CN101243775A - Ligusticum wallichii tissue cultivation fast-propagating method with stem segment as explant - Google Patents
Ligusticum wallichii tissue cultivation fast-propagating method with stem segment as explant Download PDFInfo
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- CN101243775A CN101243775A CNA2008100449871A CN200810044987A CN101243775A CN 101243775 A CN101243775 A CN 101243775A CN A2008100449871 A CNA2008100449871 A CN A2008100449871A CN 200810044987 A CN200810044987 A CN 200810044987A CN 101243775 A CN101243775 A CN 101243775A
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Abstract
The invention discloses a ligustcum chuanxiong horn tissue culture fast propagation method by taking stem segments as explants, comprising the steps of explants treatment, differentiation culture, rooting culture, seedling hardening and transplanting. The invention is characterized in that, the adopted differentiation culture medium is B5 (or MS)+6-BA1 to 3mg/L+NAA0.1 to 0.5mg/L+sucrose30g/L+agar7.0g/L; the adopted rooting culture medium is 1/2MS+IBA0.1+0.5mg/L. The method has the advantages that, ligustcum chuanxiong horn tissue culture seedlings with fast growth rate, good quality, and low pollution rate can be rapidly propagated in large amount, and the requirement to produce ligustcum chuanxiong horn on a large scale is satisfied.
Description
Technical field
The present invention relates to a kind of plant regeneration method by tissue culture technique, particularly relating to a kind of is the ligusticum wallichii tissue cultivation fast-propagating method of explant with the stem section.
Background technology
Ligusticum wallichii is one of modal traditional Chinese medicine of China, also is the situation of selling well autonomic drug of international market in recent years simultaneously, is the important source material of many health products and medicine.Because this medicinal material is subjected to the restriction of geographical conditions,, mainly be distributed in Dujiang weir, China Sichuan Province so its plantation area is limited.Ligusticum wallichii is 2 years living plants, because the solid difficulty of Ligusticum wallichii plant, always the breeding method of Ligusticum wallichii all is to adopt the cultivation of nourishing and generating of traditional stipes, and this method is educated links such as Siberian cocklebur, Ba Qu plantation owing to needing to carry out the mountain area during the breeding, so breeding cycle is longer; In addition, there is easily defective such as aging and susceptible viral dip-dye in the kind rhizome of chuanxiong germ plasm resource " Siberian cocklebur kind " that is adopted self, thereby causes Ligusticum wallichii kind sexual involution and quality to descend, and easily makes kind of rhizome of chuanxiong various serious damage by disease and insect of a large amount of generations in production and storage.Therefore the germ plasm resource quality problems of Ligusticum wallichii and breeding time length are difficult problems that perplexs Ligusticum wallichii large-scale production and improve the quality of products always.
Summary of the invention
The objective of the invention is long at the breeding time that exists in the prior art, germ plasm resource is unstable, " Siberian cocklebur kind " self is easily old and feeble and problem such as to carry disease germs and provide a kind of be the ligusticum wallichii tissue cultivation fast-propagating method of explant with the stem section, this method can breed fast growth, product are of fine quality and the microbiological contamination rate is low Ligusticum wallichii tissue cultivating seedling in a large number apace.
For achieving the above object, what the present invention adopted is that the ligusticum wallichii tissue cultivation fast-propagating method of explant comprises the steps: with the stem section
(1) processing of explant: choose and be transplanted to the Ligusticum wallichii stem section of indoor cultivation more than 14 days as explant and carry out surface sterilization and handle;
(2) differentiation culture: the explant after will sterilizing is inoculated on the differential medium, and at 24～26 ℃, illumination every day 11～13 hours, intensity of illumination are that 1500～2000Lx cultivates, and described differential medium is to adopt at B
5Or adding 6-BA 1～3mg/L, NAA0.1～0.5mg/L, sucrose 30g/L and agar 7.0g/L among the MS, pH value is 5.8～6.5;
(3) culture of rootage: the bud cutting-out of being grown is transferred on the root media, at 24～26 ℃, illumination every day 8～12 hours, intensity of illumination is that 1500～2000LX cultivates, described root media is to adopt to add IBA0.1～0.5mg/L, sucrose 15g/L and agar 7.0g/L in 1/2MS, and pH value is 5.8～6.5;
(4) hardening and transplanting: when tissue cultivating seedling grows to 2～3cm when above, open bottle cap, after 2～4 days, tissue cultivating seedling is taken out from blake bottle, wash the medium of root off, be transplanted in the soft vermiculite medium in indoor hardening.
In the such scheme can there be much the adoptable explant sterilization method of (1) step, disinfectant commonly used has 70～75% ethanol, liquor natrii hypochloritis, liquor hydrargyri perchloridi etc., preferable methods of the present invention is to use detergent immersion explant 2～5 minutes earlier, again with flowing water flushing 2～3 hours, handled 3～6 minutes 20～30 seconds, 0.1% mercuric chloride to adopt 70～75% ethanol disinfections respectively then under aseptic condition, uses sterile water wash at last 3～4 times.
Before in the such scheme bud being transferred to root media, earlier bud is cultivated more than 7 days in the MS medium, increased such transient process, more help the growth of root.
In the such scheme, tissue cultivating seedling is being transplanted to soft vermiculite medium, suitably shading, and maintain the temperature at 18～22 ℃, and relative moisture is 75～85%, and the growth of tissue cultivating seedling is faster, more healthy and stronger like this.
Explant can be the green stem section of terrestrial stem, terrestrial stem purple stem section, subterranean stem top stem section or subterranean stem bottom stem section among the present invention program, there is certain difference in their differentiation rate, wherein the purple stem segment with axillary buds differentiation rate of terrestrial stem is the highest, rhizomatous top stem section differentiation rate is taken second place, but when subterranean stem is bred as explant, degerming is difficulty slightly, so Ligusticum wallichii terrestrial stem purple stem section is best explant.
Best differential medium is B in the inventive method
5+ 6-BA2mg/L+NAA0.2mg/L+ sucrose 30g/L+ agar 7.0g/L, when adopting this differential medium to carry out differentiation culture, the differentiation rate of axillalry bud is the highest, and seedling fast growth, stalwartness.
Best root media is 1/2MS+IBA0.5mg/L+ sucrose 15g/L+ agar 7.0g/L in the inventive method, and when adopting this root media to carry out culture of rootage, rooting rate is the highest, and root differentiation speed is fast, well developed root system and root are sturdy.
The present invention adopts method for tissue culture to cultivate tissue cultivating seedling institute's time spent and compares shortening greatly with traditional breeding method institute's time spent, and the tissue cultivating seedling of cultivating is because self robust growth, well developed root system, therefore be difficult for bacteria infection, overcome can not utilize in the traditional breeding method that seminal propagation, germ plasm resource instability, breeding time are long, " Siberian cocklebur kind " self easily old and feeble and problem such as carry disease germs.Implementing this method in addition only need have simple Plant Tissue Breeding equipment to carry out, practical, and is not subject to seasonal restrictions, and therefore can satisfy the Ligusticum wallichii requirement of large-scale production.
Below in conjunction with embodiment the present invention is described in further detail.
Embodiment
Embodiment 1
(1) processing of explant: gather and be transplanted to the purple stem section of the Ligusticum wallichii terrestrial stem of indoor cultivation more than 14 days as explant, and to its disinfection, the sterilization method that is adopted is: use detergent immersion 2 minutes earlier, again with flowing water flushing 3 hours, adopting concentration then under aseptic condition respectively is that 70% ethanol disinfection 20 seconds, concentration are that 0.1% mercuric chloride was handled 3 minutes, uses sterile water wash at last 4 times;
(2) differentiation culture: on superclean bench, explant is cut into the length of 1～2cm, is inoculated into the differential medium B of axillalry bud
5On+6-BA2.0mg/L+NAA0.2mg/L+ sucrose 30g/L+ agar the 7.0g/L, and at 24～26 ℃, illumination every day 12 hours, intensity of illumination are to cultivate under the condition of 1500～2000Lx, cultivate after 3 days, stem segment with axillary buds begins to sprout successively, and growth rate is the fastest, and the axillalry bud differentiation rate reaches 92.5% after 7 days, and the seedling stalwartness, the leaf look dark green;
(3) culture of rootage: when bud length arrives the 2cm left and right sides, bud is downcut, in the MS medium, cultivate after 7 days, be transferred on the root media 1/2MS+IBA0.5mg/L+ sucrose 15g/L+ agar 7.0g/L, and be to carry out culture of rootage under the condition of 1500～2000Lx at 26～26 ℃, illumination every day 10 hours, intensity of illumination, cultivating does not have offspring (bud) eustipes part and begins to take root after 15 days, the statistics rooting rate is 95% after 30 days, observe that unrooted seedling rooting speed is fast, well developed root system and root be sturdy, the number of taking root is more than 10;
(4) hardening and transplanting: when tissue cultivating seedling grows to 3cm when above, select growth healthy and strong, have the green number of blade and open bottle cap strong sprout more and that root system is flourishing gradually, allow seedling adapt to surrounding environment at leisure, in indoor hardening after 3 days, transplant to soft vermiculite medium, suitably shading, temperature remains on about 20 ℃, relative moisture is 75～85%, and the survival rate that tissue cultivating seedling is transplanted can reach 100%.
Embodiment 2
Explant adopts the green stem section of being transplanted to the Ligusticum wallichii terrestrial stem of indoor cultivation more than 14 days, and other step is identical with embodiment 1, and the differentiation rate of statistics axillalry bud is 62.5% after 7 days.
Embodiment 3
Explant adopts has been transplanted to the rhizomatous top of the Ligusticum wallichii of indoor cultivation more than 14 days 1/2 stem section, its sterilization method is: use detergent immersion 5 minutes earlier, again with flowing water flushing 2 hours, handled 6 minutes 30 seconds, 0.1% mercuric chloride to adopt 75% ethanol disinfection respectively then under aseptic condition, uses sterile water wash at last 3 times.
Other step is added up the differentiation rate 87.5% of axillalry bud after 1,7 day with embodiment.
Embodiment 4
Explant adopts has been transplanted to the rhizomatous bottom of the Ligusticum wallichii of indoor cultivation more than 14 days 1/2 stem section, and after 3,7 days, the differentiation rate of statistics axillalry bud is 81% to other step with embodiment.
Embodiment 5
Differential medium adopts MS+6-BA2.0mg/L+NAA0.2mg/L+ sucrose 30g/L+ agar 7.0g/L, and other step is with embodiment 1.It is very fast to observe no offspring growth, but seedling does not have the strong of embodiment 1 growth.
Embodiment 6
Differential medium adopts B
5+ 6-BA1.0mg/L+NAA0.2mg/L+ sucrose 30g/L+ agar 7.0g/L, other step is with embodiment 1.The growth of observing axillalry bud is slower, and differentiation rate is 62.5%.
Embodiment 7
Differential medium adopts B
5+ 6-BA2mg/L+NAA0.5mg/L+ sucrose 30g/L+ agar 7.0g/L, other step is with embodiment 1.Observe that axillary bud growth is fast, seedling is healthy and strong, the leaf look normal, and differentiation rate is 85%.
Embodiment 8
Differential medium adopts B
5+ 6-BA3mg/L+NAA0.2mg/L+ sucrose 30g/L+ agar 7.0g/L, other step is with embodiment 1.It is fast to observe axillary bud growth, but seedling does not have embodiment 1 stalwartness.
Embodiment 9
Root media adopts 1/2MS+IBA0.1mg/L+ sucrose 15g/L+ agar 7.0g/L, and other step is with embodiment 1.The statistics rooting rate is 75%, observes the fast growth of root, and well developed root system, the number of taking root be more than 10, and embodiment's 1 is thick but root does not have.
Embodiment 10
Root media adopts 1/2MS+IBA0.3mg/L+ sucrose 15g/L+ agar 7.0g/L, and other step is with embodiment 1, and the statistics rooting rate is 85%, observes the fast growth of root, and well developed root system, the number of taking root be more than 10, and embodiment's 1 is thick but root does not have.
Claims (8)
1. one kind is the ligusticum wallichii tissue cultivation fast-propagating method of explant with the stem section, it is characterized in that it comprises the steps:
(1) processing of explant: choose and be transplanted to the Ligusticum wallichii stem section of indoor cultivation more than 14 days, and carry out surface sterilization and handle as explant;
(2) differentiation culture: the explant after will sterilizing is inoculated on the differential medium, is that 1500～2000Lx cultivates at 24～26 ℃, illumination every day 11～13 hours, intensity of illumination, and described differentiation culture medium is to adopt at B
5Or adding 6-BA 1～3mg/L, NAA 0.1～0.5mg/L, sucrose 30g/L and agar 7.0g/L among the MS, pH value is 5.8～6.5;
(3) culture of rootage: the bud cutting-out of being grown is transferred on the root media, at 24～26 ℃, illumination every day 8～12 hours, intensity of illumination is that 1500～2000LX cultivates, described differentiation culture medium is to adopt to add IBA 0.1～0.5mg/L, sucrose 15g/L and agar 7.0g/L in 1/2MS, and pH value is 5.8～6.5;
(4) hardening and transplanting: when tissue cultivating seedling grows to 2～3cm when above, open bottle cap, after 2～4 days, tissue cultivating seedling is taken out from blake bottle, wash the medium of root off, be transplanted in the soft vermiculite medium in indoor hardening.
2. according to claim 1 a kind of be the ligusticum wallichii tissue cultivation fast-propagating method of explant with the stem section, it is characterized in that: (1) sterilization method that step adopted is to use detergent immersion explant 2～5 minutes earlier, again with flowing water flushing 2～3 hours, adopting concentration then under aseptic condition respectively is that 70～75% ethanol disinfection 20～30 seconds, concentration are that 0.1% mercuric chloride was handled 3～6 minutes, uses sterile water wash at last 3～4 times.
3. according to claim 1 and 2 a kind of be the ligusticum wallichii tissue cultivation fast-propagating method of explant with the stem section, it is characterized in that: before bud is transferred to root media, earlier in the MS medium, cultivate more than 7 days bud.
4. according to claim 1 and 2 a kind of be the ligusticum wallichii tissue cultivation fast-propagating method of explant with the stem section, it is characterized in that: in (4) step tissue cultivating seedling is transplanted to soft vermiculite medium, suitably shading, and maintain the temperature at 18～22 ℃, relative moisture is 75～85%.
5. according to claim 3 a kind of be the ligusticum wallichii tissue cultivation fast-propagating method of explant with the stem section, it is characterized in that: in (4) step tissue cultivating seedling is transplanted to soft vermiculite medium, suitably shading, and guarantee temperature at 18～22 ℃, relative moisture is 75～85%.
6. according to claim 1 a kind of be the ligusticum wallichii tissue cultivation fast-propagating method of explant with the stem section, it is characterized in that: explant is for being transplanted to the purple stem section of the Ligusticum wallichii terrestrial stem of indoor cultivation more than 14 days in (1) step.
7. according to claim 1 a kind of be the kind ligusticum wallichii tissue cultivation fast-propagating method of explant with the stem section, it is characterized in that: differential medium is B in (2) step
5+ 6-BA2.0mg/L+NAA0.2mg/L+ sucrose 30g/L+ agar 7.0g/L.
8. according to claim 1 a kind of be the ligusticum wallichii tissue cultivation fast-propagating method of explant with the stem section, it is characterized in that: root media is 1/2MS+IBA0.5mg/L+ sucrose 15g/L+ agar 7.0g/L in (3) step.
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| CN2008100449871A CN101243775B (en) | 2008-03-14 | 2008-03-14 | Ligusticum wallichii tissue cultivation fast-propagating method with stem segment as explant |
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| CN2008100449871A CN101243775B (en) | 2008-03-14 | 2008-03-14 | Ligusticum wallichii tissue cultivation fast-propagating method with stem segment as explant |
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| CN101243775B CN101243775B (en) | 2012-03-21 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112931220A (en) * | 2021-04-13 | 2021-06-11 | 中国农业科学院都市农业研究所 | Plant tissue culture and rapid propagation method taking stem tip as explant |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112931220A (en) * | 2021-04-13 | 2021-06-11 | 中国农业科学院都市农业研究所 | Plant tissue culture and rapid propagation method taking stem tip as explant |
| CN112931220B (en) * | 2021-04-13 | 2022-05-27 | 中国农业科学院都市农业研究所 | Plant tissue culture and rapid propagation method taking stem tip as explant |
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