Enhanced production of asiaticoside from hairy root cultures of Centella asiatica (L.) Urban elicited by methyl jasmonate

Abstract

Transformed root ("hairy root") cultures have been shown to be a good model for the study of many secondary metabolites. However, economically important compounds such as asiaticoside and madecassoside are produced in insignificant amounts in the root of Centella asiatica (L.) Urban. To overcome this problem, C. asiatica was transformed using Agrobacterium rhizogenes strain R1000 that harbors pCAMBIA1302 encoding the hygromycin phosphotransferase (hpt) and green fluorescence protein (mgfp5) genes and the hairy culture was coupled with elicitation technique. Hairy roots were obtained at a frequency of up to 14.1% from a tissue junction between the leaf and petiole. Abundant hairy roots were observed when co-cultivation of the plant with A. rhizogenes was done for 7 days (36.1%). Transformation was confirmed by PCR and Southern blot analyses. Five weeks after inoculation, no asiaticoside was detected in the hairy root samples. However, when 0.1 mM methyl jasmonate (MJ) was applied as an elicitor to the culture medium for 3 weeks, a large quantity of asiaticoside was generated (7.12 mg/g, dry wt). In the case of gene expression, 12 h after MJ treatment the expression of the CabAS (C. asiatica putative beta-amyrin synthase) gene in the hairy roots is significantly different from that of the control and this level of transcripts was maintained for 14 days. Our results showed that production of C. asiatica hairy roots could be optimized and the resulting cultures could be elicited with MJ treatment for enhanced production of asiaticoside.

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

“…With Agrobacterium rhizogenes, the most suitable co-cultivation period was 2 days for Alpinia galanga (Rao et al 2012), 5 days for Amaranthus spinosus (Pal et al 2013) and 7 days for Centella asiatica (Kim et al 2007). These variations in the requirement for a definite co-cultivation period may perhaps be attributed to the plant tissue specificity for T-DNA transfer, choice of Agrobacterium strain and its optimal cell density that allow infectivity without compromising with survival of plant cells (Pal 2008).…”

TAP46 Plays a Positive Role in the ABSCISIC ACID INSENSITIVE5-Regulated Gene Expression in Arabidopsis

“…To confirm that the ABA-insensitive phenotype of tap46-1 is due to the T-DNA insertion into TAP46, we rescued this mutant phenotype by introducing a wildtype TAP46 gene into this mutant with a different vector, pCambia1302 mGFP5 (Kim et al, 2007). After transformation and screening, two lines that expressed the transgene transcript at relatively high level, Com1 and Com2 (Fig.…”

“…The PCR-amplified DNA fragment was cut by using the restriction enzymes NcoI and SpeI, and the DNA fragment was inserted into the binary transforming vector pCambia1302 mGFP5 vector (Kim et al, 2007). The resulting recombinant binary vector was then introduced into the A. tumefaciens GV3101, which was then used to transform the tap46-1 mutant using the floral dip method of Clough and Bent (1998).…”

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“…MTCC 2364 was less efficient (29.3%) than R1000, but much more efficient than MTCC 532 (18.6%) [31]. R1000 strain was widely used for hairy root induction in many plants such as Torenia fournieri [32], Gentiana macrophylla [33], Centella asiatica [34] and Agastache rugosa [35]. This intrinsic capacity of Ri plasmid must be higher in R1000 when compared to other strains as inferred by other reports also [36].…”