CN1554229A - Quick seedling growing tissue culture method of Baoshan violet - Google Patents
Quick seedling growing tissue culture method of Baoshan violet Download PDFInfo
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- CN1554229A CN1554229A CNA2003101174426A CN200310117442A CN1554229A CN 1554229 A CN1554229 A CN 1554229A CN A2003101174426 A CNA2003101174426 A CN A2003101174426A CN 200310117442 A CN200310117442 A CN 200310117442A CN 1554229 A CN1554229 A CN 1554229A
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- viola baoshanensis
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Abstract
The fast seedling growing process includes selecting leaf or leafstalk of Viola baoshanensis as explant, disinfection, inoculating onto MS inducing culture medium with hormone 0.5-1.0 mg/L 6-BA plus 0.5-1.5 mg/L NAA or 0.5-1 mg/L KT plus 1-1.5 mg/L 2, 4-D to induce the growth of adventitious bud and callus, rooting culture and seedling strengthening, and transplantation to obtain tissue culture seedling. The grown seedling of Viola baoshanensis can complete its life history in culture medium and soil, and has high transplantation survival rate, domestication period of 2-3 months and high propagation coefficient. The said method makes it possible to culture Viola baoshanensis as one kind of super enriched heavy metal plant.
Description
Technical field
The present invention relates to a kind of heavy metal super-enriched plant Viola baoshanensis (Viola baoshanensis) tissue culturing fast seedling-cultivating method.
Background technology
Viola baoshanensis (Viola baoshanensis) is Violaceae one novel species, also is the super enriching plant at the domestic newfound heavy metal of China.Viola baoshanensis is used for scientific research and repairs the potential value of water body or heavy metal pollution of soil aspect, has now attracted scientific circles, environmental protection circle and business people's concern.
Current, to the research and experiment of Viola baoshanensis, need to use seedling in a large number, and these directly are collected in Guiyang County Golconda mining area, Hunan Province basically with seedling.Field investigation shows, Viola baoshanensis only in the Golconda mining area two square kilometres distribute with interior concentration of local, still undiscovered in other mining area of China.Previous and ongoing Viola baoshanensis research have consumed a large amount of wild Viola baoshanensis germ plasm resource, thus artificial disturbance the procreation of Viola baoshanensis population, cause the progressively atrophy of Viola baoshanensis population.To the needs of the protection of resources of Viola baoshanensis wild germplasm and next step research, make seek a kind of new increasing with seedling source meaning.
Utilize seed sprouting to attempt showing that the seed of Viola baoshanensis is less, collect and be difficult for, and germination rate is lower, thereby be proved and be not a kind of desirable approach that obtains with seedling.
Summary of the invention
The purpose of this invention is to provide a kind of Viola baoshanensis tissue culturing fast seedling-cultivating method.
Tissue culturing fast seedling-cultivating, solved the problem of breeding of many other precious species, but each species cultivates the condition of breeding and have nothing in common with each other, need the multiplex screening test, the invention solves this problem, a kind of suitable Viola baoshanensis tissue culturing fast seedling-cultivating method is provided.The present invention is an explant by blade or the petiole of choosing wild Viola baoshanensis, after the sterilization, be inoculated on candidate's the inducing culture, respond different hormone combination inducing cultures according to the separate sources explant and produce different differentiation effects, and, filter out the Viola baoshanensis optimization and breed scheme the groping of follow-up enrichment culture, culture of rootage, transplanting condition.
The concrete steps of Viola baoshanensis tissue culturing fast seedling-cultivating method of the present invention comprise:
1) explant is selected and disinfected: selecting blade or petiole is explant, with 0.1%HgCl
2Be disinfectant, sterilize 8～12 minutes (best) with 10 minutes effects;
2) adventitious bud inducing and callus generate: the explant through above-mentioned processing is inoculated in evoking adventive bud and callus generate on the MS minimal medium of one of following interpolation hormone;
A. the hormone ratio is 0.5～1.0mg/L 6-BA+0.5～1.5mg/L NAA, and inoculating has after a week indefinite bud to generate, the indefinite bud growth rapidly, after 2～3 weeks of inoculation, high about 2～3 centimetres of indefinite bud;
B. the hormone ratio is 0.5～1mg/L KT+1～1.5mg/L 2, and 4-D inoculates adularescent callus generation after 2～3 days, after 3～4 weeks, generates tangible callus; Callus is inoculated on the MS medium of 0.5～1.0mg/L 6-BA+0.5～1.5mg/L NAA proportioning, and inoculation had indefinite bud to generate in 1～2 week;
3) culture of rootage: with the indefinite bud of above-mentioned acquisition transfer in 1/2 or the 1MS minimal medium on root induction;
Above-mentioned steps 2) and 3) condition of culture of (inoculation back to hardening) is: illumination 12～14 hours/day, 22～25 ℃ of temperature;
4) hardening and transplanting: culture of rootage is after 2～3 weeks (or treat that the Viola baoshanensis seedling is long by 4～5cm), and open the blake bottle hardening, culture transferring is in compost, cover is with transparent membrane on tissue cultivating seedling, keep 70～80% relative moisture, take transparent membrane away after 2～4 days, obtain tissue cultivating seedling; Transplanting survival rate＞80%.
When opening the blake bottle hardening, preferably earlier aseptic indoor cultivation 2 days, transfer to hot-house culture after 1 day, seedling is shifted out from blake bottle again, rinse root agar well, culture transferring is in compost.
The present invention has following outstanding effect:
1) shorten the Viola baoshanensis breeding cycle, survival rate is higher.
The solid seed sexual propagation cycle of wild Viola baoshanensis is longer, and tissue culture method of the present invention can be bred Viola baoshanensis fast, from inoculation, hardening, be transplanted to and obtain stalwartness and may be configured as only 2～3 months seedling cycle.The Viola baoshanensis seedling that utilizes prioritization scheme to breed out all can be finished the history of life on medium He in the soil, and transplanting survival rate is greater than 80%.
2) improve reproduction coefficient, growing seedlings, it is less influenced by external condition.
The Viola baoshanensis seed tool hibernation feature of open-air growth, germination rate is lower, and the indefinite bud quantity average out to 6-10 of each explant induction is individual in the tissue culture, and reproduction coefficient is higher, group training work can be at general aseptic in-house operation, thus reproductive process influenced by external condition less.
3) promote China that scientific research of heavy metal super-enriched plant Viola baoshanensis and environmental protection are used.
The method of utilizing the present invention to set up, can be simply, fast, large scale cultivating heavy metal super-enriched plant Viola baoshanensis.The method that the present invention sets up is not only bred out seedling, also obtained callus, as material, utilize Protocols in Molecular Biology and means to be improved, may optimize Viola baoshanensis rehabilitating soil or heavy metal pollution of water body uses, simultaneously also for plant provides desirable reliable material to the scientific research of toxic metals patience and enrichment mechanism aspect, thereby application of the present invention, corresponding scientific research and the environmental protection application of China will be promoted to Viola baoshanensis.
The invention will be further described below by way of embodiments and drawings.
Description of drawings
Fig. 1 is Viola baoshanensis petiole explant differentiation figure, is adding 1mg/L KT+1mg/L 2, and on the MS medium of 4-D hormone combination, explant is induced a large amount of hairy root;
Fig. 2 is Viola baoshanensis leaf explant differentiation figure, is adding 1mg/L KT+1mg/L 2, and on the MS medium of 4-D hormone combination, explant is induced tangible callus;
Fig. 3 is Viola baoshanensis leaf explant adventitious bud inducing generation figure, and leaf explant can generate indefinite bud at the MS medium of 1mg/L 6-BA+1mg/L NAA proportioning in addition;
Fig. 4 is the solid drawing of seeds of Viola baoshanensis tissue cultivating seedling, transfers in 1/2MS medium indefinite bud, can finish the history of life on medium, and the explanation group training stage has kept the genetic character of Viola baoshanensis preferably;
Fig. 5 is transplant survival figure behind the Viola baoshanensis training tissue culture seedling, and the Viola baoshanensis tissue cultivating seedling that is transplanted into after the hardening in the nutrition soil can be grown up strong and sturdy, and survival rate is greater than 80%.
Embodiment
Embodiment 1
The petiole of getting wild Viola baoshanensis is an explant, and flowing water is rinsed well, is cut into 0.5cm
2Fritter, 0.1%HgCl
2In soaked 10 minutes, then,, be inoculated on the MS minimal medium of 1mg/L 6-BA+1mg/L NAA with aseptic water washing 4～5 times, this operates on the superclean bench of ultraviolet sterilization and carries out.The back condition of culture to hardening of inoculation is: 22～25 ℃, and long 12 hours of illumination.Inoculation one week back indefinite bud generates (as Fig. 3), changes short its long root on the 1/2MS medium during high about 2 centimetres of indefinite bud over to, and culture of rootage is after 2 weeks, uncork lid hardening, in the immigration compost, on cover plastic sack to keep certain humidity, take away two days later, water every day.Survival rate is greater than 80%.Move into compost after 4～5 weeks, the tissue cultivating seedling form is identical with wild[l, and robust growth is opened latent flower, and knot is planted, and can finish the whole history of life (as Fig. 4).The Viola baoshanensis that this embodiment cultivates can be directly used in corresponding scientific experimentation, and be applied to restoration of soil polluted by heavy metal or water body.
Embodiment 2
The blade of getting wild Viola baoshanensis is an explant, and flowing water is rinsed well, is cut into 0.5cm
2Fritter, 0.1%HgCl
2The middle immersion 10 minutes then, used aseptic water washing 5 times, is inoculated in hormone than being 1mg/L KT+1mg/L 2, during 4D, inoculates adularescent callus generation after 3 days, after 3 weeks, generates tangible callus (as Fig. 2).Callus is inoculated on the MS medium of 1mg/L 6-BA+1mg/L NAA proportioning, has indefinite bud to generate in 1～2 week.The back condition of culture to hardening of inoculation is: 22～25 ℃, and long 14 hours of illumination.Change short its long root on the 1/2MS medium during high about 2 centimetres of the indefinite bud that generates over to, culture of rootage is after 3 weeks, and uncork lid hardening sprays little water, aseptic indoor cultivation 2 days, transfer to hot-house culture after 1 day, seedling is shifted out from blake bottle, rinse root agar well, culture transferring is gone in the compost, cover is kept 70～80% relative moisture with transparent membrane on tissue cultivating seedling, takes transparent membrane away after 3 days.Move into compost after 3～4 weeks, Viola baoshanensis tissue cultivating seedling morphological feature is identical with wild[l, robust growth (as Fig. 5).
Claims (3)
1. the method for a Viola baoshanensis tissue culturing fast seedling-cultivating, concrete steps comprise:
1) explant is selected and disinfected: selecting blade or petiole is explant, with 0.1%HgCl
2Be disinfectant, sterilized 8～12 minutes;
2) adventitious bud inducing and callus generate: the explant through above-mentioned processing is inoculated in evoking adventive bud and callus generate on the MS minimal medium of one of following interpolation hormone:
A. the hormone ratio is 0.5～1.0mg/L 6-BA+0.5～1.5mg/L NAA, and inoculating has after a week indefinite bud to generate, the indefinite bud growth rapidly, after 2～3 weeks of inoculation, high about 2～3 centimetres of indefinite bud;
B. the hormone ratio is 0.5～1mg/L KT+1～1.5mg/L 2, and 4-D inoculates adularescent callus generation after 2～3 days, after 3～4 weeks, generates tangible callus; Callus is inoculated on the MS medium of 0.5～1.0mg/L 6-BA+0.5～1.5mg/L NAA proportioning, and inoculation had indefinite bud to generate in 1～2 week;
3) culture of rootage: with the indefinite bud of above-mentioned acquisition transfer in 1/2 or the 1MS minimal medium on root induction; Above-mentioned steps 2) and 3) condition of culture be: illumination 12～14 hours/day, 22～25 ℃ of temperature;
4) hardening and transplanting: culture of rootage is after 2～3 weeks or treat the long 4～5cm of Viola baoshanensis seedling, opens the blake bottle hardening, and culture transferring is in compost, cover is with transparent membrane on tissue cultivating seedling, keep 70～80% relative moisture, take transparent membrane away after 2～4 days, obtain tissue cultivating seedling.
2. in accordance with the method for claim 1, it is characterized in that the time that the step 1) explant is disinfected is 10 minutes.
3. according to claim 1 or 2 described methods, when it is characterized in that step 4) is opened the blake bottle hardening, preferably, transfer to hot-house culture after 1 day earlier aseptic indoor cultivation 2 days, again seedling is shifted out from blake bottle, rinse root agar well, culture transferring is in compost.
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| CN 200310117442 CN1245868C (en) | 2003-12-22 | 2003-12-22 | Quick seedling growing tissue culture method of Baoshan violet |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103004611A (en) * | 2013-01-21 | 2013-04-03 | 四川农业大学 | Method of tissue culture and rapid propagation of viola prionantha |
| CN107900090A (en) * | 2017-12-29 | 2018-04-13 | 湖南现代环境科技股份有限公司 | A kind of Cd-polluted farmland integrates restorative procedure |
-
2003
- 2003-12-22 CN CN 200310117442 patent/CN1245868C/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103004611A (en) * | 2013-01-21 | 2013-04-03 | 四川农业大学 | Method of tissue culture and rapid propagation of viola prionantha |
| CN103004611B (en) * | 2013-01-21 | 2014-03-12 | 四川农业大学 | Method of tissue culture and rapid propagation of viola prionantha |
| CN107900090A (en) * | 2017-12-29 | 2018-04-13 | 湖南现代环境科技股份有限公司 | A kind of Cd-polluted farmland integrates restorative procedure |
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| CN1245868C (en) | 2006-03-22 |
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