CN104396753B - A kind of method of spermine breeding sugar-cane tissue culture seedlings - Google Patents
A kind of method of spermine breeding sugar-cane tissue culture seedlings Download PDFInfo
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- CN104396753B CN104396753B CN201410681991.4A CN201410681991A CN104396753B CN 104396753 B CN104396753 B CN 104396753B CN 201410681991 A CN201410681991 A CN 201410681991A CN 104396753 B CN104396753 B CN 104396753B
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Abstract
A kind of method of spermine Fast-propagation sugar-cane tissue culture seedlings, makes outer implant by In The Young Leaf of Sugarcane section, is inoculated into MS+Spm.1.5mg L-1-3.0mg·L-1Modified form MS solid medium in, adjust pH 5.8-6.0, cultivate continuously 10d-25d and induce and obtain sugarcane callus agglomerate；After being then transferred to fresh same culture medium continuation cultivation 15d, gained callus is cut into fritter and is transferred to fresh same culture medium again.Continue to cultivate 20d to 30d, regrowth is transferred to fresh same culture medium culturing 20d, obtain the test tube Seedling that plant height 5-6cm, root are complete.The present invention cultivates from outer implant and starts to building up of complete regenerated plant, only by a kind of culture medium, significantly simplify the production routine of sugar-cane tissue culture seedlings, has broken away from the toxic action of 2,4-D callus induction simultaneously, and the spermine of use is cheap, is more conducive to promote.
Description
Technical field
The invention belongs to the technical field of tissue culture of Plant Biotechnology, the method for especially a kind of spermine Fast-propagation sugar-cane tissue culture seedlings.
Technical background
" tissue cultured seedling " is also known as " test tube Seedling ", " aseptic seedling " and " clone's (Clone) Seedling ", it is that the totipotency according to cell is theoretical, utilize outer implant, under aseptic and suitable culture medium and light and temperature condition, the complete regenerated plant that the root of fast breeding acquisition, Seedling are complete.In the same time period, the reproductive speed of tissue cultured seedling can the natural propagation speed of decades of times, even hundreds of times of plants, and anosis nontoxic (Virus), colony is neat, is best suitable for the extensive industrialization plantation of crop fine breed.Tissue cultured seedling 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 tissue cultured seedling 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 implant callus induction and 2 links of embryoid outside occurs.All the time, existing must use 2,4-D, the negative harm [sugarcane tissue culture Review Study [J] Guangxi hotwork science and technology Huang favour virtue 1998, (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 Li Song and Xu Hongyuan etc. establish high-efficiency high-quality with LFS (spirit hair element) and produce the technical system [one spirit hair element carries out sugarcane tissue culture rapid propagation method China Patent No. ZL201010216192.1] of sugar-cane tissue culture seedlings, but, LFS does not have manufacturer at home, import price is sufficiently expensive, it is difficult to popularization and application.Spermine (Spermine,, Spm.) and it is that one is widely present in higher plant circle, the plant growth regulating substance of pure natural vehement 1985 (6) 63-68 of instrumentality [J] Plant Physiology Communications Pan Rui of growth and development of plants [polyamines be], and low price, convenient popularization.It is promoted the research of the physiologically actives such as plant growing, slow down aging, enhancing anti-adversity ability by people, has had many reports.But, also fail to the research data retrieving 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 above-mentioned technical problem with following technical scheme:
A kind of method of spermine Fast-propagation sugar-cane tissue culture seedlings, comprises the steps:
1. outer implant, routine disinfection are made in the In The Young Leaf of Sugarcane section obtained in conventional manner, are inoculated into MS+Spm.1.5mg L-1-3.0mg·L-1Modified form MS solid medium in, adjust pH5.8-6.0.Under conventional culture conditions, cultivate 10d-25d continuously induce and obtain sugarcane callus agglomerate.
2. above-mentioned callus agglomerate is carefully transferred to fresh MS+Spm.1.5mg L-1-3.0mg·L-1Culture medium, continues to cultivate 15d under conventional culture conditions, and the embryoid that comes in every shape successively occurs.
3. the fritter (its embryoid is existing turns green better) that the above-mentioned callus agglomerate having differentiated embryoid cuts into Semen Glycines size is transferred to fresh MS+Spm.1.5mg L-1-3.0mg·L-1Culture medium.5 fritters of every bottle graft kind.Inoculating more than 10 bottles for every batch, continue to cultivate 20d and 30d under conventional culture conditions, in the process, while original embryoid Germination And Seedling successively, callus islands also may proceed to grow up, and has new embryoid to be formed and Germination And Seedling.
4. plant height reaches the regrowth of about 2cm, and unified selecting transfers to fresh MS+Spm.1.5mg L-1-3.0mg·L-1Culture medium.Every bottle of 15 strains.Cellar culture 20d, just obtains plant height 5-6cm, the test tube Seedling that root (root) Seedling (shoot) is complete.
5. conventionally seedling exercising, bottle outlet, washes Seedling plant division, transplants to equipped with in the seedling-raising cup of seedling medium or seedling bed, Routine Management, survival rate >=90%.
The outstanding advantages of present invention spermine Fast-propagation sugar-cane tissue culture seedlings is:
(1) it is totally different from traditional method, cultivates from outer implant and start to building up of complete regenerated plant, only with a kind of culture medium MS+Spm.1.5mg L-1-3.0mg·L-1Can complete, it is not necessary to conventional art separately joins " subculture medium " and " root media " like that.Without adding the special root-inducing promoter such as NAA (naphthalene acetic acid), IBA (indolebutyric acid) in the medium.Significantly simplify the production routine of sugar-cane tissue culture seedlings.
(2) it has been completely free of in traditional method, both with 2,4-D callus induction, the helpless situation of its toxic action must have been cannot be avoided again.
(3) 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, it is easier to popularization and application.
Detailed description of the invention
Below by way of case study on implementation, technical scheme is described further.
The method of spermine of the present invention induction sugar-cane tissue culture seedlings comprises the following steps:
1. the acquisition of callus: conventionally method obtains the In The Young Leaf of Sugarcane section outer implant of work, is inoculated in containing Spm.1.5mg L after routine disinfection-1-3.0mg·L-1Modified form MS solid medium in, every batch test no less than 10 bottles, 5 every bottle sections.10d-25d is cultivated under conventional culture conditions.Take 3 bottles of statistics healing rates at random, quickly weigh fresh weight, remember 3 bottles of meansigma methodss (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 wound healing fresh weight is, after cultivating 20d and 25d, often group randomly draws 3 bottles, takes out culture materials (containing former section, callus and derivative appurtenance thereof) and carefully remove adhesion culture medium in units of bottle, quickly claim its fresh weight, remember 3 bottles of meansigma methodss.
Integrated comparative table 1 data are visible, and the optimum culture medium of callus induction is MS+Spm.2.5mg.L-1；Suitable incubation time is 20d-25d.
2. the acquisition of embryoid: above-mentioned callus agglomerate is carefully transferred to fresh MS+Spm.1.5mg L-1-2.5mg·L-1Culture medium.Kind 4-5 agglomerate of every bottle graft, inoculates more than 10 bottles for every batch.Continuing to cultivate 15d under conventional culture conditions, take 3 bottles at random, statistics embryoid incidence rate, bore hole can distinguish that embryoid number/bottle and embryoid turn green rate, remember 3 bottles of meansigma methodss (see table 2.).
The case study on implementation (cultivating 15d) of table 2. variable concentrations Spm. inducing embryoids of sugarcane
In table 2；
A. callus agglomerate number (no matter embryoid quantity)/inoculation callus agglomerate number × 100% of embryoid incidence rate (%)=differentiate embryoid.
B. " bore hole can distinguish embryoid number/bottle " refers to terminate the same day at culture period, every bottle by naked eyes can be shrewd embryoid sum (no matter developmental condition of embryoid), remember 3 bottles of meansigma methodss.
C. embryoid turns embryoid number/total embryoid number × 100% of green rate (%)=occurred green.
Integrated comparative table 2 data are visible, and Spm. concentration change is to " embryoid incidence rate " and " bore hole can distinguish that embryoid number/bottle " all presents obvious positive correlation, and " embryoid turns green rate " affected little.
3. the acquisition of tissue cultured seedling: the fritter (its embryoid is existing turns green better) that the above-mentioned callus agglomerate having differentiated embryoid cuts into Semen Glycines size is transferred to fresh MS+Spm.1.5mg L-1-2.5mg·L-1Culture medium.5 fritters of every bottle graft kind.Inoculating more than 10 bottles for every batch, continue to cultivate 20d and 30d under conventional culture conditions, in the process, while original embryoid Germination And Seedling successively, callus islands also may proceed 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, it is all the meansigma methods of stochastic sampling 3 bottles." seedling " refers to plant height >=2.0cm.
Integrated comparative table 3 data are visible, and present invention Spm. is by the process of the embryoid induction Germination And Seedling on callus agglomerate, in trial stretch, cultivate 20d, Spm. concentration increase equally and favorably improve number of seedling.Spm.3.0mg L-1Group is than Spm.1.5mg L-1Group raising 11.3%.But, then Extending culture 10d, during to 30d, number of seedling there is no and dramatically increases, and can take big quantity space and utensil when batch production on the contrary, may not favourable overall efficiency.
4. the acquisition of finished product tissue cultured seedling: unified the selecting of regrowth that plant height reaches about 2cm transfers to fresh MS+Spm.1.5mg L-1-3.0mg·L-1Culture medium.Every bottle of 15 strains.Cellar culture 20d, just obtains plant height 5-6cm, the finished product tissue cultured seedling that root (root) Seedling (shoot) is complete." finished product tissue cultured seedling " needs to reach the following standard that the Ministry of Agriculture issues: (1) culture medium and material fungus or contamination rate≤8.0%；(2) root system has white root >=2 of more than 1.0cm；(3) plant height >=4.0cm, cauloid with or without；(4) fully expanded leaves >=2 slice, leaf color is green.(5) growth is normal without variation.
The Caulis Sacchari sinensis finished product tissue cultured seedling that the present invention produces, conventionally seedling exercising, bottle outlet, wash Seedling plant division, transplant extremely equipped with the seedling-raising cup of nutrient matrix or seedling bed, Routine Management, survival rate >=90%.
In sum, the present invention is with In The Young Leaf of Sugarcane section for outer implant, with MS for minimal medium, adds Spm.1.5mg L-1-3.0mg·L-1It is configured to improvement solidified MS media, all can complete callus induction generation, inducing embryoid body generation and induction seedling three key links.To Spm. concentration preferably after, use MS+Spm.2.5mg L-1Single culture medium, about 70d just can be cultivated the finished product tissue cultured seedling of strain more than 40, transplanting survival rate >=90% by 5 In The Young Leaf of Sugarcane sections.The technical program of the present invention has all been done description in greater detail by case study on implementation by this specification.But, on the basis of the present invention, the minimal medium MS of the present invention is replaced by those skilled in the relevant art readily by other minimal medium, or the consumption of Spm. is modified, 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 in the production of sugar-cane tissue culture seedlings, with the addition of spermine (Spm.), broadly fall into the scope of protection of present invention.
Claims (2)
1. the method breeding sugar-cane tissue culture seedlings with spermine, is characterized in that comprising the steps:
(1) outer implant, routine disinfection are made in the In The Young Leaf of Sugarcane section obtained in conventional manner, are inoculated into MS+Spm.1.5mg L-1-3.0mg·L-1Modified form MS solid medium in, adjust pH5.8-6.0；Under conventional culture conditions, cultivate 10d-25d continuously induce and obtain sugarcane callus agglomerate；
(2) above-mentioned callus agglomerate is carefully transferred to fresh MS+Spm.1.5mg L-1-3.0mg·L-1Culture medium, continues to cultivate 15d under conventional culture conditions, and the embryoid that comes in every shape successively occurs；
(3) the fritter that the above-mentioned callus agglomerate having differentiated embryoid cuts into Semen Glycines size is transferred to fresh MS+Spm.1.5mg L-1-3.0mg·L-1Culture medium；5 fritters of every bottle graft kind；Inoculating more than 10 bottles for every batch, continue to cultivate 20d and 30d under conventional culture conditions, in the process, while original embryoid Germination And Seedling successively, callus islands also may proceed to grow up, and has new embryoid to be formed and Germination And Seedling；
(4) unified the selecting of regrowth that plant height reaches 2cm transfers to fresh MS+Spm.1.5mg L-1-3.0mg·L-1Culture medium；Every bottle of 15 strains；Cellar culture 20d, just obtains the test tube Seedling that plant height 5-6cm, root are complete；
(5) conventionally seedling exercising, bottle outlet, washes Seedling plant division, transplants to equipped with in the seedling-raising cup of seedling medium or seedling bed, Routine Management, survival rate >=90%.
2. the method breeding sugar-cane tissue culture seedlings with spermine as claimed in claim 1, is characterized in that modified form MS solid medium is with MS+Spm.2.5mg L-1For being best suited for the culture medium of spermine breeding sugar-cane tissue culture seedlings.
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| 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 |
| CN106359102B (en) * | 2016-10-14 | 2018-08-28 | 广西壮族自治区烟草公司百色市公司 | With the method for the direct evoking tobacco leaf regeneration plant of spermine |
| 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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| GB9703628D0 (en) * | 1997-02-21 | 1997-04-09 | Abdelrahman Layla Z | Sugar cane production |
| US6995016B2 (en) * | 2000-08-17 | 2006-02-07 | Her Majesty The Queen In Right Of Canada, As Represented By The Minister Of Agriculture And Agri-Food | Process for inducing direct somatic embryogenesis in immature scutella cells of pooideae, and rapidly regenerating fertile plants |
| CN101803574B (en) * | 2010-04-23 | 2012-02-01 | 广州甘蔗糖业研究所 | Detoxication and tissue culture rapid propagation method of chewing cane axillary buds |
| CN101904301B (en) * | 2010-07-02 | 2011-07-20 | 广西壮族自治区甘蔗研究所 | Method for inducing embryoids of sugarcane by using lingfasu |
| CN103155872B (en) * | 2013-04-12 | 2014-01-08 | 湛江师范学院 | Eucalyptus urophylla*grandis embryoid induction seedling raising method |
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