Analysis of endosperm proteins in T3 generation of GWD RNAi lines by western blotting showed that GWD protein was reduced to undetectable levels. Starch from T1, T2 and T3 generations contains less than 40% of the level of phosphate as that of non-GM lines. Amylose content and chain length distribution of debranched amylopectin are unchanged.
A secondary effect of GWD silencing has been observed in the T3 generation in glasshouse: an increase in biomass of young wheat plants and an increase in seed production per plant. Significant increase in biomass, seed number per head and seed weight was also observed in field trial under the licence DIR 092. However, under the field conditions, the GWD RNAi plants produced fewer tillers than the control plants. Therefore, no significant difference in yield was observed. The data provided by the applicant also show that while the lines display early vigour and increased seed weight in comparison to the parental cultivar, the increase is within the normal range of phenotypes observed in conventionally bred wheat lines (Bordes et al. 2008).
SBE RNAi lines (Group 3)
Comparisons of GM and non-GM plants under glasshouse conditions have shown no evidence that the introduced RNAi constructs have affected their growth habit. A sub sample of the seeds to be used in the field has been grown and the plant height and tiller number were measured and showed no significant difference to non-GM control plants. Two GM wheat lines showed a slight increase in mean grain number per spike.
One phenotype associated with increased amylose content of the grain is altered grain morphology which manifests itself as a distorted appearance of the grains which can be visualised under a scanning electron microscope. This morphology was observed for the grains obtained from all the GM lines (information provided by the applicant).
Analysis using gel filtration and iodometry11showed an increase in amylose content of the genetically modified grains compared to non-GM lines. Increased amylose content also results in higher gelatinisation12temperature of starch (Tester et al. 2001). The GM lines in Table 3 show slightly altered gelatinisation temperatures compared to their respective parent lines. This is an expected outcome as increase in amylose content results in a slowing of gelatinisation reflected by an increase in gelatinisation temperature.
A change in amylopectin content and/or branch structure results in changes in chain length distribution. Previously Regina et al (2005) showed that in GM wheat lines with reduced SBE IIa expression, there was a decrease in the proportion of amylopectin with chain length of DP 4-12 and an increase in the proportion of chain lengths larger than DP 12. The GM lines proposed containing the SBE IIa RNAi construct for this trial also show some trend in chain length distribution (Table 3).
Table . Characterization of starch from seeds of SBE IIa RNAi lines
