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 B5 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.

Keywords: Agrobacterium-mediated transformation; Lemna minor; Pharmaceutical proteins; Tissue culture; Transgenic plants.

Fig. 1

Schematic representation of the T-DNA (5.3 kb) of pCAMBIA2301 containing the β-glucuronidase (GUS) gene (uidA) with intron (3.2 kb) and neomycin phosphotransferase (nptII) gene (2.1 kb) each with CaMV35S promoter sequences. Bar line represent the region of the probe prepared for southern hybridization. The position of the HindIII is indicated on the T-DNA. No other HindIII sites are present on pCAMBIA2301 (total size: 11.6 kb). LB/RB left and right T-DNA border sequences

Fig. 2

(a) Hard nodular callus developed on the explant (b) Frond regeneration from the nodular callus (c) Transformed nodular calli showing typical GUS sectors (arrow mark represent new frond arising) (d) Transformed fronds showing GUS activity

Fig. 3

Molecular analysis of the transgenic Lemna minor plants. PCR analysis of primary transformants (a) using nptII primers. Lane M marker DNA, lane C DNA from untransformed control, Lane P positive control. lanes 1–6 transformed plants. (b) using uidA primers. Lane M marker DNA, Lane P positive control, lane C DNA from untransformed control, lanes 1–6 transformed plants (c) Southern blot analysis of genomic DNA of independent transformed and non-transformed control plants. The DNA was digested with HindIII, and the blot was probed with the PCR amplified fragment (750 bp) of nptII gene. Lane M marker DNA, lane C DNA from untransformed control, lanes 1–4 transformed plants. (d) Southern blot analysis of genomic DNA of independent transformed and non-transformed control plants. The DNA was digested with HindIII, and the blot was probed with the PCR amplified fragment (280 bp) of GUS gene. Lane M marker DNA, lane C DNA from untransformed control, lanes 1–5 transformed plants

Fig. 4

Flow chart of the transformation protocol