Augmented production of vincristine in induced tetraploids of Agrobacterium transformed shooty teratomas of Catharanthus roseus

Catharanthus roseus is well-known to produce the chemotherapeutic anticancer agents, vinblasine and vincristine. Shooty teratomas produced by genetic transformation have shown stabilized production of vindoline, but there has been no report on the increased production of vincristine. Stable diploid clones were produced in C. roseus by cocultivation of Agrobacterium tumefaciens C-58 with shootlets raised in culture. Nopaline enzyme assay of diploid clones after repeated subcultures confirmed the successful transformation. Colchicine was then used (0.01% for 2448 h) to produce stable tertraploid clones of shooty teratomas. Cytological observations showed 16 chromosomes in diploid and 32 in tetraploid clones. Although clones of shooty teratomas showed similar biomass, leaf area and stem sizes were higher in tetraploids despite a slower growth. Biochemical analysis of clones of shooty teratomas showed 2-fold higher vincristine (0.022%) in tetraploids than diploids (0.011%) and 73-fold higher than un-transformed shootlets or mature leaves of 6 month old plants in field (0.0003%) of C. roseus. These genetically transformed tetraploid clones should prove useful for metabolic engineering aimed at commercial production of vincristine.