Tissue culture and recombinant DNA technology: Developing protocols for potentially higher yielding switchgrass cultivars

The purpose of this thesis project was to, firstly, establish and optimize protocols for high-throughput callus induction and plant regeneration for the new, higher yielding switchgrass (Panicum virgatum L.) cultivars NSL and SL93. Secondly, to subclone the complementary DNA (cDNA) of the anthocyanin transcription factor, C1, from pBECKS.red into pUC18 and pBSL15 for downstream use. For the first part of this project the cultivars, NF/GA992, NF/GA993, NSL, and SL93, were tested for callus induction by plating whole dehusked caryopses on callus induction media (CIM) containing 8.5uM of the auxin dicamba. NF/GA992 responded best to the treatment with 44% of plated seeds producing callus. For the second experiment seeds of the cultivars NSL, SL93, and Alamo were plated on CIM containing various molar concentrations of dicamba (0uM, 8.5uM, 17uM, and 34uM) and 6-benzylamino purine (BAP) (0uM, 5uM, 15uM, and 45uM). This research revealed that the presence of BAP in CIM plates did not promote callus induction in Alamo, SL93, and NSL, but elevated concentrations (34uM) of dicamba significantly increased callus formation in all three cultivars. It was also found that the SL93 callus derived from CIM plates containing 34uM dicamba and 15uM BAP regenerated the most shoots, 27 shoots were regenerated from 3 calluses. Seed pretreatments were evaluated to determine their impact on callus induction and subsequent plant regeneration. For this experiment, seeds of NSL, SL93, and Alamo were plated on CIM containing 34uM of dicamba. Seeds were subjected to one of three treatments before plating: a) dehusked with H2SO4 , b) chilled for two weeks at 4°C then plated, and c) sterilized with sodium hypochlorite and ethanol. This research revealed that seed pretreatment significantly increased callus induction amongst the three tested cultivars. The second part of this experiment compared the shoot regeneration efficiencies of seed-derived calluses to inflorescence-derived calluses. Analysis showed that SL93 calluses induced from inflorescences produced significantly more shoots than any of the other explants. The final arm of this thesis project focused on sub-cloning the C1 anthocyanin regulatory cDNA. The C1 cDNA isolated from pBECKS.red was sub-cloned into pUC18 and pBSL15 using a sticky-end ligation