Rapid Generation of Barley Homozygous Transgenic Lines Based on Microspore Culture: HvPR1 Overexpression as an Example

Obtaining homozygous lines from transgenic plants is an important step for phenotypic evaluations, but the selection of homozygous plants is time-consuming and laborious. The process would be significantly shortened if anther or microspore culture could be completed in one generation. In this study, we obtained 24 homozygous doubled haploid (DH) transgenic plants entirely by microspore culture from one T-0 transgenic plant overexpressing the gene HvPR1 (pathogenesis-related-1). Nine of the doubled haploids grew to maturity and produced seeds. qRCR (quantitative real-time PCR) validation showed that the HvPR1 gene was expressed differentially even among different DH1 plants (T-2) from the same DH0 line (T-1). Phenotyping analysis suggested that the overexpression of HvPR1 inhibited nitrogen use efficiency (NUE) only under low nitrogen treatment. The established method of producing homozygous transgenic lines will enable the rapid evaluation of transgenic lines for gene function studies and trait evaluation. As an example, the HvPR1 overexpression of DH lines also could be used for further analysis of NUE-related research in barley.

Automated summary

Obtaining homozygous lines from transgenic plants is crucial for phenotype evaluation, but the current selection process is time-consuming. By using microspore culture, this study successfully obtained 24 homozygous doubled haploid (DH) transgenic plants from one T-0 transgenic plant overexpressing the HvPR1 gene. The established method allows for rapid evaluation of transgenic lines for gene function studies and trait evaluation, such as analyzing NUE-related research in barley using the HvPR1 overexpression DH lines.