Speed-breeding for trees: Engineering early flower induction via the Flowering Locus T (FT) protein in Eucalyptus.

Van Buuren, A.*¹, Kleinhans, N.¹, Goralogia, G.², Strauss, S.², Naidoo, S.¹, Myburg, A. A.¹

¹ Department of Biochemistry, Genetics and Microbiology (BGM), Forest Molecular Genetics (FMG), Forestry and Agriculture Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa
² Department of Forest Ecosystems and Society, Oregon State University, Corvallis, Oregon, USA

Early flowering induction in Eucalyptus trees will contribute to developing accelerated breeding programmes resulting in faster commercialization of improved trees. Early flowering can be induced in Eucalyptus through the transgenic expression of Flowering locus T (FT), a small, phloem-expressed, graft transmissible protein in plants. Transgenic Eucalyptus can be genetically contained through a knockout of the Eucalyptus grandis,/i> LEAFY (EgLFY) gene. We propose that this combination of modifications can be used to induce early and abundant flowering in young Eucalyptus scions through top grafting of wild-type scions onto FT/lfy rootstock. A study in citrus has achieved graft-transmissible floral induction through grafting onto rootstocks expressing Citrus clementina Flowering Locus T3 (CcFT3) under the control of the Arabidopsis thaliana phloem-specific promoter SUCROSE SYNTHASE 2 (AtSUC2). This study aimed to confirm the transgenic status of CcFT3 expressing Eucalyptus lines produced in earlier work and determine the CcFT3 expression levels in different lines. The study further aims to design CRISPR EgLFY gRNAs for transgenic containment of the rootstock lines. Our results have indicated that the transgenic plants suspected to express CcFT3 and hygromycin had stunted growth and early flowering. The CcFT3 expression analysis revealed that certain lines had higher CcFT3 expression levels, but it is not clear yet that expression level is correlated with the amount of floral induction observed. Future studies will aim to develop a multigene construct containing Eucalyptus grandis FT (EgFT) and a CRISPR EgLFY sgRNA that will allow early expression of EgFT in transgenic, but sterile rootstock. The multigene construct will be created by cloning the CRISPR EgLFY gRNAs designed in this study and EgFT into separate GAANTRY donor vectors that can be combined through repeated insertion and excision reactions by different recombinases. Additionally, future research will focus on determining the graft transmissibility of the CcFT3 expressing Eucalyptus lines produced in earlier work, and whether CcFT3 can induce early flowering in wild-type scions grafted onto the CcFT3 expressing rootstock. The successful grafting of wild-type scion onto sterile rootstocks will lead to the induction of fertile flowers and the ability to use these induced flowers in accelerated crossing and genomic selection strategies in Eucalyptus breeding.

Keywords: early flowering, Flowering locus T, LEAFY, CRISPR, GAANTRY