CN106069751B - A kind of method of nobile-type dendrobium variety ' firebird ' tissue-culturing rapid propagation - Google Patents
A kind of method of nobile-type dendrobium variety ' firebird ' tissue-culturing rapid propagation Download PDFInfo
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- CN106069751B CN106069751B CN201610412130.5A CN201610412130A CN106069751B CN 106069751 B CN106069751 B CN 106069751B CN 201610412130 A CN201610412130 A CN 201610412130A CN 106069751 B CN106069751 B CN 106069751B
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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
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Abstract
The invention belongs to plant tissue culture fields, disclose a kind of method of nobile-type dendrobium variety ' firebird ' tissue-culturing rapid propagation.Key step includes：It is explant that spring, which takes ' firebird ' current year raw stem section, carry out successively 1) explant in NaClO and HgCl2Middle disinfection, 2) it is transferred to inducing culture MS+6-BA 3mgL‑1+NAA0.2mg·L‑1Middle culture, 3) proliferated culture medium MS+TDZ 0.1mgL are changed to afterwards‑1+NAA 0.2mg·L‑1+200g·L‑1Mashed potatoes+1gL‑1Cultivated in activated carbon, 4) change strengthening seedling and rooting culture medium 1/2MS+0.5mgL‑1IBA+200g·L‑1Cultivated in mashed potatoes, 5) acclimatization and transplants, obtain nobile-type dendrobium variety ' firebird ' tissue-cultured seedling.One kind ' firebird ' tissue culture and rapid propagation method provided by the invention, the maximization of breeding coefficient can be reached in the shortest time, be conducive to the fast seedling growing of cattleya Dendrobium kind ' firebird ', shorten seedling stage and growth cycle, achievees the purpose that reproduction speed is fast, saves production cost and be suitable for scale seedling raising.The vegetable material used is few, and growth is fast, and the period is short, and repeatability is strong, can test in the anniversary or produce.
Description
Technical field
The invention belongs to agricultural biological technical fields, refer to a kind of tissue-culturing rapid propagation of nobile-type dendrobium variety ' firebird '
Method.
Background technology
Nobile-type dendrobium variety ' firebird ' (Dendrobium stardust ' Fire Bird ') is orchid family Dendrobium kind, at present
It there is no the report of the tissue-culturing rapid propagation about nobile-type dendrobium variety ' firebird ' in document.This method is the quick of nobile-type dendrobium variety ' firebird '
Breeding provides a kind of new method, and a kind of the new of breeding is provided as the industrialization of nobile-type dendrobium for nobile-type dendrobium variety ' firebird '
Method.
Invention content
In order to change current nobile-type dendrobium variety ' firebird ' breeding, cultivate undesirable situation, it is an object of the invention to accelerate
The cultivation of nobile-type dendrobium variety ' firebird ' and exploitation dynamics, improve the reproductive efficiency of nobile-type dendrobium variety ' firebird ', accelerate nobile-type dendrobium product
The propagation in scale of kind of ' firebird ' seedling is conducive to nobile-type dendrobium variety ' firebird ' as energy xylophyta and ornamental nobile-type dendrobium
Production and exploitation.The method of the present invention carries out induction Multiple Buds especially suitable for spring producing initial stage acquisition spray.
A kind of method of nobile-type dendrobium variety ' firebird ' tissue-culturing rapid propagation, includes the following steps：
Acquisition nobile-type dendrobium variety ' firebird ' current year, raw tender stem was explant, carried out 1) tender stem disinfection, 2) tender stem induction successively
Culture, 3) adventitious buds proliferation culture, 4) culture of Multiple Buds strengthening seedling and rooting, 5) acclimatization and transplants, obtain nobile-type dendrobium variety ' firebird ' group
Train seedling.
Acquisition nobile-type dendrobium variety ' firebird ' the current year raw spray is sprouted in spring acquisition nobile-type dendrobium variety ' firebird ' bud
The branch not bound formed, it is desirable that branch degree of lignification is low, it is desirable that each stem section is with 1~2 axillary bud.
1) tender stem explant disinfection is that flowing water rinses 2~3h of tender stem, on super-clean bench with 75% ethanol postincubation 30~
40s, rinsed with sterile water 3~4 times sterilize 5min with 5% sodium hypochlorite (NaClO, Sodium hypochlorite), then use
6min is sterilized in 0.3% mercuric chloride (mercuric chloride, Mercury chloride), rinsed with sterile water 3~4 times later are used in combination sterile
Filter paper sucks excessive moisture.
2) the inducing clumping bud culture：Aseptically the tender stem of acquisition is inoculated on inducing culture, 25
± 2 DEG C, 14~16h of illumination, pH value is 5.8~6.0, is cultivated under the conditions of light intensity 3000lx, and sprouting starts to sprout after being inoculated with 22d,
22~35d obtains Multiple Buds；45d rear blades are unfolded.
The inducing clumping bud culture medium is 6-BA (the 6- benzyls containing 3.0mg/L using MS culture mediums as minimal medium
Base adenine phosphate, benzylaminmopurine), the NAA (methyl α-naphthyl acetate, 1-Naphthaleneacetic acid) of 0.2mg/L,
The culture medium of 5g/L agar, 20g/L glucose.
Described to sprout new Multiple Buds, the time is to be inoculated on inducing clumping bud culture medium after 18d.
The mounted blade, time are to be inoculated on inducing clumping bud culture medium after 42d.
3) the adventitious buds proliferation squamous subculture is inoculated into Multiplying culture for that will pass through the Multiple Buds of Fiber differentiation sprouting
Squamous subculture is carried out on base, is placed in the culturing room of 25 ± 1 DEG C of temperature, and every 30~40d subcultures rolling bottle is primary, expands Multiple Buds number
Amount, and make Multiple Buds continued growth.
The adventitious buds proliferation culture medium is TDZ, 0.2mg/L containing 0.1mg/L using MS culture mediums as minimal medium
NAA, 200gL-1Mashed potatoes, 1gL-1The culture medium of activated carbon, 4.5g/L agar, 20g/L glucose.
3) strengthening seedling and rooting：The Multiple Buds that will largely be formed after subculture are transferred to root media and carry out rooting induction, promote
Into taking root, the seedling with root is grown up to after 38d.
Described to grow up to the seedling with root, the time is to be inoculated on root media after 38 days.
4) the Multiple Buds strengthening seedling and rooting culture, for the unrooted Multiple Buds for passing through abundant subculture are inoculated into strengthening seedling and rooting training
It supports on base, after cultivating 12d, the white adventitious root of base portion generation, culture 35d hair roots are apparent, form intact plant.
The Multiple Buds strengthening seedling and rooting culture medium is the IBA containing 0.5mg/L using 1/2MS culture mediums as minimal medium
The culture medium of (indolebutyric acid, Indole-3-Butytric acid), 4.5g/L agar, 20g/L glucose.
5) acclimatization and transplants are put into 4~6d of hardening in greenhouse for the tissue-cultured seedling for forming complete root system is connected bottle, increase light
According to rear bottle outlet is cleaned to be put into clean matrix and be cultivated, and higher levels of humidity is kept.
The matrix is turfy soil：Matrix obtained by perlite 3: 1 mixing by volume.
The inducing clumping bud culture medium, adventitious buds proliferation culture medium, root media pH value be preferably 5.80.
The culture, condition of culture be daily illumination 10h~12h, temperature be 25 DEG C ± 2 DEG C, illumination 12h/d, illumination
2000~3000lx of intensity.
The innovation of the present invention be disclose nobile-type dendrobium variety ' firebird ' sprout induction, the formula that is proliferated, takes root and its
A kind of method of the seedling propagation in scale of tissue-culturing rapid propagation.Similar report is had no both at home and abroad at present.
The present invention is conducive to accelerate the breeding of nobile-type dendrobium variety ' firebird ', accelerates its seedling radix, is beneficial to later stage cattleya
The universal and production of middle nobile-type dendrobium.
Description of the drawings
Fig. 1：Nobile-type dendrobium variety ' firebird ' tissue culture and hardening situation of the present invention
Wherein：a：Inducing clumping bud, b：Adventitious buds proliferation, c：Nobile-type dendrobium variety ' firebird ' is taken root, d：Nobile-type dendrobium variety ' fire
The tissue-cultured seedling being colonized after bird ' bottle outlet hardening.
Specific implementation mode
A kind of nobile-type dendrobium variety ' firebird ' tissue culture and rapid propagation method, this approach includes the following steps：(following example for pair
The further explanation of the present invention, but it is not limited to the scope of the present invention).
Embodiment 1：Nobile-type dendrobium variety ' firebird ' inducing culture is screened with the inventive method
1) explant is chosen：Nobile-type dendrobium variety ' firebird ' the branch tender stem for choosing current year raw robust growth no disease and pests harm is outer
Implant is cut into 3cm stem sections, and each stem section stays 1~2 bud, and 10min is cleaned with detergent, then rinses 3h with tap water, most
After to dry material surface moisture spare；
2) tender stem Fiber differentiation：It is dense with quality on superclean bench by the nobile-type dendrobium variety handled well ' firebird ' stem section
Degree is that 75% alcohol impregnates 30s, then sterilizes 7min with mass concentration for 0.3% mercuric chloride, finally uses aseptic water washing 3 times, then will
Tender stem, which is placed on aseptic filter paper, blots excess surface moisture, is seeded on stem section inducing culture, and the explant of inoculation is placed in
22 ± 1 DEG C of temperature, illumination 12h/d, 1500~2000lx of intensity of illumination culturing room in, cultivate 20d after bud sprout, wait for 42d
Mounted blade removes bud, is transferred on adventitious buds proliferation culture medium, is placed in culturing room, breaks up clump after 40d on callus
Sprout (by 40d cultivate induction simple bud averagely Multiple Buds growth coefficient is induced to reach 2.6) nobile-type dendrobium varieties ' firebird ' tissue-cultured seedling
It initially forms.
Embodiment 2：Nobile-type dendrobium variety ' firebird ' adventitious buds proliferation subculture is carried out with the inventive method
Multiple Buds are accessed on adventitious buds proliferation subculture medium, 25 ± 2 DEG C of temperature, illumination 12h/d, intensity of illumination are placed in
In the culturing room of 2000~3000lx, every 30~40d subcultures are primary, and the tissue-cultured seedling of the generation robust growth of subculture 3 times is (in subculture the
At 3 times 5.3) its adventitious buds proliferation coefficient averagely reaches；
3) strengthening seedling and rooting：The test tube seedling of robust growth is subjected to culture of rootage using 2/3DKW culture mediums, grows up to band after 30d
Nobile-type dendrobium variety ' firebird ' seedling of root；
4) acclimatization and transplants：Nobile-type dendrobium variety ' firebird ' seedling with root is subjected to acclimatization and transplants, that is, is trained nobile-type dendrobium product
Kind ' firebird ' tissue-cultured seedling.
Inventive method induction differentiation reaches fast breeding, and then Multiple Buds is induced to take root, and the present invention can make
Reproductive efficiency can reach 8.0, and rooting rate reaches 95%, hardening survival rate 96%.Every plant of nobile-type dendrobium variety ' firebird ' is in scale
Rearing condition under, hundreds of thousands strain can be bred within 1 year, greatly improve the breeding efficiency of nobile-type dendrobium variety ' firebird '.
Implementation example described above is only the preferred embodiment of the present invention, is the further explanation to the present invention, but not
For limiting the scope of the invention.It points out simultaneously, for the those of ordinary skill of this professional domain, is not departing from the invention
Under the premise of technical principle, several improvements and modifications can be also made, these improvements and modifications should also be regarded as the protection model of the present invention
It encloses.
Claims (6)
1. a kind of nobile-type dendrobium variety ' firebird ' tissue culture and rapid propagation method, including the following steps：
It is explant to adopt nobile-type dendrobium variety ' firebird ' tender stem, carries out 1) tender stem disinfection, 2) tender stem Fiber differentiation, 3) Multiple Buds successively
Multiplying culture, 4) culture of Multiple Buds strengthening seedling and rooting, 5) acclimatization and transplants, basin on tissue-cultured seedling bottle outlet；2) the tender stem Fiber differentiation
Method is：Aseptically the tender stem of acquisition is inoculated on inducing culture, in 25 2 DEG C, 14~16h of illumination of soil, pH value
It is 5.8~6.0, is cultivated under the conditions of light intensity 3000lx, sprouting starts to sprout after being inoculated with 22d, 22~35d, obtains Multiple Buds；45d
Rear blade is unfolded；The culture medium of tender stem induction be using MS culture mediums as minimal medium, the 6-BA containing 3.0mg/L,
NAA, 5g/L agar of 0.2mg/L, the culture medium of 20g/L glucose；The adventitious buds proliferation used medium is cultivated with MS
Base is minimal medium, NAA, 200gL of TDZ, 0.2mg/L containing 0.1mg/L-1Mashed potatoes, 1gL-1Activated carbon,
The culture medium of 4.5g/L agar, 20g/L glucose；The culture medium of the Multiple Buds strengthening seedling and rooting is using 1/2MS culture mediums as base
Basal culture medium, the culture medium of IBA, 4.5g/L agar, 20g/L glucose containing 0.5mg/L；The culture item of the acclimatization and transplants
Part is daily illumination
10h-12h, temperature are 2 DEG C, illumination 12h/d of 25 soil, intensity of illumination 2000-3000lx.
2. the tissue culture and rapid propagation method of nobile-type dendrobium variety ' firebird ' according to claim 1, it is characterised in that：1) tender stem disappears
Poison is the current year of clip nobile-type dendrobium variety ' firebird ' plant to give birth to tender stem, and flowing water rinses 2~3h, with 75% alcohol on super-clean bench
30~40s is handled, rinsed with sterile water 3~4 times is sterilized with 5% sodium hypochlorite (NaClO, Sodium hypochlorite)
5min, then with sterilizing 6min in 0.3% mercuric chloride (mercuric chloride, Mercury chloride), rinsed with sterile water 3~4 times later,
It is used in combination aseptic filter paper to suck excessive moisture.
3. ' firebird ' tissue culture and rapid propagation method according to claim 1, it is characterised in that：3) the adventitious buds proliferation culture is
The Multiple Buds that will be sprouted by Fiber differentiation, are inoculated on proliferated culture medium and carry out squamous subculture, expand Multiple Buds quantity, and make
Multiple Buds continued growth.
4. ' firebird ' tissue culture and rapid propagation method according to claim 1, it is characterised in that：4) the Multiple Buds strengthening seedling and rooting culture
Base, for the unrooted Multiple Buds for passing through abundant subculture are inoculated on strengthening seedling and rooting culture medium, after cultivating 12d, base portion generates white
Adventitious root, culture 35d hair roots are apparent, form intact plant.
5. ' firebird ' tissue culture and rapid propagation method according to claim 1, it is characterised in that：5) acclimatization and transplants, for that will be formed
The tissue-cultured seedling of complete root system connects bottle and is put into 4~6d of hardening in greenhouse, increases illumination, and rear bottle outlet is cleaned to be put into clean matrix and be planted
Training keeps higher levels of humidity.
6. the method for claim 1-5 any one of them kind ' firebird ' stem segment tissue culture fast breeding is in nobile-type dendrobium variety breeding
Using.
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| CN101180950B (en) * | 2007-12-26 | 2010-12-08 | 浙江森禾种业股份有限公司 | Tissue cultivation rapid breeding method of spring dendrobium stem |
| CN102160525A (en) * | 2011-01-29 | 2011-08-24 | 福建省金草生物科技有限公司 | Method for realizing excellent plant body induction and successive propagation culture on dendrobium candidum by virtue of plant tissue culture |
| CN102657097A (en) * | 2012-06-02 | 2012-09-12 | 上海市闵行区农业科学研究所 | In-vitro culture method for tender stem segments of dendrobium nobile |
| CN103053425B (en) * | 2013-01-22 | 2014-11-12 | 杨宝明 | Rapid propagation method for tissue cultivation of dendrobium candidum stem |
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