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The rice GA biosynthetic gene OsGA3ox1 has been proposed to regulate pollen development through the gametophytic manner, but cellular characterization of its mutant pollen is lacking. In this study, three heterozygotic biallelic variants, "-3/-19", "-3/-2" and "-3/-10", each containing one null and one 3bp-deletion allele, were obtained by the CRISPR/Cas9 technique for the functional study of OsGA3ox1. The three homozygotes, "-19/-19", "-2/-2" and "-10/-10", derived from heterozygotic variants, did not affect the development of most vegetative and floral organs but showed a significant reduction in seed-setting rate and in pollen viability. Anatomic characterizations of these mutated osga3ox1 pollens revealed defects in starch granule accumulation and pollen wall development. Additional molecular characterization suggests that abnormal pollen development in the osga3ox1 mutants might be linked to the regulation of transcription factors OsGAMYB, OsTDR and OsbHLH142 during late pollen development. In brief, the rice GA3ox1 is a crucial gene that modulates pollen starch granule accumulation and pollen wall development at the gametophytic phase. [ABSTRACT FROM AUTHOR]