GENERATION OF GRAPEFRUIT AND SWEET LIME TRANSGENIC ROOTS AND PLANTLETS VIA Agrobacterium rhizogenes

Abstract

Genetic transformation is an attractive technique for citrus improvement because it avoids long juvenile periods and it offers the ability to introduce single new characteristics into a cultivar without altering existing traits. Development of efficient genetic transformation systems that confer agronomic advantages is an indispensable step to apply this technology to citrus breeding. In this report we present the development of genetic transformation systems for grapefruit (Citrus paradisi cvs. Duncan and Rio Red) and sweet lime (C. limmettioides) using Agrobacterium rhizogenes. First, transformed roots were generated by coculturing stem segments obtained from in vitro germinated seedlings with A. rhizogenes strain A4 containing the wild-type plasmid pRiA4 and the binary vector pESC4 with nos-nptII and cabgus genes. The highest transformation efficiency, measured as the percentage of root forming explants, was obtained in Duncan grapefruit (78 %), followed by Rio Red grapefruit (68 %) and sweet lime (60 %). GUS activity was observed in 89, 92 and 76 % of Duncan and Rio Red grapefruits, and sweet lime roots, respectively. Second, adventitious buds were regenerated through organogenesis on transformed root segments cultivated in media with either cytokinins and auxins or only cytokinins. Adventitious buds were generated in 24, 14 and 8 % of the segments of Duncan and Rio Red grapefruits, and sweet lime transformed roots, respectively. Regenerated buds were transferred to medium without growth regulators to elongate and produce adventitious roots. GUS analyses confirmed that generated plants conserved its activity. Our results show that the established transformation system successfully delivered foreign DNA to grapefruit and sweet lime, and it might be useful in generation of plants expressing genes of agronomic interest.