CN104396753A - Method for rapidly propagating sugarcane tissue culture seedlings with spermine - Google Patents
Method for rapidly propagating sugarcane tissue culture seedlings with spermine Download PDFInfo
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- CN104396753A CN104396753A CN201410681991.4A CN201410681991A CN104396753A CN 104396753 A CN104396753 A CN 104396753A CN 201410681991 A CN201410681991 A CN 201410681991A CN 104396753 A CN104396753 A CN 104396753A
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Abstract
The invention relates to a method for rapidly propagating sugarcane tissue culture seedlings with spermine. The method comprises the following steps: using sugarcane immature leaf slices as explants, vaccinating the explants to an improved MS solid medium of MS+Spm.1.5 mg . L<-1>-3.0mg . L<-1>, regulating the pH to be 5.8-6.0, and performing continuous culture for 10 d-25 d to induce and obtain sugarcane callus tissue agglomerates; retransferring the agglomerates to the same fresh culture medium for subsequent culturing for 15 d, and then separating and cutting obtained callus tissues into small blocks to be revaccinated to the same fresh culture medium; performing subsequent culture for 20d -30d, transferring regeneration seedlings to the same fresh culture medium to be cultured for 20d so as to obtain test-tube seedlings of which the stem length is 5-6cm and the root seedlings are complete. According to the invention, only one culture medium is used from the starting of explant culture to the generation of the complete regenerated plantlets, so that the production process for the sugarcane tissue culture seedlings is obviously simplified, besides, the toxic action of 2,4-D induction callus tissues is removed, and the cost of the used spermine is low, so that the method is more favorable for popularization.
Description
Technical field
The invention belongs to the technical field of tissue culture of Plant Biotechnology, especially a kind of method of spermine Fast-propagation sugar-cane tissue culture seedlings.
Technical background
" plantlet in vitro " is also known as " test-tube plantlet ", " aseptic seedling " and " clone's (Clone) seedling ", theoretical according to the totipotency of cell, utilize explant, under aseptic and suitable medium and light and temperature condition, the complete regenerated plant that the root that fast breeding obtains, seedling are complete.At one time in section, the reproductive speed of plantlet in vitro can decades of times, even hundreds of times to the natural propagation speed of plant, and anosis nontoxic (Virus), colony is neat, the extensive industrialization plantation of the most applicable crop fine breed.Plantlet in vitro production technology is one of support technology of agricultural modernization, is the important component part of " test tube agricultural ".The production of Sugarcane Superior Variety plantlet in vitro and popularization, the cane planting industry of Jin20Nian Shi China there occurs earth-shaking change.The research report setting up sugar-cane tissue culture seedlings production technology is also never interrupted.But the key of its card throat remains and occurs in sugarcane explants through callus induction and embryoid 2 links.All the time, existingly must use 2,4-D, the negative harm [the yellow favour virtue 1998 of sugarcane tissue culture Review Study [J] Guangxi hotwork science and technology, (2): 33-37] of 2,4-D toxicity cannot be broken away from again; [sugarcane tissue culture progress [J] Agriculture of Anhui science willow etc. 2007,35 (12): 3490-3492].Though the LFS (spirit hair element) such as Li Song and Xu Hongyuan establishes the technical system [a kind of spirit hair element carries out the method China Patent No. ZL201010216192.1 of sugarcane tissue culture Fast-propagation] that high-efficiency high-quality produces sugar-cane tissue culture seedlings, but, LFS does not have manufacturer at home, import price is very expensive, is difficult to apply.Spermine (Spermine, Spm.) be that one is extensively present in higher plant circle, the plant growth regulating substance [polyamines is vehement 1985 (6) 63-68 of instrumentality [J] Plant Physiology Communications Pan Rui of growth and development of plants] of pure natural, and low price, convenient popularization.People, to the research of its Promoting plant growth, the physiologically actives such as anti-adversity ability that delay senility, strengthen, have had many reports.But, also fail to retrieve the research data of spermine for sugar-cane tissue culture seedlings Fast-propagation.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of method of spermine Fast-propagation sugar-cane tissue culture seedlings
The present invention solves the problems of the technologies described above with following technical scheme:
A method for spermine Fast-propagation sugar-cane tissue culture seedlings, comprises the steps:
1. the In The Young Leaf of Sugarcane section obtained in conventional manner makes explant, routine disinfection, is inoculated into MS+Spm.1.5mgL
-1-3.0mgL
-1modified form MS solid culture medium in, adjust pH 5.8-6.0.Under conventional culture conditions, continuous culture 10d-25d induces and obtains sugarcane callus agglomerate.
2. above-mentioned callus agglomerate is transferred to fresh MS+Spm1.5mgL carefully
-1-3.0mgL
-1medium, continue to cultivate 15d under conventional culture conditions, the embryoid that comes in every shape successively occurs.
3. the fritter (its embryoid is existing turns green better) the above-mentioned callus agglomerate having differentiated embryoid being cut into soya bean size is transferred to fresh MS+Spm.1.5mgL
-1-3.0mgL
-1medium.Every bottle graft kind 5 fritters.Often criticize inoculation more than 10 bottles, continue to cultivate 20d and 30d under conventional culture conditions, in the process, original embryoid is successively while Germination And Seedling, and callus islands also can continue to grow up, and has new embryoid to be formed and Germination And Seedling.
4. plant height is reached the regrowth of about 2cm, unified selecting transfers to fresh MS+Spm.1.5mgL
-1-3.0mgL
-1medium.Every bottle of 15 strains.Cellar culture 20d, just obtains plant height 5-6cm, the test-tube plantlet that root (root) seedling (shoot) is complete.
5. conveniently hardening, bottle outlet, washes seedling plant division, transplants in the seedling-raising cup that seedling medium is housed or seedling bed, Routine Management, survival rate >=90%.
The outstanding advantages of the present invention's spermine Fast-propagation sugar-cane tissue culture seedlings is:
(1) be different from traditional method completely, cultivate from explant and start to building up of complete regenerated plant, only with a kind of medium MS+Spm.1.5mgL
-1-3.0mgL
-1can complete, join " subculture medium " and " root media " without the need to conventional art is another like that.Also without the need to adding the special root-inducing promoter such as NAA (methyl α-naphthyl acetate), IBA (indolebutyric acid) in the medium.Significantly simplify the production routine of sugar-cane tissue culture seedlings.
(2) be completely free of in conventional method, both with 2,4-D evoked callus, must can not have avoided again the helpless situation of its toxic action.
(3) be also different from the new method of the Fast-propagation sugar-cane tissue culture seedlings set up with LFS, it is cheap that the price of spermine compares LFS ten points, more easily applies.
Embodiment
Below by way of case study on implementation, technical scheme of the present invention is described further.
The method of spermine induction sugar-cane tissue culture seedlings of the present invention comprises the following steps:
1. the acquisition of callus: conventionally method obtains In The Young Leaf of Sugarcane section and makes explant, is inoculated in containing Spm.1.5mgL after routine disinfection
-1-3.0mgL
-1modified form MS solid culture medium in, often criticize test and be no less than 10 bottles, 5 every bottle sections.10d-25d is cultivated under conventional culture conditions.Get 3 bottles of statistics healing rates at random, weigh fresh weight fast, remember 3 bottles of mean values (see table 1).
Table 1. variable concentrations Spm. induces In The Young Leaf of Sugarcane to form the case study on implementation of callus
In table 1:
A. healing rate (%)=go out more spire number of slices/inoculation spire number of slices × 100%.
B. the metering method of callus fresh weight is, after cultivating 20d and 25d, often group randomly draws 3 bottles, takes out culture materials (containing former section, callus and derivative adjunct thereof) and carefully remove adhesion medium in units of bottle, its fresh weight of quick title, remembers 3 bottles of mean values.
Integrated comparative table 1 data are visible, and the optimum medium of evoked callus is MS+Spm.2.5mg.L
-1; Suitable incubation time is 20d-25d.
2. the acquisition of embryoid: above-mentioned callus agglomerate is transferred to fresh MS+Spm.1.5mgL carefully
-1-2.5mgL
-1medium.Every bottle graft kind 4-5 agglomerate, often criticizes inoculation more than 10 bottles.Continue to cultivate 15d under conventional culture conditions, get 3 bottles at random, statistics embryoid incidence, bore hole can distinguish that embryoid number/bottle and embryoid turn green rate, remember 3 bottles of mean values (see table 2.).
The case study on implementation (cultivating 15d) of table 2. variable concentrations Spm. inducing embryoids of sugarcane
In table 2;
Callus agglomerate number (no matter embryoid quantity)/inoculation callus agglomerate number × 100% of A. embryoid incidence (%)=differentiate embryoid.
B. " bore hole can distinguish embryoid number/bottle " refers to terminate the same day at culture period, every bottle can be shrewd by naked eyes embryoid sum (no matter developmental condition of embryoid), remember 3 bottles of mean values.
C. embryoid turns the green embryoid number of green rate (%)=occurred/total embryoid number × 100%.
Integrated comparative table 2 data are visible, and Spm. change in concentration is to " embryoid incidence " and " bore hole can distinguish that embryoid number/bottle " all presents obvious positive correlation, and affects " embryoid turns green rate " little.
3. the acquisition of plantlet in vitro: the fritter (its embryoid is existing turns green better) the above-mentioned callus agglomerate having differentiated embryoid being cut into soya bean size is transferred to fresh MS+Spm.1.5mgL
-1-2.5mgL
-1medium.Every bottle graft kind 5 fritters.Often criticize inoculation more than 10 bottles, continue to cultivate 20d and 30d under conventional culture conditions, in the process, original embryoid is successively while Germination And Seedling, and callus islands also can continue to grow up, and has new embryoid to be formed and Germination And Seedling.
Table 3. Spm. induces the case study on implementation of sugar-cane tissue culture seedlings
In table 3: A. every bottle graft kind embryoid number, 20d number of seedling/bottle, 20d number of seedling/bottle, be all the mean value of random sampling 3 bottles." seedling " refers to plant height >=2.0cm.
Integrated comparative table 3 data are visible, and the present invention Spm., by the process of the embryoid induction Germination And Seedling on callus agglomerate, in trial stretch, cultivates 20d equally, and Spm. concentration increases favourable raising number of seedling.Spm.3.0mgL
-1group compares Spm.1.5mgL
-1group raising 11.3%.But, then Extending culture 10d, during to 30d, number of seedling there is no remarkable increase, can take large quantity space and utensil on the contrary when producing in batches, may not favourable overall efficiency.
4. the acquisition of finished product plantlet in vitro: unified the selecting of regrowth plant height being reached about 2cm transfers to fresh MS+Spm.1.5mgL
-1-3.0mgL
-1medium.Every bottle of 15 strains.Cellar culture 20d, just obtains plant height 5-6cm, the finished product plantlet in vitro that root (root) seedling (shoot) is complete." finished product plantlet in vitro " needs to reach the following standard that the Ministry of Agriculture issues: (1) medium and material fungi or contamination rate≤8.0%; (2) root system has white root >=2 of more than 1.0cm; (3) plant height >=4.0cm, false stem has or nothing; (4) fully expanded leaves >=2 slice, leaf look green.(5) growth is normal without variation.
The sugarcane finished product plantlet in vitro that the present invention produces, conveniently hardening, bottle outlet, washes seedling plant division, transplants to seedling-raising cup or seedling bed that nutrient matrix is housed, Routine Management, survival rate >=90%.
In sum, the present invention for explant, take MS as minimal medium with In The Young Leaf of Sugarcane section, adds Spm.1.5mgL
-1-3.0mgL
-1be mixed with improvement solidified MS media, all can complete evoked callus generation, inducing embryoid body generation and induction seedling three key links.After preferred to Spm. concentration, use MS+Spm.2.5mgL
-1single medium, about 70d just can be cut into slices by 5 In The Young Leaf of Sugarcanes and cultivate the finished product of strain more than 40 plantlet in vitro, transplanting survival rate>=90%.This specification has all done more detailed description by case study on implementation to technical program of the present invention.But, on basis of the present invention, those skilled in the relevant art are easy to replace minimal medium MS of the present invention with other minimal medium, or change the consumption of Spm., even Spm. and other growth regulatory substance carry out composite after be used further to the production of sugar-cane tissue culture seedlings.So all spermine (Spm.) of with the addition of in the production of sugar-cane tissue culture seedlings, all belong to the scope of protection of present invention.
Claims (2)
1., by a method for spermine Fast-propagation sugar-cane tissue culture seedlings, it is characterized in that comprising the steps:
(1) the In The Young Leaf of Sugarcane section obtained in conventional manner makes explant, routine disinfection, is inoculated into MS+Spm.1.5mgL
-1-3.0mgL
-1modified form MS solid culture medium in, adjust pH5.8-6.0; Under conventional culture conditions, continuous culture 10d-25d induces and obtains sugarcane callus agglomerate;
(2) above-mentioned callus agglomerate is transferred to fresh MS+Spm.1.5mgL carefully
-1-3.0mgL
-1medium, continue to cultivate 15d under conventional culture conditions, the embryoid that comes in every shape successively occurs;
(3) the fritter that the above-mentioned callus agglomerate having differentiated embryoid cuts into soya bean size is transferred to fresh MS+Spm.1.5mgL
-1-3.0mgL
-1medium; Every bottle graft kind 5 fritters; Often criticize inoculation more than 10 bottles, continue to cultivate 20d and 30d under conventional culture conditions, in the process, original embryoid is successively while Germination And Seedling, and callus islands also can continue to grow up, and has new embryoid to be formed and Germination And Seedling;
(4) unified the selecting of regrowth plant height being reached about 2cm transfers to fresh MS+Spm.1.5mgL
-1-3.0mgL
-1medium; Every bottle of 15 strains; Cellar culture 20d, just obtains plant height 5-6cm, the test-tube plantlet that root (root) seedling (shoot) is complete;
(5) conveniently hardening, bottle outlet, washes seedling plant division, transplants in the seedling-raising cup that seedling medium is housed or seedling bed, Routine Management, survival rate >=90%.
2. the method with spermine Fast-propagation sugar-cane tissue culture seedlings as claimed in claim 1, is characterized in that modified form MS solid culture medium is with MS+Spm.2.5mgL
-1for being best suited for the medium of spermine Fast-propagation sugar-cane tissue culture seedlings.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106258988A (en) * | 2016-10-14 | 2017-01-04 | 广西壮族自治区烟草公司百色市公司 | Method with spermine evoking tobacco callus |
| CN106359102A (en) * | 2016-10-14 | 2017-02-01 | 广西壮族自治区烟草公司百色市公司 | Method for directly inducing tobacco leaf regenerated plant by using spermine |
| CN109452123A (en) * | 2018-12-27 | 2019-03-12 | 广西民族大学 | A kind of implantation methods improving the germination of sugarcane kind and survival rate |
| CN110915654A (en) * | 2019-11-28 | 2020-03-27 | 南京林业大学 | Method for promoting fir somatic embryogenesis by using spermidine |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106258988A (en) * | 2016-10-14 | 2017-01-04 | 广西壮族自治区烟草公司百色市公司 | Method with spermine evoking tobacco callus |
| CN106359102A (en) * | 2016-10-14 | 2017-02-01 | 广西壮族自治区烟草公司百色市公司 | Method for directly inducing tobacco leaf regenerated plant by using spermine |
| CN106258988B (en) * | 2016-10-14 | 2019-02-26 | 广西壮族自治区烟草公司百色市公司 | With the method for spermine evoking tobacco callus |
| CN109452123A (en) * | 2018-12-27 | 2019-03-12 | 广西民族大学 | A kind of implantation methods improving the germination of sugarcane kind and survival rate |
| CN109452123B (en) * | 2018-12-27 | 2021-08-27 | 广西民族大学 | Planting method for improving germination and survival rate of sugarcane seeds |
| CN110915654A (en) * | 2019-11-28 | 2020-03-27 | 南京林业大学 | Method for promoting fir somatic embryogenesis by using spermidine |
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