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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.