CN101258835A - Fast reproducing method for high quality seedling of dendrobium officinale - Google Patents
Fast reproducing method for high quality seedling of dendrobium officinale Download PDFInfo
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Abstract
The invention provides a method for rapidly reproducing high quality germchit with dendrobium officinale, which is characterized in that: (1) folded sprouts of the dendrobium officinale are taken and sterilized, and stem apexes are inoculated to a solid culture medium to establish a non-bacterial system; (2) the stem sections or small basal tissue blocks of the young seedlings of the non-bacterial system are cut for inducing protocorm based on suitable hormone combination and a culture medium; (3) the protocorm is cultured alternatively on liquid and solid subculture mediums for rapid multiplication to form protocorm masses; (4) the protocorm masses are cut apart and transferred on a solid planting medium for disuniting plants into small seedlings; (5) the small seedlings are transferred on a solid strong seedling culture medium for strengthening the seedlings and taking roots to culture complete plants; (6) the non-bacterial strong seedlings are fixedly planted in suitable seedling adapting substrate to obtain the high quality germchit. The method can be used in large scale industrialization and the explants have the advantages of high soil removal efficiency, high multiplication rate, low variability, strong germchit, good quality, high survival rate after being transplanted, strong growth potential, etc.
Description
Technical field
The present invention relates to technical field of bioengineering, particularly fast reproducing method for high quality seedling of dendrobium officinale.
Background technology
Dendrobium (Dendrobium) is the big genus of of the orchid family (Orchidaceae), comprise 1600 various plants, China has 63 kinds approximately, wherein hyoscine has a kind more than 30, mainly contain dendrobium loddigesii Rolfe (D.loddigesii), stem of Eyeshaped Dendrobium (D.fimbriatum), dendrobium candidum (D.candidum), HERBA DENDROBII (D.chrysanthum) and HERBA DENDROBII (D.nobile), dendrobium candidum is the most precious person wherein.The traditional Chinese medical science is thought the effect that the stem of noble dendrobium has the yin-nourishing detoxicating, promotes the production of body fluid to quench thirst, makes eye bright and keep fit, and modern medicine study finds that the stem of noble dendrobium contains multiple alkaloid, polysaccharide and amino acid, has raise immunity, suppresses thrombosis, antitumor and antidotal multiple efficacies.Therefore at home in the tcm clinical practice prescription and the Chinese patent drug raw material of industry for export demand constantly increase, price one tunnel domestic, the international market soars, and makes the stem of noble dendrobium seem rare famous and precious day by day.But the natural propagation rate of the wild stem of noble dendrobium is low, poor growth, crop are low, does not satisfy the needs in market far away, and the long-term immoderate coyoting of people is dug excessively in addition, causes the heavy damage of wild resource.Though Dendrobium Plant Tissue Breeding and artificial cultivation stem of noble dendrobium Study on Technology are arranged at present, but all there is long, problem such as production cost is high, reproduction coefficient is low, quality is uneven of seedling production cycle, and the high quality seedling method for quickly breeding of being satisfied with large-scale industrialized production report not.
At present, the explant of the dendrobium candidum tissue culture technique interior seeds of fern fruit that adopt wild plant more, be seeded to through sterilization and form protocorm on the inducing culture, setting up sterile system, this method exists that seed obtains difficulty, maternal source is restricted, The Characters is inconsistent, the seedling quality deterioration fast, serious, the high-quality germplasm of aging phenomenon is difficult to problems such as preservation behind shoot proliferation several times.
Summary of the invention
The purpose of this invention is to provide the method for quickly breeding that a kind of short time obtains extensive dendrobium candidum high quality seedling, this method seedling explant disinfection efficiency height, medium component uniqueness and cost is low, the breeding cycle is short, the seedling of producing is best in quality, neat, the using value height.
The present invention as explant, can obtain enough explant quantity by the sprouting of taking not open up leaf at the group training initial stage from the wild or tame dendrobium candidum plant of The Characters excellence, guarantee the success rate of later stage large-scale production.Induce by protocorm promptly that---shoot proliferation of protocorm---becomes seedling differentiation culture---strengthening seedling and rooting cultivation---a series of links of test-tube seedling transplanting, can set up the aseptic high quality seedling production system of a whole set of batch production in 6 months.
Logarithm strain of the present invention is picked up from tissue culture and quick propagating technology, the training tissue culture seedling technology of the wild or tame dendrobium candidum plant in Guangxi province, Guangnan County, Yunnan Province and state, Xishuangbanna and has been carried out system research, angle in large-scale industrialized production has bigger innovation to aspects such as culture medium prescription, technological process, hardening matrix, and cover a fast reproducing method for high quality seedling of dendrobium officinale and relevant culture medium prescription, hardening matrix formulations is provided.Specifically comprise the steps:
1. material: get wild or tame dendrobium candidum (Dendrobium candidum).
2. the method for explant induction materials disinfection: in the 2-4 the first tenday period of a month month, cut wild or tame dendrobium candidum and do not open up the leaf sprouting, flowing water flushing 10 minutes, with explant on the superclean bench with 75% alcohol-pickled 45 seconds after, be the mercuric chloride solution of 1 grams per liter again with mass percent concentration, 2～3 Tween-80s soaked 8 minutes, aseptic water washing 3 times, divest outer foil with scalpel, put into the mercuric chloride solution that mass percent concentration is 1 grams per liter again, soaked 5 minutes in 2～3 Tween 80s, aseptic water washing 3 times, successively peel off blade until exposing growing point, be inoculated on the 1/2MS solid culture medium and sprout plant, set up sterile system.
3. protocorm is induced: cut sterile system seedling stem section or base portion small tissue blocks and be inoculated into the protocorm inducing culture: 1/2MS+6-BA2.0-5.0 mg/litre+NAA0.2-1.0 mg/litre+potato juice 200 grams per liters, potato juice adopts the boiling of clear soup method after filtration, adds the inductivity that potato juice can obviously improve protocorm.
4. shoot proliferation: cultivated about 30 days, protocorm is transferred to the shoot proliferation medium, solid culture and liquid culture hocket and can significantly improve the rate of increase of protocorm.Solid culture medium is 1/2MS+6-BA1.0-3.0 mg/litre+NAA0.1-0.5 mg/litre+potato juice 200 grams per liters+active carbon 0.2-0.5 grams per liter+agar 7 grams per liters; Liquid nutrient medium is 1/2MS+6-BA1.0-3.0 mg/litre+NAA0.1-0.5 mg/litre+potato juice 200 grams per liters+active carbon 0.2-0.5 grams per liter.The shaking speed of liquid nutrient medium is 50-80r/min, 25 ± 2 ℃ of cultivation temperature, luminous intensity 1500-2500lx, illumination 10 hours/day.This cultivation stage adds small amount of activated and can reduce protocorm and roll into a ball brownization, and the hormone concentration that reduces 6-BA and NAA can guarantee the possibility that morphs under certain rate of increase.
5. become seedling to cultivate: protocorm group to be inoculated into into seedling medium culture 3-4 week to form the unrooted seedling.Become the seedling medium: 1/2MS+6-BA0.1-0.5 mg/litre+NAA1.02.0 mg/litre+potato juice 200 grams per liters+active carbon 0.2-0.5 grams per liter.
6. strengthening seedling and rooting is cultivated: when 2-3 centimetre of unrooted height of seedling, be transferred on the strengthening seedling and rooting medium and cultivate.The strengthening seedling and rooting medium: 1/2MS+NAA0.5-1.5 mg/litre+banana homogenate 100 grams per liters+white sugar 30 grams per liters+active carbon 0.2-0.5 grams per liter, add banana homogenate and white sugar and can obviously improve rooting rate, and seedling is sturdy, neat.
Above solid culture medium all contains sucrose 20 grams per liters (except that the strengthening seedling and rooting medium), PH5.4-5.8, agar 7 grams per liters; Condition of culture: 28 ± 2 ℃ of cultivation temperature, luminous intensity 1500-2500lx, illumination 12 hours/day.
7. test-tube seedling transplanting: when strong seedling culture about 6 weeks, test-tube plantlet can grow to 8-10 centimetre high, transfer to the natural daylight lower refining seedling after 7 days, it is taken out from vial, clean the medium of root, in the mixed-matrix after immigration wood shavings+pig manure co-fermentation, keep suitable ventilation and enough humidity (relative moisture 80%-90%), 2 all test-tube plantlets can recover growth, and the survival rate of transplanting all can reach 95%-100%.
Advantages such as method provided by the present invention has that the wide and sterilization success rate height in explant source, growth coefficient height, aberration rate are low, sturdy neat, the quality better of seedling, transplanting survival rate height, growth potential are strong.Its explant disinfection efficiency height, medium component uniqueness and cost are low, large-scale industrialized with short production cycle, seedling first-class quality, the using value height produced.
Embodiment
Embodiment 1:
1. draw materials: the tame dendrobium candidum in state, Xishuangbanna, Yunnan Province is not opened up the leaf sprouting.
2. the method for explant induction materials disinfection: cut the dendrobium candidum sprouting of cultivating in the greenhouse with new knife blade from base portion, running water flushing 10 minutes.Explant is placed on the superclean bench, after 75% alcohol-pickled 45 seconds, be that the mercuric chloride solution of 1 grams per liter, 2～3 Tween-80s soaked 8 minutes with mass percent concentration again, aseptic water washing 3 times, divest outer foil with scalpel, put into mercuric chloride solution that mass percent concentration is 1 grams per liter again, 2～3 Tween-80s soaked aseptic water washing 3 times 5 minutes, successively peel off blade until exposing growing point, be seeded on the 1/2MS solid culture medium.
3. protocorm is induced: the stem section or the base portion small tissue blocks that cut the sterile system seedling are inoculated into the protocorm inducing culture: 1/2MS+6-BA2 mg/litre+NAA0.5 mg/litre+potato juice 200 grams per liters, cultivating had protocorm form in about 30 days.
4. shoot proliferation: inducing culture about 30 days is transferred to protocorm on the liquid nutrient medium of the solid culture medium of 1/2MS+6-BA2.0 mg/litre+NAA0.2 mg/litre+potato juice 200 grams per liters+active carbon 0.5 grams per liter+agar 7 grams per liters and 1/2MS+6-BA1.0 mg/litre+NAA0.1 mg/litre+potato juice 200 grams per liters+active carbon 0.2 grams per liter and is alternately cultivated propagation, and general about 30 days is a subculture cycle.
5. become seedling to cultivate: to be inoculated into into the seedling medium behind the extremely certain algebraically of propagation: 1/2MS+6-BA0.2 mg/litre+NAA1.5 mg/litre+potato juice 200 grams per liters+active carbon 0.5 grams per liter.Be that visible protocorm forms shoot apex after one week, can grow up to 2-3 centimetre high unrooted seedling about 4 weeks.
6. strengthening seedling and rooting is cultivated: will form about 2-3 centimetre of high unrooted seedling and be transferred to the strengthening seedling and rooting medium: 1/2MS+NAA1.0 mg/litre+banana homogenate 100 grams per liters+white sugar 30 grams per liters+active carbon 0.5 grams per liter becoming on the seedling medium to cultivate about 4 weeks; Every bottle graft kind 15 strains.The plant mean elements is 5.2 after 6 weeks of inoculation, 9.5 centimetres of average plant heights, average fresh weight 3.0 grams.
7. test-tube seedling transplanting: the bottle seedling in 6 weeks of strong seedling culture is transferred to greenhouse culture condition lower refining seedling 7 days.It is taken out from vial, clean the medium of root, in the mixed-matrix after immigration wood shavings+pig manure co-fermentation, keep suitable ventilation and enough humidity (relative moisture 80%-90%), transplanting survival rate can reach more than 95%.
Above solid culture medium all contains sucrose 20 grams per liters (except that the strengthening seedling and rooting medium), PH5.4-5.8, agar 7 grams per liters, 28 ± 2 ℃ of cultivation temperature, luminous intensity 1500-2500lx, illumination 12 hours/day.Potato juice adopts the boiling of clear soup method after filtration.
The liquid nutrient medium of above-mentioned shoot proliferation does not add agar, contains sucrose 20 grams per liters, PH5.4-5.8; Shaking speed is 60r/min, 25 ± 2 ℃ of cultivation temperature, luminous intensity 1500-2000lx, illumination 10 hours/day.
Embodiment 2:
The sprouting of getting the wild stem of noble dendrobium of Guangxi province is as explant.The basic operation method is with embodiment 1 unanimity.But its protocorm inducing culture is: 1/2MS+6-BA5.0 mg/litre+NAA1.0 mg/litre+potato juice 200 grams per liters; Protocorm shoot proliferation medium is: the liquid nutrient medium of the solid culture medium of 1/2MS+6-BA3 mg/litre+NAA0.5 mg/litre+potato juice 200 grams per liters+active carbon 0.5 grams per liter+agar 7 grams per liters and 1/2MS+6-BA3.0 mg/litre+NAA0.5 mg/litre+potato juice 200 grams per liters+active carbon 0.5 grams per liter; Become the seedling medium to be: 1/2MS+6-BA0.5 mg/litre+NAA2.0 mg/litre+potato juice 200 grams per liters+active carbon 0.2 grams per liter; The strengthening seedling and rooting medium is: 1/2MS+NAA1.5 mg/litre+banana homogenate 100 grams per liters+white sugar 30 grams per liters+active carbon 0.2 grams per liter.Strengthening seedling and rooting is cultivated after 40 days 4.8 of statistical average radicals, and average plant is high 9.2 centimetres, and average fresh weight reaches 2.8 grams.The survival rate of test-tube seedling transplanting can reach 98%.
Above solid culture medium all contains sucrose 20 grams per liters (except that the strengthening seedling and rooting medium), PH5.4-5.8, agar 7 grams per liters, 28 ± 2 ℃ of cultivation temperature, luminous intensity 1500-2500lx, illumination 12 hours/day.Potato juice adopts the boiling of clear soup method after filtration.
The liquid nutrient medium of above-mentioned shoot proliferation does not add agar, contains sucrose 20 grams per liters, PH5.4-5.8; Shaking speed is 60r/min, 25 ± 2 ℃ of cultivation temperature, luminous intensity 1500-2000lx, illumination 10 hours/day.
Claims (9)
1. fast reproducing method for high quality seedling of dendrobium officinale is characterized in that concrete steps are as follows:
1) method of explant induction materials disinfection: in the 2-4 the first tenday period of a month month, cut wild or tame dendrobium candidum and do not open up the leaf sprouting, flowing water flushing 10～15 minutes, with explant on the superclean bench with 75% alcohol-pickled 45 ± 5 seconds after, be the mercuric chloride solution of 1 grams per liter again with mass percent concentration, 2～3 Tween-80s soaked 8 minutes, aseptic water washing 3 times, divest outer foil with scalpel, put into the mercuric chloride solution that mass percent concentration is 1 grams per liter again, soaked 5 minutes in 2～3 Tween 80s, aseptic water washing 3 times, successively peel off blade until exposing growing point, be inoculated on the 1/2MS solid culture medium and sprout plant, set up sterile system;
2) protocorm is induced: the stem section or the base portion small tissue blocks that cut the sterile system seedling are inoculated into the protocorm inducing culture;
3) shoot proliferation: the protocorm that induces is inoculated into alternately cultivation on liquid and the solid multiplication medium, and enrichment culture 30 days was a subculture cycle;
4) become seedling to cultivate: protocorm group to be transferred on the seedling medium, to cultivate 2-3 week, form seedling;
5) strengthening seedling and rooting is cultivated: when 2-3 centimetre of unrooted height of seedling, be transferred on the strengthening seedling and rooting medium and cultivate;
6) test-tube seedling transplanting: when 6 weeks of strong seedling culture, test-tube plantlet can grow to 8-10 centimetre high, transfer to the natural daylight lower refining seedling after 7 days, it is taken out from vial, clean the medium of root, move in the mixed-matrix after wood shavings+pig manure co-fermentation, keeps suitable ventilation and enough humidity, 2 all test-tube plantlets can recover to grow.
2. fast reproducing method for high quality seedling of dendrobium officinale according to claim 1, it is characterized in that step 2) in the protocorm inducing culture be 1/2MS+6-BA2.0-5.0 mg/litre+NAA0.2-1.0 mg/litre+potato juice 200 grams per liters, contain sucrose 20 grams per liters, PH5.4-5.8, agar 7 grams per liters.
3. according to the fast reproducing method for high quality seedling of dendrobium officinale described in the claim 1, it is characterized in that subculture propagation solid culture medium is in the step 3): 1/2MS+6-BA1.0-3.0 mg/litre+NAA0.1-0.5 mg/litre+potato juice 200 grams per liters+active carbon 0.2-0.5 grams per liter+agar 7 grams per liters, contain sucrose 20 grams per liters, PH5.4-5.8.
4. according to the fast reproducing method for high quality seedling of dendrobium officinale described in the claim 1, it is characterized in that subculture proliferating liquid body medium is in the step 3): 1/2MS+6-BA1.0-3.0 mg/litre+NAA0.1-0.5 mg/litre+potato juice 200 grams per liters+active carbon 0.2-0.5 grams per liter, contain sucrose 20 grams per liters, PH5.4-5.8.
5. according to the fast reproducing method for high quality seedling of dendrobium officinale described in the claim 3, it is characterized in that it is 50-80r/min that the shoot proliferation liquid nutrient medium is cultivated shaking speed, 25 ± 2 ℃ of cultivation temperature, luminous intensity 1500-2000lx, illumination 10 hours/day.
6. according to the fast reproducing method for high quality seedling of dendrobium officinale described in the claim 1, it is characterized in that becoming in the step 4) seedling medium to be: 1/2MS+6-BA0.1-0.5 mg/litre+NAA1.0-2.0 mg/litre+potato juice 200 grams per liters+active carbon 0.2-0.5 grams per liter, contain sucrose 20 grams per liters, PH5.4-5.8, agar 7 grams per liters.
7. according to the fast reproducing method for high quality seedling of dendrobium officinale described in the claim 1, it is characterized in that the strengthening seedling and rooting medium is in the step 5): 1/2MS+NAA0.5-1.5 mg/litre+banana homogenate 100 grams per liters+white sugar 30 grams per liters+active carbon 0.2-0.5 grams per liter, PH5.4-5.8 contains agar 7 grams per liters.
8. according to the fast reproducing method for high quality seedling of dendrobium officinale described in the claim 1, it is characterized in that above protocorm inducing culture, shoot proliferation solid culture medium, become seedling medium, strengthening seedling and rooting medium to be: 28 ± 2 ℃ of cultivation temperature, luminous intensity 1500-2500lx, illumination 12 hours/day.
9. according to the fast reproducing method for high quality seedling of dendrobium officinale described in claim 2 or 3 or 5, it is characterized in that described potato juice adopts the boiling of clear soup method to get after filtration.
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Cited By (24)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101822211A (en) * | 2009-09-03 | 2010-09-08 | 昆明优利丰园科技开发有限公司 | Tissue cultivating method of dendrobium officinale growing in Yunnan |
| CN102138420A (en) * | 2011-02-22 | 2011-08-03 | 贵州省农作物品种资源研究所 | Method for cultivating dendrobium fimbriatum |
| CN102428870A (en) * | 2011-09-22 | 2012-05-02 | 澄思源生物科技（上海）有限公司 | Preparation method for artificial seeds of dendrobium candidum |
| CN102523881A (en) * | 2012-01-16 | 2012-07-04 | 澄思源生物科技（上海）有限公司 | Industrial cultivation method for dendrobium candidum |
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