Bazı Kavun Çeşitlerine NHX-1 Geninin Transformasyonu

Year 2016, Volume: 31 Issue: 2, 1 - 8, 29.07.2016

Tolga İzgü Başar Sevindik Özhan Şimşek Yeşim Yalçın Mendi

Abstract

Bu çalışmada, iki kavun çeşidinde (Cucumis melo var. inodorus cv. ‘Kirkagac 637’ ve Cucumis melo L. cv. Védrantais) Agrobacterium tumefaciens suşu kullanılarak sürgün rejenerasyonu ve transformasyonu için en uygun metod geliştirilmiştir. A. tumefaciens AGL1 ırkındaki pUBINHX1 plazmidinde tuzluluğa dayanıklılık geni bulunmaktadır. pUBINHX1 plazmidi içerisinde bulunan AtNHX1 (Na-H antiporter) geni, CaMV 35S promotoru kontolü altında ifade olmaktadır. T-DNA bölgesinde lokalize olan hygromisin phospotransferase geni bitki seleksiyon marker geni olarak görev almaktadır. En iyi rejenerasyon sonucu, 2.2 μM BAP ve 0.5 μM NAA içeren MS ortamından sağlanmıştır. Putatif transgenik bitkilerde, DNA izolasyonu, PCR ve jel elektroforez çalışmaları yapılmış, 200 bç bant büyüklüğü saptanmıştır. Proksimal ve kotiledon eksplantlarından elde edilmiş putative transgenik bitkilerin rejenerasyonu, her iki çeşit içerisinde farklılıklar göstermiştir.

Keywords

Cucumis melo, Transformasyon, Tuzluluk, Gen transferi, PCR

Transformation of NHX-1 Gene into Some Melon Varieties

Abstract

In the present study, an efficient method for shoot regeneration and transformation by Agrobacterium tumefaciens strains in two different  melon cultivars (Cucumis melo var. inodorus cv. ‘Kirkagac 637’ and Cucumis melo L., cv. Védrantais) have been developed. A. tumefaciens AGL1 race provide pUBINHX1 plasmid, resistant to salinity. Expression of AtNHX1 (Na-H antiporter) gene driven by CaMV 35S control in pUBINHX1 plasmid. In the structure of the plasmid hygromisin phospotransferase gene located as plant selection marker gene in T DNA region. The best regeneration result was obtained from MS medium supplemented with 2.2 μM BAP and 0.5 μM NAA. DNA isolation, PCR and gel electrophoresis were done from putative transgenic plants, 200 bp band size was obtain. Regeneration of putative transgenic plants obtained from proximal and cotyledon explants showed differences in both cultivars.

Keywords

Cucumis melo, Transformation, Salinity, Gene transfer, PCR

References

• Bezirganoglu, I., Hwang, S. Y., Shaw, J. F., Fang, T. J. 2014. Efficient production of transgenic melon via Agrobacteriummediated transformation. Genetics and molecular research: GMR, 13(2), 3218.
• Bordas, M., Moreno, V., Roig, L. 1991. Organogenic and embryogenic potential of several commercial lines of Cucumis melo. Cucurbit Genetics Cooperative, 14, 71-74.
• Bordas, M., Montesinos, C., Dabauza, M., Salvador, A., Roig, L. A., Serrano, R., Moreno, V. Icente. 1997. Transfer of the yeast salt tolerance gene HAL1 to Cucumis melo L. cultivars and in vitro evaluation of salt tolerance. Transgenic Research, 6(1), 41-50.
• Dong, J.Z., Yang, M.Z., Jia, S.R., Chua, N.H., 1991. Transformation of melon (Cucumis melo L.) and expression of the caulifower mosaic virus 35S promoter in transgenic melon plants. Biotechnology 9, 858-863.
• Edwards, A. L., Hammond, H. A., Jin, L., Caskey, C. T., Chakraborty, R. 1992. Genetic variation at five trimeric and tetrameric tandem repeat loci in four human population groups. Genomics, 12(2), 241-253.
• Fang, G., Grumet, R. 1990. Agrobacterium tumefaciens mediated transformation and regeneration of muskmelon plants. Plant Cell Reports, 9(3), 160-164.
• Guowei, F., Grumet, R. 1990. Agrobacterium tumefaciens mediated transformation and regeneration of muskmelon plants. Plant cell reports 9(3):160-164.
• Kusvuran, S., Ellialtioglu, S., Yasar, F., Abak, K. 2007. Effects of salt stress on ion accumulations and some of the antioxidant enzymes activities in melon (Cucumis melo L.), Inter. J. Food Agric. Environ., 2(5): 351- 354.
• Mendi, Y.Y., Ipek, M., Serbest-Kobaner, S., Curuk, S., Aka-Kacar, Y., Cetiner, S., Gaba, V., Grumet, R. 2004. Agrobacterium-Mediated Transformation of ‘Kırkagac 637’ a Recalcitrant Melon (Cucumis melo L.) Cultivar with ZYMV Coat Protein Encoding Gene. Europ.J.Hort.Sci., 69 (6). S. 258–262, ISSN 1611-4426.
• Mendi, Y.Y., Izgu, T., Aka-Kacar,Y., Solmaz, I., Simsek, O., Sarı, N 2012. Cucurbitaceae 2012. Transformation in Melon. Proceedings of the Xth EUCARPIA meeting on genetics and breeding of Cucurbitaceae (eds. Sari, Solmaz and Aras) Antalya (Turkey), October 15-18th.
• Murashige T, Skoog F 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497.
• Pitrat, M., P. Hanelt and K. Hammer, 2000. Some comments on infraspecific classification of cultivars of melon. Acta Horticulturae 510, 29-36.
• Serrano, R., Gaxiola, R. 1994. Microbial models and salt stress tolerance in plants. Critical Reviews in Plant Sciences, 13(2), 121-138.
• Yalcin-Mendi, N. Y., Ipek, M., SerbestKobaner, S., Curuk, S., Kacar, Y. A., Cetiner, S., Grumet, R. 2004. Agrobacterium-mediated transformation of' Kirkagac 637 recalcitrant melon (Cucumis melo L.) cultivar with ZYMV coat protein encoding gene. European Journal of Horticultural Science, 258-262.
• Zhang, H., Gao, G., Wang, X., Luan, F., 2014. An improved method of Agrobacterium tumefaciens-mediated genetic transformation system of melon (Cucumis melo L.). Journal of Plant Biochemistry and Biotechnology 23, 3: 278-283.