Cytodifferentiation and transformation of embryogenic callus lines derivedfrom anther culture of wheat

Three types of callus tissues established from anther culture of eleven dou bled haploid (DH) lines of wheat (Triticum aestivum L.) were evaluated for their ability in enhancing friable embryogenic (Type II) culture differenti ation and genetic transformation. Differences between types of callus inocu la were highly significant (P < 0.001), suggesting that the quality of the initial callus explant is of profound importance in encouraging the prolife ration of Type II cultures. Other factors found to be crucial included week ly subculture of friable embryogenic callus tissues on a maintenance medium containing 30 mu M dicamba and a predominance of amino-acid nitrogen suppl ement. Transfer and integration of the beta-glucuronidase gene was also aff ected by the type of inoculum when suitable embryogenic cell cultures were transformed using silicon carbide whiskers and high velocity microprojectil es. Expression of the hygromycin phosphotransferase selectable marker gene sequence was confirmed in all the stably transformed cell lines maintained on selection media containing lethal levels of hygromycin. Comparatively, t here were differences in the frequency of regenerable, transgenic clonal se gments between whisker-treated and microprojectile bombarded tissues mainly as a result of the fact that cultures vortexed with whiskers were more cap able of post-treatment cell proliferation and embryo differentiation than t hose bombarded with cDNA-coated microprojectiles. Conditions for obtaining these results are outlined and discussed in relation to the suitability of the two transformation strategies for producing transgenic cell aggregates of wheat.