CN105660401B - A kind of tissue culture roxburgh anoectochilus terminal bud culture medium and its preparation method and application - Google Patents
A kind of tissue culture roxburgh anoectochilus terminal bud culture medium and its preparation method and application Download PDFInfo
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Abstract
The present invention relates to a kind of tissue culture roxburgh anoectochilus terminal bud culture medium and its preparation method and application, a kind of tissue culture roxburgh anoectochilus terminal bud culture medium, tissue culture roxburgh anoectochilus terminal bud culture medium described in per 1000ml includes 0*MS~1*MS minimal mediums mother liquor, 10~1000 μ L edible mushrooms cannings processing pre-boiled liquid, 10~50g sucrose, 50~250g bananas, 6g~8g agar, 1.0g~2.0g activated carbons, surplus is distilled water, the advantage of the invention is that：Solve the emission problem of caused industrial wastewater in edible mushroom canning process, Edible mushroom processing enterprise is reached wastewater zero discharge, reduce cost of sewage disposal；The culture medium of the present invention can promote tissue culture roxburgh anoectochilus terminal bud to root, tissue culture roxburgh anoectochilus terminal bud growth cycle can also be shortened, the use of edible mushroom canning processing pre-boiled liquid reduces application of the artificial synthesized hormone in tissue culture roxburgh anoectochilus terminal bud, tissue culture roxburgh anoectochilus terminal bud is turned into a kind of green organic food.
Description
Technical field
The present invention relates to a kind of tissue culture roxburgh anoectochilus terminal bud culture medium and its preparation method and application.
Background technology
Roxburgh anoectochilus terminal bud (Anoectochilus roxburghii (Wall.) Lindl) be orchid family Anoectochilus Blume raw type for many years
Raw herbaceous plant, subtropical zone, i.e. Fujian, Guangdong, Guangxi, Zhejiang, Jiangxi, Hainan, Yunnan, four are distributed mainly in China
The provinces and regions such as river, Guizhou and southern Tibet.Roxburgh anoectochilus terminal bud is the traditional valuable ingredient of traditional Chinese medicine in China, can all herbal medicine, it is sweet and slightly bitter taste, mild-natured
Be slightly cold, have it is clearing heat and detoxicating, moisten the lung and relieve the cough with expelling wind and removing dampness and other effects, therefore by the extensive attention of pharmaceuticals industry.Due to open country
Production-goods source is increasingly exhausted, and roxburgh anoectochilus terminal bud has been listed in Fujian Province's Endangered Medicinal Herb and has been protected by.But its seed is under field conditions (factors)
Germination rate is extremely low, so carrying out rescue preservation to it using artificial propagation.The artificial propagation of roxburgh anoectochilus terminal bud at present is mainly leaned on
Field-transplanting is carried out after tissue cultivating and seedling, the roxburgh anoectochilus terminal bud of tissue cultures is mostly that promotion is adjusted using auxin
It grows, but exists using growth hormone come to adjust the conventional method of roxburgh anoectochilus terminal bud tissue cultures be unsustainable sexual development
Cost is high, influences the problems such as food security, in addition, under the social development of nowadays high speed, green organic food gradually into
For main product, original roxburgh anoectochilus terminal bud tissue culture medium (TCM) and cultural method, do not catch up with what people were pursued pollution-free food
Step.Therefore, it is necessary to a kind of green, nuisanceless and that roxburgh anoectochilus terminal bud growth can be accelerated culture medium is worked out to cultivate gold
Line lotus, in order to reach the purpose for promoting roxburgh anoectochilus terminal bud quickly to breed, to meet large market demand, while and can enough makes cultivation
The roxburgh anoectochilus terminal bud gone out reaches green and organically required.
The content of the invention
It is an object of the invention to provide a kind of tissue culture roxburgh anoectochilus terminal bud culture that is environmentally friendly, nuisanceless and can accelerating roxburgh anoectochilus terminal bud growth
Base and its preparation method and application.
The purpose of the present invention is achieved through the following technical solutions：A kind of tissue culture roxburgh anoectochilus terminal bud culture medium, including banana, sucrose,
Agar and activated carbon, it also includes 0*MS~1*MS culture medium mother liquors and edible mushroom canning processing pre-boiled liquid；Per 1000ml
Described tissue culture roxburgh anoectochilus terminal bud culture medium includes 0*MS~1*MS minimal mediums mother liquor, 10~1000 μ L edible mushrooms cannings processing in advance
Boil liquid, 10~50g sucrose, 50~250g bananas, 6g~8g agar, 1.0g~2.0g activated carbons, surplus are distilled water, wherein 0*
MS represents to be added without minimal medium mother liquor.
The preparation of described tissue culture roxburgh anoectochilus terminal bud culture medium, every 1000 milliliters of organic culture medium preparation steps are as follows：
(1) edible mushroom canning processing pre-boiled liquid is prepared, and the edible mushroom canning processing pre-boiled liquid for measuring 10~1000 μ L is treated
With；
(2) according to the volume for preparing the various mother liquors needed for 0*MS~1*MS minimal mediums, MS a great number of elements mother is measured
Liquid, MS trace elements mother liquor, MS mother liquid of iron salt and MS organic matter mother liquors are stand-by；
(3) pre-boiled liquid and the MS culture medium mother liquors measured in step (2) are processed into the edible mushroom canning measured in step (1)
It is dissolved in after being mixed in distilled water；
(4) 50~250g bananas and 1.0g~2.0g activated carbons are weighed, and is added after banana is ground into mud in step (3)
In the solution of gained, stir and evenly mix, add the activated carbon weighed up afterwards, stir；
(5) 200~300mL distilled water is added in beaker, distilled water is heated to add what is weighed up after 80~90 DEG C
6g~8g agar, agar dissolves after being heated to boiling while stirring, after treating agar dissolving, continuously adds the 10~50g weighed up
Sucrose, sucrose dissolving is heated to while stirring, then adds the mixed solution of obtained agar and sucrose obtained by step (4)
In mixed solution, after stirring plus distilled water is settled to 1000 milliliters and adds acid-base modifier and pH value is adjusted into 5.0-6.0；
(6) culture medium obtained by step (5) is sub-packed in tissue culture bottle, carries out autoclave sterilization, cooling is solidifying afterwards
Gu.
The preparation method of the edible mushroom canning processing pre-boiled liquid is as follows：
A. caused industrial wastewater in edible mushroom canning process is collected；
B. caused industrial wastewater in next edible mushroom canning process will be collected to be centrifuged, the rotating speed of centrifuge
Between 11500-12500rpm, centrifuging temperature is 15-25 DEG C, centrifugation time 10-20min, and supernatant is taken after centrifugation for control
Liquid；
C. the supernatant of gained is utilized into falling film evaporator, falling film evaporation concentration, obtains concentrate；
D. denaturant is added in concentrate, concentrate is denatured, wherein described denaturant is 80~100%
Ethanol, and the ethanol and the volume ratio of concentrate added is 2:1~6:1；
E. the concentrate after denaturation is filtered, collects filtrate；
F. filtrate is subjected to rotary evaporation and removes ethanol；
G. the solution of gained after rotary evaporation is subjected to tangential flow filtration using 5-10K filter membrane, collects filter liquor；
H. the filter liquor of gained in step g is subjected to rotary evaporation concentration, is concentrated into solution index of refraction and reaches 35~55%
.
Acid-base modifier described in step (5) is 0.1-0.5mol/L NaOH solution or 0.1-0.5mol/L HCl
Solution.
The autoclaved temperature control of step (6) high temperature is at 121-125 DEG C, and Stress control is at 111-115 kPas, sterilizing
Time is 15-20 minutes.
The application of described tissue culture roxburgh anoectochilus terminal bud culture medium, tissue culture roxburgh anoectochilus terminal bud culture medium is used to cultivate roxburgh anoectochilus terminal bud.Than existing
For having technology, the advantage of the invention is that：1) caused industrial wastewater is often rich in edible mushroom canning process
Some nutriments such as amino acid, mineral matter, carbohydrate etc., if these waste water discharged after processing, not only need costliness
Cost of sewage disposal, and cause certain wasting of resources.The present invention will be caused in edible mushroom canning process
After industrial wastewater is handled, edible mushroom canning processing pre-boiled liquid is obtained, itself and MS culture medium mother liquors and other additives are answered
It is configured to train roxburgh anoectochilus terminal bud culture medium, caused industrial wastewater in edible mushroom canning process is turned waste into wealth, solves food
With the emission problem of caused industrial wastewater in bacterium canning process, Edible mushroom processing enterprise is set to reach wastewater zero discharge,
Reduce its cost of sewage disposal；2) institute after the present invention is handled caused industrial wastewater in edible mushroom canning process
The edible mushroom canning processing pre-boiled liquid obtained, to substitute traditional plant growth regulator, can provide for roxburgh anoectochilus terminal bud and remove MS culture mediums
Nutrition in addition, while tissue culture roxburgh anoectochilus terminal bud can be promoted to root, moreover it is possible to shorten tissue culture roxburgh anoectochilus terminal bud growth cycle, edible mushroom canning
The use of processing pre-boiled liquid reduces application of the artificial synthesized hormone in tissue culture roxburgh anoectochilus terminal bud, tissue culture roxburgh anoectochilus terminal bud is turned into a kind of green
Color organic food；3) present invention prepares the raw material of tissue culture roxburgh anoectochilus terminal bud culture medium and is easy to get and cheap；4) present invention prepares tissue culture
The preparation method of roxburgh anoectochilus terminal bud culture medium is simple, suitable for industrialized production；5) tissue culture roxburgh anoectochilus terminal bud culture medium tool produced by the present invention
There is good fungistatic effect so that need not add bactericide in the culture medium for cultivating roxburgh anoectochilus terminal bud, further ensure roxburgh anoectochilus terminal bud
Quality.
Embodiment
Present invention is described in detail with reference to embodiment：
Embodiment one：The preparation of MS mother liquors
1.MS a great number of elements mother liquor (20X) weighs 20 rising amount and is dissolved in 1L distilled water.
2.MS trace elements mother liquor (200X)
200 rising amount are weighed to be dissolved in 1L distilled water.
3.MS mother liquid of iron salt (200X)
200 rising amount are weighed to be dissolved in 1L distilled water.
4.MS organic matters mother liquor (200X)
200 rising amount are weighed to be dissolved in 1L distilled water.
Embodiment two：The preparation of tissue culture roxburgh anoectochilus terminal bud culture medium
1L tissue culture roxburgh anoectochilus terminal bud culture medium (1) 1/2*MS+250 μ L edible mushroom canning processing pre-boiled liquid+30g sucrose+100g
The edible mushroom canning processing pre-boiled liquid+50g sucrose+250g bananas+8g of banana+7g agar+1.5g activated carbons, (2) MS+1000 μ L
Agar+2.0g activated carbons, (3) 1/4*MS+10 μ L edible mushroom canning processing pre-boiled liquid+10g sucrose+50g banana+6g agar+
The specific preparation method of 1.0g activated carbons is as follows：
Organic culture medium (1/2*MS+250 μ L edible mushroom canning processing pre-boiled liquid+30g sucrose of 1.1L tissue culture roxburgh anoectochilus terminal buds
+ 100g banana+7g agar+1.5g activated carbons) preparation, every 1000 milliliters of organic culture medium preparation steps are as follows：
(1) edible mushroom canning processing pre-boiled liquid is prepared, and the edible mushroom canning processing pre-boiled liquid for measuring 250 μ L is stand-by；
(2) according to the volume for preparing the various mother liquors needed for 1/2*MS minimal mediums, measure successively：
MS a great number of elements mother liquors 25mL
MS trace element mother liquors 2.5mL
MS mother liquid of iron salt 2.5mL
MS organic matter mother liquors 2.5mL is stand-by；
(3) pre-boiled liquid and the MS culture medium mother liquors measured in step (2) are processed into the edible mushroom canning measured in step (1)
It is dissolved in after being mixed in distilled water；
(4) 100g bananas and 1.5g activated carbons are weighed, and banana is ground into the solution that gained in step (3) is added after mud
In, stir and evenly mix, add the activated carbon weighed up afterwards, stir；
(5) 200mL distilled water is added in beaker, distilled water is heated to add the 7g agar weighed up, side after 80 DEG C
Agar dissolves after stirring side is heated to boiling, after treating agar dissolving, continuously adds the 30g sucrose weighed up, heats while stirring
Dissolve to sucrose, then add the mixed solution of obtained agar and sucrose in the mixed solution obtained by step (4), stirring is equal
After even plus distilled water is settled to 1000 milliliters and adds 0.1mol/L NaOH and pH value is adjusted into 5.0；
(6) culture medium obtained by step (5) is sub-packed in tissue culture bottle, carries out autoclave sterilization, cooling is solidifying afterwards
Gu wherein autoclave sterilization temperature control is at 121 DEG C, for Stress control at 111 kPas, sterilization time is 15 minutes.
Wherein, the preparation method of edible mushroom canning processing pre-boiled liquid is as follows：
A. caused industrial wastewater in edible mushroom canning process is collected；
B. caused industrial wastewater in next edible mushroom canning process will be collected to be centrifuged, the rotating speed of centrifuge
In 11500rpm, centrifuging temperature is 15 DEG C, centrifugation time 10min, and supernatant is taken after centrifugation for control；
C. the supernatant of gained is utilized into falling film evaporator, falling film evaporation concentration, obtains concentrate；
D. denaturant is added in concentrate, concentrate is denatured, wherein described denaturant is 80% ethanol,
And the ethanol and the volume ratio of concentrate added is 2:1；
E. the concentrate after denaturation is filtered, collects filtrate；
F. filtrate is subjected to rotary evaporation and removes ethanol；
G. the solution of gained after rotary evaporation is subjected to tangential flow filtration using 5K filter membrane, collects filter liquor；
H. the filter liquor of gained in step g is subjected to rotary evaporation concentration, is concentrated into solution index of refraction and reaches 35%.
2.1L tissue culture roxburgh anoectochilus terminal buds organic culture medium (MS+1000 μ L edible mushroom canning processing pre-boiled liquid+50g sucrose+
250g banana+8g agar+2.0g activated carbons) preparation, every 1000 milliliters of organic culture medium preparation steps are as follows：
(1) edible mushroom canning processing pre-boiled liquid is prepared, and the edible mushroom canning processing pre-boiled liquid for measuring 1000 μ L is stand-by；
(2) according to the volume for preparing the various mother liquors needed for 1*MS minimal mediums, measure successively：
MS a great number of elements mother liquors 50mL
MS trace element mother liquors 5mL
MS mother liquid of iron salt 5mL
MS organic matter mother liquors 5mL is stand-by；
(3) pre-boiled liquid and the MS culture medium mother liquors measured in step (2) are processed into the edible mushroom canning measured in step (1)
It is dissolved in after being mixed in distilled water；
(4) 250g bananas and 2.0g activated carbons are weighed, and banana is ground into the solution that gained in step (3) is added after mud
In, stir and evenly mix, add the activated carbon weighed up afterwards, stir；
(5) 300mL distilled water is added in beaker, distilled water is heated to add the 8g agar weighed up, side after 90 DEG C
Agar dissolves after stirring side is heated to boiling, after treating agar dissolving, continuously adds the 50g sucrose weighed up, heats while stirring
Dissolve to sucrose, then add the mixed solution of obtained agar and sucrose in the mixed solution obtained by step (4), stirring is equal
After even plus distilled water is settled to 1000 milliliters and adds 0.5mol/L NaOH and pH value is adjusted into 6.0；
(6) culture medium obtained by step (5) is sub-packed in tissue culture bottle, carries out autoclave sterilization, cooling is solidifying afterwards
Gu wherein autoclave sterilization temperature control is at 125 DEG C, for Stress control at 115 kPas, sterilization time is 20 minutes.
Wherein, the preparation method of edible mushroom canning processing pre-boiled liquid is as follows：
A. caused industrial wastewater in edible mushroom canning process is collected；
B. caused industrial wastewater in next edible mushroom canning process will be collected to be centrifuged, the rotating speed of centrifuge
In 12000rpm, centrifuging temperature is 20 DEG C, centrifugation time 15min, and supernatant is taken after centrifugation for control；
C. the supernatant of gained is utilized into falling film evaporator, falling film evaporation concentration, obtains concentrate；
D. denaturant is added in concentrate, concentrate is denatured, wherein described denaturant is 80% ethanol,
And the ethanol and the volume ratio of concentrate added is 3:1；
E. the concentrate after denaturation is filtered, collects filtrate；
F. filtrate is subjected to rotary evaporation and removes ethanol；
G. the solution of gained after rotary evaporation is subjected to tangential flow filtration using 8K filter membrane, collects filter liquor；
H. the filter liquor of gained in step g is subjected to rotary evaporation concentration, is concentrated into solution index of refraction and reaches 50%.
3.1L tissue culture roxburgh anoectochilus terminal buds organic culture medium (1/4*MS+10 μ L edible mushroom canning processing pre-boiled liquid+10g sucrose+
50g banana+6g agar+1.0g activated carbons) preparation, every 10 milliliters of organic culture medium preparation steps are as follows：
(1) edible mushroom canning processing pre-boiled liquid is prepared, and the edible mushroom canning processing pre-boiled liquid for measuring 10 μ L is stand-by；
(2) according to the volume for preparing the various mother liquors needed for 1/4*MS minimal mediums, measure successively：
MS a great number of elements mother liquors 12.5mL
MS trace element mother liquors 1.25mL
MS mother liquid of iron salt 1.25mL
MS organic matter mother liquors 1.25mL is stand-by；
(3) pre-boiled liquid and the MS culture medium mother liquors measured in step (2) are processed into the edible mushroom canning measured in step (1)
It is dissolved in after being mixed in distilled water；
(4) 50g bananas and 1.0g activated carbons are weighed, and banana is ground into the solution that gained in step (3) is added after mud
In, stir and evenly mix, add the activated carbon weighed up afterwards, stir；
(5) 250mL distilled water is added in beaker, distilled water is heated to add the 6g agar weighed up, side after 85 DEG C
Agar dissolves after stirring side is heated to boiling, after treating agar dissolving, continuously adds the 10g sucrose weighed up, heats while stirring
Dissolve to sucrose, then add the mixed solution of obtained agar and sucrose in the mixed solution obtained by step (4), stirring is equal
After even plus distilled water is settled to 1000 milliliters and adds 0.1mol/L HCl and pH value is adjusted into 5.5；
(6) culture medium obtained by step (5) is sub-packed in tissue culture bottle, carries out autoclave sterilization, cooling is solidifying afterwards
Gu wherein autoclave sterilization temperature control is at 122 DEG C, for Stress control at 113 kPas, sterilization time is 18 minutes.
Wherein, the preparation method of edible mushroom canning processing pre-boiled liquid is as follows：
A. caused industrial wastewater in edible mushroom canning process is collected；
B. caused industrial wastewater in next edible mushroom canning process will be collected to be centrifuged, the rotating speed of centrifuge
In 12500rpm, centrifuging temperature is 25 DEG C, centrifugation time 20min, and supernatant is taken after centrifugation for control；
C. the supernatant of gained is utilized into falling film evaporator, falling film evaporation concentration, obtains concentrate；
D. denaturant is added in concentrate, concentrate is denatured, wherein described denaturant is 80% ethanol,
And the ethanol and the volume ratio of concentrate added is 6:1；
E. the concentrate after denaturation is filtered, collects filtrate；
F. filtrate is subjected to rotary evaporation and removes ethanol；
G. the solution of gained after rotary evaporation is subjected to tangential flow filtration using 10K filter membrane, collects filter liquor；
H. the filter liquor of gained in step g is subjected to rotary evaporation concentration, is concentrated into solution index of refraction and reaches 55%.
According to above-mentioned preparation method or principle, following 60 groups of (A1~A20 are prepared；B1~B20；C1~C20) culture medium is (often
The volume of kind culture medium is 1L)：
First, with 0*MS, 1/4*MS, 1/2*MS, MS points are four big groups of processing, and the taken amount of edible mushroom canning processing pre-boiled liquid is divided
Not Wei 0 μ L, 250 μ L, 500 μ L, 1000 μ L, 2000 μ L, sucrose 30g, banana 100g, agar 7g, 1.5g activated carbon in addition.Wherein
0*MS represents not adding minimal medium mother liquor.
(1) 0*MS processing
A1:0*MS+0 μ L mushroom canning pre-boiled liquid+30g sucrose+100g banana+7g agar+1.5g activated carbons
A2:0*MS+250 μ L mushroom canning pre-boiled liquid+30g sucrose+100g banana+7g agar+1.5g activated carbons
A3:0*MS+500 μ L mushroom canning pre-boiled liquid+30g sucrose+100g banana+7g agar+1.5g activated carbons
A4:0*MS+1000 μ L mushroom canning pre-boiled liquid+30g sucrose+100g banana+7g agar+1.5g activated carbons
A5:0*MS+2000 μ L mushroom canning pre-boiled liquid+30g sucrose+100g banana+7g agar+1.5g activated carbons
(2) 1/4*MS processing
A6:1/4*MS+0 μ L mushroom canning pre-boiled liquid+30g sucrose+100g banana+7g agar+1.5g activated carbons
A7:1/4*MS+250 μ L mushroom canning pre-boiled liquid+30g sucrose+100g banana+7g agar+1.5g activated carbons
A8:1/4*MS+500 μ L mushroom canning pre-boiled liquid+30g sucrose+100g banana+7g agar+1.5g activated carbons
A9:1/4*MS+1000 μ L mushroom canning pre-boiled liquid+30g sucrose+100g banana+7g agar+1.5g activated carbons
A10:1/4*MS+2000 μ L mushroom canning pre-boiled liquid+30g sucrose+100g banana+7g agar+1.5g activated carbons
(3) 1/2*MS processing
A11:1/2*MS+0 μ L mushroom canning pre-boiled liquid+30g sucrose+100g banana+7g agar+1.5g activated carbons
A12:1/2*MS+250 μ L mushroom canning pre-boiled liquid+30g sucrose+100g banana+7g agar+1.5g activated carbons
A13:1/2*MS+500 μ L mushroom canning pre-boiled liquid+30g sucrose+100g banana+7g agar+1.5g activated carbons
A14:1/2*MS+1000 μ L mushroom canning pre-boiled liquid+30g sucrose+100g banana+7g agar+1.5g activated carbons
A15:1/2*MS+2000 μ L mushroom canning pre-boiled liquid+30g sucrose+100g banana+7g agar+1.5g activated carbons
(4) MS processing
A16:MS+0 μ L mushroom canning pre-boiled liquid+30g sucrose+100g banana+7g agar+1.5g activated carbons
A17:MS+250 μ L mushroom canning pre-boiled liquid+30g sucrose+100g banana+7g agar+1.5g activated carbons
A18:MS+500 μ L mushroom canning pre-boiled liquid+30g sucrose+100g banana+7g agar+1.5g activated carbons
A19:MS+1000 μ L mushroom canning pre-boiled liquid+30g sucrose+100g banana+7g agar+1.5g activated carbons
A20:MS+2000 μ L mushroom canning pre-boiled liquid+30g sucrose+100g banana+7g agar+1.5g activated carbons
2nd, with 0*MS, 1/4*MS, 1/2*MS, MS points are four big groups of processing, and the taken amount of edible mushroom canning processing pre-boiled liquid is divided
Wei not 0 μ L, 250 μ L, 500 μ L, 1000 μ L, 2000 μ L, in addition sucrose 10g, banana 50g, agar 6g, activated carbon 1.0g.
(1) 0*MS processing
B1:0*MS+0 μ L mushroom canning pre-boiled liquid+10g sucrose+50g banana+6g agar+1.0g activated carbons
B2:0*MS+250 μ L mushroom canning pre-boiled liquid+10g sucrose+50g banana+6g agar+1.0g activated carbons
B3:0*MS+500 μ L mushroom canning pre-boiled liquid+10g sucrose+50g banana+6g agar+1.0g activated carbons
B4:0*MS+1000 μ L mushroom canning pre-boiled liquid+10g sucrose+50g banana+6g agar+1.0g activated carbons
B5:0*MS+2000 μ L mushroom canning pre-boiled liquid+10g sucrose+50g banana+6g agar+1.0g activated carbons
(2) 1/4*MS processing
B6:1/4*MS+0 μ L mushroom canning pre-boiled liquid+10g sucrose+50g banana+6g agar+1.0g activated carbons
B7:1/4*MS+250 μ L mushroom canning pre-boiled liquid+10g sucrose+50g banana+6g agar+1.0g activated carbons
B8:1/4*MS+500 μ L mushroom canning pre-boiled liquid+10g sucrose+50g banana+6g agar+1.0g activated carbons
B9:1/4*MS+1000 μ L mushroom canning pre-boiled liquid+10g sucrose+50g banana+6g agar+1.0g activated carbons
B10:1/4*MS+2000 μ L mushroom canning pre-boiled liquid+10g sucrose+50g banana+6g agar+1.0g activated carbons
(3) 1/2*MS processing
B11:1/2*MS+0 μ L mushroom canning pre-boiled liquid+10g sucrose+50g banana+6g agar+1.0g activated carbons
B12:1/2*MS+250 μ L mushroom canning pre-boiled liquid+10g sucrose+50g banana+6g agar+1.0g activated carbons
B13:1/2*MS+500 μ L mushroom canning pre-boiled liquid+10g sucrose+50g banana+6g agar+1.0g activated carbons
B14:1/2*MS+1000 μ L mushroom canning pre-boiled liquid+10g sucrose+50g banana+6g agar+1.0g activated carbons
B15:1/2*MS+2000 μ L mushroom canning pre-boiled liquid+10g sucrose+50g banana+6g agar+1.0g activated carbons
(4) MS processing
B16:MS+0 μ L mushroom canning pre-boiled liquid+10g sucrose+50g banana+6g agar+1.0g activated carbons
B17:MS+250 μ L mushroom canning pre-boiled liquid+10g sucrose+50g banana+6g agar+1.0g activated carbons
B18:MS+500 μ L mushroom canning pre-boiled liquid+10g sucrose+50g banana+6g agar+1.0g activated carbons
B19:MS+1000 μ L mushroom canning pre-boiled liquid+10g sucrose+50g banana+6g agar+10g activated carbons
B20:MS+2000 μ L mushroom canning pre-boiled liquid+10g sucrose+50g banana+6g agar+1.0g activated carbons
3rd, with 0*MS, 1/4*MS, 1/2*MS, MS points are four big groups of processing, and the taken amount of edible mushroom canning processing pre-boiled liquid is divided
Not Wei 0 μ L, 250 μ L, 500 μ L, 1000 μ L, 2000 μ L, sucrose 50g, banana 250g, agar 8g, 2.0g activated carbon in addition.
(1) 0*MS processing
C1:0*MS+0 μ L mushroom canning pre-boiled liquid+50g sucrose+250g banana+8g agar+2.0g activated carbons
C2:0*MS+250 μ L mushroom canning pre-boiled liquid+50g sucrose+250g banana+8g agar+2.0g activated carbons
C3:0*MS+500 μ L mushroom canning pre-boiled liquid+50g sucrose+250g banana+8g agar+2.0g activated carbons
C4:0*MS+1000 μ L mushroom canning pre-boiled liquid+50g sucrose+250g banana+8g agar+2.0g activated carbons
C5:0*MS+2000 μ L mushroom canning pre-boiled liquid+50g sucrose+250g banana+8g agar+2.0g activated carbons
(2) 1/4*MS processing
C6:1/4*MS+0 μ L mushroom canning pre-boiled liquid+50g sucrose+250g banana+8g agar+2.0g activated carbons
C7:1/4*MS+250 μ L mushroom canning pre-boiled liquid+50g sucrose+250g banana+8g agar+2.0g activated carbons
C8:1/4*MS+500 μ L mushroom canning pre-boiled liquid+50g sucrose+250g banana+8g agar+2.0g activated carbons
C9:1/4*MS+1000 μ L mushroom canning pre-boiled liquid+50g sucrose+250g banana+8g agar+2.0g activated carbons
C10:1/4*MS+2000 μ L mushroom canning pre-boiled liquid+50g sucrose+250g banana+8g agar+2.0g activated carbons
(3) 1/2*MS processing
C11:1/2*MS+0 μ L mushroom canning pre-boiled liquid+50g sucrose+250g banana+8g agar+2.0g activated carbons
C12:1/2*MS+250 μ L mushroom canning pre-boiled liquid+50g sucrose+250g banana+8g agar+2.0g activated carbons
C13:1/2*MS+500 μ L mushroom canning pre-boiled liquid+50g sucrose+250g banana+8g agar+2.0g activated carbons
C14:1/2*MS+1000 μ L mushroom canning pre-boiled liquid+50g sucrose+250g banana+8g agar+2.0g activated carbons
C15:1/2*MS+2000 μ L mushroom canning pre-boiled liquid+50g sucrose+250g banana+8g agar+2.0g activated carbons
(4) MS processing
C16:MS+0 μ L mushroom canning pre-boiled liquid+50g sucrose+250g banana+8g agar+2.0g activated carbons
C17:MS+250 μ L mushroom canning pre-boiled liquid+50g sucrose+250g banana+8g agar+2.0g activated carbons
C18:MS+500 μ L mushroom canning pre-boiled liquid+50g sucrose+250g banana+8g agar+2.0g activated carbons
C19:MS+1000 μ L mushroom canning pre-boiled liquid+50g sucrose+250g banana+8g agar+2.0g activated carbons
C20:MS+2000 μ L mushroom canning pre-boiled liquid+50g sucrose+250g banana+8g agar+2.0g activated carbons
By 60 tissue culture roxburgh anoectochilus terminal bud culture medium (A1~A20 obtained above；B1~B20；C1~C20) it is used for the training of roxburgh anoectochilus terminal bud
Educate；
First, the culture that different culture media is induced bud
High-quality Taiwan kind roxburgh anoectochilus terminal bud sterilizable material is chosen, under super-clean bench gnotobasis, root and blade are removed with scissors,
Stem is cut into the stem section of long one section of 2.5cm bands.It is seeded on A1-A20 tissue culture roxburgh anoectochilus terminal bud culture mediums, regulation PH is 5.8.It is real
Test and set 20 processing altogether, each processing connects 6 bottles, 4 sections of every bottle of inoculation.The blake bottle connected is placed in thermostatic chamber, protected
Hold 25 ± 1 DEG C, illumination 10h/d, 800~1000Lux of intensity of illumination of room temperature.Germination degree is counted every other month
(differentiation rate=germination stem section number/inoculation stem section sum), the mouldy blake bottle of timely processing, the experimental result measured is shown in Table 1.
The influence that the different culture media of table 1 germinates to stem section
Note：Germination when table 1 is is inoculated with 90d.+ representing that bud is tiny, color is yellowish green；++ represent that bud is tiny, color is blackish green；
+++ represent that bud is sturdy, color is blackish green；Bud is sturdy for ++++represent, color is blackish green, there is clump bud growth.
It was found from the roxburgh anoectochilus terminal bud stem section germination data that the A1-A5 culture mediums in the data of table 1 are cultivated, when the basic training of no addition
When supporting base mother liquor, as the increase of pre-boiled liquid concentration is processed in edible mushroom canning, the germination sum of roxburgh anoectochilus terminal bud stem section constantly increases,
Differentiation rate and proliferation times also gradually increase, it is seen that edible mushroom canning processing pre-boiled liquid both can be that the germination of roxburgh anoectochilus terminal bud stem section carries
For required nutrition, while tissue culture roxburgh anoectochilus terminal bud can be promoted to germinate.
The roxburgh anoectochilus terminal bud stem section germination data that A6-A10 culture mediums are cultivated are understood, when addition 1/4*MS minimal medium mother liquors
Afterwards, the nutrition required for germination is provided for roxburgh anoectochilus terminal bud stem section, still, the nutrition is still not able to fully meet roxburgh anoectochilus terminal bud stem section
Needed for germination, therefore as the increase of pre-boiled liquid concentration is processed in edible mushroom canning, the germination sum of roxburgh anoectochilus terminal bud stem section constantly increases
Adding, differentiation rate and proliferation times also gradually increase, when the concentration of edible mushroom canning processing pre-boiled liquid increases to 1000 μ L/L,
Have been able to meet needed for the nutrition of roxburgh anoectochilus terminal bud stem section germination, and can also promote the germination of roxburgh anoectochilus terminal bud stem section, Zhi Houru well
Fruit continues to increase the concentration of edible mushroom canning processing pre-boiled liquid, and roxburgh anoectochilus terminal bud stem section germinative number reduces on the contrary, illustrates the food of high concentration
There will be certain inhibitory action to the germination of roxburgh anoectochilus terminal bud stem section with bacterium canning processing pre-boiled liquid.
The roxburgh anoectochilus terminal bud stem section germination data that A11-A15 culture mediums are cultivated are understood, when addition 1/2*MS minimal medium mother liquors
Afterwards, have been able to meet the needs of germination of roxburgh anoectochilus terminal bud stem section is to nutrition well, therefore edible mushroom canning processing pre-boiled liquid is dense
Degree only need to reach 250 μ L/L, just can be good at promoting the germination of roxburgh anoectochilus terminal bud stem section, and the germinative number of roxburgh anoectochilus terminal bud stem section and increasing
Grow multiple and reach maximum, afterwards if continuing to improve the concentration of edible mushroom canning processing pre-boiled liquid, roxburgh anoectochilus terminal bud stem section germinative number
Reduce on the contrary.
Knowable to the roxburgh anoectochilus terminal bud stem section germination data that A16-A20 culture mediums are cultivated, after MS minimal medium mother liquors are added,
Through can be good at meeting the needs of germination of roxburgh anoectochilus terminal bud stem section is to nutrition, therefore the concentration of edible mushroom canning processing pre-boiled liquid only needs
Reach 250 μ L/L, just can be good at promoting the germination of roxburgh anoectochilus terminal bud stem section, but germinative number of the germinative number now not as good as A12.
It can thus be appreciated that the best results of A12 culture mediums, are best able to promote the germination of roxburgh anoectochilus terminal bud stem section.
Roxburgh anoectochilus terminal bud stem section is also seeded on B1-B20 and C1-C20 tissue culture roxburgh anoectochilus terminal bud culture mediums by inventor, obtained reality
Being substantially the same for data and table 1 is tested, but utilizes the roxburgh anoectochilus terminal bud stem of B1-B20 and C1-C20 tissue culture roxburgh anoectochilus terminal bud medium cultures
The roxburgh anoectochilus terminal bud stem section that the germinative number of section is still cultivated not as good as A12 culture mediums, it can thus be appreciated that sucrose, banana, agar and activated carbon
The influence germinateed to roxburgh anoectochilus terminal bud stem section of concentration it is little.
2nd, influence of the different culture media to root growth
Selection is grown fine and growing way is tried one's best close anoectochilus blume seedling, under super-clean bench gnotobasis, by under the base portion of seedling
End (about 2cm length) is cut, then is transferred on A11-A15 culture mediums, and regulation PH is 5.8.Experiment sets 5 processing altogether, each processing
4 bottles of inoculation, every bottle is inoculated with 6 plants.The blake bottle connected is placed in thermostatic chamber, keeps 25 ± 1 DEG C, illumination 10h/d of room temperature, light
According to intensity 1800Lux.Root of hair number and root growth situation are counted after cultivating 60d.It the results are shown in Table 2
Influence of the different culture media of table 2 to taking root
From the data of table 2, roxburgh anoectochilus terminal bud can be promoted to take root when the moderate concentration of edible mushroom canning processing pre-boiled liquid,
And it is most to be seeded in the tissue-cultured seedling root of hair bar number on A12 culture mediums, sum is 98, and rooting rate has reached 4.08, and experiment is also sent out
Now when edible mushroom canning processing pre-boiled liquid concentration is low, the root system of anoectochilus blume seedling is elongated, and root hair is more, and root color is milky.
When edible mushroom canning processing pre-boiled liquid concentration reaches more than 1500 μ L/L, root system becomes sturdy, and root length elongation is slow, and root hair subtracts
It is few, it is the obvious aging of yellow green with color, in addition, the easily downright bad growth for influenceing root of the roxburgh anoectochilus terminal bud tip of a root.
3rd, influence of the different culture media to roxburgh anoectochilus terminal bud growth of seedling
The Taiwan kind roxburgh anoectochilus terminal bud seedling that selection is grown fine and growing way is tried one's best close, will be small under super-clean bench gnotobasis
Seedling is inoculated on A11-A15, B11-B15, C11-C15 culture medium, and regulation PH is 5.8.Experiment sets 15 processing altogether, each processing
4 bottles of inoculation, every bottle is inoculated with 6 plants (the seedling growing way of same bottle inoculation is as far as possible close).The blake bottle connected is placed on thermostatic chamber
It is interior, keep 25 ± 1 DEG C, 10~12h/d of illumination, 1500~2000Lux of intensity of illumination of room temperature.Count being averaged after growing 3 months
Plant height, average individual plant increase the number of blade and average single-strain fresh weight newly.
In addition, the Fujian kind roxburgh anoectochilus terminal bud seedling that selection is grown fine and growing way is tried one's best close, under super-clean bench gnotobasis,
Seedling is inoculated on A11-A15, B11-B15, C11-C15 culture medium, regulation PH is 5.8.Experiment sets 15 processing altogether, each
4 bottles of processing inoculation, every bottle is inoculated with 6 plants (the seedling growing way of same bottle inoculation is as far as possible close).The blake bottle connected is placed on perseverance
In greenhouse, 25 ± 1 DEG C, 10~12h/d of illumination, 1500~2000Lux of intensity of illumination of room temperature is kept.After statistics growth 3 months
Average plant height, average individual plant increase the number of blade and average single-strain fresh weight newly.Experimental result is shown in Table 3：
Influence of the different culture media of table 3 to roxburgh anoectochilus terminal bud growth of seedling
The data of A12-A15 culture mediums and data, the data and B11 of B12-B15 culture mediums of A11 culture mediums from table 3
The data of culture medium and the data of C12-C15 culture mediums are understood compared with the data of C11 culture mediums, added with debita spissitudo
The culture medium of edible mushroom canning processing pre-boiled liquid (is not added with the culture medium of edible mushroom canning processing pre-boiled liquid such as with common culture medium
A11, B11, C11) compare, it can more promote the growth of roxburgh anoectochilus terminal bud added with the culture medium of edible mushroom canning processing pre-boiled liquid, can be obvious
Shorten tissue culture roxburgh anoectochilus terminal bud growth cycle, and the best results of A12 culture mediums, be best medium.Other A11-A15 culture mediums
Data are knowable to compared with the data of B11-B15 culture mediums, the data of C11-C15 culture mediums, sucrose, banana, agar and activity
Growth of the concentration of charcoal to roxburgh anoectochilus terminal bud has a certain impact, but influences little.In addition, it is observed that added with the edible of debita spissitudo
The roxburgh anoectochilus terminal bud that the culture medium of bacterium canning processing pre-boiled liquid is cultivated is more healthy and strong compared to the roxburgh anoectochilus terminal bud plant that ordinary culture medium is cultivated,
Blade is unfolded, is abundant, and color is more bright-coloured.
In order to verify the culture medium of the present invention if appropriate for the culture for other plant, inventor is also by the 60 of the present invention
Group (A1~A20；B1~B20；C1~C20) cultivation of the culture medium for plants such as radish, cauliflower, tomato, blueberries, find
Radish, cauliflower, tomato and conventional medium (i.e. the culture medium without pre-boiled liquid) contrast, growing way difference unobvious, blueberry with
Conventional medium contrasts, and growing way indifference, the use for illustrating culture medium of the present invention is targetedly, to be not necessarily suitable for owning
The component proportion relation of the cultivation of plant or above-mentioned culture medium is not appropriate for the cultivation of other plant.
Claims (6)
1. a kind of tissue culture roxburgh anoectochilus terminal bud culture medium, including banana, sucrose, agar and activated carbon, it is characterised in that：It also includes 0*
MS~1*MS culture medium mother liquors and edible mushroom canning processing pre-boiled liquid；Tissue culture roxburgh anoectochilus terminal bud culture medium described in per 1000ml includes
0*MS~1*MS minimal mediums mother liquor, 10~1000 μ L edible mushrooms cannings processing pre-boiled liquid, 10~50g sucrose, 50~250g
Banana, 6g~8g agar, 1.0g~2.0g activated carbons, surplus are distilled water, and wherein 0*MS represents to be added without minimal medium mother
Liquid.
2. the preparation of tissue culture roxburgh anoectochilus terminal bud culture medium according to claim 1, it is characterised in that：Every 1000 milliliters of organic training
It is as follows to support base preparation process：
(1) edible mushroom canning processing pre-boiled liquid is prepared, and the edible mushroom canning processing pre-boiled liquid for measuring 10~1000 μ L is stand-by；
(2) according to the volume for preparing the various mother liquors needed for 0*MS~1*MS minimal mediums, MS a great number of elements mother liquor, MS are measured
Micro- mother liquor, MS mother liquid of iron salt and MS organic matter mother liquors are stand-by；
(3) the edible mushroom canning measured in step (1) is processed into pre-boiled liquid to carry out with the MS culture medium mother liquors measured in step (2)
It is dissolved in after mixing in distilled water；
(4) 50~250g bananas and 1.0g~2.0g activated carbons are weighed, and banana is ground into after mud and adds gained in step (3)
Solution in, stir and evenly mix, add the activated carbon weighed up afterwards, stir；
(5) 200~300mL distilled water is added in beaker, distilled water is heated to adding after 80~90 DEG C the 6g that weighs up~
8g agar, agar dissolves after being heated to boiling while stirring, after treating agar dissolving, continuously adds the 10~50g sucrose weighed up,
Sucrose dissolving is heated to while stirring, and the mixed solution of obtained agar and sucrose is then added into the mixing obtained by step (4)
In solution, after stirring plus distilled water is settled to 1000 milliliters and adds acid-base modifier and pH value is adjusted into 5.0-6.0；
(6) culture medium obtained by step (5) is sub-packed in tissue culture bottle, carries out autoclave sterilization, cooled and solidified is afterwards
Can.
3. the preparation of tissue culture roxburgh anoectochilus terminal bud culture medium according to claim 2, it is characterised in that：The edible mushroom canning processing
The preparation method of pre-boiled liquid is as follows：
A. caused industrial wastewater in edible mushroom canning process is collected；
B. caused industrial wastewater in next edible mushroom canning process will be collected to be centrifuged, the rotating speed control of centrifuge
Between 11500-12500rpm, centrifuging temperature is 15-25 DEG C, centrifugation time 10-20min, and supernatant is taken after centrifugation；
C. the supernatant of gained is utilized into falling film evaporator, falling film evaporation concentration, obtains concentrate；
D. denaturant is added in concentrate, concentrate is denatured, wherein described denaturant is 80~100% second
Alcohol, and the ethanol and the volume ratio of concentrate added is 2:1~6:1；
E. the concentrate after denaturation is filtered, collects filtrate；
F. filtrate is subjected to rotary evaporation and removes ethanol；
G. the solution of gained after rotary evaporation is subjected to tangential flow filtration using 5-10K filter membrane, collects filter liquor；
H. the filter liquor of gained in step g is subjected to rotary evaporation concentration, is concentrated into solution index of refraction and reaches 35~55%.
4. the preparation of organic culture medium of tissue culture roxburgh anoectochilus terminal bud according to claim 2, it is characterised in that：Institute in step (5)
The acid-base modifier stated is 0.1-0.5mol/L NaOH solution or 0.1-0.5mol/L HCl solution.
5. the preparation of tissue culture roxburgh anoectochilus terminal bud culture medium according to claim 2, it is characterised in that：Step (6) high temperature high pressure
The temperature control of sterilizing is at 121-125 DEG C, and for Stress control at 111-115 kPas, sterilization time is 15-20 minutes.
6. the application of tissue culture roxburgh anoectochilus terminal bud culture medium according to claim 1, it is characterised in that：For cultivating roxburgh anoectochilus terminal bud.
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