Establishment of transformation system in wheat close relatives under assistance of TaWOX5

Abstract

Wheat relative species are important for agriculture production, functional genomics study and wheat improvement as useful genetic resources. In this study, a regeneration related wheat gene TaWOX5 was applied to establish the Agrobacterium-mediated transformation systems of Triticum monococcum, hexaploid triticale, and rye (Secale cereale L.) using their immature embryos. Transgenic plants were efficiently generated. During the transformation process, the Agrobacterium infection efficiency was assessed by histochemical staining for β-glucuronidase (GUS). Finally, the transgenic nature of regenerated plants was verified by polymerase chain reaction (PCR)-based genotyping for presence of the GUS and bialaphos resistance (bar) genes, histochemical staining for GUS protein, and QuickStix strip assay for bar protein. The transformation efficiency of T. monococcum genotype PI428182 was 94.4%; the efficiencies of four hexaploid triticale genotypes Lin456, ZS3297, ZS1257, and ZS3224 were 52.1, 41.2, 19.4, and 16.0%, respectively; the transformation efficiency of rye cultivar Lanzhou Heimai was 7.8%. Fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH) analysis indicated that the GUS transgenes were integrated into the distal or near centromere (proximal) region of the chromosomes in transgenic T. monococcum and hexaploid triticale plants; in the transgenic hexaploid triticale plants, the foreign DNA fragment was randomly integrated into the AABB genome and RR genome. Furthermore, the transgene was proved to be almost stably inherited in the next generation by Mendel’s law. The findings in this study will promote genetic improvement for grain or forage production of the three plant species and for functional genomics study of cereal species including wheat.