CN100512631C - Method and technique of the separated tissue culture of Kava - Google Patents
Method and technique of the separated tissue culture of Kava Download PDFInfo
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- CN100512631C CN100512631C CNB2006101469498A CN200610146949A CN100512631C CN 100512631 C CN100512631 C CN 100512631C CN B2006101469498 A CNB2006101469498 A CN B2006101469498A CN 200610146949 A CN200610146949 A CN 200610146949A CN 100512631 C CN100512631 C CN 100512631C
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Abstract
The invention relates to a slip pepper isolated organism cultivating technique and method, which uses two kinds of organism cultivate techniques to realize the slip pepper regeneration. The invention is characterized in that it overcomes the technique bottleneck of slip pepper isolated organism cultivation as endotrophic mycorrhiza pollution, with which the slip pepper can be cultivated via various methods, to realize the regeneration and quick cultivation of slip pepper and harmless seed via germ cultivation and callus induction. The slip pepper seed cultivated by the invention is germ free and harmless while th cultivate index in each month can reach 7-8, the root rate can reach 100%, and the transplant survival rate can reach 87.2%. The invention has significant social and economic benefits.
Description
[technical field]
The present invention relates to the technology and method that the kawakaw in vitro tissue is cultivated, comprise by the numerous bud of bud, callus induction two approach that sprout and successfully realized the regeneration of aseptic, the nontoxic seedling plant of kawakaw and numerous soon.
[background technology]
Kawakaw (Piper Methysticum Forst.f), be commonly called as slips, perennial Piperaceae (Piperaceae) Piper (Piper) shrub class plant, it is a kind of famous medicinal plant, mainly be distributed in all island countries in ocean, Nan Ping such as Vanuatu, Fiji, Tonga, Papua-New Guinea and Solomon Islands, introduce China in recent years and plant experimentally.
Kawakaw is at the South Sea Islands state medicinal, the cultivation history in existing more than 3000 year.Utilize the beverage of kawakaw root or rhizome preparation to have to relax one's muscles and mood, regain one's strength, hypnotic, lenitive effect.The pharmacology analysis of components shows, the main pharmacology composition of kawakaw root comprise slips because of, dihydro slips because of α-pyrone derivative such as, kawa bitter principle, dihydro kawa bitter principle, yangonin and the high sheep of de-methoxy be peaceful, be referred to as the slips lactone.Pathology experiment and clinical trial confirm that the kawakaw root can be used for treating depression, neurasthenia, autonomic nerve disorder etc.
Kawakaw has the fruitless biological property of only blooming, and mainly breeds in the cuttage mode at present.Cottage propagation need consume a large amount of healthy stem sections, influences the plant normal growth and grows, and long-term cottage propagation causes also that seedling carries disease germs, the seedling endophyte is serious, causes seedling to be degenerated.The technology and method that development kawakaw in vitro tissue is cultivated, will for extensive, commercialization produce kawakaw aseptic nontoxic seedling, extensive artificial cultivation kawakaw lay the first stone.
Because the long-term cottage propagation that relies on, carry disease germs in kawakaw plant surface, endophyte is comparatively serious.How effectively overcoming the endophytic bacterial contamination problem has become the key of development kawakaw in vitro tissue culture technique and method.Taylor and Taufa attempted in 1998, but not success.Calendar year 2001 Briskin etc. add multiple ways such as antibiotic by surface sterilization, in medium, also only obtained the callus of kawakaw blade, stem section, not see so far have the report that obtains complete regeneration kawakaw plant by the in vitro tissue culture technique.
The present invention has successfully overcome technical bottleneck---the endophytic bacterial contamination problem in the cultivation of kawakaw in vitro tissue, broken the single propagation method that kawakaw can only rely on cuttage to breed, successfully realized the regeneration of aseptic, the nontoxic seedling plant of kawakaw and numerous soon by the numerous bud of bud, callus induction two approach that sprout.
[summary of the invention]
The present invention has overcome technical bottleneck---the endophytic bacterial contamination in the cultivation of kawakaw in vitro tissue, broken the single propagation method that kawakaw can only rely on cuttage to breed, successfully realized the regeneration of aseptic, the nontoxic seedling plant of kawakaw and numerous soon by the numerous bud of bud, callus induction two approach that sprout.
Technology path of the present invention and basic step are as follows:
(1) explant is selected and sterilization: selecting annual and the kawakaw stem that has sleeping bud is initial explant, cutting the long stem explants of 1cm that has a sleeping bud after the preliminary treatment places 70% alcohol to soak 30 seconds, in 0.1% mercuric chloride solution constantly agitation treatment 10-15 minute then, aseptic water washing 3-4 time, each 5 minutes;
(2) inoculation and induced dormancy bud sprout
Explant after the sterilization is inoculated on the MS+BA 1.0mg/L+NAA 0.1mg/L+ sucrose 30g/L+ agar 7g/L inducing culture, place under the intensity of illumination environment of 26-28 ℃ and 1500Lx, cultivated 15 days under the condition of 12 hours illumination/12 hour dark, the induced dormancy bud sprouts;
(3) cut leader and induced bundle is sprouted
Cut the long bud point of 15 days 1mm of growth as secondary explant, be inoculated on the MS+BA 1.0mg/L+NAA0.1mg/L+ sucrose 30g/L+ agar 7g/L inducing culture, place under the intensity of illumination environment of 26-28 ℃ and 1500Lx, under the condition of 12 hours illumination/12 hour dark, induced 20 days, on this basis, cutting the aseptic bud that induces is inoculated on the MS+BA 0.5mg/L+NAA 0.5mg/L+ sucrose 30g/L+ agar 7g/L inducing culture, place under the intensity of illumination environment of 26-28 ℃ and 1500Lx, induced bundle is sprouted under hour dark condition in 12 hours illumination/12;
(4) evoked callus
With the aseptic seedling blade inoculation evoked callus on MS+BA 1.0mg/L+NAA 0.1mg/L+ sucrose 30g/L+ agar 7g/L medium that obtains; Well-grown callus lines is inoculated on the inducing clumping bud medium MS+BA 1.0mg/L+IAA 0.5mg/L+ sucrose 30g/L+ agar 7g/L medium, place under the intensity of illumination environment of 26-28 ℃ and 1500Lx, evoked callus sprouts under hour dark condition in 12 hours illumination/12;
(5) root induction
From the bud of growing thickly, cut well-grown single bud, be inoculated on the 1/2MS+IBA 0.75mg/L+ sucrose 30g/L+ agar 7g/L medium, place under the intensity of illumination environment of 26-28 ℃ and 1500Lx root induction under hour dark condition in 12 hours illumination/12.
Preferably, method of the present invention select annual and the healthy kawakaw stem section that has a sleeping bud as initial explant.
Preferably, method of the present invention is carried out pretreated method to initial explant and is: with the flushing 12 hours down of streamlined running water normal temperature condition.
In addition, method of the present invention can also be from the bud of growing thickly that step (3) obtains, cut well-grown single bud, be inoculated on the 1/2MS+IBA 0.75mg/L+ sucrose 30g/L+ agar 7g/L medium, place under the intensity of illumination environment of 26-28 ℃ and 1500Lx root induction under hour dark condition in 12 hours illumination/12.
Interpretation of result:
1, explant sterilization: streamlined running water flushing is after 12 hours, place 70% alcohol to soak 30 seconds with cutting into the long stem section (having 1 sleeping bud) of 1cm, in 0.1% mercuric chloride constantly agitation treatment 10-15 minute, on medium, cultivated 10 days, visible pollution rate only has 41%, and this illustrates that this surface sterilization processing all has certain effect to kawakaw stem section surface silt particle, bacterium and fungal spore removal thereof.Because endophytic bacterial contamination, when cultivating 30 days, pollution rate is near 100%.
2, the removal of endophyte and aseptic bud obtain
Because long-term cottage propagation, the kawakaw endophytic bacterial contamination is serious, is the technical bottleneck that the kawakaw in vitro tissue is cultivated.This research is secondary explant by the bud point (from sleeping bud) of selecting certain growth time, certain-length, has successfully overcome this difficulty, has obtained the bud of integral asepsis.We studies show that, select the bud point of growth 15 days, 0.5mm to carry out the numerous bud of bud as secondary explant, and the secondary pollution rate can be reduced to 64%.
3, obtain higher reproduction coefficient by the numerous bud of bud
By the numerous bud approach of bud, every month growth coefficient of kawakaw reaches 7-8.
4, utilize the aseptic seedling blade to obtain embryo callus and induced bundle is sprouted for explant
With the aseptic seedling blade that obtains is explant, and by induced embryonic callus, the induced bundle approach of sprouting has also obtained higher reproduction coefficient then, is up to 4.5.
5, high rooting rate: the aseptic bud cutting that will obtain, go to and urge the root medium, obtained the highest efficient of taking root, can reach 100%.
6, higher transplanting survival rate: it is essential that the seedling of growing under the normal condition carries out before moving into the land for growing field crops that domestication about 10-15 days cultivates.According to the proportioning test of this laboratory to different culture matrixes, find at sand: limestone soil: the little shoot survival percent of growing in the matrix of coconut palm chaff=1:2:1 is up to 87.2%.
This research has overcome technical bottleneck---the endophytic bacterial contamination in the cultivation of kawakaw in vitro tissue in the world first, broken the single propagation method that kawakaw can only rely on cuttage to breed, successfully realized the regeneration of aseptic, the nontoxic seedling plant of kawakaw and numerous soon by the numerous bud of bud, callus induction two approach of bud of growing thickly.The kawakaw seedling that this method is cultivated is aseptic, nontoxic seedling, and every month index of reproduction is up to 7-8, rooting rate 100%, and transplanting survival rate reaches 87%.By this research, we have grasped from being sampled to and have transplanted the operating technology that waits a whole set of, this technology has been broken the single propagation method that kawakaw can only rely on cuttage to breed, can satisfy commercialization, the extensive requirement of producing aseptic, the nontoxic cultivation seedling of kawakaw plant fast of batch production, have bigger commercial application prospect.
[description of drawings]
The in vitro tissue of kawakaw is cultivated: A. induced dormancy bud sprouts; B. induce bud propagation; C. callus induction group Knit; D. induced bundle is sprouted; E. urge root; F. group is trained the seedling whole plant.
[embodiment]
The present invention is described in detail below in conjunction with embodiment:
1, material source: to introduce a fine variety, to plant the annual stem of kawakaw (Piper MethysticumForst.f) from South Pacific Ocean island countries such as Cook as initial explant in the National Key Laboratory of Tropical Plant Bio-technology, Chinese Academy of Tropical Agricultural Sciences greenhouse.
2, method:
(1) material preliminary treatment and the explant 1. preliminary treatment of sterilizing: get healthy annual stem and carry out after preliminary treatment, wash with streamlined running water and spend the night or be not less than 12 hours; 2. explant sterilization: initial explant is cut into the long stem explants of 1cm, and each explant (stem section) has 1 sleeping bud, handles 30-60 second with 70% alcohol, 0.1% mercuric chloride sterilization 10-15 minute, aseptic water washing 3-4 time, each 5 minutes.
(2) the induced dormancy bud sprouts
Explant after the sterilization is inoculated on MS+BA 1.0mg/L+NAA 0.1mg/L+30g/L sucrose+7g/L agar medium (pH=5.8), places 26-28 ℃, 12 hours illumination/12 hour dark conditions to cultivate down, intensity of illumination is 15001x.
(3) cut leader and induced bundle is sprouted
The most advanced and sophisticated 0.5mm bud point of the sleeping bud of cut growth 15 days, sprouting is inoculated in to induce on the MS+BA1.0mg/L+NAA0.1mg/L+ sucrose 30g/L+ agar 7g/L inducing culture (PH=5.8) and sprouts for secondary explant.On this basis, cutting the bud that induces is inoculated on MS+BA1.0mg/L+NAA0.1mg/L+ sucrose 30g/L+ agar 7g/L inducing culture (PH=5.8) medium, placing 26-28 ℃, 12 hours illumination/12 hour dark, intensities of illumination is under the 15001x condition, is that basic induced bundle is sprouted with bud.
(4) evoked callus and the bud of growing thickly
Get the aseptic seedling blade that the numerous bud approach of bud obtains, be inoculated in MS+BA1.0mg/L+NAA0.1mg/L+ sucrose 30g/L+ agar 7g/L medium (pH=5.8) and go up evoked callus, then well-grown callus is inoculated on the inducing clumping bud medium MS+BA1.0mg/L+IAA0.5mg/L+ sucrose 30g/L+ agar 7g/L medium (pH=5.8) soon, placing 26-28 ℃, 12 hours illumination/12 hour dark, intensities of illumination is evoked callus sprout (bud of growing thickly) under the 15001x condition.
(5) root induction
Cut well-grown single bud from the bud of growing thickly, be inoculated on the 1/2MS+IBA0.75mg/L+ sucrose 30g/L+ agar 7g/L medium (pH=5.8), illumination/12 hour dark, intensity of illumination were root induction under the 15001x condition in 26-28 ℃, 12 hours.
(6) domestication of test-tube plantlet seedling and transplanting
The seedling of will taking root places under the natural daylight after 2-3 days hardening, the agar on the flush away root, and (sand: volcano rock and soil: in the coconut palm chaff=1:2:1), 26-28 ℃, every day, illumination cultivation was 12 hours, and intensity is 8001x, is transplanted into the land for growing field crops after 1-15 days to transplant matrix in sterilization.
Attached: related data and form:
Table one: the growth time of sleeping bud source bud and the relation between the pollution rate
(use 0.5-1.0mm sprout point be explant)
Table two: hormone is to the influence of bud proliferation-inducing
Table three: hormone is to the influence (callus fresh weight g) of callus induction
Table four: the influence that 4 hormones sprout to callus induction (bud number/every callus)
Table five: variable concentrations IBA is to urging the influence of root effect.
Claims (4)
1. the method for tissue culture of a kawakaw is characterized in that:
(1) explant is selected and sterilization: selecting annual and the kawakaw stem that has sleeping bud is initial explant, cutting the long stem explants of 1cm that has a sleeping bud after the preliminary treatment places 70% alcohol to soak 30 seconds, in 0.1% mercuric chloride solution constantly agitation treatment 10-15 minute then, aseptic water washing 3-4 time, each 5 minutes;
(2) inoculation and induced dormancy bud sprout
Explant after the sterilization is inoculated on the MS+BA 1.0mg/L+NAA 0.1mg/L+ sucrose 30g/L+ agar 7g/L inducing culture, place under the intensity of illumination environment of 26-28 ℃ and 1500Lx, cultivated 15 days under the condition of 12 hours illumination/12 hour dark, the induced dormancy bud sprouts;
(3) cut leader and induced bundle is sprouted
Cut the long bud point of 15 days 1mm of growth as secondary explant, be inoculated on the MS+BA 1.0mg/L+NAA0.1mg/L+ sucrose 30g/L+ agar 7g/L inducing culture, place under the intensity of illumination environment of 26-28 ℃ and 1500Lx, under the condition of 12 hours illumination/12 hour dark, induced 20 days, on this basis, cutting the aseptic bud that induces is inoculated on the MS+BA 0.5mg/L+NAA 0.5mg/L+ sucrose 30g/L+ agar 7g/L inducing culture, place under the intensity of illumination environment of 26-28 ℃ and 1500Lx, induced bundle is sprouted under hour dark condition in 12 hours illumination/12;
(4) evoked callus
With the aseptic seedling blade inoculation evoked callus on MS+BA 1.0mg/L+NAA 0.1mg/L+ sucrose 30g/L+ agar 7g/L medium that obtains; Well-grown callus lines is inoculated on the inducing clumping bud medium MS+BA 1.0mg/L+IAA 0.5mg/L+ sucrose 30g/L+ agar 7g/L medium, place under the intensity of illumination environment of 26-28 ℃ and 1500Lx, evoked callus sprouts under hour dark condition in 12 hours illumination/12;
(5) root induction
From the bud of growing thickly, cut well-grown single bud, be inoculated on the 1/2MS+IBA 0.75mg/L+ sucrose 30g/L+ agar 7g/L medium, place under the intensity of illumination environment of 26-28 ℃ and 1500Lx root induction under hour dark condition in 12 hours illumination/12.
2. the method for tissue culture of a kind of kawakaw according to claim 1, it is characterized in that selecting annual and the healthy kawakaw stem section that has a sleeping bud as initial explant.
3. the method for tissue culture of a kind of kawakaw according to claim 1 is characterized in that initial explant is carried out pretreated method is: washed 12 hours down with streamlined running water normal temperature condition.
4. the method for tissue culture of a kind of kawakaw according to claim 1, it is characterized in that, from the bud of growing thickly that step (3) obtains, cut well-grown single bud, be inoculated on the 1/2MS+IBA 0.75mg/L+ sucrose 30g/L+ agar 7g/L medium, place under the intensity of illumination environment of 26-28 ℃ and 1500Lx root induction under hour dark condition in 12 hours illumination/12.
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| CN105706881A (en) * | 2016-03-16 | 2016-06-29 | 沈素香 | Little pepper fast propagation method |
| CN108077072A (en) * | 2016-11-21 | 2018-05-29 | 巴中精致现代农业开发有限公司 | Windbell green pepper tissue cultures culture medium and rapid propagation method |
| CN106577286B (en) * | 2016-12-20 | 2018-05-08 | 三明市农业科学研究院 | A kind of method of pipex kadsura tissue-culturing rapid propagation |
| CN106613273B (en) * | 2017-01-04 | 2020-06-16 | 福建千佰億农业科技发展有限公司 | Method for establishing seed garden of Piper hancei |
| CN106688852A (en) * | 2017-01-04 | 2017-05-24 | 福建千佰億农业科技发展有限公司 | Pipex kadsura microbody cutting seedling raising method |
| CN106718932B (en) * | 2017-01-22 | 2019-02-12 | 三明市农业科学研究院 | A kind of caulis piperis futokadsurae method for in-vitro rapid propagation |
| CN111771721A (en) * | 2020-07-21 | 2020-10-16 | 佛山市三水阳特园艺有限公司 | Tissue culture propagation method for red Japanese pepper grass |
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Non-Patent Citations (6)
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| 卡瓦胡椒引种栽培及药用价值. 杨卫东，张金萍，王敬文.浙江农业科学，第5期. 2005 |
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