Sustained root culture for generation and vegetative propagation of transgenic Arabidopsis thaliana

Excised roots of wild-type and nitrate-reductase deficient mutant Arabidopsis thaliana (L.) HEYNH. can be propagated as sustained root cultures in liquid medium. Culture initiation from a single seedling required a two-day indoleacetic acid treatment at 0.05 mg/l concentration. Indoleacetic acid facilitated subculture but was not essential for sustained growth. This procedure has allowed the clonal propagation of roots derived from individual wildtype and mutant seedlings for more than 21 months. The cultured roots retained their shoot regeneration ability; however, a controlled desiccation treatment was required to restore it to the level of freshly excised roots. The chromosome number remained diploid and no evidence for the accumulation of recessive mutations was observed. The cultured roots are competent for Agrobacterium-mediated transformation. The sustained root culture technology allowed the maintenance of transgenic tissues in which expression of a dominant, seed-lethal gene (seed-specific pea vicilin promoter fused to diphtheria toxin A chain gene) precluded generative propagation.