CN101889551A - Tissue culture method of huperizia serrata - Google Patents
Tissue culture method of huperizia serrata Download PDFInfo
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Abstract
The invention discloses a method for tissue culture method of huperizia serrata. The method comprises the following steps of: selecting an explant; killing surface bacteria; performing inoculation and cultivation; killing endophyte; performing propagation cultivation; and performing rooting cultivation. The huperizia serrata is a precious medicinal pteridophyte which has a tough requirement on a habitat, grows slowly under a natural condition and has a long production period. In a conventional method, propagation coefficient and biomass are low and requirements on medicament production cannot be met. Due to the adoption of the method, the differentiation rate of a huperizia serrata test tube plantlet is up to 90 percent, monthly multiplication multiple is up to 2.1 to 3.5, rooting rate is up to 100 percent, a root system grows quickly, root number is up to 1.6 to 3.4 per plant, a regeneration seedling grows healthily and the root system is developed. The method has the advantages of effectively restraining the pollution and browning of a huperizia serrata explant, promoting the survival and growth of the explant and providing important technical support for the realization of manual mass planting of the huperizia serrata along with simpleness, convenience, practicability, high efficiency and low cost.
Description
Technical field
The invention belongs to field of plant tissue culture technique, be specifically related to the method that a kind of Huperzia serrata tissue culturing system sets up.
Background technology
Huperzia serrata [Huperzia serrata (Thunb.) Trev.] has another name called help king, invaluable, serrate clubmoss herb, mountain sesame, feet added to a snake by an ignorant artist grass, belongs to fern Huperziaceae (Huperiaceae) stone araucaria (Huperzia Bemh.), is a kind of medicinal pteridophyte of preciousness.Diseases such as its herb treatment traumatic injury commonly used among the people, extravasated blood pyogenic infections, schizophrenia.Its contained huperzine A (Huperzine A) but proved low toxicity, efficient, reversible and highly selective acetylcholine esterase inhibition (AchE) by pharmacological evaluation, developed multiple patent medicine, can be used for memory function for the treatment of myasthenia gravis, improve learning efficiency and improve the elderly etc., be the specific drug of diseases such as present treatment senile dementia, market prospects are wide.Because the huperzine category-A of synthetic is big and cost is high like the thing side effect, therefore is used for the huperzine A of patent medicine at present all from wild resource.The wild resource of huperzine is mainly derived from Huperziaceae plants of Huperzia Huperzia serrata, Huperziaceae horse hair araucaria and stone araucaria other plant.The huperzine A that the present overwhelming majority is used for patent medicine all extracts from wild Huperzia serrata.
Because Huperzia serrata is grown in unfrequented mountain region thick forest or the dark and damp soil of cheuch more, the habitat is required harshness, poor growth, the production cycle is long; Huperzine content in herb is very little in addition, and at present, pharmaceutical production relies on wild resource fully, costs an arm and a leg, and excavates for a long time and causes resource sharply to reduce, and its wild resource total amount is very limited.Therefore, it is imperative to seek the huperzine regenerated resources.Present studies show that: though it is feasible to carry out the artificial cultivation of Huperzia serrata by modes of nourishing and generating such as gemma, cuttage, press strip and plant division, reproduction coefficient and biomass are lower, carry out extensive tame condition is that all right ripe.And advantage such as tissue culture has that the material of needs is few, condition is controlled, be not subject to seasonal restrictions, the cycle is short, repeatability is strong is the lycopod desirable means of breeding and large-scale industrialized production fast.By tissue culture technique Huperzia serrata is carried out expanding propagation, be intended to seek one and conveniently breed approach, so that, lay the first stone for meeting the need of market for artificial a large amount of cultivations provide technical support.The application organizes culture technique is bred Huperzia serrata, both can enlarge crude drug source, can protect wild resource again.
Exist the pick-up rate of explant sterilization difficulty, aseptic explant low in the Huperzia serrata tissue culture procedures, explant inoculation after stain and browning are serious, can not normal growth, differentiation and propagation, and problems such as the extremely low and propagation difficulty of the inductivity of callus.Though the researcher has also carried out the trial of some related fields both at home and abroad, does not also have the report of Huperzia serrata tissue culture success so far.
Summary of the invention
Low at there being the pick-up rate of explant sterilization difficulty, aseptic explant in the Huperzia serrata tissue culture procedures, explant inoculation after stain and browning are serious, can not normal growth, problem such as differentiation and propagation, the present invention has realized the lower pollution rate of Huperzia serrata tissue culture, low brownization rate, high-servival rate, high growth rate, high differentiation rate and high rooting rate by the research to sterilizing methods, medium and condition of culture; The present invention aims to provide a kind of method of Huperzia serrata tissue culture.
Concrete operations step of the present invention is as follows:
A kind of method of Huperzia serrata tissue culture may further comprise the steps:
(1) selects explant
Sporophyte from field acquisition Huperzia serrata [Huperzia serrata (Thunb.) Trev.] cuts stem apex, rinses well through running water;
(2) kill surperficial bacterium
The stem apex of cleaning is immersed the ethanolic solution 30-50s of concentration 70% earlier in super-clean bench; Then place the mercuric chloride (HgCl of concentration 0.1%
2) solution soaking 7-10min, change the hydrogen peroxide (H of concentration 7% again over to
2O
2) soak 10-15min in the solution, use aseptic water washing 3-5 time at last, as the explant of Huperzia serrata tissue culture;
(3) inoculated and cultured
Described explant is seeded on the modified MS medium that promotes the stem apex growth, places under 22-27 ℃, light application time 12-14h/d, intensity of illumination 800-2000lx condition and cultivate 2-3 week;
(4) kill endophyte
Explant after the inoculated and cultured is gone on the modified MS medium that contains malachite green and antibiotic AAS, continue to cultivate 2-3 week, the same step of condition of culture (3); Endophyte is killed.
(5) expand numerous cultivation
Change step (4) sterile explant after endophyte is killed over to the bud differentiation of growing thickly and expand numerous modified MS medium continuation cultivation a period of time, when stem apex terminal bud length behind 5-8mm, cut the about 3-5mm of explant terminal bud that newly grows and be transferred to differentiation and the enrichment culture that carries out tissue cultivating seedling in the numerous modified MS medium of the bud differentiation expansion of growing thickly, the same step of condition of culture (3) produces the seedling of growing thickly from the differentiation of tissue cultivating seedling basal part of stem;
(6) culture of rootage
Cut the seedling of growing thickly that produces from the differentiation of tissue cultivating seedling basal part of stem in the step (5) and go to the root media, place culturing room to carry out culture of rootage, the same step of condition of culture (3);
Described modified MS medium is made up of following material: 950mg/L potassium nitrate (KNO
3), 825mg/L ammonium nitrate (NH
4NO
3), 185mg/L magnesium sulfate (MgSO
47H
2O), 220mg/L calcium chloride (CaCl
22H
2O), 85mg/L potassium dihydrogen phosphate (KH
2PO
4), 8.45mg/L manganese sulphate (MnSO
4H
2O), 4.3mg/L zinc sulphate (ZnSO
47H
2O), 3.1mg/L boric acid (H
3BO
3), 0.415mg/L potassium iodide (KI), 0.125mg/L sodium molybdate (Na
2MoO
42H
2O), 0.025mg/L copper sulphate (CuSO
45H
2O), 0.025mg/L cobalt chloride (CoCl
26H
2O), 50mg/L inositol, 1mg/L glycine, 0.5mg/L puridoxine hydrochloride (VB
6), 0.5mg/L nicotinic acid (VB
5), 0.1mg/L thiamine hydrochloride (VB
1), 9.31mg/L disodium ethylene diamine tetraacetate (Na
2-EDTA2H
2O), 6.96mg/L ferrous sulfate (FeSO
47H
2O), 30g/L sucrose, 5.6g/L agar;
The described modified MS medium that contains malachite green and antibiotic AAS is made up of following material: add 100mg/L antibiotic (AAS) and 0.5mg/L malachite green in modified MS medium;
The described bud differentiation of growing thickly is expanded numerous modified MS medium and is made up of following material: add 2 μ mol/L 6-benzyl aminopurines (6-BA) in modified MS medium;
Described root media is made up of following material: add 10 μ mol/L 3-indolebutyric acids (IBA) in modified MS medium.
Described step cuts stem apex 1.5-2.0cm in (1), rinses well with running water, changes in every liter of running water that drips the 0.2ml liquid detergent to soak rinsing 20min, rinses well with running water again.
After need blotting surface moisture with aseptic filter paper, the stem apex explant behind the surface sterilizing is used further to inoculated and cultured in the step (3).
Described 100mg/L antibiotic AAS contains 30mg/L penicillin, 50mg/L streptomycin and 125 μ g/L amphotericin Bs, and AAS is now with the current for the 100mg/L antibiotic, adds after the equal filtration sterilization of AAS and malachite green.
The seedling of growing thickly that culture of rootage described in the step (6) is used is: the bud of growing thickly that produces from the differentiation of tissue cultivating seedling basal part of stem in step (5) grows to 5-8mm.
The differentiation of described modified MS medium, the bud of growing thickly is expanded before numerous modified MS medium and the root media sterilization all under 50-60 ℃, regulates pH to 5.8, and all in 121 ℃, 104kPa sterilization 17-20min down.
Malachite green in the described modified MS medium that contains malachite green and antibiotic AAS and antibiotic AAS be earlier through 0.22 μ m filtering with microporous membrane degerming, and then add to through autoclave sterilization and be cooled to 50-60 ℃ modified MS medium.
Useful technique effect of the present invention embodies in the following areas:
1, since the sporophyte stem apex of having chosen Huperzia serrata [Huperzia serrata (Thunb.) Trev.] as explant, having adopted ethanol, mercuric chloride and hydrogen peroxide to cooperate kills the microorganism of outer planting surface, combine with antibiotic by malachite green again and kill the method for endophyte, obtained about 50% aseptic explant, improved the yield of aseptic explant, effectively control brownization and the albefaction of explant, reached the lower pollution rate of explant and the technique effect of brownization rate.
2, owing to selected the minimal medium of modified MS medium as tissue culture, and under 22-27 ℃, light application time 12-14h/d, intensity of illumination 800-2000lx condition, cultivate, brownization of explant is light, the survival rate that has realized the stem apex explant is up to 90%, growth rate reaches the 6-8mm/ month, well-grown technique effect.
3, owing to selected the additional 2 μ mol/L 6-benzyl aminopurines (6-BA) of modified MS medium to expand breeding culture medium as the bud differentiation of growing thickly, and under 22-27 ℃, light application time 12-14h/d, intensity of illumination 800-2000lx condition, cultivate, the differentiation rate that has realized tissue cultivating seedling reaches 90%, breed multiple by the moon and reach 2.1-3.5, there be not brownization, the technique effect that growth result is good.
4, owing to selected modified MS medium to add 10 μ mol/L 3-indolebutyric acids (IBA) as root media, and under 22-27 ℃, light application time 12-14h/d, intensity of illumination 800-2000lx condition, cultivate, the rooting rate of seedling of having realized growing thickly reaches 100%, fast and the several 1.6-3.4/ strains that reach of taking root of root growth, the seedling growth rate reaches the 9.4mm/ month, young leaves forms number and reaches 4 slices/month, and biomass increases by 5.1 times, the flourishing technique effect of seedling robust growth and root system of growing thickly.
Embodiment
Below in conjunction with specific embodiment the present invention is further described.Characteristics of the present invention and advantage will be clearer along with description, but this exemplary embodiment only is used for illustrating the present invention, scope of the present invention is not constituted any restriction.
Embodiment
(1) selects explant
Sporophyte from field acquisition Huperzia serrata [Huperzia serrata (Thunb.) Trev.], cutting stem apex 1.5-2.0cm rinses well with running water, change in every liter of running water that drips 0.2ml (2-3 drips) liquid detergent and soak rinsing 20min, it is subject to sterilization as the explant of tissue culture to rinse the back well with running water again; Experimental result shows, the stem apex that adopts appropriate length is as explant, and brownization gently, easily survive.
Sporophyte: in the heterogamous history of life, produce the plant corpus of spore and tool 2 diploids plant.Grow by fertilized egg (zygote).Sporangium of bryophyte and accessory structure thereof (seta and base foot), fern and the phanerogamous plant corpus of commonly seeing all are sporophytes.
After the Huperzia serrata sporophyte is adopted back from the field, in 15-22 ℃ of environment, preserve moisture at most and placed 7-8 days; Sporophyte long sterilization and the survival that is unfavorable for stem apex explant standing time, sporophyte are adopted Hui Houyi and are cut stem apex immediately and be used for sterilization and cultivate.
(2) kill surperficial bacterium
Stem apex after cleaning is immersed the ethanolic solution 30-50s of concentration 70% earlier in super-clean bench; Then place the mercuric chloride (HgCl of concentration 0.1%
2) solution soaking 7-10min, change over to again in the hydrogen peroxide solution of concentration 7% and soak 10-15min, use aseptic water washing 3-5 time at last, as the explant of Huperzia serrata tissue culture; Experimental result shows, adopts the ethanolic solution of concentration 70%, the mercuric chloride (HgCl of concentration 0.1%
2) and the hydrogen peroxide of concentration 7%, surface sterilizing is effective, and pollution rate is 36% only, and pollution rate is lower.
Mercuric chloride (the HgCl of the ethanolic solution of concentration 70%, concentration 0.1%
2) hydrogen peroxide (H of solution and concentration 7%
2O
2) solution prepares with sterile water.
(3) inoculated and cultured
The explant of Huperzia serrata tissue culture is seeded on the modified MS medium that promotes the stem apex growth, places under 22-27 ℃, light application time 12-14h/d, intensity of illumination 800-2000lx condition and cultivate; Experimental result shows, is cultivating 2-3 on this medium after week, and the survival rate of aseptic explant reaches 90%, and brownization is light.
Modified MS medium is made up of following material: 950mg/L potassium nitrate (KNO
3), 825mg/L ammonium nitrate (NH
4NO
3), 185mg/L magnesium sulfate (MgSO
47H
2O), 220mg/L calcium chloride (CaCl
22H
2O), 85mg/L potassium dihydrogen phosphate (KH
2PO
4), 8.45mg/L manganese sulphate (MnSO
4H
2O), 4.3mg/L zinc sulphate (ZnSO
47H
2O), 3.1mg/L boric acid (H
3BO
3), 0.415mg/L potassium iodide (KI), 0.125mg/L sodium molybdate (Na
2MoO
42H
2O), 0.025mg/L copper sulphate (CuSO
45H
2O), 0.025mg/L cobalt chloride (CoCl
26H
2O), 50mg/L inositol, 1mg/L glycine, 0.5mg/L puridoxine hydrochloride (VB
6), 0.5mg/L nicotinic acid (VB
5), 0.1mg/L thiamine hydrochloride (VB
1), 9.31mg/L disodium ethylene diamine tetraacetate (Na
2-EDTA2H
2O), 6.96mg/L ferrous sulfate (FeSO
47H
2O), 30g/L sucrose, 5.6g/L agar.
(4) kill endophyte
Explant after the inoculated and cultured is gone on the modified MS medium that contains 0.5mg/L malachite green and 100mg/L antibiotic AAS, under 22-27 ℃, light application time 12-14h/d, intensity of illumination 800-2000lx condition, continue to cultivate 2-3 week, endophyte is killed; Experimental result shows, by adding bactericide in medium, after the explant through cultivating absorbs in the body again endophyte is killed, and sterilization effect is good, and the pollution rate of explant only is 3% after 3 weeks.
The modified MS medium that contains 0.5mg/L malachite green and 100mg/L antibiotic AAS is to add 100mg/L antibiotic (AAS) and 0.5mg/L malachite green composition in above-mentioned modified MS medium.
Concrete operations are as follows: contain malachite green in the modified MS medium of malachite green and antibiotic AAS and antibiotic AAS earlier through 0.22 μ m filtering with microporous membrane degerming, and then add to through autoclave sterilization and be cooled in 50-60 ℃ the modified MS medium.
(5) expand numerous cultivation
With in the step (4) after endophyte is killed sterile explant change the bud differentiation of growing thickly over to and expand numerous modified MS medium and continue to cultivate a period of time, when stem apex terminal bud length behind 5-8mm, cut the about 3-5mm of explant terminal bud that newly grows and be transferred on the modified MS medium of having added 2 μ mol/L 6-benzyl aminopurines (6-BA), the differentiation and the enrichment culture of the bud of under 22-27 ℃, light application time 12-14h/d, intensity of illumination 800-2000lx condition, in culturing room, growing thickly; Experimental result shows that the differentiation rate of tissue cultivating seedling reaches 90%, and a month propagation multiple reaches 2.1-3.5, does not have brownization.
Numerous modified MS medium is expanded in the bud of growing thickly differentiation, adds 2 μ mol/L 6-benzyl aminopurines (6-BA) and form in above-mentioned modified MS medium.
(6) culture of rootage
Cut in (5) seedling of growing thickly that produces from the differentiation of tissue cultivating seedling basal part of stem and go to the root media, under 22-27 ℃, light application time 12-14h/d, intensity of illumination 800-2000lx condition, in culturing room, carry out culture of rootage; Experimental result shows that the rooting rate of the seedling of growing thickly reaches 100%, and the fast and number of taking root of root growth reaches the 1.6-3.4/ strain, and the seedling growth rate reaches the 9.4mm/ month, and young leaves forms number and reaches 4 slices/month, and biomass increases by 5.1 times, and grow thickly seedling robust growth and root system are flourishing.
Root media adds 10 μ mol/L 3-indolebutyric acids (IBA) and forms in above-mentioned modified MS medium.
The differentiation of above-mentioned modified MS medium, the bud of growing thickly is expanded before numerous modified MS medium and the root media sterilization all under 50-60 ℃, regulates pH to 5.8, and all in 121 ℃, 104kPa sterilization 17-20min down.
Claims (7)
1. the method for a Huperzia serrata tissue culture is characterized in that may further comprise the steps:
(1) selects explant
Sporophyte from field acquisition Huperzia serrata [Huperzia serrata (Thunb.) Trev.] cuts stem apex, rinses well through running water;
(2) kill surperficial bacterium
The stem apex of cleaning is immersed the ethanolic solution 30-50s of concentration 70% earlier in super-clean bench; Then place the mercuric chloride (HgCl of concentration 0.1%
2) solution soaking 7-10min, change the hydrogen peroxide (H of concentration 7% again over to
2O
2) soak 10-15min in the solution, use aseptic water washing 3-5 time at last, as the explant of Huperzia serrata tissue culture;
(3) inoculated and cultured
Described explant is seeded on the modified MS medium that promotes the stem apex growth, places under 22-27 ℃, light application time 12-14h/d, intensity of illumination 800-2000lx condition and cultivate 2-3 week;
(4) kill endophyte
Explant after the inoculated and cultured is gone on the modified MS medium that contains malachite green and antibiotic AAS, continue to cultivate 2-3 week, the same step of condition of culture (3); Endophyte is killed.
(5) expand numerous cultivation
Change step (4) sterile explant after endophyte is killed over to the bud differentiation of growing thickly and expand numerous modified MS medium continuation cultivation a period of time, when stem apex terminal bud length behind 5-8mm, cut the about 3-5mm of explant terminal bud that newly grows and be transferred to differentiation and the enrichment culture that carries out tissue cultivating seedling in the numerous modified MS medium of the bud differentiation expansion of growing thickly, the same step of condition of culture (3) produces the seedling of growing thickly from the differentiation of tissue cultivating seedling basal part of stem;
(6) culture of rootage
Cut the seedling of growing thickly that produces from the differentiation of tissue cultivating seedling basal part of stem in the step (5) and go to the root media, place culturing room to carry out culture of rootage, the same step of condition of culture (3);
Described modified MS medium is made up of following material: 950mg/L potassium nitrate (KNO
3), 825mg/L ammonium nitrate (NH
4NO
3), 185mg/L magnesium sulfate (MgSO
47H
2O), 220mg/L calcium chloride (CaCl
22H
2O), 85mg/L potassium dihydrogen phosphate (KH
2PO
4), 8.45mg/L manganese sulphate (MnSO
4H
2O), 4.3mg/L zinc sulphate (ZnSO
47H
2O), 3.1mg/L boric acid (H
3BO
3), 0.415mg/L potassium iodide (KI), 0.125mg/L sodium molybdate (Na
2MoO
42H
2O), 0.025mg/L copper sulphate (CuSO
45H
2O), 0.025mg/L cobalt chloride (CoCl
26H
2O), 50mg/L inositol, 1mg/L glycine, 0.5mg/L puridoxine hydrochloride (VB
6), 0.5mg/L nicotinic acid (VB
5), 0.1mg/L thiamine hydrochloride (VB
1), 9.31mg/L disodium ethylene diamine tetraacetate (Na
2-EDTA2H
2O), 6.96mg/L ferrous sulfate (FeSO
47H
2O), 30g/L sucrose, 5.6g/L agar;
The described modified MS medium that contains malachite green and antibiotic AAS is made up of following material: add 100mg/L antibiotic (AAS) and 0.5mg/L malachite green in modified MS medium;
The described bud differentiation of growing thickly is expanded numerous modified MS medium and is made up of following material: add 2 μ mol/L 6-benzyl aminopurines (6-BA) in modified MS medium;
Described root media is made up of following material: add 10 μ mol/L 3-indolebutyric acids (IBA) in modified MS medium.
2. the method for a kind of Huperzia serrata tissue culture according to claim 1, it is characterized in that: described step cuts stem apex 1.5-2.0cm in (1), rinse well with running water, change in every liter of running water that drips the 0.2ml liquid detergent and soak rinsing 20min, rinse well with running water again.
3. the method for a kind of Huperzia serrata tissue culture according to claim 1 is characterized in that: be used further to inoculated and cultured in the step (3) after the stem apex explant behind the surface sterilizing need blot surface moisture with aseptic filter paper.
4. the method for a kind of Huperzia serrata tissue culture according to claim 1, it is characterized in that: described 100mg/L antibiotic AAS contains 30mg/L penicillin, 50mg/L streptomycin and 125 μ g/L amphotericin Bs, AAS is now with the current for the 100mg/L antibiotic, adds after the equal filtration sterilization of AAS and malachite green.
5. the method for a kind of Huperzia serrata tissue culture according to claim 1 is characterized in that: the seedling of growing thickly that the culture of rootage described in the step (6) is used is: the bud of growing thickly that produces from the differentiation of tissue cultivating seedling basal part of stem in step (5) grows to 5-8mm.
6. the method for a kind of Huperzia serrata tissue culture according to claim 1, it is characterized in that: the differentiation of described modified MS medium, the bud of growing thickly is expanded before numerous modified MS medium and the root media sterilization all under 50-60 ℃, regulate pH to 5.8, and all in 121 ℃, the following sterilization of 104kPa 17-20min.
7. the method for a kind of Huperzia serrata tissue culture according to claim 1, it is characterized in that: malachite green in the described modified MS medium that contains malachite green and antibiotic AAS and antibiotic AAS be earlier through 0.22 μ m filtering with microporous membrane degerming, and then add to through autoclave sterilization and be cooled to 50-60 ℃ modified MS medium.
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2010
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| CN102754595A (en) * | 2011-04-28 | 2012-10-31 | 张宗申 | Huperzia serrata hairy root system preparation and cultivation method |
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| CN106613938A (en) * | 2016-09-09 | 2017-05-10 | 江西农业大学 | Effective huperzia serrata callus culture method |
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| CN110663547A (en) * | 2018-07-03 | 2020-01-10 | 中国科学院上海生命科学研究院 | Culture method of cryptomeria draconis and picea mucronata explants |
| CN109258148A (en) * | 2018-09-29 | 2019-01-25 | 西北大学 | A kind of method of Huperzia serrata rooting of cuttings |
| CN109258424A (en) * | 2018-09-29 | 2019-01-25 | 西北大学 | A kind of Huperzia serrata plant artificial graft's method |
| CN109496861A (en) * | 2018-12-05 | 2019-03-22 | 湖北省农业科学院经济作物研究所 | A method of inhibiting mulberry tree tissue cultures endophytic bacterial contamination |
| 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 |
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