CN104351058A - Method for inducing sugarcane embryoids by spermine - Google Patents
Method for inducing sugarcane embryoids by spermine Download PDFInfo
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- CN104351058A CN104351058A CN201410679435.3A CN201410679435A CN104351058A CN 104351058 A CN104351058 A CN 104351058A CN 201410679435 A CN201410679435 A CN 201410679435A CN 104351058 A CN104351058 A CN 104351058A
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Abstract
The invention relates to a method for inducing sugarcane embryoids by spermine. The method takes a young sugarcane leaf section as an explant, and comprises the steps of enabling the young sugarcane leaf section to be inoculated into a solid medium of 2.5mg/L of MS spermine (Spm.) with the pH value of 5.8-6.0, culturing for 20 days and inducing to obtain sugarcane callus agglomerates; under the condition of no change, continuously culturing for 15 days and 20 days to successively obtain embryoids with various shapes; or transferring the agglomerates which are cultured for 20 days and are induced to have callus into a fresh original medium, and culturing for 10 days and 15 days to obtain a great deal of embryoids. The method is high in induction speed, high in efficiency and good in quality when being used for inducing the sugarcane embryoids; the obtained sugarcane embryoids easily generate organs such as roots and leaves by continuous redifferentiation, and complete regeneration plants can be obtained; the total effect of the method is superior to the traditional technology for inducing the sugarcane embryoids by 2, 4-D and the new technology for inducing the sugarcane embryoids by LFS; the method is lower in production cost.
Description
Technical field
The invention belongs to the technical field of tissue culture of Plant Biotechnology, especially a kind of method of spermine inducing embryoids of sugarcane.
Technical background
" embryoid " be by the part parenchyma cell in callus without sexual reproduction, be directly differentiated to form the structure of the function with embryo again, be also called " somatic embryo " or " body embryo ", to be different from " zygotic embryo "." embryoid " can be directly used in manufacture of intraocular seed and produce regeneration plant (test-tube plantlet) in production; In theoretical research, be explore Cell Differentiation, Apparatuses formation, ontogeny, and the important materials of relevant anatomy and physiological acoustic signals process.Therefore, how obtaining embryoid is one of hot technology field of plant educational circles care.Research for embryoids of sugarcane has some reports, but derivant is all use 2,4-D (2,4-dichlorphenoxyacetic acid), after acquisition callus, then transfer to low concentration 2,4-D, not even containing 2, in the medium of 4-D, to reduce the failure that the murder by poisoning of 2,4-D to culture materials causes embryoid induction.Even so, inductivity also only have about 10% [there is [J] in the embryoid in sugarcane Somatic Cell Culture. plant physiology journal Zeng Jishu 1979,5 (4): 411-416]; [there is [J] GUIHAIA Li Chun precious jade etc. in the somatic embryo in In The Young Leaf of Sugarcane segment cultivation, 1989,9 (3): 243-246].Somebody is composite 6-BA (N again
6-benzyladenine), NAA (methyl α-naphthyl acetate) or milk protein, inductivity is brought up to impact [J] Jilin Normal University journal (natural science edition) Sun Ying 2004,8 (3): 93-94 that 30%-50%[hormone occurs sugarcane lobus cardiacus embryoid].Li Song and Xu Hongyuan etc. once used LFS (spirit hair element, code name PGR-08) that the inductivity of embryoids of sugarcane is brought up to more than 90% [a kind of method Chinese patent ZL201010216154.6 of spirit hair element inducing embryoids of sugarcane].Regrettably, spirit hair element does not have manufacturer production at home, and import price is very expensive, causes application to be extremely restricted.Spermine (Spermine; Spm.) be the one of polyamines; be prevalent in higher plant circle; that [polyamines is instrumentality [J] Plant Physiology Communications of growth and development of plants for the plant growth regulating substance of pure natural; Pan Rui vehement 1985 (6): 63-68], and cheap, be easy to apply in scientific research and large-scale production.People to its Promoting plant growth, delay senility, strengthen anti-adversity ability, wait the research of physiologically active, had many reports.But, also fail to retrieve the research data of spermine for inducing embryoids of sugarcane.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of method of spermine inducing embryoids of sugarcane.
The present invention solves the problems of the technologies described above with following technical scheme:
A method for spermine inducing embryoids of sugarcane, comprises the steps:
Explant is made in the In The Young Leaf of Sugarcane section obtained in conventional manner, after routine disinfection, is inoculated into MS+ spermine (Spm.) 2.5mgL
-1(this concentration be preferably after suitable concentration), the solid culture medium of pH 5.8-6.0, cultivates 20d and induces and obtain sugarcane callus agglomerate under conventional culture conditions.
By above-mentioned callus agglomerate when not doing any variation, then continuing to cultivate 15d and 20d, amounting to incubation time and reaching 35d and 40d respectively.The embryoid come in every shape is had to occur successively.
Or cultivated 20d and the agglomerate inducing callus carefully transfers to fresh former medium by above-mentioned, avoid hurt culture, every bottle of 4-5 rolls into a ball culture, often criticizes test and is no less than 6 bottles as far as possible.Under conventional culture conditions, cultivate 10d and 15d again, amount to incubation time and reach 30d and 35d respectively.Namely a large amount of embryoid is had to occur.The generation speed of embryoid is faster, and sum is more.
Outstanding advantages of the present invention is:
The present invention is used for the induction of embryoids of sugarcane, and speed is fast, and efficiency is high, quality better.Gained embryoid easily continues to differentiate root, foliage organ and complete regenerated plant again, and general effect had both been better than the conventional art with 2,4-D inducing embryoids of sugarcane, and be also better than the new technology with LFS inducing embryoids of sugarcane, production cost is lower.
Embodiment
Below by way of case study on implementation, technical scheme of the present invention is described further.
The method of spermine inducing embryoids of sugarcane of the present invention comprises the following steps:
Conventionally method strips sugarcane tip yolk yellow spire close to the inner portion, cuts into the section of about 0.5cm × 0.5cm, is inoculated into containing Spm.2.5mgL
-11(this concentration be preferably after suitable concentration) modified form MS solid culture medium in, often criticize test and be no less than 10 bottles, every bottle of 4-5 sheet.Under conventional culture conditions, cultivate 20d induce and obtain sugarcane callus agglomerate.When not doing any variation, continuing to cultivate 15d and 20d, amounting to incubation time and reaching 35d and 40d respectively.The embryoid come in every shape is had to occur successively.
Or cultivated 20d and the agglomerate inducing callus carefully transfers to fresh former medium by above-mentioned, avoid hurt culture, every bottle of 4-5 rolls into a ball culture, often criticizes test and is no less than 6 bottles as far as possible.Under conventional culture conditions, cultivate 10d and 15d again, amount to incubation time and reach 30d and 35d respectively, namely have a large amount of embryoid to occur, the generation speed of embryoid is faster, and efficiency is higher, and cost is lower.
In subordinate list, routine 1-4 is at MS+Spm.2.5mgL
-1on medium, different incubation time cultivation program different from 2 kinds, to the result of inducing embryoids of sugarcane.Each case explant material used, medium and condition of culture are all identical.
Table 1. case study on implementation of spermine inducing embryoids of sugarcane
Note: in subordinate list, Spm. is that the present invention innovates embryoids of sugarcane derivant spermine.
Callus group's number (no matter embryoid quantity)/inoculation callus group number × 100% of embryoid incidence (%)=differentiate again embryoid is one of quality index of callus.
" bore hole can distinguish that embryoid breaks up number again " refers to terminate the same day at culture period, every bottle can be shrewd by naked eyes the embryoid overall mean turning green (showing have chloroplast to break up again), Cheng Ye and seedling, be the important quality index of embryoid.
By the contrast of example in subordinate list 1 and example 2, the contrast of example 3 and example 4 is known, and under ensureing that medium has the prerequisite of enough nutrition supply cultures, proper extension culture period, can improve incidence and the results sum of embryoid.
By the contrast of example in subordinate list 1 and example 3, the contrast of example 2 and example 4 is known, timely replacement medium, both can shorten incubation time (5d), can significantly improve again incidence and the results sum of embryoid, make production efficiency higher.
In subordinate list, listed each case study on implementation has done comparatively detailed description to technical system 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 the consumption of spermine is changed, even spermine and other growth regulatory substance carry out composite after be used further to the embryoid of inducing embryoids of sugarcane and other plant.So, allly in the cultivation program of induction sugarcane and other plant embryoid, with the addition of spermine, all belong to the scope of protection of present invention.
Claims (1)
1., by a method for spermine inducing embryoids of sugarcane, it is characterized in that the steps include:
(1) explant is made in the In The Young Leaf of Sugarcane section obtained in conventional manner, after routine disinfection, is inoculated into MS+ spermine Spm.2.5mgL
-1, pH 5.8-6.0 solid culture medium, under conventional culture conditions, cultivate 20d induce and obtain sugarcane callus agglomerate;
By above-mentioned callus agglomerate when not doing any variation, then continue to cultivate 15d and 20d, amount to incubation time and reach 35d and 40d respectively, have the embryoid that comes in every shape to occur successively;
Or cultivated 20d and the agglomerate inducing callus carefully transfers to fresh former medium by above-mentioned, avoid hurt culture, every bottle of 4-5 rolls into a ball culture, often criticizes test and is no less than 6 bottles as far as possible; Under conventional culture conditions, cultivate 10d and 15d again, amount to incubation time and reach 30d and 35d respectively; Namely a large amount of embryoid is had to occur; The generation speed of embryoid is faster, and sum is more.
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| CN201410679435.3A CN104351058A (en) | 2014-11-24 | 2014-11-24 | Method for inducing sugarcane embryoids by spermine |
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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 |
| CN106613944A (en) * | 2016-10-14 | 2017-05-10 | 广西壮族自治区烟草公司百色市公司 | Method of using spermine to induce tobacco embryoid |
| CN114027197A (en) * | 2021-12-09 | 2022-02-11 | 广西大学 | Application of passion fruit tissue culture medium |
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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 |
| CN106613944A (en) * | 2016-10-14 | 2017-05-10 | 广西壮族自治区烟草公司百色市公司 | Method of using spermine to induce tobacco embryoid |
| CN106613944B (en) * | 2016-10-14 | 2018-12-11 | 广西壮族自治区烟草公司百色市公司 | With the method for spermine evoking tobacco embryoid |
| 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 |
| CN114027197A (en) * | 2021-12-09 | 2022-02-11 | 广西大学 | Application of passion fruit tissue culture medium |
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