Agrobacterium mediated gene transformation of rice: Comparison of callus and shoot apex derived plants

Two serious drawbacks have limited the use of Agrobacterium in crop improvement: a host-range that appeared to exclude the cereals, and a practice of genotype-dependent plant regeneration from callus. Agrobacterium has recently been accepted for use in rice transformation; however, plant regeneration methods in use remain callus-based, and perpetuate many tissue culture problems. Regenerating plants from callus imposes specificity to regenerable genotypes and induces a wide range of permanent genetic mutations that are inherited by subsequent generations. Use of shoot apical meristems, in Agrobacterium-mediated transformations was proposed to overcome many of these problems since plant regeneration from shoots is simple and direct (Gould and Smith, 1988; Ulian et al., 1988; Gould and Smith, 1989). Use of shoot apex inoculations for Agrobacterium -mediated transformation of a cereal, corn (Zea mays L.), was immediately successful and permitted genotype-independent recovery of plants in 4–6 weeks (Gould et al., 1991). However, Hiei (1994) while proving that rice sustained genetic transformation by A. tumefaciens , also reported that inoculation of rice shoots failed to produce transgenic plants, and stated that shoot-based transformations were a fantasy. The objectives of this study, therefore, were to transform rice using Agrobacterium both shoot and callus-based approaches; and compare the transformation efficiencies, inheritance and somaclonal variation in the transgenic plants produced. Rice varieties Taipei309 and Texas rice cultivar Dixiebelle were transformed using A. tumefaciens LBA4404 (pTOK233) used by Hiei (1994), courtesy of Japan Tobacco. Transferred genes were detected in high molecular weight DNA of shoot derived plants up to three generations and callus derived plants up to two generations. A finding of this study was that homologous recombination occurred within the transferred T-DNA region after incorporation into plant DNA. Our conclusions are contrary to published reports, rice shoots were transformed by A. tumefaciens and by the LBA4404 (pTOK233) vector. The rate of transformation of shoots is similar to, but definitely not lower than, that of callus. Somaclonal variation appears in both shoot and callus-derived lines, but may be less severe in shoot-derived lines.