A Convenient Method for Screening Genes Related to Fertilization and Embryogenesis in Arabidopsis

Research on early embryogenesis and fertilization is a central issue for understanding sexual reproduction in higher plants. The forward genetic method e.g. mutation, is commonly used to explore the physiological functions of genes in plants. However, the high false positive rate in mutant libraries based on regular mutagenesis methods, for example T-DNA insertion, transposon, ethylmethane sulfonate and high-energy rays, retard the screening of mutants related to fertilization and early embryogenesis. To solve this problem, we developed a new method to construct mutant stock. We introduced a pollen-specific fluorescence marker (pLAT52::EGFP) into the T-DNA element of the plasmid pCAMBIA1302, then transferred it into the qrt1-2 mutant, in which the four products of microsporogenesis remained fused and pollen grains were released as tetrads, to construct a T-DNA insertion mutant library. This allowed us to discard the false positive mutants rapidly by simply visualizing the fluorescence of pollen. In addition, we could determine the mutated gene locus of candidate mutants through inverse-PCR. Results showed that this method could help screen mutants related to fertilization and early embryo development rapidly and identify the mutated gene locus efficiently.