CN105660401A - Culture medium for tissue-culture anoectochilus roxburghii (Wall.)Lindl as well as preparation method and application of culture medium - Google Patents
Culture medium for tissue-culture anoectochilus roxburghii (Wall.)Lindl as well as preparation method and application of culture medium Download PDFInfo
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- CN105660401A CN105660401A CN201610055520.1A CN201610055520A CN105660401A CN 105660401 A CN105660401 A CN 105660401A CN 201610055520 A CN201610055520 A CN 201610055520A CN 105660401 A CN105660401 A CN 105660401A
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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
Abstract
The invention relates to a culture medium for tissue-culture anoectochilus roxburghii (Wall.)Lindl as well as a preparation method and an application of the culture medium. Every 1,000 ml of the culture medium for the tissue-culture anoectochilus roxburghii (Wall.)Lindl comprises 0*MS-1*MS of a basic culture medium mother solution, 10-1,000 mu L of an edible fungus canning processing precooking fluid, 10-50 g of saccharose, 50-250 g of bananas, 6-8 g of agar, 1.0-2.0 g of activated carbon and the balance of distilled water. The culture medium has the advantages that the problem about discharging of industrial wastewater produced in the edible fungus canning processing process is solved, edible fungus processing enterprises can achieve zero discharge of wastewater, and the sewage treatment cost is reduced; the culture medium can promote rooting and germination of tissue-culture anoectochilus roxburghii (Wall.)Lindl and can shorten the growth cycle of tissue-culture anoectochilus roxburghii (Wall.)Lindl; artificially synthesized hormone applied to tissue-culture anoectochilus roxburghii (Wall.)Lindl is reduced due to use of the edible fungus canning processing precooking fluid, and accordingly, tissue-culture anoectochilus roxburghii (Wall.)Lindl becomes green and organic food.
Description
Technical field
The present invention relates to a kind of group training Herba Anoectochili roxburghii culture medium and its preparation method and application.
Background technology
Herba Anoectochili roxburghii (Anoectochilusroxburghii (Wall.) Lindl) is the raw type herbaceos perennial in the orchid family Anoectochilus Blume ground, it is distributed mainly on subtropical zone, i.e. the provinces and regions such as Fujian, Guangdong, Guangxi, Zhejiang, Jiangxi, Hainan, Yunnan, Sichuan, Guizhou and southern Tibet in China. Herba Anoectochili roxburghii is the valuable ingredient of Chinese medicine that China is traditional, can all herbal medicine, sweet and slightly bitter taste, property are flat to be slightly cold, and has the effects such as heat-clearing and toxic substances removing, nourishing the lung to arrest cough and expelling wind and removing dampness, therefore suffers from the extensive attention of pharmaceuticals industry. owing to wild resource is increasingly exhausted, Herba Anoectochili roxburghii has been listed in Fujian Province's Endangered Medicinal Herb and has been protected by. but its seed germination rate under field conditions (factors) is extremely low, so utilizing artificial propagation that it is carried out rescue preservation. the artificial propagation of current Herba Anoectochili roxburghii mainly carries out field-transplanting by after tissue cultivating and seedling, the Herba Anoectochili roxburghii of tissue culture is mostly utilize auxin to be adjusted promoting that it grows, but utilizing growth hormone is unsustainable sexual development to regulate the traditional method of Herba Anoectochili roxburghii tissue culture, there is cost height, affect the problems such as food safety, in addition, under the social development of nowadays high speed, green organic food is increasingly becoming main product, original Herba Anoectochili roxburghii tissue culture medium (TCM) and cultural method, do not catch up with the step that pollution-free food is pursued by people. therefore, be necessary to work out a kind of environmental protection, nuisanceless and the culture medium of Herba Anoectochili roxburghii growth can be accelerated to cultivate Herba Anoectochili roxburghii, so that reaching to promote the purpose of Herba Anoectochili roxburghii Fast-propagation to meet large market demand, the Herba Anoectochili roxburghii cultivated can be made again to reach green organically requirement simultaneously.
Summary of the invention
It is an object of the invention to provide a kind of environmental protection, nuisanceless and group training Herba Anoectochili roxburghii culture medium and its preparation method and application of Herba Anoectochili roxburghii growth can be accelerated.
The purpose of the present invention is achieved through the following technical solutions: a kind of group training Herba Anoectochili roxburghii culture medium, and including Fructus Musae, sucrose, agar and activated carbon, it also includes 0*MS～1*MS culture medium mother solution and liquid of precooking is processed in edible fungi canning;Group training Herba Anoectochili roxburghii culture medium described in every 1000ml includes 0*MS～1*MS minimal medium mother solution, liquid, 10～50g sucrose, 50～250g Fructus Musae, 6g～8g agar, 1.0g～2.0g activated carbon are precooked in 10～1000 μ L edible fungi canning processing, surplus is distilled water, and wherein 0*MS represents and is added without minimal medium mother solution.
The preparation of described group training Herba Anoectochili roxburghii culture medium, organic culture medium preparation steps of every 1000 milliliters is as follows:
(1) prepare edible fungi canning processing to precook liquid, and the edible fungi canning processing measuring 10～1000 μ L liquid of precooking is stand-by;
(2) according to the volume of the various mother solutions needed for preparation 0*MS～1*MS minimal medium, MS a great number of elements mother solution, MS trace element mother solution, MS mother liquid of iron salt and MS Organic substance mother solution are measured stand-by;
(3) the edible fungi canning measured in step (1) processing is precooked after liquid mixes with the MS culture medium mother solution measured in step (2) and is dissolved in distilled water;
(4) weigh 50～250g Fructus Musae and 1.0g～2.0g activated carbon, and add in step (3) in the solution of gained after Fructus Musae is ground into mud, stirring and evenly mixing, add the activated carbon weighed up afterwards, stir;
(5) in beaker, add the distilled water of 200～300mL, distilled water heating is added, after 80～90 DEG C, the 6g～8g agar weighed up, after the extremely boiling of heating while stirring, agar dissolves, after treating that agar dissolves, continuously add the 10～50g sucrose weighed up, heat while stirring to sucrose dissolving, then the mixed solution of the agar that obtains with sucrose is added in the mixed solution of step (4) gained, add distilled water after stirring and be settled to 1000 milliliters and add acid-base modifier pH value is adjusted to 5.0-6.0;
(6) culture medium obtained for step (5) is sub-packed in tissue culture bottle, carries out autoclave sterilization, afterwards cooled and solidified.
The preparation method that liquid of precooking is processed in described edible fungi canning is as follows:
A. produced industrial wastewater in the edible fungi canning course of processing is collected;
B. being centrifuged collecting produced industrial wastewater in the edible fungi canning course of processing come, the rotating speed of centrifuge controls between 11500-12500rpm, and centrifuging temperature is 15-25 DEG C, and centrifugation time is 10-20min, is centrifuged and takes supernatant afterwards;
C. the supernatant of gained being utilized falling film evaporator, falling film evaporation concentrates, and obtains concentrated solution;
D. adding denaturant in concentrated solution, concentrated solution carries out degeneration, wherein said denaturant is the ethanol of 80～100%, and the volume ratio of affiliated ethanol and concentrated solution is 2:1～6:1;
E. the concentrated solution after degeneration is filtered, collects filtrate;
F. filtrate is rotated evaporating ethanol;
G., the solution of gained after rotary evaporation utilizes the filter membrane of 5-10K carry out tangential flow filtration, collects filter liquor;
H. the filter liquor of gained in step g is rotated evaporation and concentration, be concentrated into solution index of refraction and reach 35～55%.
The HCl solution of the NaOH solution that acid-base modifier is 0.1-0.5mol/L described in step (5) or 0.1-0.5mol/L.
In step (6), the temperature of autoclave sterilization controls at 121-125 DEG C, and Stress control is at 111-115 kPa, and sterilization time is 15-20 minute.
The application of described group training Herba Anoectochili roxburghii culture medium, trains Herba Anoectochili roxburghii culture medium by group and is used for cultivating Herba Anoectochili roxburghii. Compared to prior art, it is an advantage of the current invention that: 1) in the edible fungi canning course of processing produced industrial wastewater often rich in some nutrient substance such as aminoacid, mineral, saccharide etc., if by these waste water after treatment in discharge, not only need the cost of sewage disposal of costliness, and cause certain wasting of resources. After industrial wastewater produced in the edible fungi canning course of processing is processed by the present invention, obtain edible fungi canning and process liquid of precooking, its makes composite with MS culture medium mother solution and other additives is trained Herba Anoectochili roxburghii culture medium, industrial wastewater produced in the edible fungi canning course of processing is turned waste into wealth, solves the emission problem of produced industrial wastewater in the edible fungi canning course of processing, make Edible mushroom processing enterprise reach wastewater zero discharge, reduce its cost of sewage disposal;2) after industrial wastewater produced in the edible fungi canning course of processing is processed by the present invention, the edible fungi canning processing of gained is precooked liquid, replace traditional plant growth regulator, the nutrition except MS culture medium can be provided for Herba Anoectochili roxburghii, can root by promotion group training Herba Anoectochili roxburghii simultaneously, group training Herba Anoectochili roxburghii growth cycle can also be shortened, edible fungi canning is processed the use of liquid of precooking and is decreased the application in group training Herba Anoectochili roxburghii of the synthetic hormone, makes group training Herba Anoectochili roxburghii become a kind of green organic food; 3) raw material of preparation group of the present invention training Herba Anoectochili roxburghii culture medium is easy to get and cheap; 4) preparation method of preparation group of the present invention training Herba Anoectochili roxburghii culture medium is simple, it is adaptable to industrialized production; 5) the group training Herba Anoectochili roxburghii culture medium that the present invention prepares has good fungistatic effect so that without adding antibacterial in the culture medium cultivating Herba Anoectochili roxburghii, further ensure the quality of Herba Anoectochili roxburghii.
Detailed description of the invention
Below in conjunction with embodiment, present invention is described in detail:
The preparation of embodiment one: MS mother solution
1.MS a great number of elements mother solution (20X) weighs 20 rising amount and is dissolved in 1L distilled water.
2.MS trace element mother solution (200X)
Weigh 200 rising amount to be dissolved in 1L distilled water.
3.MS mother liquid of iron salt (200X)
Weigh 200 rising amount to be dissolved in 1L distilled water.
4.MS Organic substance mother solution (200X)
Weigh 200 rising amount to be dissolved in 1L distilled water.
Embodiment two: the preparation of group training Herba Anoectochili roxburghii culture medium
The edible fungi canning processing of 1L group training Herba Anoectochili roxburghii culture medium (1) 1/2*MS+250 μ L precook liquid+30g sucrose+100g Fructus Musae+7g agar+1.5g activated carbon, (2) MS+1000 μ L edible fungi canning processing precook liquid+50g sucrose+250g Fructus Musae+8g agar+2.0g activated carbon, (3) 1/4*MS+10 μ L the precook concrete preparation method of liquid+10g sucrose+50g Fructus Musae+6g agar+1.0g activated carbon of edible fungi canning processing as follows:
The preparation of organic culture medium (liquid+30g sucrose+100g Fructus Musae+7g agar+1.5g activated carbon of precooking is processed in the edible fungi canning of 1/2*MS+250 μ L) of 1.1L group training Herba Anoectochili roxburghii, organic culture medium preparation steps of every 1000 milliliters is as follows:
(1) prepare edible fungi canning processing to precook liquid, and the edible fungi canning processing measuring 250 μ L liquid of precooking is stand-by;
(2) according to the volume of the various mother solutions needed for preparation 1/2*MS minimal medium, measure successively:
MS a great number of elements mother solution 25mL
MS trace element mother solution 2.5mL
MS mother liquid of iron salt 2.5mL
MS Organic substance mother solution 2.5mL is stand-by;
(3) the edible fungi canning measured in step (1) processing is precooked after liquid mixes with the MS culture medium mother solution measured in step (2) and is dissolved in distilled water;
(4) weigh 100g Fructus Musae and 1.5g activated carbon, and add in step (3) in the solution of gained after Fructus Musae is ground into mud, stirring and evenly mixing, add the activated carbon weighed up afterwards, stir;
(5) in beaker, add the distilled water of 200mL, distilled water heating is added, after 80 DEG C, the 7g agar weighed up, after the extremely boiling of heating while stirring, agar dissolves, after treating that agar dissolves, continuously add the 30g sucrose weighed up, heat while stirring and dissolve to sucrose, then the mixed solution of the agar that obtains with sucrose is added in the mixed solution of step (4) gained, add distilled water after stirring and be settled to 1000 milliliters and add the NaOH of 0.1mol/L pH value is adjusted to 5.0;
(6) being sub-packed in tissue culture bottle by culture medium obtained for step (5), carry out autoclave sterilization, afterwards cooled and solidified, wherein autoclave sterilization temperature controls at 121 DEG C, and Stress control is at 111 kPas, and sterilization time is 15 minutes.
Wherein, the precook preparation method of liquid of edible fungi canning processing is as follows:
A. produced industrial wastewater in the edible fungi canning course of processing is collected;
B. being centrifuged collecting produced industrial wastewater in the edible fungi canning course of processing come, the rotating speed of centrifuge controls at 11500rpm, and centrifuging temperature is 15 DEG C, and centrifugation time is 10min, is centrifuged and takes supernatant afterwards;
C. the supernatant of gained being utilized falling film evaporator, falling film evaporation concentrates, and obtains concentrated solution;
D. adding denaturant in concentrated solution, concentrated solution carries out degeneration, wherein said denaturant is the ethanol of 80%, and the volume ratio of affiliated ethanol and concentrated solution is 2:1;
E. the concentrated solution after degeneration is filtered, collects filtrate;
F. filtrate is rotated evaporating ethanol;
G., the solution of gained after rotary evaporation utilizes the filter membrane of 5K carry out tangential flow filtration, collects filter liquor;
H. the filter liquor of gained in step g is rotated evaporation and concentration, be concentrated into solution index of refraction and reach 35%.
The preparation of organic culture medium (liquid+50g sucrose+250g Fructus Musae+8g agar+2.0g activated carbon of precooking is processed in the edible fungi canning of MS+1000 μ L) of 2.1L group training Herba Anoectochili roxburghii, organic culture medium preparation steps of every 1000 milliliters is as follows:
(1) prepare edible fungi canning processing to precook liquid, and the edible fungi canning processing measuring 1000 μ L liquid of precooking is stand-by;
(2) according to the volume of the various mother solutions needed for preparation 1*MS minimal medium, measure successively:
MS a great number of elements mother solution 50mL
MS trace element mother solution 5mL
MS mother liquid of iron salt 5mL
MS Organic substance mother solution 5mL is stand-by;
(3) the edible fungi canning measured in step (1) processing is precooked after liquid mixes with the MS culture medium mother solution measured in step (2) and is dissolved in distilled water;
(4) weigh 250g Fructus Musae and 2.0g activated carbon, and add in step (3) in the solution of gained after Fructus Musae is ground into mud, stirring and evenly mixing, add the activated carbon weighed up afterwards, stir;
(5) in beaker, add the distilled water of 300mL, distilled water heating is added, after 90 DEG C, the 8g agar weighed up, after the extremely boiling of heating while stirring, agar dissolves, after treating that agar dissolves, continuously add the 50g sucrose weighed up, heat while stirring and dissolve to sucrose, then the mixed solution of the agar that obtains with sucrose is added in the mixed solution of step (4) gained, add distilled water after stirring and be settled to 1000 milliliters and add the NaOH of 0.5mol/L pH value is adjusted to 6.0;
(6) being sub-packed in tissue culture bottle by culture medium obtained for step (5), carry out autoclave sterilization, afterwards cooled and solidified, wherein autoclave sterilization temperature controls at 125 DEG C, and Stress control is at 115 kPas, and sterilization time is 20 minutes.
Wherein, the precook preparation method of liquid of edible fungi canning processing is as follows:
A. produced industrial wastewater in the edible fungi canning course of processing is collected;
B. being centrifuged collecting produced industrial wastewater in the edible fungi canning course of processing come, the rotating speed of centrifuge controls at 12000rpm, and centrifuging temperature is 20 DEG C, and centrifugation time is 15min, is centrifuged and takes supernatant afterwards;
C. the supernatant of gained being utilized falling film evaporator, falling film evaporation concentrates, and obtains concentrated solution;
D. adding denaturant in concentrated solution, concentrated solution carries out degeneration, wherein said denaturant is the ethanol of 80%, and the volume ratio of affiliated ethanol and concentrated solution is 3:1;
E. the concentrated solution after degeneration is filtered, collects filtrate;
F. filtrate is rotated evaporating ethanol;
G., the solution of gained after rotary evaporation utilizes the filter membrane of 8K carry out tangential flow filtration, collects filter liquor;
H. the filter liquor of gained in step g is rotated evaporation and concentration, be concentrated into solution index of refraction and reach 50%.
The preparation of organic culture medium (liquid+10g sucrose+50g Fructus Musae+6g agar+1.0g activated carbon of precooking is processed in the edible fungi canning of 1/4*MS+10 μ L) of 3.1L group training Herba Anoectochili roxburghii, organic culture medium preparation steps of every 10 milliliters is as follows:
(1) prepare edible fungi canning processing to precook liquid, and the edible fungi canning processing measuring 10 μ L liquid of precooking is stand-by;
(2) according to the volume of the various mother solutions needed for preparation 1/4*MS minimal medium, measure successively:
MS a great number of elements mother solution 12.5mL
MS trace element mother solution 1.25mL
MS mother liquid of iron salt 1.25mL
MS Organic substance mother solution 1.25mL is stand-by;
(3) the edible fungi canning measured in step (1) processing is precooked after liquid mixes with the MS culture medium mother solution measured in step (2) and is dissolved in distilled water;
(4) weigh 50g Fructus Musae and 1.0g activated carbon, and add in step (3) in the solution of gained after Fructus Musae is ground into mud, stirring and evenly mixing, add the activated carbon weighed up afterwards, stir;
(5) in beaker, add the distilled water of 250mL, distilled water heating is added, after 85 DEG C, the 6g agar weighed up, after the extremely boiling of heating while stirring, agar dissolves, after treating that agar dissolves, continuously add the 10g sucrose weighed up, heat while stirring and dissolve to sucrose, then the mixed solution of the agar that obtains with sucrose is added in the mixed solution of step (4) gained, add distilled water after stirring and be settled to 1000 milliliters and add the HCl of 0.1mol/L pH value is adjusted to 5.5;
(6) being sub-packed in tissue culture bottle by culture medium obtained for step (5), carry out autoclave sterilization, afterwards cooled and solidified, wherein autoclave sterilization temperature controls at 122 DEG C, and Stress control is at 113 kPas, and sterilization time is 18 minutes.
Wherein, the precook preparation method of liquid of edible fungi canning processing is as follows:
A. produced industrial wastewater in the edible fungi canning course of processing is collected;
B. being centrifuged collecting produced industrial wastewater in the edible fungi canning course of processing come, the rotating speed of centrifuge controls at 12500rpm, and centrifuging temperature is 25 DEG C, and centrifugation time is 20min, is centrifuged and takes supernatant afterwards;
C. the supernatant of gained being utilized falling film evaporator, falling film evaporation concentrates, and obtains concentrated solution;
D. adding denaturant in concentrated solution, concentrated solution carries out degeneration, wherein said denaturant is the ethanol of 80%, and the volume ratio of affiliated ethanol and concentrated solution is 6:1;
E. the concentrated solution after degeneration is filtered, collects filtrate;
F. filtrate is rotated evaporating ethanol;
G., the solution of gained after rotary evaporation utilizes the filter membrane of 10K carry out tangential flow filtration, collects filter liquor;
H. the filter liquor of gained in step g is rotated evaporation and concentration, be concentrated into solution index of refraction and reach 55%.
According to above-mentioned preparation method or principle, the following 60 groups of (A1～A20 of preparation; B1～B20; C1～C20) culture medium (volume of every kind of culture medium is 1L):
One, being divided into four big groups to process with 0*MS, 1/4*MS, 1/2*MS, MS, edible fungi canning processing is precooked taken amount respectively 0 μ L, 250 μ L, 500 μ L, 1000 μ L, the 2000 μ L of liquid, additionally sucrose 30g, Fructus Musae 100g, agar 7g, 1.5g activated carbon. Wherein 0*MS represents and does not add minimal medium mother solution.
(1) 0*MS process
A1:0*MS+0 μ L mushroom canning is precooked liquid+30g sucrose+100g Fructus Musae+7g agar+1.5g activated carbon
A2:0*MS+250 μ L mushroom canning is precooked liquid+30g sucrose+100g Fructus Musae+7g agar+1.5g activated carbon
A3:0*MS+500 μ L mushroom canning is precooked liquid+30g sucrose+100g Fructus Musae+7g agar+1.5g activated carbon
A4:0*MS+1000 μ L mushroom canning is precooked liquid+30g sucrose+100g Fructus Musae+7g agar+1.5g activated carbon
A5:0*MS+2000 μ L mushroom canning is precooked liquid+30g sucrose+100g Fructus Musae+7g agar+1.5g activated carbon
(2) 1/4*MS process
A6:1/4*MS+0 μ L mushroom canning is precooked liquid+30g sucrose+100g Fructus Musae+7g agar+1.5g activated carbon
A7:1/4*MS+250 μ L mushroom canning is precooked liquid+30g sucrose+100g Fructus Musae+7g agar+1.5g activated carbon
A8:1/4*MS+500 μ L mushroom canning is precooked liquid+30g sucrose+100g Fructus Musae+7g agar+1.5g activated carbon
A9:1/4*MS+1000 μ L mushroom canning is precooked liquid+30g sucrose+100g Fructus Musae+7g agar+1.5g activated carbon
A10:1/4*MS+2000 μ L mushroom canning is precooked liquid+30g sucrose+100g Fructus Musae+7g agar+1.5g activated carbon
(3) 1/2*MS process
A11:1/2*MS+0 μ L mushroom canning is precooked liquid+30g sucrose+100g Fructus Musae+7g agar+1.5g activated carbon
A12:1/2*MS+250 μ L mushroom canning is precooked liquid+30g sucrose+100g Fructus Musae+7g agar+1.5g activated carbon
A13:1/2*MS+500 μ L mushroom canning is precooked liquid+30g sucrose+100g Fructus Musae+7g agar+1.5g activated carbon
A14:1/2*MS+1000 μ L mushroom canning is precooked liquid+30g sucrose+100g Fructus Musae+7g agar+1.5g activated carbon
A15:1/2*MS+2000 μ L mushroom canning is precooked liquid+30g sucrose+100g Fructus Musae+7g agar+1.5g activated carbon
(4) MS process
A16:MS+0 μ L mushroom canning is precooked liquid+30g sucrose+100g Fructus Musae+7g agar+1.5g activated carbon
A17:MS+250 μ L mushroom canning is precooked liquid+30g sucrose+100g Fructus Musae+7g agar+1.5g activated carbon
A18:MS+500 μ L mushroom canning is precooked liquid+30g sucrose+100g Fructus Musae+7g agar+1.5g activated carbon
A19:MS+1000 μ L mushroom canning is precooked liquid+30g sucrose+100g Fructus Musae+7g agar+1.5g activated carbon
A20:MS+2000 μ L mushroom canning is precooked liquid+30g sucrose+100g Fructus Musae+7g agar+1.5g activated carbon
Two, being divided into four big groups to process with 0*MS, 1/4*MS, 1/2*MS, MS, edible fungi canning processing is precooked taken amount respectively 0 μ L, 250 μ L, 500 μ L, 1000 μ L, the 2000 μ L of liquid, additionally sucrose 10g, Fructus Musae 50g, agar 6g, activated carbon 1.0g.
(1) 0*MS process
B1:0*MS+0 μ L mushroom canning is precooked liquid+10g sucrose+50g Fructus Musae+6g agar+1.0g activated carbon
B2:0*MS+250 μ L mushroom canning is precooked liquid+10g sucrose+50g Fructus Musae+6g agar+1.0g activated carbon
B3:0*MS+500 μ L mushroom canning is precooked liquid+10g sucrose+50g Fructus Musae+6g agar+1.0g activated carbon
B4:0*MS+1000 μ L mushroom canning is precooked liquid+10g sucrose+50g Fructus Musae+6g agar+1.0g activated carbon
B5:0*MS+2000 μ L mushroom canning is precooked liquid+10g sucrose+50g Fructus Musae+6g agar+1.0g activated carbon
(2) 1/4*MS process
B6:1/4*MS+0 μ L mushroom canning is precooked liquid+10g sucrose+50g Fructus Musae+6g agar+1.0g activated carbon
B7:1/4*MS+250 μ L mushroom canning is precooked liquid+10g sucrose+50g Fructus Musae+6g agar+1.0g activated carbon
B8:1/4*MS+500 μ L mushroom canning is precooked liquid+10g sucrose+50g Fructus Musae+6g agar+1.0g activated carbon
B9:1/4*MS+1000 μ L mushroom canning is precooked liquid+10g sucrose+50g Fructus Musae+6g agar+1.0g activated carbon
B10:1/4*MS+2000 μ L mushroom canning is precooked liquid+10g sucrose+50g Fructus Musae+6g agar+1.0g activated carbon
(3) 1/2*MS process
B11:1/2*MS+0 μ L mushroom canning is precooked liquid+10g sucrose+50g Fructus Musae+6g agar+1.0g activated carbon
B12:1/2*MS+250 μ L mushroom canning is precooked liquid+10g sucrose+50g Fructus Musae+6g agar+1.0g activated carbon
B13:1/2*MS+500 μ L mushroom canning is precooked liquid+10g sucrose+50g Fructus Musae+6g agar+1.0g activated carbon
B14:1/2*MS+1000 μ L mushroom canning is precooked liquid+10g sucrose+50g Fructus Musae+6g agar+1.0g activated carbon
B15:1/2*MS+2000 μ L mushroom canning is precooked liquid+10g sucrose+50g Fructus Musae+6g agar+1.0g activated carbon
(4) MS process
B16:MS+0 μ L mushroom canning is precooked liquid+10g sucrose+50g Fructus Musae+6g agar+1.0g activated carbon
B17:MS+250 μ L mushroom canning is precooked liquid+10g sucrose+50g Fructus Musae+6g agar+1.0g activated carbon
B18:MS+500 μ L mushroom canning is precooked liquid+10g sucrose+50g Fructus Musae+6g agar+1.0g activated carbon
B19:MS+1000 μ L mushroom canning is precooked liquid+10g sucrose+50g Fructus Musae+6g agar+10g activated carbon
B20:MS+2000 μ L mushroom canning is precooked liquid+10g sucrose+50g Fructus Musae+6g agar+1.0g activated carbon
Three, being divided into four big groups to process with 0*MS, 1/4*MS, 1/2*MS, MS, edible fungi canning processing is precooked taken amount respectively 0 μ L, 250 μ L, 500 μ L, 1000 μ L, the 2000 μ L of liquid, additionally sucrose 50g, Fructus Musae 250g, agar 8g, 2.0g activated carbon.
(1) 0*MS process
C1:0*MS+0 μ L mushroom canning is precooked liquid+50g sucrose+250g Fructus Musae+8g agar+2.0g activated carbon
C2:0*MS+250 μ L mushroom canning is precooked liquid+50g sucrose+250g Fructus Musae+8g agar+2.0g activated carbon
C3:0*MS+500 μ L mushroom canning is precooked liquid+50g sucrose+250g Fructus Musae+8g agar+2.0g activated carbon
C4:0*MS+1000 μ L mushroom canning is precooked liquid+50g sucrose+250g Fructus Musae+8g agar+2.0g activated carbon
C5:0*MS+2000 μ L mushroom canning is precooked liquid+50g sucrose+250g Fructus Musae+8g agar+2.0g activated carbon
(2) 1/4*MS process
C6:1/4*MS+0 μ L mushroom canning is precooked liquid+50g sucrose+250g Fructus Musae+8g agar+2.0g activated carbon
C7:1/4*MS+250 μ L mushroom canning is precooked liquid+50g sucrose+250g Fructus Musae+8g agar+2.0g activated carbon
C8:1/4*MS+500 μ L mushroom canning is precooked liquid+50g sucrose+250g Fructus Musae+8g agar+2.0g activated carbon
C9:1/4*MS+1000 μ L mushroom canning is precooked liquid+50g sucrose+250g Fructus Musae+8g agar+2.0g activated carbon
C10:1/4*MS+2000 μ L mushroom canning is precooked liquid+50g sucrose+250g Fructus Musae+8g agar+2.0g activated carbon
(3) 1/2*MS process
C11:1/2*MS+0 μ L mushroom canning is precooked liquid+50g sucrose+250g Fructus Musae+8g agar+2.0g activated carbon
C12:1/2*MS+250 μ L mushroom canning is precooked liquid+50g sucrose+250g Fructus Musae+8g agar+2.0g activated carbon
C13:1/2*MS+500 μ L mushroom canning is precooked liquid+50g sucrose+250g Fructus Musae+8g agar+2.0g activated carbon
C14:1/2*MS+1000 μ L mushroom canning is precooked liquid+50g sucrose+250g Fructus Musae+8g agar+2.0g activated carbon
C15:1/2*MS+2000 μ L mushroom canning is precooked liquid+50g sucrose+250g Fructus Musae+8g agar+2.0g activated carbon
(4) MS process
C16:MS+0 μ L mushroom canning is precooked liquid+50g sucrose+250g Fructus Musae+8g agar+2.0g activated carbon
C17:MS+250 μ L mushroom canning is precooked liquid+50g sucrose+250g Fructus Musae+8g agar+2.0g activated carbon
C18:MS+500 μ L mushroom canning is precooked liquid+50g sucrose+250g Fructus Musae+8g agar+2.0g activated carbon
C19:MS+1000 μ L mushroom canning is precooked liquid+50g sucrose+250g Fructus Musae+8g agar+2.0g activated carbon
C20:MS+2000 μ L mushroom canning is precooked liquid+50g sucrose+250g Fructus Musae+8g agar+2.0g activated carbon
By above-mentioned 60 groups of prepared training Herba Anoectochili roxburghii culture medium (A1～A20; B1～B20; C1～C20) for the cultivation of Herba Anoectochili roxburghii;
One, the different culture media cultivation to bud inducement
Choose the Taiwan kind Herba Anoectochili roxburghii sterilizable material of high-quality, under super-clean bench gnotobasis, remove root and blade with shears, stem is cut into the long 2.5cm stem section with a joint. Being seeded in A1-A20 group training Herba Anoectochili roxburghii culture medium, regulating PH is 5.8. Experiment sets 20 process altogether, and each process connects 6 bottles, 4 sections of every bottle graft kind. The culture bottle connected is placed in thermostatic chamber, keeps room temperature 25 ± 1 DEG C, illumination 10h/d, intensity of illumination 800～1000Lux. Germination degree being added up (differentiation rate=germination stem hop count/inoculation stem section sum) every other month, processes mouldy culture bottle in time, the experimental result recorded is in Table 1.
The impact that stem section is germinateed by table 1 different culture media
Note: germination when table 1 is for inoculation 90d. + representing that bud is tiny, color is yellowish green; ++ representing that bud is tiny, color is blackish green; +++ representing that bud is sturdy, color is blackish green; ++++represent, bud was sturdy, and color is blackish green, had clump bud growth.
The Herba Anoectochili roxburghii stem section germination data that A1-A5 culture medium from table 1 data is cultivated are known, when not adding minimal medium mother solution, along with the increase of liquid concentration of precooking is processed in edible fungi canning, the germination sum of Herba Anoectochili roxburghii stem section constantly increases, differentiation rate and proliferation times are also gradually increased, liquid of precooking is processed in visible edible fungi canning both can for the nutrition needed for the germination offer of Herba Anoectochili roxburghii stem section, and energy promotion group trains Herba Anoectochili roxburghii germination simultaneously.
The Herba Anoectochili roxburghii stem section germination data that A6-A10 culture medium is cultivated are known, after adding 1/4*MS minimal medium mother solution, required nutrition of germinateing is provided for Herba Anoectochili roxburghii stem section, but, needed for this nutrition is still not able to fully meet the germination of Herba Anoectochili roxburghii stem section, therefore along with the increase of liquid concentration of precooking is processed in edible fungi canning, the germination sum of Herba Anoectochili roxburghii stem section constantly increases, differentiation rate and proliferation times are also gradually increased, when the concentration that liquid of precooking is processed in edible fungi canning increases to 1000 μ L/L, needed for having been able to meet the nutrition that Herba Anoectochili roxburghii stem section is germinateed, and also can well promote the germination of Herba Anoectochili roxburghii stem section, if continuing to increase edible fungi canning afterwards to process the concentration of liquid of precooking, Herba Anoectochili roxburghii stem section germinative number reduces on the contrary, illustrate that the edible fungi canning of high concentration is processed liquid of precooking and the germination of Herba Anoectochili roxburghii stem section will be had certain inhibitory action.
The Herba Anoectochili roxburghii stem section germination data that A11-A15 culture medium is cultivated are known, after adding 1/2*MS minimal medium mother solution, have been able to the demand well meeting the germination of Herba Anoectochili roxburghii stem section to nutrition, therefore the precook concentration of liquid of edible fungi canning processing only need to reach 250 μ L/L, just can be good at promoting the germination of Herba Anoectochili roxburghii stem section, and the germinative number of Herba Anoectochili roxburghii stem section and proliferation times reach maximum, if continuing to improve edible fungi canning afterwards to process the concentration of liquid of precooking, Herba Anoectochili roxburghii stem section germinative number reduces on the contrary.
The Herba Anoectochili roxburghii stem section germination data that A16-A20 culture medium is cultivated are known, after adding MS minimal medium mother solution, have been able to the demand well meeting the germination of Herba Anoectochili roxburghii stem section to nutrition, therefore the precook concentration of liquid of edible fungi canning processing only need to reach 250 μ L/L, just can be good at promote Herba Anoectochili roxburghii stem section germination, but the germinative number that germinative number now is not as good as A12. It can thus be appreciated that the best results of A12 culture medium, it is best able to promote the germination of Herba Anoectochili roxburghii stem section.
Herba Anoectochili roxburghii stem section is also seeded in B1-B20 and C1-C20 group training Herba Anoectochili roxburghii culture medium by inventor, the experimental data obtained is substantially the same with table 1, but utilize the Herba Anoectochili roxburghii stem section that the germinative number of the Herba Anoectochili roxburghii stem section of B1-B20 and C1-C20 group training Herba Anoectochili roxburghii culture medium culturing is still cultivated not as good as A12 culture medium, it can thus be appreciated that the impact that Herba Anoectochili roxburghii stem section is germinateed by the concentration of sucrose, Fructus Musae, agar and activated carbon is little.
Two, the different culture media impact on root growth
Choosing and grow fine and growing way is tried one's best close anoectochilus blume seedling, under super-clean bench gnotobasis, cut the base portion lower end (about 2cm length) of Seedling, then be transferred in A11-A15 culture medium, regulating PH is 5.8. Experiment sets 5 process altogether, and each process inoculates 4 bottles, the strain of every bottle graft kind 6. The culture bottle connected is placed in thermostatic chamber, keeps room temperature 25 ± 1 DEG C, illumination 10h/d, intensity of illumination 1800Lux. Root of hair number and root growth situation is added up after cultivating 60d. Result is in Table 2
The impact on taking root of table 2 different culture media
Data from table 2, can promote that Herba Anoectochili roxburghii is taken root when the moderate concentration of liquid of precooking is processed in edible fungi canning, and the tissue cultured seedling root of hair bar number that is seeded in A12 culture medium is maximum, add up to 98, rooting rate has reached 4.08, experiment also find when edible fungi canning processing precook liquid concentration low time, the root system of anoectochilus blume seedling is elongated, root hair is more, and root color is milky. When edible fungi canning processing precook liquid concentration reach 1500 more than μ L/L time, root system becomes sturdy, and root length elongation is slow, and root hair reduces, and is that yellow green is substantially aging with color, and in addition, the Herba Anoectochili roxburghii tip of a root is the downright bad growth affecting root easily.
Three, the different culture media impact on Herba Anoectochili roxburghii growth of seedling
Choosing and grow fine and Taiwan kind Herba Anoectochili roxburghii seedling that growing way is tried one's best close, under super-clean bench gnotobasis, be inoculated into by seedling in A11-A15, B11-B15, C11-C15 culture medium, regulating PH is 5.8. Experiment sets 15 process altogether, and each process inoculates 4 bottles, every bottle graft kind 6 strain (the seedling growing way of same bottle graft kind is as far as possible close). The culture bottle connected is placed in thermostatic chamber, keeps room temperature 25 ± 1 DEG C, illumination 10～12h/d, intensity of illumination 1500～2000Lux. Add up the average plant height after growing 3 months, the newly-increased number of blade of average individual plant and average single-strain fresh weight.
Growing fine and Fujian kind Herba Anoectochili roxburghii seedling that growing way is tried one's best close it addition, choose, under super-clean bench gnotobasis, be inoculated into by seedling in A11-A15, B11-B15, C11-C15 culture medium, regulating PH is 5.8. Experiment sets 15 process altogether, and each process inoculates 4 bottles, every bottle graft kind 6 strain (the seedling growing way of same bottle graft kind is as far as possible close). The culture bottle connected is placed in thermostatic chamber, keeps room temperature 25 ± 1 DEG C, illumination 10～12h/d, intensity of illumination 1500～2000Lux. Add up the average plant height after growing 3 months, the newly-increased number of blade of average individual plant and average single-strain fresh weight. Experimental result is in Table 3:
The impact on Herba Anoectochili roxburghii growth of seedling of table 3 different culture media
The data of the data of A12-A15 culture medium and A11 culture medium from table 3, the data of B12-B15 culture medium and the data of the data of B11 culture medium and C12-C15 culture medium are known compared with the data of C11 culture medium, process the culture medium of liquid of precooking and common culture medium added with the edible fungi canning of debita spissitudo (to be not added with edible fungi canning processing to precook the culture medium such as A11 of liquid, B11, C11) compare, the growth of Herba Anoectochili roxburghii more can be promoted added with the precook culture medium of liquid of edible fungi canning processing, substantially shortening group can train Herba Anoectochili roxburghii growth cycle, and the best results of A12 culture medium, for best medium. additionally it can be seen that the growth of Herba Anoectochili roxburghii is had a certain impact by the concentration of sucrose, Fructus Musae, agar and activated carbon compared with the data of the data of A11-A15 culture medium and the data of B11-B15 culture medium, C11-C15 culture medium, but impact is little. it addition, it be observed that and the edible fungi canning added with debita spissitudo process the Herba Anoectochili roxburghii that the culture medium of liquid of precooking cultivates to compare the Herba Anoectochili roxburghii plant that ordinary culture medium cultivates more healthy and strong, blade unfolds, abundant, and color is more bright-coloured.
In order to verify that the culture medium of the present invention is if appropriate for the cultivation for other plant, inventor is also by the 60 of the present invention groups of (A1～A20; B1～B20; C1～C20) culture medium is for the cultivation of the plants such as Radix Raphani, Brassica oleracea L. var. botrytis L., Fructus Lycopersici esculenti, blue berry, find Radix Raphani, Brassica oleracea L. var. botrytis L., Fructus Lycopersici esculenti and conventional medium (i.e. the culture medium without liquid of precooking) contrast, growing way difference is inconspicuous, blue berry and conventional medium contrast, growing way zero difference, the use that culture medium of the present invention is described is targetedly, be not necessarily suitable for all plants cultivate or the component proportion relation of above-mentioned culture medium is not appropriate for the cultivation of other plant.
Claims (6)
1. a group training Herba Anoectochili roxburghii culture medium, including Fructus Musae, sucrose, agar and activated carbon, it is characterised in that: it also includes 0*MS～1*MS culture medium mother solution and liquid of precooking is processed in edible fungi canning; Group training Herba Anoectochili roxburghii culture medium described in every 1000ml includes 0*MS～1*MS minimal medium mother solution, liquid, 10～50g sucrose, 50～250g Fructus Musae, 6g～8g agar, 1.0g～2.0g activated carbon are precooked in 10～1000 μ L edible fungi canning processing, surplus is distilled water, and wherein 0*MS represents and is added without minimal medium mother solution.
2. the preparation of according to claim 1 group of training Herba Anoectochili roxburghii culture medium, it is characterised in that: organic culture medium preparation steps of every 1000 milliliters is as follows:
(1) prepare edible fungi canning processing to precook liquid, and the edible fungi canning processing measuring 10～1000 μ L liquid of precooking is stand-by;
(2) according to the volume of the various mother solutions needed for preparation 0*MS～1*MS minimal medium, MS a great number of elements mother solution, MS trace element mother solution, MS mother liquid of iron salt and MS Organic substance mother solution are measured stand-by;
(3) the edible fungi canning measured in step (1) processing is precooked after liquid mixes with the MS culture medium mother solution measured in step (2) and is dissolved in distilled water;
(4) weigh 50～250g Fructus Musae and 1.0g～2.0g activated carbon, and add in step (3) in the solution of gained after Fructus Musae is ground into mud, stirring and evenly mixing, add the activated carbon weighed up afterwards, stir;
(5) in beaker, add the distilled water of 200～300mL, distilled water heating is added, after 80～90 DEG C, the 6g～8g agar weighed up, after the extremely boiling of heating while stirring, agar dissolves, after treating that agar dissolves, continuously add the 10～50g sucrose weighed up, heat while stirring to sucrose dissolving, then the mixed solution of the agar that obtains with sucrose is added in the mixed solution of step (4) gained, add distilled water after stirring and be settled to 1000 milliliters and add acid-base modifier pH value is adjusted to 5.0-6.0;
(6) culture medium obtained for step (5) is sub-packed in tissue culture bottle, carries out autoclave sterilization, afterwards cooled and solidified.
3. the preparation of according to claim 2 group of training Herba Anoectochili roxburghii culture medium, it is characterised in that: the preparation method that liquid of precooking is processed in described edible fungi canning is as follows:
A. produced industrial wastewater in the edible fungi canning course of processing is collected;
B. being centrifuged collecting produced industrial wastewater in the edible fungi canning course of processing come, the rotating speed of centrifuge controls between 11500-12500rpm, and centrifuging temperature is 15-25 DEG C, and centrifugation time is 10-20min, is centrifuged and takes supernatant afterwards;
C. the supernatant of gained being utilized falling film evaporator, falling film evaporation concentrates, and obtains concentrated solution;
D. adding denaturant in concentrated solution, concentrated solution carries out degeneration, wherein said denaturant is the ethanol of 80～100%, and the volume ratio of affiliated ethanol and concentrated solution is 2:1～6:1;
E. the concentrated solution after degeneration is filtered, collects filtrate;
F. filtrate is rotated evaporating ethanol;
G., the solution of gained after rotary evaporation utilizes the filter membrane of 5-10K carry out tangential flow filtration, collects filter liquor;
H. the filter liquor of gained in step g is rotated evaporation and concentration, be concentrated into solution index of refraction and reach 35～55%.
4. the preparation of organic culture medium of according to claim 2 group of training Herba Anoectochili roxburghii, it is characterised in that: the HCl solution of the NaOH solution that acid-base modifier is 0.1-0.5mol/L described in step (5) or 0.1-0.5mol/L.
5. the preparation of according to claim 2 group of training Herba Anoectochili roxburghii culture medium, it is characterised in that: in step (6), the temperature of autoclave sterilization controls at 121-125 DEG C, and Stress control is at 111-115 kPa, and sterilization time is 15-20 minute.
6. the application of according to claim 1 group of training Herba Anoectochili roxburghii culture medium, it is characterised in that: it is used for cultivating Herba Anoectochili roxburghii.
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