Protocol for screening and expression studies of T-DNA and tagging-based insertional knox mutants in Arabidopsis thaliana.

KNOTTED1-like homeobox (KNOX) genes serve important roles in meristem function and many developmental processes in all higher plants. In Arabidopsis, studies of KNOX genes especially among members of class II KNOX genes remain limited and functional data are largely lacking. In the present study, we established a reproducible protocol that is important for genetic studies of KNOX genes using Arabidopsis insertional mutants. This protocol contains a reproducible and serial procedure containing detailed and step-by-step laboratory and field works covering all experiment steps from the screening of homozygous mutant lines to the KNOX expression analysis using qRT-PCR in a single paper. The troubleshooting and challenges that might occur are also presented and discussed. T-DNA insertion mutants for all Arabidopsis KNOX genes (except for knat4) were isolated based on kanamycin screening, phenotype selection, and PCR genotyping. Surprisingly, the insertions resulted in strong repression of the respective KNOX genes. However, no gene suppression was observed for the positively selected knat5 mutant. Moreover, qRT-PCR was effective for transcript analysis among the knox mutant samples. The use of different relative expression quantification produces a similar indication of expression level. Overall, the proposed procedure is highly effective for expression studies of KNOX genes in Arabidopsis mutants and will serve as a fundamental work protocol to open opportunities for genetic studies of genes involving insertional mutants in Arabidopsis. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02868-8. © King Abdulaziz City for Science and Technology 2021.