Effects associated with insertion of cryptogein gene utilizing Ri and Ti plasmids on morphology and secondary metabolites are stable in Bacopa monnieri-transformed plants grown in vitro and ex vitro

Abstract

Bacopa monnieri (Linn.) Wettst. (family Scrophulariaceae), a therapeutically important perennial herb, was transformed to study the stability of the effects associated with the insertion of a gene encoding b-cryptogein, a proteinaceous elicitor, either alone or in addition to the T-DNA genes in long-term in vitro culture as well as after transfer to the greenhouse. Plant lines BmAr-IXcrypt obtained via transformation by LBA 9402-crypt (pRi1855?pBin19??crypt) showed integration and expression of rolA, rolB, rolC, and rolD genes after 4 years of maintenance in vitro. The plant line BmAt-ncrypt obtained via transformation with A. tumefaciens strain LBA4404-crypt (harboring pBin19??crypt) as well as plant line BmAr-IXcrypt showed stable integration and expression of nptII gene and crypt gene even after transfer to greenhouse. The clones of Ri-crypt-transformed plants differed morphologically from Ri-transformed plants in that the typical ''hairy root syndrome'' was not observed in plants expressing crypt gene in the presence of rol genes. Except plant line BmAr-IXcrypt, none of the other transgenic plant lines showed any alteration in floral morphology and onset of flowering. The crypt-transformed plant lines (BmAr-IXcrypt and BmAt-ncrypt), maintained under long-term culture, showed significantly higher (p B 0.05) amount of bacoside production in vitro (1.66-to 2.05-fold, with respect to non-transformed plants). In addition, accumulation of all the four bacosides was significantly higher (p B 0.05) in crypt-transformed plants as compared to non-transformed plants grown for 1 year in greenhouse. Thus, the crypt-transformed plant lines of B. monnieri may be considered as the potential source for sustainable bioproduction of bacosides.

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

“…Leaves (1-1.5 cm) and internodes (~0.9 cm) excised from 15-day-old in vitro-grown plants cultured on MS basal medium were used as explants. Transformation was done following the protocol as reported earlier (Paul et al 2015). For each experiment, 50 leaves and 50 internode explants excised from 15 different in vitro propagated plants were infected and the experiment was repeated thrice.…”

“…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.…”

“…The crypt-transformed plants were also developed in this medicinal herb through A. tumefaciens mediated transformation to examine the role of crypt gene on plant morphology and bacoside accumulation (Majumdar et. 2013;Paul et al 2015). To our knowledge, there are no reports till date on the establishment of transgenic B. monnieri plants harbouring individual rol genes as well as on the role of rol genes on morphogenesis.…”

Morphogenesis, Genetic Stability, and Secondary Metabolite Production in Untransformed and Transformed Cultures

“…There are contradictory reports regarding the extent of stability in transformed roots maintained in vitro during prolonged culture. Stability in growth and morphological characters over prolonged culture is reported in different species like Lycopersicon esculentum [518], Cinchona officinalis "Ledgeriana" [519], Aesculus hippocastanum [520], Pimpinella anisum [521], Plumbago zeylanica [522], Rauvolfia serpentina [522], Tylophora indica [523], Bacopa monnieri [524], etc. However, instability in morphological phenotype and growth kinetics of hairy roots in prolonged in vitro culture is reported in certain species like carrot, Brugmansia candida, Duboisia myoporoides, Hyoscyamus muticus, etc.…”

Tobacco plantlets ameliorate oxidative stress upon expression of a cryptogein gene

“…Expression of cryptogein gene in plants and organ cultures using either induced promoter or constitutive promoter were carried out by many research groups (Sil et al 2015;Vuković et al 2013). Transgenic plants expressing cryptogein gene showed increased growth and enhanced secondary metabolite accumulation (Chaudhuri et al 2009;Majumdar et al 2012;Vuković et al 2013;Paul et al 2015).…”