Haploid-inducer development by CRISPR/Cas9 mediated CenH3 gene modification in Helianthus annuus (Sunflower)

Abstract

Sunflower is the third most important oil seed crop globally following soybean and rapeseed. Climate change threatens crop production with various limitations, including unpredictable temperature fluctuations, altered rainfall patterns and, novel diseases and pests. Conventional, mutation and accelerated breeding technologies, such as haploidization, have been able to address some of these challenges through the production of resistant cultivars. The production of sunflower doubled haploid lines through in vitro approaches such as anther, pollen and unpollinated ovule culturing have been tested with little success, mainly due to sunflower being recalcitrant to tissue culture regeneration. A universal in vivo technique based on the modification of the CenH3 gene leads to uniparental chromosome elimination during hybridisation with a wild type line. This technique has been successfully demonstrated to induce a small number of haploid progenies in Arabidopsis thaliana, maize and wheat. Given the universal function and conserved nature of the CENH3 protein, this approach promises to be a breakthrough in haploid induction technology. The aim of the current study was to facilitate the production of a sunflower haploid inducer line through CENH3 protein modification using the CRISPR/Cas9 technology. Two CRISPR/Cas9 constructs were designed to target three different amino acids (P51S, G52E and A55V) in the histone fold domain region of the sunflower CENH3 through homology directed repair (HDR). The individual constructs (pDe-Cas-258-nptII, pDe-Cas-1185-bar) were introduced to sunflower mature cotyledons by Agrobacterium-mediated transformation while the geminiviral construct (IL-60-BS-Cas9-1185) was mechanically introduced into sunflower seedlings. Next generation sequencing revealed that only four of the transgenics obtained via Agrobacterium transformation contained two of the targeted amino acid conversions (P51S and A55V) and the other three did not contain any mutations. 20% of the plants inoculated with the geminiviral construct displayed mutations, with one plant displaying complete homology directed repair. Furthermore, a slight increase in HDR was observed with the use of a geminiviral vector when compared to Agrobacterium mediated transformation. Sunflower mutant plants were tested for their ability to act as haploid inducers using flow cytometry. Flow cytometric analysis revealed 71% and 12.5% aneuploidy in the T1 and T2 generations, respectively. No haploid progeny was obtained in either generation. The overall results of this study indicate that uniparental chromosome elimination coupled with the use of viral vectors for the delivery of CRISPR/Cas9 components into plant cells, have the potential to improve double haploid technology in sunflower. The research carried out in this study will enhance the improvement of sunflower through genome editing and the production of doubled haploid lines.