CN106258981A - Candidum tissue culturing factorial seedling-culturing method - Google Patents
Candidum tissue culturing factorial seedling-culturing method Download PDFInfo
- Publication number
- CN106258981A CN106258981A CN201610805264.3A CN201610805264A CN106258981A CN 106258981 A CN106258981 A CN 106258981A CN 201610805264 A CN201610805264 A CN 201610805264A CN 106258981 A CN106258981 A CN 106258981A
- Authority
- CN
- China
- Prior art keywords
- seedling
- acid
- calcium
- culture medium
- improve
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of candidum tissue culturing factorial seedling-culturing method, relate to biological technical field, mixed and disorderly kind of field matter is screened by this candidum tissue culturing factorial seedling-culturing method by simple method, filter out high yield, disease-resistant, outward appearance, good mouthfeel and quality and meet the Herba Dendrobii stem section of Chinese Pharmacopoeia standard, shorten the screening varieties time greatly；Incubation increases in the improvement of a great number of elements, additive in MS culture medium fulvic acid calcium and novel foodstuff oxidation preventive isoascorbic acid sodium or calcium ascorbate, condition of culture use colour temperature 4000K LED light source illumination and during producing strong sprout the seedling of circulation switching certain specification, improve the speed of growth of tissue cultured seedling, decrease seedling brownization in production process, yellow, dysgonic situation and the number of turnover of tissue cultured seedling, thus improve factorial praluction efficiency.
Description
Technical field
The present invention relates to biological technical field, it is more particularly related to a kind of candidum tissue culturing batch production is educated
Seedling-growing method.
Background technology
Herba Dendrobii clinically and is widely used in Chinese medicine compound.The application of Herba Dendrobii is mainly to gather wild money
Source is main, and along with the day by day exhaustion of wild resource, the plantation of artificial Herba Dendrobii is a large amount of rises.And current most of Herba Dendrobii people
Work post is planted Seedling and both is from breeding in plant division, cuttage and aseptically sowing seeds, and first two mode consumes ample resources on foot and develops
Slowly, rear kind mode belongs to sexual propagation, has certain production efficiency, but the method exists seedling variability greatly, property list
Existing the most inconsistent cause the fluctuation of medical material physical and chemical index the biggest, it is impossible to meet the stationarity that quality of medicinal material is controlled by Chinese crude drug GAP.Field
Screening high yield, Herba Dendrobii Dan Cong disease-resistant, outward appearance, good mouthfeel carry out physical and chemical index analysis, and physical and chemical index meets middle traditional Chinese medicines
Allusion quotation utilizes its individual plant stem section to clone after requiring, has weight for the production of high quality seedling commercialization and the Appropriate application of resource
The meaning wanted.Existing dendrobe tissue culture industrial seedling rearing belongs to culture medium, additive and the cultivation produced substantially with reference to other the orchid families
Condition, although produce and have certain efficiency, but seedling incubation still exists protocorm, clump bud and seedling brownization, yellow etc.
The phenomenons such as death.
Summary of the invention
Problem to be solved by this invention is to provide a kind of candidum tissue culturing factorial seedling-culturing method.By a series of letters
Field kind matter is screened by single easy method, operational approach, culture medium, additive and condition of culture in incubation
Improve current dendrobium candidum tissue and cultivate the shortcoming produced.
To achieve these goals, the technical scheme that the present invention takes is: a kind of candidum tissue culturing industrial seedling rearing side
Method, it is characterised in that comprise the steps:
(1) source screening is planted: Herba Dendrobii growth busy season 6～JIUYUE pest and disease damage few to outward appearance, single clump of seedling of robust growth is carried out
Labelling one by one, monthly sprays three EM bacterium, fulvic acid calcium and brassin, collecting season 11～March binds institute's labelling before collection
Ripe seedling carries out the corresponding physical and chemical index of Chinese Pharmacopoeia and makees full inspection, and the seedling meeting standards of pharmacopoeia is carried out stem section collection；
(2) outer implant pretreatment: fresh for Herba Dendrobii bar stem section is put in the 35～45 DEG C of warm water adding appropriate liquid detergent and soak
30～60 minutes, being peeled off by the sheath of soaked stem of Dendrobium section and can not injure the upper resting bud of joint, the stem section that sheath is peeled off is cut into
One section of each joint, soaks 30～60 minutes with sodium erythorbate or calcium ascorbate 50～200mg/L, then clear with clear water
Washing absorbent paper, to blot surface moisture stand-by；
(3) outer implant sterilizing: stem section soaked 10～15 seconds with 75% ethanol, is immediately placed in 0.1% mercuric chloride 8～12 points
Clock, and the gentliest shaking, finally takes out stem section sterile water wash 4～5 times, then it is stand-by to blot surface moisture with aseptic absorbent paper；
(4) stem segment with axillary buds induction: will cut stem section and access and improve in No. 1 culture medium light culture 10～15 days, axillalry bud starts
Sprout, use colour temperature 4000K LED illumination lamp illumination 8～10 hour/day, intensity of illumination 1000～1200lex, temperature 23 afterwards
～26 DEG C；
(5) clump bud inducement: when axillalry bud grows to 0.5～1cm, cuts sprout and puts into containing sodium erythorbate or Vitamin C
Acid calcium 50～200mg/L sterilized water soaks 20～30 minutes, within every 5 minutes, rocks once, each one minute, then use aseptic suction
Water paper blot surface moisture proceed to improve in No. 2 culture medium cultivate within 40～50 days, i.e. may occur in which little Cong bud.Condition of culture: use color
Temperature 4000K LED illumination lamp illumination 10～12 hour/day, intensity of illumination 1800～2000lex, temperature 23～26 DEG C；
(6) clump Shoot propagation: proceed to clump bud improve in No. 3 culture medium cultivate 40～50 days, clump bud almost breed cover with whole
Individual inoculation bottle.Condition of culture: use colour temperature 4000K LED illumination lamp illumination 10～12 hour/day, intensity of illumination 1800～
2000lex, temperature 23～26 DEG C；
(7) strong sprout: the clump bud of high more than 0.3cm is accessed in No. 4 culture medium of improvement and cultivate 50～60 days, 1.5cm in bottle
The circulation of below 1.5cm seedling, between 7: 3～5: 5, is proceeded to improve No. 4 trainings by the seedling ratio of above seedling and below 1.5cm
Support and base can realize the anniversary keep a number of production.Condition of culture: use colour temperature 4000K LED illumination lamp illumination 10～
12 hours/day, intensity of illumination 1800～2000lex, temperature 23～26 DEG C；
(8) take root and emerge: high more than 1.5cm seedling is accessed in No. 5 culture medium of improvement and cultivate 80～90 days, Seedling mean height
Degree 4～5cm, well developed root system, leaf color are dense can open domestication.Condition of culture: use colour temperature 4000K LED illumination lamp illumination 12
～14 hours/day, intensity of illumination 2000～2500lex, temperature 23～26 DEG C.
Improved culture medium formula is as follows:
Improve No. 1 culture medium: calcium nitrate 200mg/L, ammonium nitrate 500mg/L, potassium nitrate 760mg/L, magnesium sulfate 180mg/
L, potassium dihydrogen phosphate 100mg/L, chelated iron 35mg/L, manganese sulfate 22.3mg/L, zinc sulfate 8.6mg/L, cobaltous chloride 0.025mg/
L, copper sulfate 0.025mg/L, sodium molybdate 0.025mg/L, potassium iodide 0.83mg/L, boric acid 6.2mg/L, nicotinic acid 0.5mg/L, VB6
0.5mg/L, VB1 0.1mg/L, inositol 100mg/L, glycine 2mg/L, full potato starch 10-15g/L, fulvic acid calcium 0.5g/L,
Naphthalene acetic acid 0.05-0.2mg/L, 6-benzyladenine 0.2-0.6mg/L, sodium erythorbate or calcium ascorbate 20-100mg/
L, white sugar 20g/L, agar 4-5g/L, PH:5.6-6.0, water are dechlorination tap water；
Improve No. 2 culture medium: calcium nitrate 200mg/L, ammonium nitrate 500mg/L, potassium nitrate 760mg/L, magnesium sulfate 180mg/
L, potassium dihydrogen phosphate 100mg/L, chelated iron 35mg/L, manganese sulfate 22.3mg/L, zinc sulfate 8.6mg/L, cobaltous chloride 0.025mg/
L, copper sulfate 0.025mg/L, sodium molybdate 0.025mg/L, potassium iodide 0.83mg/L, boric acid 6.2mg/L, nicotinic acid 0.5mg/L, VB6
0.5mg/L, VB1 0.1mg/L, inositol 100mg/L, glycine 2mg/L, full potato starch 10-15g/L, fulvic acid calcium 0.5g/L,
Naphthalene acetic acid 0.2-0.3mg/L, 6-benzyladenine 2-3mg/L, sodium erythorbate or calcium ascorbate 20-100mg/L, white sugar
20g/L, agar 4-5g/L, PH:5.6-6.0, water are dechlorination tap water；
Improve No. 3 culture medium: calcium nitrate 200mg/L, ammonium nitrate 500mg/L, potassium nitrate 760mg/L, magnesium sulfate 180mg/
L, potassium dihydrogen phosphate 100mg/L, chelated iron 35mg/L, manganese sulfate 22.3mg/L, zinc sulfate 8.6mg/L, cobaltous chloride 0.025mg/
L, copper sulfate 0.025mg/L, sodium molybdate 0.025mg/L, potassium iodide 0.83mg/L, boric acid 6.2mg/L, nicotinic acid 0.5mg/L, VB6
0.5mg/L, VB1 0.1mg/L, inositol 100mg/L, glycine 2mg/L, full potato starch 10-15g/L, fulvic acid calcium 0.5g/L,
Naphthalene acetic acid 0.2-0.5mg/L, 6-benzyladenine 0.5-1.5mg/L, sodium erythorbate or calcium ascorbate 20-100mg/L,
White sugar 20g/L, agar 4-5g/L, PH:5.6-6.0, water are dechlorination tap water；
Improve No. 4 culture medium: calcium nitrate 200mg/L, ammonium nitrate 500mg/L, potassium nitrate 760mg/L, magnesium sulfate 180mg/
L, potassium dihydrogen phosphate 100mg/L, chelated iron 35mg/L, manganese sulfate 22.3mg/L, zinc sulfate 8.6mg/L, cobaltous chloride 0.025mg/
L, copper sulfate 0.025mg/L, sodium molybdate 0.025mg/L, potassium iodide 0.83mg/L, boric acid 6.2mg/L, nicotinic acid 0.5mg/L, VB6
0.5mg/L, VB1 0.1mg/L, inositol 100mg/L, glycine 2mg/L, full potato starch 10-15g/L, fulvic acid calcium 0.5-
0.1g/L, naphthalene acetic acid 0.2-0.5mg/L, 6-benzyladenine 0.05-0.2mg/L, sodium erythorbate or calcium ascorbate 20-
100mg/L, white sugar 20g/L, agar 4-5g/L, PH:5.6-6.0, water are dechlorination tap water；
Improve No. 5 culture medium: calcium nitrate 200mg/L, ammonium nitrate 500mg/L, potassium nitrate 950mg/L, magnesium sulfate 180mg/
L, potassium dihydrogen phosphate 100mg/L, chelated iron 35mg/L, manganese sulfate 22.3mg/L, zinc sulfate 8.6mg/L, cobaltous chloride 0.025mg/
L, copper sulfate 0.025mg/L, sodium molybdate 0.025mg/L, potassium iodide 0.83mg/L, boric acid 6.2mg/L, nicotinic acid 0.5mg/L, VB6
0.5mg/L, VB1 0.1mg/L, inositol 100mg/L, glycine 2mg/L, full potato starch 10-15g/L, Fructus Musae lyophilized powder 7-10g/
L, fulvic acid calcium 0.5-0.1g/L, naphthalene acetic acid 0.2-0.5mg/L, 6-benzyladenine 0mg/L, sodium erythorbate or Vitamin C
Acid calcium 20-100mg/L, activated carbon 0.7-1g/L, white sugar 15g/L, agar 4-5g/L, PH:5.6-6.0, water are dechlorination tap water.
Beneficial effect: this candidum tissue culturing factorial seedling-culturing method by simple method to mixed and disorderly kind of field matter
Screen, filter out high yield, disease-resistant, outward appearance, good mouthfeel and quality and meet the Herba Dendrobii stem section of Chinese Pharmacopoeia standard, pole
Big shortens the screening varieties time；Incubation increases in the improvement of a great number of elements, additive in MS culture medium fulvic acid
Calcium and novel foodstuff oxidation preventive isoascorbic acid sodium or calcium ascorbate, condition of culture use the LED light source of colour temperature 4000K
Illuminate and the seedling of circulation switching certain specification during producing strong sprout, improve the speed of growth of tissue cultured seedling, decrease
Seedling brownization, yellow, dysgonic situation and the number of turnover of tissue cultured seedling in production process, improve factorial praluction effect
Rate.
Detailed description of the invention
Embodiment 1:
This candidum tissue culturing factorial seedling-culturing method comprises the steps:
(1) source screening is planted: Herba Dendrobii growth busy season 6～JIUYUE pest and disease damage few to outward appearance, single clump of seedling of robust growth is carried out
Labelling one by one, monthly sprays three EM bacterium, fulvic acid calcium and brassin, collecting season 11～March binds institute's labelling before collection
Ripe seedling carries out the corresponding physical and chemical index of Chinese Pharmacopoeia and makees full inspection, and the seedling meeting standards of pharmacopoeia is carried out stem section collection；
(2) outer implant pretreatment: fresh for Herba Dendrobii bar stem section is put in the 35 DEG C of warm water adding appropriate liquid detergent and soak 60 points
Clock, peels off the sheath of soaked stem of Dendrobium section and can not injure the upper resting bud of joint, and the stem section that sheath is peeled off is cut into each joint one
Individual section, soaks 60 minutes with sodium erythorbate or calcium ascorbate 50mg/L, then blots surface with clean water absorbent paper
Moisture is stand-by；
(3) outer implant sterilizing: stem section soaked 10 seconds with 75% ethanol, is immediately placed in 0.1% mercuric chloride 8 minutes, and frequently
Light rolling, finally takes out stem section sterile water wash 5 times, then it is stand-by to blot surface moisture with aseptic absorbent paper；
(4) stem segment with axillary buds induction: will cut stem section and access and improve light culture 10 days in No. 1 culture medium, axillalry bud starts to sprout,
Use colour temperature 4000K LED illumination lamp illumination 10 hour/day, intensity of illumination 1000lex, temperature 26 DEG C afterwards；
(5) clump bud inducement: when axillalry bud grows to 0.5cm, cuts sprout and puts into containing sodium erythorbate or ascorbic acid
Calcium 50mg/L sterilized water soaks 30 minutes, within every 5 minutes, rocks once, each one minute, then blot table with aseptic absorbent paper
Face moisture proceeds to improve cultivate in No. 2 culture medium and i.e. may occur in which little Cong bud in 50 days.Condition of culture: use colour temperature 4000K LED to shine
Bright lamp illumination 12 hour/day, intensity of illumination 1800lex, temperature 23 DEG C；
(6) clump Shoot propagation: proceeding to clump bud improve in No. 3 culture medium and cultivate 40 days, clump bud is almost bred and covered with whole connecing
Plant bottle.Condition of culture: use colour temperature 4000K LED illumination lamp illumination 10 hour/day, intensity of illumination 2000lex, temperature 26 DEG C；
(7) strong sprout: the clump bud of high more than 0.3cm is accessed in No. 4 culture medium of improvement and cultivate 50 days, more than 1.5cm in bottle
The seedling ratio of seedling and below 1.5cm is 7: 3, and the circulation of below 1.5cm seedling is proceeded to improve in No. 4 culture medium and can be realized
Anniversary keeps a number of production.Condition of culture: use colour temperature 4000K LED illumination lamp illumination 12 hour/day, intensity of illumination
1900lex, temperature 24 DEG C；
(8) take root and emerge: high more than 1.5cm seedling is accessed in No. 5 culture medium of improvement and cultivate 82 days, Seedling average height
4cm, well developed root system, leaf color are dense can open domestication.Condition of culture: use colour temperature 4000K LED illumination lamp illumination 13 hours/
My god, intensity of illumination 2200lex, temperature 25 DEG C.
Embodiment 2:
This candidum tissue culturing factorial seedling-culturing method comprises the steps:
(1) source screening is planted: Herba Dendrobii growth busy season 6～JIUYUE pest and disease damage few to outward appearance, single clump of seedling of robust growth is carried out
Labelling one by one, monthly sprays three EM bacterium, fulvic acid calcium and brassin, collecting season 11～March binds institute's labelling before collection
Ripe seedling carries out the corresponding physical and chemical index of Chinese Pharmacopoeia and makees full inspection, and the seedling meeting standards of pharmacopoeia is carried out stem section collection；
(2) outer implant pretreatment: fresh for Herba Dendrobii bar stem section is put in the 35～45 DEG C of warm water adding appropriate liquid detergent and soak
35 minutes, being peeled off by the sheath of soaked stem of Dendrobium section and can not injure the upper resting bud of joint, the stem section that sheath is peeled off is cut into each
Save a section, soak 50 minutes with sodium erythorbate or calcium ascorbate 160mg/L, then blot with clean water absorbent paper
Surface moisture is stand-by；
(3) outer implant sterilizing: stem section soaked 14 seconds with 75% ethanol, is immediately placed in 0.1% mercuric chloride 11 minutes, and frequently
Light rolling, finally takes out stem section sterile water wash 5 times, then it is stand-by to blot surface moisture with aseptic absorbent paper；
(4) stem segment with axillary buds induction: will cut stem section and access and improve light culture 13 days in No. 1 culture medium, axillalry bud starts to sprout,
Use colour temperature 4000K LED illumination lamp illumination 9 hour/day, intensity of illumination 1100lex, temperature 25 DEG C afterwards；
(5) clump bud inducement: when axillalry bud grows to 0.9cm, cuts sprout and puts into containing sodium erythorbate or ascorbic acid
Calcium 160mg/L sterilized water soaks 27 minutes, within every 5 minutes, rocks once, each one minute, then blot table with aseptic absorbent paper
Face moisture proceeds to improve cultivate in No. 2 culture medium and i.e. may occur in which little Cong bud in 48 days.Condition of culture: use colour temperature 4000K LED to shine
Bright lamp illumination 11 hour/day, intensity of illumination 2000lex, temperature 25 DEG C；
(6) clump Shoot propagation: proceeding to clump bud improve in No. 3 culture medium and cultivate 48 days, clump bud is almost bred and covered with whole connecing
Plant bottle.Condition of culture: use colour temperature 4000K LED illumination lamp illumination 12 hour/day, intensity of illumination 1900lex, temperature 26 DEG C；
(7) strong sprout: the clump bud of high more than 0.3cm is accessed in No. 4 culture medium of improvement and cultivate 58 days, more than 1.5cm in bottle
The circulation of below 1.5cm seedling, between 5: 5, is proceeded to improve in No. 4 culture medium by the seedling ratio of seedling and below 1.5cm
The anniversary that realizes keeps a number of production.Condition of culture: use colour temperature 4000K LED illumination lamp illumination 12 hour/day, illumination
Intensity 1800lex, temperature 23 DEG C；
(8) take root and emerge: high more than 1.5cm seedling is accessed in No. 5 culture medium of improvement and cultivate 87 days, Seedling average height
5cm, well developed root system, leaf color are dense can open domestication.Condition of culture: use colour temperature 4000K LED illumination lamp illumination 13 hours/
My god, intensity of illumination 2500lex, temperature 25 DEG C.
Embodiment 3:
This candidum tissue culturing factorial seedling-culturing method comprises the steps:
(1) source screening is planted: Herba Dendrobii growth busy season 6～JIUYUE pest and disease damage few to outward appearance, single clump of seedling of robust growth is carried out
Labelling one by one, monthly sprays three EM bacterium, fulvic acid calcium and brassin, collecting season 11～March binds institute's labelling before collection
Ripe seedling carries out the corresponding physical and chemical index of Chinese Pharmacopoeia and makees full inspection, and the seedling meeting standards of pharmacopoeia is carried out stem section collection；
(2) outer implant pretreatment: fresh for Herba Dendrobii bar stem section is put in the 35～45 DEG C of warm water adding appropriate liquid detergent and soak
55 minutes, being peeled off by the sheath of soaked stem of Dendrobium section and can not injure the upper resting bud of joint, the stem section that sheath is peeled off is cut into each
Save a section, soak 40 minutes with sodium erythorbate or calcium ascorbate 200mg/L, then blot with clean water absorbent paper
Surface moisture is stand-by；
(3) outer implant sterilizing: stem section soaked 12 seconds with 75% ethanol, is immediately placed in 0.1% mercuric chloride 9 minutes, and frequently
Light rolling, finally takes out stem section sterile water wash 4 times, then it is stand-by to blot surface moisture with aseptic absorbent paper；
(4) stem segment with axillary buds induction: will cut stem section and access and improve light culture 10 days in No. 1 culture medium, axillalry bud starts to sprout,
Use colour temperature 4000K LED illumination lamp illumination 10 hour/day, intensity of illumination 1200lex, temperature 25 DEG C afterwards；
(5) clump bud inducement: when axillalry bud grows to 0.8cm, cuts sprout and puts into containing sodium erythorbate or ascorbic acid
Calcium 180mg/L sterilized water soaks 27 minutes, within every 5 minutes, rocks once, each one minute, then blot table with aseptic absorbent paper
Face moisture proceeds to improve cultivate in No. 2 culture medium and i.e. may occur in which little Cong bud in 42 days.Condition of culture: use colour temperature 4000K LED to shine
Bright lamp illumination 11 hour/day, intensity of illumination 1900lex, temperature 25 DEG C；
(6) clump Shoot propagation: proceeding to clump bud improve in No. 3 culture medium and cultivate 48 days, clump bud is almost bred and covered with whole connecing
Plant bottle.Condition of culture: use colour temperature 4000K LED illumination lamp illumination 11 hour/day, intensity of illumination 1800lex, temperature 25 DEG C；
(7) strong sprout: the clump bud of high more than 0.3cm is accessed in No. 4 culture medium of improvement and cultivate 58 days, more than 1.5cm in bottle
The circulation of below 1.5cm seedling, between 7: 3, is proceeded to improve in No. 4 culture medium by the seedling ratio of seedling and below 1.5cm
The anniversary that realizes keeps a number of production.Condition of culture: use colour temperature 4000K LED illumination lamp illumination 11 hour/day, illumination
Intensity 1800lex, temperature 24 DEG C；
(8) take root and emerge: high more than 1.5cm seedling is accessed in No. 5 culture medium of improvement and cultivate 81 days, Seedling average height
4cm, well developed root system, leaf color are dense can open domestication.Condition of culture: use colour temperature 4000K LED illumination lamp illumination 12 hours/
My god, intensity of illumination 2500lex, temperature 23 DEG C.
Improved culture medium formula is as follows:
Improve No. 1 culture medium: calcium nitrate 200mg/L, ammonium nitrate 500mg/L, potassium nitrate 760mg/L, magnesium sulfate 180mg/
L, potassium dihydrogen phosphate 100mg/L, chelated iron 35mg/L, manganese sulfate 22.3mg/L, zinc sulfate 8.6mg/L, cobaltous chloride 0.025mg/
L, copper sulfate 0.025mg/L, sodium molybdate 0.025mg/L, potassium iodide 0.83mg/L, boric acid 6.2mg/L, nicotinic acid 0.5mg/L, VB6
0.5mg/L, VB1 0.1mg/L, inositol 100mg/L, glycine 2mg/L, full potato starch 10-15g/L, fulvic acid calcium 0.5g/L,
Naphthalene acetic acid 0.05-0.2mg/L, 6-benzyladenine 0.2-0.6mg/L, sodium erythorbate or calcium ascorbate 20-100mg/
L, white sugar 20g/L, agar 4-5g/L, PH:5.6-6.0, water are dechlorination tap water；
Improve No. 2 culture medium: calcium nitrate 200mg/L, ammonium nitrate 500mg/L, potassium nitrate 760mg/L, magnesium sulfate 180mg/
L, potassium dihydrogen phosphate 100mg/L, chelated iron 35mg/L, manganese sulfate 22.3mg/L, zinc sulfate 8.6mg/L, cobaltous chloride 0.025mg/
L, copper sulfate 0.025mg/L, sodium molybdate 0.025mg/L, potassium iodide 0.83mg/L, boric acid 6.2mg/L, nicotinic acid 0.5mg/L, VB6
0.5mg/L, VB1 0.1mg/L, inositol 100mg/L, glycine 2mg/L, full potato starch 10-15g/L, fulvic acid calcium 0.5g/L,
Naphthalene acetic acid 0.2-0.3mg/L, 6-benzyladenine 2-3mg/L, sodium erythorbate or calcium ascorbate 20-100mg/L, white sugar
20g/L, agar 4-5g/L, PH:5.6-6.0, water are dechlorination tap water；
Improve No. 3 culture medium: calcium nitrate 200mg/L, ammonium nitrate 500mg/L, potassium nitrate 760mg/L, magnesium sulfate 180mg/
L, potassium dihydrogen phosphate 100mg/L, chelated iron 35mg/L, manganese sulfate 22.3mg/L, zinc sulfate 8.6mg/L, cobaltous chloride 0.025mg/
L, copper sulfate 0.025mg/L, sodium molybdate 0.025mg/L, potassium iodide 0.83mg/L, boric acid 6.2mg/L, nicotinic acid 0.5mg/L, VB6
0.5mg/L, VB1 0.1mg/L, inositol 100mg/L, glycine 2mg/L, full potato starch 10-15g/L, fulvic acid calcium 0.5g/L,
Naphthalene acetic acid 0.2-0.5mg/L, 6-benzyladenine 0.5-1.5mg/L, sodium erythorbate or calcium ascorbate 20-100mg/L,
White sugar 20g/L, agar 4-5g/L, PH:5.6-6.0, water are dechlorination tap water；
Improve No. 4 culture medium: calcium nitrate 200mg/L, ammonium nitrate 500mg/L, potassium nitrate 760mg/L, magnesium sulfate 180mg/
L, potassium dihydrogen phosphate 100mg/L, chelated iron 35mg/L, manganese sulfate 22.3mg/L, zinc sulfate 8.6mg/L, cobaltous chloride 0.025mg/
L, copper sulfate 0.025mg/L, sodium molybdate 0.025mg/L, potassium iodide 0.83mg/L, boric acid 6.2mg/L, nicotinic acid 0.5mg/L, VB6
0.5mg/L, VB1 0.1mg/L, inositol 100mg/L, glycine 2mg/L, full potato starch 10-15g/L, fulvic acid calcium 0.5-
0.1g/L, naphthalene acetic acid 0.2-0.5mg/L, 6-benzyladenine 0.05-0.2mg/L, sodium erythorbate or calcium ascorbate 20-
100mg/L, white sugar 20g/L, agar 4-5g/L, PH:5.6-6.0, water are dechlorination tap water；
Improve No. 5 culture medium: calcium nitrate 200mg/L, ammonium nitrate 500mg/L, potassium nitrate 950mg/L, magnesium sulfate 180mg/
L, potassium dihydrogen phosphate 100mg/L, chelated iron 35mg/L, manganese sulfate 22.3mg/L, zinc sulfate 8.6mg/L, cobaltous chloride 0.025mg/
L, copper sulfate 0.025mg/L, sodium molybdate 0.025mg/L, potassium iodide 0.83mg/L, boric acid 6.2mg/L, nicotinic acid 0.5mg/L, VB6
0.5mg/L, VB1 0.1mg/L, inositol 100mg/L, glycine 2mg/L, full potato starch 10-15g/L, Fructus Musae lyophilized powder 7-10g/
L, fulvic acid calcium 0.5-0.1g/L, naphthalene acetic acid 0.2-0.5mg/L, 6-benzyladenine 0mg/L, sodium erythorbate or Vitamin C
Acid calcium 20-100mg/L, activated carbon 0.7-1g/L, white sugar 15g/L, agar 4-5g/L, PH:5.6-6.0, water are dechlorination tap water.
The foregoing is only embodiments of the invention, not thereby limit the scope of the claims of the present invention, every utilize this
Equivalent structure or equivalence flow process that bright description and embodiment content are made convert, or it is relevant to be directly or indirectly used in other
Technical field, be the most in like manner included in the scope of patent protection of the present invention.
Claims (2)
1. a candidum tissue culturing factorial seedling-culturing method, it is characterised in that comprise the steps:
(1) source screening is planted: Herba Dendrobii growth busy season 6～JIUYUE pest and disease damage few to outward appearance, single clump of seedling of robust growth is carried out one by one
Labelling, monthly sprays three EM bacterium, fulvic acid calcium and the maturation that brassin, collecting season 11～March binds institute's labelling before collection
Seedling carry out the corresponding physical and chemical index of Chinese Pharmacopoeia and make full inspection, the seedling meeting standards of pharmacopoeia is carried out stem section collection；
(2) outer implant pretreatment: fresh for Herba Dendrobii bar stem section is put into add appropriate liquid detergent 35～45 DEG C of warm water in soak 30～
60 minutes, being peeled off by the sheath of soaked stem of Dendrobium section and can not injure the upper resting bud of joint, the stem section that sheath is peeled off is cut into each
Save a section, soak 30～60 minutes with sodium erythorbate or calcium ascorbate 50～200mg/L, then inhale by clean water
It is stand-by that water paper blots surface moisture；
(3) outer implant sterilizing: stem section soaked 10～15 seconds with 75% ethanol, is immediately placed in 0.1% mercuric chloride 8～12 minutes, and
The gentliest shake, finally take out stem section sterile water wash 4～5 times, then it is stand-by to blot surface moisture with aseptic absorbent paper；
(4) stem segment with axillary buds induction: will cut stem section and access and improve in No. 1 culture medium light culture 10～15 days, axillalry bud starts to sprout,
Use colour temperature 4000K LED illumination lamp illumination 8～10 hour/day, intensity of illumination 1000～1200lex, temperature 23～26 afterwards
℃；
(5) clump bud inducement: when axillalry bud grows to 0.5～1cm, cuts sprout and puts into containing sodium erythorbate or calcium ascorbate
50～200mg/L sterilized water soak 20～30 minutes, within every 5 minutes, rocks once, each one minute, then use aseptic absorbent paper
Blot surface moisture proceed to improve in No. 2 culture medium cultivate within 40～50 days, i.e. may occur in which little Cong bud.Condition of culture: use colour temperature
4000K LED illumination lamp illumination 10～12 hour/day, intensity of illumination 1800～2000lex, temperature 23～26 DEG C；
(6) clump Shoot propagation: proceeding to clump bud improve in No. 3 culture medium and cultivate 40～50 days, clump bud is almost bred and covered with whole connecing
Plant bottle.Condition of culture: use colour temperature 4000K LED illumination lamp illumination 10～12 hour/day, intensity of illumination 1800～2000lex,
Temperature 23～26 DEG C；
(7) strong sprout: the clump bud of high more than 0.3cm is accessed in No. 4 culture medium of improvement and cultivate 50～60 days, more than 1.5cm in bottle
The circulation of below 1.5cm seedling, between 7: 3～5: 5, is proceeded to improve No. 4 culture medium by the seedling ratio of seedling and below 1.5cm
In can realize the anniversary keep a number of production.Condition of culture: use colour temperature 4000K LED illumination lamp illumination 10～12 little
Time/sky, intensity of illumination 1800～2000lex, temperature 23～26 DEG C；
(8) take root and emerge: high more than 1.5cm seedling is accessed in No. 5 culture medium of improvement and cultivate 80～90 days, Seedling average height 4
～5cm, well developed root system, leaf color are dense can open domestication.Condition of culture: use colour temperature 4000K LED illumination lamp illumination 12～
14 hours/day, intensity of illumination 2000～2500lex, temperature 23～26 DEG C.
2. according to a kind of candidum tissue culturing factorial seedling-culturing method described in claim 1, it is characterised in that: described improvement training
Support based formulas as follows:
Improve No. 1 culture medium: calcium nitrate 200mg/L, ammonium nitrate 500mg/L, potassium nitrate 760mg/L, magnesium sulfate 180mg/L, phosphorus
Acid dihydride potassium 100mg/L, chelated iron 35mg/L, manganese sulfate 22.3mg/L, zinc sulfate 8.6mg/L, cobaltous chloride 0.025mg/L, sulfur
Acid copper 0.025mg/L, sodium molybdate 0.025mg/L, potassium iodide 0.83mg/L, boric acid 6.2mg/L, nicotinic acid 0.5mg/L, VB6
0.5mg/L, VB1 0.1mg/L, inositol 100mg/L, glycine 2mg/L, full potato starch 10-15g/L, fulvic acid calcium 0.5g/L,
Naphthalene acetic acid 0.05-0.2mg/L, 6-benzyladenine 0.2-0.6mg/L, sodium erythorbate or calcium ascorbate 20-100mg/
L, white sugar 20g/L, agar 4-5g/L, PH:5.6-6.0, water are dechlorination tap water；
Improve No. 2 culture medium: calcium nitrate 200mg/L, ammonium nitrate 500mg/L, potassium nitrate 760mg/L, magnesium sulfate 180mg/L, phosphorus
Acid dihydride potassium 100mg/L, chelated iron 35mg/L, manganese sulfate 22.3mg/L, zinc sulfate 8.6mg/L, cobaltous chloride 0.025mg/L, sulfur
Acid copper 0.025mg/L, sodium molybdate 0.025mg/L, potassium iodide 0.83mg/L, boric acid 6.2mg/L, nicotinic acid 0.5mg/L, VB6
0.5mg/L, VB1 0.1mg/L, inositol 100mg/L, glycine 2mg/L, full potato starch 10-15g/L, fulvic acid calcium 0.5g/L,
Naphthalene acetic acid 0.2-0.3mg/L, 6-benzyladenine 2-3mg/L, sodium erythorbate or calcium ascorbate 20-100mg/L, white sugar
20g/L, agar 4-5g/L, PH:5.6-6.0, water are dechlorination tap water；
Improve No. 3 culture medium: calcium nitrate 200mg/L, ammonium nitrate 500mg/L, potassium nitrate 760mg/L, magnesium sulfate 180mg/L, phosphorus
Acid dihydride potassium 100mg/L, chelated iron 35mg/L, manganese sulfate 22.3mg/L, zinc sulfate 8.6mg/L, cobaltous chloride 0.025mg/L, sulfur
Acid copper 0.025mg/L, sodium molybdate 0.025mg/L, potassium iodide 0.83mg/L, boric acid 6.2mg/L, nicotinic acid 0.5mg/L, VB6
0.5mg/L, VB1 0.1mg/L, inositol 100mg/L, glycine 2mg/L, full potato starch 10-15g/L, fulvic acid calcium 0.5g/L,
Naphthalene acetic acid 0.2-0.5mg/L, 6-benzyladenine 0.5-1.5mg/L, sodium erythorbate or calcium ascorbate 20-100mg/L,
White sugar 20g/L, agar 4-5g/L, PH:5.6-6.0, water are dechlorination tap water；
Improve No. 4 culture medium: calcium nitrate 200mg/L, ammonium nitrate 500mg/L, potassium nitrate 760mg/L, magnesium sulfate 180mg/L, phosphorus
Acid dihydride potassium 100mg/L, chelated iron 35mg/L, manganese sulfate 22.3mg/L, zinc sulfate 8.6mg/L, cobaltous chloride 0.025mg/L, sulfur
Acid copper 0.025mg/L, sodium molybdate 0.025mg/L, potassium iodide 0.83mg/L, boric acid 6.2mg/L, nicotinic acid 0.5mg/L, VB6
0.5mg/L, VB1 0.1mg/L, inositol 100mg/L, glycine 2mg/L, full potato starch 10-15g/L, fulvic acid calcium 0.5-
0.1g/L, naphthalene acetic acid 0.2-0.5mg/L, 6-benzyladenine 0.05-0.2mg/L, sodium erythorbate or calcium ascorbate 20-
100mg/L, white sugar 20g/L, agar 4-5g/L, PH:5.6-6.0, water are dechlorination tap water；
Improve No. 5 culture medium: calcium nitrate 200mg/L, ammonium nitrate 500mg/L, potassium nitrate 950mg/L, magnesium sulfate 180mg/L, phosphorus
Acid dihydride potassium 100mg/L, chelated iron 35mg/L, manganese sulfate 22.3mg/L, zinc sulfate 8.6mg/L, cobaltous chloride 0.025mg/L, sulfur
Acid copper 0.025mg/L, sodium molybdate 0.025mg/L, potassium iodide 0.83mg/L, boric acid 6.2mg/L, nicotinic acid 0.5mg/L, VB6
0.5mg/L, VB1 0.1mg/L, inositol 100mg/L, glycine 2mg/L, full potato starch 10-15g/L, Fructus Musae lyophilized powder 7-10g/
L, fulvic acid calcium 0.5-0.1g/L, naphthalene acetic acid 0.2-0.5mg/L, 6-benzyladenine 0mg/L, sodium erythorbate or Vitamin C
Acid calcium 20-100mg/L, activated carbon 0.7-1g/L, white sugar 15g/L, agar 4-5g/L, PH:5.6-6.0, water are dechlorination tap water.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610805264.3A CN106258981A (en) | 2016-08-31 | 2016-08-31 | Candidum tissue culturing factorial seedling-culturing method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610805264.3A CN106258981A (en) | 2016-08-31 | 2016-08-31 | Candidum tissue culturing factorial seedling-culturing method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106258981A true CN106258981A (en) | 2017-01-04 |
Family
ID=57710316
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610805264.3A Pending CN106258981A (en) | 2016-08-31 | 2016-08-31 | Candidum tissue culturing factorial seedling-culturing method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106258981A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109169220A (en) * | 2018-08-16 | 2019-01-11 | 镇江陶氏健康源生物科技有限公司 | A kind of cultural method of the dendrobium candidum containing selenium |
| CN109169277A (en) * | 2018-09-17 | 2019-01-11 | 江苏强农农业技术服务有限公司 | A kind of germ plasm resource in-vitro conservation method of dendrobium candidum |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102369881A (en) * | 2011-09-22 | 2012-03-14 | 澄思源生物科技（上海）有限公司 | Rapid propagation technique of Dendrobium candidum axillary buds |
| CN102388797A (en) * | 2011-07-21 | 2012-03-28 | 玉林市林业科学研究所 | Cross breeding method for Dendrobium officinale Kimura et Migo |
| CN102972299A (en) * | 2012-12-10 | 2013-03-20 | 黄怀毅 | Rapid propagation method for dendrobium officinale stem tissue culture |
| CN103548681A (en) * | 2013-10-31 | 2014-02-05 | 浙江农林大学天目学院 | Culture medium for rapidly inducing adventitious buds in tissue culture of dendrobium nobile |
| CN104585032A (en) * | 2014-12-31 | 2015-05-06 | 安徽康久生物科技有限公司 | Nutrient solution for promoting rooting of dendrobium officinale |
| CN104855293A (en) * | 2015-06-15 | 2015-08-26 | 泉州正和堂生物科技有限公司 | Clone production process for dendrobium officinale kmuraet migo officinal protocorm by adopting large-scale bioreactor |
| CN105230489A (en) * | 2015-11-02 | 2016-01-13 | 大新县生产力促进中心 | Tissue culture fast propagation method for dendrobium candidum |
| CN105409780A (en) * | 2015-12-30 | 2016-03-23 | 云南飞熊农业科技有限公司 | Plant tissue culture medium without disinfection and preparation method therefor |
| CN105850690A (en) * | 2016-06-27 | 2016-08-17 | 广西农业职业技术学院 | Dendrobium officinale culture medium |
-
2016
- 2016-08-31 CN CN201610805264.3A patent/CN106258981A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102388797A (en) * | 2011-07-21 | 2012-03-28 | 玉林市林业科学研究所 | Cross breeding method for Dendrobium officinale Kimura et Migo |
| CN102369881A (en) * | 2011-09-22 | 2012-03-14 | 澄思源生物科技（上海）有限公司 | Rapid propagation technique of Dendrobium candidum axillary buds |
| CN102972299A (en) * | 2012-12-10 | 2013-03-20 | 黄怀毅 | Rapid propagation method for dendrobium officinale stem tissue culture |
| CN103548681A (en) * | 2013-10-31 | 2014-02-05 | 浙江农林大学天目学院 | Culture medium for rapidly inducing adventitious buds in tissue culture of dendrobium nobile |
| CN104585032A (en) * | 2014-12-31 | 2015-05-06 | 安徽康久生物科技有限公司 | Nutrient solution for promoting rooting of dendrobium officinale |
| CN104855293A (en) * | 2015-06-15 | 2015-08-26 | 泉州正和堂生物科技有限公司 | Clone production process for dendrobium officinale kmuraet migo officinal protocorm by adopting large-scale bioreactor |
| CN105230489A (en) * | 2015-11-02 | 2016-01-13 | 大新县生产力促进中心 | Tissue culture fast propagation method for dendrobium candidum |
| CN105409780A (en) * | 2015-12-30 | 2016-03-23 | 云南飞熊农业科技有限公司 | Plant tissue culture medium without disinfection and preparation method therefor |
| CN105850690A (en) * | 2016-06-27 | 2016-08-17 | 广西农业职业技术学院 | Dendrobium officinale culture medium |
Non-Patent Citations (1)
| Title |
|---|
| 郭洪波等: "铁皮石斛茎节离体培养的研究 ", 《时珍国医国药》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109169220A (en) * | 2018-08-16 | 2019-01-11 | 镇江陶氏健康源生物科技有限公司 | A kind of cultural method of the dendrobium candidum containing selenium |
| CN109169277A (en) * | 2018-09-17 | 2019-01-11 | 江苏强农农业技术服务有限公司 | A kind of germ plasm resource in-vitro conservation method of dendrobium candidum |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104429938B (en) | A kind of dendrobium candidum tissue cultural method | |
| CN104186295B (en) | Pocket orchid seed germination medium and cultural method | |
| CN107047320A (en) | A kind of bigflower centranthera root method for tissue culture | |
| CN103026884B (en) | Artificial planting method for producing truffles at high yield | |
| CN102823503B (en) | Tissue culture medium for propagating anthurium buds by using buds | |
| CN103798145A (en) | Culture medium for tissue culture of vernonia amygdalina del. | |
| CN101953307A (en) | Method for producing Fujian jewel orchid by plant tissue culture | |
| CN107047316A (en) | A kind of iris tissue culture method and culture medium | |
| CN103202232A (en) | Method for efficiently and industrially producing sweet potato detoxification tissue culture seedlings | |
| CN102228003B (en) | Culture method for brassica oleracea L. var. acephala microspore regeneration plant | |
| CN104938184A (en) | Method for cultivating pakchoi which is highly rich in vitamin C | |
| CN102160525A (en) | Method for realizing excellent plant body induction and successive propagation culture on dendrobium candidum by virtue of plant tissue culture | |
| CN106258981A (en) | Candidum tissue culturing factorial seedling-culturing method | |
| CN102499034B (en) | Soilless cultivation method capable of reducing content of nitrate in curled mallow and curled mallow nutrient solution | |
| CN103636506B (en) | method for performing plant culture by utilizing shepherdia argentea caulicle regenerated plant induction culture medium and | |
| CN103782912B (en) | Culture medium for red cassia tree tissue culture | |
| CN106386495A (en) | Novel Herba Dendrobii culturing method | |
| CN106359101A (en) | Tissue culture and rapid propagation method of ficus deltoidea | |
| CN105284606A (en) | South China organic blueberry planting technology | |
| CN108849451B (en) | Method for improving quality of hydroponic lettuce | |
| CN103598093B (en) | A kind of abductive approach of blueberry embryoid | |
| CN105230492B (en) | A kind of scythian lamb rhizome spore tissue culture mating system | |
| CN104686348A (en) | Tissue culture rapid propagation technique of moringa oleifera Lam. | |
| CN109042666A (en) | A kind of foliage-spray liquid improving sugarcane test tube seedling survival rate | |
| CN107466850A (en) | Blueberry planting and seedling rapid cultivation method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20170104 |
|
| WD01 | Invention patent application deemed withdrawn after publication |