Results and Discussion
Effect of mannose on shoot formation of explants
As pBCAMBIA-PMI contains the pmi gene which confers mannose resistance, we used the mannose as a selection agent in our transformation experiments. Nine different concentrations and combinations of mannose and sucrose in shoot regeneration medium were used to evaluate the effect of mannose on the organogenesis of non-transformed tobacco leaf explants (Fig. 2). After 3 weeks cultivation, we found that no explants produced shoots on media without any tested carbon source (Fig. 2A). When cultivated on 30 gl-1 sucrose (0/30), 100% of the tobacco leaf explants regenerated. The organogenesis response decreased progressively ...view middle of the document...
We had chosen as the most appropriate concentration of mannose 20 gl-1 without sucrose for subsequent genetic transformation experiments, for which the inhibitory effect of mannose on organogenesis was more evident.
Figure 3. Effect of various concentrations of mannose and sucrose on regeneration of Nicotinia tobacum. M: Mannose, S: Sucrose. A) , B) . The bars represent means ±SE. Bars followed by the same letter are not significantly different (p ˂ 0.01) according to Duncan’s multiple range test.
Agrobacterium-mediated transformation and selection of putative transgenic plants
The pBCAMBIA-PMI containing pmi gene was transformed into N. tabacum leaf explants via A. tumefaciens-mediated transformation. For positive mannose selection, on the basis of a previous evaluation of the inhibitory effects of mannose on organogenesis, leaf explants were cultured on selection/regeneration MS modified media containing 20 gl-1 mannose, 1.0 mgl-1 BAP and 0.1 mgl-1 IBA. From 400 transformed explants used for transformation experiment, 92 putative transgenic plants were regenerated. These transgenic plants exhibited a normal phenotype, and rooted on MS medium without any PGRs. Well-developed rooted plantlets were hardened-off in the greenhouse and then under outdoor conditions (Fig. 3). 100% survival was achieved when the rooted shoots were transferred to pots containing a mixture of peat and perlit (3:1) under regular irrigation with tap water and their growth in natural environments was satisfactory. The presence of the transgenes was confirmed by PCR analysis of genomic DNA.
Molecular analysis of transgenic plants
To confirm the transgenic status of putative transformants of tobacco plants, total DNA and RNA were isolated and analyzed from the leaves of both non transformed (as a control) and transgenic plants. By PMI/mannose selection and PCR screening we were able to recover a total of 92 putative transgenic tobacco plants from a single transformation experiment of 400 explants. All transgenic lines showed the specific fragments of the pmi gene (836 bp) detected by PCR, while no fragment was amplified in non-transformed wild type plants (Fig. 5). Some of the tested plants did not show the presence of transgenes, i.e. the regenerants presented escapes, or they revealed clonal origin, despite our attempt to avoid collecting regenerated shoots harboring the same transformation events by careful marking of shoot positions on the leaf explants. Basing on these results, the transformation efficiency was 21.75 % in experiment after mannose/sucrose 20/0 gl-1 (M20+S0) selection. The average selection efficiency, calculated as the number of T0 plants containing the selectable marker gene divided by the total number of regenerated plantlets, was 94.56%, showing that mannose was an effective selection agent. Recently, Baharia et al. (2012) have been reported that at 30 g l-1 concentration of mannose, shoot and root formation was completely inhibited in...