CN101707981A - Rubber tree cotyledon embryo high-efficiency embryonic callus induction and regeneration method - Google Patents
Rubber tree cotyledon embryo high-efficiency embryonic callus induction and regeneration method Download PDFInfo
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- CN101707981A CN101707981A CN200910226023A CN200910226023A CN101707981A CN 101707981 A CN101707981 A CN 101707981A CN 200910226023 A CN200910226023 A CN 200910226023A CN 200910226023 A CN200910226023 A CN 200910226023A CN 101707981 A CN101707981 A CN 101707981A
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Abstract
The invention relates to a rubber tree cotyledon embryo high-efficiency embryonic callus induction and regeneration method; anther or inner integument dedifferentiation callus is chosen to generate cotyledon embryo which is used as an explant through embryonic induction, and then the explant is sliced, and then the slice is planted to an embryonic callus induction culture medium to carry out dark culture, so as to obtain the dedifferentiation callus; the dedifferentiation callus is connected to the embryonic induction culture medium to carry out dark culture, so as to generate the cotyledon embryo; the cotyledon embryo is connected on an own-rooted tree culture medium to carry out light culture. In the invention, on the basis of the tissue culture of the anther or the inner integument of the rubber tree, the high-efficiency embryonic callus of the cotyledon embryo is utilized to induce embryo generation, so as to greatly improving the efficiency of the cotyledon embryo; in addition, no limit of natural conditions such as flowering phase, fruit period and weather exists, and the method has the characteristics of simple process, low production cost, high cotyledon embryo reproduction rate and short tissue culture period, thereby having high application value and opening up new technical line for the building of a rubber conversion system.
Description
Technical field
The present invention relates to a kind of method for plant tissue culture, be specifically related to that the efficient embryo callus subculture of a kind of bamboo grows somatic embryo is induced and the method for regenerate embryo plant.
Background technology
Para rubber tree (Hevea brasiliensis) belongs to the Hevea Euphorbiaceae, is the extensively important economic crops of plantation of hot-zone, and is good because of its latex output height, quality, be easy to obtain and have business development and be worth, and becomes the main source of natural rubber in the world.Because Para rubber tree is perennial arbor, the conventional breeding cycle is long, and the seed selection kind is very restricted.In order to widen Para rubber tree breeding approach, improve its quality, from eighties of last century since the fifties, rubber breeding scholars promptly take up to study its tissue culture technique, particularly eighties of last century is since the seventies, China, Malaysia, France, India, Indonesia, states such as Sri Lanka drop into great amount of manpower, material resources, financial resources are carried out extensive studies to this technology, at present in anther culture, ovule and the ovary of not pollinating cultivated, stem apex and tender stem are cultivated, suspension cell culture, protoplast is cultivated, aspects such as clonal expansion of somatic cell plant and genetic transformation have all obtained certain progress.
It is to study in the Para rubber tree tissue culture early and more direction that flower pesticide and inner integument are cultivated, its basic process is: 1) the allocation rubber inner integument that is in the male flower anther in monokaryon late period or cuts young tender seeds of rubber tree carries out dedifferentiation and cultivates about 30～60 days; 2) change the dedifferentiation callus that forms over to rubber embryonal induction medium, carry out the embryonal induction incubation, about 3～4 months; 3) after the embryonal induction process, can form embryo callus subculture from part dedifferentiation callus, embryo callus subculture can progressively form bar-shaped, heart-shaped, torpedo-shape embryo (above is mature embryo) etc. through further growth, and these all are embryonal connective tissues; 4) mature embryo is changed over to successively bud induce with the root induction medium in carry out the cultivation of bud and root, change natural environment again over to behind the suitable height and take exercise and field planting when long.
So far, existing nearly thousand mu of the field production area of China's flower pesticide and inner integument plant, compare with traditional budling, flower pesticide and inner integument plant plant have the characteristics of fast growth, dried glue output height, strong stress resistance, and may become main planting material future.At present, the subject matter that exists during flower pesticide and inner integument plant are cultivated is: explant is drawn materials and bloomed and the restriction of time as a result by rubber, the dedifferentiation callus becomes the embryo rate low and be difficult to long-term subculture and preserve, thereby plant regeneration frequency is low, the production cost height, the later stage also be difficult to satisfy produce by the little propagating technology of body embryo plants stems section in to the wilderness demand of tissue cultivating seedling with set up the needs of Biotechnology Experiment such as efficient genetic conversion system.
Summary of the invention
The purpose of this invention is to provide the efficient embryo callus subculture of a kind of bamboo grows somatic embryo induces and renovation process, it is to select suitable cotyledonary embryos to carry out the embryo callus subculture of slicing treatment inducement efficient, induce a large amount of cotyledonary embryos again by embryo callus subculture, and the process of Cheng Miao, this method can a large amount of long-term subculture propagation be preserved good embryo material, improved the tissue culture efficient of bamboo grows greatly, for producing and test the good regenerating system method that provides.
The technical solution adopted in the present invention:
A kind of rubber tree cotyledon embryo high-efficiency embryonic callus is induced and renovation process, comprises that choosing with induce process, the efficient embryo callus subculture of processing procedure, the efficient embryo callus subculture of somatic embryo of embryonal connective tissue induce choosing of embryogenetic process, mature embryo and induce into the seedling process.
1, choosing and processing procedure of embryonal connective tissue: on the basis of bamboo grows flower pesticide or inner integument tissue culture, selection flower pesticide or inner integument dedifferentiation callus are fresh, healthy through the generation of embryonal induction process, the above cotyledonary embryos of long 1cm is an explant, carry out slicing treatment, be cut into the grain of rice shape section of wide 2～3mm, long 3～5mm.The quality influence callus of induce efficient of section, it is too small cut into slices, is difficult to induce callus, when cutting into slices too big waste material also difficulty induce desirable callus.
2, the efficient embryo callus subculture of somatic embryo induce process: the section of grain of rice shape cotyledonary embryos is inoculated on the embryo callus subculture inducing culture secretly cultivated 20～40 days in 26～28 ℃.Wherein to go out embryo most effective for 20～30 days callus, body embryo callus is observed after cultivating 45 days on the embryonal induction medium, go out embryo callus rate and can reach 40～60%, germ extraction rate can reach 200～300%, callus amount before 20 days seldom, 40 days later callus go out embryo efficient and obviously reduce, and all should not be chosen as the callus material of embryonal induction.Described embryo callus subculture inducing culture is to be basal medium with the MS medium, and add 0.5～2ppm 2,4-D, 0.5～2ppm KT, 0～1ppm BA, 0～2ppm NAA, 0～2ppm ZT, sucrose 70～90g/L, Sucus Cocois 50ml/L, 0.1～2g/L asparagine and 0.1～0.2g/L inositol.
3, efficient embryo callus subculture is induced embryogenetic process: the dedifferentiation callus that step 2 is obtained is received on the embryonal induction medium and is secretly cultivated in 24～26 ℃.Can be observed the growth course of body embryo in 20～30 days, can form cotyledonary embryos in a large number in the time of 40～60 days.The circulation that the long inducement efficient embryo callus subculture of just can further cutting into slices when above to 1cm of these cotyledonary embryos carries out the body embryo is bred, and perhaps inserts into and induces the formation own-rooted tree in the seedling medium.Described embryonal induction medium is to be basal medium with the MS medium, wherein macroelement reduces 10～50%, trace element increases by 0.5～2 times, and adds 0.5～3ppm BA, 0.5～3ppm KT, 0～0.5ppmNAA, 0.2～1ppmGA, 0～.5ppm ABA, hydrolysis tyrosinase 15 0～300mg/l, adds 0.1～0.2g/L inositol, maltose 0～20g/L, sucrose 50～70g/L and Sucus Cocois 50ml/L.
4, the choosing and induce into the seedling process of mature embryo: choose step 3 and induce the health of generation, the above cotyledonary embryos of complete, high 1.5cm to receive to carry out light in 28～30 ℃ on the own-rooted tree medium and cultivate.About 10 days cotyledonary embryos can be extracted the main root of strong shape out, and about 20 days cotyledonary embryos can be extracted the strong shape stem section of 10cm out.Described own-rooted tree medium is to be basal medium with the MS medium, and wherein macroelement reduces 10～30%, and adds 0～2ppm BA, 0.5～2ppm KT, 0.2～1ppm NAA, 0.5～1.5ppmGA, sugar 30～50g/L and Sucus Cocois 50ml/L.
Advantage of the present invention:
1, the cotyledonary embryos inductivity is low is the subject matter that exists in the training of bamboo grows group, it also is the bottleneck that the batch production of restriction bamboo grows somatic cell plant is produced, utilize the efficient embryo callus subculture of cotyledonary embryos to induce embryo, can improve the efficient that forms cotyledonary embryos greatly, solve bamboo grows and become the low and low problem of own-rooted tree planting percent of embryo rate.The embryogenetic process of rubber, need pass through globular embryo, heart-shape embryo, torpedo embryo and cotyledonary embryos four-stage from big aspect, the inventor finds that white embryonal connective tissue beyond the cotyledonary embryos comprises that lopsided embryo also can be cut into slices and induces the callus with higher embryonal induction efficient in the experiment, but the embryo that these embryonal connective tissues induce, the embryo instability, the ratio that produces lopsided embryo is very high; Simultaneously, cotyledonary embryos is less with the white embryonal connective tissue individuality of last stage, and embryoid is difficult to section, and waste material, thereby should not be chosen as the explant sliced materials.The cotyledonary embryos section is easy, section is even, and the callus that induces goes out embryo efficient height, and does not almost have the appearance of lopsided embryo, can well preserve the ability that forms cotyledonary embryos, and the ripe laggard one-step inducing planting percent of cotyledonary embryos can reach more than 80%.
2, flower pesticide or inner integument the inoculation take a lot of work, expensive, also be subjected to the very big restriction of natural conditions such as florescence, fruit phase and weather, utilize cotyledonary embryos section callus induction to breed and can remedy above deficiency, long preservation health can utilization group training material, improve experiment and production efficiency.
3, traditional bamboo grows tissue culture was inoculated into the general needs of the cotyledonary embryos that induces maturation 150 days from flower pesticide or inner integument, and the present invention only needed 100 days just can obtain ripe cotyledonary embryos, had shortened the one-tenth embryo time greatly, had improved tissue culture efficient.
4, embryonal connective tissue success shoot proliferation provides new technology path for the rubber transformation technology.Since utilizing particle bombardment rifle bombardment paragutta flower pesticide dedifferentiation callus to obtain the first strain transgenosis rubber plant from Arokiaraj in 1994 etc., utilized flower pesticide or inner integument dedifferentiation callus to change into the basic fundamental route that paragutta transforms.This class transforms route and realizes that conversion process is long, only become embryo just to need 3～4 months from the dedifferentiation callus of induce, particularly antibacterial, a large amount of antibiotic of the medium-term and long-term use of screening process of Agrobacterium-mediated Transformation, inducing of the growth of callus, embryonal connective tissue etc. produced bigger harmful effect, and these factors have caused the callus after transforming to become at the bottom of the embryo rate utmost point.Yet, the efficient embryo callus subculture germ extraction rate of cotyledonary embryos height, preliminary Agrobacterium-mediated Transformation experimental results show that its anti-transformation is strong, still can comparatively fast become embryo after the conversion.Therefore, started for the foundation of rubber transformation system and opened up new technology path.
On the basis of training of bamboo grows flower pesticide group or the training of inner integument group, technological process of the present invention is simple, and production cost is low, cotyledonary embryos rate of increase height, and the tissue culture cycle is short, has very big using value.
Description of drawings
Fig. 1 is callus of induce fate of the present invention and the graph of a relation that goes out embryo callus number.
Fig. 2 is the graph of a relation of callus of induce fate of the present invention and germ extraction rate.
Embodiment
The invention will be further described below in conjunction with embodiment.
Embodiment
1, on the basis of bamboo grows flower pesticide or inner integument tissue culture, selection flower pesticide or inner integument dedifferentiation callus are fresh, healthy through the generation in 3 months of embryonal induction process, the above cotyledonary embryos of long 1～3cm is an explant, carry out slicing treatment on superclean bench, first rip cutting crosscut again becomes the little section of grain of rice shape of wide 2～3mm, long 3～5mm.
2, cotyledonary embryos section is inoculated on the embryo callus subculture inducing culture cultivates, with the culture dish of 9cm, each culture dish connects 10～15 sections, and section is evenly placed, and culturing room's temperature remains on 26～28 ℃, the dark cultivation.Experiment shows: the callus of induce fate with go out embryo callus number and germ extraction rate linear (seeing Fig. 1, Fig. 2), it is higher that wherein 20～30 days callus goes out embryo efficient, should be chosen as the callus material that induces embryo, section callus amount before 20 days seldom, 40 days later callus go out the obvious reduction of embryo efficient and all should not be chosen as the callus material that induces embryo.
3, the dedifferentiation callus that will cultivate 25 days on the efficient embryo callus subculture inducing culture of somatic embryo is received on the efficient embryonal induction medium and is secretly cultivated in 24～26 ℃, just can be observed the growth course of body embryo about 20 days, can form cotyledonary embryos in a large number in the time of 40～60 days, count the embryo situation in the time of 45 days, wherein going out embryo callus rate is 47.5%, and the cotyledonary embryos germ extraction rate is 221.3%.
4, choose step 3 and receive on the own-rooted tree medium through the health of body embryonal induction process generation, the cotyledonary embryos more than complete, the high 1.5cm, about 10 days cotyledonary embryos can be extracted the main root of strong shape out, and about 20 days cotyledonary embryos can be extracted the strong shape stem section of 10cm out.Culturing room's temperature remains on 28～30 ℃, and light is cultivated, illumination 12 hours every days.
Claims (2)
1. a rubber tree cotyledon embryo high-efficiency embryonic callus is induced and renovation process, it is characterized in that: comprise that choosing with induce process, the efficient embryo callus subculture of processing procedure, the efficient embryo callus subculture of somatic embryo of embryonal connective tissue induce choosing of embryogenetic process, mature embryo and induce into the seedling process;
1), choosing and processing procedure of embryonal connective tissue: on the basis of bamboo grows flower pesticide or inner integument tissue culture, selection flower pesticide or inner integument dedifferentiation callus are fresh, healthy through the generation of embryonal induction process, the above cotyledonary embryos of long 1cm is an explant, carry out slicing treatment, be cut into the grain of rice shape section of wide 2～3mm, long 3～5mm;
2), the efficient embryo callus subculture of somatic embryo induce process: the section of grain of rice shape cotyledonary embryos is inoculated on the embryo callus subculture inducing culture secretly cultivated 20～40 days in 26～28 ℃; Described embryo callus subculture inducing culture is to be basal medium with the MS medium, and add 0.5～2ppm2,4-D, 0.5～2ppm KT, 0～1ppm BA, 0～2ppm NAA, 0～2ppm ZT, sucrose 70～90g/L, Sucus Cocois 50ml/L, 0.1～2g/L asparagine and 0.1～0.2g/L inositol;
3), efficient embryo callus subculture is induced embryogenetic process: the dedifferentiation callus that step 2 is obtained is received on the embryonal induction medium and is secretly cultivated in 24～26 ℃; Described embryonal induction medium is to be basal medium with the MS medium, wherein macroelement reduces 10～50%, trace element increases by 0.5～2 times, and adds 0.5～3ppm BA, 0.5～3ppm KT, 0～0.5ppmNAA, 0.2～1ppm GA, 0～.5ppm ABA, hydrolysis tyrosinase 15 0～300mg/L, adds 0.1～0.2g/L inositol, maltose 0～20g/L, sucrose 50～70g/L and Sucus Cocois 50ml/L;
4), the choosing and induce into the seedling process of mature embryo: choose step 3 and induce the health of generation, the above cotyledonary embryos of complete, high 1.5cm to receive to carry out light in 28～30 ℃ on the own-rooted tree medium and cultivate; Described own-rooted tree medium is to be basal medium with the MS medium, and wherein macroelement reduces 10～30%, and adds 0～2ppm BA, 0.5～2ppm KT, 0.2～1ppm NAA, 0.5～1.5ppmGA, sugar 30～50g/L and Sucus Cocois 50m1/L.
2. rubber tree cotyledon embryo high-efficiency embryonic callus according to claim 1 is induced and renovation process, it is characterized in that: the long circulation of inducing the generation embryo callus subculture to carry out the body embryo of can cutting into slices again when above to 1cm of the cotyledonary embryos of step 3 gained is bred, and perhaps inserts into and induces the formation own-rooted tree in the seedling medium.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107517851A (en) * | 2017-09-08 | 2017-12-29 | 海南大学 | The Cord blood culture medium and its Cryopreservation of gum tree embryonic callus |
| CN112088774A (en) * | 2020-10-28 | 2020-12-18 | 中国热带农业科学院橡胶研究所 | Method for inducing polyploid plant |
| CN115500261A (en) * | 2022-09-20 | 2022-12-23 | 中国热带农业科学院橡胶研究所 | High-efficiency generation method and application of rubber tree secondary embryos |
| CN117178897A (en) * | 2023-10-26 | 2023-12-08 | 中国热带农业科学院橡胶研究所 | Method for rejuvenating mature somatic embryos of rubber trees and regenerating plants |
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2009
- 2009-11-13 CN CN200910226023A patent/CN101707981A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107517851A (en) * | 2017-09-08 | 2017-12-29 | 海南大学 | The Cord blood culture medium and its Cryopreservation of gum tree embryonic callus |
| CN107517851B (en) * | 2017-09-08 | 2020-01-10 | 海南大学 | Low-temperature preservation culture medium for rubber tree embryonic callus and low-temperature preservation method thereof |
| CN112088774A (en) * | 2020-10-28 | 2020-12-18 | 中国热带农业科学院橡胶研究所 | Method for inducing polyploid plant |
| CN115500261A (en) * | 2022-09-20 | 2022-12-23 | 中国热带农业科学院橡胶研究所 | High-efficiency generation method and application of rubber tree secondary embryos |
| CN117178897A (en) * | 2023-10-26 | 2023-12-08 | 中国热带农业科学院橡胶研究所 | Method for rejuvenating mature somatic embryos of rubber trees and regenerating plants |
| CN117178897B (en) * | 2023-10-26 | 2024-05-07 | 中国热带农业科学院橡胶研究所 | Method for rejuvenating mature somatic embryos of rubber trees and regenerating plants |
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Open date: 20100519 |