Variation in floral form of CRISPR knock-outs of the poplar homologs of LEAFY and AGAMOUS after FT heat-induced early flowering

Abstract

Plant migration and gene flow from genetically modified or exotic trees to nearby lands or by crossing with wild relatives is a major public and regulatory concern. Many genetic strategies exist to mitigate potential gene flow; however, the long delay in onset of flowering is a severe constraint to research progress. We used heat-induced FT overexpression to speed assessment of the expected floral phenotypes after CRISPR knockout of poplar homologs of the key floral genes, LEAFY and AGAMOUS. We selected events with previously characterized CRISPR-Cas9 induced biallelic changes then re-transformed them with the Arabidopsis thaliana FLOWERING LOCUS T (AtFT) gene under control of either a strong constitutive promoter or a heat-inducible promoter. We successfully obtained flowering in both a male and female clone of poplar, observing a wide range of inflorescence and floral forms among flowers, ramets, and insertion events. Overall, flowers obtained from the selected LFY and AG targeted events were consistent with what would be predicted for loss-of-function of these genes. LFY-targeted events showed small catkins with leaf-like organs, AG-targeted events had nested floral organs consistent with reduction in floral determinacy and absence of well-formed carpels or anthers. These findings demonstrate the great developmental plasticity of Populus flowers during genetically accelerated flowering, which may be of horticultural value. They also provide an informative early view of floral phenotypes and apparent sterility from knockouts of both these gene targets.

Acknowledgements

We thank the Tree Biosafety and Genomics Research Cooperative (TBGRC) at Oregon State University, the USDA (award 2011-68005-30407, System for Advanced Biofuels Production From Woody Biomass In The Pacific Northwest), USDA Biotechnology Risk Assessment Grants (2011-68005-30407 and 2010-33522-21736), and the NSF I/UCRC Center for Advanced Forestry (0736283).

Author Contributions

S.H.S. designed and supervised the project. E.E. and C.M. performed the experiments. E.E., and A.L.K. analyzed the data. A.L.K. and E.E. created the figures. A.L.K. wrote the original manuscript. A.L.K., E.E., C.M., and S.H.S. reviewed and revised the manuscript. All authors reviewed and approved the manuscript.

Data availability

The data underlying this article are available in the article and in its online supplementary material.

Conflicts of interest statement

None declared.

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