Pradeep K. Chand

Hairy root cultures (HRCs) of Boerhaavia diffusa L., an ethno-medicinally important herb, were established via explant co-cultivation with Agrobacterium rhizogenes. Several key factors influencing transformation efficiency were optimized. Rhizoclones showing prolific growth were maintained through successive subcultures in solidified Murashige & Skoog's medium without growth regulators (MS0) under diffused illumination. PCR analysis of the rhizoclones demonstrated positive amplification of rolB and rolC genes of pRi T_L-DNA. Reverse transcription-PCR (RT-PCR) and opine assay revealed the presence and expression of the pRi T_R-DNA borne ags gene. The methanol and dichloromethane (M:DM) extracts of hairy roots (HRs) and natural roots (NRs) were screened against a wide range of bacterial pathogens causing human urinary tract infections (UTIs). The MIC and MBC values of HRs were markedly lower when compared to NRs against microorganisms causing UTIs. Extracts of HRs were more effective than those of NRs not only against wild type strains but also, and more importantly, against multi-drug resistant strains. Establishment of fast-proliferating renewable HRCs of B. diffusa would provide a direct source of anti-microbial phytocompounds against several pathogenic bacteria, including resistant strains, causing UTIs.

Independent transformed root somaclones (rhizoclones) of butterfly pea (Clitoria ternatea L.) were established using explant co-cultivation with Agrobacterium rhizogenes. Rhizoclones capable of sustained growth were maintained under low illumination in auxin-free agar-solidified MS medium through subcultures at periodic intervals. Integration of T _L -DNA rolB gene in the transformed rhizoclone genome was verified by Southern blot hybridization, and the transcript expression of T _R -DNA ags and man2 genes was ascertained by reverse transcription polymerase chain reaction analysis. The major compound isolated and purified from the transformed root extracts was identified as the pentacyclic triterpenoid compound taraxerol using IR, ¹H-NMR, and ¹³C-NMR spectroscopy. The taraxerol yield in cultured hairy roots, as quantified by HPTLC analysis, was up to 4-fold on dry weight basis compared to that in natural roots. Scanning of bands from cultured transformed roots and natural roots gave super-imposable spectra with standard taraxerol, suggesting a remarkable homology in composition. To date, this is the first report claiming production of the cancer therapeutic phytochemical taraxerol in genetically transformed root cultures as a viable alternative to in vivo roots of naturally occurring plant species.

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 OD₆₆₀ ≅ 0.6, diluted to a density of 10⁹ 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.

The past 30 years have witnessed a series of systematic biotechnological advances made in pomegranate. These encompass optimization and establishment of in vitro culture techniques including micropropagation, somatic embryogenesis, synthetic seed production, plant regeneration via callus-mediated shoot organogenesis, adventitious shoot regeneration, anther culture, tetraploid induction and genetic transformation. This review attempts to provide a comprehensive account on the tissue culture-mediated biotechnological interventions made in pomegranate aimed at complementing conventional programmes for improvement of this nutraceutically important fruit crop.