CN109197586A - A kind of dendrobium candidum Protocorm base and its application - Google Patents
A kind of dendrobium candidum Protocorm base and its application Download PDFInfo
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- CN109197586A CN109197586A CN201810880368.XA CN201810880368A CN109197586A CN 109197586 A CN109197586 A CN 109197586A CN 201810880368 A CN201810880368 A CN 201810880368A CN 109197586 A CN109197586 A CN 109197586A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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Abstract
The invention discloses a kind of dendrobium candidum Protocorm base, including induced medium, proliferation and differential medium；The induced medium is composed of the following components: MS minimal medium, 6- benzyl 1.6~2.5mg/L of aminoadenine, 0.6~0.8mg/L of methyl α-naphthyl acetate, 0.5~1.5umol/L of PQQ, 16~28mg/L of botanical extract mixture, 80~120mg/L of sucrose, 5~8mg/L of culture medium functional liquid；The proliferation and differential medium are composed of the following components: 1/2MS minimal medium, 6- benzyl 1.6~2.5mg/L of aminoadenine, 0.6~0.8mg/L of methyl α-naphthyl acetate, 0.5~1.5umol/L of PQQ, 16~28mg/L of botanical extract mixture, 40~60mg/L of sucrose, 0.2 ~ 0.3 mg/L of vitamin B2；5~8mg/L of culture medium functional liquid.Botanical extract mixture includes mulberry-leaf extract, Baical Skullcap root P.E and tannin and special ratios in culture medium provided by the invention, it can adapt to the intensity of illumination during Proliferation, Differentiation, to which planting percent is high, after transplantation of seedlings, sunlight is adaptable, the intensity of illumination for completing adaptation In Nanjing dog days, not will cause phenomena such as burning.
Description
Technical field
The invention belongs to dendrobium nobile culture fields, and in particular to a kind of Proliferation, Differentiation culture medium of dendrobium candidum protocorm and its
Using.
Background technique
Dendrobium candidum (Deudrobium officiuale) is the perennial herbaceous plant that grows nonparasitically upon another plant of orchid family Dendrobium, is China
Distinctive rare medicinal herbs, Li Shizhen (1518-1593 A.D.) evaluates dendrobium candidum in Compendium of Material Medica, and " strong yin strengthening the essence, thick stomach, benefit is interior never sufficient, puts down
Stomach Qi, long muscle, intelligence development is except frightened, macrobiosis of making light of one's life by commiting suicide "；It is civil to be called " help mesona ".It is distributed in the mountainous region of the nearly km of height above sea level more
On half dark and damp rock, in addition long-term excavation, so that dendrobium officinale resource is increasingly exhausted.Because its seed is without endosperm, need to combine
Fungal component could normally sprout, therefore the cultivation difficulty of seedling is big.Traditional vegetative manner breeding coefficient is low, protocorm
By induction, differentiation, proliferation three phases, the stem section thickness that the quality of the culture of protocorm directly affects dendrobium candidum is more for culture
Less, rooting rate and planting percent.
The culture of protocorm needs to carry out common film solid media, will cause stem's damage, and seedling pair when taking stem
What is generated after the drawing of nutrient solution is unevenly distributed；Fluid nutrient medium will cause anoxic；In addition, common ball stem Proliferation, Differentiation
Poor at commonly occurring resistance after transplantation of seedlings afterwards, sunlight adaptability difference, that is, sunlight easily causes burn by force, and sunlight is few easily to breed very
Bacterium.
Summary of the invention
Goal of the invention: in order to overcome the deficiencies in the prior art, the present invention provides a kind of dendrobium candidum protocorms
Culture medium.
Technical solution: in order to solve the above technical problems, the present invention provides a kind of dendrobium candidum Protocorm base, feature
It is: including induced medium, proliferation and differential medium；
The induced medium is composed of the following components: MS minimal medium, 6- benzyl 1.6~2.5mg/L of aminoadenine,
Methyl α-naphthyl acetate 0.6~0.8mg/L, PQQ 0.5~1.5umol/L, 16~28mg/L of botanical extract mixture, sucrose 80~
120 mg/L, 5~8mg/L of culture medium functional liquid；
The proliferation and differential medium are composed of the following components: 1/2MS minimal medium, and 6- benzyl aminoadenine 1.6~
2.5mg/L, methyl α-naphthyl acetate 0.6~0.8mg/L, PQQ 0.5~1.5umol/L, 16~28mg/L of botanical extract mixture,
40~60 mg/L of sucrose, 0.2 ~ 0.3 mg/L of vitamin B2；5~8mg/L of culture medium functional liquid；
The MS minimal medium is composed of the following components:
Ammonium nitrate NH4NO3 600 mg/L
Calcium nitrate Ca (NO3)2·4H2O 656 mg/L
Calcium chloride CaCl2·2H2O 196 mg/L
Magnesium sulfate MgSO4·7H2O 370 mg/L
Potassium dihydrogen phosphate KH2PO4 170 mg/L
Potassium sulfate K2SO4 990 mg/L
Disodium ethylene diamine tetraacetate Na2·EDTA 37.3 mg/L
Ferrous sulfate FeSO4·7H2O 27.8 mg/L
Manganese sulfate MnSO4·H2O 22.3 mg/L
Zinc sulfate ZnSO4·7H2O 8.6 mg/L
Boric acid H3BO3 8.2 mg/L
Sodium molybdate Na2MoO4·2H2O 0.25 mg/L
Copper sulphate CuSO4·5H2O 0.05mg/L
Cobalt chloride CoCl2.6 H2O 2 0.05 mg/L
Inositol C6H12O6·2H2O 500 mg/L
Glycine NH2·CH2·COOH 5mg/L
Thiamine hydrochloride C12H17ClOS·2HCl 2 mg/L
Puridoxine hydrochloride C8H10NO5P 0.8 mg/L
Niacin NC5H4COOH 0.8mg/L；
The 1/2MS minimal medium is composed of the following components:
Ammonium nitrate NH4NO3 300 mg/L
Calcium nitrate Ca (NO3)2·4H2O 328 mg/L
Calcium chloride CaCl2·2H2O 98mg/L
Magnesium sulfate MgSO4·7H2O 185 mg/L
Potassium dihydrogen phosphate KH2PO4 85mg/L
Potassium sulfate K2SO4 495 mg/L
Disodium ethylene diamine tetraacetate Na2·EDTA 37.3 mg/L
Ferrous sulfate FeSO4·7H2O 27.8 mg/L
Manganese sulfate MnSO4·H2O 22.3 mg/L
Zinc sulfate ZnSO4·7H2O 8.6 mg/L
Boric acid H3BO3 8.2 mg/L
Sodium molybdate Na2MoO4·2H2O 0.25 mg/L
Copper sulphate CuSO4·5H2O 0.05mg/L
Cobalt chloride CoCl2.6 H2O 2 0.05 mg/L
Inositol C6H12O6·2H2O 500 mg/L
Glycine NH2·CH2·COOH 5mg/L
Thiamine hydrochloride C12H17ClOS·2HCl 2 mg/L
Puridoxine hydrochloride C8H10NO5P 0.8 mg/L
Niacin NC5H4COOH 0.8mg/L
Further, the plant extraction liquid is made of mulberry-leaf extract, Baical Skullcap root P.E and tannin；Mulberry-leaf extract
The mulberry leaf powder of the 1st~3 young leaves processing on mulberry branch is raw material, dries in the shade, crushes, uses n-butanol, 90% second alcohol and water respectively
Heating extraction, and be spray-dried and obtain；Flaxen powder made from the extracted drying of Baical Skullcap root P.E radix scutellariae.
Further, the weight part ratio between the mulberry-leaf extract, Baical Skullcap root P.E and tannin three is successively distinguished
For 5:4:1.
Further, the culture medium functional liquid is made of aloe gel, saponin and palmitinic acid.
Further, the weight ratio of the aloe gel, saponin and palmitinic acid respectively is 6:4:1.
Further, the culture medium applying step is as follows:
1) tender stem segments that long 2~5cm is cut from dendrobium candidum plant, rinse under flowing water, dip in a little washing powder with brush
Aqueous solution is gently scrubbed, and is sufficiently cleaned through tap water.Stem section is placed in 75% alcohol on superclean bench and is sterilized
After 30s, then with 0.1% HgCl2 8~10min of aqueous solution soaking, aseptic water washing 5~6 times；
2) Fiber differentiation: the tender stem segments of dendrobium candidum are cut with scalpel, is seeded in induced medium and is cultivated；15
After~20 days, explant starts to sprout, and stem section is expanded, and section and top organize the formation of open texture；After continuing culture 20 days, turn
In a subtle way in holding chamber, the surface that tissue is expanded at section and top forms rough particulate matter, with the extension of incubation time,
Particulate matter grows up to form the protocorms particulate matter of 1~2mm of diameter；
3) Proliferation, Differentiation culture: the protocorm of induced synthesis is transferred in Proliferation, Differentiation culture medium, and Protocorm Multiplication speed is very
Fastly, proliferation times are up to 8~10 times, while part protocorm starts to differentiate bud point and grows 1~2 leaflet, carry out certain
Condition illumination；The proliferation of protocorm carries out simultaneously with bud differentiation.
Further, the illumination condition is that light quality is feux rouges or red, blue mixed light.
Further, the illumination condition are as follows: light intensity is 90-100 μm of olm-2s-1,9 hour/day of light application time.
The utility model has the advantages that the present invention has following advantages in terms of existing technologies:
1) culture medium functional liquid includes aloe gel, saponin and palmitinic acid and passes through specific in culture medium provided by the invention
Ratio so that culture medium in liquid and it is fixed between, culture medium is in suspended state, is in microfluidic state between liquid, so that training
Support base in component between uniformly, will not because dendrobium nobile stem eye develop due to caused by component be unevenly distributed；And when finally at transplantation of seedlings,
It is easier to extract, is not easy to hurt stem root.
2) botanical extract mixture includes mulberry-leaf extract, Baical Skullcap root P.E and tannin in culture medium provided by the invention
And special ratios, it can adapt to the intensity of illumination during Proliferation, Differentiation, so that planting percent is high, after transplantation of seedlings, sunlight is adapted to
Ability is strong, completes the intensity of illumination for adapting to In Nanjing dog days, not will cause phenomena such as burning.
3) the improved 1/2MS minimal medium of the present invention and MS minimal medium, cooperate with culture medium other components
Effect, can increase growth coefficient, for proliferation times up to 8~10 times, cultivation cycle is short significantly.
Specific embodiment
The configuration consistency of minimal medium in the present embodiment, specific as follows:
In MS minimal medium and 1/2MS minimal medium: including following composition:
The component of macroelement is as follows: ammonium nitrate (NH4NO3), calcium nitrate (Ca (NO3)2·4H2O), calcium chloride (CaCl2·
2H2O), magnesium sulfate (MgSO4·7H2O), potassium dihydrogen phosphate (KH2PO4), potassium sulfate (K2SO4)；
The component of microelement is corresponding with its as follows using concentration: manganese sulfate (MnSO4·H2O), zinc sulfate (ZnSO4·
7H2O), boric acid (H3BO3), sodium molybdate (Na2MoO4·2H2O), copper sulphate (CuSO4·5H2O), cobalt chloride (CoCl2.62 )；
The component of molysite is as follows: disodium ethylene diamine tetraacetate (Na2EDTA), ferrous sulfate (FeSO4·7H2O)；
The component of organic principle is as follows: inositol, glycine (Gly), thiamine hydrochloride (VB1), puridoxine hydrochloride (VB6), niacin
(VPP).
MS minimal medium is composed of the following components:
Ammonium nitrate NH4NO3 600 mg/L
Calcium nitrate Ca (NO3)2·4H2O 656 mg/L
Calcium chloride CaCl2·2H2O 196 mg/L
Magnesium sulfate MgSO4·7H2O 370 mg/L
Potassium dihydrogen phosphate KH2PO4 170 mg/L
Potassium sulfate K2SO4 990 mg/L
Disodium ethylene diamine tetraacetate Na2·EDTA 37.3 mg/L
Ferrous sulfate FeSO4·7H2O 27.8 mg/L
Manganese sulfate MnSO4·H2O 22.3 mg/L
Zinc sulfate ZnSO4·7H2O 8.6 mg/L
Boric acid H3BO3 8.2 mg/L
Sodium molybdate Na2MoO4·2H2O 0.25 mg/L
Copper sulphate CuSO4·5H2O 0.05mg/L
Cobalt chloride CoCl2.6 H2O 2 0.05 mg/L
Inositol C6H12O6·2H2O 500 mg/L
Glycine NH2·CH2·COOH 5mg/L
Thiamine hydrochloride C12H17ClOS·2HCl 2 mg/L
Puridoxine hydrochloride C8H10NO5P 0.8 mg/L
Niacin NC5H4COOH 0.8mg/L；
1/2MS minimal medium is composed of the following components:
Ammonium nitrate NH4NO3 300 mg/L
Calcium nitrate Ca (NO3)2·4H2O 328 mg/L
Calcium chloride CaCl2·2H2O 98mg/L
Magnesium sulfate MgSO4·7H2O 185 mg/L
Potassium dihydrogen phosphate KH2PO4 85mg/L
Potassium sulfate K2SO4 495 mg/L
Disodium ethylene diamine tetraacetate Na2·EDTA 37.3 mg/L
Ferrous sulfate FeSO4·7H2O 27.8 mg/L
Manganese sulfate MnSO4·H2O 22.3 mg/L
Zinc sulfate ZnSO4·7H2O 8.6 mg/L
Boric acid H3BO3 8.2 mg/L
Sodium molybdate Na2MoO4·2H2O 0.25 mg/L
Copper sulphate CuSO4·5H2O 0.05mg/L
Cobalt chloride CoCl2.6 H2O 2 0.05 mg/L
Inositol C6H12O6·2H2O 500 mg/L
Glycine NH2·CH2·COOH 5mg/L
Thiamine hydrochloride C12H17ClOS·2HCl 2 mg/L
Puridoxine hydrochloride C8H10NO5P 0.8 mg/L
Niacin NC5H4COOH 0.8mg/L
Embodiment 1:
A kind of dendrobium candidum Protocorm base, including induced medium, proliferation and differential medium；
The induced medium is composed of the following components: MS minimal medium, 1.6 mg/L of 6- benzyl aminoadenine, methyl α-naphthyl acetate
0.8mg/L, PQQ 1.5umol/L, botanical extract mixture 28mg/L, 120 mg/L of sucrose, culture medium functional liquid 8mg/L；
The proliferation and differential medium are composed of the following components: 1/2MS minimal medium, 6- benzyl aminoadenine 2.5mg/
L, methyl α-naphthyl acetate 0.6 mg/L, PQQ 0.5umol/L, 16 mg/L of botanical extract mixture, 40 mg/L of sucrose, vitamin B2 0.2
mg/L；Culture medium functional liquid 5mg/L；
Wherein, plant extraction liquid is made of mulberry-leaf extract, Baical Skullcap root P.E and tannin；On mulberry-leaf extract mulberry branch
The mulberry leaf powder of 1st~3 young leaves processing is raw material, is dried in the shade, and is crushed, and heats extraction with n-butanol, 90% second alcohol and water respectively, and
It is spray-dried and obtains；Flaxen powder made from the extracted drying of Baical Skullcap root P.E radix scutellariae.The mulberry-leaf extract, radix scutellariae
Weight part ratio between extract and tannin three respectively is 5:4:1.The culture medium functional liquid by aloe gel,
Saponin and palmitinic acid composition.The weight ratio of the aloe gel, saponin and palmitinic acid respectively is 6:4:1.
Above-mentioned culture medium applying step is as follows:
1) tender stem segments that long 2~5cm is cut from dendrobium candidum plant, rinse under flowing water, dip in a little washing powder with brush
Aqueous solution is gently scrubbed, and is sufficiently cleaned through tap water.Stem section is placed in 75% alcohol on superclean bench and is sterilized
After 30s, then with 0.1% HgCl2 8~10min of aqueous solution soaking, aseptic water washing 5~6 times；
2) Fiber differentiation: the tender stem segments of dendrobium candidum are cut with scalpel, is seeded in induced medium and is cultivated；15
After~20 days, explant starts to sprout, and stem section is expanded, and section and top organize the formation of open texture；After continuing culture 20 days, turn
In a subtle way in holding chamber, the surface that tissue is expanded at section and top forms rough particulate matter, with the extension of incubation time,
Particulate matter grows up to form the protocorms particulate matter of 1~2mm of diameter；
3) Proliferation, Differentiation culture: the protocorm of induced synthesis is transferred in Proliferation, Differentiation culture medium, and Protocorm Multiplication speed is very
Fastly, while part protocorm starts to differentiate bud point and grows 1~2 leaflet, carries out certain condition illumination；The proliferation of protocorm
It is carried out simultaneously with bud differentiation.The illumination condition is that light quality is red, blue mixed light.The illumination condition are as follows: light intensity is 90 μ
Molm-2s-1,9 hour/day of light application time.
Embodiment 2:
A kind of dendrobium candidum Protocorm base, it is characterised in that: including induced medium, proliferation and differential medium；
The induced medium is composed of the following components: MS minimal medium, 6- benzyl aminoadenine 2.5mg/L, methyl α-naphthyl acetate
0.6 mg/L, PQQ 0.5 umol/L, botanical extract mixture 16mg/L, sucrose 80mg/L, 5 mg/L of culture medium functional liquid；
The proliferation and differential medium are composed of the following components: 1/2MS minimal medium, 1.6 mg/ of 6- benzyl aminoadenine
L, methyl α-naphthyl acetate 0.8mg/L, PQQ 1.5umol/L, botanical extract mixture 28mg/L, 60 mg/L of sucrose, vitamin B2 0.3
mg/L；Culture medium functional liquid 8mg/L；
Wherein, plant extraction liquid is made of mulberry-leaf extract, Baical Skullcap root P.E and tannin；On mulberry-leaf extract mulberry branch
The mulberry leaf powder of 1st~3 young leaves processing is raw material, is dried in the shade, and is crushed, and heats extraction with n-butanol, 90% second alcohol and water respectively, and
It is spray-dried and obtains；Flaxen powder made from the extracted drying of Baical Skullcap root P.E radix scutellariae.The mulberry-leaf extract, radix scutellariae
Weight part ratio between extract and tannin three respectively is 5:4:1.The culture medium functional liquid by aloe gel,
Saponin and palmitinic acid composition.The weight ratio of the aloe gel, saponin and palmitinic acid respectively is 6:4:1.
Above-mentioned culture medium applying step is as follows:
1) tender stem segments that long 2~5cm is cut from dendrobium candidum plant, rinse under flowing water, dip in a little washing powder with brush
Aqueous solution is gently scrubbed, and is sufficiently cleaned through tap water.Stem section is placed in 75% alcohol on superclean bench and is sterilized
After 30s, then with 0.1% HgCl2 8~10min of aqueous solution soaking, aseptic water washing 5~6 times；
2) Fiber differentiation: the tender stem segments of dendrobium candidum are cut with scalpel, is seeded in induced medium and is cultivated；15
After~20 days, explant starts to sprout, and stem section is expanded, and section and top organize the formation of open texture；After continuing culture 20 days, turn
In a subtle way in holding chamber, the surface that tissue is expanded at section and top forms rough particulate matter, with the extension of incubation time,
Particulate matter grows up to form the protocorms particulate matter of 1~2mm of diameter；
3) Proliferation, Differentiation culture: the protocorm of induced synthesis is transferred in Proliferation, Differentiation culture medium, and Protocorm Multiplication speed is very
Fastly, while part protocorm starts to differentiate bud point and grows 1~2 leaflet, carries out certain condition illumination；The proliferation of protocorm
It is carried out simultaneously with bud differentiation.The illumination condition is, light quality is feux rouges or red, blue mixed light, the illumination condition are as follows: light intensity is
95 μm of olm-2s-1,9 hour/day of light application time.
Embodiment 3:
A kind of dendrobium candidum Protocorm base, including induced medium, proliferation and differential medium；
The induced medium is composed of the following components: MS minimal medium, methyl α-naphthyl acetate 0.7mg/L, PQQ 0.8umol/L plants
Object extraction mixture 23mg/L, 110 mg/L of sucrose, culture medium functional liquid 6mg/L；
The proliferation and differential medium are composed of the following components: 1/2MS minimal medium, 6- benzyl aminoadenine 1.8mg/
L, methyl α-naphthyl acetate 0.7mg/L, PQQ 0.7umol/L, botanical extract mixture 22mg/L, 50 mg/L of sucrose, vitamin B2 0.25
mg/L；Culture medium functional liquid 6mg/L；
Wherein, plant extraction liquid is made of mulberry-leaf extract, Baical Skullcap root P.E and tannin；On mulberry-leaf extract mulberry branch
The mulberry leaf powder of 1st~3 young leaves processing is raw material, is dried in the shade, and is crushed, and heats extraction with n-butanol, 90% second alcohol and water respectively, and
It is spray-dried and obtains；Flaxen powder made from the extracted drying of Baical Skullcap root P.E radix scutellariae.The mulberry-leaf extract, radix scutellariae
Weight part ratio between extract and tannin three respectively is 5:4:1.The culture medium functional liquid by aloe gel,
Saponin and palmitinic acid composition.The weight ratio of the aloe gel, saponin and palmitinic acid respectively is 6:4:1.
Above-mentioned culture medium applying step is as follows:
1) tender stem segments that long 2~5cm is cut from dendrobium candidum plant, rinse under flowing water, dip in a little washing powder with brush
Aqueous solution is gently scrubbed, and is sufficiently cleaned through tap water.Stem section is placed in 75% alcohol on superclean bench and is sterilized
After 30s, then with 0.1% HgCl2 8~10min of aqueous solution soaking, aseptic water washing 5~6 times；
2) Fiber differentiation: the tender stem segments of dendrobium candidum are cut with scalpel, is seeded in induced medium and is cultivated；15
After~20 days, explant starts to sprout, and stem section is expanded, and section and top organize the formation of open texture；After continuing culture 20 days, turn
In a subtle way in holding chamber, the surface that tissue is expanded at section and top forms rough particulate matter, with the extension of incubation time,
Particulate matter grows up to form the protocorms particulate matter of 1~2mm of diameter；
3) Proliferation, Differentiation culture: the protocorm of induced synthesis is transferred in Proliferation, Differentiation culture medium, and Protocorm Multiplication speed is very
Fastly, while part protocorm starts to differentiate bud point and grows 1~2 leaflet, carries out certain condition illumination；The proliferation of protocorm
It is carried out simultaneously with bud differentiation.The illumination condition is that light quality is feux rouges or red, blue mixed light.The illumination condition are as follows: light intensity is
100 μm of olm-2s-1,9 hour/day of light application time.
Embodiment 4:
A kind of dendrobium candidum Protocorm base, including induced medium, proliferation and differential medium；
The induced medium is composed of the following components: MS minimal medium, 6- benzyl aminoadenine 2.3mg/L, methyl α-naphthyl acetate
0.7mg/L, PQQ 1.3umol/L, botanical extract mixture 26mg/L, 115 mg/L of sucrose, culture medium function liquid 6mg/L；
The proliferation and differential medium are composed of the following components: 1/2MS minimal medium, 6- benzyl aminoadenine 1.9mg/
L, methyl α-naphthyl acetate 0.7mg/L, PQQ 0.7umol/L, botanical extract mixture 26mg/L, 55 mg/L of sucrose, vitamin B2 0.26
mg/L；Culture medium functional liquid 6mg/L；
Wherein, plant extraction liquid is made of mulberry-leaf extract, Baical Skullcap root P.E and tannin；On mulberry-leaf extract mulberry branch
The mulberry leaf powder of 1st~3 young leaves processing is raw material, is dried in the shade, and is crushed, and heats extraction with n-butanol, 90% second alcohol and water respectively, and
It is spray-dried and obtains；Flaxen powder made from the extracted drying of Baical Skullcap root P.E radix scutellariae.The mulberry-leaf extract, radix scutellariae
Weight part ratio between extract and tannin three respectively is 5:4:1.The culture medium functional liquid by aloe gel,
Saponin and palmitinic acid composition.The weight ratio of the aloe gel, saponin and palmitinic acid respectively is 6:4:1.
Above-mentioned culture medium applying step is as follows:
1) tender stem segments that long 2~5cm is cut from dendrobium candidum plant, rinse under flowing water, dip in a little washing powder with brush
Aqueous solution is gently scrubbed, and is sufficiently cleaned through tap water.Stem section is placed in 75% alcohol on superclean bench and is sterilized
After 30s, then with 0.1% HgCl2 8~10min of aqueous solution soaking, aseptic water washing 5~6 times；
2) Fiber differentiation: the tender stem segments of dendrobium candidum are cut with scalpel, is seeded in induced medium and is cultivated；15
After~20 days, explant starts to sprout, and stem section is expanded, and section and top organize the formation of open texture；After continuing culture 20 days, turn
In a subtle way in holding chamber, the surface that tissue is expanded at section and top forms rough particulate matter, with the extension of incubation time,
Particulate matter grows up to form the protocorms particulate matter of 1~2mm of diameter；
3) Proliferation, Differentiation culture: the protocorm of induced synthesis is transferred in Proliferation, Differentiation culture medium, and Protocorm Multiplication speed is very
Fastly, while part protocorm starts to differentiate bud point and grows 1~2 leaflet, carries out certain condition illumination；The proliferation of protocorm
It is carried out simultaneously with bud differentiation.The illumination condition is that light quality is feux rouges or red, blue mixed light.The illumination condition are as follows: light intensity is
100 μm of olm-2s-1,9 hour/day of light application time.
Comparative example:
1, induced medium :+0.8% agar of+3% sucrose of MS+2.0mg/L 6- benzyl aminoadenine；
2, immortalizing point of culture medium :+0.8% agar of+3% sucrose of MS+20% potato；
Other condition of culture are identical.
The present embodiment 1 ~ 3 passes through nearly ten thousand plants of dendrobium nobile culture experiments, and acquisition data are as follows:
| Embodiment | It burns rate | Average coefficient of proliferation |
| Embodiment 1 | 0.03% | 8 |
| Embodiment 2 | 0.02% | 8 |
| Embodiment 3 | 0.01% | 9 |
| Embodiment 4 | 0.01% | 9 |
| Comparative example | 15% | 4 |
The above is only a preferred embodiment of the present invention, it should be pointed out that: for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of dendrobium candidum Protocorm base, it is characterised in that: including induced medium, proliferation and differential medium；
The induced medium is composed of the following components: MS minimal medium, 6- benzyl 1.6~2.5mg/L of aminoadenine,
Methyl α-naphthyl acetate 0.6~0.8mg/L, PQQ 0.5~1.5umol/L, 16~28mg/L of botanical extract mixture, sucrose 80~
120 mg/L, 5~8mg/L of culture medium functional liquid；
The proliferation and differential medium are composed of the following components: 1/2MS minimal medium, and 6- benzyl aminoadenine 1.6~
2.5mg/L, methyl α-naphthyl acetate 0.6~0.8mg/L, PQQ 0.5~1.5umol/L, 16~28mg/L of botanical extract mixture,
40~60 mg/L of sucrose, 0.2 ~ 0.3 mg/L of vitamin B2；5~8mg/L of culture medium functional liquid.
2. dendrobium candidum Protocorm base according to claim 1, it is characterised in that: the MS minimal medium is by following
Group is grouped as:
Ammonium nitrate NH4NO3 600 mg/L
Calcium nitrate Ca (NO3)2·4H2O 656 mg/L
Calcium chloride CaCl2·2H2O 196 mg/L
Magnesium sulfate MgSO4·7H2O 370 mg/L
Potassium dihydrogen phosphate KH2PO4 170 mg/L
Potassium sulfate K2SO4 990 mg/L
Disodium ethylene diamine tetraacetate Na2·EDTA 37.3 mg/L
Ferrous sulfate FeSO4·7H2O 27.8 mg/L
Manganese sulfate MnSO4·H2O 22.3 mg/L
Zinc sulfate ZnSO4·7H2O 8.6 mg/L
Boric acid H3BO3 8.2 mg/L
Sodium molybdate Na2MoO4·2H2O 0.25 mg/L
Copper sulphate CuSO4·5H2O 0.05mg/L
Cobalt chloride CoCl2.6 H2O 2 0.05 mg/L
Inositol C6H12O6·2H2O 500 mg/L
Glycine NH2·CH2·COOH 5mg/L
Thiamine hydrochloride C12H17ClOS·2HCl 2 mg/L
Puridoxine hydrochloride C8H10NO5P 0.8 mg/L
Niacin NC5H4COOH 0.8mg/L。
3. dendrobium candidum Protocorm base according to claim 1, it is characterised in that: the 1/2MS minimal medium by
Following components composition:
Ammonium nitrate NH4NO3 300 mg/L
Calcium nitrate Ca (NO3)2·4H2O 328 mg/L
Calcium chloride CaCl2·2H2O 98mg/L
Magnesium sulfate MgSO4·7H2O 185 mg/L
Potassium dihydrogen phosphate KH2PO4 85mg/L
Potassium sulfate K2SO4 495 mg/L
Disodium ethylene diamine tetraacetate Na2·EDTA 37.3 mg/L
Ferrous sulfate FeSO4·7H2O 27.8 mg/L
Manganese sulfate MnSO4·H2O 22.3 mg/L
Zinc sulfate ZnSO4·7H2O 8.6 mg/L
Boric acid H3BO3 8.2 mg/L
Sodium molybdate Na2MoO4·2H2O 0.25 mg/L
Copper sulphate CuSO4·5H2O 0.05mg/L
Cobalt chloride CoCl2.6 H2O 2 0.05 mg/L
Inositol C6H12O6·2H2O 500 mg/L
Glycine NH2·CH2·COOH 5mg/L
Thiamine hydrochloride C12H17ClOS·2HCl 2 mg/L
Puridoxine hydrochloride C8H10NO5P 0.8 mg/L
Niacin NC5H4COOH 0.8mg/L。
4. dendrobium candidum Protocorm base according to claim 1, it is characterised in that: the plant extraction liquid is mentioned by mulberry leaf
Take object, Baical Skullcap root P.E and tannin composition.
5. dendrobium candidum Protocorm base according to claim 2, it is characterised in that: the mulberry-leaf extract, radix scutellariae mention
The weight part ratio between object and tannin three is taken respectively to be 5:4:1.
6. dendrobium candidum Protocorm base according to claim 1, it is characterised in that: the culture medium functional liquid is by aloe
Gel, saponin and palmitinic acid composition.
7. dendrobium candidum Protocorm base according to claim 1, it is characterised in that: the aloe gel, saponin and
The weight ratio of palmitinic acid respectively is 6:4:1.
8. a kind of application of dendrobium candidum Protocorm base according to claim 1, it is characterised in that: the applying step
It is as follows:
1) tender stem segments that long 2~5cm is cut from dendrobium candidum plant, rinse under flowing water, dip in a little washing powder with brush
Aqueous solution is gently scrubbed, and is sufficiently cleaned through tap water；Stem section is placed in 75% alcohol on superclean bench and is sterilized
After 30s, then with 0.1% HgCl2 8~10min of aqueous solution soaking, aseptic water washing 5~6 times；
2) Fiber differentiation: the tender stem segments of dendrobium candidum are cut with scalpel, is seeded in induced medium and is cultivated；15
After~20 days, explant starts to sprout, and stem section is expanded, and section and top organize the formation of open texture；After continuing culture 20 days, turn
Entering in holding chamber, the surface that tissue is expanded at section and top forms rough particulate matter, with the extension of incubation time,
Grain object grows up to form the protocorms particulate matter of 1~2mm of diameter；
3) Proliferation, Differentiation culture: the protocorm of induced synthesis is transferred in Proliferation, Differentiation culture medium, while part protocorm is opened
Beginning differentiates bud point and grows 1~2 leaflet, carries out certain condition illumination；The proliferation of protocorm carries out simultaneously with bud differentiation.
9. a kind of application of dendrobium candidum Protocorm base according to claim 6, it is characterised in that: the illumination condition
For light quality is feux rouges or red, blue mixed light.
10. a kind of application of dendrobium candidum Protocorm base according to claim 6, it is characterised in that: the illumination item
Part are as follows: light intensity is 90-100 μm of olm-2s-1,9 hour/day of light application time.
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