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The Establishment of an Agrobacterium-Mediated Transformation Platform for the Non-Embryogenic Calli of Vitis vinifera L
Year:
2011
Source of publication :
Agricultural Sciences in China
Authors :
Perl, Avi
;
.
Volume :
10
Co-Authors:
Zhao, F.-X., College of Food Science and Nutrition Engineering, China Agricultural University, Beijing 100083, China
Chen, S.-W., College of Food Science and Nutrition Engineering, China Agricultural University, Beijing 100083, China
Perl, A., Department of Fruit Tree Sciences, Volcani Center, Bet Dagan 50250, Israel
Dai, R., College of Agriculture and Biotechnology, China Agricultural University, Beijing 100193, China
Xu, H.-Y., Institute of Forestry and Fruit Tree Sciences, Beijing Academy of Agricultural Sciences, Beijing 100093, China
Ma, H.-Q., College of Agriculture and Biotechnology, China Agricultural University, Beijing 100193, China
Facilitators :
From page:
686
To page:
694
(
Total pages:
9
)
Abstract:
Non-embryogenic calli (NEC) was inevitably and heavily produced when grape embryogenic calli (EC) was induced from explants or during the subculture of EC. A stable and highly efficient NEC transformation platform is required to further sort out and verify key genes which determine/switch the identity of NEC and EC. In this research, a vector pA5 containing a chitinase signal sequence fused to gfp (green fluorescent protein) and an HDEL motive was used to target and immobilize into Agrobacterium strain EHA105 to establish a transformation platform for Vitis vinifera L. cv. Chardonnay NEC. It was determined that NEC 10 d after subculture was the best target tissue; 30 min for inoculation followed by 3 d co-cultivation with the addition of 200 μmol L -1 acetosyringone (AS) was optimized as protocol. The use of bacterial densities as 1.0 at OD 600 did not result in serious tissue hypersensitive reaction and it had higher efficiency. Kanamycin at 200 mg L -1 was picked for positive expression selection. The stable transformation of NEC was proved by reverse transcription-polymerase chain reaction techniques (RT-PCR) and fluorescent microscopy after three sub-cultures of the selected cell line. Highly efficient genetic transformation protocol of grape NEC was achieved and some of the optimized parameters were different from that reported for EC. This transformation platform could facilitate the verification of candidate somatic embryogenesis (SE) decisive genes, and the successfully transformed NEC with certain genes can also be used as bioreactors for the production of functional products, as NEC not only proliferates fast, but also keeps in a rather stable condition. © 2011 Chinese Academy of Agricultural Sciences.
Note:
Related Files :
Agrobacterium
Agrobacterium tumefaciens
Non-embryogenic calli
Rhizobium
transformation
Vitaceae
Vitis vinifera L.
Show More
Related Content
More details
DOI :
10.1016/S1671-2927(11)60051-5
Article number:
Affiliations:
Database:
Scopus
Publication Type:
article
;
.
Language:
English
Editors' remarks:
ID:
27465
Last updated date:
02/03/2022 17:27
Creation date:
17/04/2018 00:31
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Scientific Publication
The Establishment of an Agrobacterium-Mediated Transformation Platform for the Non-Embryogenic Calli of Vitis vinifera L
10
Zhao, F.-X., College of Food Science and Nutrition Engineering, China Agricultural University, Beijing 100083, China
Chen, S.-W., College of Food Science and Nutrition Engineering, China Agricultural University, Beijing 100083, China
Perl, A., Department of Fruit Tree Sciences, Volcani Center, Bet Dagan 50250, Israel
Dai, R., College of Agriculture and Biotechnology, China Agricultural University, Beijing 100193, China
Xu, H.-Y., Institute of Forestry and Fruit Tree Sciences, Beijing Academy of Agricultural Sciences, Beijing 100093, China
Ma, H.-Q., College of Agriculture and Biotechnology, China Agricultural University, Beijing 100193, China
The Establishment of an Agrobacterium-Mediated Transformation Platform for the Non-Embryogenic Calli of Vitis vinifera L
Non-embryogenic calli (NEC) was inevitably and heavily produced when grape embryogenic calli (EC) was induced from explants or during the subculture of EC. A stable and highly efficient NEC transformation platform is required to further sort out and verify key genes which determine/switch the identity of NEC and EC. In this research, a vector pA5 containing a chitinase signal sequence fused to gfp (green fluorescent protein) and an HDEL motive was used to target and immobilize into Agrobacterium strain EHA105 to establish a transformation platform for Vitis vinifera L. cv. Chardonnay NEC. It was determined that NEC 10 d after subculture was the best target tissue; 30 min for inoculation followed by 3 d co-cultivation with the addition of 200 μmol L -1 acetosyringone (AS) was optimized as protocol. The use of bacterial densities as 1.0 at OD 600 did not result in serious tissue hypersensitive reaction and it had higher efficiency. Kanamycin at 200 mg L -1 was picked for positive expression selection. The stable transformation of NEC was proved by reverse transcription-polymerase chain reaction techniques (RT-PCR) and fluorescent microscopy after three sub-cultures of the selected cell line. Highly efficient genetic transformation protocol of grape NEC was achieved and some of the optimized parameters were different from that reported for EC. This transformation platform could facilitate the verification of candidate somatic embryogenesis (SE) decisive genes, and the successfully transformed NEC with certain genes can also be used as bioreactors for the production of functional products, as NEC not only proliferates fast, but also keeps in a rather stable condition. © 2011 Chinese Academy of Agricultural Sciences.
Scientific Publication
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