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

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Abstract
Agrobaclerium-mediated transformation of suspension cells of Citrus 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 sinensis OSBECK), 'Ohta' ponkan and 'Kara' mandarin (C. reticulata BLANCO), and a pollen embyoid callus of 'Trovita' orange (C. sinelesis) were cocultlvated with two strains of A. tumefaciens, A415 or GV3010, for 3, 5 and 7 days. The former strain harbors a binary vector pTRA415 containing a neomycin phosphotransferase II (npt-II) gene. The later strain harbors a cointegrative vector pGV3850 : pMPK110-1lpt containing a hygromycin phosphotransferase (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.

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