Engineering early floral induction in Eucalyptus by manipulating the Flowering Locus T (FT) gene

Abstract

Eucalyptus species and hybrids include some of the most widely cultivated trees in the forestry industry. They are primarily used to produce pulp and paper, and other bio-based products. Eucalyptus trees are ideal for plantation forestry because of their rapid growth rate and wide adaptability. However, Eucalyptus species have a relatively long time to flower with widely cultivated species such as Eucalyptus grandis and E. urophylla, and their hybrids, only flowering at approximately three to five years. Other species such as E. dunnii and E. nitens only flower after ten years. These long generation times present a bottleneck in Eucalyptus breeding programs and the speed with which new cultivars and elite lines can be produced. This MSc study aimed to identify the functional FT ortholog in Eucalyptus trees and test its functionality in Arabidopsis plants. The putative Eucalyptus FT ortholog was selected based on phylogenetic analysis and subsequent 3D protein prediction and comparisons. The gene identified was cloned and transformed into Arabidopsis using Agrobacterium transformation. Transgenic plants were grown under non-inducing, short-day conditions to test the ability of the Eucalyptus FT protein to induce flowering ectopically. To further assess the localisation and graft transmission potential of the Eucalyptus FT protein, the GFP protein was translationally fused with the Eucalyptus FT protein. Wild-type Arabidopsis plants were grafted with transgenic tissues and subjected to fluorescent confocal microscopy. We found that the selected Eucalyptus FT gene induced significantly early flowering in Arabidopsis when ectopically expressed under short day conditions. We also show that the Eucalyptus FT protein is localised to the plant’s vasculature and is capable of graft transmissible movement. An additional aim of this MSc was to determine graft transmissibility of heterologously expressed Arabidopsis FT protein and its ability to induce floral development in wild-type Eucalyptus tissue and how the application of paclobutrazol affected the transport and floral induction. To assess the graft transmission of Arabidopsis FT protein in Eucalyptus plants, Arabidopsis FT overexpressing Eucalyptus lines were grown to a graftable size in naturally lit phytotrons. When the main stem diameters of the plants were approximately 5 cm, a reciprocal grafting trial was performed. After successful grafts produced shoots, a subset of these was treated with paclobutrazol, which is a known floral inducer. We found that the paclobutrazol treated plants had significantly shorter internodal lengths compared to plants which were not treated with paclobutrazol. As of writing this thesis no floral induction has been seen in wildtype Eucalyptus tissue. The work completed in this MSc study has led to the identification of the functional Eucalyptus FT ortholog. We confirmed that this protein is capable of graft transmission in a heterologous system. The Eucalyptus FT gene can be used in future floral induction studies in Eucalyptus, and the graft transmission of the protein assessed in Eucalyptus plants. We also showed that the Arabidopsis FT protein is capable of inducing early flowering in Eucalyptus. However, we found that the Arabidopsis FT was not capable of causing early flowering in wild-type tissue grafted onto transgenic material in Eucalyptus. We also found that the application of paclobutrazol only resulted in the shortening of the internodal lengths of new shoots on grafted scion material. These results provide a step towards accelerated breeding in Eucalyptus.