Knockout of floral and meiosis genes using CRISPR/Cas9 produces male-sterility in Eucalyptus without impacts on vegetative growth

Eucalyptus spp. are widely cultivated for the production of pulp, energy, essential oils, and as ornamentals. However, their dispersal from plantings, especially when grown as an exotic, can cause ecological disruptions. To provide new tools for prevention of sexual dispersal by pollen as well as to induce male-sterility for hybrid breeding, we studied the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated knockout of three floral genes in both FT-expressing (early-flowering) and non-FT genotypes. We report male-sterile phenotypes resulting from knockout of the homologs of all three genes, including one involved in meiosis and two regulating early stages of pollen development. The targeted genes were Eucalyptus homologs of REC8 (EREC8), TAPETAL DEVELOPMENT AND FUNCTION 1 (ETDF1), and HECATE3 (EHEC3-like). The erec8 knockouts yielded abnormal pollen grains and a predominance of inviable pollen, whereas the etdf1 and ehec3-like knockouts produced virtually no pollen. In addition to male-sterility, both erec8 and ehec3-like knockouts may provide complete sterility because the failure of erec8 to undergo meiosis is expected to be independent of sex, and ehec3-like knockouts produce flowers with shortened styles and no visible stigmas. When comparing knockouts to controls in wild-type (non-early-flowering) backgrounds, we did not find visible morphological or statistical differences in vegetative traits, including average single-leaf mass, stem volume, density of oil glands, or chlorophyll in leaves. Loss-of-function mutations in any of these three genes show promise as a means of inducing male- or complete sterility without impacting vegetative development.