Genetic transformation of Indian isolate of Lemna minor mediated by Agrobacterium tumefaciens and recovery of transgenic plants

Abstract

Transgenic plants of an Indian isolate of Lemna minor have been developed for the first time using Agrobacterium tumefaciens and hard nodular cell masses 'nodular calli' developed on the BAP -pretreated daughter frond explants in B 5 medium containing sucrose (1.0 %) with 2,4-D (5.0 μM) and 2-iP (50.0 μM) or 2,4-D (50.0 μM) and TDZ (5.0 μM) under light conditions. These calli were co-cultured with A. tumefaciens strain EHA105 harboring a binary vector that contained genes for β-glucuronidase with intron and neomycin phosphortransferase. Transformed cells selected on kanamycin selection medium were regenerated into fronds whose transgenic nature was confirmed by histochemical assay for GUS activity, PCR analysis and Southern hybridization. The frequency of transformation obtained was 3.8 % and a period of 11-13 weeks was required from initiation of cultures from explants to fully grown transgenic fronds. The pretreatment of daughter fronds with BAP, use of non-ionic surfactant, presence of acetosyringone in co-cultivation medium, co-culture duration of 3 d and 16 h photoperiod during culture were found crucial for callus induction, frond regeneration and transformation of L. minor. This transformation system can be used for the production of pharmaceutically important protein and in bioremediation.

Detection of uncoupled circadian rhythms in individual cells of Lemna minor using a dual-color bioluminescence monitoring system

“…Recently, methods for the stable transformation of L. minor have been established (Chhabra et al, 2011). Thus, spatiotemporal analysis for circadian rhythm can be performed using a transgenic duckweed carrying a circadian bioluminescent reporter.…”

“…Among duckweed species, Lemna gibba has long been used for studies on circadian rhythms (Muranaka and Oyama, 2018). Recently, Lemna minor, which is phylogenetically close to L. gibba, has been used for various studies because an efficient genetic transformation system has been established and the genome sequence information is available (Chhabra et al, 2011;Van Hoeck et al, 2015).…”

Callus induction and frond regeneration in Spirodela polyrhiza

“…Hurfeish, L. minor and W. arrhiza, when sugar supplements and specific PGRs were critical determinants for callus induction, callus growth and frond regeneration (Li et al 2004;Cox et al 2006;Vunsh et al 2007;Rival et al 2008;Chhabra et al 2011;Nguyen et al 2012;Khvatkov et al 2015).…”

“…The commercial application of Lemnaceae for a plant expression system requires an efficient callus induction, plant regeneration and genetic transformation technology (Chhabra et al 2011;Nguyen et al 2012). Due to the limitation of the genus Lemna, among the 38 species of Lemnaceae, a frond regeneration system from callus has so far been established in L. gibba, L. minor, L. perpusilla, S. oligorrhiza and W. arrhiza (Li et al 2004;Cox et al 2006;Vunsh et al 2007;Rival et al 2008;Chhabra et al 2011;Nguyen et al 2012;Khvatkov et al 2015).…”

First Report of Agrobacterium tumefaciens mediated genetic transformation of aquatic Rice paddy herb (Limnophila aromatica)

“…Agrobacterium mediated genetic transformation studies in aquatic plants started late and first report on genetic transformation was reported in Lemna gibba and Lemna minor in 2001 (Yamamoto et al, 2001). To date, a number of optimization of Agrobacterium mediated genetic transformation studies in aquatic plants has been reported including Ipomea aquatica (Khamwanı et al, 2003;Meerak et al, 2006), Typha latifolia (Nandakumar et al, 2005), Cryptocoryne willisii (Wong et al, 2013), L. minor (Chhabra et al, 2011), Mentha aquatica (Hajian et al, 2011) and Bacopa monnieri (Nisha et al, 2004;Ramesh et al, 2011;Mahender et al, 2012;Yadav et al, 2014;Kumari et al, 2015).…”