CN109601392A - A kind of Huperzia serrata excised cotyledon, breeding and preservation method - Google Patents
A kind of Huperzia serrata excised cotyledon, breeding and preservation method 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
- A01H4/001—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 Huperzia serrata excised cotyledons, the method for breeding and preservation, establish best rapid propagation system in Huperzia serrata tissue incubation, the selection method including optimum explant, efficient multiple sterilizations method；Solve the Huperzia serrata isolated growth breeding of different genotype and the key technical problems such as save, it is final realize Huperzia serrata it is a large amount of in a short time, quickly, the breeding and preservation of high quality.The present invention can Regeneration in Vitro source difference province, different genotype Huperzia serrata, in vitro Huperzia serrata tissue can produce huperzine.This can effectively meet the needs of people are growing to Huperzia serrata, and then reduce destruction of the people to wild Huperzia serrata, be conducive to the protection of wild Huperzia serrata, have huge scientific research value, economic value and the ecological value.
Description
Technical field
The invention belongs to Plant Biotechnology and cell engineering field, are related to the breeding of Huperzia serrata excised cotyledon
Method.
Background technique
Huperzia serrata (Huperzia serrate (Thunb) Trev.) also known as serrate clubmoss herb, serrate clubmoss herb, feet added to a snake by an ignorant artist grass etc.,
For Huperziaceae stone araucaria pteridophyte, herb has effects that removing toxic substances, analgesic, eliminating stasis to subdue swelling.Huperzia serrata has under field conditions (factors)
Two ways, one by sporogenesis, and it is long that spore in underground forms the sprouting period needed for gametophyte, from spore germination to plant at
It is ripe to need 6-15 mature；Two are bred by gemma, but are planted mode and bred few discovery, thus the regeneration of wild resource by
Greatly limitation.
Report that alkaloid-huperzine (Huperzine A) contained in Huperzia serrata exists from first Chinese in 1972
After playing the role of loose striated muscle in animal experiment, abundant experimental results show Huperzine A be it is a kind of efficiently, low toxicity, can
Inverse, highly selective acetylcholinesterase inhibitor can be used for improving memory, treatment myasthenia gravis and senile dementia etc.
Disease, and have certain effect to inhibiting organic phosphoric acid to be poisoned.The chemical synthesis of huperzine is the hot spot of chemical synthesis research, but
The synthetic method reported at present all has certain limitation, can not the artificial synthesized huperzine with efficient bio-active
First is extracted from plants huperzine and remains most effective approach.
However, under field conditions (factors), Huperzia serrata natural propagation is extremely difficult, and slow growth, from spore germination to
Grow up to a complete plant and needed for about 15 years, plant height is only several centimetres to more than ten centimetres, is unable to satisfy people to snake
The growing demand of sufficient stone China fir, causes Huperzia serrata plant by extensive predatory acquisition, and wild natural resources increasingly subtracts
It is few, so that the regeneration of this kind of herb resource is received very big limitation, so that the artificial propagation problem of Huperzia serrata is concerned.Sheng Shu
The research and probes such as army etc., the Qin great Ji cuttage and seedling culture of Huperzia serrata or the Lycopersicum esculentum system of domestication are Huperzia serrata
The Sustainable Development and Utilization of resource has established certain technical foundation, but could not also industrialization promotion.Shen Xiaoxia etc. uses feet added to a snake by an ignorant artist
Stone China fir stem apex be explant carry out tissue cultures, it is found that its sterilizing is very difficult, although repeatedly sterilizing, be still difficult acquisition at
Function.In recent years, although Tu Yisheng seminar has obtained the huperzia serrata prothallium for being derived from Lushan Mountain, people by tissue cultures
Find that the repeatability of Huperzia serrata vitro Regeneration System is still very low, and due to different regions, the feet added to a snake by an ignorant artist stone of different genotype
Huperzine content in China fir has notable difference.Research also indicates that the difference of genotype has Plant Tissue Breeding Regeneration in Vitro
Have a great impact.So establish different regions source, different genotype Huperzia serrata in vitro breeding system have very
Important meaning, to lay the foundation from the basic bottleneck for solving huperzine raw materials for production.
Bibliography:
(1) Guo Bin, Xu Lingling, Wei Yahui, wait progress [J] CHINA JOURNAL OF CHINESE MATERIA MEDICA of serrate clubmoss herb, and 2009,34
(16):2018-2023.
(2) Sheng Shu army, Xu Jianzhong, Wang Zhian wait research [J] the development of resources and market of Cuttage Propagation of Huperzia Serratum Thunb, and 2000,
16(5):268-269.
(3) Qin great Ji, Yang Yongkang wait serrate clubmoss herb NFT cuttage raise seedling technique to study [J] Hubei Institute For Nationalities to pole pricker
Journal (natural science edition) 2010,28 (1): 18-21
(4) Ji Zhidan, Tu Yisheng, Chen Man wait Huperzia serrata in vitro culture to produce the condition optimizing and dynamics of huperzine
Study [J] Chinese herbal medicine, 2016,47 (3): 488-492.
(5) research [J] CHINA JOURNAL OF CHINESE MATERIA MEDICA of Shen Xiaoxia, Yu Xuping serrate clubmoss herb stem tip tissue culture sterilizing methods,
2002,27(6):458-459.
(6) Wang Deli, Gan Ping Chun, Qi Yaodong wait the ground of huperzine content in Huperzia serrata different stages of growth plant
Comparative observation [J] Chinese Journal of New Drugs that area's difference and habitat influence, 2014,23 (3): 326-332.
(7) Chen Jingfeng edits Plant Tissue Breeding and biotechnology Science Press, 2018, pp 22-23.
(8)Schenk RU,Hildebrandt AMedium and techniques for induction and
growth of monocotyledonous and dicotyledonous plant cell
cultures.Can.J.Bot.1972,50:199-204.
(9)Veliky I A,Martin S M,Afermenter for plant cell suspension
cultures Canadian Journal of Microbiology,1970,16(4),223-226.
Summary of the invention
In order to determine the optimum culture of its growth and breeding to explant effective sterilizing in Huperzia serrata tissue incubation
Base component etc., and then the rapid propagation system and preservation system of different genotype Huperzia serrata are established, finally improve in vitro culture
Thallus hupzine A in Huperzia serrata content (or enriched output), the present invention provides a kind of training of Huperzia serrata in vitro tissue
It supports, the method for breeding and preservation.
A kind of Huperzia serrata excised cotyledon, breeding and preservation method, comprising the following steps:
(1) tender stem segments of Huperzia serrata are chosen as explant；
(2) by explant with aseptic water washing 3-5 all over after, with ethyl alcohol surface sterilizing 30 seconds of 75% volume ratio, be used in combination
10% oxydol H2O2Surface sterilizing 8min, then with aseptic water washing 2-3 times, then with 0.15% HgC12Sterilize 5min,
It finally uses aseptic water washing 3-5 times, obtains aseptic explant；
(3) aseptic explant is inoculated on the SH training sample base of improvement or 6, the 7-V culture medium of improvement, is 5.8- with pH value
6.0, temperature is 25 ± 2 DEG C, relative humidity 60%-70%, intensity of illumination 2000-2500Lx, light application time 13h/d, skill
Art index is the CMC model that explant pollution rate is lower than 10%-20%, the explant after obtaining dedifferentiation；
(4) explant after dedifferentiation is removed into old leaf, then is transferred to differentiation and expands differentiation and proliferation, culture on breeding culture medium
Conditional synchronization is rapid (3), carries out Multiplying culture 30-40d, differentiation rate and proliferation rate 100%, obtains the leaf of Huperzia serrata Regeneration in Vitro
Shape body；
(5) thallus of Huperzia serrata Regeneration in Vitro is inoculated on Storaged media and is cultivated, subculture is primary within 50-80 days；
If you need in vitro breeding again, then it is inoculated into differentiation again and expands differentiation and proliferation on breeding culture medium；
Wherein,
The SH training sample base of improvement includes following components: SH minimal medium (Schenk RU, Hildebrandt AMedium
and techniques for induction and growth of monocotyledonous and
Dicotyledonous plant cell cultures.Can.J.Bot.1972,50:199-204), NAA1.0mg/L, sucrose
20g/L, agar 6.0g/L；
The 6,7-V culture medium of improvement includes following components: 1/2 6,7-V minimal medium (Veliky I A, Martin S
M,A fermenter for plant cell suspension cultures Canadian Journal of
Microbiology, 1970,16 (4), 223-226), NAA 1.0mg/L, sucrose 20g/L, agar 6.0g/L；
It includes following components: 1/2SH minimal medium or 1/2 6,7-V minimal medium, NAA that breeding culture medium is expanded in differentiation
0.5mg/L, sucrose 20g/L, agar 6.0g/L；
Storaged media includes following components: SH minimal medium, sucrose 20g/L, agar 6.0g/L.
Preferably, in step (1), the tender stem segments conduct for growing Huperzia serrata that is vigorous, containing mature spore is chosen
Regeneration rate can be improved in explant.
Preferably, in step (1), the tender stem segments of Huperzia serrata are cut into the segment of 1-4cm, cut off the leaf in stem section
Piece.
Preferably, in step (1), the Huperzia serrata is acquired from Wuyi Mountain, fujian or Hubei Wulin tomb nature reserve area
(they are the Huperzia serratas of two kinds of different genotypes).
Preferably, in step (2), 10% oxydol H2O2With 0.15% HgC12In contain 0.5% polysorbas20.
The thallus of Huperzia serrata Regeneration in Vitro obtained by the above method can be used for extracting huperzine, can be used for
Construct transformation system.
The solution have the advantages that: the present invention solves how to find explant in Huperzia serrata tissue incubation and have
The key technical problems such as the optimum media components of the sterilizing methods of effect and its growth and breeding, obtain two kinds of different genotypes
Huperzia serrata Regeneration in Vitro tissue, and then set up the rapid propagation system of Huperzia serrata, it is final to realize Huperzia serrata short
A large amount of in time, quick, high quality breeding, this can effectively meet the needs of people are growing to Huperzia serrata, into
And destruction of the people to wild Huperzia serrata is reduced, be conducive to the protection of wild Huperzia serrata, there is huge scientific research value, warp
Ji value and the ecological value.
Detailed description of the invention
Fig. 1 is the photo of the Huperzia serrata plant of the field grown as explant.
Fig. 2 is the relation schematic diagram of different sterilization types and Huperzia serrata in vitro culture survival rate.Sterilization type 1 indicates
75% alcohol 30s+ mercuric chloride 5min；2 indicate 75% alcohol 30s+ mercuric chloride 5min+450mg/L cephalo solution 5min；3 indicate 75%
Alcohol 30s+ hydrogen peroxide 8min+ mercuric chloride 5min；4 indicate 75% alcohol 30s+ hydrogen peroxide 8min+ mercuric chloride 5min+AAS+ malachite
It is green.
Fig. 3 is the photo of the in vitro Initial culture of Huperzia serrata.
Fig. 4 is the photo of callus regeneration during Huperzia serrata in vitro culture.
Fig. 5 is Huperzia serrata in vitro culture process Direct Regeneration thallus photo from the spore in stem section.
Fig. 6 is the thallophytic photo of Huperzia serrata after proliferation.
Fig. 7 is the result data of the content of huperzine in the Huperzia serrata tissue of high performance liquid chromatography detection Regeneration in Vitro
Figure；There is apparent absorption peak in Fig. 7, at 308nm, shows containing huperzine.
Specific embodiment
Below in conjunction with figure embodiment beneficial effect possessed by the present invention will be described in detail, it is intended to help reader more preferable
Ground understands essence of the invention, but cannot constitute any restriction to implementation of the invention and protection scope.
The invention is realized in this way the described method comprises the following steps:
(1) it selects explant: acquiring Wuyi Mountain, fujian and two kinds of Hubei Wulin tomb nature reserve area genotype feet added to a snake by an ignorant artist stone respectively
China fir, choose growth it is vigorous, containing mature spore by wild Huperzia serrata plant (Fig. 1), the tender stem segments of Huperzia serrata are cut
At the segment of 1-4cm, the blade in stem section is cut off.Explant is put into aseptic bottle on the super-clean bench with after sterile water wash 3 times
It is spare.It is low using the wild explant pollution rate of drying, it is seriously polluted if using wet wild Huperzia serrata for explant
(pollution rate is up to 80% or more).
(2) it kills surface bacterium: in experimentation of the present invention, successively using different explant sterilizing methods and (be detailed in figure
2) it, finally determines following best sterilizing process: the explant rinsed well is used into 75% (V/V) ethyl alcohol 30 seconds on the super-clean bench,
Then with 10% hydrogen peroxide (H2O2) (polysorbas20 containing 0.5%) surface sterilizing 8min, aseptic water washing 2-3 times, followed in turn by
0.1% mercuric chloride (HgC12) (polysorbas20 containing 0.5%) sterilizing 5min, finally use aseptic water washing 3-5 times, it is outer after sterilizing
Implant is spare.The explant pollution rate of the multiple sterilizations method is lower than 20%-30%.And use 75% (V/V) ethyl alcohol 30 seconds,
0.1% mercuric chloride (HgC12) sterilizing 5min method explant pollution rate up to 90% or more；Using 75% (V/V) ethyl alcohol 30 seconds,
10% hydrogen peroxide (H2O2) sterilizing 5min, 0.1% mercuric chloride (HgC12) sterilizing 8min, 450mg/L cephalo solution 5min side
Method explant pollution rate 70%-80%.
1 Huperzia serrata of table breeds various culture mediums in vitro
(3) Initial culture: being seeded in different initial culture bases (table 1, Fig. 3) for the explant that size is 1-2cm,
20-40d statistics discovery, SH minimal medium (or 1/2 6,7-V minimal medium)+NAA1.0mg/L of improvement, culture medium
Dosage of sucrose 20g/L, agar are that 6.0g/L dedifferentiation and inductivity are high (Fig. 4 and Fig. 5).PH value is 5.8-6.0, and culture parameters are
25 ± 2 DEG C, relative humidity 60-70%, intensity of illumination 2000-2500Lx, light application time 13h/d.
(4) Multiplying culture: by the lobate body tissue after proliferation in step (3), removing old leaf, takes fresh tender tissue about
1*1cm is transferred to differentiation and expands breeding culture medium (1/2SH minimal medium or 1/2 6,7-V minimal medium+NAA 0.5mg/L+ sugarcane
Sugared 20g/L+ agar 6.0g/L) on differentiation and proliferation, carry out Multiplying culture 30-40d, differentiation rate and proliferation rate 100% (Fig. 6),
The same step of condition of culture (3).Finally obtain Wuyi Mountain, fujian and two kinds of Hubei Wulin tomb nature reserve area genotype Huperzia serrata from
The regenerated thallus culture of body.
(5) Plantlet in vitro of different genotype Huperzia serrata: the Huperzia serrata Regeneration in Vitro thallus of different genotype connects
Kind is in culture (SH minimal medium+sucrose 20g/L+ agar 6.0g/L) on Storaged media.Subculture is primary within 50-80 days.If you need to
In vitro breeding again, is inoculated on proliferated culture medium, condition of culture is identical as step (4).
(6) in Regeneration in Vitro Huperzia serrata huperzine base content detection: after the thallus of tissue cultures is cleaned out
Drying to constant weight at 54 DEG C, after being ground into fine powder with mortar, accurately weighs powder 0.5g in the tool plug test tube of 10ml, adds
Enter 10ml2% tartaric acid solution, sealing, extraction for 24 hours, then in 70MHz, 50 DEG C of ultrasound 0.5h, filter out acid solution, sequentially add
2% tartaric acid solution of 10ml, 7ml, 7ml repeats to extract 3 times, filtering, merges gained extracting solution and filters 2 times；Gained filtrate adds
Ammonium hydroxide tune pH to 9-10, by the collection liquid of modulated PH, 40 DEG C of drying.Draw the dedicated methanol reagent of 3ml chromatography and above-mentioned drying
It in extract, volatilizees in draught cupboard methanol after extraction for 24 hours, draws 0.25ml into centrifuge tube；2ml first is added in remaining filter residue
Alcohol impregnates 2h, and 0.25ml is drawn after volatilization into centrifuge tube.It is repeated 3 times, merges filtrate several times, with 0.22 μm of membrane filtration degerming,
Filtrate is obtained to detect as efficient liquid phase point sample.By huperzine standard sample, wild Huperzia serrata plant powder liquid phase first
Alcohol processing, and with 0.22 μm of membrane filtration degerming, the sample as liquid phase point sample.Chromatographic column: waters C18 (250mm ×
4.6mm, 5 μm)；Mobile phase: methanol, 0.08mol/L ammonium acetate (pH 6.00) (3: 7)；Flow velocity 1mL/min；Detection wavelength
308nm；25 DEG C of column temperature；40 μ L of sample volume.Through detecting, in the Regeneration in Vitro thallus culture of two kinds of genotype Huperzia serratas
Contain huperzine (Fig. 7).
Embodiment described above only describe the preferred embodiments of the invention, not to model of the invention
It encloses and is defined, without departing from the spirit of the design of the present invention, those of ordinary skill in the art are to technical side of the invention
The various changes and improvements that case is made should all be fallen into the protection scope that claims of the present invention determines.
Claims (8)
1. a kind of method of Huperzia serrata excised cotyledon, breeding and preservation, comprising the following steps:
(1) tender stem segments of Huperzia serrata are chosen as explant；
(2) by explant with aseptic water washing 3-5 after, with ethyl alcohol surface sterilizing 30 seconds of 75% volume ratio, and with 10%
Oxydol H2O2Surface sterilizing 8min, then with aseptic water washing 2-3 times, then with 0.15% HgC12Sterilize 5min, finally uses
Aseptic water washing 3-5 times, obtain aseptic explant；
(3) aseptic explant is inoculated on the SH training sample base of improvement or 6, the 7-V culture medium of improvement, with pH value be 5.8-6.0,
Temperature is 25 ± 2 DEG C, relative humidity 60%-70%, intensity of illumination 2000-2500Lx, light application time 13h/d, technology refer to
It is designated as the CMC model that explant pollution rate is lower than 10%-20%, the explant after obtaining dedifferentiation；
(4) explant after dedifferentiation is removed into old leaf, then is transferred to differentiation and expands differentiation and proliferation, condition of culture on breeding culture medium
Same step (3) carries out Multiplying culture 30-40d, differentiation rate and proliferation rate 100%, obtains the lobate of Huperzia serrata Regeneration in Vitro
Body；
(5) thallus of Huperzia serrata Regeneration in Vitro is inoculated on Storaged media and is cultivated, subculture is primary within 50-80 days；If you need to
In vitro breeding again is then inoculated into differentiation again and expands differentiation and proliferation on breeding culture medium；
Wherein,
The SH training sample base of improvement includes following components: SH minimal medium, NAA 1.0mg/L, sucrose 20g/L, agar 6.0g/L；
The 6,7-V culture medium of improvement include following components: 1/2 6,7-V minimal medium, NAA 1.0mg/L, sucrose 20g/L,
Agar 6.0g/L；
It includes following components: 1/2 SH minimal medium or 1/2 6,7-V minimal medium, NAA that breeding culture medium is expanded in differentiation
0.5mg/L, sucrose 20g/L, agar 6.0g/L；
Storaged media includes following components: SH minimal medium, sucrose 20g/L, agar 6.0g/L.
2. according to the method described in claim 1, it is characterized by: selection growth is vigorous in step (1), contains mature spore
The tender stem segments of the Huperzia serrata of son are as explant.
3. according to the method described in claim 1, it is characterized by: the tender stem segments of Huperzia serrata are cut into step (1)
The segment of 1-4cm cuts off the blade in stem section.
4. according to the method described in claim 1, it is characterized by: the Huperzia serrata is acquired from Fujian in step (1)
Wuyi Mountain or Hubei Wulin tomb nature reserve area.
5. according to the method described in claim 1, it is characterized by: in step (2), 10% oxydol H2O2With 0.15%
HgC12In contain 0.5% polysorbas20.
6. the thallus for the Huperzia serrata Regeneration in Vitro that method according to claims 1 to 5 obtains.
7. the thallophytic application of Huperzia serrata Regeneration in Vitro according to claim 6.
8. application according to claim 7, it is characterised in that: the thallus of the Huperzia serrata Regeneration in Vitro is for mentioning
Take huperzine or for constructing transformation system.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110393152A (en) * | 2019-07-26 | 2019-11-01 | 西北大学 | A kind of improvement MS fluid nutrient medium and the sterile Shoot Tip Culture method of Huperzia serrata |
| CN111893136A (en) * | 2020-08-12 | 2020-11-06 | 江西师范大学 | Method for establishing agrobacterium-mediated huperzia serrata thallus genetic transformation system |
-
2019
- 2019-02-20 CN CN201910127856.8A patent/CN109601392B/en active Active
Non-Patent Citations (2)
| Title |
|---|
| 刘江海等: "蛇足石杉侧芽的发生与培养研究", 《中草药》 * |
| 吉枝单等: "蛇足石杉离体培养产生有效成分的研究", 《天然产物研究与开发》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110393152A (en) * | 2019-07-26 | 2019-11-01 | 西北大学 | A kind of improvement MS fluid nutrient medium and the sterile Shoot Tip Culture method of Huperzia serrata |
| CN111893136A (en) * | 2020-08-12 | 2020-11-06 | 江西师范大学 | Method for establishing agrobacterium-mediated huperzia serrata thallus genetic transformation system |
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