CN104429962A - Cultivation method of dendrobium nobile tissue culture seedlings - Google Patents
Cultivation method of dendrobium nobile tissue culture seedlings Download PDFInfo
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- CN104429962A CN104429962A CN201410745002.3A CN201410745002A CN104429962A CN 104429962 A CN104429962 A CN 104429962A CN 201410745002 A CN201410745002 A CN 201410745002A CN 104429962 A CN104429962 A CN 104429962A
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- herba dendrobii
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- seedling
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- 240000004638 Dendrobium nobile Species 0.000 title abstract description 3
- 238000012364 cultivation method Methods 0.000 title abstract 3
- 230000004069 differentiation Effects 0.000 claims abstract description 10
- 244000061456 Solanum tuberosum Species 0.000 claims abstract description 6
- 235000002595 Solanum tuberosum Nutrition 0.000 claims abstract description 6
- 229920001817 Agar Polymers 0.000 claims abstract description 4
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims abstract description 4
- 229930006000 Sucrose Natural products 0.000 claims abstract description 4
- 239000008272 agar Substances 0.000 claims abstract description 4
- 239000000843 powder Substances 0.000 claims abstract description 4
- 239000005720 sucrose Substances 0.000 claims abstract description 4
- 238000000338 in vitro Methods 0.000 claims description 22
- 238000009395 breeding Methods 0.000 claims description 14
- 235000013399 edible fruits Nutrition 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 238000005286 illumination Methods 0.000 claims description 8
- 230000001954 sterilising effect Effects 0.000 claims description 6
- 238000004659 sterilization and disinfection Methods 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- RCTYPNKXASFOBE-UHFFFAOYSA-M chloromercury Chemical compound [Hg]Cl RCTYPNKXASFOBE-UHFFFAOYSA-M 0.000 claims description 3
- 230000003816 axenic effect Effects 0.000 claims description 2
- 230000035784 germination Effects 0.000 claims description 2
- 238000000034 method Methods 0.000 abstract description 8
- 239000001963 growth medium Substances 0.000 abstract description 6
- 238000004519 manufacturing process Methods 0.000 abstract description 6
- 241000196324 Embryophyta Species 0.000 abstract description 5
- 239000002775 capsule Substances 0.000 abstract description 3
- 238000010899 nucleation Methods 0.000 abstract description 2
- 230000004083 survival effect Effects 0.000 abstract description 2
- 239000002609 medium Substances 0.000 description 15
- 238000005516 engineering process Methods 0.000 description 4
- 229940088597 hormone Drugs 0.000 description 4
- 239000005556 hormone Substances 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 238000011534 incubation Methods 0.000 description 3
- 230000008859 change Effects 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 2
- 239000006259 organic additive Substances 0.000 description 2
- 244000060011 Cocos nucifera Species 0.000 description 1
- 235000013162 Cocos nucifera Nutrition 0.000 description 1
- 241001523681 Dendrobium Species 0.000 description 1
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 1
- 240000008790 Musa x paradisiaca Species 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 238000004500 asepsis Methods 0.000 description 1
- 235000021015 bananas Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 235000020415 coconut juice Nutrition 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 238000009400 out breeding Methods 0.000 description 1
- 238000004161 plant tissue culture Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000034655 secondary growth Effects 0.000 description 1
- 238000009331 sowing Methods 0.000 description 1
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a cultivation method of dendrobium nobile tissue culture seedlings. The cultivation method comprises the following steps: selecting ripe capsules; sterilely seeding the capsules into a 1/2MS+20% potato+30g/L sucrose+4g/L agar powder culture medium; transferring grown protocorms into the same culture medium to carry out protocorm multiplication and differentiation culture; and transferring formed seedlings into the same culture medium to carry out strong seedling and rooting culture. The seedlings which grown consistently are transferred once every 2 months; and when the plant heights of the tissue culture seedlings are about 6.0cm-8.0cm, each tissue culture seedling at least has 8-10 developed root systems. The method is simple and practical; the obtained tissue culture seedlings have more roots and a high survival rate; the production cost is low, and the pollution rate is low; and the industrialized production level is reached.
Description
Technical field
The invention belongs to field of plant tissue culture technique, be specifically related to a kind of breeding method of HERBA DENDROBII plantlet in vitro.
Background technology
HERBA DENDROBII (Dendrobium nobile) is grown nonparasitically upon another plant for the orchid family Dendrobium is perennial type herbaceous plant, it is China's tradition rare Chinese medicine, there is higher medical value, add that its flower pattern is peculiar, pattern is gorgeous, has higher ornamental value, the huge market demand, but it is more special that HERBA DENDROBII requires habitat, self-sow is relatively slower, and supply falls short of demand for wild resource.In recent years, China scientific worker utilizes the method for Plant Tissue Breeding to carry out the production of HERBA DENDROBII medicinal material, and successful incubation goes out plantlet in vitro.
Current HERBA DENDROBII tissue cultures mainly uses stem section, basal part of stem and the seed of being with lateral bud as explant, be minimal medium with MS, 1/2MS and N6, add 6-BA, NAA, IBA, KT and 2, the organic additives such as the hormones such as 4-D and coconut palm breast, bananas juice, Coconut Juice, potato juice carry out differentiation-inducing, propagation, culture of rootage, final acquisition HERBA DENDROBII plantlet in vitro.But existing technology exists following defect: the hormone kind added in minimal medium is many, and concentration is higher, affect growing of explant; The kinds of culture medium selected is many, and process more complicated, adds workload; Explant generally selects stem section, causes reproduction speed very slow, cannot industrialization produce; Often, incubation time is long, easily produces sudden change, and pollution rate also rises in switching; The procurement price of the Organic additives added in medium is more expensive, causes production cost to improve a lot.
Summary of the invention
The present invention is directed to the defect that prior art exists, aim to provide a kind of breeding method of HERBA DENDROBII plantlet in vitro.
The present invention adopts the seed asepsis sprouting of HERBA DENDROBII, the formation of protocorm and seedling differentiation, strong sprout and culture of rootage technology.Choose ripe capsule, by aseptically sowing seeds on medium, after 30 days, the protocorm grown is proceeded in same medium and carry out Protocorm Multiplication and seedling differentiation cultivation, proceeding in same medium by forming about 1.0cm seedling after 1 first quarter moon, carrying out strong sprout and culture of rootage.Plant height is that the plantlet in vitro of 6.0 ~ 8.0 centimetres carries out acclimatization and transplants by every 2 months consistent seedlings of switching 1 secondary growth, the root system that every plantlet in vitro at least 8 ~ 10 is flourishing.
The present invention realizes especially by following technical scheme:
A breeding method for HERBA DENDROBII plantlet in vitro, comprises the following steps:
1) selection of HERBA DENDROBII fruit and surface sterilization;
2) axenic germination of HERBA DENDROBII seed;
3) Protocorm Multiplication and seedling differentiation are cultivated;
4) strong sprout and culture of rootage.
Further,
Step (1) is specially selects HERBA DENDROBII fruit, and after clear water rinses, at 70% alcohol-pickled 1 minute, aseptic water washing 3 times, then use 0.1% mercury chloride surface sterilization 10 minutes, after aseptic water washing 5 times, blots surface moisture with aseptic filter paper.
Step (2) is specially and is cut in half by HERBA DENDROBII fruit, removes pericarp, is implanted by seed on medium, cultivates HERBA DENDROBII protocorm.
Step (3) is specially and proceeds on fresh culture by HERBA DENDROBII protocorm, Protocorm Multiplication and differentiation is cultivated.
Step (4) is specially and seedling is proceeded to fresh culture, and switching in every 2 months 1 seedling, when seedling reaches 6.0 ~ 8.0cm height after 2 ~ 3 times, carries out hardening, transplanting.
Culture medium prescription of the present invention all adopts: 1/2MS+20% potato+30g/L sucrose+4g/L agar powder, pH value is 5.8.
In of the present invention group of training cultivating process, condition of culture is: temperature 26 ± 2 DEG C, illumination 12h/d, intensity of illumination 1500 ~ 2500Lx.
Beneficial effect of the present invention is:
1) medium that the present invention adopts is 1/2MS minimal medium, without the need to adding hormone;
2) whole cultivation link all adopts unified medium, changes kinds of culture medium, greatly promoting working processes efficiency without the need to each link;
3) explant adopts seed, and reproduction speed is fast, and easy industrialization is produced;
4) the switching number of times of seedling is few, and incubation time is short, effectively reduces mutation rate and pollution rate;
5) the potato market price of adding in medium is comparatively cheap, effectively reduces costs in industrialization is produced.
Embodiment
Below in conjunction with embodiment, the present invention is described further, the following stated, only to preferred embodiment of the present invention, not do other forms of restriction to the present invention, any those skilled in the art may utilize the technology contents of above-mentioned announcement to be changed to the Equivalent embodiments of equal change.Everyly do not depart from the present invention program's content, any simple modification done following examples according to technical spirit of the present invention or equivalent variations, all drop in protection scope of the present invention.
Embodiment 1
A breeding method for HERBA DENDROBII plantlet in vitro, comprises the following steps:
1) fruit surface sterilization
First scrub HERBA DENDROBII fruit gently with banister brush, running water is clean, then 70% alcohol-pickled 1 minute, aseptic water washing 3 times, then use 0.1% mercury chloride surface sterilization 10 minutes, after aseptic water washing 5 times, fruit is placed on aseptic filter paper, blots surface moisture.
2) aseptic seeding of seed is cultivated
After HERBA DENDROBII fruit being cut in half with sterile scalpel, pericarp is pinched with tweezers, shake gently, seed is scattering on dry aseptic filter paper, with aseptic medicine spoon, seed is sown on medium 1/2MS+20% potato+30g/L sucrose+4g/L agar powder (pH5.8) (lower same), shake blake bottle a little, make seed dispersal even.Cultivate seed after 30 days to start to sprout, form green protocorm gradually.Condition of culture is: temperature (26 ± 2) DEG C, illumination 12h/d, intensity of illumination 1500 ~ 2500Lx.
3) differentiation of protocorm, strong sprout and culture of rootage
After HERBA DENDROBII protocorm is formed, proceed in fresh culture and carry out breeding and break up cultivation.In the medium, now protocorm is transferred on identical fresh culture, allow protocorm differentiation become a strain or tufted seedling, some seedling base portion also produces new protocorm to can be observed Protocorm Multiplication accumulation in bottle after 20 days.
Proceeded to by seedling in fresh culture, carry out culture of rootage and strong seedling culture, this step will note the uniformity of seedling, will eliminate seedling inferior simultaneously.Every 2 months are as stated above, on identical medium, 1 seedling of transferring, after corotation connects 2 ~ 3 times, seedling reaches the bottle seedling of 6.0 ~ 8.0 centimetres high, grows comparatively thick longer simultaneously, now bottle seedling can bottle outlet hardening, transplanting.
Condition of culture is: temperature (26 ± 2) DEG C, illumination 12h/d, intensity of illumination 1500 ~ 2500lx.
This HERBA DENDROBII group training breeding method, 1 HERBA DENDROBII fruit can turn out 10000 ~ 12500 clumps of plantlet in vitro.Practical Calculation is as follows: 1 fruit can connect 8 ~ 10 female bottles, every 1 female bottle can connect female bottle in 5, in every 1, female bottle can connect female bottle in 5 secondaries, female bottle graft 5 sub-bottles in every 1 secondary, finally cultivate 1000 ~ 1250 bottles of plantlet in vitro altogether, there are 10 clumps of plantlet in vitro in one bottle, namely cultivate 10000 ~ 12500 clumps of plantlet in vitro, 1 clump of plantlet in vitro 3 ~ 5 strain HERBA DENDROBII.
Above process, needs 6 ~ 8 months altogether.
Compared with HERBA DENDROBII modes of reproduction in the past, this method adopts without hormone cultivation, and overall cultivation stage all adopts identical medium, greatly improve sapling multiplication speed and reduce workload, the plantlet in vitro robust growth of acquisition, root of hair is many, transplanting survival rate is high, production cost is low, and pollution rate is low, reaches the industrialization level of production.
Claims (7)
1. a breeding method for HERBA DENDROBII plantlet in vitro, is characterized in that comprising the following steps: the 1) selection of HERBA DENDROBII fruit and surface sterilization; 2) axenic germination of HERBA DENDROBII seed; 3) Protocorm Multiplication and differentiation are cultivated; 4) strong sprout and culture of rootage.
2. the breeding method of a kind of HERBA DENDROBII plantlet in vitro according to claim 1, it is characterized in that: step (1) is specially selects HERBA DENDROBII fruit, after clear water rinses, at 70% alcohol-pickled 1 minute, aseptic water washing 3 times, use 0.1% mercury chloride surface sterilization 10 minutes again, after aseptic water washing 5 times, blot surface moisture with aseptic filter paper.
3. the breeding method of a kind of HERBA DENDROBII plantlet in vitro according to claim 1, is characterized in that: step (2) is specially and is cut in half by HERBA DENDROBII fruit, removes pericarp, is implanted by seed on medium, cultivates HERBA DENDROBII protocorm.
4. the breeding method of a kind of HERBA DENDROBII plantlet in vitro according to claim 1, is characterized in that: step (3) is specially and proceeds on fresh culture by HERBA DENDROBII protocorm, carries out Protocorm Multiplication and differentiation cultivation.
5. the breeding method of a kind of HERBA DENDROBII plantlet in vitro according to claim 1, it is characterized in that: step (4) is specially and seedling is proceeded to fresh culture, switching in every 2 months 1 seedling, when seedling reaches 6.0 ~ 8.0cm height after 2 ~ 3 times, carries out hardening, transplanting.
6. the breeding method of a kind of HERBA DENDROBII plantlet in vitro according to any one of Claims 1 to 5, is characterized in that: described medium is: 1/2MS+20% potato+30g/L sucrose+4g/L agar powder, pH value is 5.8.
7. the breeding method of a kind of HERBA DENDROBII plantlet in vitro according to claim 1, is characterized in that: described condition of culture is: temperature 26 ± 2 DEG C, illumination 12h/d, intensity of illumination 1500 ~ 2500Lx.
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| CN201410745002.3A CN104429962A (en) | 2014-12-10 | 2014-12-10 | Cultivation method of dendrobium nobile tissue culture seedlings |
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| CN201410745002.3A CN104429962A (en) | 2014-12-10 | 2014-12-10 | Cultivation method of dendrobium nobile tissue culture seedlings |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104938338A (en) * | 2015-06-17 | 2015-09-30 | 临沧市云瑞堂生物科技有限公司 | Culture medium series for quick breeding and seedling of dendrobium chrysanthum and tissue culture method |
| CN105028192A (en) * | 2015-06-17 | 2015-11-11 | 临沧市云瑞堂生物科技有限公司 | Culture medium series for rapidly breeding dendrobium nobile seedlings and tissue culture method |
| CN110972944A (en) * | 2019-12-18 | 2020-04-10 | 南京师范大学 | Method for intermittently immersing dendrobium nobile seedlings |
| CN113025498A (en) * | 2021-02-05 | 2021-06-25 | 遵义医科大学 | Method for co-culturing endophyte and dendrobium nobile protocorm with stable and high dendrobine yield |
| CN113598045A (en) * | 2021-07-16 | 2021-11-05 | 上海植物园 | Culture medium for dendrobium yunnanense and rapid propagation method |
| CN113951143A (en) * | 2021-11-23 | 2022-01-21 | 韶关学院 | In-vitro propagation method of Dendrobii officmalis caulis with Danxia landform |
| 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 |
-
2014
- 2014-12-10 CN CN201410745002.3A patent/CN104429962A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104938338A (en) * | 2015-06-17 | 2015-09-30 | 临沧市云瑞堂生物科技有限公司 | Culture medium series for quick breeding and seedling of dendrobium chrysanthum and tissue culture method |
| CN105028192A (en) * | 2015-06-17 | 2015-11-11 | 临沧市云瑞堂生物科技有限公司 | Culture medium series for rapidly breeding dendrobium nobile seedlings and tissue culture method |
| CN110972944A (en) * | 2019-12-18 | 2020-04-10 | 南京师范大学 | Method for intermittently immersing dendrobium nobile seedlings |
| CN110972944B (en) * | 2019-12-18 | 2022-12-06 | 南京师范大学 | Method for intermittently immersing dendrobium nobile seedlings |
| CN113025498A (en) * | 2021-02-05 | 2021-06-25 | 遵义医科大学 | Method for co-culturing endophyte and dendrobium nobile protocorm with stable and high dendrobine yield |
| CN113598045A (en) * | 2021-07-16 | 2021-11-05 | 上海植物园 | Culture medium for dendrobium yunnanense and rapid propagation method |
| CN113951143A (en) * | 2021-11-23 | 2022-01-21 | 韶关学院 | In-vitro propagation method of Dendrobii officmalis caulis with Danxia landform |
| CN113951143B (en) * | 2021-11-23 | 2023-11-21 | 韶关学院 | In-vitro propagation method of dendrobium candidum with geomorphic form of danxia |
| 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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