OPTIMIZATION OF BIOLISTIC TRANSFORMATION OF EMBRYOGENIC GRAPE CELL-SUSPENSIONS

Abstract. Embryogenic suspensions of 'Chancellor' (Vitis L. complex in terspecific hybrid) were bombarded with tungsten particles coated with plasmid pBI426 encoding beta-glucuronidase (GUS) and neomycin phospho transferase (NPTII) which results in kanamycin resistance. Two d after bombardment, cultures were placed on semi-solid medium containing eit her 8.6 or 17.2 muM kanamycin. Factors that affect biolistic transform ation rates were studied. Tungsten microprojectiles with a mean diamet er of 1.07 mum (M10) resulted in more transient gene expression than 0 .771 mum diameter particles. Using M10 particles, helium pressures of 1000 and 1200 psi yielded more GUS-expressing colonies per plate than did 800 psi 2 d following bombardment. The number of transformants pre sent after 34 d was not affected by the helium pressure. The distance between the particle launch site and the target cells, and the number of days between the last cell subculture and bombardment, did not affe ct the numbers of transient and long term GUS expressing colonies. The addition of 3 g/l of activated charcoal to the post-bombardment mediu m increased long term GUS expression four fold. Wrapping the plates af ter bombardment with Parafilm increased long term GUS expression three fold compared with plates wrapped with a porous venting tape. With up to 850 transformed callus colonies per plate 23 d after bombardment, the biolistic device holds much promise as a method to achieve stable transformation of grapevines.