CN103548683B - Embryoid induction method taking polyphylla bud axis as explant - Google Patents
Embryoid induction method taking polyphylla bud axis as explant Download PDFInfo
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- CN103548683B CN103548683B CN201310529396.4A CN201310529396A CN103548683B CN 103548683 B CN103548683 B CN 103548683B CN 201310529396 A CN201310529396 A CN 201310529396A CN 103548683 B CN103548683 B CN 103548683B
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Abstract
The invention discloses an embryoid induction method taking a polyphylla bud axis as an explant. The method comprises the following steps: firstly, carrying out disinfection and pre-cultivation on an un-germinated bud on polyphylla rhizome, and then cutting off coleoptile of the bud of which the volume obviously expands after pre-cultivation; inserting the bud axis into a callus-inducing medium to cultivate from the center after cutting in length, and then cultivating the callus tissue generated by the bud axis to obtain polyphylla embryonic tissues; finally carrying out induction cultivation on the embryonic tissues, so as to generate white spherical embryoids. By adopting the embryoid induction method, a new path is supplied for cultivation of excellent variety of the polyphylla and massive and rapid propagation of tissue culture seedlings; mass production of polyphylla plants at low cost within a short period of time can be achieved.
Description
Technical field
The present invention relates to the method for tissue culture of a kind of Paris polyphylla plant, particularly relate to a kind of embryoid induction method being explant with polyphylla bud axle.
Background technology
The Rhizoma Paridis that Chinese Pharmacopoeia specifies is the subterranean stem of Yunnan Paris polyphylla (Paris polyphylla var.yunnanensis) and magnificent Paris polyphylla (Paris polyphylla var.chinensis), the former main product is in Yunnan, and the latter's main product is in Sichuan.The flea that Shennong's Herbal is recorded the earliest stops i.e. magnificent Paris polyphylla.Paris polyphylla has clearing heat and detoxicating, anti-inflammatory analgetic, activates blood circulation and disperses blood clots, effect of cool liver arresting convulsion.
Because finding that Paris polyphylla has antiviral, antitumor action, demand increases in recent years.Paris polyphylla traditional Chinese medicine always provides medicinal with wild resource, in recent years due to excessively excavating wild resource, causes wild Paris polyphylla resource to suffer destructive destruction, and existing own endangered, thus Devoting Major Efforts To Developing Paris polyphylla plant resources tool is of great significance.
The subject matter of current restriction Paris polyphylla plant scale plantation is the shortage of Paris polyphylla seedling.Paris polyphylla seed is incomplete owing to there is embryo development, endosperm is hard, plumular axis after-ripening etc. are " dual dormancy " characteristic obviously, therefore after planting usually need the summer in experience two winter one could Germination And Seedling, typical " biennial seed ", and in its natural state, even if having passed through long-time dormancy, the emergence rate of Paris polyphylla seed is not high yet, therefore only carries out by Paris polyphylla seed the needs that sexual propagation cannot meet the plantation of Paris polyphylla plant large area at present.
Plant embryoid is by forming the cell of plant corpus under conditions of tissue culture, and the zygote formed by being similar to fertilized egg develops into the process of embryo and the somatic cell idiosome that formed.Embryoid has the characteristic of plant embryos, can be grown to regeneration plant fast when conditions permit, effectively can solve the problem of Paris polyphylla seedling shortage.
Summary of the invention
The object of the present invention is to provide a kind of embryoid induction method being explant with polyphylla bud axle, the embryoid that the method induction produces, regeneration plant can be grown to fast under suitable condition, thus the Fast-propagation of Paris polyphylla regeneration plant can be realized.
The embryoid induction method being explant with polyphylla bud axle provided by the invention comprises the following steps:
(1) robust growth on rhizoma paris rhizome is got and the sprout do not sprouted, dry in the shade after first clean with tap water, then disinfection, then with after sterile water concussion washing, repeatedly blot the moisture on sprout surface with sterilized filter paper, be then inoculated in pre-culture medium MS+6-BA0.5 ~ 3.0mgL
-1+ salicylic acid 20 ~ 80mgL
-1in, cultivation temperature be 16 ~ 22 DEG C, illumination every day 6 ~ 14 hours, intensity of illumination carry out preculture under being the condition of 1000 ~ 2000lx;
(2) sprout that after choosing preculture, volume obviously expands, bud scale first cuts by superclean bench, then bud axle is carried out rip cutting, and the vertical section of the bud axle cut is accessed callus inducing culture MS+6-BA0.5 ~ 2.0mgL
-1+ NAA0.5 ~ 2.0mgL
-1+ 2,4-D1.0 ~ 4.0mgL
-1in, cultivation temperature be 12 ~ 26 DEG C, illumination every day 4 ~ 16 hours, intensity of illumination cultivate under being the condition of 1000 ~ 2000lx;
(3) tissue of the callus produced by bud axle is cut into 1 ~ 3cm
3size access LS+6-BA0.5 ~ 2.0mgL
-1+ NAA0.5 ~ 4.0mgL
-1in medium, cultivation temperature be 16 ~ 28 DEG C, illumination every day 8 ~ 12 hours, intensity of illumination cultivate under being the condition of 1200 ~ 2500lx, cultivate 25 ~ 45 days subcultures once, carry out 2 ~ 4 squamous subculture continuously, obtain Paris polyphylla embryonal connective tissue;
(4) Paris polyphylla embryonal connective tissue is cut into 0.5 ~ 2.5cm
3size access LS+6-BA2.0 ~ 4.0mg+IAA0.5 ~ 2.0mgL
-1+ Cefotaxime Sodium 100 ~ 400mgL
-1in medium, cultivation temperature be 5 ~ 10 DEG C, illumination every day 2 ~ 6 hours, intensity of illumination cultivate under being the condition of 400 ~ 1000lx, cultivate after 50 ~ 70 days, produce the spherical embryoid of white circular on embryonal connective tissue surface.
Above-mentioned all medium also comprise agar 5.0 ~ 7.0gL
-1with sucrose 20 ~ 60gL
-1, the pH value of medium is 5.6 ~ 6.5,
Disinfect described in above-mentioned steps (1) and refer to first with alcohol disinfecting 10 ~ 60s that concentration is 70% ~ 75% on super-clean bench, then be the mercuric chloride sterilization 4 ~ 10min of 0.1% ~ 0.15% by concentration.
The embryoid induction method that it is explant that the present invention proposes with polyphylla bud axle first, the method to some extent solves the problem to provenance demand when Paris polyphylla plant is planted on a large scale, for the cultivation of Paris polyphylla improved seeds and a large amount of Fast-propagations of plantlet in vitro provide a new way, the Paris polyphylla short time can be realized, produce low-costly and in high volume.
Below in conjunction with embodiment, the present invention is described in further detail.
Embodiment
Embodiment 1
(1) preculture: get robust growth on rhizoma paris rhizome, sprout diameter at more than 0.5cm and the sprout do not sprouted, dry in the shade after first clean with tap water, then be the alcohol disinfecting 10s of 70% in super-clean bench concentration, be the mercuric chloride sterilization 4min of 0.1% again by concentration, then with after sterile water concussion washing 3 times, repeatedly blot the moisture on sprout surface with sterilized filter paper, be then inoculated in pre-culture medium MS+6-BA0.5mgL
-1+ salicylic acid 20mgL
-1in, cultivation temperature be 16 DEG C, illumination every day 6 hours, intensity of illumination carry out preculture under being the condition of 1000lx, cultivate 20 days statistics, the sprout rate of expanding is 78.93%, and microbiological contamination rate is 20.23%;
(2) bud axoblast callusization induction: the sprout that after choosing preculture, volume obviously expands, bud scale first cuts by superclean bench, then bud axle is carried out rip cutting from central authorities, then the vertical section of the bud axle cut is accessed callus inducing culture MS+6-BA0.5mgL
-1+ NAA0.5mgL
-1+ 2,4-D1.0mgL
-1in, cultivation temperature be 12 DEG C, illumination every day 4 hours, intensity of illumination cultivate 50 days under being the condition of 1000lx, to observe and the bud axle counting 72.84% creates callus;
(3) generation of embryonal connective tissue: the tissue of the callus produced by bud axle is cut into 1cm
3size access LS+6-BA0.5mgL
-1+ NAA0.5mgL
-1in medium, cultivation temperature be 16 DEG C, illumination every day 8 hours, intensity of illumination cultivate under being the condition of 1200lx, cultivate 25 days subcultures once, carry out 2 squamous subculture continuously, have the culture materials of 41.31% to create embryonal connective tissue;
(4) generation of embryoid: the Paris polyphylla embryonal connective tissue that the yellowish white that selecting step (3) produces, splitting ability are strong, is cut into 0.5cm
3size after access LS+6-BA2.0mgL
-1+ IAA0.5mgL
-1+ Cefotaxime Sodium 100mgL
-1in medium, cultivation temperature be 5 DEG C, illumination every day 2 hours, intensity of illumination cultivate under being the condition of 400lx, cultivate after 55 days, have the embryonal connective tissue of 61.86% surface to create the spherical embryoid of white circular;
The pH value of above-mentioned all medium is 5.6, agar 5.0gL
-1, sucrose 20gL
-1.
Embodiment 2
(1) preculture: get robust growth on rhizoma paris rhizome, sprout diameter at more than 0.5cm and the sprout do not sprouted, dry in the shade after first clean with tap water, then be first the alcohol disinfecting 40s of 75% by concentration at super-clean bench, be the mercuric chloride sterilization 8min of 0.1% again by concentration, then with after sterile water concussion washing 5 times, repeatedly blot the moisture on sprout surface with sterilized filter paper, be then inoculated in the pre-culture medium MS+6-BA2.0mgL that pH value is 5.8
-1+ salicylic acid 60mgL
-1+ sucrose 40gL
-1+ agar 6.0gL
-1in, cultivation temperature be 20 DEG C, illumination every day 8 hours, intensity of illumination carry out preculture under being the condition of 1500lx, cultivate 20 days statistics, the sprout rate of expanding is 90.3%, and microbiological contamination rate is 0%;
(2) bud axoblast callusization induction: the sprout that after choosing preculture, volume obviously expands, bud scale first cuts by superclean bench, then bud axle is carried out rip cutting from central authorities, then be the callus inducing culture MS+6-BA1.0mgL of 5.8 by the vertical section of the bud axle of incision access pH value
-1+ NAA1.0mgL
-1+ 2,4-D2.0mgL
-1+ sucrose 20gL
-1+ agar 6.0gL
-1in, cultivation temperature be 20 DEG C, illumination every day 8 hours, intensity of illumination cultivate 60 days under being the condition of 1500lx, to observe and the bud axle counting 85% creates callus;
(3) generation of embryonal connective tissue: the tissue of the callus produced by bud axle is cut into 2cm
3size access pH value be 6 medium LS+6-BA1.0mgL
-1+ NAA2.0mgL
-1+ sucrose 30gL
-1+ agar 6.0gL
-1in, cultivation temperature be 20 DEG C, illumination every day 10 hours, intensity of illumination cultivate under being the condition of 2000lx, cultivate 30 days subcultures once, carry out 3 squamous subculture continuously, through microexamination, have the culture materials of 67.8% to create embryonal connective tissue;
(4) generation of embryoid: the Paris polyphylla embryonal connective tissue that the yellowish white that selecting step (3) produces, splitting ability are strong, is cut into 2.0cm
3size after access the medium LS+6-BA3.0mgL that pH value is 6
-1+ IAA1.0mgL
-1+ Cefotaxime Sodium 200mgL
-1+ sucrose 30gL
-1+ agar 6.0gL
-1in, cultivation temperature be 6 ~ 10 DEG C, illumination every day 4 hours, intensity of illumination cultivate under being the condition of 600lx, cultivate after 60 days, the embryonal connective tissue of 87.6% surface is had to create the spherical embryoid of white circular, average each embryonal connective tissue material produces 4 ~ 8 embryoids, observe through microsection, the embryoid of formation is not connected with the vascular bundle of maternal tissue, has independently fibrovascular system.
Embodiment 3
(1) preculture: get robust growth on rhizoma paris rhizome, sprout diameter at more than 0.5cm and the sprout do not sprouted, dry in the shade after first clean with tap water, then be the alcohol disinfecting 60s of 75% in super-clean bench concentration, be the mercuric chloride sterilization 8min of 0.15% again by concentration, then with after sterile water concussion washing 6 times, repeatedly blot the moisture on sprout surface with sterilized filter paper, be then inoculated in pre-culture medium MS+6-BA3.0mgL
-1+ salicylic acid 80mgL
-1in, cultivation temperature be 22 DEG C, illumination every day 14 hours, intensity of illumination carry out preculture under being the condition of 2000lx, cultivate 20 days statistics, the sprout rate of expanding is 82.53%, and microbiological contamination rate is 0%;
(2) bud axoblast callusization induction: the sprout that after choosing preculture, volume obviously expands, bud scale first cuts by superclean bench, then bud axle is carried out rip cutting from central authorities, then the vertical section of the bud axle cut is accessed callus inducing culture MS+6-BA2.0mgL
-1+ NAA2.0mgL
-1+ 2,4-D4.0mgL
-1in, cultivation temperature be 26 DEG C, illumination every day 16 hours, intensity of illumination cultivate 70 days under being the condition of 2000lx, to observe and the bud axle counting 83.54% creates callus;
(3) generation of embryonal connective tissue: the tissue of the callus produced by bud axle is cut into 3cm
3size access LS+6-BA2.0mgL
-1+ NAA4.0mgL
-1in medium, cultivation temperature be 28 DEG C, illumination every day 12 hours, intensity of illumination cultivate under being the condition of 2500lx, cultivate 45 days subcultures once, carry out 4 squamous subculture continuously, have the culture materials of 68.26% to create embryonal connective tissue;
(4) generation of embryoid: the Paris polyphylla embryonal connective tissue that the yellowish white that selecting step (3) produces, splitting ability are strong, is cut into 2.5cm
3size after access LS+6-BA4.0mgL
-1+ IAA2.0mgL
-1+ Cefotaxime Sodium 400mgL
-1in medium, cultivation temperature be 10 DEG C, illumination every day 6 hours, intensity of illumination cultivate under being the condition of 1000lx, cultivate after 65 days, have the embryonal connective tissue of 84.15% surface to create the spherical embryoid of white circular;
The pH value of above-mentioned all medium is 6.5, agar 7.0gL
-1, sucrose 60gL
-1.
Embodiment 4
Change the pre-culture medium described in embodiment 2 step (1) into MS+6-BA0.5mgL that pH value is 6
-1+ salicylic acid 30mgL
-1+ sucrose 30gL
-1+ agar 5.8gL
-1, other step is with embodiment 2; Cultivate after 20 days, the sprout rate of expanding is 81.13%.
Embodiment 5
Change the bud axle callus inducing culture in embodiment 2 step (2) into MS+6-BA2.0mgL that pH value is 5.8
-1+ NAA0.5mgL
-1+ 2,4-D1.0mgL
-1+ sucrose 20gL
-1+ agar 6.0gL
-1, other step, with embodiment 2, is cultivated after 60 days, observes and the bud axle counting 77% creates callus.
Embodiment 6
Change the medium that the induction Paris polyphylla embryoid in embodiment 2 step (4) produces into LS+6-BA4.0mgL that pH value is 6
-1+ IAA1.0mgL
-1+ Cefotaxime Sodium 400mgL
-1+ sucrose 30gL
-1+ agar 6.0gL
-1, cultivate after 60 days, have the embryonal connective tissue of 85.53% surface to produce the spherical embryoid of white circular, average each embryonal connective tissue material produces 5 ~ 8 embryoids.
Comparing embodiment 1
Sprout in embodiment 2 step (1) is changed into the sprout that employing is obviously sprouted, other step is with embodiment 2; Cultivate 20 days statistics, sprout expands rate 40.39%, and microbiological contamination rate is 61.71%;
Comparing embodiment 2
Change the pre-culture medium described in embodiment 2 step (1) into B5+6-BA1.0mgL that pH value is 5
-1+ 2,4-D1.5mgL
-1+ sucrose 20gL
-1+ agar 6gL
-1, other step is with embodiment 2; Cultivate 20 days statistics, sprout expands rate 43.15%.
Comparing embodiment 3
In embodiment 2 step (2), adopt and do not cut the bud scale of expanding on sprout, but will access in medium after the sprout rip cutting of band bud scale, other step be with embodiment 2; Cultivate 60 days statistics, only have the bud axle culture materials of 43.52% to create callus.
Comparing embodiment 4
Change the cutting mode of bud axle in embodiment 2 step (2) into crosscut, after being cut to upper and lower two parts by bud axle, then access in medium by cross section, other step is with embodiment 2; Cultivate 60 days observe find, there is not callus in the bud axle upper part of access, but show as to a certain degree take out seedling; And the bud axle bottom of access has 35% to create callus;
Comparing embodiment 5
Change the cells,primordial inducing culture in embodiment 2 step (3) into MS+6-BA2.0mgL that pH value is 6.5
-1+ NAA1.0mgL
-1+ sucrose 30gL
-1+ agar 6.0gL
-1, other step is with embodiment 2; Through microexamination after cultivation, the culture materials of 23.18% is only had to create embryonal connective tissue.
Comparing embodiment 6
Change the medium that the induction Paris polyphylla embryoid in embodiment 2 step (4) produces into 1/2MS+6-BA1.0mgL that pH value is 5
-1+ IAA2.0mgL
-1+ sucrose 30gL
-1+ agar 6.0gL
-1, other step is with embodiment 2; Cultivate after 60 days, have the embryonal connective tissue of 9.87% surface to produce the spherical embryoid of white circular, average each embryonal connective tissue material produces 1 ~ 3 embryoid.
Claims (2)
1., with the embryoid induction method that polyphylla bud axle is explant, it is characterized in that comprising the steps:
(1) robust growth on rhizoma paris rhizome is got and the sprout do not sprouted, dry in the shade after first clean with tap water, then disinfection, then with after sterile water concussion washing, repeatedly blot the moisture on sprout surface with sterilized filter paper, be then inoculated in pre-culture medium MS+6-BA0.5 ~ 3.0mgL
-1+ salicylic acid 20 ~ 80mgL
-1in, cultivation temperature be 16 ~ 22 DEG C, illumination every day 6 ~ 14 hours, intensity of illumination carry out preculture under being the condition of 1000 ~ 2000lx;
(2) sprout that after choosing preculture, volume obviously expands, bud scale first cuts by superclean bench, then bud axle is carried out rip cutting, and the vertical section of the bud axle cut is accessed callus inducing culture MS+6-BA0.5 ~ 2.0mgL
-1+ NAA0.5 ~ 2.0mgL
-1+ 2,4-D1.0 ~ 4.0mgL
-1in, cultivation temperature be 12 ~ 26 DEG C, illumination every day 4 ~ 16 hours, intensity of illumination cultivate under being the condition of 1000 ~ 2000lx;
(3) tissue of the callus produced by bud axle is cut into 1 ~ 3cm
3size access LS+6-BA0.5 ~ 2.0mgL
-1+ NAA0.5 ~ 4.0mgL
-1in medium, cultivation temperature be 16 ~ 28 DEG C, illumination every day 8 ~ 12 hours, intensity of illumination cultivate under being the condition of 1200 ~ 2500lx, cultivate 25 ~ 45 days subcultures once, carry out 2 ~ 4 squamous subculture continuously, obtain Paris polyphylla embryonal connective tissue;
(4) Paris polyphylla embryonal connective tissue is cut into 0.5 ~ 2.5cm
3size access LS+6-BA2.0 ~ 4.0mgL
-1+ IAA0.5 ~ 2.0mgL
-1+ Cefotaxime Sodium 100 ~ 400mgL
-1in medium, cultivation temperature be 5 ~ 10 DEG C, illumination every day 2 ~ 6 hours, intensity of illumination cultivate under being the condition of 400 ~ 1000lx, cultivate after 50 ~ 70 days, produce the spherical embryoid of white circular on embryonal connective tissue surface;
Above-mentioned all medium also comprise agar 5.0 ~ 7.0gL
-1with sucrose 20 ~ 60gL
-1, the pH value of medium is 5.6 ~ 6.5.
2. a kind of embryoid induction method that is explant with polyphylla bud axle according to claim 1, it is characterized in that: disinfect described in step (1) and refer to first with alcohol disinfecting 10 ~ 60s that concentration is 70% ~ 75% on super-clean bench, then be the mercuric chloride sterilization 4 ~ 10min of 0.1% ~ 0.15% by concentration.
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| CN104774800B (en) * | 2015-04-08 | 2017-11-07 | 成都大学 | A kind of suspension culture method of Paris polyphylla nucellar cell |
| CN106417036B (en) * | 2016-12-21 | 2018-04-20 | 西南科技大学 | A kind of method for tissue culture for inducing Paris polyphylla immature zygotic embryos to produce somatic embryo |
| CN106613983A (en) * | 2016-12-26 | 2017-05-10 | 江西宜信堂医疗科技有限公司 | Paris polyphylla var chinensis induction culture medium and application thereof |
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| US5350689A (en) * | 1987-05-20 | 1994-09-27 | Ciba-Geigy Corporation | Zea mays plants and transgenic Zea mays plants regenerated from protoplasts or protoplast-derived cells |
| SK281132B6 (en) * | 1992-11-04 | 2000-12-11 | Ústav Genetiky Rastlín Sav | Method of induction of creation of somatic embryos |
| WO2000036911A2 (en) * | 1998-12-18 | 2000-06-29 | Monsanto Technology Llc | Method for the regeneration of cotton |
| JP4222810B2 (en) * | 2002-10-28 | 2009-02-12 | 株式会社 タカマ | Gastric mucosa protective agent |
| CN100527941C (en) * | 2007-06-14 | 2009-08-19 | 恩施济源药业科技开发有限公司 | Paris polyphylla var. pseudothibetica rhizome multiple shoot asexual reproduction method |
| CN101773071A (en) * | 2010-01-19 | 2010-07-14 | 烟台汇鹏生物科技有限公司 | Callus culture method of snow lotus herb |
| CN102301956B (en) * | 2011-07-26 | 2012-11-14 | 成都大学 | Callus induction method taking paris polyphylla coleoptiles as explants |
| CN102301957B (en) * | 2011-07-26 | 2013-01-09 | 四川农业大学 | Callus culture method by taking Rhizoma Paridis roots and stems as explants |
| CN102511388B (en) * | 2011-11-25 | 2013-09-25 | 贵州师范大学 | Method for breeding anti-reversible tissue culture seedlings of anoectochilus roxburghii |
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