Figure 6
Flow chart representation of the steps taken to come to a maker-free crambe GM plant.
The (chart A,B) exemplifies the strategy engaged in this approach to obtain individual recombinant shoots from a two rooting plants (Step1) of line 2 with the 15 μM DEX treatment. There were 5 axillary buds obtained from the shoot in total and 4 from A and 1 from B. As showed in the right corner, the blue arrows mean a round of regeneration and selection with 5-FC; plants in pale represent those killed by the selection and green plants indicate the survivors. The drawn numbers are the actual number of plants handled. Gus-staining at the end of Step 6 and 7 were all positive for the green individuals. The ‘PCR Test’ showes the results of two PCR analyses on the surviving shoots above. ‘O’ means recombinant and ‘X’ means non recombinant. So, finally from this specific starting plant, 9 surviving shoots were obtained and within them, there were 4 double-confirmed recombinantindivial shoots, 1 single-confirmed recombinant shoots and 4 non-recombinantshoots. Seeds from two of these double-confirmed recombinantshoots were germinated to establish seedlings for further PCR, gfp fluorescence and GUS staining analysis (Table 4) followed by performance studies on kanamycin containing media. And the chart B showed the flow-diagram of the other one recombinant regeneration shoot originated from another single rooting plant of line 2 with 15 μM DEX treatment as well.
