Vegetative effects of RNAi-suppression of the poplar homolog of LEAFY in the field

Genetic containment technology is critical for the research and development of genetically-modified trees in forestry. A research trial with limited replication (two independent observations per insertion event) demonstrated that an RNAi-LFY construct induced sterility in 6K10, an early-flowering female variety of Populus alba, but was inconclusive regarding whether there were also vegetative effects from LFY suppression. We therefore created a more highly replicated field (averaging 32 independent observations per insertion event) to examine possible vegetative impacts. This trial explored vegetative differences between three groups: transgenic sterile (TS, 2 events, 77 ramets), transgenic normal-flowering (TN, 4 events, 114 ramets), and a non-transgenic poplar control (C, 30 trees) organized into four blocks. The TN events contained the same RNAi-LFY construct as did the TS events, but were not sterile, presumably due to differences in location, expression, and/or copy number of the insertion event affecting the degree of gene LFY suppression. The field trial was planted in the summer of 2016; productivity (height, diameter) and morphology traits (chlorophyll content, petiole length, leaf area) were measured each year through the end of the 2018 growing season, when the trees were an average of 1.7 m in height. Due to transplant shock and deer grazing, data from 2016 were considered of low quality and excluded from main analyses. Contrasts from an analysis of variance of models that included block and event as the main effects showed that TS trees grew significantly (P<0.05) slower (25.5%) than TN trees in volume index (height * diameter squared) averaged over 2017-2018. Among morphology traits, only leaf area (2017) and petiole length (2016-2018) differed significantly among groups. Leaf area of TS trees ranged from 10.4% (P=0.117) to 25.6% (P<0.001) smaller than TN trees, and 12.7% (P=0.345) to 31.3% (P<0.05) smaller than C trees over 2017-2018. Petioles from the TS group also showed a consistent reduction in length–but not width–when compared to TN and C groups; TS petioles were 17.1% (P<0.05) shorter than C and 18% (P<.001) smaller than TN averaged across 2017-2018. We also analyzed principle components to reduce the number and similarity of traits, and found that PC1, which summarized 39.1% of the trait variation, was mainly determined by productivity traits and showed similar results to that of volume index. PC2, summarizing 15.5% and PC4 summarizing 5.5%, represented the 2018 leaf traits. No statistical differences were found for PC2; however, in PC4 the TS tree were significantly smaller than TN (P<0.001) and C trees (P=0.002). PC3, representing the 2017 leaf traits, accounted for 12.7% of the variation and showed significant differences (P<0.05) between all three groups. Variation of trees and events within groups varied widely among traits. When a trait showed significant differences between groups, the events were often clustered within their groups. However, as shown in PC centroid plots, individual trees were also widely scattered within events and groups. If an event was not clustered with the rest of its group, it was nearly always TN4, which grew poorly and had very few trees compared to the other events. This was mainly due to a very low survival rate in the greenhouse; only 9 ramets survived to be planted in the field. Our results suggest that LFY suppression substantially impacts vegetative growth and morphology in poplar, which would be a concern to using LFY suppression or CRISPR gene knock-outs in commercial forestry. However, our results are tempered by the small number of events studied, only two of which had sufficient suppression to show a sterility phenotype; the substantial variation among our transgenic and wild-type controls; variation in results among years; and the extensive environmental and event within group variation observed. We recommend further years of study, preferably, with many more insertion events or knock-outs, and more control of environment, to confirm these findings.