Analysis of propagation of Bacopa monnieri (L.) from hairy roots, elicitation and Bacoside A contents of Ri transformed plants

Abstract

Agrobacterium rhizogenes mediated transformation has been experimented in leaf explants of the memory herb Bacopa monnieri in order to assess the regeneration potential of hairy roots (HR) followed by the elicitation of transformed plants for increased Bacoside A production. Out of the four strains tested, A4 and MTCC 532 derived HR exhibited regrowth in MS basal medium while MTCC 2364 derived HR showed regeneration in MS medium supplemented with suitable phyto hormones. R1000 derived HR possessed no regeneration potential. Comparable to A4, MTCC 532 derived HR displayed maximum regrowth frequency of about 85.71 ± 1.84 % with an increase in biomass to threefold. Therefore, five HR plant lines (MTCC 532 derived) were generated and maintained in MS basal liquid medium in which HR3 topped the others in producing a huge biomass of about 67.09 ± 0.66 g FW. PCR amplification and southern hybridization analysis of rol A gene (280 bp) has been performed in order to confirm the transformation process. Moreover, HR3 plant line has accumulated highest total phenolic content of about 165.68 ± 0.82 mg GAE/g DW and highest total flavonoid content of about 497.78 ± 0.57 mg QRE/g DW when compared to other lines and untransformed controls. In addition, HR3 plant extract showed 85.58 ± 0.14 % of DPPH (2, 2-diphenyl-1-picryl hydrazyl) inhibition displaying its reliable anti oxidant potential. Further on elicitation with 10 mg/L chitosan for 2 weeks, HR3 has produced 5.83 % of Bacoside A which is fivefold and threefold increased production when compared to untransformed and transformed unelicited controls respectively. This is the first report on eliciting HR plants for increased metabolite accumulation in B. monnieri.

Effects associated with insertion of rol genes on morphogenic potential in explants derived from transgenic Bacopa monnieri (L.) Wettst

“…Several researchers have established Ri-transformed roots and plants following A. rhizogenes infection in B. monnieri and demonstrated the effect of T-DNA genes on growth, biomass accumulation and bacosides production in transformed tissues (Majumdar et al 2011;Bansal et al 2014;Paul et al 2015;Largia et al 2016). However, few attempts were made on A. tumefaciens mediated transformation where hygromycin/ kanamycin resistant transgenic plants expressing GUS gene were produced to develop an e cient protocol in this species (Ramesh et al 2011;Mahender et al 2012;Aggarwal et al 2013;Yadav et al 2014).…”

“…Establishment of hairy root culture and Ri-transformed plant regeneration from Ri-transformed roots following A. rhizogenes mediated genetic transformation have been reported in B. monnieri which highlighted the stimulatory role of T-DNA genes on secondary metabolite production (Majumdar et al 2011;Bansal et al 2014;Paul et al 2015). Spontaneous shoot regeneration was noted either directly or from dedifferentiated Ri-transformed calli in growth-hormone free basal medium in Ri-transformed root explants but not in control non-transformed root explants (Majumdar et al 2011;Paul et al 2015;Largia et al 2016). This suggests that Ri-transformed roots have higher competence to undergo shoot organogenesis due to the regulation of morphogenic pathways in transformed tissues by T-DNA oncogenes specifying auxin and cytokinin synthesis.…”

Rheological and Microstructural Features of Plant Culture Media Doped with Biopolymers: Influence on the Growth and Physiological Responses of In Vitro-Grown Shoots of Thymus lotocephalus

“…[21], and Ipomoea purpurea (L.) Roth [22]. However, Largia et al [26] also found that an increased concentration and an increased period of exposure to chitosan had a negative effect on the growth of Bacopa monnieri (L.). Nonetheless, the medium with lower chitosan concentration, Chit100, allowed obtaining similar plants as in the control medium.…”

“…Chitosan, a linear polysaccharide derived from chitin, is a very versatile biomaterial, when comparing to other natural and synthetic polymers [18]. This biopolymer has been used to improve plant growth [19][20][21][22] and seed germination [21,23], as an elicitor of secondary metabolites [24][25][26], and as a disinfectant agent [27]. Conventionally, agar, a natural polysaccharide, is used to solidify in vitro plant culture media due to its gelling properties, stability, resistance to plant metabolic enzymes and high clarity [28].…”

Saponins as chemosensitizing substances that improve effectiveness and selectivity of anticancer drug—Minireview of in vitro studies

“…As saponins usually occur in plants in very low levels, substantial amounts of biomass would be required to obtain large quantities of the drug. An alternative could be obtaining saponins in biotechnological processes (Largia, Satish, Johnsi, Shilpha, & Ramesh, 2016) or by chemical semisynthetic routes (Yang, Laval, & Yu, 2014); however, current technologies require definite improvements for mass-scale production.…”