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.

To explore the use of defensins for transgenic plant disease resistance, expression by agroinfiltration of plants, stable transformation of plants and stable transformation of yeast were tested for porcine β-defensin 1 (pbd-1) and Nicotiana tabacum defensin 1 (Ntdef1). Attempts to screen constructs by agroinfiltration of Nicotiana benthamiana leaves revealed that agroinfiltration alone induced localized resistance against Colletotrichum destructivum. A comparison of Agrobacterium tumefaciens strains showed that the induced resistance required the transfer of type IV effectors into plant cells and was independent of salicylic acid or ethylene signaling. Stable expression of pbd-1 in N. tabacum and Pichia pastoris showed that PBD-1 purified from P. pastoris had varying degrees of antimicrobial activity against a broad range of microbes, including P. syringae pv. tabaci, C. destructivum and C. orbiculare, but in transgenic N. tabacum, the protein could not be detected and resistance increased only slightly to P. syringae pv. tabaci but not to C. destructivum or C. orbiculare. Stable expression of Ntdef1 in P. pastoris yielded a protein with no or little antimicrobial activity, and stable expression in N. tabacum did not result in detectable Ntdef1 or increased resistance to those pathogens. Although PBD-1 had strong antimicrobial activity against plant pathogens, plant disease resistance likely did not increase because of the low level of the protein in the plants, whereas resistance did not increase with Ntdef1 likely because of low antimicrobial activity and low levels of the protein in the plant. This research demonstrates that agroinfiltration is not appropriate for testing genes for antimicrobial activity in planta, while the P. pastoris expression system is useful for producing protein for in vitro tests of a gene prior to its transfer to plants.

The objective of this study was to optimize regeneration parameters of immature inflorescence culture of Triticum aestivum cv. Y&uuml / regir-89. The effects of dark incubation period and explant region on regeneration success were tested. Immature inflorescences were cut into 3 pieces as tip, mid, base and put onto 2mg /L 2,4-dichlorophenoxyacetic acid containing callus induction medium. These explants were taken to regeneration after 6, 9, 13 weeks of dark incubation period. The regeneration capacities of calli were determined as rooting and shooting percentages. Shooting percentages were found to be 72.0 % for 6 weeks of dark incubation and 64.1 % for 9 weeks of dark incubation while it decreases to 26.1 % in 13 weeks of dark incubation period. This showed that prolonged dark incubation period decreased regeneration capacity of the callus. There was no significant difference in regeneration capacities of tip, mid and base regions of immature inflorescences, which reveals the potential of every region of inflorescence to be used as explant source in further transformation studies. Besides regeneration studies, optimization of transformation parameters for Turkish wheat cultivar Y&uuml / regir by using Agrobacterium tumefaciens AGLI containing binary vector pALl56 was performed. Transformation efficiencies were determined by monitoring the transient expression of uidA gene via histochemical GUS assay. Three to four weeks old calli were found to be more responsive to Agrobacterium-mediated transformation. Different media were tested for utilization during co-cultivation period. It was found that including phenolic compound acetosyringone along with ascorbic acid as an antioxidant was essential for succesful transformation.

The objective of this study was to optimize an efficient selection system and Agrobacterium mediated transformation of chickpea (Cicer arietinum L.). Cotyledonary node explants of Turkish chickpea cultivar G&ouml / k&ccedil / e were used to determine the effects of selective agents, two antibiotics (Kanamycin, Hygromycin) and two herbicides (PPT, Glyphosate) as well as four antibiotics (Augmentin, Carbenicillin, Cefotaxime, Timentin) for eliminating Agrobacterium on multiple shoot and root induction. Selective agents and antibiotics were applied to explants at different concentrations for one month and numbers of regenerated shoots and roots were recorded. Kanamycin at 100 mg/L, Hygromycin at 20 mg/L, PPT at 3 mg/L and Glyphosate at 5 mg/L were found to be appropriate to select chickpea transformants. Lowest concentrations of all selective agents (50 mg/L Kanamycin, 10 mg/L Hygromycin, 3 mg/L PPT, 1 mg/L Glyphosate) totally inhibited rooting of the regenerated shoots. Among the Agrobacterium-eliminating antibiotics, Cefotaxime and Augmentin each up to 600 mg/L had no adverse effect on shoot induction, whereas Timentin (300 mg/L) significantly increased and Carbenicillin (300 mg/L) significantly decreased shoot induction after four weeks of culture. Augmentin was determined to have no effect on rooting capacities of chickpea shoots. However Cefotaxime at all concentrations significantly decreased root induction. On the other hand only high concentrations of Carbenicillin (300 mg/L) and Timentin (200 mg/L) significantly decreased rooting. Sulbactam in combination with Carbenicillin and Cefotaxime displayed effective inhibition of bacterial growth. Furthermore, Agrobacterium mediated transformation procedure for cotyledonary node explants of G&ouml / k&ccedil / e, was also optimized by monitoring transient uidA expression on 4th, 9th, and 16th days after transformation. Transformation procedure was improved via mechanical injury of axillary region of explants and application of vacuum infiltration at 200 mmHg for 40 minutes.

iv The objective of this study was to optimize tissue culture and regeneration parameters of immature inflorescence culture of Triticum aestivum cv. Bezostaja- 01 and Triticum durum cv. Kiziltan-91. The effects of callus age and vernalisation time of explants on regeneration success were evaluated. For determination of optimum vernalisation time of immature inflorescence, plants subjected to 4 &deg / C for 1, 2, 3, 4, and 5 weeks, respectively. Tillers containing immature inflorescences were collected at the same time. Percentage of inflorescence formed tillers over total explants were reached the highest value, 79 %, at 4 weeks cold treated Kiziltan cultivar and, 73 %, at 5 weeks cold treated Bezostaja cultivar. Isolated immature inflorescences were put onto 2mg /L 2,4-dichlorophenoxyacetic acid and picloram containing callus induction medium for Kiziltan and Bezostaja cultures, respectively. Callus induction rate were found to be 100 % for Kiziltan and Bezostaja. These explants were taken to regeneration after 6, 9, 12 and 15 weeks of dark incubation period. The regeneration capacities of calli were determined as shooting percentage and data were collected after 4, 8, 12, and 15 week regeneration period. The highest shooting percentage of 69 %, were obtained from 6 weeks old calli produced from 4 weeks vernalised explants in Kiziltan cultures at the end of 15 weeks regeneration period. However, shooting percentage was 57.2 % for 9 weeks old calli while it decreases to 37.6 % in 12 weeks old calli and 44.2 % in 15 weeks old calli at the end of 15 weeks regeneration period. This showed that prolonged dark incubation period decreased regeneration capacity of the callus. However, there was no significant difference in regeneration capacities of calli produced from Bezostaja immature inflorescence and the highest shooting percentage was obtained from 9 weeks old calli produced from 5 weeks vernalised explants, 27.4 %. Besides regeneration studies, optimization of transformation parameters for winter wheat cultivars Kiziltan and Bezostaja by using Agrobacterium tumefaciens AGLI containing binary vector pALl56 was performed. Transformation efficiencies were determined by monitoring the transient expression of uidA gene via histochemical GUS assay. Three to four weeks old calli were found to be more responsive to Agrobacterium-mediated transformation in Kiziltan cultures. However, four to five weeks old calli were found to be more responsive to Agrobacterium-mediated transformation in Bezostaja cultures. Different transformation protocols were used. It was found that MGL based and MMA based protocols could be used for Bezostaja and Kiziltan transformation, respectively. The highest GUS expression, 84%, was obtained from 28 weeks old calli produced from 5 weeks vernalised explants in Bezostaja cultures.

Diss. (sammanfattning) Alnarp : Sveriges lantbruksuniv. / Härtill 4 uppsatser.

Heidelberg, Univ., Diss., 2007. / Online publiziert: 2008.

Universiẗat, Diss.--Halle, 1994.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (p. 84-109).

Thesis (Ph. D.)--Rijksuniversiteit te Leiden. / eContent provider-neutral record in process. Description based on print version record.