of sucrose, 17 ~ 21g/L of glycine betaine, 2.2 ~ 2.6mg/L of calcium propionate, peppermint 22 ~ 28mL/L of leaching liquor, coconut palm
30 ~ 36mL/L of cream, plant gel 2 ~ 4g/L, pH are 5.6 ~ 6.0.
5. the germplasm Plantlet in vitro and rejuvenation method of a kind of peppermint according to claim 1, which is characterized in that the step
(4) method that bottleneck is sealed up in is to be sealed with the masking foil bilayer with a thickness of 0.01mm plus sealed membrane.
6. the germplasm Plantlet in vitro and rejuvenation method of a kind of peppermint according to claim 1, which is characterized in that the step
(4) subinoculation is no more than 4 times in.
7. a kind of germplasm in-vitro conservation method of peppermint according to claim 1, which is characterized in that in the step (5)
The formula of rejuvenation culture medium are as follows: MS(is free of sucrose, agar)+glutathione 11 ~ 15mg/L+ humic acid 3.6 ~ 4.0mg/L+ carboxylic first
2.7 ~ 3.3g/L+ of base sodium cellulosate is to bromobenzene fluoroacetic acid 1.3 ~ 1.7mg/L+2-(3,4- dichlorophenoxy) triethylamine 0.8 ~
1.0mg/L+ maltose 23 ~ 25g/L+ fructose 13 ~ 16g/L+ plant gel 2 ~ 4g/L, pH 5.8.
8. the germplasm Plantlet in vitro and rejuvenation method of a kind of peppermint according to claim 1, which is characterized in that the step
(5) formula of the root media in are as follows: 1/2MS+NAA0.5mg/L+ active carbon 0.6g/L, pH 5.8.
9. the germplasm Plantlet in vitro and rejuvenation method of a kind of peppermint according to claim 4, which is characterized in that the germplasm
In Storaged media, peppermint leaching liquor the preparation method comprises the following steps: fresh mint blade is taken to smash to pieces, be in mass ratio the material-water ratio of 1:3
It is put into heat preservation extraction 1h in 60-70 DEG C of hot water, obtains leaching liquor after the filtering of 200 mesh filter screens.
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