Genetic Containment of Forest Trees by RNAi Suppression of LEAFY

Gene flow into wild and feral populations of forest trees present a significant barrier to field studies and commercial use of exotic and recombinant DNA modified trees.  Both male and female sexual reproduction are significant concerns in most species.  To provide bisexual containment, we used RNA-interference (RNAi) to suppress the poplar ortholog of the LEAFY (LFY) gene, which is essential for development of floral primordia in male and female sexual organs.  We transformed this construct into male and female clones of poplar; here we present results from early-flowering female clone 6K10 (Populus alba).  We obtained 15 independent transformed events in clone 6K10 and examined their floral and vegetative traits over 4 seasons of growth in an APHIS-approved field trial.  Rooted trees were planted in 2011 and began flowering in 2014.  Floral phenotypes were initially assessed through indoor flushing of dormant floral buds followed by observation of field-opened buds.  We found that suppression of the poplar orthologs of LFY gave rise to complete and stable sexual sterility in the field.  Of the 15 RNAi-LFY events, 2 had extremely small inflorescences that lacked functional sexual organs and had reduced expression of LFY in developing floral buds.  The floral phenotype was repeated over two growing seasons, and the trees showed normal survival, seasonal dormancy, vegetative morphology, and growth rate.  Suppression or mutation of this gene should greatly facilitate field research, regulatory approval, and public acceptance of exotic and recombinant DNA modified forest trees.