A mature, sweet orange cultivar derived from 'Valencia' with high Agrobacterium transformation efficiency
Michel Canton | Orlene Peraza-Guerra | Hao Wu | Jude Grosser | Zhonglin Mou | Janice Zale
Transformation efficiencies of sweet orange cultivars 'Florida EV1' and 'Valencia', recalcitrant to Agrobacterium transformation, were investigated using liquid culture in We-V™ vessels. The two mature cultivars were transformed using Agrobacterium with a vector containing selectable markers, nptII and TIPS-EPSPS, and the GFP reporter. Transgenics were identified with GFP in liquid culture at 0, 100, and 200 mg·L−1 kanamycin or in the semi-solid control with 100 mg·L−1 kanamycin. For 'Florida EV1', there were significant differences in the mean transformation efficiency based on the number of shoots screened (TES) at all kanamycin concentrations. Selection at 200 mg·L−1 was better than at lower concentrations in liquid or semi-solid control medium with 100 mg·L−1 kanamycin. The variable TEE, transformation efficiency based on the number of explants, did not discern differences. The means ± standard errors for TES at 200 mg·L−1 were 7.9% ± 2.7% for 'Florida EV1' and 2.4% ± 1.7% for 'Valencia'. In total, 74 transgenics were produced in 'Florida EV1', whereas seven were generated in 'Valencia'. Obtaining transgenics in 'Florida EV1' was easy; fewer shoots were screened at 200 mg·L−1. 'Florida EV1' exhibited better regeneration ability, and all transgenics survived on glyphosate medium, suggesting the TIPS-EPSPS selectable marker could be useful in transformation. Molecular analyses confirmed their transgenic nature. 'Florida EV1' trees produced fruit earlier than 'Valencia' in less than two years. 'Florida EV1' could accelerate the production of HLB disease-resistant trees.
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