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LAPORAN AKHIR PENELITIAN UNGGULAN PERGURUAN TINGGI
PERAKITAN VARIETAS KENTANG TAHAN PHYTOPTHORA INFESTANS SECARA MOLEKULER
Tahun ke-1 dari rencana 3 tahun Oleh: TIM PENGUSUL : Ketua
: Dr.Ir.Syamsuddin Djauhari MS (NIDN 0022055509) Prof.Dr.Liliek Setyawati (NIDN 0012125508) Dr. Istiyono Kirnoprasetyo SP.MP (NIDN 0001024602) Mochammad Roviq SP.MP (NIDN 0005017503)
Dibiayai oleh: Direktorat Jenderal Pendidikan Tinggi, Kementrian Pendidikan dan Kebudayaan, Melalui DIPA Universitas Brawijaya Nomor: 023.04.2.414989/2014, Tanggal 5 Desember 2013 dan berdasarkan SK Rektor Univesitas Brawijaya Nomor 157 Tahun 2014 Tanggal 10 April 2014
UNIVERSITAS BRAWIJAYA NOPEMBER 2014
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RINGKASAN
Penyakit utama kentang (Solanum tuberosum) adalah Phytopthora infestans yang dapat menghancurkan tanaman sampai 100% hanya dalam waktu semalam. Selama ini petani hanya mengandalkan fungisida yang diberikan setiap 2-3 hari, sehingga biayanya mahal dan juga menyebabkan pencemaran lingkungan yang dampaknya tidak hanya hewan dan manusia tetapi juga microba. Salah satu upaya untuk menghindari serangan penyakit tersebut adalah menghasilkan tanaman tahan Phytopthora infestans yang dapat dihasilkan melalui pemuliaan konvensional maupun melalui pemuliaan modern yaitu transformasi genetic. Ada beberapa cara insersi gen kedalam sel tanaman antara lain yaitu melalui protoplast. Penelitian akan dilakukan untuk 3 tahun yaitu tahun pertama insersi gen melalui kalus dan penyiapan protoplast kemudian insersi gen Glu kedalam protoplast. Hasil penelitian tahun pertama diperoleh beberapa kalus yang telah berhasil diinsersi oleh gen Glu dan protoplast yang telah berhasil diisolasi, walaupun belum semurna. Pada penelitian tahun kedua ini akan dilakukan insersi gen melalui protoplast yang akan dilanjutkan dengan regenerasi sel menjadi planlet. Pada saat yang sama planlet hasil insersi gen Glu melalui kalus atau protoplasi akan dilakukan pengujian ekspresi melalui imunohistoassay, ELISA dan PCR. Planlet kemudian diaklimatisasi menjadi tanaman kentang dan diuji ketahanannya terhadap Phytophthora infestans dalam skala laboratorium dan lapang pada tahun ketiga.
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DAFTAR PUSTAKA ABSP II, 2008; Hawar Daun. Single Project Report.http://www.absp2.comel.edu/project. 13 Januari 2008 Andreu, A., C. Tono´ n, M. Van Damme, M. Huarte and G. Daleo. 1998. Effect of glucans from different races of Phytophthora infestans on defense reactions in potato tuber. Eur. J. Plant Pathol. 104:777–783. Anfoka, G. and H. Buchenauer. 1997. Systemic acquired resistance in tomato against Phytophthora infestans by pre-inoculation with tobacco necrosis virus. Physiol. Mol. Plant Pathol. 50: 85–101. Awade A, L. DeTapla, Diderjean and L. Burkard. 1989. Biological function of bean pathogenesis- related proteins. Plant Science 63: 121-130. Aziz, N. and G.C. Machray. 2003. Efficient male germ line transformation for transgenic tobacco production without selection. Plant Molecular Biology, 51(2): p. 203-211 Baldauf,S.L. 2003. The deep roots of eucaryotes. Science 300 : 1703-1706. Bartnicki Garcia, S and M.C. Wang, 1983. Biochemical aspewcts of morphogenesis in Phytopthora. Pages 121-123 in : Phytopthora . edited by D.C.Erwin,S.BartnickiGarcia, and P.H.Tsao. American Phytopathological Society Press. St.Paul,MN USA. Beerhues, L. and E. Kombrink. 1994. Primary structure and expression of mRNAs encoding basic chitinase and 1,3-beta-glucanase in potato. Plant Mol Biol. 24(2):353-67. Birch, R.G. 1997. Plant transformation: Problems and strategies for practical application. Annu Rev Plant Physiol. Plant Mol Biol 48: 297-326. Birch P.R.J. and S.C.Whisson. 2001. Phytopthiora infestans enters the genomic era.Moleculer Plant Pathology 2(5) : 257-263 Bowles, D.J. 1990. Defence-related proteins in higher plants. Annu. Rev. Biochem. 59: 873– 907 BPS 2011. Statistik Pertanian. Balai Pusat Statistik. Bucciaglia P. A. and A.G. Smith. 1994. Cloning and characterization of Tag1, a tobacco anther β-1,3-glucanase expressed during tetrad dissolution. Plant Mol. Biol. 24: 903– 914. Chen,Q., L.M.Kawchuk, D.R.Lynch , M.S.Goettel, and D.K.Fujimatto. 2003.Identification of late blight, Colorado potato beetle, and blackleg resistance in three mexican and two south American wild solanum species. American J.of Potato Res.80 : 9-19 Chen, W.S., C.C. Chiu, H.Y. Liu, T.L. Lee, J.T. Chen,C.C. Lin, Y.J. Wu, and H.Y. Chang. 1998. Gene transfer via pollen-tube pathway for anti-fusarium wilt in watermelon. Biochem Mol Biol Int 46: 1201-1209. Chilton, M.D., M.H. Drummond, D.J. Merio, D. Sciaky, A.L. Montoya, M.P. Gordon and E.W. Nester. 1977. Stable incorporation of plasmid DNA into higher plant cells: the molecular basis of crown gall tumorigenesis. Cell 11: 263–271 Christou, P. 1995. Strategies for variety-independent genetic transformation of important cereals, legumes, and woody species utilizing particle bombardment. Euphytica 85:1327. Cocking, E.C. and M.R. Davey. 1987. Gene transfer in cereals. Science 236: 1259-1262
25
Côté, F., and M.G. Hahn. 1994. Oligosaccharins: Structures and signal transduction. Plant Mol. Biol. 26: 1379–1411 DeBonte, L.R. and Loh W.H.T. 1991. Method for the manipulation of pollen in plants. http://www.google.com/patents/US5066594 Ebel, J., and E.G. Cosio. 1994. Elicitors of plant defense responses. Int. Rev. Cytol. 148: 1– 36 FAO, 2010. FAO Statistical.http://faostat.fao.org/site/567. 17 Juli 2010. Felix G. and F. Meins Jr. 1986. Developmental and hormonal regulation of β-1,3-glucanase in tobacco. Planta 167: 206–211. Fromm, M., L. Taylor, and V. Walbot. 1985. Expression of genes transferred into monocot and dicot plant cells by electroporation. Proc. Natl. Acad. Sci. USA 82:5824-5828. Joosten, M.H.A.J., and P.J.G.M. De Wit. 1989. ldentification of severa1 pathogenesis-related proteins in tomato leaves inoculated with Cladosporium fulvum (syn. Fulvia fulva) as 1,3-pglucanases and chitinases. Plant Physiol. 89, 945-951. Kauffmann S., M. Legrand, P. Geoffroy and B. Fritig.1987. Biological function of ‘pathogenesis-related’ proteins: four PR proteins of tobacco have 1,3-β-glucanase activity.EMBO J. 6: 3209–3212. Kim, S.-S.; Shin, D-II. and Park, H.-S. 2007. Transient β-glucuronidase expression in lily (Lilium longflorumL.) pollen via wounding-assisted Agrobacterium-mediated transformation.Biotechnology Letters, vol. 29, no. 6, p. 965-969. Kim, Y. and B. Hwang. 1997. Isolation of a 34 kiloDalton b-1,3-glucanase with inhibitory activity against Phytophthora capsici from pepper stems. Physiol. Mol. Plant Pathol. 50: 103–115. Kombrink, E., M. Schröder and K. Hahlbrock. 1988. Several "pathogenesis-related" proteins in potato are 1,3-beta-glucanases and chitinases. Proc Natl Acad Sci U S A. 85(3):7826. Krens, F. A., L. Molendijk, G. J. Wullens and R. A. Schilperoort, 1982. In vitro transformation of plant protoplasts with Ti-plasmid DNA. Nature 296: 72-74. Kustiari,R.,P.Simatupang,D.Ketut Sndra, Wahida, A.Purwoto, H.J.purba dan T.Nurasa. 2009. Model Proyeksi jangka pendek permintaan dan penawaran komoditas pertanian utama. Pusat Analisis Kajian Ekonomi dan Kebijakan Pertanian, Departemen Pertanian. Lei, B.J, C.H. Lu, H. Qian, X.C. Li, Y.B. Lu, K. Zhao, G.Y. Liu, and X.Y. Yang. 1995. New soybean strains of high protein and double high content obtained from introduction of exogenous total DNA. Soybean Sci 14: 203-208. Linthorst, H.J. 1991. Pathogenesis-related proteins of plants. Crit. Rev. Plant Sci. 10: 123– 150. Liu, D., K. G. Raghothama, P. M. Hasegawa, R. A. Bressan. 1994. Osmotin overexpression in potato delays development of disease symptoms. Proc. Natl Acad. Sci. USA 91: 1888– 1892. Lörz, H., Baker, B. and Schell, J. (1985). Gene transfer to cereal cells mediated by protoplast transformation. Molecular General Genetics 199: 473-497. Luo, Z.X. and R. Wu. 1989. A simple method for the transformation of rice via the pollentube pathway. Plant Mol Biol Rep 7: 69-77
26
Mauch, F., B. Mauch-Mani, and T. Boller. 1988. Antifungal hydrolases in pea tissue. II. Inhibition of fungal growth by combinations of chitinase and β-1,3-glucanase. Plant Physiol. 88: 936–942. Nasser, W., M. De Tapia and G. Burkard. 1990. Maize pathogenesisrelated proteins: characterization and cellular distribution of b-1,3-glucanases and chitinases induced by brome mosaic virus infection or mercuric chloride treatment. Physiol. Mol. Plant Pathol. 36, 1–14. Neumann, E., M. Schäefer-Ridder, M. Wang and P.H. Hofschneider. 1982. Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J. 1:841-845 Nickoloff, J.A. (Ed.). 1995. Electroporation Protocols for Microorganisms. Methods in Molecular Biology, vol. 47. Humana Press. Totowa. Niderman, T., I. Genetet, T. Bruye´ re, R. Gees, A. Stintzi, M. Legrand, B. Fritig and E. Mosinger. 1995. Pathogenesis-related PR-1 proteins are antifungal. Plant Physiol. 108: 17–27. Niedz, R.P., W. L. McKendree, R. G. Shatters Jr. 2003. Electroporation of embryogenic protoplasts of sweet orange (Citrus sinensis (l.) Osbeck) and regeneration of transformed plants. Plant 39(6):586-594 Ori N., G. Sessa, T. Lotan, S. Himmelhoch and R. Fluhr. 1990. A major stylar polypeptide (sp41) is a member of the pathogenesis-related proteins superclass. Embo J. 9:3429– 3436. Payne G., E. Ward, T. Gaffney, P. Ahl Goy and M. Moyer. 1990. Evidence for a third structural class of β-1,3-glucanase in tobacco. Plant Mol. Biol. 15: 797–808 Purwanti, H.2002. Penyakit hawar daun (Phytopthora infestans (Mont deBary) pada kentang dan tomat: Identifikasi permasalahan di Indonesia. Buletin AgroBio 5(2) : 67-72 Rose, J.K.C., KS Ham, A.G. Darvill and P. Albersheim. 2002. Molecular Cloning and Characterization of Glucanase Inhibitor Proteins.The Plant Cell 14(6): 1329-1345 Stinzi, A., T. Heitz, V. Prasad, S. Weidemann-Merdinoglu, S. Kauffmann, P. Geoffroy, M. Legrand, and B. Fritig. 1993. Plant “pathogenesis-related” proteins and their role in defense against pathogens.Biochimie 75: 687–706 Tada, Y., M. Sakamoto and T. Fujimura T. 1990. Efficient gene introduction into rice by electroporation and analysis of transgenic plants: use of electroporation buffer lacking chloride ions. Theor. Appl. Genet. 80:475-480 Tonon, G., G. Guevara, G. Oliva and G. Daleo. 2003. Isolation of a Potato Acidic 39 kDa β1,3glucanase with Antifungal Activity against Phytophthora infestans and Analysis of its Expression in Potato Cultivars Differing in their Degrees of Field Resistance. Van den Bulcke M., G. Bauw, C. Castresana, M. Van Montagu and J. Vandekerckhove. 1989. Characterization of vacuolar and extracellular β-(1,3)-glucanases of tobacco: evidence for a strictly compartmentalized plant defense system. Proc. Natl. Acad. Sci. USA 86: 2673–2677. Van Wert, S.L. and J.A. Saunders. 1992. Electrofusion and Electroporation of Plants. Plant Physiol . 99: 365-367
27
Vieira, M.L.C., B. Jones, E.C. Cocking and M.R. Davey. 1990. Plant regeneration from protoplasts isolated from seedling cotyledons of Stylosanthes guianensis, S. macrocephala and S. scabra. Plant Cell Rep. 9:289-292 Vogelsang, R.., and W. Barz. 1993. Purification, characterization and differential hormonal regulation of a beta-1,3-glucanase and two chitinases from chickpea (Qcerarierinum L). Planta 189, 60-69. Walbot V. 1993. Gene pulser electroprotocols, survey number 181, Bio-Rad Laboratories, Hercules-CA. Wang, W., J. Wang, C. Yang, Y. Li, L. Liu and J. Xu.2007. Pollen-mediated transformation of Sorghum bicolor plants. Biotechnol. Appl. Biochem. 48, 79–83 Wessels, J.G.H. and J.H. Sietsma. 1981. Significance of linkages between chitin and ßGlucan in fungal walls. Annual Volume on Microbiology. Microbiology: 232-234. Yu, H.X., J.J. Liu, Z.L. Feng, and J.D. Dong. 1999. Study on introduction of verminresistance gene (CpTI) into wheat through pollen-tube pathway method. J ShanDong Agric Sci 99: 5-8. Zhang, Z., X. Aiqiu, P. Staswick and T.E. Clemente. 1999. The use of glufosinate as a selective agent in Agrobacterium-mediated transformation of soybean. Plant Cell Tissue Org Cult 56: 37-46. Zhou, G.Y., J. Weng , Y.S. Zhen, J.G. Huang, S.Y. Qian, and G.L. Liu. 1983. Introduction of exogenous DNA into cotton embryos. In: Wu R, Grossman L and Molddave K (eds), Methods in enzymology, Vol 101, Recombination DNA, Part C, pp 433-481. Academic Press, New York.
