An efficient protocol for transient transformation of intact fruit and transgene expression inCitrus

Abstract

In this study, conditions were optimized for transient gene expression in Rough Lemon (Citrus jambhiri Lush.), a major rootstock used in the citrus growing regions of Pakistan.Agrobacterium tumefaciens carrying the binary vector p35GUSINT, containingNPT II andGUS genes, was used in the study. The transformation method was based on injection ofAgrobacterium intoCitrus fruits followed by histochemical assay ofGUS activity in different tissues. Different tissues of mature fruits exhibited significantly different percentages of transientGUS expression: in rind (76%), spongy tissue (92%), juice vesicles (0%) and seeds (83%) (P<0.01)., The incubation period after injecting theAgrobacterium culture also showed a significant (P<0.01) effect on the transient expression ofGUS in these tissues. An incubation period of 48 h was found to be the best (72%) for transformation of whole fruit, followed by 72 h (67%) and 96 h (49%). TransientGUS expression also varied significantly (P<0.01) in juice vesicles and seeds as fruit matured. Juice vesicles from mature fruits showed no transientGUS expression, while those from immature fruits showed 50% expression. Furthermore, transformation of seeds had no effect on their germination capability. Germinating seeds from mature fruits injected withAgrobacterium culture showed tolerance to kanamycin (100 mg/L), which varied with the incubation period (55% at 48 h, 25% at 72 h and 23% at 96 h). This report offers an easy protocol for transient expression studies of transgenes and has the potential to be used for stable transformation ofCitrus.