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

Abstract

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) ≅ 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.

Agrobacterium rhizogenes-mediated transformation of a multi-medicinal herb,Boerhaavia diffusaL.: optimization of the process and anti-microbial activity against bacterial pathogens causing urinary tract infections

“…In our study, it was found that immersing pre-pricked explants of B. diffusa in A. rhizogenes suspension was the most suitable method for achieving the highest transformation efficiency, regardless of Agrobacterium strains employed. This was in conformity with that for C. ternatea (Swain et al 2012). The influence of phenolic compounds secreted from the wound sites of explants on throughput of transformed tissues has been discussed earlier.…”

“…Internode segments of garden-grown explants were preferred to those from in vitro cultures; this is in conformity with another important medicinal plant, butterfly pea (Clitoria ternatea; Swain et al 2012). The transformation efficiency could also vary in response to different pathogenic strains of A. rhizogenes (Pal et al 2013;Rao et al 2012).…”

Hairy Root Induction on Justicia gendarussa by Various Density of Agrobacterium rhizogenes strain LB 510

“…The hairy root induction are influenced by several factors, such as Agrobacterium rhizogenes strains, the addition of growth regulators on the media, parts of the plant are used as explants, and the density of bacteria (Ermayanti et al, 2009;Lee et al, 2010;Swain et al, 2011). The research was carried out by Chakravarty & Pruski (2010) showed that OD 600 = 0.2 could improve significantly the average efficiency transformation of Agrobacterium rhizogenes on potato.…”

Advances in Molecular Approaches for the Integrative Genetic Transformation of Highly Important Climbers

“…Transgenic shoots were obtained on the selective media after 4 months in tissue culture and their transgenic status was confi rmed through GUS assay and PCR analysis. During further study efforts were made to optimise various factors governing the maximum transformation effi ciency during hairy root cultures in C. ternatea (Swain et al 2012a ). Among the four different strains of A. rhizogenes (A4, A4T, 8196, LBA 9402) employed, the highest transformation efficiency was obtained with A4T type of strain.…”