Transformation of broccoli (Brassica oleracea var. italica) with isopentenyltransferase gene via Agrobacterium tumefaciens for post-harvest yellowing retardation.
Chen, L. F. O., Hwang J.Y., Charng Y.Y., Sun C.W. and Yang S.F.
Molecular Breeding, Volume 7, Number 3, 2001. pp. 243-257
2001
บทคัดย่อ
Transgenic plantlets with a retarding effect on postharvest yellowing in broccoli cv. Green King have been generated via Agrobacterium tumefaciens (strain LBA4404)-mediated transformation of cytokinin synthesizing ipt (isopentenyltransferase) gene. The ipt gene is constructed under the control of senescence-associated gene promoters from Arabidopsis thaliana in the forms of pSG529(+) and pSG766A. Evidence of transgene integration was confirmed by assays on neomycin phosphotransferase II [kanamycin kinase] (NPTII) activity of selection markers, polymerase chain reaction (PCR) and Southern hybridization. Based on the chlorophyll retention rate (>50%) after 4 days of postharvest storage at 25 deg 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. Reverse transcriptase-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 optimum 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 preculture was established for explants of hypocotyl, cotyledon, and peduncle. Most of the ipt transformants with a retarding effect on yellowing had a chimaeric 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 postharvest yellowing in broccoli should be feasible.