Agrobacterium-mediated transformation and regeneration of Citrus spp. from suspension cells.

Abstract

Agrobacterium.-medi:J.ted transformation of suspension cells of Citms and regeneration of transgenic plants have been achieved using a binary vector and a cointegrative vector systems. Three embryo callus lines of 'Washington navel' orange- (Citrus sil/('I/sis OSBECK), 'Ohta' ponkan and 'Kara' mandarin (c. reticulata BLA;-;CO), and a pollen embyoid callus of 'Trovita' orange (c. sinensis) were cocultivated with two strains of A. tume.faciens. A415 or GV3010. for 3. 5 and 7 days. The former strain harbors a binary vector pTRA415 containing a neomycin phosphotransferase II (npt-If) gene. The later strain harbors a cointegrative vector pGV3850: pMPKllO-hpt containing a hygromycin phospho transferase (hpt) gene. Callus colonies were formed in calli of 'Washington navel' orange and 'Trovita' orange on selection media containing kanamycin or hygromycin. A relatively high frequency of transformation was observed with 3-day-cocultivation but a lower frequency was observed with 7-day-cocultivation. Embryoids were formed from kanamycin or hygromycin resistant calli and developed into plantlets. Transformation was confirmed on calli and a plantlet by Southern hybrydization.