Increased Availability of Tryptophan in 5-Methyltryptophan-Tolerant Shoots of Catharanthus roseus and Their Postharvest in vivo Elicitation Induces Enhanced Vindoline Production

Abstract

Ten 5-methyltryprophan (5-MT)-resistant multiple shoot culture lines in three genotypes of Catharanthus roseus were selected in vitro. The variant shoot lines displayed a differential threshold tolerance limit against the analogue stress, ranged from 20 to 70 mg/l 5-MT in the medium. The lines tolerant to 40 mg/l 5-MT stress were most stable and fast proliferating. All the selected lines in the presence of 5-MT stress recorded increased level of tryptophan in their free amino acid pool. Highest tryptophan accumulation occurred in lines P40, P30, D40, and N40 (i.e., 296.5, 241.0, 200.6, and 202.0 μg/g dry wt., respectively). A concomitant increase in the total alkaloid content (2.3-3.8 % dry wt.) under the analogue stress was also noticed in these lines when compared to 1.0-1.58 % dry wt. in the respective wild-type shoot maintained on a stress-free medium. The HPLC analysis of the alkaloid extracts of the 5-MT-tolerant lines grown under analogue stress also revealed vindoline as a major constituent with maximum accumulation in lines N40, N30, D30, D40, and P40 (0.046, 0.032, 0.034, and 0.022 % dry wt., respectively). The rooted shoots of 5-MT-tolerant lines were successfully acclimatized under glasshouse environment wherein they grew normally and set seeds. Flowering twigs or leaves excised from 1-year-old glasshouse-grown plants of 5-MT variant lines upon postharvest in vivo elicitation with 30 mg/l 5-MT or 5.0 mg/l tryptophan registered an eight-to-tenfold increment in their vindoline content within 24-48 h.

Tryptophan metabolism and evaluation of morphological, biochemical and molecular variations in a field grown plant population derived via direct adventitious shoot bud regeneration from pre-plasmolysed leaves of Catharanthus roseus

“…Feeding such lines with tryptophan and terpenoid intermediates showed that they have the capacity for high alkaloid production (Whitmer et al 2002), indicating that the terpenoid branch of pathway is limiting. It was shown earlier also that plants/ cell lines that can over synthesize and accumulate tryptophan in the cells were capable of diverting the pathway flux towards TIA production (Verma et al 2012c(Verma et al , 2013. This observation has provided support to hypothesis that cells with better tryptophan availability can effectively counter-balance the inhibitory effects of 5MT.…”

“…Limitations in pushing the metabolic flux towards the TIAs synthesis have been shown to operate both at the level of tryptophan as well as secologanin. Tryptophan availability is likely to be more crucial because of its low occurrence in the amino acid pool and its high demand in other life sustaining pathways like auxin metabolism (Verma et al 2012c). Higher concentration of tryptophan results in feedback inhibition of its synthesis from chorismate by anthranilate synthase.…”