CN102422811B - Dendrobium officinale tissue culture reproduction method - Google Patents
Dendrobium officinale tissue culture reproduction method Download PDFInfo
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Abstract
The invention belongs to the field of biotechnology, and relates to a dendrobium officinale aseptic seedling hormone-free culture and rapid reproduction method. The content comprises culture technology of germination of dendrobium officinale aseptic seedlings, formation and differentiation of protocorms, and seedling strengthening and rooting. Mature capsules are selected, and are aseptically sown in a protocorm culture medium; after 1.5 months, the grown protocorms are put in a differentiation culture medium for seedling differentiation; after 1.5 months, the formed 1-cm seedlings are put in a seedling strengthening and rooting culture medium; after 2 months, container seedlings with consistent growth and a plant height of 6-8 cm can be observed, and each container seedling has at least 5 developed root systems. Compared with traditional reproduction modes, the method adopts hormone-free culture, and greatly increases the reproduction speed of the seedlings; the seedlings grow robustly, has obvious pseudobulbs, and a lot of hairy roots; the transplanting survival rate is high; the production cost is low; no food safety problem such as mutagenesis or pollution is caused; the reproduction speed is high; the rooted seedlings are robust; the transplanting survival rate is high; and the method reaches the industrial production level.
Description
Affiliated technical field:
The invention belongs to plant biotechnology field, specifically, relate to a kind of no hormone tissue cultivation rapid breeding method of dendrobium candidum.
Background technology:
Dendrobium candidum (Dendrobium officinale); The another name ribbed hedyotis herb; Being famous and precious medicinal plant in the orchid family (Orchidaceae) Dendrobium (Dendrobium), is first of the Chinese nine immortal grass, and extremely abundant medical value is arranged; Wild resource is few, mainly is distributed in ground such as Yunnan, Guangxi, Anhui.According to Compendium of Material Medica record, dendrobium candidum have " cure mainly in the wound, tonifying five zang organs consumptive disease, reinforcing yin essence benefit essence, thick stomach, mend in never sufficient, flat stomach Qi, longue meat, intelligence development remove the effect of shying, making light of one's life by commiting suicide and prolonging life ".Because its stem is used as medicine, pharyngolaryngitis, raising body immunity and even tumour are all had significant curative effect, become the health-care good product that has won fame both at home and abroad.Because excavate in the long-term field of people, wild dendrobium candidum has arrived situation in imminent danger.The dendrobium candidum fruit-setting rate is very low, and seed is difficult for germinateing, and the natural propagation rate is extremely low.Setting up the tissue-culturing rapid propagation system can fundamentally address this problem.
So far, it is explant that stem apex, sprouting, sleeping bud are all selected in the fast numerous legislation of candidum tissue culturing mostly for use, cultivates through intending the protocorm approach.Produce the protocorm tissue culture method with aseptic seed and rarely have report.Although these methods have been optimized the medium component of traditional mode of production, and are easy and simple to handle, production cost is low, and medicinal ingredient exists reproduction rate lower near wild dendrobium candidum, and differentiation is slow, is difficult to produce in batches and problem such as batch production production.
Goal of the invention:
Above-mentioned deficiency to the prior art existence; The present invention aims to provide a kind of no hormone tissue rapid propagation method of dendrobium candidum; Each stage is adopted different optimization culture medium prescriptions, makes it under the situation that the wild species source lacks and reproduction rate is low, can multiply fast, and the preservation of the merit of these species can be protected; Avoided simultaneously through intending the long shortcoming of cycle that the protocorm approach causes; Shorten cultured in vitro time and production cost, improve rooting rate and seedling replanting rate, establish to organize for the sustainable use of dendrobium candidum and cultivate seedling and breed the basis.
Above-mentioned purpose of the present invention is to realize with following technical scheme:
The no hormone tissue culture propagation method of dendrobium candidum comprises that capsule is sterilized, protocorm is induced, and differentiation and strengthening seedling and rooting are cultivated; The test-tube seedling transplanting step; The ripe capsule of getting dendrobium candidum carries out that protocorm is induced, differentiation, strong sprout, culture of rootage, and the temperature of cultivation is 20-23 ℃, and humidity is 30-45%; Illumination condition is the artificial fill-in light 1500-2000LUX of natural daylight 2000-2500LUX+, and light application time is 12h/d; Described protocorm inducing culture is improvement MS+10% banana+10% potato+peptone 1%, and differential medium is B
5+ 10% banana+10% potato, strengthening seedling and rooting medium are 1/2 improvement MS+20% banana+active carbon 0.5%; Improvement MS uses KNO
3Reduce by half NH
4NO
3Reduce by half.
In the described tissue culture propagation method, capsule sterilization is earlier with 5% washing powder water logging bubble cleaning 20min, again with aseptic water washing to non-foam; Adopt 75% alcohol sterilization 3min then; Carry out surface sterilization 10min-15min with 0.1% mercuric chloride solution again behind the aseptic water washing 3 times, carry out at twice, 5-8min sterilizes for the first time; Constantly the concussion back is with sterile water wash 3-5 time, sterilizes behind the 5-7min with sterile water wash 3-5 time with 0.1% mercuric chloride again.
Described tissue culture propagation method, transplanting are that the tissue cultivating seedling of taking root is cultivated 45 days refining seedlings 30 days later in blake bottle, transplant in green house again.
Described tissue culture propagation method, transplanting medium are plain boiled water tongue or sawdust, pine bark, and pH is 5.8.
Described tissue culture propagation method is transplanted 8-11 point in the morning, carries out under temperature 18-25 ℃.
Particularly, tissue culture propagation method of the present invention is: choose the ripe capsule of dendrobium candidum, clean 20min with 5% washing powder water logging bubble earlier; Again with aseptic water washing to non-foam, adopt 75% alcohol sterilization 3min then, carry out surface sterilization 10min-15min with 0.1% mercuric chloride solution again behind the aseptic water washing 3 times; Carry out at twice; The 5-8min that sterilizes for the first time, constantly the concussion back is with sterile water wash 3-5 time, sterilizes behind the 5-7min with sterile water wash 3-5 time with 0.1% mercuric chloride again.Capsule is placed on the filter paper after the sterilization, cut rip cutting behind the two ends, seed broadcasting is promptly improved in MS+10% banana+10% potato+peptone 1% in the protocorm inducing culture, pH is 5.8, and improvement MS uses KNO
3Reduce by half NH
4NO
3Reduce by half, after 1.5 months the protocorm that grows being put into differential medium is B
5Carry out the seedling differentiation in+10% banana+10% potato; 1.5 after individual month the seedling that forms is put into i.e. 1/2 improvement, the 1/2 improvement MS+20% banana+active carbon 0.5% of strong sprout and root media; Cultivate the laggard capable training tissue culture seedling 30 days of taking root in 45 days in the bottle; Transplant on plain boiled water tongue or sawdust, pine bark, humidity 40%-50%, pH are 5.8 matrix seedbed, air humidity is 60%-80%, and every day, each water spray was once sooner or later.
Compared with prior art, the advantage of tissue culture method of the present invention is:
The inventive method is cultivating the medium that different phase has adopted different formulations, to adapt to plant in the demand of different times to nutriment, at the protocorm induction period; Added 1% peptone; Effectively raise the inductivity of seed,, adopted B in protocorm differential period
5Medium makes value-added coefficient bring up to 10, has fundamentally solved the low problem of dendrobium candidum tissue culture differentiation rate.In rooting process, added 0.5% active carbon, make that taking root the bar number does not add doubling of active carbon than having, and the sturdy prosperity of root system, improved transplanting survival rate.
Set up effective batch production candidum tissue culturing quick-breeding method of the present invention, solved prior seed breeding difficulty, the situation that breeding is obstructed.Through the seedling that tissue-culturing rapid propagation of the present invention obtains, become seedling easy, uniformity is strong, robust growth, the leaf look dark green, and seldom damage by disease and insect is easy to management.Do not receive season limit, whenever can organize training.Can keep the species characteristic, this kind can be continued and sustainable use.Because no hormone adds, and does not have food-safety problems such as mutagenesis, pollution, has practiced thrift production cost, and differentiation rate is high in the production process, and reproduction speed is fast.
Through tissue culture and rapid propagation method of the present invention; But the dendrobium candidum of breeding growth coefficient in January is 10, and average rooting rate is 98%, and average transplanting survival rate is 95%; Greatly improved the reproduction coefficient of dendrobium candidum; Stoped because open-air plant lacks seed and seed viability is low less, caused the danger of this species extinction, for the preservation and the sustainable use of these species provides very effective propagation method.
Utilize capsule aseptic seed propagation method of the present invention; It is low to solve the protocorm differentiation rate that runs into when all the time using capsule to breed, and the rate of increase is low, and algebraically is low; The problem that the test-tube plantlet growing way is weak; The prescription that uses the present invention to optimize, the medicinal ingredient of the dendrobium candidum of production and quite wild or a little more than the active ingredient of wild dendrobium candidum has reached the purpose of high efficiency quick breeding.
The present invention has formed the special no hormone dendrobium candidum tissue culture propagating technical system of a cover through constantly exploring experiment, and the patent medium component is complicated before having overcome; Incubation is loaded down with trivial details, does not have actual industrialized propagation experience, for mass artificial plantation dendrobium candidum provides guarantee; Present stage has used batch production of the present invention to produce dendrobium candidum bottle seedling 4 years, and problem is few in the production process, and differentiation rate is high; Reproduction speed is fast; Subculture algebraically is many, and composition and wild dendrobium candidum have proved that this propagation technique is to be fit to the tissue culture method that batch production is produced so far quite or a little more than the active constituent content of wild dendrobium candidum.
Embodiment:
Following examples are used for further specifying essentiality content of the present invention.Based on the description of technical scheme of the present invention and embodiment, perhaps the same domain technical staff can also carry out some modifications and improvement to technical scheme of the present invention on basis of the present invention.Therefore, do not depart from modification and the improvement of being made on the main technical schemes of the present invention basis, all should belong to the present invention's scope required for protection.
Embodiment 1:
Ripe capsule with dendrobium candidum (Dendrobium officinale) is that explant exploration protocorm is induced optimum medium.Clean 20min with 5% washing powder water logging bubble earlier; Again with aseptic water washing to non-foam, adopt 75% alcohol sterilization 3min then, carry out surface sterilization 10min-15min with 0.1% mercuric chloride solution again behind the aseptic water washing 3 times; Carry out at twice; The 5-8min that sterilizes for the first time, constantly the concussion back is with sterile water wash 3-5 time, sterilizes behind the 5-7min with sterile water wash 3-5 time with 0.1% mercuric chloride again.Capsule is placed on the filter paper after the sterilization, cut rip cutting behind the two ends, seed broadcasting is promptly improved in MS+10% banana+10% potato+peptone 1% in the protocorm inducing culture, pH is 5.8, and improvement MS uses KNO
3Reduce by half NH
4NO
3Reduce by half.1.5 after individual month the protocorm that grows being put into differential medium is B
5Carry out the seedling differentiation in+10% banana+10% potato; 1.5 after individual month the seedling that forms is put into i.e. 1/2 improvement, the 1/2 improvement MS+20% banana+active carbon 0.5% of strong sprout and root media; Cultivate the laggard capable training tissue culture seedling 30 days of taking root in 45 days in the bottle; Transplant on plain boiled water tongue or sawdust, pine bark, humidity 40%-50%, pH are 5.8 matrix seedbed, humidity is 60%-80% in the booth, and every day, each water spray was once sooner or later.
The inventive method is cultivating the medium that different phase has adopted different formulations, to adapt to plant in the demand of different times to nutriment, at the protocorm induction period; Added 1% peptone; Effectively raise the inductivity of seed,, adopted B in protocorm differential period
5Medium makes value-added coefficient bring up to 10, has fundamentally solved the low problem of dendrobium candidum tissue culture differentiation rate.In rooting process, added 0.5% active carbon, make that taking root the bar number does not add doubling of active carbon than having, and the sturdy prosperity of root system, improved transplanting survival rate.
The culture medium prescription of optimization of the present invention has not only improved coefficient of differentiation but also has promoted to take root, and subculture is after 30 days, each blastogenesis linear leaf 7-8 sheet, and plant height 3cm, a large amount of fibrous roots are green in vain.In addition, temperature has also played very crucial effect to inducing with the growth of tissue cultivating seedling and taking root of clump bud, and temperature is at 21-23 ℃, and illumination is the artificial fill-in light 1500-2000LUX of natural daylight 2000-2500LUX+, and light application time 12h/d helps the protocorm differentiation.The leaf look dark green with this understanding, robust growth, and the growth coefficient of test-tube plantlet is higher, and average rooting rate is 98%, and average transplanting survival rate is 95%, has greatly improved the reproduction coefficient of dendrobium candidum.
In bottle, take root closelyer when every strain dendrobium candidum, root is long can do to transplant preparation during for 1-3cm.At first bottle cap is opened and refined seedling, simultaneously, prepare to transplant preparation in the greenhouse, transplanting medium is plain boiled water tongue or sawdust pine bark etc., pH5.8.Transplanting medium is packed in the transplanting dish; Clear water soaks into, and the refining tissue cultivating seedling of seedling after 30 days transplanted in the transplanting seedlings dish in the greenhouse, and transplanting time is preferably in morning 8-11 point to carry out; Temperature is at 18-25 ℃; Cover and water 1-2 time film and every day, note in the morning the 8-10 point with the front opening film with ventilation, humidity is between 50-60%.Can take film off later on, and carry out normal management in 10 days.
Tissue culture and rapid propagation method through the invention described above; But the dendrobium candidum of breeding growth coefficient in January is 10, and average rooting rate is 98%, and average transplanting survival rate is 95%; Greatly improved the reproduction coefficient of dendrobium candidum; Stoped because open-air plant lacks seed and seed viability is low less, caused the danger of this species extinction, for the preservation and the sustainable use of these species provides very effective propagation method.
Utilize capsule aseptic seed propagation method of the present invention; It is low to solve the protocorm differentiation rate that runs into when all the time using capsule to breed, and the rate of increase is low, and algebraically is low; The problem that the test-tube plantlet growing way is weak; The prescription that uses the present invention to optimize, the medicinal ingredient of the dendrobium candidum of production and quite wild or a little more than the active ingredient of wild dendrobium candidum has reached the purpose of high efficiency quick breeding.
The present invention has formed the special no hormone dendrobium candidum tissue culture propagating technical system of a cover through constantly exploring experiment, and the patent medium component is complicated before having overcome; Incubation is loaded down with trivial details, does not have actual industrialized propagation experience, for mass artificial plantation dendrobium candidum provides guarantee; Present stage has used batch production of the present invention to produce dendrobium candidum bottle seedling 4 years, and problem is few in the production process, and differentiation rate is high; Reproduction speed is fast, does not add any hormone, does not have food-safety problems such as mutagenesis, pollution; The seedling stalwartness of taking root; Transplanting survival rate is high, and medicinal ingredient and wild dendrobium candidum have proved that this propagation technique is the tissue culture method that is fit to batch production production so far quite or a little more than the active constituent content of wild dendrobium candidum.
Claims (6)
1. the no hormone tissue culture propagation method of dendrobium candidum comprises that capsule is sterilized, protocorm is induced, and differentiation and strengthening seedling and rooting are cultivated; The test-tube seedling transplanting step; The capsule that it is characterized in that getting dendrobium candidum cuts that the seed that takes out carries out that protocorm is induced, differentiation, strong sprout, culture of rootage, and the temperature of cultivation is 20-23 ℃, and humidity is 30-45%; Illumination condition is natural daylight 2000-2500 LUX+artificial fill-in light 1500-2000 LUX, and light application time is 12h/d; The protocorm inducing culture is improvement MS+10% banana+10% potato+peptone 1%, and differential medium is B
5+ 10% banana+10% potato, strengthening seedling and rooting medium are 1/2 improvement MS+20% banana+active carbon 0.5%; Wherein improve MS and use KNO
3Reduce by half NH
4NO
3Reduce by half.
2. tissue culture propagation method according to claim 1 is characterized in that the sterilization of said capsule earlier with 5% washing powder water logging bubble cleaning 20min, again with aseptic water washing to non-foam; Adopt 75% alcohol sterilization 3min then; Carry out surface sterilization 10min-15min with 0.1% mercuric chloride solution again behind the aseptic water washing 3 times, carry out at twice, 5-8min sterilizes for the first time; Constantly the concussion back is with sterile water wash 3-5 time, sterilizes behind the 5-7min with sterile water wash 3-5 time with 0.1% mercuric chloride again.
3. tissue culture propagation method according to claim 1, it is characterized in that transplanting is that the tissue cultivating seedling of taking root is cultivated in blake bottle and refined seedling 30 days later in 45 days, transplants in green house again.
4. according to the said tissue culture propagation method of claim 1, it is characterized in that transplanting medium is plain boiled water tongue or sawdust, pine bark, pH is 5.8.
5. tissue culture propagation method according to claim 1 is characterized in that transplanting 8-11 point in the morning, carries out under temperature 18-25 ℃.
6. according to any described tissue culture propagation method among claims 1-5, it is characterized in that said capsule sterilization earlier with 5% washing powder water logging bubble cleaning 20min, again with aseptic water washing to non-foam; Adopt 75% alcohol sterilization 3min then, carry out surface sterilization 10min-15min with 0.1% mercuric chloride solution again behind the aseptic water washing 3 times, carry out at twice; 5-8min sterilizes for the first time; Constantly the concussion back is with sterile water wash 3-5 time, sterilizes behind the 5-7min with sterile water wash 3-5 time with 0.1% mercuric chloride again, and capsule is placed on the filter paper after the sterilization; Cut rip cutting behind the two ends; The seed uniform broadcasting is promptly improved in MS+10% banana+10% potato+peptone 1% in the protocorm inducing culture, and pH is 5.8, and improvement MS uses KNO
3Reduce by half NH
4NO
3Reduce by half; 1.5 after individual month the protocorm that grows being put into differential medium is B
5Carry out the seedling differentiation in+10% banana+10% potato; 1.5 after individual month the seedling that forms is put into the i.e. 1/2 improvement MS+20% banana+active carbon 0.5% of strong sprout and root media; Cultivate the laggard capable training tissue culture seedling 30 days of taking root in 45 days in the bottle; Transplant on plain boiled water tongue or sawdust, pine bark, humidity 40%-50%, pH are 5.8 matrix seedbed, humidity is 60%-80% in the booth, and every day, each water spray was once sooner or later.
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| CN102696482B (en) * | 2012-06-18 | 2013-06-12 | 湖南省林业科学院 | Method for detoxifying clonal tissue culture explant of Camellia oleifera Abel |
| CN102948368B (en) * | 2012-11-13 | 2014-03-12 | 安徽新津铁皮石斛开发有限公司 | Tissue culture method of dendrobium candidum |
| CN103181316A (en) * | 2013-03-11 | 2013-07-03 | 安徽新津铁皮石斛开发有限公司 | Precursor culture medium for wild dendrobium candidum |
| CN103250639B (en) * | 2013-04-19 | 2015-08-12 | 浙江韵芝堂生物科技有限公司 | A kind of method of seed culture of dendrobium candidum |
| CN103202234B (en) * | 2013-04-28 | 2014-05-14 | 陕西师范大学 | Rapid propagation method of dendrobium candidum |
| CN104221827B (en) * | 2013-06-06 | 2016-06-08 | 上海中意农业科技发展有限公司 | A kind of candidum tissue culturing seedling directly transplants soilless planting method |
| CN105325290A (en) * | 2014-08-13 | 2016-02-17 | 云南省德宏热带农业科学研究所 | Hormone-free tissue culture method for dendrobium lituiflorum |
| CN104145795B (en) * | 2014-08-15 | 2016-08-24 | 湖南省农业信息与工程研究所 | A kind of dendrobe cultivation method improving facility utilization rate |
| CN104322371A (en) * | 2014-10-29 | 2015-02-04 | 安徽牧龙山生态旅游开发股份有限公司 | Tissue culture medium and tissue culture method of dendrobium officinale |
| CN104542289A (en) * | 2014-12-31 | 2015-04-29 | 安徽康久生物科技有限公司 | Method for performing tissue culture of dendrobium candidum seedlings |
| CN104813936B (en) * | 2015-05-11 | 2017-04-26 | 福建农林大学 | Method for inducing Chinese pholidota herb plant to regenerate and propagate by using pseudobulbs |
| CN106359044A (en) * | 2016-08-30 | 2017-02-01 | 柳州市泓吉农业科技有限公司 | Breeding and planting method of dendrobium officinale |
| CN107278554A (en) * | 2017-05-27 | 2017-10-24 | 霍山宝信园石斛开发有限公司 | A kind of method for culturing seedlings of the stem of noble dendrobium |
| CN109906940A (en) * | 2019-04-22 | 2019-06-21 | 重庆市渝东南农业科学院 | A kind of Seeds of Dendrobium Candidum aseptic seeding method |
| CN113575399A (en) * | 2021-09-02 | 2021-11-02 | 南京农业大学 | Dendrobium direct seeding seedling method |
| CN115804342A (en) * | 2022-12-05 | 2023-03-17 | 贵州康旅食斛开发有限公司 | Tissue culture method for dendrobium officinale |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101564008B (en) * | 2008-04-24 | 2011-03-23 | 中国医学科学院药用植物研究所 | Hormone-free cultivation and rapid propagation method of dendrobium candidum axenic seedlings |
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