PRODUCTION OF DIPLOID AND HAPLOID TRANSGENIC ATROPA-BELLADONNA PLANTS- MORPHOLOGICAL TRAITS AND TROPANE ALKALOID PRODUCTION

Hairy root cultures from diploid and haploid Atropa belladonna plants were established by co-culture method, using the Ri plasmid of Agrobac terium rhizogenes (strain A4) and its insertion and deletion mutants. The hairy roots integrated beta-glucuonidase and neomycin phosphotrans ferase genes were also established by binary vector method. Diploid ha iry roots grew well in phytohormone-free Murashige and Skoog (HF MS) l iquid medium, especially those induced from the bacterium with the ins ertion mutation at rol c, Haploid hairy roots could be maintained in H F MS liquid medium though their growth was inferior to that of diploid s. Both, diploid and haploid hairy roots, contained hyoscyamine (ca 0. 2-0.6% dry weight) as the main alkaloid. A higher yield of hyoscyamine was obtained in the diploid hairy roots (400-1300 mu g/100 ml flask f or diploids against 200-400 mu g/100 ml flask for haploids). Transgeni c plantlets were regenerated on MS solid medium with or without phytoh ormones at 25 degrees C under 16 h day light, Except for the rol b and rol c insertion mutants, the diploid plantlets showed the typical hai ry root syndrome, while the haploids exhibited much more dwarf feature s, The co-existence of the two rol loci b and c determined the hairy r oot syndrome expression, Alkaloid concentrations in roots and leaves o f normal and transgenic plantlets were much lower than those of the co rresponding hairy roots (less than 0.09% dry weight hyoscyamine), and no relationship in alkaloid concentration was found between the regene rated plantlets and the original hairy roots.