CN104012401A - Rapid propagation method of dendrobium huoshanense - Google Patents
Rapid propagation method of dendrobium huoshanense Download PDFInfo
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- CN104012401A CN104012401A CN201310061665.9A CN201310061665A CN104012401A CN 104012401 A CN104012401 A CN 104012401A CN 201310061665 A CN201310061665 A CN 201310061665A CN 104012401 A CN104012401 A CN 104012401A
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Abstract
The invention relates to a rapid propagation method of dendrobium huoshanense. The method comprises the steps of obtaining seeds, stem tips or caulicles with axillary buds in dendrobium huoshanense fruits, soaking the fruits in absolute ethanol, and burning on a flame of an alcohol lamp, and inoculating; washing the stem tips or caulicles by clear water, carrying out surface sterilization respectively by 75% alcohol and 0.2% mercuric chloride (mercury bichloride) solutions, washing for 3-4 times by sterile water, putting a product on a piece of sterilized filter paper, and absorbing the water thoroughly; shearing the stem section into 0.5-1.5cm of small sections with joints under the aseptic condition, and inoculating the small sections into a culture medium; after the seeds germinate or the stem tips and the caulicles grow into protocorm-like bodies, carrying out subculture for mass multiplication; putting the protocorms into a differentiation bud culture medium; when the budling height is 2-3cm, transferring into a rooting medium; and when a test-tube plantlet has 4-5 stretching leaves, the root system is normal, and the height is 6cm or higher, taking the test-tube plantlet outside a bottle, and transplanting.
Description
Affiliated field: the present invention relates to biological technical field, particularly, relate to the method for quickly breeding of Dendrobidium huoshanness.
Background technology: Dendrobidium huoshanness is that the orchid family stone solution belongs to medicinal plant, and under field conditions (factors), seed need could be sprouted with mycosymbiosis, and germination rate is extremely low.Long-term immoderate the excavating of extremely low reproduction rate and people makes wild dendrobium candidum resource day by day exhausted, and market is anxious to be lacked.The effective way that solves Dendrobidium huoshanness market demand problem will expand cultivated area and plantation region exactly, therefore source of seedling especially the screening of high quality seedling become letter problem to be solved.Effective method carries out artificial quick numerous good seed by plant tissue culture technique exactly.
Summary of the invention: the object of the present invention is to provide a kind of stem of noble dendrobium seedling method for quickly breeding, to save stem of noble dendrobium class species in imminent danger, make it to become renewable medicine source, and plant Technical Reference and practical value are provided for stem of noble dendrobium class GAP kind.In order to realize above-mentioned purpose of the present invention, the invention provides following technical scheme:
The method for quickly breeding of Dendrobidium huoshanness: get seed or stem apex or the young stem with axillalry bud in stem of noble dendrobium fruit, fruit is soaked in straight alcohol then burns on alcolhol burner flame, then for inoculation, stem apex or young stem are after clear water rinses, use respectively 75% alcohol, 0.2% mercury chloride (mercuric chloride) solution carries out surface sterilization, aseptic water washing 3-4 time, be placed in suck dry moisture on the filter paper after sterilizing, under aseptic condition, stem section is cut into the band joint segment of 0.5-1.5cm, be seeded in medium, treat that seed germination or stem apex and young stem grow up to plan protocorm, carry out subculture and cultivate a large amount of propagation, then protocorm is placed on the medium of Bud Differentiation, in the time of the high 2-3cm of budling, proceed to root media, treat that test-tube plantlet tool 4-5 sheet stretches leaf, root system is normal, highly reach 6cm and when above, bottle outlet is transplanted.
Particularly, get the fruit that field acquisition or hybridization pollination are tied, after alcolhol burner burning sterilizing, under aseptic condition, pericarp is opened, planting seed is being spent on the agar medium of precious No. 1 (N: P: K=7: 6: 19) 3g/L, and cracking fruit wraps up with nylon cloth the seed that is scattered, at 0.2%HgCl
2middle sterilization 6-8min, after aseptic water washing is clean, sowing is being spent on the agar medium of precious No. 1 (N: P: K=7: 6: 19) 3g/L, in immature fruit, seed can be sowed and spend on the agar medium of precious No. 1 3g/L+NAA1mg/L, 25 ± 2 DEG C of culturing room's temperature, intensity of illumination is 1500-2000Lx, illumination 12h/d, medium PH is 5, cultivate 8-12 days seeds and turn green by yellow, increase spherical in shape, base portion has the protocorm of filiform hair, subculture is cultivated on the agar medium of 1/2MS+NAA1+ mashed potatoes 3%+ banana puree 15%, after 30 days, differentiate 1cm budlet and 0.5cm clump bud, can obtain high about 2-3cm budling through the cultivation of 1-2 month again, the Rooting and hardening-off culture base cultivation that proceeds to 1/2MS+NAA1+ mashed potatoes 3%+ banana puree 15% added intense light irradiation to 4000Lx after 1 month, cultivate one month, when test-tube plantlet tool 4-5 sheet stretches leaf, root system is normal, while highly reaching 6cm, can supply to transplant by bottle outlet, when bottle outlet, remove the agar on root system, one clump of plantation of 3-5 strain, planting matrix has sawdust, bark, tongue Xue and haydite etc., the water of 20: 1 for matrix: formaldehyde sealing sterilization 24h or sterilize by vapours, after cultivation, be placed in shade rate 70% booth, temperature of shed is controlled at 20 DEG C-28 DEG C, note moisturizing and draining, within 8-16 days, spray once with 1/10MS liquid, just can field planting after test-tube plantlet grows Xin Gen or young leaves.
Particularly, by field acquisition to stem of noble dendrobium stem apex or young stem segment sterilizing with axillalry bud after be seeded on the agar medium of 1/2MS+NAA1mg/L+ mashed potatoes 3%0 banana puree 15%, culturing room's temperature remains 25 ± 2 DEG C, intensity of illumination is 1500-2000Lx, illumination 12h/d, medium PH is 5, cultivate 30-60 days, axillalry bud place can pumping shoot, shoot can be cut and repeat to cultivate, the short clump bud shape that is of stem apex pumping shoot, on above-mentioned medium, continue to cultivate, grow up to complete test-tube plantlet for transplanting, when bottle outlet, remove the agar on root system, one clump of plantation of 3-5 strain, planting matrix has sawdust, bark, fresh and the haydite of tongue etc., the water of 20: 1 for matrix: formaldehyde sealing sterilization 24h or sterilize by vapours, after cultivation, be placed on shade rate 70% in booth, temperature of shed is controlled at 20 DEG C-28 DEG C, note moisturizing and draining, within 8-16 days, spray once with 1/10MS liquid.
Embodiment:
Embodiment 1:
1 materials and methods
1.1 for examination material
Dendrobidium huoshanness: be selected from Guangnan, Simao, Zhejiang, Yingjiang
1.2 method
25 ± 2 DEG C of culturing room's temperature, preserving chamber temperature 15-20 DEG C, intensity of illumination is 1500-2000Lx, illumination 12h/d.Medium is different with cultivating object and different developmental phases.Medium PH is 5.
Explant derives from seed or stem apex in the various fruit of Dendrobium, and fruit is soaked in straight alcohol, then on alcolhol burner flame, burns, then for inoculation; Stem apex or young stem are after clear water rinses, use respectively 75% alcohol, 0.2% mercury chloride (mercuric chloride) solution to carry out surface sterilization, aseptic water washing 3-4 time, be placed in suck dry moisture on the filter paper after sterilizing, under aseptic condition, the band joint segment that stem section is cut into 0.5-1.5cm, is seeded in medium.
2 results
2.1 tissues are cultivated and quick propagating technology
The present invention, in order to keep genetic stability and large-scale production object, has adopted seed asepsis cultivation and stem apex, stipes meristematic tissue to cultivate two kinds of modes.
2.1.1 seed asepsis is cultivated
The fruit that seed source is tied in field acquisition and own hybridization pollination.The cracking fruit of field acquisition, collects seed and wraps up with nylon cloth, the 7min that sterilizes in 0.2% mercury chloride, and after aseptic water washing is clean, sowing is being spent on the agar medium of precious No. 1 (N: P: K=7: 6: 19) 3g/L.Complete fruit can be opened pericarp after alcolhol burner burning sterilizing, by planting seed on above-mentioned medium.It is yellow that Dendrobium seed is mostly, cultivates about approximately 10 days and can turn green, increases, and then grows up to the bud that there is blade tip on top or emerges, and base portion has the protocorm of filiform hair, shows seed germination and germination rate higher (reaching more than 95%).
2.1.2 meristematic tissue is cultivated
By field acquisition to stone solution be seeded on the agar medium of 1/2MS+NAA1mg/L+ mashed potatoes 3%+ banana puree 15% after belonging to stem apex or the young stem segment sterilizing with axillalry bud, cultivate about 30 days, axillalry bud place can pumping shoot, and shoot can be cut and repeat to cultivate, the short clump bud shape that is of stem apex pumping shoot.On this medium, they can grow up to complete test-tube plantlet, for transplanting.If stem apex and young stem are seeded on the agar medium of 1/2MS or 1/2MS+BA0.5+NAA0.2, explant base portion can differentiate a large amount of plan protocorms.Intend protocorm on the agar medium of 1/2MS+NAA1+ mashed potatoes 3%+ banana puree 15%, also can grow up to seedlings for transplanting.
2.1.3 successive propagation
Dendrobium Protocorm Multiplication speed 25 days to 50 days (according to kind of an amount) on average can reach 1: 5-10 doubly.Previous step protocorm and plan Protocorm base are cultivated about 30 days, can obtain 10%1cm budlet and 10%0.5cm clump bud, can obtain high about 2-3cm budling (accounting for 50% left and right) through the cultivation of 1-2 month again, proceeding to Rooting and hardening-off culture base (1/2MS ten NAA1+ mashed potatoes 3%+ banana puree 15%) cultivates and adds intense light irradiation (4000Lx and more than) after 1 month, cultivate about one month, test-tube plantlet can reach the standard of transplanting seedlings.4.5 months to 6 months whole moon production cycle.
2.1.4 test-tube seedling transplanting
When test-tube plantlet, at least 4-5 sheet stretching, extension blade root system is normal, healthy, while highly reaching 6cm left and right, can supply to transplant by bottle outlet, when bottle outlet, being careful can not injured blade and the tip of a root, removes the agar on root system, one clump of plantation of 3-5 strain, planting matrix has the fresh and haydite of sawdust, bark, tongue etc., the water of 20: 1 for matrix: formaldehyde sealing sterilization 2411 or sterilize by vapours.After cultivation, be placed on shade rate approximately 70% in booth, temperature of shed is controlled at 20 DEG C-28 DEG C, notes moisturizing and draining.After test-tube plantlet grows Xin Gen or young leaves, within 8-16 days, use 1/10MS liquid, spray once, promote growth of seedling.
Claims (3)
1. a method for quickly breeding for Dendrobidium huoshanness, is characterized in that getting seed or stem apex or the young stem with axillalry bud in stem of noble dendrobium fruit, and fruit is soaked in straight alcohol, then on alcolhol burner flame, burns, then for inoculation, stem apex or young stem are after clear water rinses, use respectively 75% alcohol, 0.2% mercury chloride (mercuric chloride) solution carries out surface sterilization, aseptic water washing 3-4 time, be placed in suck dry moisture on the filter paper after sterilizing, under aseptic condition, stem section is cut into the band joint segment of 0.5-1.5cm, be seeded in medium, treat that seed germination or stem apex and young stem grow up to plan protocorm, carry out subculture and cultivate a large amount of propagation, then protocorm is placed on the medium of Bud Differentiation, in the time of the high 2-3cm of budling, proceed to root media, treat that test-tube plantlet tool 4-5 sheet stretches leaf, root system is normal, highly reach 6cm and when above, bottle outlet is transplanted.
2. the method for claim 1, it is characterized in that getting the fruit that field acquisition or hybridization pollination are tied, complete fruit is after alcolhol burner burning sterilizing, under aseptic condition, pericarp is opened, planting seed is being spent precious No. 1 (on the agar medium of (N: P: K=7: 6: 19) 3g/L, cracking fruit, with the nylon cloth parcel seed that is scattered, at 0.2%HgCl
2middle sterilization 6-8min, after aseptic water washing is clean, sowing is being spent on the agar medium of precious No. 1 (N: P: K=7: 6: 19) 3g/L, in immature fruit, seed can be sowed and spend on the agar medium of precious No. 1 3g/L+NAA1mg/L, 25 ± 2 DEG C of culturing room's temperature, intensity of illumination is 1500-2000Lx, illumination 12h/d, medium PH is 5, cultivate 8-12 days seeds and turn green by yellow, increase spherical in shape, base portion has the protocorm of filiform hair, subculture is cultivated on the agar medium of 1/2MS+NAA1+ mashed potatoes 3%+ banana puree 15%, after 30 days, differentiate 1cm budlet and 0.5cm clump bud, can obtain high about 2-3cm budling through the cultivation of 1-2 month again, the Rooting and hardening-off culture base cultivation that proceeds to 1/2MS+NAA1+ mashed potatoes 3%+ banana puree 15% adds intense light irradiation to 4000Lx cultivation one month after 1 month, when test-tube plantlet tool 4-S sheet stretches leaf, root system is normal, while highly reaching 6cm, can supply to transplant by bottle outlet, when bottle outlet, remove the agar on root system, one clump of plantation of 3-5 strain, planting matrix has sawdust, bark, fresh and the haydite of tongue etc., the water of 20: 1 for matrix: formaldehyde sealing sterilization 24h or sterilize by vapours, after cultivation, be placed in shade rate 70% booth, temperature of shed is controlled at 20 DEG C-28 DEG C, note moisturizing and draining, within 8-16 days, spray once with 1/1OMS liquid, just can field planting after test-tube plantlet grows Xin Gen or young leaves.
3. the method for claim 1, it is characterized in that by field acquisition to stem of noble dendrobium stem apex or young stem segment sterilizing with axillalry bud after be seeded on the agar medium of 1/2MS+NAA1mg/L+ mashed potatoes 3%+ banana puree 15%, culturing room's temperature remains 25 ± 2 DEG C, intensity of illumination is 1500-2000Lx, illumination 12h/d, medium PH is 5, cultivate 30-60 days, axillalry bud place can pumping shoot, shoot can be cut and repeat to cultivate, the short clump bud shape that is of stem apex pumping shoot continues to cultivate on above-mentioned medium, grows up to complete test-tube plantlet for transplanting, also can be seeded on the agar medium of 1/2MS or 1/2MS+BA0.5+NAA0.2, explant base portion can differentiate a large amount of plan protocorms, intend protocorm cultivates after 30 days on the agar medium of 1/2MS+NAA1+ mashed potatoes 3%+ banana puree 15%, differentiate 1cm budlet and 0.5cm clump bud, can obtain high 2-3cm budling through the cultivation of 1-2 month again, the Rooting and hardening-off culture base that proceeds to again 1/2MS+NAA1+ mashed potatoes 3%+ banana puree 15% is cultivated 1 month, now illumination is strengthened to 4000Lx, when test-tube plantlet tool 4-5 sheet stretches leaf, root system is normal, while highly reaching 6em, can supply to transplant by bottle outlet, when bottle outlet, remove the agar on root system, one clump of plantation of 3-5 strain, planting matrix has sawdust, bark, liquor-saturated and the haydite of tongue etc., the water of 20: 1 for matrix: formaldehyde sealing sterilization 24h or sterilize by vapours, after cultivation, be placed on shade rate 70% in booth, temperature of shed is controlled at 20 DEG C-28 DEG C, note moisturizing and draining, within 8-16 days, spray once with 1/10MS liquid.
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| CN201310061665.9A CN104012401A (en) | 2013-02-28 | 2013-02-28 | Rapid propagation method of dendrobium huoshanense |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104642107A (en) * | 2015-01-05 | 2015-05-27 | 安徽省霍山县五峰山石斛开发有限公司 | Tissue culture propagation method for dendrobium huoshanense |
| CN105359874A (en) * | 2015-10-31 | 2016-03-02 | 余大春 | Pinelliacordata seedling culture medium and seedling culture method |
| CN107114223A (en) * | 2017-05-12 | 2017-09-01 | 贵州济生农业科技有限公司 | A kind of cultural method of dendrobium candidum |
| CN108029553A (en) * | 2017-11-29 | 2018-05-15 | 徐小毛 | Biological engineering's method of rice dry measure used in former times |
| CN108633735A (en) * | 2018-05-02 | 2018-10-12 | 中国热带农业科学院热带作物品种资源研究所 | Dendrobium breeding of new variety method |
| CN109042339A (en) * | 2018-10-18 | 2018-12-21 | 九仙尊霍山石斛股份有限公司 | A kind of Dendrobidium huoshanness tissue culture breeding method |
| CN109328985A (en) * | 2018-11-22 | 2019-02-15 | 安徽祥峰生物科技有限公司 | It is a kind of promote Dendrobidium huoshanness seed sprout culture medium and type of seeding |
| CN110214702A (en) * | 2019-07-22 | 2019-09-10 | 平顶山学院 | Dendrobidium huoshanness tissue-cultured seedling is cultivated and hardening off method |
-
2013
- 2013-02-28 CN CN201310061665.9A patent/CN104012401A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104642107A (en) * | 2015-01-05 | 2015-05-27 | 安徽省霍山县五峰山石斛开发有限公司 | Tissue culture propagation method for dendrobium huoshanense |
| CN105359874A (en) * | 2015-10-31 | 2016-03-02 | 余大春 | Pinelliacordata seedling culture medium and seedling culture method |
| CN107114223A (en) * | 2017-05-12 | 2017-09-01 | 贵州济生农业科技有限公司 | A kind of cultural method of dendrobium candidum |
| CN108029553A (en) * | 2017-11-29 | 2018-05-15 | 徐小毛 | Biological engineering's method of rice dry measure used in former times |
| CN108633735A (en) * | 2018-05-02 | 2018-10-12 | 中国热带农业科学院热带作物品种资源研究所 | Dendrobium breeding of new variety method |
| CN108633735B (en) * | 2018-05-02 | 2021-12-17 | 中国热带农业科学院热带作物品种资源研究所 | Method for breeding new variety of dendrobium |
| CN109042339A (en) * | 2018-10-18 | 2018-12-21 | 九仙尊霍山石斛股份有限公司 | A kind of Dendrobidium huoshanness tissue culture breeding method |
| CN109328985A (en) * | 2018-11-22 | 2019-02-15 | 安徽祥峰生物科技有限公司 | It is a kind of promote Dendrobidium huoshanness seed sprout culture medium and type of seeding |
| CN110214702A (en) * | 2019-07-22 | 2019-09-10 | 平顶山学院 | Dendrobidium huoshanness tissue-cultured seedling is cultivated and hardening off method |
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Application publication date: 20140903 |