Hairy root cultures of butterfly pea (Clitoria ternatea L.): Agrobacterium × plant factors influencing transformation.

Transformed rhizoclones were developed from Agrobacterium-treated explants of the medicinally important twinning legume Clitoria ternatea L. Several key factors influencing transformation events were optimized. A4T was the most infectious among the strains employed. Internode segments were more responsive than leaves, outdoor-grown explants preferred to those from in vitro cultures. High frequency transformation, resulting in up to 85.8% rhizogenesis, was attained using pre-pricked internodal explants for immersion (10 min) in Agrobacterium rhizogenes suspension grown overnight with acetosyringone (100 µM) to an OD(660) â‰… 0.6, diluted to a density of 10(9) cells ml(-1), followed by 5-day co-cultivation. Roots were individually cultured in MS0 supplemented with the bacteriostatic antibiotic cefotaxime (500 µg ml(-1)). Rhizoclones were renewed through successive subcultures in MS0 under diffused illumination. The T ( L )-DNA rolB and rolC ORF were detected in rhizoclones through PCR amplification. The T ( R )-DNA gene encoding mannopine synthase (man2) was revealed by positive amplification and opine gene expression substantiated by agropine and mannopine biosynthesis in all selected transformed rhizoclones. The implication of such findings is discussed on the context of utilization of such genetically transformed root cultures towards sustainable production of medicinally useful phytocompounds, besides providing a means for plant conservation.

ED-XRF spectrometric analysis of comparative elemental composition of in vivo and in vitro roots of Andrographis paniculata (Burm.f.) Wall. ex Nees--a multi-medicinal herb.

The multi-elemental composition of in vitro--proliferated root tissues of Andrographis paniculata (Burm.f.) Wall. ex Nees was compared with that of the naturally grown in vivo plants. Trace elements namely Cr, Mn, Fe, Co, Ni, Cu, Zn, Se, Rb, Sr and Pb in addition to two macro-elements K and Ca were identified and quantified in root tissues of both sources using the energy dispersive X-ray fluorescence (ED-XRF) technique. ED-XRF analysis was performed using Mo K X-rays generated from a secondary molybdenum target. The elemental content of in vitro roots was found to be at par with that of naturally grown plants of the same species. This opens up a possibility of exploiting in vitro root cultures as a viable, alternative and renewable source of phytochemicals of relevance, besides providing a means for conservation of the valuable natural resources.

Transgenic grasspea (Lathyrus sativus L.): factors influencing agrobacterium-mediated transformation and regeneration.

A reproducible procedure was developed for genetic transformation of grasspea using epicotyl segment co-cultivation with Agrobacterium. Two disarmed Agrobacterium tumefaciens strains, EHA 105 and LBA 4404, both carrying the binary plasmid p35SGUSINT with the neomycin phosphotransferase II (nptII) gene and the beta-glucuronidase (gus)-intron, were studied as vector systems. The latter was found to have a higher transforming ability. Several key factors modifying the transformation rate were optimized. The highest transformation rate was achieved using hand-pricked explants for infection with an Agrobacterium culture corresponding to OD(600) congruent with 0.6 and diluted to a cell density of 10(9) cells ml(-1) for 10 min, followed by co-cultivation for 4 days in a medium maintained at pH 5.6. Putative transformed explants capable of forming shoots were selected on regeneration medium containing kanamycin (100 mug ml(-1)). We achieved up to 36% transient expression based on the GUS histochemical assay. Southern hybridization of genomic DNA of the kanamycin-resistant GUS-expressive shoots to a gus-intron probe substantiated the integration of the transgene. Transformed shoots were rooted on half-strength MS containing 0.5 mg l(-1) indole-3-acetic acid, acclimated in vermi-compost and established in the experimental field. Germ-line transformation was evident through progeny analysis. Among T(1) seedlings of most transgenic plant lines, kanamycin-resistant and -sensitive plants segregated in a ratio close to 3:1.