K. Yoshimatsu et al., PRODUCTION OF DIPLOID AND HAPLOID TRANSGENIC ATROPA-BELLADONNA PLANTS- MORPHOLOGICAL TRAITS AND TROPANE ALKALOID PRODUCTION, Belgian journal of botany, 130(1), 1997, pp. 38-46
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.