CN103477976A - Stem tissue culture seedling method of dendrobium candidum - Google Patents
Stem tissue culture seedling method of dendrobium candidum Download PDFInfo
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- CN103477976A CN103477976A CN201310291949.7A CN201310291949A CN103477976A CN 103477976 A CN103477976 A CN 103477976A CN 201310291949 A CN201310291949 A CN 201310291949A CN 103477976 A CN103477976 A CN 103477976A
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Abstract
The invention discloses a stem tissue culture seedling method of dendrobium candidum. The stem tissue culture seedling method comprises following steps: (1) disinfection; (2) induced germination; (3) culturing of lateral buds; (4) culture propagation; and (5) transplantation of seedlings. The stem tissue culture seedling method comprises following advantages: (1) inoculation method is changed in stem tissue culturing processes, and medium is adjusted, so that stem cultivate proliferation rate is more than 4, and a problem of low increase rate in stem culturing processes is solved; and (2) five times of seedling transplantation and preparation of different medium are not needed, so that culture efficiency is increased, and labor amount is reduced.
Description
Technical field
The present invention relates to the seedling production technical field, be specifically related to a kind of tissue culture method that dendrobium candidum stem bar is material of take.
Background technology
Dendrobium candidum is the orchid family epiphyte, mainly be distributed in the ground such as Jiangxi, Guangxi, Guangdong, Guizhou, Yunnan, Anhui, Zhejiang, Hunan, Shaanxi, Henan, Fujian, its habitat uniqueness, to microclimate, require very strict, often grow between 500～1500 meters of height above sea level, grow nonparasitically upon another plant on overhanging cliff or the trunk of broad-leaved tree on and on limestone, like environment warm, moistening, half light.Under wild state, the dendrobium candidum proliferative speed is low, poor growth, and predatoriness is excavated in addition, and its wild resource is reduced day by day, can not meet domestic and international market economic development and clinical medical needs far away, so the scale of dendrobium candidum artificial planting enlarges rapidly.Extremely tiny, the natural survival rate of Seeds of Dendrobium Candidum is low, therefore cultivates the first problem that must solve when a large amount of stem of noble dendrobium seedlings are artificial cultivation, and the seedlings of Dendrobium officinale mode of production mainly solves by method for tissue culture now.
Candidum tissue culturing grow seedlings now general adopt the dendrobium candidum capsule after sterilization by planting seed vernalization in the MS medium-ball stem differentiation and proliferation-induce process of the root-hardening of sprouting-induce-domestication, the method efficiency is high, and the enterprise that produces seedlings of Dendrobium officinale by great majority is adopted; But owing to utilizing seed as propagating materials, belong to the sexual propagation mode, inevitably there will be gene impure, the phenomenon that seedling mixes.Also have in addition report choose dendrobium candidum stem section be explant by inducing the report that sprouts and bred, but, because the rate of increase is not high, efficiency is too low, causes this training method substantially to be confined to the use of research unit's research, can not get the application in production.
For example, the invention that application number is 200810058316.0, disclose a kind of fast reproducing method for high quality seedling of dendrobium officinale, and its method is: (1) is got dendrobium candidum and do not launched the leaf sprouting, through sterilizing, stem apex is inoculated into to solid culture medium, sets up sterile system; (2) cut stem section or the base portion small tissue blocks of sterile system seedling, on suitable hormone combinations and medium, induce protocorm; (3) protocorm is alternately cultivated on liquid and solid shoot proliferation medium, and fast breeding forms protocorm group; (4) protocorm group is cut, be transferred on solid seedling medium and break up plant, form seedling; (5) seedling is transferred to strong sprout on solid strong seedling culture base, takes root, cultivate complete plant; (6) by aseptic field planting in strong sprout in suitable hardening matrix, obtain high quality seedling.The present invention can be used for large-scale industrialized production, has that the explant soil removal efficiency is high, the rate of increase is high, aberration rate is low, seedling is sturdy, an advantage such as survival rate is high after quality better, transplanting, growth potential is strong.But, the technical scheme of this disclosure of the invention, shoot proliferation, seedling are cultivated, seedling is cultivated, the strengthening seedling and rooting cultivation stage, transplants seedlings for five times, needs different medium, and the efficiency of cultivation is low, staff's the amount of labour is large.And the shoot proliferation of protocorm need to be through alternately cultivation on liquid and solid multiplication medium, operation trouble.
The invention that application number is 200910154225.1, a kind of light regulate and control method of tissue cultivation of seedlings of Dendrobium officinale is disclosed, it is characterized in that in the tissue culture procedures of seedlings of Dendrobium officinale, the LED plant growth lighting system of usining is implemented light treatment as light source, and seed germination stage light quality is that white light, light intensity are 10-15 μ molm
-2s
-1, light application time 12 hours/day; The first stage of seedling early growth is the 1st day to the 30th day from the embryogenesis plumule, and it is ruddiness that light treatment is selected light quality, and light intensity is 45-50 μ molm
-2s
-1; After second stage is the 31st day after the embryogenesis plumule, light quality is selected red, blue mixed light, ruddiness: blue light is 4: 1, light intensity is 45-50 μ molm
-2s
-1.
Summary of the invention
The purpose of this invention is to provide a kind of method that dendrobium candidum stem section tissue is cultivated, the method is to change vaccination ways in stem section incubation, in conjunction with the adjustment of medium, make the stem section cultivate the rate of increase and reach more than 4, solved the not high problem of the rate of increase in the cultivation of stem section.
For solving the problems of the technologies described above, the invention discloses a kind of dendrobium candidum stem section tissue culture method, the step of described dendrobium candidum stem section tissue culture method is:
(1) sterilization: will give birth to then fresh dendrobium candidum stem bar and get off by clean scissor cut, then with disinfectant, rinse and blot the globule on the stem bar well, then use 75% alcohol-pickled sterilization 1 minute, pick up dry stand-by; Again by described dendrobium candidum stem bar on superclean bench with 0.1% mercuric chloride sterilization 10 minutes, and then by sterile water wash three times;
(2) induce and sprout: the stem section that the described dendrobium candidum stem bar disinfected is cut into to two stem section internodes, and be inoculated in the medium of 1/2Ms+6-BA1.0mg/L+NAA0.3mg/L+10% potato+30% sucrose to cultivate to induce and sprout, cultivation temperature is 25 ℃, intensity of illumination is 2000Lx, light application time is 12 hours, and after cultivating 40 days, described stem section internode starts to sprout lateral bud, cultivate after 60 days the described lateral bud 1～2cm that can grow tall;
(3) lateral bud culture: described lateral bud cutout is got off to be inoculated in the medium of 1/2Ms+NAA0.3mg/L+10% potato+30% sucrose, after cultivating 60 days, described lateral bud just can continue to grow tall 4～5cm and with between 4～5 stems;
(4) cultivation is expanded numerous: previous step is cultivated out the lateral bud that described 4～5cm is high and cut away terminal bud and/or root, become new stem section, mode with tiling is inoculated in the cultivation of 1/2Ms+NAA0.3mg/L+10% potato+30% sucrose again, cultivate after 30 days, each internode of described new stem section can sprout 1～2 sprouting, cultivate after 90 days, it is the stem section that 4～5cm is high that described sprouting grows up to;
(5) emerge: select the above described sprouting of 3cm to cut separately, with terminal bud, to straight cutting, inoculate in the medium of 1/2Ms+NAA0.3mg/L+10% banana+active carbon 2g/L+30% sucrose, cultivate and within 30 days, start long root, cultivate after 60 days, transfer in the hardening booth that intensity of illumination is stronger and cultivate, within 90 days, just grow into afterwards the healthy and strong seedling that can transplant, in the suitable matrix be transplanted to after cleaning.
Described disinfectant is washing powder water.
The stage is bred in cultivation in described dendrobium candidum stem section tissue culture method, after described sprouting grows up to and is the stem section that 4～5cm is high, repeat this step continue to cultivate expand numerous.
A kind of dendrobium candidum stem of the present invention section tissue culture method compared to the prior art, its advantage is: the first, change vaccination ways in stem section incubation, in conjunction with the adjustment of medium, make the stem section cultivate the rate of increase and reach more than 4, solved the not high problem of the rate of increase in the cultivation of stem section; The second, at shoot proliferation, seedling cultivation, seedling cultivation, strengthening seedling and rooting cultivation stage, no longer need to transplant seedlings, configure different medium five times, but only need to transplant seedlings for three times, improved the efficiency of cultivating, reduced staff's the amount of labour.
Embodiment
For making the technical problem to be solved in the present invention, technical scheme and advantage clearer, below will further illustrate specific embodiments of the invention.
The invention discloses a kind of dendrobium candidum stem section tissue culture method, the step of described dendrobium candidum stem section tissue culture method is:
(1) sterilization: will give birth to then fresh dendrobium candidum stem bar and get off by clean scissor cut, and then with disinfectants such as washing powder water, rinse and blot the globule on the stem bar well, then use 75% alcohol-pickled sterilization 1 minute, pick up dry stand-by; Again by described dendrobium candidum stem bar on superclean bench with 0.1% mercuric chloride sterilization 10 minutes, and then by sterile water wash three times;
(2) induce and sprout: the stem section that the described dendrobium candidum stem bar disinfected is cut into to two stem section internodes, and be inoculated in the medium of 1/2Ms+6-BA1.0mg/L+NAA0.3mg/L+10% potato+30% sucrose to cultivate to induce and sprout, cultivation temperature is 25 ℃, intensity of illumination is 2000Lx, light application time is 12 hours, and after cultivating 40 days, described stem section internode starts to sprout lateral bud, cultivate after 60 days the described lateral bud 1～2cm that can grow tall;
(3) lateral bud culture: described lateral bud cutout is got off to be inoculated in the medium of 1/2Ms+NAA0.3mg/L+10% potato+30% sucrose, after cultivating 60 days, described lateral bud just can continue to grow tall 4～5cm and with between 4～5 stems;
(4) cultivation is expanded numerous: previous step is cultivated out the lateral bud that described 4～5cm is high and cut away terminal bud and/or root, become new stem section, mode with tiling is inoculated in the cultivation of 1/2Ms+NAA0.3mg/L+10% potato+30% sucrose again, cultivate after 30 days, each internode of described new stem section can sprout 1～2 sprouting, cultivate after 90 days, it is the stem section that 4～5cm is high that described sprouting grows up to; After described sprouting grows up to and is the stem section that 4～5cm is high, repeat this step continue to cultivate expand numerous.
(5) emerge: select the above described sprouting of 3cm to cut separately, with terminal bud, to straight cutting, inoculate in the medium of 1/2Ms+NAA0.3mg/L+10% banana+active carbon 2g/L+30% sucrose, cultivate and within 30 days, start long root, cultivate after 60 days, transfer in the hardening booth that intensity of illumination is stronger and cultivate, within 90 days, just grow into afterwards the healthy and strong seedling that can transplant, in the suitable matrix be transplanted to after cleaning.
The above is the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite that does not break away from principle of the present invention and spirit; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (3)
1. a dendrobium candidum stem section tissue culture method, is characterized in that, the step of described dendrobium candidum stem section tissue culture method is:
(1) sterilization: will give birth to then fresh dendrobium candidum stem bar and get off by clean scissor cut, then with disinfectant, rinse and blot the globule on the stem bar well, then use 75% alcohol-pickled sterilization 1 minute, pick up dry stand-by; Again by described dendrobium candidum stem bar on superclean bench with 0.1% mercuric chloride sterilization 10 minutes, and then by sterile water wash three times;
(2) induce and sprout: the stem section that the described dendrobium candidum stem bar disinfected is cut into to two stem section internodes, and be inoculated in the medium of 1/2Ms+6-BA1.0mg/L+NAA0.3mg/L+10% potato+30% sucrose to cultivate to induce and sprout, cultivation temperature is 25 ℃, intensity of illumination is 2000Lx, light application time is 12 hours, and after cultivating 40 days, described stem section internode starts to sprout lateral bud, cultivate after 60 days the described lateral bud 1～2cm that can grow tall;
(3) lateral bud culture: described lateral bud cutout is got off to be inoculated in the medium of 1/2Ms+NAA0.3mg/L+10% potato+30% sucrose, after cultivating 60 days, described lateral bud just can continue to grow tall 4～5cm and with between 4～5 stems;
(4) cultivation is expanded numerous: previous step is cultivated out the lateral bud that described 4～5cm is high and cut away terminal bud and/or root, become new stem section, mode with tiling is inoculated in the cultivation of 1/2Ms+NAA0.3mg/L+10% potato+30% sucrose again, cultivate after 30 days, each internode of described new stem section can sprout 1～2 sprouting, cultivate after 90 days, it is the stem section that 4～5cm is high that described sprouting grows up to;
(5) emerge: select the above described sprouting of 3cm to cut separately, with terminal bud, to straight cutting, inoculate in the medium of 1/2Ms+NAA0.3mg/L+10% banana+active carbon 2g/L+30% sucrose, cultivate and within 30 days, start long root, cultivate after 60 days, transfer in the hardening booth that intensity of illumination is stronger and cultivate, within 90 days, just grow into afterwards the healthy and strong seedling that can transplant, in the suitable matrix be transplanted to after cleaning.
2. a kind of dendrobium candidum stem section tissue culture method according to claim 1, is characterized in that, described disinfectant is washing powder water.
3. a kind of dendrobium candidum stem section tissue culture method according to claim 1 and 2, it is characterized in that, the stage is bred in cultivation in described dendrobium candidum stem section tissue culture method, after described sprouting grows up to and is the stem section that 4～5cm is high, repeat this step continue to cultivate expand numerous.
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| 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 |
| CN104429973A (en) * | 2014-12-25 | 2015-03-25 | 安徽同创现代农业投资发展有限公司 | Method of culturing dendrobium officinale plantlets |
| CN104719159A (en) * | 2015-03-13 | 2015-06-24 | 广东省农业科学院作物研究所 | Nutrient solution and method for applying nutrient solution to stem tissue-cultured seedling of dendrobium officinale |
| CN109105265A (en) * | 2018-11-09 | 2019-01-01 | 翁源县天下泽雨农业科技有限公司 | A kind of stem section induction Multiple Buds rapid propagation method of dendrobium candidum |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN104429973A (en) * | 2014-12-25 | 2015-03-25 | 安徽同创现代农业投资发展有限公司 | Method of culturing dendrobium officinale plantlets |
| CN104719159A (en) * | 2015-03-13 | 2015-06-24 | 广东省农业科学院作物研究所 | Nutrient solution and method for applying nutrient solution to stem tissue-cultured seedling of dendrobium officinale |
| CN109105265A (en) * | 2018-11-09 | 2019-01-01 | 翁源县天下泽雨农业科技有限公司 | A kind of stem section induction Multiple Buds rapid propagation method of dendrobium candidum |
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