Tissue culture recalcitrance is overcome by a trial and error approach in which the different combinations of explant, growth regulators and culture conditions are evaluated; however empirical identification of the different parameters that contribute to efficient regeneration is time consuming and inefficient, as only a few parameters can be tested at one time. Moreover, no major innovation in the choice of growth regulators and culture media has taken place in the last decades.
Given the current state of the art, there is an urgent need within the green sector to develop novel, generic tools to improve this plant regeneration processes in a germplasm-independent manner.
Plant regeneration can also be enhanced by ectopic expression of a number of embryo-expressed transcription factors, including the AP2 domain protein BABY BOOM and the CAAT-box binding factor LEAFY COTYLEDON1 (LEC1). BBM and LEC1 proteins have been used to enhance plant regeneration in a wide range of model and crop plants. These approaches rely on generation of transgenic lines that express the transcription factor from a constitutive promoter, together with a system for conditional expression to allow recovery of fertile plants. Although successful, the transgenic approach is limited to plants that can be genetically transformed and also precludes routine commercial utilization.
In this project we will examine the extent to which the LEC1 and BBM transcription factors can be used to transiently promote in vitro regeneration without genomic integration of nucleic acids and without genomic DNA mutation.
We will use three approaches to transiently boost LEC1/BBM protein in plant cells:
1) introduction of LEC1/BBM protein with cell penetrating peptides;
2) activation of endogenous LEC/BBM gene expression using CRISPR-dCas9 technology and
3) activation of endogenous BBM/LEC1 gene expression using small compounds.
We will focus on improving two types of in vitro regeneration: haploid embryo induction for doubled-haploid production and somatic embryogenesis for clonal propagation. This project will deliver transient, and in part, non-GM methods for improving plant regeneration in crop and model plants.