CN106665366A - Tissue culture method of dendrobium unicum - Google Patents
Tissue culture method of dendrobium unicum Download PDFInfo
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- CN106665366A CN106665366A CN201710138761.7A CN201710138761A CN106665366A CN 106665366 A CN106665366 A CN 106665366A CN 201710138761 A CN201710138761 A CN 201710138761A CN 106665366 A CN106665366 A CN 106665366A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
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Abstract
The invention belongs to the technical field of plant tissue culture, and particularly relates to a tissue culture method of dendrobium unicum. The tissue culture method of the dendrobium unicum comprises the following steps: (1) disinfecting a dendrobium unicum capsule serving as an explant; (2) enabling the explant to be germinated to obtain a germfree test-tube plantlet; (3) culturing the germfree test-tube plantlet to obtain test-tube plantlet cluster buds; (4) putting the test-tube plantlet cluster buds into a seedling strengthening culture medium, and carrying out culture in the seedling strengthening culture medium to obtain a test-tube plantlet; (5) putting the test-tube plantlet into a rooting culture medium to obtain a tissue culture seedling; (6) after the tissue culture seedling is hardened, transferring the tissue culture seedling into a matrix for culture. The growth coefficient of the dendrobium unicum cluster buds obtained by the culture method disclosed by the invention is increased by 20 to 30 times; the tissue culture method can provide robust and high-quality dendrobium unicum seedlings within short time, so that the problem of large-scale seedling culture of the dendrobium unicum is effectively solved.
Description
Technical field
The invention belongs to field of plant tissue culture technique, relates in particular to a kind of tissue culture side of only keratite dry measure used in former times
Method.
Background technology
Only keratite dry measure used in former times (Dendrobium unicum), orchid family Dendrobium Sw.Its lip is inverted like Cornu rhinocerotis, is furnished with Fructus Citri tangerinae
Red reticulate pattern, it is very unique.Although it does not have the flower pattern of full rounding, the orange red something lost with tendon shape net vein on lip of petal
Pass and it is a dark horse, gradually pursued.Vietnam, Laos, Burma and Thailand's (height above sea level 800-1550 rice) are distributed in, are grown
On the rock and undershrub of low land forest.Spring at florescence to early summer, flower life-span length, up to more than 1 month.It is deep to receive people's
Like.So far, have no the report of only keratite dry measure used in former times tissue culture.
Yet with the natural propagation of orchid family seed mainly by division propagation, germination is originally difficult in the wild, and with society
Development, the excessive harvesting of people causes wild only angle Dendrobium Plants to be destroyed mostly, almost becomes extinct, therefore find a kind of
The only keratite dry measure used in former times of method culture of tissue culture, makes only angle Rapid Propagation of Dendrobium, realizes that the batch production of only keratite dry measure used in former times high quality seedling is educated
Seedling, meets the demand of people, becomes the problem that urgently people solve.
The information for being disclosed in the background section is merely intended to increase the understanding of the general background to the present invention, and should not
When the prior art for being considered to recognize or imply the information structure in any form well known to persons skilled in the art.
The content of the invention
It is an object of the invention to provide a kind of method for tissue culture of only keratite dry measure used in former times, can keep the Optimality of original kind
Shape, cultivates only keratite dry measure used in former times seedling of a large amount of suitable cultivating and growings at short notice, and simple bud growth coefficient reaches more than 20-30 times,
And test tube seedling Multiple Buds are healthy and strong, easily take root in root media, after transplanting medium, survival rate is more than 90%.
The technical scheme that the present invention is provided is as follows：
A kind of method for tissue culture of only keratite dry measure used in former times, comprises the following steps：
(1) Fruit pod for taking only keratite dry measure used in former times carries out disinfection and pretreatment, first with the water-soluble immersion of liquid detergent that volume fraction is 2%
After bubble 5min, 15-30min is rinsed with flowing water, then by the Fruit pod calcination 2 times of only keratite dry measure used in former times, after each calcination 5-10s, obtain
Explant；
(2) explant is cut, is inoculated in the first culture medium after taking out seed, cultivated in the first culture medium
20-30 days, obtain in vitro cuttings；
(3) in vitro cuttings are placed in the second culture medium, cultivate 50~70 days in the second culture medium, tried
Pipe Seedling Multiple Buds；
(4) the test tube seedling Multiple Buds are placed in strong seedling culture base, cultivate 20-40 days in strong seedling culture base, obtain
Test tube seedling；
(5) test tube seedling is placed in root media, cultivates 30-50 days in root media, obtain tissue cultured seedling；
(6) seedling exercising：The culture bottle of the tissue cultured seedling and the culture tissue cultured seedling is removed into culturing room together, culture bottle is opened
Bottle cap, after the tap water of 30mL is added in culture bottle, natural lighting 3-5d；
(7), after seedling exercising terminates, the tissue cultured seedling is taken out from culture bottle, will the root of the tissue cultured seedling clean after transplant to
In substrate.
Preferably, in step (1) by the concrete grammar of the Fruit pod calcination 2 times of only keratite dry measure used in former times being：In aseptic super-clean bench
The Fruit pod of only keratite dry measure used in former times is lived with aseptic tweezer, in the ethanol that whole Fruit pod surface contamination volume fraction is 75%, then calcination,
Calcination 1 time is repeated after ethanol is burnt out.
Preferably, the first culture medium described in step (2) includes：The 6- benzyl ammonia purine of MS, 3.0mg/L,
The activated carbon of the naphthalene acetic acid and 3g/L of 2.0mg/L.
Preferably, the second culture medium described in step (3) includes：The first mercapto of MS, 0.5-2.0mg/L up to piperazine,
The kinetins of the naphthalene acetic acid and 0.2-0.5mg/L of 0.5-1.5mg/L.
Preferably, the strong seedling culture base described in step (4) includes：The 6- benzyl amino glands of MS, 1.0-4.0mg/L are fast
The naphthalene acetic acid of purine and 1.0-4.0mg/L.
Preferably, the root media described in step (5) includes：The mashed potatoes of 1/2MS and 20g/L, it is described
Mashed potatoes be Rhizoma Solani tuber osi is blended after obtain.
Preferably, the first described culture medium, the second described culture medium, described strong seedling culture base and described
The also agar containing 5g/L and 30g/L sucrose in root media, and it is first culture medium, second culture medium, described
The original ph of strong seedling culture base and the root media is 5.8.
Preferably, the culture in step (2)-(5) is and controls illumination cultivation condition and be：Cultivation temperature 23-27 DEG C, light
According to intensity 1500lux, light application time is 8-10h/d.
Preferably, the volume ratio of bark, humus soil and sphagna is 1 in substrate described in step (7):1:1.
Compared with prior art, the present invention has the advantages that：
(1) method of the present invention carries out tissue-culturing quick-propagation to only keratite dry measure used in former times using biotechnology, maintains original
The merit of kind, can cultivate only keratite dry measure used in former times seedling of a large amount of suitable cultivating and growings at short notice, and cultivate it is only
Keratite dry measure used in former times seedling quality is good, can accomplish scale production, and meets the needs in production.In the present invention, simple bud growth coefficient reaches
More than 20-30 times, and test tube seedling Multiple Buds are healthy and strong, easily take root after being inoculated into root media, after transplanting medium, survival rate exists
More than 90%.
(2) explant is sterilized in the way of calcination in the method for the present invention, with traditional employing mercuric chloride sterilizing side
Formula is compared, and has both avoided secondary pollution, and nonhazardouss are more environmentally friendly.
(3) add activated carbon in the first culture medium that experiment is sprouted in the method for the present invention so that in the first culture medium
Environment imitates dark culturing, is more beneficial for embryo germination.The mashed potatoes smashed is added in root media, containing rich in mashed potatoes
Rich carbohydrate, protein, also abundant potassium, calcium, phosphorus, ferrum and vitamin A, B and C etc., can be provided needed for taking root
Nutrient substance, and B12 vitamin energy hestening rootings are conducive to tissue culture seedling rooting.
(4) solely in the Fruit pod of keratite dry measure used in former times containing abundant carbohydrate, aminoacid, protein, also abundant potassium,
Calcium, phosphorus, ferrum, magnesium etc. and various trace elements, it is broken to add Fruit pod in the first culture medium, after only keratite dry measure used in former times Fruit pod is crushed and swallow
Nutrient substance in wheat is mutually complementary, common to provide nutrient substance for endosperm, promotes germination.
Specific embodiment
Below the specific embodiment of the present invention is described in detail, it is to be understood that protection scope of the present invention is not
Limited by specific embodiment.
Embodiment 1：
A kind of method for tissue culture of only keratite dry measure used in former times, comprises the following steps：
(1) Fruit pod for taking only keratite dry measure used in former times carries out disinfection and pretreatment, with the liquid detergent aqueous solution soaking that volume fraction is 2%
After 5min, 15min is rinsed with flowing water, then by the Fruit pod calcination 2 times of only keratite dry measure used in former times, after each calcination 5s, obtain explant；
(2) explant is cut, is inoculated in the first culture medium after taking out seed, trained in first culture medium
Support 30 days, obtain in vitro cuttings；First culture medium includes：The 6- benzyl ammonia purine of MS, 3.0mg/L and 2.0mg/L
Naphthalene acetic acid, the activated carbon of 3g/L；
(3) in vitro cuttings are placed in the second culture medium, cultivate 30 days in second culture medium, tried
Pipe Seedling Multiple Buds；Second culture medium includes：The first mercapto of MS, 1.0mg/L reaches piperazine, the naphthalene acetic acid of 0.5mg/L and 0.2mg/L
Kinetins；
(4) the test tube seedling Multiple Buds are placed in strong seedling culture base, cultivate 30 days in the strong seedling culture base, obtain
Test tube seedling；The strong seedling culture base includes：The 6- benzyls aminoadenine and the naphthalene acetic acid of 4.0mg/L of MS, 3.0mg/L；
(5) test tube seedling is placed in root media, cultivates 30 days in the root media, obtain complete group
Seedlings cultivating；The root media includes：The mashed potatoes of 1/2MS and 20g/L, the mashed potatoes be Rhizoma Solani tuber osi is blended after obtain；
(6) seedling exercising, the concrete grammar of seedling exercising is：The culture bottle of the tissue cultured seedling and the culture tissue cultured seedling is removed together
Culturing room, opens culture bottle cap, after the tap water of 30mL is added in culture bottle, natural lighting 3d；
(7), after seedling exercising terminates, the tissue cultured seedling is taken out from culture bottle, will the root of the tissue cultured seedling clean after transplant to
In substrate, in the substrate, the volume ratio of bark, humus soil and sphagna is 1:1:1；
Described the first culture medium, the second described culture medium, described strong seedling culture base and described root culture
The also agar containing 5g/L and 30g/L sucrose in base, and first culture medium, second culture medium, the strong seedling culture
The original ph of base and the root media is 5.8；
Culture in step (2)-(5) is and controls illumination cultivation condition and be：23 DEG C of cultivation temperature, intensity of illumination
1500lux, light application time are 8-10h/d.
Embodiment 2：
A kind of method for tissue culture of only keratite dry measure used in former times, comprises the following steps：
(1) Fruit pod for taking only keratite dry measure used in former times carries out disinfection and pretreatment, with the liquid detergent aqueous solution soaking that volume fraction is 2%
After 5min, 30min is rinsed with flowing water, then by the Fruit pod calcination 2 times of only keratite dry measure used in former times, after each calcination 10s, obtain explant；
(2) explant is cut, is inoculated in the first culture medium after taking out seed, trained in first culture medium
Support 30 days, obtain in vitro cuttings；First culture medium includes：The 6- benzyl ammonia purine of MS, 3.0mg/L and 2.0mg/L
Naphthalene acetic acid, the activated carbon of 3g/L；
(3) in vitro cuttings are placed in the second culture medium, cultivate 30 days in second culture medium, tried
Pipe Seedling Multiple Buds；Second culture medium includes：The first mercapto of MS, 1.5mg/L reaches piperazine, the naphthalene acetic acid of 0.5mg/L and 0.2mg/L
Kinetins；
(4) the test tube seedling Multiple Buds are placed in strong seedling culture base, cultivate 30 days in the strong seedling culture base, obtain
Test tube seedling；The strong seedling culture base includes：The 6- benzyls aminoadenine and the naphthalene acetic acid of 4.0mg/L of MS, 3.0mg/L；
(5) test tube seedling is placed in root media, cultivates 30 days in the root media, obtain complete group
Seedlings cultivating；The root media includes：The mashed potatoes of 1/2MS and 20g/L, the mashed potatoes be Rhizoma Solani tuber osi is blended after obtain；
(6) seedling exercising, the concrete grammar of seedling exercising is：The culture bottle of the tissue cultured seedling and the culture tissue cultured seedling is removed together
Culturing room, opens culture bottle cap, after the tap water of 30mL is added in culture bottle, natural lighting 5d；
(7), after seedling exercising terminates, the tissue cultured seedling is taken out from culture bottle, will the root of the tissue cultured seedling clean after transplant to
In substrate, in the substrate, the volume ratio of bark, humus soil and sphagna is 1:1:1；
Described the first culture medium, the second described culture medium, described strong seedling culture base and described root culture
The also agar containing 5g/L and 30g/L sucrose in base, and first culture medium, second culture medium, the strong seedling culture
The original ph of base and the root media is 5.8；
Culture in step (2)-(5) is and controls illumination cultivation condition and be：27 DEG C of cultivation temperature, intensity of illumination
1500lux, light application time are 8-10h/d.
Embodiment 3：
A kind of method for tissue culture of only keratite dry measure used in former times, comprises the following steps：
(1) Fruit pod for taking only keratite dry measure used in former times carries out disinfection and pretreatment, with the liquid detergent aqueous solution soaking that volume fraction is 2%
After 5min, 20min is rinsed with flowing water, then by the Fruit pod calcination 2 times of only keratite dry measure used in former times, after each calcination 8s, obtain explant；
(2) explant is cut, is inoculated in the first culture medium after taking out seed, trained in first culture medium
In vitro cuttings are obtained after supporting 30 days；First culture medium includes：The 6- benzyl ammonia purine of MS, 3.0mg/L and 2.0mg/L
Naphthalene acetic acid, the activated carbon of 3g/L；
(3) in vitro cuttings are placed in the second culture medium, cultivate 30 days in second culture medium, tried
Pipe Seedling Multiple Buds；Second culture medium includes：The first mercapto of MS, 2.0mg/L reaches piperazine, the naphthalene acetic acid of 0.5mg/L and 0.2mg/L
Kinetins；
(4) the test tube seedling Multiple Buds are placed in strong seedling culture base, cultivate 30 days in the strong seedling culture base, obtain
Test tube seedling；The strong seedling culture base includes：The 6- benzyls aminoadenine and the naphthalene acetic acid of 4.0mg/L of MS, 3.0mg/L；
(5) test tube seedling is placed in root media, after cultivating 30 days in the root media, obtains complete
Tissue cultured seedling；The root media includes：The mashed potatoes of 1/2MS and 20g/L, the mashed potatoes be Rhizoma Solani tuber osi is blended after
Arrive；
(6) seedling exercising, the concrete grammar of seedling exercising is：The culture bottle of the tissue cultured seedling and the culture tissue cultured seedling is removed together
Culturing room, opens culture bottle cap, after the tap water of 30mL is added in culture bottle, natural lighting 4d；
(7), after seedling exercising terminates, the tissue cultured seedling is taken out from culture bottle, will the root of the tissue cultured seedling clean after transplant to
In substrate, in the substrate, the volume ratio of bark, humus soil and sphagna is 1:1:1；
Described the first culture medium, the second described culture medium, described strong seedling culture base and described root culture
The also agar containing 5g/L and 30g/L sucrose in base, and first culture medium, second culture medium, the strong seedling culture
The original ph of base and the root media is 5.8；
Culture in step (2)-(5) is and controls illumination cultivation condition and be：25 DEG C of cultivation temperature, intensity of illumination
1500lux, light application time are 8-10h/d.
The transplanting survival rate of the tissue cultured seedling of the breeding coefficient and gained of simple bud in embodiment 1-3 is investigated and is counted,
The results are shown in Table 1.
The transplanting survival rate of the tissue cultured seedling of the breeding coefficient and gained of 1 method of the present invention simple bud of table is determined
As shown in Table 1, in the method for the present invention simple bud growth coefficient up to 20-30 times, and test tube seedling Multiple Buds are healthy and strong, connect
Plant and easily take root to after root media, survival rate is more than 95% after transplanting medium.
It is aforementioned to the present invention specific illustrative embodiment description be in order to illustrate and illustration purpose.These descriptions
It is not wishing to limit the invention to disclosed precise forms, and it will be apparent that according to above-mentioned teaching, much can be changed
And change.The purpose of selecting and describing the exemplary embodiment is that explaining that the certain principles and its reality of the present invention should
With so that those skilled in the art can realize and using the present invention a variety of exemplaries and
A variety of selections and change.The scope of the present invention is intended to be limited by claims and its equivalents.
Claims (9)
1. a kind of method for tissue culture of only keratite dry measure used in former times, it is characterised in that comprise the following steps：
(1) Fruit pod for taking only keratite dry measure used in former times carries out disinfection and pretreatment, first with the liquid detergent aqueous solution soaking that volume fraction is 2%
After 5min, 15-30min is rinsed with flowing water, then by the Fruit pod calcination 2 times of only keratite dry measure used in former times, after each calcination 5-10s, obtain outer
Implant；
(2) explant is cut, be inoculated in the first culture medium after taking out seed, 20-30 cultivated in the first culture medium
My god, obtain in vitro cuttings；
(3) in vitro cuttings are placed in the second culture medium, cultivate 50~70 days in the second culture medium, obtain test tube seedling
Multiple Buds；
(4) the test tube seedling Multiple Buds are placed in strong seedling culture base, cultivate 20-40 days in strong seedling culture base, obtain test tube
Seedling；
(5) test tube seedling is placed in root media, cultivates 30-50 days in root media, obtain tissue cultured seedling；
(6) seedling exercising：The culture bottle of the tissue cultured seedling and the culture tissue cultured seedling is removed into culturing room together, culture bottle bottle is opened
Lid, after the tap water of 30mL is added in culture bottle, natural lighting 3-5d；
(7) after seedling exercising terminates, the tissue cultured seedling is taken out from culture bottle, is transplanted to substrate after the root of the tissue cultured seedling is cleaned
In.
2. the method for tissue culture of only keratite dry measure used in former times according to claim 1, it is characterised in that by only keratite in step (1)
The concrete grammar of the Fruit pod calcination 2 times of dry measure used in former times is：The Fruit pod of only keratite dry measure used in former times is lived with aseptic tweezer in aseptic super-clean bench, whole
Fruit pod surface contamination volume fraction is 75% ethanol, and then calcination repeats calcination 1 time after ethanol is burnt out.
3. the method for tissue culture of only keratite dry measure used in former times according to claim 1, it is characterised in that described in step (2)
One culture medium includes：The activated carbon of the 6- benzyl ammonia purine, the naphthalene acetic acid of 2.0mg/L and 3g/L of MS, 3.0mg/L.
4. the method for tissue culture of only keratite dry measure used in former times according to claim 1, it is characterised in that described in step (3)
Two culture medium include：The first mercapto of MS, 0.5-2.0mg/L swashing up to piperazine, the naphthalene acetic acid of 0.5-1.5mg/L and 0.2-0.5mg/L
Therbligs.
5. the method for tissue culture of only keratite dry measure used in former times according to claim 1, it is characterised in that strong described in step (4)
Seedling culture medium includes：The 6- benzyls aminoadenine and the naphthalene acetic acid of 1.0-4.0mg/L of MS, 1.0-4.0mg/L.
6. the method for tissue culture of only keratite dry measure used in former times according to claim 1, it is characterised in that the life described in step (5)
Root culture medium includes：The mashed potatoes of 1/2MS and 20g/L, described mashed potatoes be Rhizoma Solani tuber osi is blended after obtain.
7. the method for tissue culture of only keratite dry measure used in former times according to claim 1, it is characterised in that described the first culture medium,
The also agar containing 5g/L and 30g/ in described the second culture medium, described strong seedling culture base and described root media
L sucrose, and first culture medium, second culture medium, the strong seedling culture base and the root media is initial
PH value is 5.8.
8. the method for tissue culture of only keratite dry measure used in former times according to claim 1, it is characterised in that the training in step (2)-(5)
Support to be and control illumination cultivation condition and be：Cultivation temperature 23-27 DEG C, intensity of illumination 1500lux, light application time are 8-10h/d.
9. the method for tissue culture of only keratite dry measure used in former times according to claim 1, it is characterised in that the base described in step (7)
In matter, the volume ratio of bark, humus soil and sphagna is 1:1:1.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710138761.7A CN106665366A (en) | 2017-03-09 | 2017-03-09 | Tissue culture method of dendrobium unicum |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710138761.7A CN106665366A (en) | 2017-03-09 | 2017-03-09 | Tissue culture method of dendrobium unicum |
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| CN201710138761.7A Pending CN106665366A (en) | 2017-03-09 | 2017-03-09 | Tissue culture method of dendrobium unicum |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107333653A (en) * | 2017-07-31 | 2017-11-10 | 广西大学 | A kind of common wild-rice tissue cultivation rapid breeding method |
| CN108541594A (en) * | 2018-06-28 | 2018-09-18 | 广西壮族自治区农业科学院农产品质量安全与检测技术研究所 | A kind of tissue culture and rapid propagation method of polygonatum cirrhifolium Royle |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1541518A (en) * | 2003-11-04 | 2004-11-03 | 中国科学院华南植物研究所 | Dendrobium unicum aseptic seeding and test tube seedling tecnnology |
| CN105432470A (en) * | 2015-12-08 | 2016-03-30 | 广西壮族自治区药用植物园 | Tissue culture method for dendrobium moniliforme |
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2017
- 2017-03-09 CN CN201710138761.7A patent/CN106665366A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1541518A (en) * | 2003-11-04 | 2004-11-03 | 中国科学院华南植物研究所 | Dendrobium unicum aseptic seeding and test tube seedling tecnnology |
| CN105432470A (en) * | 2015-12-08 | 2016-03-30 | 广西壮族自治区药用植物园 | Tissue culture method for dendrobium moniliforme |
Non-Patent Citations (1)
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| 陈之林等: ""独角石斛的离体繁殖"", 《植物生理学通讯》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107333653A (en) * | 2017-07-31 | 2017-11-10 | 广西大学 | A kind of common wild-rice tissue cultivation rapid breeding method |
| CN107333653B (en) * | 2017-07-31 | 2019-11-22 | 广西大学 | A kind of common wild-rice tissue cultivation rapid breeding method |
| CN108541594A (en) * | 2018-06-28 | 2018-09-18 | 广西壮族自治区农业科学院农产品质量安全与检测技术研究所 | A kind of tissue culture and rapid propagation method of polygonatum cirrhifolium Royle |
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Application publication date: 20170517 |