iv ABSTRACT GENETIC TRANSFORMATION OF LENTIL ( Lens culinaris M. cv.Sultan.1) WITH A TRANSCRIPTION FACTOR REGULATOR (MBF1c) AND ANALYSIS OF TRANSGENIC PLANTS KAM&Ccedil / I, Hamdi Ph.D., Biotechnology, Institute of Natural ad Applied Sciences Supervisor Prof. Dr. Meral Y&Uuml / CEL Co-Supervisor: Dr. Ufuk &Ccedil / elikkol AK&Ccedil / AY September 2011, 252 pages In this study, 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.

ABSTRACT GENETIC TRANSFORMATION OF LENTIL ( Lens culinaris M. cv.Sultan.1) WITH A TRANSCRIPTION FACTOR REGULATOR (MBF1c) AND ANALYSIS OF TRANSGENIC PLANTS KAM&Ccedil / I, Hamdi Ph.D., Biotechnology, Institute of Natural ad Applied Sciences Supervisor Prof. Dr. Meral Y&Uuml / CEL Co-Supervisor : Dr. Ufuk &Ccedil / elikkol AK&Ccedil / AY September 2011, 252 pages In this study, 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.

The effects of cytokinin-like Thidiazuron growth regulator on the regeneration responses of callus cultures of Turkish bread Triticum aestivum L. cv. (BaSak 95, Gerek 79, and Bezostaja 1) and durum Triticum durum Desf. cv. (Kunduru, &Ccedil / akmak 79, and Kirmizi 5132) wheat varieties have been investigated in this study. High callus induction frequencies are found to be independent of bread and durum wheat varieties ( &rsaquo / 96%) whereas the callus weight is found to be variety-dependent. For bread wheat, BaSak 95 and for the durum wheat Kunduru is found to be the best performers. TDZ treatments are found to be negatively affecting the regeneration capacity of all the tested bread wheat varieties whereas for the durum wheat variety of Kunduru positive effect is observed. Since the culture efficiency is a derivation from the regeneration capacity, this parameter yielded very similar results as in the case of regeneration capacity for both bread and durum wheat varieties. In bread wheat varieties, the TDZ treatments increased the number of regenerated plants more than 2-fold when compared with the control and likewise very similar results were obtained from durum wheat varieties. Unfortunately, following their transfer to soil, plants that were treated with various concentrations of TDZ displayed reduced vigor probably due to underdeveloped roots. In addition, majority of these plants did not sufficiently develop above the ground parts when compared with the control plants. The simplicity and rapid development of shoots using mature embryos could potentially be used for regenerating superior plants following gene transfer studies in the future.