Figure 9

Analysis of transgenic TaNHX2 events. Schematic representation of the T-DNA regions of pGFPGUSPlus (upper panel) and pCMTaNHX2 (lower panel) (A). The relative location of GUS Plus, HPT II and TaNHX2 are shown. LB, left border; T, polyA site; 2×35S, double CaMV35S promoter; N, nopaline synthase (NOS) terminator region; RB, right border. Southern blot analysis of regenerated transgenic lines using a 728-bp TaNHX2 fragment as a probe (B). P, EcoR I-digested pCMTaNHX2 plasmid DNA; 1, negative control plant; 2–7, randomly selected transgenic regenerated plants. Transgenic TaNHX2 lines were identified by GUS staining in regenerated transgenic TaNHX2 plant (left) and a negative control (right) (C). TaNHX2 expression was analyzed by RT-PCR in L. corniculatus cv. Superroot transgenic lines (D). A specific PCR product of 728 bp (upper panel) was detected in four randomly selected TaNHX2 (1–4) transgenic lines. 5, negative control; 6, PCR on a mixture of 1–5 RNA samples without reverse transcription. A 252 bp beta-tubulin fragment was amplified as an internal control (lower panel). Phenotypes of representative TaNHX2 transgenic (35S::TaNHX2) and control (CK) L. corniculatus plants after treatment with 150 mM NaCl for 15 days (E). All negative control plants were regenerated from hairy roots developed by A. rhizogenes harbouring no binary vector.