Korungaya (Onobrychis viciifolia Scop.) Partikül Bombardımanı İle Gen Aktarımında Fiziksel ve Kimyasal Parametrelerin Etkisi

Yıl 2005, Cilt: 18 Sayı: 2, 241 - 244, 01.09.2005

M. A. Birsin S. Önde M. Özgen

Öz

Bu çalışma partikül bombardımanı sistemi kullanılarak korunga bitkisine (Onobrychis viciifolia Scop) gen aktarımında uygun parametrelerin belirlenmesi amacıyla Ankara Üniversitesi, Ziraat Fakültesi, Tarla Bitkileri Bölümü Biyoteknoloji laboratuarında yürütülmüştür. Çalışmada, eksplant olarak kotiledonlar, mikrotaşıyıcı olarak 1.0 µm çapındaki altın parçacıkları ve belirleyici (markör) gen olarak ise “pB1221.23”plazmidinde bulunan ßglucuronidase (GUS) geni kullanılmıştır. Bombardıman, üç ayrı yöntemle hazırlanan altın partikülleri ile, farklı iki fırlatılma basıncı (1100 ve 1350 psi) ve 6 cm uzaklıktan Bio-Rad®/PDS-1000/He gen transformasyon sistemi kullanılarak gerçekleştirilmiştir. Elde edilen veriler değerlendirildiğinde, korunga kotiledonlarına partikül bombardımanı ile gen aktarımında, doğrudan gliserol ile hazırlanan mikrotaşıyıcıların 1350 psi’lik basınçta fırlatılması ile en iyi sonucu verdiği saptanmıştır

Anahtar Kelimeler

Korunga, Partikül Bombardımanı, Gen Aktarımı, GUS

Effect of Physical and Chemical Parameters on Transient Gene Expression via Particle Bombardment in Sainfoin (Onobrychis viciifolia Scop.)

Öz

This study was carried out at the biotechnology laboratories of Department of Field Crops, Faculty of Agriculture, Ankara University to determine suitable transformation parameters and investigate marker gene transfer possibilities to sainfoin (Onobrychis viciifolia Scop.) using the particle bombardment technique. Sainfoin plant cotyledons were used as plant material; 1.0 µm diameter gold particles as microprojectiles; the plasmid “pB1221.23” containing the ß-glucuronidase (GUS) as marker gene were used in this study. Microprojectiles were prepared in three different methods (directly glycerol, alcohol + water, 3 alcohol + water) Bio-Rad®/PDS-1000 /He gene transfer system was used for bombardment in 1100 and 1350 psi pressures and 6 cm distance. The statistical analysis of results revealed that microprojectiles directly prepared with glycerol and 1350 psi pressure were suitable for particle bombardment delivery marker gene transfer to sainfoin cotyledons.

Anahtar Kelimeler

Onobrychis viciifolia Scop., Particle Bombardment, Transformation, GUS

Kaynakça

• Düzgüneş, O., Kesici, T., Kavuncu, O. ve Gürbüz, F. 1987. Araştırma ve Deneme Metodları. Ankara Üniv. Zir. Fak. Yay. 1021, Ankara.
• Folling, L. and Olesen, A. 2002. Transformation of wheat (Triticum aestivum L.) microspore– derived calluse and microspores by particle bombardment. P. Cell Rep., 20: 1098–1105.
• Golds, T. J., Lee, J. Y., Husnain, T., Ghose, T. K. and Daver, M. R. 1991. Agrobacterium rhizogenesis mediated transformation of the forage legumes Medicago sativa and Onobrychis viciifolia. J. Exp. Bot., 42: 1147- 1157.
• Ingram, H. M., Power, J. B., Lowe, K. C. and Davey, M. R. 1999. Optimisation of procedures for microprojectile bombardment of microspore- derived embryos in wheat. Plant Cell, Tissue and Organ Culture, 56: 207-210.
• Klein, T. M., Fromm, M. E., Wessinger, A., Tomes, D., Schaaf, S., Sleeten, M. and Sanford, J. C. 1988. Transfer of foreign genes into intact maize cells using high velocity microprojectiles. PNAS, 85: 4305–4309.
• Lindsey, K. 1992. Genetic manuplations of crop plants. J. Biotec., 26: 1–28.
• Lonsdale, D., Önde, S. and Cuming, A. 1990. Transit expression of exogenus DNA in intact viable wheat embryos following particle bombardment. J. Exp. Bot., 14: 1161–1165.
• Manners, J. M. and Way, H. 1989. Efficient transformation with regeneration of the tropical pasture legume Stylosanthes humilis using Agrobacterium rhizogenesis and a Ti plasmid binary vector system. P. Cell Rep., 8: 39–51.
• Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Phsiol. Plant, 15: 473–497.
• Önde, S., Sancak, C., Altınok, S., Birsin, M. and Özgen, M. 2001. Transient expression of β- glucuronidase reporter gene in sainfoin (Onobrychis viciifolia Scop.) cotyledons via microprojectile bombardment. Tr. J. Biol., 25: 171-176
• Özcan, S., Yıldız, M., Sancak, C. and Özgen, M. 1996. Advetitious shoot regeneration in sainfoin (Onobrychis viciifolia Scop.). Tr. J. of Bot., 20: 497-501.
• Özgen, M., Özcan, S., Sevimay, C. S., Sancak, C. and Yıldız, M. 1998. High frequency adventitious shoot regeneration in sainfoin. Plant Cell Tissue and Organ Culture, 52: 205-208.
• Rasco–Gaunt, S., Riley, A., Barcelo, P. and Lazzeri, P.A. 1999. Analysis of particle bombardment parameters to optimise DNA delivery into wheat tissues. P. Cell Rep., 19: 118–127.
• Romano, A., Raemarkers, K., Visser, R. and Mooibroek, H. 2001. Transformation of potato (Solanum tuberosum) using patricle bombardment. P. Cell Rep., 20: 198–204.
• Schopke, C., Taylor, N. J., Carcamo, R., Beachy, R. N. and Fauquet, C. 1997. Optimization of parameters for particle bombardment of embriyogenic suspension cultures of cassava (Manihot esculenta Crantz) using computer image analysis. P. Cell Rep., 16: 526-530.
• Somers, D. A., Rines, H. V., Gu, W., Kaeppler, H. F. and Bushnell, W. R. 1992. Fertile transgenic oat plants. Bio-Technology, 10: 1598–1594.
• Spano, L., Mariotte, D., Pezzotti, M., Damiana, F. and Arcioni, S. 1987. Hairy root transformation in alfaalfa (Medicago sativa L.) Theor. App. Genet., 73: 523–530.
• Stougaard, J., Marcker, K. A., Otten, L. and Schell, J. 1986. Nodule specific expression of a chimaeric soybean leghaemoglobin gene in transgenic Lotus corniculatus plant. Nature, 321: 669–674.
• Tadesse, Y., Sagi , L., Swennen, R. and Jacobs, M. 2003. Optimisation of transformation conditions and production of transgenic sorghum (Sorghum bicolor) via microparticle bombardment. Pl. Cell Tiss. Org. Culture, 75: 1-18.
• Tee, C. S. and Maziah, M. 2005. Optimization of biolistic bombardment parameters for Dendrobium sonia 17 calluses using GFP and GUS as the reporter system. Plant Cell Tissue Organ Culture, 80: 77–89.
• Wang, Y. C., Klein, T. M., Fromm, M., Cao, J., Sanford, J. C. and Wu, R. 1988. Transformation of rice, wheat and soybean by the particle bombardment method. Plant. Mol. Bio., 11: 433-439.