CN101775408A - Efficient genetic transformation method for embryogenic callus of cotyledon embryo of rubber tree - Google Patents
Efficient genetic transformation method for embryogenic callus of cotyledon embryo of rubber tree Download PDFInfo
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Abstract
The invention belongs to the field of biotech, and relates to an efficient genetic transformation method for embryogenic callus of cotyledon embryo of rubber tree. The method comprises the following steps: conducting the tissue culture of the anther or the internal integument of rubber trees, using the cotyledon embryo as explant and carrying out induction and genetic transformation; then, inducing the transformed embryogenic callus into embryo and inducing the selected mature embryos into sprouts. The invention provides a simple-flow, extra-high efficiency, low-cost transformation method which overcomes the serious defects like low frequency of embryo production rate in rubber tree genetic transformation system and the long induction time of the embryogenic callus. Meanwhile, the invention also compensates the defect that the obtainment of transformed rubber tree acceptor materials is prone to natural conditions limitations and greatly shortens the transformation circle, thus making it a high-efficient genetic transformation method for rubber free. The invention is of great scientific as well as using value, and also provides a new technical route for the formation of transformation systems for rubber tree.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of plant genetic transformation method, being specifically related to a kind of is the rubber tree high-efficiency genetic transforming method of transformation receptor material with rubber tree somatocyte cotyledonary embryos section embryo callus.
Background technology
Para rubber tree (Hevea brasiliensis Muell.Arg) is the main source of the natural rubber of one of four big industrial raw material, is again energy-source plant simultaneously, and the chemical ingredients rubber hydrocarbon of natural rubber can be used as the substitute of fossil energy through biological modification.Rubber tree is perennial, cross-pollinatd plant, and the conventional breeding cycle is long, general 30a, and gene is narrow in the rubber seeds simultaneously, and cross-breeding is difficult to that big breakthrough is arranged.Therefore genetic engineering breeding becomes the core content of rubber tree biotechnology research, breed improvement, by the orientable improvement proterties of transgenic technology, remedies the deficiency in the conventional breeding, shortens breeding cycle greatly, saves human and material resources.The genetic transformation of rubber tree has great importance: the one, and improvement natural rubber tree performance improves the multiple proterties such as output, wind resistance, winter resistance, disease resistance of its latex, and by the transgenosis approach breeding cycle is shortened greatly.The 2nd, rubber tree has special latex dust system, can obtain a large amount of latex by rubber tapping, and rubber tree can be used as a kind of natural biological reactor, makes its expression have the exogenous protein of high value.
Arokiaraj in 1991 etc. are first by Agrobacterium inoculation rubber subcutis and obtain containing by Agrobacterium and induce the octopine that forms, the formation of tumour has comprised the transfer of plasmid, and the gene that shows some metabolite of control that plasmid carries (synthetic as opine, plant hormone) obtains expressing in tumour cell, and this is the earliest about the engineered report of rubber tree.Afterwards, applying gene marksmanship and agrobacterium-mediated transformations such as 1994-1998 Arokiaraj, all successfully change beta-glucuronidase (GUS) and neomycin phosphotransferase gene (NPTII) in the rubber (GL 1 clone), normal expression still in the 3rd generation clone of multiplication culture, show the stability of height, and this required key character that possesses of transgenic plant just, tentatively set up the genetic conversion system of rubber tree, from then on utilize the dedifferentiation callus to be transformation receptor, and utilize NPT II to change into the basic skills that Para rubber tree transforms for screening-gene, but transformation efficiency is extremely low.The Montoro of French international farming research in 2000 and centre of development etc. utilizes the frangible inner integument callus and the NPT II screening-gene of subculture, study the effect of calcium in rubber tree (PB 260 clone) Agrobacterium-mediated Transformation, but do not obtained transfer-gen plant.Li Wei state was an acceptor with flower pesticide dedifferentiation callus in 2000, and NPT II is a screening-gene, the cassava sod gene is imported rubber, and obtain transgenosis embryoid (2clone is cultivated in the sea).The Jayashree of India rubber institute in 2003 etc. are acceptor with flower pesticide dedifferentiation callus, utilize agrobacterium-mediated transformation successfully to change gus gene, sod gene, NPT II screening-gene in the rubber tree (RRII 105clone), and in transgenic progeny stably express.Montoro in 2006 etc. are by the frangible callus of rubber tree internal integument, and carry out suspension culture, and utilize agrobacterium-mediated transformation to change gus gene and NPT II screening-gene over to rubber tree (PB260clone), obtain transfer-gen plant, but have a large amount of vitrifying seedlings to produce in the conversion process, relevant tissue culture technique is imperfection also.Transformation experiment is carried out with particle gun bombardment rubber flower pesticide dedifferentiation callus in Chen Xiong front yards in 2006 etc., gus gene, NPT II gene, GAI are downgraded gene import Para rubber tree (2clone is cultivated in the sea), the method that particle gun is transformed the rubber tree callus is explored, and determine that tentatively the GAI gene has been incorporated in the rubber genome, but after the rubber callus bombards through particle gun, brownization degree is more serious, and embryoid regeneration rate and seedling rate are all lower.Zhao Hui in 2007 etc. obtain a large amount of frangible embryo callus by the rubber anther culture, and carry out long-term subculture propagation and plant regeneration system is studied, and this regeneration system is applied to Agrobacterium-mediated Transformation, by agrobacterium-mediated transformation with freeze proof regulatory gene CBF of the little Arabidopis thaliana in Xinjiang and downstream functional gene COR15a thereof, and NPT II gene changes in the rubber tissue, obtains a large amount of transgenosis embryoids (heat is ground 7-33-97clone).
A large amount of rubber-based prove that because of transformation experiment transformation receptor and corresponding regeneration system are to transform successful key factor.In the transformation tissue culture system of rubber tree, it is to study in the Para rubber tree tissue culture early and more direction that flower pesticide and inner integument are cultivated, its primary 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 substratum, carry out the embryonal induction culturing process, 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 substratum in carry out the cultivation of bud and root, change physical 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 heavy demand of tissue cultured seedling with set up the needs of Biotechnology Experiment such as efficient genetic conversion system.
Zhao Hui in 2007 etc. have invented a kind of rubber tree friable embryogenic callus induction and shoot proliferation method, and be applied to rubber tree gene transformation technology research, remedied the draw materials shortcoming of restriction such as the natural condition that are subjected to season of rubber flower pesticide and inner integument group training explant, for the rubber transformation technology provides new technological line, also provide a large amount of good materials for setting up the cultivation of rubber flower pesticide suspension culture and rubber protoplastis.
Zhao Hui in 2008 etc. have invented the efficient embryo callus subculture of rubber tree somatocyte cotyledonary embryos and have induced and renovation process, and utilize this regeneration system to carry out gene transformation technology research, and experiment shows that this regeneration system is the efficient transformation tissue culture system of suitable gene transformation.Embryo callus has the ovum characteristic, and the ability of accepting foreign gene is strong, and its inductive embryoid is that unicellular origin mosaic can not occur, is ideal gene transformation receptor system.
Summary of the invention
The purpose of this invention is to provide a kind of embryogenic callus of cotyledon embryo of rubber tree high-efficiency genetic transforming method, it is to utilize rubber tree flower pesticide or inner integument tissue culture, rubber tree cotyledon embryo high-efficiency embryonic callus is induced and renovation process, and success improves the method for rubber tree genetic transformation efficiency greatly in conjunction with particle gun and agrobacterium-mediated transformation.
The technical solution adopted in the present invention:
A kind of embryogenic callus of cotyledon embryo of rubber tree high-efficiency genetic transforming method comprises: embryonal connective tissue choose with treating processes, the efficient embryo callus of somatic embryo induce with chosen process, genetic transformation process, embryo callus subculture after transforming induces choosing of embryogenetic process, mature embryo and induces into the seedling process.
1, choosing and treating processes of embryonal connective tissue: on the basis of rubber tree 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, carries out slicing treatment.The slicing treatment of described cotyledonary embryos is the grain of rice shape section of being cut into wide 2～3mm, long 3～5mm.The quality influence callus of induce efficient of section, it is too small to cut into slices, and is difficult to induce callus, when cutting into slices too big waste material also difficulty induce the ideal callus.
2, inducing and chosen process of the efficient embryo callus of somatic embryo: grain of rice shape cotyledonary embryos section is inoculated on the embryo callus subculture inducing culture secretly cultivated 20～30 days in 26～28 ℃.Wherein to go out embryo most effective for 20～30 days callus, should be chosen as the acceptor material of conversion, body embryo callus is observed after cultivating 45 days on the embryonal induction substratum, 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 transformation receptor material.Described embryo callus subculture inducing culture is to be basic medium with the MS substratum, 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, genetic transformation process: utilizing particle gun or agrobacterium-mediated transformation, is that transformation receptor carries out genetic transformation with 20～30 days cotyledonary embryos embryo callus of dark cultivation.
4, the embryo callus subculture after the conversion is induced embryogenetic process: the cotyledonary embryos embryo callus subculture after will transforming is received on the embryonal induction substratum and secretly is cultured to the generation cotyledonary embryos in 24～26 ℃.Can be observed the growth of body embryo in 20～30 days through antibacterial or screening process, can form cotyledonary embryos in a large number in the time of 40～60 days, 60～90 can obtain many mature embryos.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 Seedling in the seedling substratum.Described embryonal induction substratum is to be basic medium with the MS substratum, 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.
5, the choosing and induce into the seedling process of mature embryo: choose the health, the cotyledonary embryos more than complete, the high 1.5cm that produce through screening process and receive on the Seedling substratum and to carry out light in 28～30 ℃ and be cultured to and extract strong shape stem section out.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 Seedling substratum is to be basic medium with the MS substratum, 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, be that transformation receptor has overcome the wretched insufficiency of dedifferentiation callus in conversion with the efficient embryo callus of somatocyte cotyledonary embryos, the effect of cotyledonary embryos embryo callus tolerance transforming factor is strong, and become the embryo rate high, solved the low problem of embryonal induction rate in the conversion process.
2, embryo callus has the ovum characteristic, and the ability of accepting foreign gene is strong, and its inductive embryoid is that unicellular origin mosaic can not occur, is ideal gene transformation receptor system.Generally induced dedifferentiation callus needs 50～60 days from flower pesticide or inner integument, inducing the friable embryogenic callus again from the dedifferentiation callus again needs 3～4 months; And can directly induce embryo callus efficiently from the section of somatocyte cotyledonary embryos, and only need 20～30 days, this has shortened the induction time of embryo callus greatly, can be very fast for gene transformation provides efficient embryo acceptor material, therefore can improve transformation efficient greatly.
3, after the cotyledonary embryos embryo callus carries out gene transformation, directly entering the embryoid induction stage carries out antibacterial or screening process, except that the influence that is subjected to fungistat or selective agent, the subculture process does not influence the formation of body embryo, can keep the efficient one-tenth embryo rate of rubber tree cotyledonary embryos high-efficiency regeneration system to greatest extent.
4, often strip flower pesticide or inner integument induce the dedifferentiation callus take a lot of work, expensive, also be subjected to the very big restriction of natural condition such as florescence, fruit phase and weather, utilize somatocyte cotyledonary embryos high-efficiency regeneration system to breed cotyledonary embryos by long-term subculture, cut the cotyledonary embryos induced embryonic callus repeatedly and carry out gene transformation, this can remedy above deficiency, improves experiment and production efficiency.
5, the resistance embryoid of getting after the conversion is used for Molecular Detection, and experiment shows that it is the transgenosis embryoid that a large amount of resistance embryoids are arranged, and has illustrated that the embryogenic callus of cotyledon embryo of rubber tree high-efficiency genetic transforming method not only has to become the embryo rate efficiently, also has high transformation efficiency.
Technical process of the present invention is simple, and cost is low, and it is all high to transform back one-tenth embryo rate and transformation efficiency, has very big scientific research and using value.
Embodiment
The invention will be further described below in conjunction with embodiment.
Embodiment
1, on the basis that the rubber tree anther tissue is cultivated, selection flower pesticide dedifferentiation callus is 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 Bechtop, 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 cotyledonary embryos 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.Wherein to go out embryo most effective for 20～30 days callus, should be chosen as the transformation receptor material, the 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 acceptor material.
3, genetic transformation process: utilizing particle gun or agrobacterium-mediated transformation, is that acceptor carries out genetic transformation with 25 days cotyledonary embryos embryo callus.Show with the contrast experiment of rubber flower pesticide dedifferentiation callus, the embryogenic callus of cotyledon embryo of rubber tree tissue is the good material of conversion preferably, transforming, antibacterial, screening, becoming each link of embryo all to show suitable obvious superiority, be embodied in: cotyledonary embryos embryo callus regenerative power and anti-brownization ability are strong, it is still very strong to induce into the embryo ability after the conversion, is subjected to the side effect of conversion processes such as antibacterial, screening little; The cotyledonary embryos that produces by screening process can further carry out slicing treatment, induces and renovation process reaches the purpose of embryonal connective tissue circulation propagation by rubber tree cotyledon embryo high-efficiency embryonic callus, can obtain a large amount of transfer-gen plants like this; The arrangement of transformation is more random, and operating process does not receive time limitation greatly.At DO
6000.8～1.0,25 ℃, infect 10～15 minutes, cultivate under 2 days, antibacterial good situation and carry out Agrobacterium-mediated Transformation altogether, the embryo rate of inducing into of dedifferentiation callus is 0, yet transform with the cotyledonary embryos embryo callus, inducing into the embryo rate can reach more than 80%.
4, choose the health that produces through body embryonal induction process, the cotyledonary embryos more than complete, the high 1.5cm is received on the Seedling substratum, about 10 days cotyledonary embryos can be extracted the main root of strong shape out, 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. embryogenic callus of cotyledon embryo of rubber tree high-efficiency genetic transforming method is characterized in that: comprise embryonal connective tissue choose with treating processes, the efficient embryo callus of somatic embryo induce with chosen process, genetic transformation process, embryo callus subculture after transforming induces choosing of embryogenetic process, mature embryo and induces into the seedling process;
1), choosing and treating processes of embryonal connective tissue: on the basis of rubber tree 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, carries out slicing treatment;
2), inducing and chosen process of the efficient embryo callus of somatic embryo: cotyledonary embryos section is inoculated on the embryo callus subculture inducing culture secretly cultivated 20～30 days in 26～28 ℃; Described embryo callus subculture inducing culture is to be basic medium with the MS substratum, 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), genetic transformation process: utilizing particle gun or agrobacterium-mediated transformation, is that transformation receptor carries out genetic transformation with 20～30 days cotyledonary embryos embryo callus of dark cultivation;
4), the embryo callus subculture after the conversion is induced embryogenetic process: the cotyledonary embryos embryo callus subculture after will transforming is received on the embryonal induction substratum and secretly is cultured to the generation cotyledonary embryos in 24～26 ℃; Described embryonal induction substratum is to be basic medium with the MS substratum, 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;
5), the choosing and induce into the seedling process of mature embryo: choose the health, the cotyledonary embryos more than complete, the high 1.5cm that produce through screening process and receive on the Seedling substratum and to carry out light in 28～30 ℃ and be cultured to and extract strong shape stem section out; Described Seedling substratum is to be basic medium with the MS substratum, 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.
2. embryogenic callus of cotyledon embryo of rubber tree high-efficiency genetic transforming method according to claim 1 is characterized in that: the slicing treatment of described cotyledonary embryos is the grain of rice shape section of being cut into wide 2～3mm, long 3～5mm.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102344934A (en) * | 2010-07-23 | 2012-02-08 | 兰州大学 | Chorispora bungeana transformation method adopting suspended embryogenic callus |
| CN102893862A (en) * | 2012-07-16 | 2013-01-30 | 中国热带农业科学院橡胶研究所 | Oil palm embryonic callus induction method |
| 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 |
| CN110169358A (en) * | 2019-06-24 | 2019-08-27 | 西安同人五凤农业有限公司 | A kind of embryoid culture medium and its method for preparing honey raisin tree artificial seed |
| CN110607323A (en) * | 2019-09-24 | 2019-12-24 | 四川育良生物科技有限公司 | Agrobacterium tumefaciens-mediated rice genetic transformation method |
| 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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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102344934A (en) * | 2010-07-23 | 2012-02-08 | 兰州大学 | Chorispora bungeana transformation method adopting suspended embryogenic callus |
| CN102893862A (en) * | 2012-07-16 | 2013-01-30 | 中国热带农业科学院橡胶研究所 | Oil palm embryonic callus induction method |
| CN102893862B (en) * | 2012-07-16 | 2013-12-18 | 中国热带农业科学院橡胶研究所 | Oil palm embryonic callus induction method |
| 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 |
| CN110169358A (en) * | 2019-06-24 | 2019-08-27 | 西安同人五凤农业有限公司 | A kind of embryoid culture medium and its method for preparing honey raisin tree artificial seed |
| CN110607323A (en) * | 2019-09-24 | 2019-12-24 | 四川育良生物科技有限公司 | Agrobacterium tumefaciens-mediated rice genetic transformation method |
| 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: 20100714 |