CN102499092A - Method for cultivating and breeding cell tissue of tissue culture seedling of Dendrobium candidum - Google Patents
Method for cultivating and breeding cell tissue of tissue culture seedling of Dendrobium candidum Download PDFInfo
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- CN102499092A CN102499092A CN201110365328XA CN201110365328A CN102499092A CN 102499092 A CN102499092 A CN 102499092A CN 201110365328X A CN201110365328X A CN 201110365328XA CN 201110365328 A CN201110365328 A CN 201110365328A CN 102499092 A CN102499092 A CN 102499092A
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Abstract
A method for cultivating and breeding cell tissue of the tissue culture seedling of Dendrobium candidum comprises the steps of mother plant treatment, soaking disinfection, cleaning and cutting, callus induction, callus crushing, shake cultivation, protocorm cultivation, adventitious bud cultivation, plantlet cultivation and sapling cultivation. According to the invention, good characters of the previous generation are stably inherited by the next generation through a method of asexual reproduction, so that the phenomenon of character segregation of the filial generation, brought by sexual reproduction, can be avoided effectively, the characters of the mother plant can be reserved best, and the production of tissue culture seedling has purposiveness and pertinence.
Description
Technical field
The cellular tissure that the present invention relates to a kind of candidum tissue culturing seedling is cultivated mating system.
Background technology
The stem of noble dendrobium has another name called dendrobium, is the orchid family Dendrobium plant, is one of orchid of putting down in writing the earliest during China's ancient Chinese prose is offered.Dendrobium candidum is one of category the famousst and precious in the stem of noble dendrobium, and medical value is high, and China's successive dynasties pharmacy works all has detailed record to it, and Compendium of Material Medica thinks that it removes the numbness therapeutic method to keep the adverse qi flowing downward, the win of benefit internal organ consumptive disease is thin, and the reinforcing yin essence benefit is smart.Obey for a long time, thick stomach, never sufficient in the benefit.Come stomach Qi, longue meat, intelligence development removes frightened, makes light of one's life by commiting suicide and prolongs life.In the Taoism medical science classical " Taoist Scriptures " dendrobium candidum is classified as first of China's " nine immortals grass ".Its reinforcing stomach reg fluid of Pharmacopoeia of the People's Republic of China record is nourishing Yin and clearing heat.Be used for the moon and hinder body fluid deficiency, the dry polydipsia, food is few retches, abnormal heat after being ill, order is secretly not clear.
In recent years,, add people and excavate without limit for a long time, cause this species wild resource seriously poor, classified as the national secondary endangered protective plant at present because dendrobium candidum natural propagation rate is extremely low.
It is ripe that the candidum tissue culturing technology is tending towards at present gradually, but most tissue cultivating seedling all is to adopt the dendrobium candidum seed as the initial provenance of breeding.There is the maternal difference of male parent in seed owing to be amphigenetic product on genetics, the proterties segregation phenomenon appears in the filial generation meeting of breeding out, is unfavorable for preserving improved seeds.
Summary of the invention
The objective of the invention is to propose a kind of method of utilizing modern biotechnology to cultivate seedlings of Dendrobium officinale; Its technique effect is to make the merit genetic stability of previous generation give the next generation through vegetative method; Effectively avoided the filial generation proterties segregation phenomenon that sexual reproduction brought; The proterties of reservation maternal plant that can be best makes tissue cultivating seedling produce purposive and specific aim.
Embodiment of the present invention is:
1, maternal plant is handled: select target dendrobium candidum plant, clean up with pure water, and remove blade, with tweezers the outer sheath skin of plant is peeled off totally.
2, soaking disinfection: the mercuric chloride solution soaking disinfection 15min that on toilet's superclean bench, plant is put into concentration 0.1%.
3, clean segment: sterilization finishes to take out plant, and is clean with aseptic water washing, with the aseptic operation cutter with the plant segment, every segment length 2.0-3.0cm.
4, evoked callus: above-mentioned plant section is seeded on the callus culture base, at temperature 20-28 ℃, humidity 30%-45%, illuminance 1800-2000Lx cultivates under the environment of illumination every day 8-12h.Incubation time 45-90 days, till callus grows.
Callus culture based formulas (in 1L):
1/2MS+20g white granulated sugar+5g agar+5.0mg 6-BA+0.5mg NAA.
5, callus pulverizes: callus is downcut on superclean bench with the aseptic operation cutter, pulverize dispersion with aseptic nipper.
6, shaking table is cultivated: the callus that will pulverize is put into cell culture fluid and is placed on the shaking table normal temperature to cultivate 15-25 days.
Cell culture formula of liquid (in 1L):
1/2MS+20 g white granulated sugar+5.0mg 6-BA+0.5mg NAA.
7, the ball stem is cultivated: with breeding the cell inoculation that in the cell culture fluid on ball stem medium, and at temperature 20-28 ℃, humidity 30%-45%, illuminance 1800-2000Lx cultivated 45-60 days under the environment of illumination every day 8-12h, grew until the ball stem.
Ball stem culture medium prescription (in 1L):
1/2MS+4.0mg 6-BA+0.4mg NAA+50g potato+50g banana+25 g white granulated sugar+5g agar.
8, the differentiation seedling is cultivated: with the ball stem of turning out, evenly be seeded on the differentiation seedling medium, and at temperature 20-28 ℃, humidity 30%-45%, illuminance 2000-2200Lx cultivated 40-60 days under the environment of illumination every day 8-12h, grew until the differentiation seedling.
Differentiation seedling culture medium prescription (in 1L):
1/2MS+3.0mg 6-BA+50g potato+50g banana+25 g white granulated sugar+5g agar.
9, seedling is cultivated: the differentiation seedling that will turn out evenly is seeded on the seedling medium, at temperature 20-28 ℃, and humidity 30%-45%, illuminance 2000-2200Lx cultivated 40-60 days under the environment of illumination every day 8-12h, grew until seedling.
Seedling culture medium prescription (in 1L):
1/2MS+ 3.0mg 6-BA+0.3mg NAA+50g banana+50g potato+25 g white granulated sugar+5g agar.
10, seedlings are cultivated: the seedling of turning out evenly is seeded on the seedlings medium, and at temperature 20-28 ℃, humidity 30%-45%; Illuminance 2000-2500Lx, the environment of illumination every day 8-12h was cultivated 50-90 days down, the bottle seedling rooting; During to 2-4 bar root/strain, but bottle seedling bottle outlet refining seedling.
Seedlings culture medium prescription (in 1L):
1/2MS+0.5mg NAA+50g potato+50g banana+25g white granulated sugar+5g agar+1.0g active carbon.
Technical scheme of the present invention has following effect:The present invention has effectively avoided the sexual reproduction meeting to produce this drawback that proterties is separated; The characteristic that has Almightiness type according to biological cell; The plant callus that utilization is cultivated is as breeding provenance, and it is of future generation through vegetative method the merit genetic stability of previous generation to be given, and in filial generation, has farthest kept the proterties of maternal plant; Make tissue cultivating seedling produce purposive and specific aim, for the genetic stability of dendrobium candidum merit provides a kind of effective method.
Embodiment
Embodiment 1
The dendrobium candidum plant is cleaned up with pure water, remove blade, the outer sheath skin of plant is peeled off totally with tweezers.On toilet's superclean bench, plant put into the mercuric chloride solution soaking disinfection 15min of concentration 0.1%.Plant finish is taken out in sterilization, and is clean with aseptic water washing, with the aseptic operation cutter with the plant segment, every segment length 2.5cm.The plant section is seeded on the callus culture base, 23 ℃ of temperature, humidity 35%, illuminance 1800Lx cultivates under the environment of illumination every day 10h.Incubation time 70 days, callus grows.Callus is downcut on superclean bench with the aseptic operation cutter, pulverize dispersion with aseptic nipper.The callus that will pulverize is put into cell culture fluid and is placed on the shaking table normal temperature to cultivate 15 days.Shaking table cultivate to finish breeding the cell inoculation that in the cell culture fluid on ball stem medium, 23 ℃ of temperature, and humidity 35%, illuminance 1800Lx cultivated 50 days under the environment of illumination every day 10h, and the ball stem grows.With the ball stem of turning out, evenly be seeded on the differentiation seedling medium, 23 ℃ of temperature, humidity 35%, illuminance 2200Lx cultivated 55 days under the environment of illumination every day 10h, and the differentiation seedling grows.The differentiation seedling of turning out evenly is seeded on the seedling medium, 23 ℃ of temperature, humidity 35%, illuminance 2200Lx cultivated 55 days under the environment of illumination every day 12h, and seedling grows.The seedling of turning out evenly is seeded on the seedlings medium, 23 ℃ of temperature, humidity 35%, illuminance 2300Lx, the environment of illumination every day 10h was cultivated 60 days down, bottle seedling rooting, every strain 2-4 bar root, bottle seedling bottle outlet refining seedling.
Employed medium is:
Callus culture based formulas (in 1L):
1/2MS+20g white granulated sugar+5g agar+5.0mg 6-BA+0.5mg NAA.
Cell culture formula of liquid (in 1L):
1/2MS+20 g white granulated sugar+5.0mg 6-BA+0.5mg NAA.
Ball stem culture medium prescription (in 1L):
1/2MS+4.0mg 6-BA+0.4mg NAA+50g potato+50g banana+25 g white granulated sugar+5g agar.
Differentiation seedling culture medium prescription (in 1L):
1/2MS+3.0mg 6-BA+50g potato+50g banana+25 g white granulated sugar+5g agar.
Seedling culture medium prescription (in 1L):
1/2MS+ 3.0mg 6-BA+0.3mg NAA+50g banana+50g potato+25 g white granulated sugar+5g agar.
Seedlings culture medium prescription (in 1L):
1/2MS+0.5mg NAA+50g potato+50g banana+25g white granulated sugar+5g agar+1.0g active carbon.
Embodiment 2
The dendrobium candidum plant is cleaned up with pure water, remove blade, the outer sheath skin of plant is peeled off totally with tweezers.On toilet's superclean bench, plant put into the mercuric chloride solution soaking disinfection 15min of concentration 0.1%.Plant finish is taken out in sterilization, and is clean with aseptic water washing, with the aseptic operation cutter with the plant segment, every segment length 3.0cm.The plant section is seeded on the callus culture base, 25 ℃ of temperature, humidity 40%, illuminance 1800Lx cultivates under the environment of illumination every day 10h.Incubation time 65 days, callus grows.Callus is downcut on superclean bench with the aseptic operation cutter, pulverize dispersion with aseptic nipper.The callus that will pulverize is put into cell culture fluid and is placed on the shaking table normal temperature to cultivate 20 days.Shaking table cultivate to finish breeding the cell inoculation that in the cell culture fluid on ball stem medium, 25 ℃ of temperature, and humidity 40%, illuminance 1800Lx cultivated 55 days under the environment of illumination every day 10h, and the ball stem grows.With the ball stem of turning out, evenly be seeded on the differentiation seedling medium, 25 ℃ of temperature, humidity 40%, illuminance 2000Lx cultivated 57 days under the environment of illumination every day 12h, and the differentiation seedling grows.The differentiation seedling of turning out evenly is seeded on the seedling medium, 25 ℃ of temperature, humidity 40%, illuminance 2200Lx cultivated 60 days under the environment of illumination every day 10h, and seedling grows.The seedling of turning out evenly is seeded on the seedlings medium, 25 ℃ of temperature, humidity 40%, illuminance 2500Lx, the environment of illumination every day 10h was cultivated 77 days down, bottle seedling rooting, every strain 2-4 bar root, bottle seedling bottle outlet refining seedling.
Employed medium is:
Callus culture based formulas (in 1L):
1/2MS+20g white granulated sugar+5g agar+5.0mg 6-BA+0.5mg NAA.
Cell culture formula of liquid (in 1L):
1/2MS+20 g white granulated sugar+5.0mg 6-BA+0.5mg NAA.
Ball stem culture medium prescription (in 1L):
1/2MS+4.0mg 6-BA+0.4mg NAA+50g potato+50g banana+25 g white granulated sugar+5g agar.
Differentiation seedling culture medium prescription (in 1L):
1/2MS+3.0mg 6-BA+50g potato+50g banana+25 g white granulated sugar+5g agar.
Seedling culture medium prescription (in 1L):
1/2MS+ 3.0mg 6-BA+0.3mg NAA+50g banana+50g potato+25 g white granulated sugar+5g agar.
Seedlings culture medium prescription (in 1L):
1/2MS+0.5mg NAA+50g potato+50g banana+25g white granulated sugar+5g agar+1.0g active carbon.
Claims (7)
1. the cellular tissure of a candidum tissue culturing seedling is cultivated mating system, it is characterized in that carrying out as follows:
(1) maternal plant is handled: select target dendrobium candidum plant, clean up with pure water, and remove blade, the outer sheath skin of plant is peeled off totally;
(2) soaking disinfection: the mercuric chloride solution soaking disinfection 15min that on toilet's superclean bench, plant is put into concentration 0.1%;
(3) clean segment: sterilization finishes to take out plant, and is clean with aseptic water washing, plant is cut to the plant section of every segment length 2.0-3.0cm with the aseptic operation cutter;
(4) evoked callus: above-mentioned plant section is seeded on the callus culture base; Under the environment of temperature 20-28 ℃, humidity 30%-45%, illuminance 1800-2000Lx and illumination every day 8-12h, cultivated 45-90 days, till callus grows;
(5) callus pulverizes: callus is downcut on superclean bench with the aseptic operation cutter, pulverize dispersion with aseptic nipper;
(6) shaking table is cultivated: the callus that will pulverize is put into cell culture fluid and is placed on the shaking table normal temperature to cultivate 15-25 days;
(7) the ball stem is cultivated: with breeding the cell inoculation that in the cell culture fluid on ball stem medium; Under the environment of temperature 20-28 ℃, humidity 30%-45%, illuminance 1800-2000Lx and illumination every day 8-12h, cultivated 45-60 days, grow until the ball stem;
(8) the differentiation seedling is cultivated: with the ball stem of turning out, evenly be seeded on the differentiation seedling medium, under the environment of temperature 20-28 ℃, humidity 30%-45%, illuminance 2000-2200Lx and illumination every day 8-12h, cultivated 40-60 days, grow until the differentiation seedling;
(9) seedling is cultivated: the differentiation seedling that will turn out evenly is seeded on the seedling medium, under the environment of temperature 20-28 ℃, humidity 30%-45%, illuminance 2000-2200Lx and illumination every day 8-12h, cultivates 40-60 days, grows until seedling;
(10) seedlings are cultivated: the seedling of turning out evenly is seeded on the seedlings medium; Under the environment of temperature 20-28 ℃, humidity 30%-45%, illuminance 2000-2500Lx and illumination every day 8-12h, cultivated 50-90 days; The bottle seedling rooting, during to 2-4 bar root/strain, bottle seedling bottle outlet refining seedling.
2. the cellular tissure of a kind of candidum tissue culturing seedling as claimed in claim 1 is cultivated mating system, and it is characterized in that: step (4) callus culture based formulas, (in 1L) is: 1/2MS+20g white granulated sugar+5g agar+5.0mg 6-BA+0.5mg NAA.
3. the cultivation mating system of a kind of candidum tissue culturing seedling as claimed in claim 1, it is characterized in that: step (6) cell culture formula of liquid (in 1L) is: 1/2MS+20 g white granulated sugar+5.0mg 6-BA+0.5mg NAA.
4. the cultivation mating system of a kind of candidum tissue culturing seedling as claimed in claim 1, it is characterized in that: step (7) ball stem culture medium prescription (in 1L) is: 1/2MS+4.0mg 6-BA+0.4mg NAA+50g potato+50g banana+25 g white granulated sugar+5g agar.
5. the cultivation mating system of a kind of candidum tissue culturing seedling as claimed in claim 1 is characterized in that: step (8) differentiation seedling culture medium prescription (in 1L) is: 1/2MS+3.0mg 6-BA+50g potato+50g banana+25 g white granulated sugar+5g agar.
6. the cultivation mating system of a kind of candidum tissue culturing seedling as claimed in claim 1 is characterized in that: step (9) seedling culture medium prescription (in 1L): 1/2MS+ 3.0mg 6-BA+0.3mg NAA+50g banana+50g potato+25 g white granulated sugar+5g agar.
7. the cultivation mating system of a kind of candidum tissue culturing seedling as claimed in claim 1, it is characterized in that: step (10) seedlings culture medium prescription (in 1L) is: 1/2MS+0.5mg NAA+50g potato+50g banana+25g white granulated sugar+5g agar+1.0g active carbon.
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103039368A (en) * | 2013-01-27 | 2013-04-17 | 云南天泉生物科技股份有限公司 | Sugar-free tissue culture method of dendrobium officinale |
| CN103120123A (en) * | 2013-02-04 | 2013-05-29 | 四川深达生物科技有限公司 | Culture medium for improving dendrobium officinale maturing rate and method for preparing culture medium |
| CN104094851A (en) * | 2014-07-23 | 2014-10-15 | 上海农业科技种子有限公司 | Dendrobium candidum test-tube seedling rooting medium and preparation method thereof |
| CN104396751A (en) * | 2014-11-19 | 2015-03-11 | 湖北神农架国家级自然保护区管理局 | Method for culturing dendrobium candidum in liquid culture medium |
| CN105010138A (en) * | 2015-06-29 | 2015-11-04 | 广西健宝石斛有限责任公司 | Tissue culture propagation method of Musa balbisiana Colla |
| CN105075859A (en) * | 2015-07-07 | 2015-11-25 | 南京师范大学 | Culture medium for promoting dendrobium candidum to take root by using bryozoatum |
| CN107041302A (en) * | 2017-01-10 | 2017-08-15 | 中国林业科学研究院林业研究所 | A kind of culture medium of dendrobium aseptic seeding and sprouting and rooting |
| CN109197586A (en) * | 2018-08-03 | 2019-01-15 | 南京龙源生态农业有限公司 | A kind of dendrobium candidum Protocorm base and its application |
| CN109197585A (en) * | 2018-08-03 | 2019-01-15 | 南京龙源生态农业有限公司 | A kind of method of dendrobium candidum sprouting and rooting |
| CN109197584A (en) * | 2018-08-03 | 2019-01-15 | 南京龙源生态农业有限公司 | A kind of dendrobium candidum root media and its application |
| CN111066656A (en) * | 2020-01-16 | 2020-04-28 | 贵州师范大学 | Culture medium group and method for dendrobium officinale tissue culture |
| CN115136891A (en) * | 2022-06-28 | 2022-10-04 | 广东金颖花卉苗木有限公司 | Method for inducing dendrobium nobile pseudoprotocorm |
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Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103039368A (en) * | 2013-01-27 | 2013-04-17 | 云南天泉生物科技股份有限公司 | Sugar-free tissue culture method of dendrobium officinale |
| CN103039368B (en) * | 2013-01-27 | 2014-02-05 | 云南天泉生物科技股份有限公司 | Sugar-free tissue culture method of dendrobium officinale |
| CN103120123A (en) * | 2013-02-04 | 2013-05-29 | 四川深达生物科技有限公司 | Culture medium for improving dendrobium officinale maturing rate and method for preparing culture medium |
| CN103120123B (en) * | 2013-02-04 | 2014-06-25 | 四川深达生物科技有限公司 | Culture medium for improving dendrobium officinale maturing rate |
| CN104094851A (en) * | 2014-07-23 | 2014-10-15 | 上海农业科技种子有限公司 | Dendrobium candidum test-tube seedling rooting medium and preparation method thereof |
| CN104396751A (en) * | 2014-11-19 | 2015-03-11 | 湖北神农架国家级自然保护区管理局 | Method for culturing dendrobium candidum in liquid culture medium |
| CN105010138A (en) * | 2015-06-29 | 2015-11-04 | 广西健宝石斛有限责任公司 | Tissue culture propagation method of Musa balbisiana Colla |
| CN105010138B (en) * | 2015-06-29 | 2017-05-24 | 广西健宝石斛有限责任公司 | Tissue culture propagation method of Musa balbisiana Colla |
| CN105075859A (en) * | 2015-07-07 | 2015-11-25 | 南京师范大学 | Culture medium for promoting dendrobium candidum to take root by using bryozoatum |
| CN107041302A (en) * | 2017-01-10 | 2017-08-15 | 中国林业科学研究院林业研究所 | A kind of culture medium of dendrobium aseptic seeding and sprouting and rooting |
| CN109197586A (en) * | 2018-08-03 | 2019-01-15 | 南京龙源生态农业有限公司 | A kind of dendrobium candidum Protocorm base and its application |
| CN109197585A (en) * | 2018-08-03 | 2019-01-15 | 南京龙源生态农业有限公司 | A kind of method of dendrobium candidum sprouting and rooting |
| CN109197584A (en) * | 2018-08-03 | 2019-01-15 | 南京龙源生态农业有限公司 | A kind of dendrobium candidum root media and its application |
| CN111066656A (en) * | 2020-01-16 | 2020-04-28 | 贵州师范大学 | Culture medium group and method for dendrobium officinale tissue culture |
| CN115136891A (en) * | 2022-06-28 | 2022-10-04 | 广东金颖花卉苗木有限公司 | Method for inducing dendrobium nobile pseudoprotocorm |
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Denomination of invention: Method for cultivating and breeding cell tissue of tissue culture seedling of Dendrobium candidum Effective date of registration: 20160108 Granted publication date: 20130417 Pledgee: Yunnan sincere financing Company limited by guarantee Pledgor: Gold nine ground bio tech ltd, Yunnan Registration number: 2015530000014 |
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