Abstract
To improve transgene expression level, we synthesized a truncated insecticidal gene m-cry1Ac by increasing its GC content from 37.4 to 54.8%, based on the codon usage pattern of sugarcane genes, and transferred it into two sugarcane cultivars (ROC16 and YT79-177) by microprojectile bombardment. The integration sites and expression pattern of the transgene were determined, respectively, by Southern, northern and western blot analyses. The transgenic sugarcane lines produced up to 50 ng Cry1Ac protein per mg soluble proteins, which was about fivefold higher than that produced by the partially modified s-cry1Ac (GC% = 47.5%). In greenhouse plant assay, about 62% of the transgenic lines exhibited excellent resistance to heavy infestation by stem borers. In field trials, the m-cry1Ac transgenic sugarcane lines expressing high levels of Cry1Ac were immune from insect attack. In contrast, expression of s-cry1Ac in transgenic sugarcane plants resulted in moderately decreased damages in internodes (0.4–1.7%) and stalks (13.3–26.7%) in comparison with the untransformed sugarcane controls, which showed about 4 and 26–40% damaged internodes and stalks, respectively. Significantly, these transgenic sugarcane lines with high levels of insect resistance showed similar agronomic and industrial traits as untransformed control plants. Taken together, the findings from this study indicate a promising potential of engineering an insect-resistant gene to tailor its protein expression levels in transgenic sugarcane to combat insect infestations.
Similar content being viewed by others
References
Adang MJ (1991) Bacillus thuringiensis insecticidal crystal proteins. In: Maramorosch K (ed) Gene structure, action and utilization. CRC Press, Boca Raton
Arencibia A, Molina PR, Delariva G, Selmanhousein G (1995) Production of transgenic sugarcane (Saccharum officinarum L) plants by intact cell electroporation. Plant Cell Rep 14:305–309
Arencibia A, Vazquez RI, PrietoD TellezP, Carmona ER, Coego A, Hernandez L, DelaRiva GA, SelmanHousein G (1997) Transgenic sugarcane plants resistant to stem borer attack. Mol Breed 3:247–255
Arencibia AD, Carmona ER, Cornide MT, Castiglione S, O’Relly J, Chinea A, Oramas P, Sala F (1999) Somaclonal variation in insect-resistant transgenic sugarcane (Saccharum hybrid) plants produced by cell electroporation. Transgenic Res 8:349–360
Arvinth S, Arun S, Selvakesavan RK, Srikanth J, Mukunthan N, Ananda Kumar P, Premachandran MN, Subramonian N (2010) Genetic transformation and pyramiding of aprotinin-expressing sugarcane with cry1Ab for shoot borer (Chilo infuscatellus) resistance. Plant Cell Rep 29:383–395
Bower R, Birch RG (1992) Transgenic sugarcane plants via microprojectile bombardment. Plant J 2:409–416
Bower R, Elliott AR, Potier BAM, Birch RG (1996) High-efficiency, microprojectile-mediated cotransformation of sugarcane, using visible or selectable markers. Mol Breed 2:239–249
Chong BF, Bonnett GD, Glassop D, O’Shea MG, Brumbley SM (2007) Growth and metabolism in sugarcane are altered by the creation of a new hexose-phosphate sink. Plant Biotechnol J 5:240–253
Chong BF, Abeydeera WPP, Glassop D, Bonnett GD, O’Shea MG, Brumbley SM (2010) Co-ordinated synthesis of gentiobiitol and sorbitol, evidence of sorbitol glycosylation in transgenic sugarcane. Phytochemistry 71:736–741
Christy LA, Arvinth S, Saravanakumar M, Kanchana M, Mukunthan N, Srikanth J, Thomas G, Subramonian N (2009) Engineering sugarcane cultivars with bovine pancreatic trypsin inhibitor (aprotinin) gene for protection against top borer (Scirpophaga excerptalis Walker). Plant Cell Rep 28:175–184
Enríquez GA, Trujillo LE, Menéndez C, Vázquez-Padrón R I, Tiel K, Dafhnis F, et al (2000) Sugarcane (Saccharum hybrid) genetic transformation mediated by Agrobacterium tumefaciens: production of transgenic plants expressing proteins with agronomic and industrial value. In: Arencibia A (ed) Developments in plant genetics and breeding. Elsevier, Amsterdam, pp 76–81
Estruch JJ, Carozzi NB, Desai N, Duck NB, Warren GW, Koziel MG (1997) Transgenic plants: an emerging approach to pest control. Nat Biotechnol 15:137–141
Falco MC, Silva-Filho MC (2003) Expression of soybean proteinase inhibitors in transgenic sugarcane plants: effects on natural defense against Diatraea saccharalis. Plant Physiol Biochem 41:761–766
Fauconnier R (1993) Sugar cane. Macmillan, London
Gilbert RA, Glynn NC, Comstock JC, Davis MJ (2009) Agronomic performance and genetic characterization of sugarcane transformed for resistance to sugarcane yellow leaf virus. Field Crops Res 111:39–46
Herbert A, Rich A (1999) Left-handed Z-DNA: structure and function. Genetica 106:37–47
Hillocks RJ, Waller JM (1997) Soilborne diseases of tropical crops. CAB International, Wallingford
Ingelbrecht IL, Irvine JE, Mirkov TE (1999) Posttranscriptional gene silencing in transgenic sugarcane. Dissection of homology-dependent virus resistance in a monocot that has a complex polyploid genome. Plant Physiol 119:1187–1197
Jain M, Chengalrayan K, Abouzid A, Gallo M (2007) Prospecting the utility of a PMI/mannose selection system for the recovery of transgenic sugarcane (Saccharum spp. hybrid) plants. Plant Cell Rep 26:581–590
Kim S, Kim C, Li W, Kim T, Li Y, Zaidi MA, Altosaar I (2008) Inheritance and field performance of transgenic Korean Bt rice lines resistant to rice yellow stem borer. Euphytica 164:829–839
McQualter RB, Dale JL, Harding RM, McMahon JA, Smith GR (2004) Production and evaluation of transgenic sugarcane containing a Fiji disease virus (FDV) genome segment S9-derived synthetic resistance gene. Aust J Agric Res 55:139–145
Murray EE, Rocheleau T, Eberle M, Stock C, Sekar V, Adang M (1991) Analysis of unstable RNA transcripts of insecticidal crystal protein genes of Bacillus-thuringiensis in transgenic plants and electroporated protoplasts. Plant Mol Biol 16:1035–1050
Perlak FJ, Deaton RW, Armstrong TA, Fuchs RL, Sims SR, Greenplate JT, Fischhoff DA (1990) Insect resistant cotton plants. Bio-Technology 8:939–943
Perlak FJ, Fuchs RL, Dean DA, McPherson SL, Fischhoff DA (1991) Modification of the coding sequence enhances plant expression of insect control protein genes. Proc Natl Acad Sci USA 88:3324–3328
Perlak FJ, Stone TB, Muskopf YM, Petersen LJ, Parker GB, McPherson SA, Wyman J, Love S, Reed G, Biever D, Fischhoff DA (1993) Genetically improved potatoes––protection from damage by Colorado potato beetles. Plant Mol Biol 22:313–321
Prodromou C, Pearl LH (1992) Recursive PCR––a novel technique for total gene synthesis. Protein Eng 5:827–829
Ricaud C, Egan BT, Gillaspie AG, Hughes CG (1989) Diseases of sugarcane. Elsevier, Netherlands
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring
Tekaia F, Yeramian E, Dujon B (2002) Amino acid composition of genomes, lifestyles of organisms, and evolutionary trends: a global picture with correspondence analysis. Gene 297:51–60
Valderrama AM, Velasquez N, Rodriguez E, Zapata A, Zaidi MA, Altosaar I, Arango R (2007) Resistance to Tecia solanivora (Lepidoptera : Gelechiidae) in three transgenic Andean varieties of potato expressing Bacillus thuringiensis Cry1Ac protein. J Econ Entomol 100:172–179
Verwoerd TC, Dekker BMM, Hoekema A (1989) A small-scale procedure for the rapid isolation of plant RNAs. Nucleic Acids Res 17:2362
Vinogradov AE (2003) DNA helix: the importance of being GC-rich. Nucleic Acids Res 31:1838–1844
Weng LX, Deng HH, Xu JL, Li Q, Wang LH, Jiang ZD, Zhang HB, Li QW, Zhang LH (2006) Regeneration of sugarcane elite breeding lines and engineering of stem borer resistance. Pest Manag Sci 62:178–187
Zhang LH, Xu JL, Birch RG (1998) High affinity binding of albicidin phytotoxins by the AlbA protein from Klebsiella oxytoca. Microbiology 144:555–559
Zhang LH, Xu JL, Birch RG (1999) Engineered detoxification confers resistance against a pathogenic bacterium. Nat Biotechnol 17:1021–1024
Zhang LH, Weng LX, Jiang ZD (2007) Sugarcane. Springer, Berlin
Acknowledgments
We are grateful for funding from the Agency of Science, Technology, and Research (A*Star), Singapore; and for the funds provided by the Industry–University Research Foundation of Guangdong province and the Ministry of Education in China under the grant 2008B090500227, the National Natural Science Foundation of China under the grant 90813010, and the Shanghai Natural Science Foundation under the grant 08ZR1401400.
Author information
Authors and Affiliations
Corresponding authors
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Weng, LX., Deng, HH., Xu, JL. et al. Transgenic sugarcane plants expressing high levels of modified cry1Ac provide effective control against stem borers in field trials. Transgenic Res 20, 759–772 (2011). https://doi.org/10.1007/s11248-010-9456-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11248-010-9456-8