CN1308454C - Culture of indica rice tran-gene tree from agrobacterium medium - Google Patents
Culture of indica rice tran-gene tree from agrobacterium medium Download PDFInfo
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- CN1308454C CN1308454C CNB2004100133442A CN200410013344A CN1308454C CN 1308454 C CN1308454 C CN 1308454C CN B2004100133442 A CNB2004100133442 A CN B2004100133442A CN 200410013344 A CN200410013344 A CN 200410013344A CN 1308454 C CN1308454 C CN 1308454C
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Abstract
The present invention belongs to the technical field of plant transgene, which discloses a novel method for culturing transgene hsien rice plants by agrobacterium tumefaciens. A plurality of special culture mediums are screened, a genetic conversion test is carried out to explants of kinds of Minghui 63, Zhenshan 97B, zhong 419, W9864S, etc. of hsien rice with representativeness, and high-efficiency transgene plantlets are obtained. The conversion rates are respectively that Minghui 63 reaches 23.4%, Zhenshan 97B reaches 9.3%, zhong 419 reaches 8.5%, and W9864S reaches 22.2%.
Description
Technical field
The invention belongs to the plant transgenic technology field, be specifically related to utilize the method for agriculture bacillus mediated cultivation long-grained nonglutinous rice transfer-gen plant.The invention still further relates to a kind of long-grained nonglutinous rice callus subculture medium of improvement and a kind of application of division culture medium in the transgenosis long-grained nonglutinous rice of improvement, and the method for programming of the long-grained nonglutinous rice gene transformation of the highly effective agrobacterium mediating of setting up.
Background technology
Agriculture bacillus mediated gene transformation method with its transformation efficiency height, insert copy number few, to advantages such as the plant and instrument dependency are little and extremely plant gene transformation person's favor, thereby become the most successful and the most effective plant gene method for transformation of present application.According to statistics, in the transgenic plant of success, have more than 80% to obtain by this method.
Since Hiei in 1994 sets up the japonica rice gene transformation method of highly effective agrobacterium mediating, japonica rice and java rice have been obtained very ten-strike (Abedinia etc. (1997) An efficienttransformation system for the Australia rice cultiva Jarrah by agriculture bacillus mediated gene transformation research and application, Aust J Plant Physiol, 24:133-141; Balconi etc. (1998) Agrobacterium tumefaciens-mediated transformation of rice (oryza sativaL ssp.Japonica) Italian cultiva.1.Interaction among Agrobacterium strains and rice genotypesin embryogenic callus of somatic and gametic origin, J Genet ﹠amp; Breed, 52:313-323; Cheng etc. (1998) Agrobacterium-transformed rice plants expressing synthetic cryIA (b) and cryIA (c) genes are highly toxic to striped stem borer and yellow stem borer, Proc Natl Acad Sci USA, 95:2767-2772; Dong etc. (1996) Agrobacterium-mediated transformation of japonica rice, Mol.Breeding, 2:267-276; Hiei etc. (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA, Plant J, 6:271-282; Or the like).But so far, long-grained nonglutinous rice is still the difficult species of something lost of gene transformation, successful report is (Aldemita and Hodges (1996) Agrobacterium tumefaciens-mediated transformation of japonica and indicarice varieties seldom, Planta, 199:612-617; Khanna and Raina (1999) Agrobacterium-mediatedtransformation of indica rice cultivars using binary and superbinary vectors, Aust J PlantPhysiol, 26:311-324; Rashid etc. (1996) Transgenic plant production mediated byAgrobacterium in indica rice, Plant Cell Rep 15:727-730), and transformation efficiency low (＜5%), is difficult to satisfy the needs of real work.The major cause that causes this difficult situation is also really not set up the tissue culture method of a suitable long-grained nonglutinous rice gene transformation at present.Some is different for tissue culture procedures in gene transformation (the particularly agriculture bacillus mediated gene transformation) method and general tissue culture procedures, the former relates to the problem of differentiation of calli behind the screening subculture of callus of long period and the long-time subculture, and this is the place of long-grained nonglutinous rice gene transformation difficulty just also.The method that solves must be started with from the subculture and the division culture medium that improve existing callus, and then sets up the long-grained nonglutinous rice genetic conversion system of highly effective agrobacterium mediating.
Paddy rice is one of most important food crop of China, is the staple food grain of China's 2/3 population.Long-grained nonglutinous rice is the main cultivated form of China, seems actual and more meaningful more by gene transformation improvement rice variety in China, sets up the high efficiency gene method for transformation and has bigger economic benefit and social benefit.The present invention starts with from the screening of callus subculture and regeneration culture medium, is intended to set up the method for programming of high efficiency agriculture bacillus mediated long-grained nonglutinous rice gene transformation.
Summary of the invention
The invention reside in the defective that overcomes prior art, develop the method for a kind of cultivation transgenosis long-grained nonglutinous rice plant of efficient agrobacterium mediation, study a kind of long-grained nonglutinous rice gene transformation program that is applicable to, to improve the efficient of long-grained nonglutinous rice gene transformation.The change method.
The present invention is achieved through the following technical solutions:
A kind of method of utilizing agriculture bacillus mediated cultivation transgenosis long-grained nonglutinous rice plant, it may further comprise the steps:
The first step: the inducing of callus
Mature embryo, rataria or the flower pesticide of long-grained nonglutinous rice are made explant, earlier with the described explant of 75% alcohol wash 1 minute, soaked explant 10～15 minutes with 0.15% mercuric chloride again, the sterilized water washing, the explant of bacterium of will going out then inserts inducing culture, 26 ℃ of dark down cultivations, induce callus;
Second step: the succeeding transfer culture of callus
Picking flushes from the callus that the first step obtains, the callus of surface drying changes subculture medium over to, places under 26 ℃ of dark culture condition succeeding transfer culture 20 days;
The 3rd step: the pre-cultivation of callus
With the callus behind the succeeding transfer culture be cut into about 0.2 centimetre of size in last tissue block, insert pre-culture medium, in 26 ℃ of down dark cultivations 4 days;
The 4th step: the common cultivation of callus and Agrobacterium
The fritter callus that obtains after cultivating is in advance added Agrobacterium work bacterium liquid immersion 30min, insert culture medium altogether then, in 19～20 ℃ of dark down cultivations 3 days;
The 5th step: the screening of resistant calli
Clean with sterilized water cultivating the callus that obtains altogether, add the aseptic aqueous solution that contains 400mg/L Pyocianil or cephamycin and soak 15min, blot the back with aseptic filter paper and insert screening culture medium, in 26 ℃ of dark down cultivations, per two all subcultures 1 time, screen subculture altogether 3 times, up to growing required kanamycin-resistant callus tissue;
The 6th step: the pre-differentiation of resistant calli
The resistant calli that the 5th step was obtained inserts pre-differentiation substratum, the dark cultivation 7 days under 26 ℃;
The 7th step: resistant calli regeneration plant
Resistant calli after pre-differentiation changes division culture medium over to, and 25 ℃, 2000Lux, illumination cultivation, regeneration of transgenic plant.
In the present invention, described from explant the nutrient media components of evoked callus be: MS minimum medium+2,4-D 3mg/L+ glutamine 300mg/L+ proline(Pro) 500mg/L+ sucrose 30g/L+ vegetable jelly 3g/L, pH5.9.
The component of described subculture medium is as follows: KNO
31900～2000mg/L, NH
4NO
31500～1650mg/L, KH
2PO
4150～170mg/L, MgSO
47H
2O 350～370mg/L, CaCl
22H
2O 350～400mg/L, FeSO47H
2O 35～42mg/L, Na
2EDTA 50～56mg/L, MnSO
44H
2O 80～100mg/L, ZnSO
47H
2O15～25mg/L, H
3BO
325～30mg/L, KI 6.0～8.0mg/L, CuSO
45H
2O 0.2～0.3mg/L, Na
2MoO
42H
2O 2.0～3.0mg/L, CoCl
26H
2O 0.2～0.3mg/L, glycine 2.0～3.0mg/L, V
B10.5～1.0mg/L, V
B61.0～1.5mg/L, nicotinic acid 1.0～1.5mg/L, inositol 100～150mg/L, maltose 30g/L, glutamine 300mg/L, proline(Pro) 500mg/L, 2,4-D 3.0mg/L, vegetable jelly 3g/L; PH:5.9.
The component of the pre-culture medium of described long-grained nonglutinous rice callus is as follows: KNO
31900～2000mg/L, NH
4NO
31500～1650mg/L, KH
2PO
4150～170mg/L, MgSO
47H
2O 350～370mg/L, CaCl
22H
2O350～400mg/L, FeSO47H
2O 35～42mg/L, Na
2EDTA 50～56mg/L, MnSO
44H
2O80～100mg/L, ZnSO
47H
2O 15～25mg/L, H
3BO
325～30mg/L, KI 6.0～8.0mg/L, CuSO
45H
2O0.2～0.3mg/L, Na
2MoO
42H
2O 2.0～3.0mg/L, CoCl
26H
2O 0.2～0.3mg/L, glycine 2.0～3.0mg/L, V
B10.5～1.0mg/L, V
B61.0～1.5mg/L, nicotinic acid 1.0～1.5mg/L, inositol 100～150mg/L, maltose 30g/L, glucose 1%, glutamine 300mg/L, proline(Pro) 500mg/L, 2,4-D 3.0mg/L, vegetable jelly 3g/L, Syringylethanone 200uMpH, pH5.9.
Agrobacterium is infected the suspension culture base that uses and is the AAM substratum, adds 2,4-D 3.0mg/L, Syringylethanone 200 μ M, maltose 2g/L, glucose 1g/L; PH 5.4, and the Agrobacterium work bacterial concentration of use is 0.8～1.0OD.
The common culture medium of described long-grained nonglutinous rice callus and Agrobacterium is: KNO
31900～2000mg/L, NH
4NO
31500～1650mg/L, KH
2PO
4150～170mg/L, MgSO
47H
2O 350～370mg/L, CaCl
22H
2O350～400mg/L, FeSO47H
2O 35～42mg/L, Na
2EDTA 50～56mg/L, MnSO
44H
2O80～100mg/L, ZnSO
47H
2O 15～25mg/L, H
3BO
325～30mg/L, KI 6.0～8.0mg/L, CuSO
45H
2O0.2～0.3mg/L, Na
2MoO
42H
2O 2.0～3.0mg/L, CoCl
26H
2O 0.2～0.3mg/L, glycine 2.0～3.0mg/L, V
B10.5～1.0mg/L, V
B61.0～1.5mg/L, nicotinic acid 1.0～1.5mg/L, inositol 100～150mg/L, maltose 30g/L, glutamine 300mg/L, proline(Pro) 500mg/L, 2,4-D 3.0mg/L, vegetable jelly 3g/L, Syringylethanone 200 μ M, glucose 1%, pH5.6, culture temperature is 19～20 ℃.
Described resistant calli screening culture medium is: KNO
31900～2000mg/L, NH
4NO
31500～1650mg/L, KH
2PO
4150～170mg/L, MgSO
47H
2O 350～370mg/L, CaCl
22H
2O350～400mg/L, FeSO47H
2O 35～42mg/L, Na
2EDTA 50～56mg/L, MnSO
44H
2O80～100mg/L, ZnSO
47H
2O 15～25mg/L, H
3BO
325～30mg/L, KI 6.0～8.0mg/L, CuSO
45H
2O0.2～0.3mg/L, Na
2MoO
42H
2O 2.0～3.0mg/L, CoCl
26H
2O 0.2～0.3mg/L, glycine 2.0～3.0mg/L, V
B10.5～1.0mg/L, V
B61.0～1.5mg/L, nicotinic acid 1.0～1.5mg/L, inositol 100～150mg/L, maltose 30g/L, glutamine 300mg/L, proline(Pro) 500mg/L, 2,4-D 3.0mg/L, vegetable jelly 3g/L, Pyocianil or cephamycin 250～400mg/l, Totomycin 50mg/l, pH5.9.The Pyocianil or the cephamycin concentration of subculture medium are 400mg/L for the first time, and Pyocianil that uses thereafter or cephamycin concentration are 250mg/L.
The pre-differentiation substratum of described resistant calli is: KNO
32800～3000mg/L, (NH
4)
2SO
4350～450mg/L, KH
2PO
4300～400mg/L, MgSO
47H
2O 180～200mg/L, CaCl
22H
2O170～200mg/L, FeSO47H
2O 45～55mg/L, Na
2EDTA 60～74mg/L, MnSO
44H
2O80～100mg/L, ZnSO
47H
2O 15～25mg/L, H
3BO
325～30mg/L, KI 6.0～8.0mg/L, CuSO
45H
2O0.2～0.3mg/L, Na
2MoO
42H
2O 2.0～3.0mg/L, CoCl
26H
2O 0.2～0.3mg/L, glycine 2.0～3.0mg/L, V
B10.5～1.0mg/L, V
B61.0～1.5mg/L, nicotinic acid 1.0～1.5mg/L, inositol 100～150mg/L, maltose 50g/L, glutamine 300mg/L, proline(Pro) 500mg/L, caseinhydrolysate 800mg/L, 6-benzylaminopurine 2mg/L, kinetin 2mg/L, indolylacetic acid 0.2mg/L, naphthylacetic acid 0.2mg/L, vegetable jelly 3.5g/L; Pyocianil 250mg/l, Totomycin 50mg/l, pH6.0 is in 26 times ℃ of dark cultivations 7 days.
The substratum that described plant regeneration uses is: KNO
32800～3000mg/L, (NH
4)
2SO
4350～450mg/L, KH
2PO
4300～400mg/L, MgSO
47H
2O 180～200mg/L, CaCl
22H
2O 170～200mg/L, FeSO47H
2O 45～55mg/L, Na
2EDTA 60～74mg/L, MnSO
44H
2O 80～100mg/L, ZnSO
47H
2O15～25mg/L, H
3BO
325～30mg/L, KI 6.0～8.0mg/L, CuSO
45H
2O 0.2～0.3mg/L, Na
2MoO
42H
2O 2.0～3.0mg/L, CoCl
26H
2O 0.2～0.3mg/L, glycine 2.0～3.0mg/L, V
B10.5～1.0mg/L, V
B61.0～1.5mg/L, nicotinic acid 1.0～1.5mg/L, inositol 100～150mg/L, maltose 50g/L, glutamine 300mg/L, proline(Pro) 500mg/L, caseinhydrolysate 800mg/L, 6-benzylaminopurine 2mg/L, kinetin 2mg/L, indolylacetic acid 0.2mg/L, naphthylacetic acid 0.2mg/L, vegetable jelly 3.5g/L; PH6.0.
The invention has the advantages that:
It is test materials that the present invention utilizes four rice varieties that tool is extensively representative, extensively plantation is gone up in productions [bright extensive 63 (three is to recover system), Zhenshan 97B (three is maintenance line), in 419 (three be recover be) and W9864S (two-line sterile line)], comparing with the agriculture bacillus mediated long-grained nonglutinous rice gene transformation method of having reported, and the agriculture bacillus mediated japonica rice gene transformation method of setting up with reference to this research department (woods is supported the army, Chen Hao etc.The foundation of the agriculture bacillus mediated No. 8 high-efficient transgenic systems in Mudanjiang.Acta Agronomica Sinica, 2002, on basis 28:294-300) and obtain, thereby it has than extensive applicability and high efficiency.
Description of drawings
Fig. 1: the Southern results of hybridization that is the positive transfer-gen plant of rice variety " bright extensive 63 "
Concrete embodiment
Embodiment 1: agriculture bacillus mediated long-grained nonglutinous rice gene transformation method
The first step: callus induction
Getting the mature embryo of rice variety or rataria or flower pesticide is explant, explant is sterilized according to a conventional method [earlier with 75% alcohol wash 1 minute, soak 10～15 minutes (in the soak time that not have will shorten as much as possible under the prerequisite of polluting explant) with 0.15% mercuric chloride then], wash access substratum J behind this explant with sterilized water at last
0(seeing appendix 1) in 26 ℃ of dark down cultivations, induces callus;
Second step: callus subculture
Picking flushes from second callus that obtain of step, the callus of surface drying changes J over to
3(seeing appendix 2) substratum places 26 ℃ of dark cultivations, subculture 20 days;
The 3rd step: callus is cultivated in advance
Callus behind the subculture is cut into the callus lines of about 0.2 centimetre of size, inserts substratum (J
3+ 200 μ M Syringylethanone+1% glucose), under 26 ℃, dark culture condition is pre-the cultivation 4 days down;
The 4th step: callus and Agrobacterium (the Agrobacterium strain of present embodiment is EHA105, from commercial acquisition) are cultivated altogether
Fritter callus after cultivating is in advance added Agrobacterium work bacterium liquid soak 30min, insert culture medium (J altogether
3+ 200 μ M Syringylethanone+1% glucose, pH5.6), 19～20 ℃ of dark cultivations 3 days;
The 5th step: the screening of resistant calli
The callus of cultivating is altogether cleaned with sterilized water; Add the aseptic aqueous solution that contains 400mg/L Pyocianil or cephamycin again and soak 15min; Aseptic filter paper blots callus, inserts screening culture medium (J
3+ 250～400mg/L Pyocianil or cephamycin+50mg/L Totomycin, pH5.9), 26 ℃ of dark cultivations; Per 14 days subcultures once screen subculture 3 times altogether, up to growing needed kanamycin-resistant callus tissue;
The 6th step: the pre-differentiation of resistant calli
The resistant calli that screening is obtained inserts pre-differentiation substratum [DL
3(seeing attached list 3)+250mg/L Pyocianil or cephamycin+50mg/L Totomycin], 26 ℃ of dark cultivations 7 days;
The 7th step: resistant calli regeneration plant
Resistant calli after pre-differentiation changes the not DL of added with antibiotic over to
3Division culture medium, 25 ℃, 2000Lux, illumination cultivation, regeneration of transgenic plant.
Embodiment 2: the resistant calli transformation efficiency test of agriculture bacillus mediated gene transformation method
Four rice varieties that apparatus is extensively representative, extensively plantation is gone up in productions [bright extensive 63 (three is to recover system), Zhenshan 97B (three is maintenance line), in 419 (three is that recovery is) and W9864S (two-line sterile line)] material of doing experiment, the long-grained nonglutinous rice gene transformation method of introducing by embodiment 1 carries out gene transformation, obtains resistant calli.Statistics sees Table 1.
Table 1 shows that four experimental cultivars all have higher kanamycin-resistant callus tissue rate and the positive callus rate of GUS: bright extensive 63 are respectively 45.3% and 31.3%, and W9864S is 59.0% and 40.6%, in 419 be 23.3% and 12.7%, Zhenshan 97B is 18.5% and 13.7%.Can find out also that therefrom adopt identical parameters after agriculture bacillus mediated gene transformation, the resistant calli transformation efficiency of different varieties and GUS are positive, and the callus transformation efficiency has than big-difference; The W9864S that the resistant calli transformation efficiency is the highest is 59.0%, and minimum Zhenshan 97B is 18.5%; The highest W9864S of the positive callus transformation efficiency of GUS is 40.6%, 419 is 12.7% in minimum.
The resistant calli quantity and the frequency of the different rice varieties of table 1
| Experimental cultivar | Inoculation callus number | The kanamycin-resistant callus tissue number | Kanamycin-resistant callus tissue rate (%) | The positive kanamycin-resistant callus tissue number of GUS | The positive kanamycin-resistant callus tissue rate (%) of GUS |
| 419 W9864S in bright extensive 63 Zhenshan 97Bs | 201 227 189 234 | 91 42 44 138 | 45.3％ 18.5％ 23.3％ 59.0％ | 63 31 24 95 | 31.3％ 13.7％ 12.7％ 40.6％ |
Annotate: (1) kanamycin-resistant callus tissue rate=kanamycin-resistant callus tissue number/inoculation callus number; (2) the positive kanamycin-resistant callus tissue rate of the GUS=positive kanamycin-resistant callus tissue number of GUS/inoculation callus number
Embodiment 3: the resistant calli differentiation efficiency of agriculture bacillus mediated gene transformation method and the test of total transformation efficiency
The resistant calli that screening is obtained breaks up and the differentiation culture regeneration plant through pre-.The result adds up in table 2.
The differentiation efficiency of the kanamycin-resistant callus tissue of the different rice varieties of table 2
| Experimental cultivar | The kanamycin-resistant callus tissue number | The kanamycin-resistant callus tissue number of differentiation | The kanamycin-resistant callus tissue differentiation rate | The positive independent transgenic line number of PCR | Genetic transformation efficiency |
| 419 W9864S in bright extensive 63 Zhenshan 97Bs | 91 42 44 138 | 60 35 21 82 | 65.9 83.3 47.3 59.4 | 47 21 16 52 | 23.4％ 9.3％ 8.5％ 22.2％ |
Table 2 shows: the resistant calli of four experimental cultivars all has higher differentiation rate, and the resistant calli differentiation rate of the highest Zhenshan 97B has reached 83.3%, minimum in 419 resistant calli differentiation rate also have 47.3%; All be higher than the long-grained nonglutinous rice resistant calli differentiation rate of having reported at present.And four experimental cultivars all obtain higher transformation efficiency, and bright extensive 63 transformation efficiency is 23.4%, and precious Shan 97 is 9.3%, in 419 be 8.5%, W9864S is 22.2%.Illustrate that agriculture bacillus mediated long-grained nonglutinous rice gene transformation method of the present invention is very effective.
Embodiment 4: the Southern hybridization check and analysis of transfer-gen plant
Get 3～4 μ g transfer-gen plant DNA and cut with SpeI (there is a point of contact in the T-DNA district) enzyme, the gus gene fragment is that probe is done Southern hybridization.Results of hybridization shows that the frequency that gus gene list copy is integrated is 25/33, and on average integrating copy number is 1.4.As shown in Figure 1.
Appendix:
1, J
0Medium component
(Murashige T.Skoog is revised medium for rapid growth andbioassays with tobacco cultures.Plant Physiol F.1962.A for the MS minimum medium, 15:473-493), 2,4-D 3mg/L, glutamine 300mg/L, proline(Pro) 500mg/L, sucrose 30g/L, vegetable jelly (Phytagel) 3g/L; PH5.9.
2, J
3Substratum (succeeding transfer culture of long-grained nonglutinous rice callus)
Macronutrient: KNO
31900～2000mg/L, NH
4NO
31500～1650mg/L, KH
2PO
4150～170mg/L, MgSO
47H
2O 350～370mg/L, CaCl
22H
2O 350～400mg/L
Molysite: FeSO47H
2O 35～42mg/L, Na
2EDTA 50～56mg/L
Micro-nutrient: MnSO
44H
2O 80～100mg/L, ZnSO
47H
2O 15～25mg/L, H
3BO
325～30mg/L, KI6.0～8.0mg/L, CuSO
45H
2O 0.2～0.3mg/L, Na
2MoO
42H
2O 2.0～3.0mg/L, CoCl
26H
2O0.2～0.3mg/L
Organotrophy composition: glycine 2.0～3.0mg/L, V
B10.5～1.0mg/L, V
B61.～and 1.5mg/L, nicotinic acid 1.0～1.5mg/L, inositol 100～150mg/L, maltose 30g/L
Organic additive: glutamine 300mg/L, proline(Pro) 500mg/L
Plant hormone: 2,4-D 3.0mg/L
Peptizer: vegetable jelly (Phytagel) 3g/L
pH：5.9
3, DL
3Substratum (cultivation of long-grained nonglutinous rice differentiation of calli)
A large amount of nutrition: KNO
32800～3000mg/L, (NH
4)
2SO
4350～450mg/L, KH
2PO
4300～400mg/L, MgSO
47H
2O 180～200mg/L, CaCl
22H
2O 170～200mg/L
Molysite: FeSO47H
2O 45～55mg/L, Na
2EDTA 60～74mg/L
Micro-nutrient: MnSO
44H
2O 80～100mg/L, ZnSO
47H
2O 15～25mg/L, H
3BO
325～30mg/L, KI 6.0～8.0mg/L, CuSO
45H
2O 0.2～0.3mg/L, Na
2MoO
42H
2O 2.0～3.0mg/L, CoCl
26H
2O0.2～0.3mg/L
Organotrophy composition: glycine 2.0～3.0mg/L, V
B10.5～1.0mg/L, V
B61.0～1.5mg/L, nicotinic acid 1.0～1.5mg/L, inositol 100～150mg/L, maltose 50g/L
Organic additive: glutamine 300mg/L, proline(Pro) 500mg/L, caseinhydrolysate 800mg/L
Plant hormone: 6-benzylaminopurine 2mg/L, kinetin 2mg/L, indolylacetic acid 0.2mg/L, naphthylacetic acid 0.2mg/L
Peptizer: vegetable jelly (Phytagel) 3.5g/L
pH：6.0
4, AAM substratum
AA (Toriyama K, Hinata be suspension and protoplast culture in rice.Plant Sci K.1985.Cell, 46:179-183), 2,4-D 3.0mg/L, 200 μ M Syringylethanones, maltose 2g/L, glucose 1g/L; PH 5.45, LB substratum (content in every liter of substratum)
Peptone 10 grams, yeast extract juice 5 grams, NaCl 10 grams, agar 15 grams; PH 7.0
Claims (8)
1, a kind of method of utilizing agriculture bacillus mediated cultivation transgenosis long-grained nonglutinous rice plant, it may further comprise the steps:
The first step: the inducing of callus
Mature embryo, rataria or the flower pesticide of long-grained nonglutinous rice are made explant, earlier with the described explant of 75% alcohol wash 1 minute, soaked explant 10～15 minutes with 0.15% mercuric chloride again, the sterilized water washing, the explant of bacterium of will going out then inserts inducing culture, 2 ℃ of dark down cultivations, induce callus;
Second step: the succeeding transfer culture of callus
Picking flushes from the callus that the first step obtains, the callus of surface drying changes subculture medium J3 over to, places under 26 ℃ of dark culture condition succeeding transfer culture 20 days;
The 3rd step: the pre-cultivation of callus
Callus behind the succeeding transfer culture is cut into the callus lines of about 0.2 centimetre of size, inserts pre-culture medium, in 26 ℃ of dark down cultivations 4 days;
The 4th step: the common cultivation of callus and Agrobacterium
The fritter callus that obtains after cultivating is in advance added Agrobacterium work bacterium liquid immersion 30min, insert culture medium altogether then, in 19～20 ℃ of dark down cultivations 3 days;
The 5th step: the screening of resistant calli
Clean with sterilized water cultivating the callus that obtains altogether, add the aseptic aqueous solution that contains 400mg/L Pyocianil or cephamycin and soak 15min, blot the back with aseptic filter paper and insert screening culture medium, in 26 ℃ of dark down cultivations, per two all subcultures 1 time, screen subculture altogether 3 times, up to growing required resistant calli;
The 6th step: the pre-differentiation of resistant calli
The resistant calli that the 5th step was obtained inserts pre-differentiation substratum, the dark cultivation 7 days under 26 ℃;
The 7th step: resistant calli regeneration plant
Resistant calli after pre-differentiation changes division culture medium over to, and 25 ℃, 2000Lux illumination cultivation, regeneration of transgenic plant.
2, method according to claim 1, it is characterized in that, described from explant the nutrient media components of evoked callus be: MS minimum medium+2,4-D 3mg/L+ glutamine 300mg/L+ proline(Pro) 500mg/L+ sucrose 30g/L+ vegetable jelly 3g/L, pH5.9.
3, method according to claim 1 and 2 is characterized in that, subculture medium J
3Component as follows: KNO
31900～2000mg/L, NH
4NO
31500～1650mg/L, KH
2PO
4150～170mg/L, MgSO
47H
2O 350～370mg/L, CaCl
22H
2O 350～400mg/L, FeSO
47H
2O 35～42mg/L, Na
2EDTA 50～56mg/L, MnSO
44H
2O80～100mg/L, ZnSO
47H
2O 15～25mg/L, H
3BO
325～30mg/L, KI 6.0～8.0mg/L, CuSO
45H
2O0.2～0.3mg/L, Na
2MoO
42H
2O 2.0～3.0mg/L, CoCl
26H
2O 0.2～0.3mg/L, glycine 2.0～3.0mg/L, V
B10.5～1.0mg/L, V
B61.0～1.5mg/L, nicotinic acid 1.0～1.5mg/L, inositol 100～150mg/L, maltose 30g/L, glutamine 300mg/L, proline(Pro) 500mg/L, 2,4-D 3.0mg/L, vegetable jelly 3g/L; PH:5.9.
4, method according to claim 1 is characterized in that, the component of the pre-culture medium of described long-grained nonglutinous rice callus is as follows: KNO
31900～2000mg/L, NH
4NO
31500～1650mg/L, KH
2PO
4150～170mg/L, MgSO
47H
2O350～370mg/L, CaCl
22H
2O 350～400mg/L, FeSO
47H
2O 35～42mg/L, Na
2EDTA 50～56mg/L, MnSO
44H
2O 80～100mg/L, ZnSO
47H
2O 15～25mg/L, H
3BO
325～30mg/L, KI 6.0～8.0mg/L, CuSO
45H
2O 0.2～0.3mg/L, Na
2MoO
42H
2O 2.0～3.0mg/L, CoCl
26H
2O 0.2～0.3mg/L, glycine 2.0～3.0mg/L, V
B10.5～1.0mg/L, V
B61.0～1.5mg/L, nicotinic acid 1.0～1.5mg/L, inositol 100～150mg/L, maltose 30g/L, glucose 1%, glutamine 300mg/L, proline(Pro) 500mg/L, 2,4-D 3.0mg/L, vegetable jelly 3g/L, Syringylethanone 200 μ M.
5, method according to claim 1 is characterized in that, the common culture medium of described long-grained nonglutinous rice callus and Agrobacterium is: KNO
31900～2000mg/L, NH
4NO
31500～1650mg/L, KH
2PO
4150～170mg/L, MgSO
47H
2O350～370mg/L, CaCl
22H
2O 350～400mg/L, FeSO
47H
2O 35～42mg/L, Na
2EDTA 50～56mg/L, MnSO
44H
2O 80～100mg/L, ZnSO
47H
2O 15～25mg/L, H
3BO
325～30mg/L, KI 6.0～8.0mg/L, CuSO
45H
2O 0.2～0.3mg/L, Na
2MoO
42H
2O 2.0～3.0mg/L, CoCl
26H
2O 0.2～0.3mg/L, glycine 2.0～3.0mg/L, V
B10.5～1.0mg/L, V
B61.0～1.5mg/L, nicotinic acid 1.0～1.5mg/L, inositol 100～150mg/L, maltose 30g/L, glutamine 300mg/L, proline(Pro) 500mg/L, 2,4-D 3.0mg/L, vegetable jelly 3g/L, Syringylethanone 200 μ M, glucose 1%, pH5.6.
6, method according to claim 1 is characterized in that, described resistant calli screening culture medium is: KNO
31900～2000mg/L, NH
4NO
31500～1650mg/L, KH
2PO
4150～170mg/L, MgSO
47H
2O 350～370mg/L, CaCl
22H
2O 350～400mg/L, FeSO
47H
2O 35～42mg/L, Na
2EDTA 50～56mg/L, MnSO
44H
2O80～100mg/L, ZnSO
47H
2O 15～25mg/L, H
3BO
325～30mg/L, KI 6.0～8.0mg/L, CuSO
45H
2O0.2～0.3mg/L, Na
2MoO
42H
2O 2.0～3.0mg/L, CoCl
26H
2O 0.2～0.3mg/L, glycine 2.0～3.0mg/L, V
B10.5～1.0mg/L, V
B61.0～1.5mg/L, nicotinic acid 1.0～1.5mg/L, inositol 100～150mg/L, maltose 30g/L, glutamine 300mg/L, proline(Pro) 500mg/L, 2,4-D 3.0mg/L, vegetable jelly 3g/L, Pyocianil or cephamycin 250～400mg/l, Totomycin 50mg/l, pH5.9; The Pyocianil or the cephamycin concentration of subculture medium are 400mg/L for the first time, and Pyocianil that uses thereafter or cephamycin concentration are 250mg/L.
7, method according to claim 1 is characterized in that, the pre-differentiation substratum of described resistant calli is: KNO
32800～3000mg/L, (NH
4)
2SO
4350～450mg/L, KH
2PO
4300～400mg/L, MgSO
47H
2O180～200mg/L, CaCl
22H
2O 170～200mg/L, FeSO
47H
2O 45～55mg/L, Na
2EDTA 60～74mg/L, MnSO
44H
2O 80～100mg/L, ZnSO
47H
2O 15～25mg/L, H
3BO
325～30mg/L, KI 6.0～8.0mg/L, CuSO
45H
2O 0.2～0.3mg/L, Na
2MoO
42H
2O 2.0～3.0mg/L, COCl
26H
2O 0.2～0.3mg/L, glycine 2.0～3.0mg/L, V
B10.5～1.0mg/L, V
B61.0～1.5mg/L, nicotinic acid 1.0～1.5mg/L, inositol 100～150mg/L, maltose 50g/L, glutamine 300mg/L, proline(Pro) 500mg/L, caseinhydrolysate 800mg/L, 6-benzylaminopurine 2mg/L, kinetin 2mg/L, indolylacetic acid 0.2mg/L, naphthylacetic acid 0.2mg/L, vegetable jelly 3.5g/L; Pyocianil 250mg/l, Totomycin 50mg/l, pH6.0.
8, method according to claim 1 is characterized in that the substratum that described plant regeneration uses is: KNO
32800～3000mg/L, (NH
4)
2SO
4350～450mg/L, KH
2PO
4300～400mg/L, MgSO
47H
2O 180～200mg/L, CaCl
22H
2O 170～200mg/L, FeSO
47H
2O 45～55mg/L, Na
2EDTA 60～74mg/L, MnSO
44H
2O80～100mg/L, ZnSO
47H
2O 15～25mg/L, H
3BO
325～30mg/L, KI 6.0～8.0mg/L, CuSO
45H
2O0.2～0.3mg/L, Na
2MoO
42H
2O 2.0～3.0mg/L, COCl
26H
2O 0.2～0.3mg/L, glycine 2.0～3.0mg/L, V
B10.5～1.0mg/L, V
B61.0～1.5mg/L, nicotinic acid 1.0～1.5mg/L, inositol 100～150mg/L, maltose 50g/L, glutamine 300mg/L, proline(Pro) 500mg/L, caseinhydrolysate 800mg/L, 6-benzylaminopurine 2mg/L, kinetin 2mg/L, indolylacetic acid 0.2mg/L, naphthylacetic acid 0.2mg/L, vegetable jelly 3.5g/L; PH6.0.
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| WO2002057407A2 (en) * | 2001-01-17 | 2002-07-25 | Avestha Gengraine Technologies Pvt. Ltd. | Novel method for transgenic plants by transformation & regeneration of indica rice plant shoot tips |
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| WO2002057407A2 (en) * | 2001-01-17 | 2002-07-25 | Avestha Gengraine Technologies Pvt. Ltd. | Novel method for transgenic plants by transformation & regeneration of indica rice plant shoot tips |
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