Genetic transformation of citrus species aiming to increase tolerance to drought stress

Most of the citrus trees grown in Brazil are extensively managed with no irrigation, leaving trees more vulnerable to extended dry season, which may reach 90 days without rain in some areas. Thus, utilization of rootstocks more tolerant to drought, such as ‘Rangpur’ lime (Citrus limonia), is widely employed, while other rootstocks are not. Our laboratory has produced ‘Carrizo’ and ‘Swingle’ rootstocks that accumulate the osmoprotectant aminoacid proline, and the present work has continued those efforts aimed to obtain new transgenic rootstocks and scions more tolerant to drought stress. We employed Agrobacterium tumefaciens carrying a gene encoding a mutant enzyme of P5CS (D1-pyrroline-5-carboxylate synthetase), a key enzyme in the biosynthesis of proline, from Vigna aconitifolia, under control of either the constitutive CaMV 35S promoter, or the stress induced RD29A promoter. We worked with sweet orange cultivars (Citrus sinensis) and trifoliate rootstocks (Poncirus trifoliata cv. ‘Flying Dragon’ and P. trifoliata cv. ‘Rubidoux’). Two sweet orange plantlets were micropropagated and further acclimated in greenhouse conditions. One of these trees was PCR positive for the transgenes and presented a concentration of free proline 2.6 times higher than its non-transgenic control plant. Four plantlets of juvenile ‘Flying Dragon’ were also obtained and trifoliate ‘Rubidoux’ plantlets are now under selection procedures.