Somaclonal variation in transformed roots and protoplast-derived hairy root clones of Hyoscyamus muticus.

Substantial somaclonal variation in growth rate, morphology, and alkaloid production of Hyoscyamus muticus L. hairy root clones obtained by transformation with four Agrobacterium strains was shown. The hyoscyamine content of the root clones (n = 100) obtained from the same origin varied from 0.03 to 0.59% of dry weight. The clones produced 25-320 times less scopolamine than hyoscyamine. The best producing root clone was used as a starting material for protoplast isolation. The hyoscyamine content of protoplast-derived hairy root clones (n = 171) ranged from 0.04 to 1.45 % of dry weight. Most clones showed improved alkaloid production in relation to the parent clone, but the mean hyoscyamine content of the clones was the same as that of the parent clone. All the studied hairy root clones showed relatively stable alkaloid production during long-term cultivation. No correlation was found between alkaloid production and growth rate or morphology of the clones.

Characterization of transgenic plants derived from hairy roots ofHyoscyamus muticus.

Mature plants were regenerated via protoplasts fromAgrobacterium rhizogenes-transformed root cultures ofHyoscyamus muticus L., and chemical analyses were performed on 34 individual plants. The regenerated plants showed strong phenotypic differences from clone to clone as well as from the control plants. Polymerase chain reaction studies revealed that the plants exhibiting the strongest phenotypic alterations contained therol (A, B and C) genes, whereas the plants with fewer alterations had lost them. The plants produced hyoscyamine, scopolamine and a range of different calystegins, and considerable somaclonal variation was observed. Alkaloid production in the plants transgenic for therol genes was clearly reduced. The pattern of calystegins was similar within all the regenerated plants lackingrol genes. Among the plants withrol genes, the calystegin B1 was not detectable. It seems clear that the presence ofrol genes is detrimental to the alkaloid accumulation in the transgenic plants in contrast to hairy root cultures.

Efficient plant regeneration from hairy root-derived protoplasts of Hyoscyamus muticus.

Successful plant regeneration was achieved for the first time from hairy root-derived protoplasts of Hyoscyamus muticus. High yields (7 × 10(6) / g fresh weight) of protoplasts were isolated directly from the transformed roots of Hyoscyamus muticus using an enzyme mixture comprising 1 % macerozyme and 2 % cellulase in an osmoticum consisting of 0.2 M CaCl2 and 0.6 M mannitol. Protoplasts were first cultured in liquid NT/PRO I medium and further on semi-solid NT/PRO II agar medium. The procedure permits highly efficient formation of colonies. The plating efficiency varied from 1-9 %. The small individual colonies regenerated easily into shoots and roots at frequencies of 18 % and 70 %, respectively. The time required for the development of small plantlets from protoplasts was 8-11 weeks. The regenerated plants contained rolB from Ri-T-DNA and exhibited an altered phenotype compared to the control plants.