Engineering Synthetic Apomixis in Hybrid Rice

The transfer of apomixis, a clonal mode of reproduction by seeds, to crops has the potential to revolutionize agriculture by enabling the generation of one-line F1 hybrids that propagate clonally by seeds from one generation to the next. However, despite nearly four decades of intensive research, all the attempts to transfer the identified genetic determinants of apomixis from naturally apomictic wild plants to their crop relatives have failed to produce apomictic crops. Engineering of apomixis, mimicking the key features of a natural form of apomixis known as gametophytic diplosporous apomixis, has recently been achieved in rice and further improvements by the introduction of a single "all-in-one" T-DNA construct into calli derived from F1 hybrid seed embryos resulted in high frequency of clonal seeds. The T-DNA encodes a constitutively expressed Cas9 protein guided by sgRNAs designed to knock-out the functions of three genes essential for regulating crucial steps involved in meiosis, thereby eliminating meiosis and creating the Mitosis instead of Meiosis (MiMe) triple mutant. Additionally, the T-DNA contains another gene expression cassette consisting of a parthenogenetic trigger, the BABY BOOM1 (BBM1) transcription factor driven by an egg cell-specific (ECS) promoter. Inactivation of the three MiMe genes converts meiosis into mitosis-apomeiosis-yielding unrecombined and unreduced male and female spores developing into gametophytes. The BBM1 expression triggers parthenogenetic development of an embryo from the diploid egg cell of the female gametophyte. The endosperm develops sexually by the fusion of a diploid sperm cell and the central cell of the female gametophyte, which contains two diploid polar nuclei, resulting in an initially hexaploid endosperm. To date, the "all-in-one" T-DNA method has proved to be the most efficient for achieving high frequency (95-100% clonal seeds) synthetic apomixis in rice. Since the original publication, we have successfully generated synthetic apomictic events in three additional F1 hybrids. Here, we describe the methods for designing T-DNA constructs, analyzing mutations in first-generation (T0) MiMe mutant plants of F1 hybrids, and ascertaining the apomictic nature of the progenies from confirmed MiMe F1 hybrids, which relies on the egg cell-specific accumulation of BBM1.