In this study, the effects of different plant growth regulators on regeneration responses of various lentil explants through direct and indirect organogenesis and through somatic embryogenesis from calli and cell suspension cultures were investigated. Shoot regeneration was obtained in low frequencies from longitudinal embryonic axis explants and nodal buds of epicotyls, however whole plant regeneration was unsuccessful. Conditions provided for indirect organogenesis resulted only in swelling of hypocotyls and root directed ends of internodes and weak callus formation on leaves which were followed by tissue browning and necrosis. In somatic embryogenesis studies, the explants longitudinal embryonic axis and cotyledonary petioles produced soft and friable calli on MS media with Gamborg&rsquo / s vitamins supplemented with 0.75mg/L 2,4-D+0.5mg/L BA. The highest average number of embryos per explant, 12.36 was observed on media containing 0.75mg/L BA +0.5mg/L 2,4-D for cotyledonary petiole explants, whereas 3mg/L BA+1mg/L NAA was the only hormone combination that allowed embryo development to some extent, in both explants. Somatic callus failed to regenerate despite globular embryo formation and embryo development to some extent. Combination of sonication treatment with Agrobacterium transformation of three lentil explants / cotyledonary nodes, half cotyledons and cotyledonary nodes with intact shoots, had no effect on the improvement of transient gus gene expression on explants. Sonication treatment was also unable to form localized wounds on the petiole axils. The best gus gene expression on the axil region was obtained when cotyledonary nodes and KYRT1 strain were used in combination with vacuum infiltration and scalpel wounding of the axils. Gradual selection and repeated removal of regenerated shoots between selection cycles increased the number of gus expressing shoots significantly. The regenerated shoots were grafted on root stocks and whole plant regeneration was achieved in greenhouse conditions. By the use of the optimized Agrobacterium-mediated transformation protocol, 4 independent lines were obtained with 2.3% transformation efficiency. Southern blot analysis confirmed the integration of the gus gene into the genome of lentil plants. T0 plants were fertile and all plants showed Mendelian segregation of the gus gene in 3:1 ratio to their progenies except one line which carries three copies of the gene. Reverse transcription PCR has confirmed the expression of the genes in T0 and T1 generations. T0 plants and the following three generations strongly expressed gus gene uniformly in their tissues and the PCR amplifications of both gus and npt-II genes was successful through generations.

The objective of this study was to optimize tissue culture, transformation and regeneration parameters of mature embryo based culture of Triticum durum cv. Mirzabey 2000 and Triticum aestivum cv. Y&uuml / regir 89. The effects of auxin type of hormone at different concentrations and dark incubation periods on regeneration capacity were evaluated. Two different hormone types 2,4- dichlorophenoxyacetic acid and picloram were used at three different concentrations 2, 4 and 8 mg/l. Mature embryo derived calli were incubated in 6 different induction media at dark for 4 and 6 weeks for initiation of primary callus induction. After dark incubation periods, average callus fresh weight and primary callus induction rate were determined. The primary callus induction rates for 4 weeks and 6 weeks old dark adapted Mirzabey calli incubated was found to be 91 % and 93.25 % respectively. Y&uuml / regir primary callus induction rate was 92.5 % for 6 weeks old calli in 6W2D medium and 86.75 % for 4 weeks old calli in 4W8P medium. The primary calli were transferred to embryogenic callus induction medium. The embryogenic callus formation was 94.88 in 6W2D medium for Mirzabey cultivar. The necrosis was observed at high concentration of 2,4-D for both of cultivars. After embryogenic callus induction, embryogenic calli were transferred into hormone free regeneration medium. The maximum regeneration rate (62.31 %) and culture efficiency (44.13 %) were observed in 4W2D medium for Mirzabey. However, the low regeneration rate was observed for Y&uuml / regir (5 %) in 6W2D medium. The transformation studies were performed by using Obitek Biolab Gene Transfer System. The old and the modified loading units were used for optimization of bombardment pressure and distance for mature embryo based calli transformation. After bombardment of pAHC25 coated gold particles, histochemical GUS assay was performed and blue spots were counted. The transformation efficiency increased to 0.65 fold for 30 bar bombardment pressure and 5.5 fold for 35 bar bombardment by the modified loading unit. The modified loading unit could be used for further transformation studies.

Ethylene (C2H4) is a very simple molecule, a gas, and has numerous effects on the growth, development and storage life of many fruits, vegetables and ornamental crops. In higher plants, ethylene is produced from L-methionine in essentially all tissues and ACC Synthase and ACC Oxidase are the two key enzymes in the biosynthesis of ethylene. The objective of the present study was to transform tobacco (Nicotiana tabacum L. cv. Samsun) plant with partial sequence of torenia acc oxidase gene in antisense and sense orientations via Agrobacterium-mediated gene transfer system, and to analyze its effect on ethylene production in transgenic plants. Six antisense and seven sense T0 putative transgenic lines were obtained and were further analyzed with several assays. Leaf disc assay and chlorophenol red assay under selection (75 mg/L kanamycin) revealed positive results compared to the non-transformed plant. T1 generations were obtained from all putative transgenic lines. PCR analysis and Northern Blot Hybridization results confirmed the transgenic nature of T1 progeny. Furthermore, ethylene amount produced by flowers were measured with gas chromatography, which resulted in an average of 77% reduction in S7 line and 72% reduction in A1 line compared with the control flowers. These results indicated that, transgenic tobacco plants carrying torenia acc oxidase transgene both in antisense and sense orientations showed reduced ethylene production thus a possibility of flower life extension.

In this study, optimization of a transformation and regeneration system via indirect and direct organogenesis in cotyledon, hypocotyl, petiole, leaf and shoot base tissues of sugar beet (Beta vulgaris L. cv. ELK 345 and 1195) was investigated. Two different germination, three different callus induction and shoot induction medium was used for indirect organogenesis of sugar beet cultivar ELK 345. Except cotyledon, other explants (hypocotyl, petiole and leaf) produced callus. However no shoot development was observed from callus of these explants. Shoot base tissue of sugar beet cultivar 1195 was employed for direct organogenesis. Shoot development was achieved via direct organogenesis using 0.1 mg/L IBA and 0.25 mg/L BA. Root development and high acclimatization rate were accomplished from shoot base tissue. Different concentrations of kanamycin and PPT were applied to leaf blade explants to find out optimum dose for selection of transformants. Kanamycin at 150 mg/L and PPT at 3 mg/L totally inhibited shoot development from leaf blades. Moreover, an Agrobacterium mediated transformation procedure for leaf explants of ELK 345 was also optimized by monitoring transient uidA expression 3rd days after transformation. Effects of different parameters (vacuum infiltration, bacterial growth medium, inoculation time with bacteria, Agrobacterium strains and L-cysteine application in co-cultivation medium) were investigated to improve transformation procedure. Vacuum infiltration and Agrobacterium strains were significantly improved transformation procedure. Percentage of GUS expressing areas on leaves increased three folds from the beginning of the study.