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

Abstract

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 T0 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 (T2) from the same DH0 line (T1). 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.

Real-Time Quantitative PCR: Primer Design, Reference Gene Selection, Calculations and Statistics

“…Previously, Rieu and Powers [ 4 ] provided a good reference in the calculation and statistics of qPCR analysis. Through years of work, the whole technique has also been refined, including primer design and reference gene selection [ 5 , 6 , 7 , 8 , 9 ]. Here, a workflow for qPCR analysis was proposed ( Figure 1 ), and it will be beneficial to the study of gene expressions, especially for beginners.…”