Agrobacterium–mediated genetic engineering system for the C4 apomictic grass model species Cenchrus ciliaris L.

Abstract

Cenchrus ciliaris L. (buffel grass) is a popular tropical range grass known for its perenniality and high palatability. Owing to its apomictic mode of reproduction, breeding this polymorphic grass is restricted to in vitro approaches of genetic manipulation. Apomixis facilitates genetic improvement by fixing heterosis as well as the changes made to the genome through transgenesis. Towards its genetic improvement, a robust protocol of plant transformation is desirable. Hence, first attempt to develop a rapid and efficient Agrobacterium– mediated transformation system for Cenchrus ciliaris genotype IG-3108 has been made. In this study, direct multiple shoot induction protocol using shoot apex explants was found to be suitable for transformation. Several factors such as inoculum of bacteria, co–culture time, co–cultivation duration, concentration of acetosyringone and effect of vacuum infiltration were optimized for achieving high transformation frequency using shoot apex explants. The shoot apex explants were co-cultured with EHA 105 harbouring the binary vectors pCAMBIA 1301 and 1305.1 containing the hptII gene as a selectable marker and GUSA as a reporter gene. The highest transformation frequency with pCAMBIA 1301 vector was 1.42% while with pCAMBIA 1305.1 vector, it was 1.37% when the explants were co-cultured for 30 min with cells of Agrobacterium at OD600 = 1.0 under vacuum (0.5 X 105 Pa) followed by co-cultivation for 3 days on MS with 3 mg/L TDZ and 400 µM acetosyringone. The status of transgene and its integration in the genome of regenerated putative transformed plants of Cenchrus were confirmed by PCR and Southern blot analysis.