Construction of a weight-based seed sorting system for the third-generation hybrid rice.

The third-generation hybrid rice technology can be constructed by transforming a recessive nuclear male sterile (NMS) mutant with a transgenic cassette containing three functional modules: the wild type male fertility gene to restore the fertility of the mutant, the pollen killer gene that specifically kills the pollen grains carrying the transgene, and the red fluorescence protein (RFP) gene to mark the transgenic seed (maintainer). The transgenic plant produces 1:1 NMS seeds and maintainer seeds that can be distinguished by the RFP signal. However, the RFP signals in the partially filled or pathogen-infected maintainer seeds are often too weak to be detected by RFP-based seed sorting machine, resulting in intermingling of the maintainer seeds with NMS seeds. Here we constructed a weight-based seed sorting system for the third-generation hybrid rice technology by silencing the genes encoding ADP-glucose pyrophosphorylase (AGP) essential for endosperm starch biosynthesis via endosperm-specific expression of artificial microRNAs (amiRNAs). In this system, the NMS seeds have normal endosperm and are heavy, but the maintainer seeds have shrunken endosperms and are light-weighted. The maintainer seeds can be easily and accurately sorted out from the NMS seeds by weight-sorting machines, so pure and fully filled NMS seeds are available. The weight-based seed sorting system shows obvious advantages over the RFP-based seed sorting system in accuracy, efficiency, and cost for propagation of pure male sterile seeds. These characteristics will significantly increase the value and transgenic safety of the third-generation hybrid rice technology.