An investigation into the tissue culture and transformation of embryonic callus from mature and immature embryos of the model grass species Brachypodium distachyon and Brachypodium sylvaticum

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Hanley, Martina
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University College Cork
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In recent years, the genus Brachypodium has become of great interest to researchers, for use as a model grass system for cereal crops. Exploring the traits of a model species provides researchers with a broader understanding of internal genetic operations of evolutionarily related plant species, leading to advances in selective breeding of crop plants for profitable and sustainable phenotypic traits. Model species process many favorable traits including rapid life cycles, ease of transformation, among others. Many advances have been made to optimise the Brachypodium genus as a model species, including whole-genome sequencing, BAC libraries, EST sequences, availability of many natural accessions. The most widely studied species in the Brachypodium genus, B. distachyon has rapidly soared to the frontline as a model species due it its short life cycle (annual), rapid generation time, small stature, known genetic sequence and amenability in tissue culture and field conditions. In contrast, the perennial relative B. sylvaticum is favoured for being a model species for perennial bioenergy crops including switchgrass and Miscanthus. Although not as widely studied as B. distachyon at present, B. sylvaticum is diploid, self-fertile, with a short generation time. These traits encourage the development of B. sylvaticum into a perennial crop model species. This thesis outlines tissue culture conditions needed to produce compact embryogenic callus (CEC) from both mature and immature embryos from the accessions, Bd21 of B. distachyon and Ain-1 of B. sylvaticum. Induction and propagation of embryogenic callus were promoted by different concentrations of 2,4-Dichlorophenoxyacetic acid (2,4-D) and 6-Benzylaminopurine (BAP) in the callus induction medium (CIM). An Agrobacterium-mediated transformation system was developed for immature and mature embryonic callus using the virulent strain AGL1 carrying the pCAMBIA 1305.2 vector. This vector possesses the nptII gene for bacterial selection with kanamycin, the β-glucuronidase GusPlus gene for histochemical staining and the hptII gene for plant hygromycin phosphotransferase II selection. The reporter beta-glucuronidase (GUS) gene was driven by the CaMV35S promoter and terminated by the CaMV35S polyA signal. Successful transformation was detected using polymerase chain reaction (PCR) technology. Detecting a positive result for the T-DNA hptII gene and a negative result for the intercistronic region between Vir B and Vir G genes indicated positive tissue transformation. A positive result intercistronic region would indicate the AGL1 was present on the outside of the plant tissue as this region would not be integrated into the plant tissue during transformation. Embryonic callus production, transformation efficiency (up to 22%) and regeneration efficiency (up to 26%) were compared between both species using this protocol. Production and transformation of mature and immature embryos of both species, along with regeneration of transformed plantlets, is another positive step in the journey to making B. distachyon and B. sylvaticum into model species.
Model grass species , Immature embryo , Mature embryo , Compact embryonic callus , Callus induction medium , Agrobacterium-dependent transformation
Hanley M. 2020. An investigation into the tissue culture and transformation of embryonic callus from mature and immature embryos of the model grass species Brachypodium distachyon and Brachypodium sylvaticum. MRes Thesis, University College Cork.