Genetic transformation of lentil ( Lens culinaris m. cv.Sultan.1) with a transcription factor regulator (MBF1c) and analysis of transgenic plants

Kamçı, Hamdi
Agrobacterium mediated genetic transformation of lentil Sultan 1 cultivar with MBF1c and evaluation of transgenic plants was aimed. The study was initially based on optimized protocol with Agrobacterium tumefaciens KYRT1 strain and pTJK136 binary plasmid. Based on this protocol and transient marker gene expression in embryo apex, 15% stable transformation efficiency was aimed. However limited knowledge about pTJK136 and problem with curing KYRT1 leaded us to use Agrobacterium tumefaciens C58C1 strain and also to engineer an alternative binary plasmid; pPZP101. Hence, scope of this study became construction of a plant binary transformation vector and lentil transformation optimization with C58C1 strain. First plant transformation vector designed in this study was pPZP101ManA-MBF1c. Transformations with C58C1::pPZP101ManA-MBF1c were carried out with a reformulated co-cultivation media. Cotyledonary nodes were isolated from three days old lentil seedlings germinated with phytormone (BAP/TDZ) induction. Isolated nodes were either injured and pre-incubated in co-cultivation media or pre-incubated and then injured prior to transformation. Regeneration and necrosis behaviors of the transformed explants leaded us to the conclusion that explant preparation is the critical step of transformation. And data suggest that explants isolated from 2mg/l BAP, pre-incubated two days in co-cultivation media, injured and transformed performed significantly better scores for necrosis shoot regeneration and callus formation parameters. Transformed explants that survived in subsequent sub-cultures in mannose selection raised shoots. These shoots were grafted and regenerated into plantlets. The putative transgenic plantlets were screened for transgene with PCR. Initial amplification signals fainted and lost as grafts grew. In order to make a diagnosis of this fainting behavior the second plant transformation vector pPZP101ManA-GUSint-MBF1c was constructed and transient GUS expression analysis were made.