IN PLANTA TRANSFORMATION OF ARABIDOPSIS-THALIANA

Transformants of Arabidopsis thaliana can be generated without using t issue culture techniques by cutting primary and secondary inflorescenc e shoots at their bases and inoculating the wound sites with Agrobacte rium tumefaciens suspensions. After three successive inoculations, tre ated plants are grown to maturity, harvested and the progeny screened for transformants on a selective medium. We have investigated the repr oducibility and the overall efficiency of this simple in planta transf ormation procedure. In addition, we determined the T-DNA copy number a nd inheritance in the transformants and examined whether transformed p rogeny recovered from the same Agrobacterium-treated plant represent o ne or several independent transformation events. Our results indicate that in planta transformation is very reproducible and yields stably t ransformed seeds in 7-8 weeks. Since it does not employ tissue culture , the in planta procedure may be particularly valuable for transformat ion of A, thaliana ecotypes and mutants recalcitrant to in vitro regen eration. The transformation frequency was variable and was not affecte d by lower growth temperature, shorter photoperiod or transformation v ector. The majority of treated plants gave rise to only one transforma nt, but up to nine siblings were obtained from a single parental plant . Molecular analysis suggested that some of the siblings originated fr om a single transformed cell, while others were descended from multipl e, independently transformed germ-line cells. More than 90% of the tra nsformed progeny exhibited Mendelian segregation patterns of NPTII and GUS reporter genes. Of those, 60% contained one functional insert, 16 % had two T-DNA inserts and 15% segregated for T-DNA inserts at more t han two unlinked loci. The remaining transformants displayed non-Mende lian segregation ratios with a very high proportion of sensitive plant s among the progeny. The small numbers of transformants recovered from individual T-1 plants and the fact that none of the T-2 progeny were homozygous for a specific T-DNA insert suggest that transformation occ urs late in floral development.