DsDNA and protein co-delivery in triticale microspores

Abstract

The efficiency of cell-penetrating peptide (CPP)-mediated dsDNA transfection in triticale microspores was investigated through transient and stable integration of the β-glucoronidase (GUS) reporter gene and expression assays in microspore-derived embryos and plantlets. The RecA protein, usually associated with prokaryote homologous recombination, was also tested for its capacity to protect the linear transgene from degradation. Transfections mediated by the CPP nanocarriers Tat2 and Pep1 reduced the number of regenerated embryos from 158 in the control to 122 and 100, respectively. The co-delivery of CPP-dsDNA with RecA protein also resulted in fewer embryos, 87 and 104 for Tat2 and Pep1, respectively. Delivery of dsDNA with Tat2 or Pep1, without RecA, resulted in the highest frequencies of GUS activity in regenerated embryos, at 26%. Co-delivery with RecA decreased the percentage of GUS-positive embryos to 16%. Interestingly, co-delivered RecA-dsDNA reduced the loss of integrity of inserted genetic construct, as observed by polymerase chain reaction (PCR) amplification of the 5′ and 3′ ends. GUS activity was also detected in mature haploid and diploid plants. Of all treatments, 31 T0 plants tested positive for the GUS gene by quantitative PCR, although 50% were derived from the single treatment dsDNA-Tat2. The estimated copy number of the GUS transgene varied between four and eight. This study provides the foundations for CPP-mediated co-delivery of dsDNA and protein RecA in haploid microspore nuclei for functional genomic studies in crop species.