CN105724249A - Exogenous additive culture medium for promoting differentiation of anoectochilus formosanus protocorm - Google Patents
Exogenous additive culture medium for promoting differentiation of anoectochilus formosanus protocorm Download PDFInfo
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- CN105724249A CN105724249A CN201610098329.5A CN201610098329A CN105724249A CN 105724249 A CN105724249 A CN 105724249A CN 201610098329 A CN201610098329 A CN 201610098329A CN 105724249 A CN105724249 A CN 105724249A
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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 an exogenous additive culture medium for promoting differentiation of anoectochilus formosanus protocorm. The culture medium is composed of a basic culture medium body and an exogenous additive, and by means of the culture medium, differentiation of the anoectochilus formosanus protocorm into seedlings can be obviously promoted. By means of the exogenous additive culture medium, tissue culture seedlings can be provided for large-scale planting of anoectochilus formosanus, and it is beneficial to protect anoectochilus formosanus wild resources.
Description
Technical field:
The invention belongs to biological field, in particular to a kind of additives culture medium promoting Herba Anoectochili roxburghii protocorms to break up.
Background technology:
Herba Anoectochili roxburghii (Anoectochilusroxburghii (Wall.) Lindl), has another name called Anoectochilus nefiliforme (Nakai) hara, Herba Melicae Scabrosae, is the orchid family Anoectochilus Blume herbaceos perennial.Have the good name such as the king of medicine, gold grass, bird Radix Ginseng, be widely used valuable ingredient of Chinese medicine among the people, its plant type is small and exquisite, and vein is golden yellow, in meshy arrangement, leaf attractive in appearance.It is mainly distributed on the ground such as Fujian, Taiwan, Guangdong, Guizhou, Sichuan, Yunnan.Liking dark and damp, be grown in the sough of height above sea level 300-1200 rice evergreen broadleaf forest, temperature is 20-25 DEG C, avoids direct sunlight.Gold line, with all herbal medicine, has clearing away heat and cooling blood, reduces the effects such as blood glucose blood pressure, antiinflammatory, is mainly used in treatment diabetes, hypertension and tumor etc. clinically.
In recent years, owing to irrational mad adopting is dug excessively in addition to growing environment requirement harshness, Herba Anoectochili roxburghii wild resource was endangered, and artificial culture difficulty is big, and the demand and supply contraction highlights.Plant tissue culture technique is utilized to realize Fast-propagation, it it is an important channel of this resource of sustainable use, but Herba Anoectochili roxburghii tissue culture general stem section is bred at present, its breeding coefficient is relatively low, therefore the breeding coefficient of Herba Anoectochili roxburghii tissue cultured seedling is significantly improved by the differentiation potency of protocorms, it is to meet the market demand, reduces the effective way of production cost.
Protocorms is the complex that embryo occurs with allelotaxis, is the artificial seed in group training process.In tissue-culturing rapid propagation system, utilize propagation and the differentiation of protocorms, the growth coefficient of gold link group seedlings cultivating can be improved significantly, provide technical support for implant mass Herba Anoectochili roxburghii.
Summary of the invention:
It is an object of the invention to provide a kind of additives culture medium promoting Herba Anoectochili roxburghii protocorms to break up, to improve the differentiation rate of protocorms.The composition of this culture medium includes: basal medium and additives.
The component of described basal medium and content is:
KNO380-2900mg/L, NH4NO3105-1520mg/L, KH2PO419-410mg/L, MgSO4·7H2O240-750mg/L, CaCl2·2H2O145-460mg/L, KCl0-67mg/L, KI0.7-1.7mg/L, H3BO30.7-6.3mg/L, MnSO4·4H2O4-25mg/L, ZnSO4·7H2O1.5-8.6mg/L, Na2MoO4·2H2O0-0.28mg/L, CuSO4·5H2O0-0.35mg/L, CoCl2·6H2O0-0.03mg/L, Na2-EDTA0-38mg/L, FeSO4·7H2O24-29mg/L, inositol 0-100mg/L, glycine 0-2.5mg/L, VB10.1-11.0mg/L, VB60.4-1.2mg/L, nicotinic acid 0.3-1.5mg/L, 6-benzyladenine (hereinafter referred to as 6-BA) 0.2-3.0mg/L, naphthalene acetic acid (hereinafter referred to as NAA) 0.2-2.0mg/L, sucrose 20-30g/L, agar 4.0-7.0g/L, activated carbon 0.5-4.0g/L.
Described additives is the mixed extract of Rhizoma Solani tuber osi extracting solution, Fructus Musae extracting solution or the two composition, and total volume fraction is 0.1-20%, and the Volume fraction of mixing additive extracting solution is Rhizoma Solani tuber osi extracting solution: Fructus Musae extracting solution=1-35: 1-25.
A kind of additives culture medium promoting Herba Anoectochili roxburghii protocorms to break up of the present invention is decided to be A culture medium, and it is formulated as existing compound method, and wherein the compound method of additives is as follows:
Rhizoma Solani tuber osi is peeled clean, weigh 300g Rhizoma Solani tuber osi and be cut into about 2cm2Bulk, add the pure water of 800ml, micro-30min that boils, by filtered through gauze and get final product, be settled to 1 liter, be made into Rhizoma Solani tuber osi extracting solution mother solution；Fructus Musae peeling is weighed 300g and is cut into about 2cm2Bulk, add the pure water of 800ml, micro-30min that boils, by filtered through gauze and get final product, be settled to 1 liter, be made into Fructus Musae extracting solution mother solution.After being made into mother solution, draw correspondingly volume.
The compound method of the additives culture medium of Herba Anoectochili roxburghii protocorms differentiation: take the distilled water of 60%, add MS dry powder 4.74g/L (without sucrose and agar), sucrose 30g/L, agar 4g/L, add the concentration (0.2mg/L-3.0mg/L) of plant growth regulator 6-BA, the concentration (0.2mg/L-2.0mg/L) of NAA, the extracting solution total volume fraction (0.1%-20%) of additives and activated carbon (0.5mg/L-4.0mg/L), boil once, the pH value of solution is regulated between 5.8-6.2 with 1mol/LNaOH solution after constant volume, it is distributed in tissue culture bottle, with sterilizing 20min under high-pressure sterilizing pot 121MPa, division culture medium is obtained after cooling.
Sterile working inoculates: on super-clean bench, takes the Herba Anoectochili roxburghii protocorms of growth 30d, adopts aseptic technique that protocorms is cut into 2cm2Size, is inoculated in division culture medium, and 45d observes its differentiating phenomenon.Condition of culture is illumination 20001x, light application time 12h/d, temperature 25 ± 1 DEG C, and air humidity keeps 50%-60%.
Herba Anoectochili roxburghii protocorms differentiation rate=(sum of the outer implant number/outer implant of inoculation of differentiation) × 100%
Usefulness of the present invention: adopt basal culture medium that Herba Anoectochili roxburghii protocorms can be made to break up, can provide seedling for Herba Anoectochili roxburghii implant mass and can protect Herba Anoectochili roxburghii wild resource.
Detailed description of the invention
Embodiment 1
The stem section 2cm of depletion Anoectochilus roxburghii belt segment, 6min is soaked with washing powder solution, flowing water rushes 2h, with 75% alcohol disinfecting 20s on aseptic super-clean bench, aseptic washing 3 times, sterilize 8min in 0.1% mercuric chloride, aseptic washing 6 times, with dissecting knife by stem section two tip cut-off after sterilizing, is inoculated in MS culture medium, this culture medium contains the NAA of 6-BA and the 0.2mg/L of 2.0mg/L, cultivation temperature is 25 ± 1 DEG C, illumination 15001x, light application time 12h/d, air humidity grows protocorms at the two ends of stem section after keeping 50%-60%, 30d.
After being grown 30 days in above-mentioned culture medium by the Herba Anoectochili roxburghii protocorms subculture obtained, it is cut to 2cm2Size, is inoculated in division culture medium, and 45d observes its differentiating phenomenon.Consisting of of A culture medium:
KNO380mg/L, NH4NO3105mg/L, KH2PO419mg/L, MgSO4·7H2O240mg/L, CaCl2·2H2O145mg/L, KI0.7mg/L, H3BO30.7mg/L, MnSO4·4H2O4mg/L, ZnSO4·7H2O1.5mg/L, Na2MoO4·2H2O0.12mg/L, CuSO4·5H2O0.13mg/L, CoCl2·6H2O0.01mg/L, Na2-EDTA25mg/L, FeSO4·7H2O24mg/L, inositol 20mg/L, glycine 0.5mg/L, VB10.1mg/L, VB60.4mg/L, nicotinic acid 0.3mg/L, 6-benzyladenine 2.0mg/L, naphthalene acetic acid 0.2mg/L, sucrose 20g/L, agar 4.0g/L, activated carbon 1g/L, added natural additive is Rhizoma Solani tuber osi extracting solution (the following table of Rhizoma Solani tuber osi extracting liq fraction).Cultivation temperature 25+1 DEG C, illumination 2000lx, light application time 12h/d, air humidity keeps 50%-60%, and cultivation cycle is 45d.
Cultivating the differentiation rate calculating Herba Anoectochili roxburghii protocorms after terminating, result is in Table 1.
The impact that Herba Anoectochili roxburghii protocorms is broken up by the Rhizoma Solani tuber osi extracting solution of table 1 different volumes mark
Embodiment 2
The stem section 2cm of depletion Anoectochilus roxburghii belt segment, 6min is soaked with washing powder solution, flowing water rushes 2h, with 75% alcohol disinfecting 20s on aseptic super-clean bench, aseptic washing 3 times, sterilize 8min in 0.1% mercuric chloride, aseptic washing 6 times, with dissecting knife by stem section two tip cut-off after sterilizing, is inoculated in MS culture medium, this culture medium contains the NAA of 6-BA and the 0.2mg/L of 2.0mg/L, cultivation temperature is 25+1 DEG C, illumination 1500lx, light application time 12h/d, air humidity grows protocorms at the two ends of stem section after keeping 50%-60%, 30d.
After being grown 30 days in above-mentioned culture medium by the Herba Anoectochili roxburghii protocorms subculture obtained, it is cut to 2cm2Size, is inoculated in division culture medium, and 45d observes its differentiating phenomenon.Consisting of of A culture medium:
KNO380mg/L, NH4NO3105mg/L, KH2PO419mg/L, MgSO4·7H2O240mg/L, CaCl2·2H2O145mg/L, KI0.7mg/L, H3BO30.7mg/L, MnSO4·4H2O4mg/L, ZnSO4·7H2O1.5mg/L, Na2MoO4·2H2O0.12mg/L, CuSO4·5H2O0.13mg/L, CoCl2·6H2O0.01mg/L, Na2-EDTA25mg/L, FeSO4·7H2O24mg/L, inositol 20mg/L, glycine 0.5mg/L, VB10.1mg/L, VB60.4mg/L, nicotinic acid 0.3mg/L, BA1.0mg/L, NAA0.4mg/L, sucrose 20g/L, agar 4.0g/L, activated carbon 1g/L, the extracting solution of added natural additive is Fructus Musae extracting solution (the following table of Fructus Musae extracting liq fraction).Cultivation temperature 25 ± 1 DEG C, illumination 2000lx, light application time 12h/d, air humidity keeps 50%-60%, and cultivation cycle is 45d.
Cultivating the differentiation rate calculating Herba Anoectochili roxburghii protocorms after terminating, result is in Table 2.
The impact that Herba Anoectochili roxburghii protocorms is broken up by the Fructus Musae extracting solution of table 2 different volumes mark
Embodiment 3
The stem section 2cm of depletion Anoectochilus roxburghii belt segment, 6min is soaked with washing powder solution, flowing water rushes 2h, with 75% alcohol disinfecting 20s on aseptic super-clean bench, aseptic washing 3 times, sterilize 8min in 0.1% mercuric chloride, aseptic washing 6 times, with dissecting knife by stem section two tip cut-off after sterilizing, is inoculated in MS culture medium, this culture medium contains the NAA of 6-BA and the 0.2mg/L of 2.0mg/L, cultivation temperature is 25 ± 1 DEG C, illumination 1500lx, light application time 12h/d, air humidity grows protocorms at the two ends of stem section after keeping 50%-60%, 30d.
After being grown 30 days in above-mentioned culture medium by the Herba Anoectochili roxburghii protocorms subculture obtained, it is cut to 2cm2Size, is inoculated in division culture medium, and 45d observes its differentiating phenomenon.Consisting of of A culture medium:
KNO380mg/L, NH4NO3105mg/L, KH2PO419mg/L, MgSO4·7H2O240mg/L, CaCl2·2H2O145mg/L, KI0.7mg/L, H3BO30.7mg/L, MnSO4.4H2O4mg/L, ZnSO4.7H2O1.5mg/L, Na2MoO4·2H2O0.12mg/L, CuSO4·5H2O0.13mg/L, CoCl2·6H2O0.01mg/L, Na2-EDTA25mg/L, FeSO4·7H2O24mg/L, inositol 20mg/L, glycine 0.5mg/L, VB10.1mg/L, VB60.4mg/L, nicotinic acid 0.3mg/L, BA2.0mg/L, NAA0.5mg/L, sucrose 20g/L, agar 4.0g/L, activated carbon 1g/L, the extracting solution of added additives is Rhizoma Solani tuber osi extracting solution and Fructus Musae extracting solution, and total volume fraction is 15%, added ratio such as following table.Cultivation temperature 25 ± 1 DEG C, illumination 2000lx, light application time 12h/d, air humidity keeps 50%-60%, and cultivation cycle is 45d.
Cultivating the differentiation rate calculating Herba Anoectochili roxburghii protocorms after terminating, result is in Table 3.
The impact that Herba Anoectochili roxburghii protocorms is broken up by the mixing additives of table 3 different proportion
Embodiment 4
The stem section 2cm of depletion Anoectochilus roxburghii belt segment, 6min is soaked with washing powder solution, flowing water rushes 2h, with 75% alcohol disinfecting 20s on aseptic super-clean bench, aseptic washing 3 times, sterilize 8min in 0.1% mercuric chloride, aseptic washing 6 times, with dissecting knife by stem section two tip cut-off after sterilizing, is inoculated in MS culture medium, this culture medium contains the NAA of 6-BA and the 0.2mg/L of 2.0mg/L, cultivation temperature is 25 ± 1 DEG C, illumination 15001x, light application time 12h/d, air humidity grows protocorms at the two ends of stem section after keeping 50%-60%, 30d.
After being grown 30 days in above-mentioned culture medium by the Herba Anoectochili roxburghii protocorms subculture obtained, it is cut to 2cm2Size, is inoculated in division culture medium, and 45d observes its differentiating phenomenon.Consisting of of A culture medium:
KNO380mg/L, NH4NO3105mg/L, KH2PO419mg/L, MgSO4·7H2O240mg/L, CaCl2·2H2O145mg/L, KI0.7mg/L, H3BO30.7mg/L, MnSO4·4H2O4mg/L, ZnSO4·7H2O1.5mg/L, Na2MoO4·2H2O0.12mg/L, CuSO4·5H2O0.13mg/L, CoCl2·6H2O0.01mg/L, Na2-EDTA25mg/L, FeSO4·7H2O24mg/L, inositol 20mg/L, glycine 0.5mg/L, VB10.1mg/L, VB60.4mg/L, nicotinic acid 0.3mg/L, BA1.5mg/L, NAA0.5mg/L, sucrose 20g/L, agar 4.0g/L, this basal medium has been separately added into coconut juice, Fructus Mali pumilae extracting solution, Rhizoma Solani tuber osi extracting solution, the mixed extract (ratio is 30: 19) of Fructus Musae extracting solution and Rhizoma Solani tuber osi Fructus Musae composition, volume fraction is following table such as, investigate the impact that Herba Anoectochili roxburghii protocorms is broken up by different types of additives extracting solution, it may be preferable to go out the extracting solution of the additives that Herba Anoectochili roxburghii protocorms can be promoted to break up.Cultivation temperature 25 ± 1 DEG C, illumination 2000lx, light application time 12h/d, air humidity keeps 50%-60%, and cultivation cycle is 45d.
Cultivating the differentiation rate calculating Herba Anoectochili roxburghii protocorms after terminating, result is in Table 4.When adding the extracting solution of the Rhizoma Solani tuber osi extracting solution of appropriate volume mark, Fructus Musae extracting solution or the two mixing as seen from Table 4 in basal medium, it is remarkably improved the differentiation rate of Herba Anoectochili roxburghii protocorms.
Cultivating the differentiation rate calculating Herba Anoectochili roxburghii protocorms after terminating, result is in Table 4.
The impact that Herba Anoectochili roxburghii protocorms is broken up by the extracting solution of the different types of additives of table 4
Claims (3)
1. the additives culture medium promoting Herba Anoectochili roxburghii protocorms to break up, it is characterised in that the composition of this culture medium includes: basal medium and additives.
2. a kind of additives culture medium promoting Herba Anoectochili roxburghii protocorms to break up as claimed in claim 1, it is characterised in that the component of described basal medium and content is:
KNO380-2900mg/L, NH4NO3105-1520mg/L, KH2PO419-410mg/L, MgSO4·7H2O240-750mg/L, CaCl2·2H20145-460mg/L, KCl0-67mg/L, KI0.7-1.7mg/L, H3BO30.7-6.3mg/L, MnSO4·4H2O4-25mg/L, ZnSO4·7H2O1.5-8.6mg/L, Na2MoO4·2H2O0-0.28mg/L, CuSO4·5H2O0-0.35mg/L, CoCl26H2O0-0.03mg/L, Na2-EDTA0-38mg/L, FeSO4·7H2O24-29mg/L, inositol 0-100mg/L, glycine 0-2.5mg/L, VB10.1-11.0mg/L, VB60.4-1.2mg/L, nicotinic acid 0.3-1.5mg/L, 6-benzyladenine 0.2-3.0mg/L, naphthalene acetic acid 0.2-2.0mg/L, sucrose 20-30g/L, agar 4.0-7.0g/L, activated carbon 0.5-4.0g/L.
3. a kind of additives culture medium promoting Herba Anoectochili roxburghii protocorms to break up as claimed in claim 1, it is characterized in that, described additives is the mixed extract of Rhizoma Solani tuber osi extracting solution, Fructus Musae extracting solution or the two composition, total volume fraction is 0.1-20%, and the Volume fraction of mixing additive extracting solution is Rhizoma Solani tuber osi extracting solution: Fructus Musae extracting solution=1-35: 1-25.
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Cited By (5)
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| CN106305431A (en) * | 2016-11-09 | 2017-01-11 | 唐春艳 | Anoectochilus roxburghii tissue culture seedling culture medium |
| CN106386503A (en) * | 2016-11-09 | 2017-02-15 | 唐春艳 | Tissue culture seedling culture medium capable of increasing content of anoectochilus roxburghii polysaccharides |
| CN106538389A (en) * | 2016-11-09 | 2017-03-29 | 唐春艳 | A kind of plantlet in vitro culture medium for improving roxburgh anoectochilus terminal bud flavone compound |
| CN106550873A (en) * | 2016-11-09 | 2017-04-05 | 唐春艳 | A kind of new Herba Anoectochili roxburghii tissue cultured seedling culture medium |
| CN106718871A (en) * | 2016-11-09 | 2017-05-31 | 唐春艳 | A kind of tissue-cultured seedling culture medium for improving roxburgh anoectochilus terminal bud alkaloid |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106538389A (en) * | 2016-11-09 | 2017-03-29 | 唐春艳 | A kind of plantlet in vitro culture medium for improving roxburgh anoectochilus terminal bud flavone compound |
| CN106550873A (en) * | 2016-11-09 | 2017-04-05 | 唐春艳 | A kind of new Herba Anoectochili roxburghii tissue cultured seedling culture medium |
| CN106718871A (en) * | 2016-11-09 | 2017-05-31 | 唐春艳 | A kind of tissue-cultured seedling culture medium for improving roxburgh anoectochilus terminal bud alkaloid |
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