TY - JOUR
T1 - Transformation of broccoli (Brassica oleracea var. italica) with isopentenyltransferase gene via Agrobacterium tumefaciens for post-harvest yellowing retardation
AU - Chen, Long Fang O.
AU - Hwang, Jia Yuan
AU - Charng, Yee Yung
AU - Sun, Chi Wen
AU - Yang, Shang Fa
N1 - Funding Information:
This work was supported by research grants from the National Science Council (NSC-85-2321-B-001-019-A18, NSC-86-2311-B001-030-A18, NSC-87-2311-B001-003-A18) and Academia Sinica to L.-F.O.C. We thank Dr R. A. Amasino (University of Wisconsin, Madison) for supplying the pSG766A and pSG529(+) plasmids and Dr Yu-Ming Chang (Taiwan Agricultural Research Institute, Wu-Feng, Taichung, Taiwan) for providing broccoli seeds and valuable suggestions. Gratitude is also extended to Ms H.G. Lee, Ms M.H. Chen, and Mr C.S. Tou for assistance in transformant screening, transplanting, and plantlet maintenance. We are also grateful to Dr S.C. Brown (ISV, CNRS, Gif-sur-Yvette, France) and Dr C. Bergounioux (IBP, University of Paris Sud, Orsay, France) for sharing their knowledge of flow cytometric analysis.
PY - 2001
Y1 - 2001
N2 - Transgenic plantlets with a retarding effect on post-harvest yellowing in broccoli have been generated via Agrobacterium tumefaciens-mediated transformation of cytokinin synthesizing ipt (isopentenyltransferase) gene. The ipt gene is constructed under the control of senescence-associated gene promoters from Arabidopsis in the forms of pSG529(+) and pSG766A, which were the gifts from Dr R.M. Amasino at University of Wisconsin, Madison. Evidence of transgene integration was confirmed by assays on neomycin phosphotransferase II (NPTII) activity of selection markers, PCR and Southern hybridization. Based on the chlorophyll retention rate (>50%) after 4 days of post-harvest storage at 25 °C, it was found that 31% of transformants exhibited the effect of retarding yellowing in detached leaves, with 16% having the effect on florets and 7.2% on both leaves and florets. RT-PCR revealed that ipt gene expression occurred early on the day of detachment. Factors such as vacuum aid infiltration, plasmid differences, explant types, seedling ages and kanamycin concentrations were also studied. Putative transformation frequencies tended to vary with plasmids and explant types. The advantage of vacuum aid infiltration depended on explant types. The optimal kanamycin concentration should be determined experimentally for each study to avoid the high escape rate of kanamycin selection. Flow cytometric analysis of explant nuclear DNA phases was found to be helpful for selecting suitable explants for transformation and minimizing the polyploid transformants. A reproducible transformation protocol without any pre-culture was established for explants of hypocotyl, cotyledon, and peduncle. Most of the ipt transformants with a retarding effect on yellowing had a chimeric nature but showed little or no serious morphological abnormality in comparison with their parental line. Through proper selection, transformation lines with the capability of retarding post-harvest yellowing in broccoli should be feasible.
AB - Transgenic plantlets with a retarding effect on post-harvest yellowing in broccoli have been generated via Agrobacterium tumefaciens-mediated transformation of cytokinin synthesizing ipt (isopentenyltransferase) gene. The ipt gene is constructed under the control of senescence-associated gene promoters from Arabidopsis in the forms of pSG529(+) and pSG766A, which were the gifts from Dr R.M. Amasino at University of Wisconsin, Madison. Evidence of transgene integration was confirmed by assays on neomycin phosphotransferase II (NPTII) activity of selection markers, PCR and Southern hybridization. Based on the chlorophyll retention rate (>50%) after 4 days of post-harvest storage at 25 °C, it was found that 31% of transformants exhibited the effect of retarding yellowing in detached leaves, with 16% having the effect on florets and 7.2% on both leaves and florets. RT-PCR revealed that ipt gene expression occurred early on the day of detachment. Factors such as vacuum aid infiltration, plasmid differences, explant types, seedling ages and kanamycin concentrations were also studied. Putative transformation frequencies tended to vary with plasmids and explant types. The advantage of vacuum aid infiltration depended on explant types. The optimal kanamycin concentration should be determined experimentally for each study to avoid the high escape rate of kanamycin selection. Flow cytometric analysis of explant nuclear DNA phases was found to be helpful for selecting suitable explants for transformation and minimizing the polyploid transformants. A reproducible transformation protocol without any pre-culture was established for explants of hypocotyl, cotyledon, and peduncle. Most of the ipt transformants with a retarding effect on yellowing had a chimeric nature but showed little or no serious morphological abnormality in comparison with their parental line. Through proper selection, transformation lines with the capability of retarding post-harvest yellowing in broccoli should be feasible.
KW - Agrobacteruim-mediated transformation
KW - Brassica oleracea L. var. italica
KW - Flow cytometric analysis
KW - Isopentenyltransferase gene
KW - Post-harvest senescence
KW - Senescence-associated gene promoter
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U2 - 10.1023/A:1011357320259
DO - 10.1023/A:1011357320259
M3 - Article
AN - SCOPUS:0034921528
SN - 1380-3743
VL - 7
SP - 243
EP - 257
JO - Molecular Breeding
JF - Molecular Breeding
IS - 3
ER -