Optimization of tissue culture, regeneration and Agrobacterium mediated transformation parameters in winter wheat cultivars (Kızıltan-91 and Bezostaja-01)

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Tezin Türü: Yüksek Lisans

Tezin Yürütüldüğü Kurum: Orta Doğu Teknik Üniversitesi, Fen Edebiyat Fakültesi, Biyolojik Bilimler Bölümü, Türkiye

Tezin Onay Tarihi: 2005

Tezin Dili: İngilizce

Öğrenci: MUSA KAVAS

Danışman: HÜSEYİN AVNİ ÖKTEM

Özet:

ABSTRACT OPTIMIZATION OF TISSUE CULTURE, REGENERATION AND AGROBACTERIUM MEDIATED TRANSFORMATION PARAMETERS IN WINTER WHEAT CULTIVAR (Kızıltan-91 and Bezostaja-01) Kavas, Musa M. Sc. Department of Biology Supervisor: Prof. Dr. Meral Yücel Co-supervisor: Prof. Dr. Hüseyin Avni Öktem September 2005, 146 pages 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. Kızıltan-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 ° 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 Kızıltan 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 Kızıltan and Bezostaja cultures, respectively. Callus induction rate were found to be 100 % for Kızıltan IVand 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 Kızıltan cultures at the end of 1 5 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 Kızıltan and Bezostaja by using Agrobacterium tumefaciens AGLI containing binary vector pAL156 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 Kızıltan 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 Kızıltan transformation, respectively. The highest GUS expression, 84%, was obtained from 28 weeks old calli produced from 5 weeks vernalised explants in Bezostaja cultures. Keywords: Wheat immature inflorescence; Regeneration; Agrobacterium tumefaciens; GUS; Transient gene expression, Vernalization.