Manipulation of post-harvest physiology in broccoli through an optimised Agrobacterium rhizogenes-mediated transformation protocol
The aim of this project was to down-regulate ACC oxidase (ACO) 1 and 2 and ACC synthase (ACS) in broccoli (Brassica oleracea var. italica) to lengthen post-harvest shelf-life. The ACO 1 and 2 and ACS cDNAs of broccoli were ligated into pSCV1.0 in sense and antisense orientations in relation to a CaMV 35S promoter and nos terminator. They were electroporated into the Agrobacterium rhizogenes co-integrate strain LBA 9402 pRi1855::GFP, and used to co-transform GDDH33, a doubled haploid line derived from the calabrese cultivar Green Duke. 150 transgenic hairy roots were identified by GFP fluorescence, and 18 were regenerated into whole plants. Four of these lines showed severe rol phenotype, which did not appear to be related to TL₋ DNA insert copy number. The floral buds from T₀ broccoli heads were assayed for post-harvest production of ethylene and chlorophyll levels. T₀ lines with ACC oxidase 1 and 2 constructs produced significantly less ethylene than the control plants, and chlorophyll loss was significantly reduced. A positive correlation between post-harvest bud ethylene production and chlorophyll loss was described by the equation y= 0.2386x-23.041. There were two copies of aco1 and aco2 in the Brassica oleracea genome of which one was mapped for each to linkage group 1 and 8, respectively.