Improvement of transformation efficiency in citrus

Internodal stem segments from citrus seedlings have been widely used as explants in transformation research, due to their ability of easy regeneration. Since 1990, genetic transformation has been applied on different citrus species including sweet oranges (Citrus sinensis), mandarins (C. reticulata), trifoliate orange (Poncirus trifoliata), sour orange (C. aurantium), pummelo (C. grandis), citrange (C. sinensis × P. trifoliate), and lemon (C. limon) in our biotechnology program. But the successful transformation rates are low, thus impeding the realization of transgenic cultivar breeding. Consequently, the development of an efficient transformation system becomes essential. A series of factors was tested to improve transformation rates in sweet orange and kumquat (Fortunella crassifolia). The results indicated that using seedlings grown 20 to 25 days in dark and 10 days in light as explants could provide higher frequencies of GUS positive regenerated buds. Adding auxin and placing sterile filter paper during the co-cultivation stimulated the growth of regenerated buds, and increased about 13% the frequency of GUS positive regenerated buds. Lower pH and co-cultivation temperature under acetosyringone induction enhanced transformation competence. With all the improved factors, the transformation rate was in average 18.6%, and in the best case, the transformation rate reached 23%.