Improved Genetic Transformation of Sugarcane (Saccharum spp.) Embryogenic Callus Mediated by Agrobacterium tumefaciens

Marcos Fernando Basso1, Bárbara Andrade Dias Brito da Cunha1, Ana Paula Ribeiro1, Polyana Kelly Martins1, Wagner Rodrigo de Souza1, Nelson Geraldo de Oliveira1, Thiago Jonas Nakayama1, Raphael Augusto das Chagas Noqueli Casari1, Thais Ribeiro Santiago1, Felipe Vinecky1, Letícia Jungmann Cançado1, Carlos Antônio Ferreira de Sousa1, Patricia Abrão de Oliveira1, Silvana Aparecida Creste Dias de Souza2, Geraldo Magela de Almeida Cançado3, Adilson Kenji Kobayashi1, Hugo Bruno Correa Molinari1

1 Genetics and Biotechnology Laboratory, National Center for Agroenergy Research (CNPAE), Brazilian Agricultural Research Corporation (EMBRAPA), Brasília, Distrito Federal, 2 Agronomic Institute of Campinas (IAC), Ribeirão Preto, São Paulo, 3 The Joint Research Unit for Genomics Applied to Climate Change (UMIP GenClima), National Center for Agricultural Informatics (CNPTIA), Brazilian Agricultural Research Corporation (EMBRAPA), Campinas, São Paulo
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20055
Online Posting Date:  September, 2017
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


Sugarcane (Saccharum spp.) is a monocotyledonous semi‐perennial C4 grass of the Poaceae family. Its capacity to accumulate high content of sucrose and biomass makes it one of the most important crops for sugar and biofuel production. Conventional methods of sugarcane breeding have shown several limitations due to its complex polyploid and aneuploid genome. However, improvement by biotechnological engineering is currently the most promising alternative to introduce economically important traits. In this work, we present an improved protocol for Agrobacterium tumefaciens‐mediated transformation of commercial sugarcane hybrids using immature top stalk‐derived embryogenic callus cultures. The callus cultures are transformed with preconditioned A. tumefaciens carrying a binary vector that encodes expression cassettes for a gene of interest and the bialaphos resistance gene (bar confers resistance to glufosinate‐ammonium herbicide). This protocol has been used to successfully transform a commercial sugarcane cultivar, SP80‐3280, highlighting: (i) reduced recalcitrance and oxidation; (ii) high yield of embryogenic callus; (iii) improved selection; and (iv) shoot regeneration and rooting of the transformed plants. Altogether, these improvements generated a transformation efficiency of 2.2%. This protocol provides a reliable tool for a routine procedure for sugarcane improvement by genetic engineering. © 2017 by John Wiley & Sons, Inc.

Keywords: genetic engineering; transgenic bioenergy crops; agrotransformation; immature top stalks; cultivar SP80‐3280

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Transformation of Sugarcane (Saccharum spp.) Embryogenic Callus Mediated by Agrobacterium tumefaciens and Selection via bar Gene
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
PDF or HTML at Wiley Online Library


Basic Protocol 1: Transformation of Sugarcane (Saccharum spp.) Embryogenic Callus Mediated by Agrobacterium tumefaciens and Selection via bar Gene

  • Spindle sections from field‐grown sugarcane cultivars SP80‐3280 or RB855156
  • Immature top stalks from six to ten‐month old, field‐grown sugarcane plants
  • 70% (v/v) ethanol
  • Sugarcane callus induction medium (SCIM 3; see recipe)
  • Agrobacterium tumefaciens EHA105 strain harboring binary vectorThe binary vector encodes the following: (i) a plant selectable‐marker gene (i.e., the bar gene from Streptomyces hygroscopicus) under the control of the constitutive Oryza sativa actin promoter (OsAct) and the 3′ rbcsE9 transcription termination signal; (ii) the gene of interest controlled by the constitutive Zea mays ubiquitin promoter (ZmUbi) interrupted by an intron (Christensen & Quail, ) and the nopaline synthase transcription termination signal (Tnos); (iii) ColE1 and pVS1 origins of replication; and (iv) the spectinomycin/streptomycin (Sp/Sm) resistance gene.
  • Yeast Extract Broth (YEB) Agrobacterium growth medium (see recipe)
  • Agrobacterium (AB) minimal medium for bacteria preconditioning (see recipe)
  • 100 mM acetosyringone stock solution (see recipe)
  • Half‐strength Murashige and Skoog basal liquid medium (see recipe)
  • 10% Poloxamer 188 solution (Sigma‐Aldrich, cat. no. P5556)
  • Solid co‐cultivation medium (SCCM; see recipe)
  • Callus pre‐selection medium (CPSM) with or without herbicide (see recipe)
  • Ticarcillin stock solution (see recipe)
  • Glufosinate‐ammonium stock solution (see recipe)
  • Callus regeneration medium containing 6‐benzylaminopurine (CRMBAP; see recipe)
  • Sugarcane rooting medium (SRM; see recipe)
  • Soil, substrate, and vermiculite mixture
  • 1‐liter glass bottles, for disinfection of top stalks
  • Laminar air flow cabinet
  • Orbital shaker
  • 150‐mm × 20‐mm Petri dishes, glass and polystyrene
  • Sterile Whatman filter paper
  • Sterile stainless steel scalpel, blade no. 24
  • 90‐mm × 20‐mm polystyrene Petri dishes for leaf disc cultures (callus induction)
  • Growth chamber set at 25° to 27°C, 16/8 hr light/dark photoperiod (luminous intensity of at least 500 µmol m−2 s−1) and with controlled relative humidity 65%
  • Light‐emitting diode (LED) lamp: e.g., GreenPower TLED (tubular), Philips, Model 9290008431, 20W, 220‐240V, 50/60 Hz, Photon flux: 24 µmol/s, Red:Blue 2:1
  • 90‐mm × 15‐mm polystyrene Petri dishes for callus culture
  • Stereomicroscope
  • 50‐ml conical tubes
  • Orbital shaker with temperature control
  • Spectrophotometer to measure optical density
  • 200‐ml Erlenmeyer flask for Agrobacterium growth
  • Magenta vessel GA‐7 (Sigma‐Aldrich, cat. no. V8505)
  • Plastic pots for plant growth
  • Greenhouse
PDF or HTML at Wiley Online Library



Literature Cited

  Anderson, D. J., & Birch, R. G. (2012). Minimal handling and super‐binary vectors facilitate efficient, Agrobacterium‐mediated, transformation of sugarcane (Saccharum spp. hybrid). Tropical Plant Biology, 5, 183‐192. doi: 10.1007/s12042‐012‐9101‐1.
  Arencibia, A. D., Carmona, E. R., Tellez, P., Chan, M.‐T., Yu, S.‐M., Trujillo, L. E., & Oramas, P. (1998). An efficient protocol for sugarcane (Saccharum spp. L.) transformation mediated by Agrobacterium tumefaciens. Transgenic Research, 7, 213–222. doi: 10.1023/A:1008845114531.
  Armstrong, C., Duncan, D., Kemper, E. L., & Oliveira, S. B. F. (2015). Sugarcane regeneration and transformation methods. WO2015099674 A1.
  Armstrong, C., Duncan, D., & Sidorov, V. (2005). A novel method for agrobacterium transformation for dihaploid corn plants. US20040210959 A1.
  Barros, G. O., Ballen, M. A., Woodard, S. L., Wilken, L. R., White, S. G., Damaj, M. B., … Nikolov, Z. L. (2013). Recovery of bovine lysozyme from transgenic sugarcane stalks: Extraction, membrane filtration, and purification. Bioprocess and Biosystems Engineering, 36, 1407–1416. doi: 10.1007/s00449‐012‐0878‐y.
  Basnayake, S. W., Moyle, R., & Birch, R. G. (2011). Embryogenic callus proliferation and regeneration conditions for genetic transformation of diverse sugarcane cultivars. Plant Cell Reports, 30, 439–448. doi: 10.1007/s00299‐010‐0927‐4.
  Bewg, W. P., Poovaiah, C., Lan, W., Ralph, J., & Coleman, H. D. (2016). RNAi downregulation of three key lignin genes in sugarcane improves glucose release without reduction in sugar production. Biotechnology for Biofuels, 9, 270. doi: 10.1186/s13068‐016‐0683‐y.
  Bower, R., & Birch, R. G. (1992). Transgenic sugarcane plants via microprojectile bombardment. The Plant Journal, 2, 409–416. doi: 10.1111/j.1365‐313X.1992.00409.x.
  Cheavegatti‐Gianotto, A., de Abreu, H. M., Arruda, P., Bespalhok Filho, J. C., Burnquist, W. L., Creste, S., … Cesar Ulian, E. (2011). Sugarcane (Saccharum X Officinarum): A reference study for the regulation of genetically modified cultivars in Brazil. Tropical Plant Biology, 4, 62–89. doi: 10.1007/s12042‐011‐9068‐3.
  Chen, W. H., Gartland, K. M., Davey, M. R., Sotak, R., Gartland, J. S., Mulligan, B. J., … Cocking, E. C. (1987). Transformation of sugarcane protoplasts by direct uptake of a selectable chimaeric gene. Plant Cell Reports, 6, 297–301. doi: 10.1007/BF00272003.
  Christensen, A. H., & Quail, P. H. (1996). Ubiquitin promoter‐based vectors for high‐level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgenic Research, 5, 213–218. doi: 10.1007/BF01969712.
  Dong, S., Delucca, P., Geijskes, R.J., Ke, J., Mayo, K., Mai, P., … Dunder, E. (2014). Advances in Agrobacterium‐mediated sugarcane transformation and stable transgene expression. Sugar Tech, 16, 366‐371.doi: 10.1007/s12355‐013‐0294‐x.
  Enríquez‐Obregón, G. A., Vázquez‐Padrón, R. I., Prieto‐Samsonov, D. L., De la Riva, G. A., & Selman‐Housein, G. (1998). Herbicide‐resistant sugarcane (Saccharum officinarum L.) plants by Agrobacterium‐mediated transformation. Planta, 206, 20–27. doi: 10.1007/s004250050369.
  Figueira, T. R. S., Okura, V., Rodrigues da Silva, F., Jose da Silva, M., Kudrna, D., Ammiraju, J. S. S., … Arruda, P. (2012). A BAC library of the SP80‐3280 sugarcane variety (Saccharum sp.) and its inferred microsynteny with the sorghum genome. BMC Research Notes, 5, 185–185. doi: 10.1186/1756‐0500‐5‐185.
  Fouad, W. M., Hao, W. U., Xiong, Y., Steeves, C., Sandhu, S. K., & Altpeter, F. (2015). Generation of transgenic energy cane plants with integration of minimal transgene expression cassette. Current Pharmaceutical Biotechnology, 16, 407–413. doi: 10.2174/1389201016666150303151559.
  Fronzes, R., Christie, P. J., & Waksman, G. (2009). The structural biology of type IV secretion systems. Nature Reviews Microbiology, 7, 703–714. doi: 10.1038/nrmicro2218.
  Gelvin, S. B. (2006). Agrobacterium virulence gene induction. Methods in Molecular Biology, 343, 77–84. doi: 10.1385/1‐59745‐130‐4:77.
  Goldemberg, J. (2008). The Brazilian biofuels industry. Biotechnology for Biofuels, 1, 1–7. doi: 10.1186/1754‐6834‐1‐6.
  Guo, J., Gao, S., Lin, Q., Wang, H., Que, Y., & Xu, L. (2015). Transgenic sugarcane resistant to Sorghum mosaic virus based on coat protein gene silencing by RNA interference. BioMed Research International, 2015, 861907. doi: 10.1155/2015/861907.
  Kalunke, R. M., Kolge, A. M., Babu, K. H., & Prasad, D. T. (2009). Agrobacterium mediated transformation of sugarcane for borer resistance using Cry 1Aa3 gene and one‐step regeneration of transgenic plants. Sugar Tech, 11, 355–359. doi: 10.1007/s12355‐009‐0061‐1.
  Kang, Q., Appels, L., Tan, T., & Dewil, R. (2014). Bioethanol from lignocellulosic biomass: Current findings determine research priorities. The Scientific World Journal, 2014, 1–14. doi: 10.1155/2014/298153.
  Kumar, T., Uzma Khan, M. R., Abbas, Z., & Ali, G. M. (2014). Genetic improvement of sugarcane for drought and salinity stress tolerance using Arabidopsis vacuolar pyrophosphatase (AVP1) gene. Molecular Biotechnology, 56, 199–209. doi: 10.1007/s12033‐013‐9695‐z.
  Limayem, A., & Ricke, S. C. (2012). Lignocellulosic biomass for bioethanol production: Current perspectives, potential issues and future prospects. Progress in Energy and Combustion Science, 38, 449–467. doi: 10.1016/j.pecs.2012.03.002.
  Manickavasagam, M., Ganapathi, A., Anbazhagan, V. R., Sudhakar, B., Selvaraj, N., Vasudevan, A., & Kasthurirengan, S. (2004). Agrobacterium‐mediated genetic transformation and development of herbicide‐resistant sugarcane (Saccharum species hybrids) using axillary buds. Plant Cell Reports, 23, 134–143. doi: 10.1007/s00299‐004‐0794‐y.
  Martins, M. T. B., de Souza, W. R., da Cunha, B. A. D.B., Basso, M. F., de Oliveira, N. G., Vinecky, F., … Molinari, H. B. C. (2016). Characterization of sugarcane (Saccharum spp.) leaf senescence: Implications for biofuel production. Biotechnology for Biofuels, 9, 153. doi: 10.1186/s13068‐016‐0568‐0.
  Matias de Oliveira, D., Finger‐Teixeira, A., Rodrigues Mota, T., Salvador, V. H., Moreira‐Vilar, F. C., Correa Molinari, H. B., … Dantas dos Santos, W. (2014). Ferulic acid: A key component in grass lignocellulose recalcitrance to hydrolysis. Plant Biotechnology Journal, 13, 1224–1232. doi: 10.1111/pbi.12292.
  Mayavan, S., Subramanyam, K., Arun, M., Rajesh, M., Kapil Dev, G., Sivanandhan, G., … Ganapathi, A. (2013). Agrobacterium tumefaciens‐mediated in planta seed transformation strategy in sugarcane. Plant Cell Reports, 32, 1557–1574. doi: 10.1007/s00299‐013‐1467‐5.
  Mayavan, S., Subramanyam, K., Jaganath, B., Sathish, D., Manickavasagam, M., & Ganapathi, A. (2015). Agrobacterium‐mediated in planta genetic transformation of sugarcane setts. Plant Cell Reports, 34, 1835–1848. doi: 10.1007/s00299‐015‐1831‐8.
  Molinari, H. B. C., Marur, C. J., Daros, E., de Campos, M. K. F., de Carvalho, J. F. R.P., Bespalhok, J. C., … Vieira, L. G. E. (2007). Evaluation of the stress‐inducible production of proline in transgenic sugarcane (Saccharum spp.): Osmotic adjustment, chlorophyll fluorescence and oxidative stress. Physiology Plantarum, 130, 218–229. doi: 10.1111/j.1399‐3054.2007.00909.x.
  Mudge, S. R., Basnayake, S. W., Moyle, R. L., Osabe, K., Graham, M. W., Morgan, T. E., & Birch, R. G. (2013). Mature‐stem expression of a silencing‐resistant sucrose isomerase gene drives isomaltulose accumulation to high levels in sugarcane. Plant Biotechnology Journal, 11, 502–509. doi: 10.1111/pbi.12038.
  Murashige, T., & Skoog, F. (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiology Plantarum, 15, 473–497. doi: 10.1111/j.1399‐3054.1962.tb08052.x.
  Ogawa, Y., & Mii, M. (2007). Meropenem and moxalactam: Novel β‐lactam antibiotics for efficient Agrobacterium‐mediated transformation. Plant Science, 172, 564–572. doi: 10.1016/j.plantsci.2006.11.003.
  Petrasovits, L. A., Zhao, L., McQualter, R. B., Snell, K. D., Somleva, M. N., Patterson, N. A., … Brumbley, S. M. (2012). Enhanced polyhydroxybutyrate production in transgenic sugarcane. Plant Biotechnology Journal, 10, 569–578. doi: 10.1111/j.1467‐7652.2012.00686.x.
  Rathus, C., & Birch, R. G. (1992). Stable transformation of callus from electroporated sugarcane protoplasts. Plant Science, 82, 81–89. doi: 10.1016/0168‐9452(92)90010‐J.
  Reis, R. R., Andrade Dias Brito da Cunha, B., Martins, P. K., Martins, M. T. B., Alekcevetch, J. C., Chalfun‐Júnior, A., … Molinari, H. B. C. (2014). Induced over‐expression of AtDREB2A CA improves drought tolerance in sugarcane. Plant Science, 221‐222, 59–68. doi: 10.1016/j.plantsci.2014.02.003.
  Ribeiro, A. P. (2016). Transformação de Setaria viridis e Saccharum spp. e com o gene MATE visando tolerância ao alumínio, Programa de Pós‐Graduação em Biotecnologia Vegetal Universidade Federal de Lavras, 105.
  Savage, N. (2011). Fuel options: The ideal biofuel. Nature, 474, S9–11. doi: 10.1038/474S09a.
  Souza, G. M., Berges, H., Bocs, S., Casu, R., D'Hont, A., Ferreira, J. E., … Paterson, A. H. (2011). The sugarcane genome challenge: Strategies for sequencing a highly complex genome. Tropical Plant Biology, 4, 145–156. doi: 10.1007/s12042‐011‐9079‐0.
  Taparia, Y., Fouad, W. M., Gallo, M., & Altpeter, F. (2012). Rapid production of transgenic sugarcane with the introduction of simple loci following biolistic transfer of a minimal expression cassette and direct embryogenesis. In Vitro Cellular & Developmental Biology ‐ Plant, 48, 15–22. doi: 10.1007/s11627‐011‐9389‐9.
  Thompson, C. J., Movva, N. R., Tizard, R., Crameri, R., Davies, J. E., Lauwereys, M., & Botterman, J. (1987). Characterization of the herbicide‐resistance gene bar from Streptomyces hygroscopicus. The EMBO Journal, 6, 2519–2523.
  van der Vyver, C., Conradie, T., Kossmann, J., & Lloyd, J. (2013). In vitro selection of transgenic sugarcane callus utilizing a plant gene encoding a mutant form of acetolactate synthase. In Vitro Cellular & Developmental Biology, 49, 198–206. doi: 10.1007/s11627‐013‐9493‐0.
  Weng, L. X., Deng, H. H., Xu, J. L., Li, Q., Zhang, Y. Q., Jiang, Z. D., … Zhang, L. H. (2011). Transgenic sugarcane plants expressing high levels of modified cry1Ac provide effective control against stem borers in field trials. Transgenic Research, 20, 759–772. doi: 10.1007/s11248‐010‐9456‐8.
  Wu, H., & Altpeter, F. (2015). Sugarcane (Saccharum spp. hybrids). Methods in Molecular Biology, 1224, 307‐316. doi: 10.1007/978‐1‐4939‐1658‐0_24.
  Zale, J., Jung, J. H., Kim, J. Y., Pathak, B., Karan, R., Liu, H., … Altpeter, F. (2016). Metabolic engineering of sugarcane to accumulate energy‐dense triacylglycerols in vegetative biomass. Plant Biotechnology Journal, 14, 661–669. doi: 10.1111/pbi.12411.
  Zhu, Y. J., McCafferty, H., Osterman, G., Lim, S., Agbayani, R., Lehrer, A., … Komor, E. (2011). Genetic transformation with untranslatable coat protein gene of Sugarcane yellow leaf virus reduces virus titers in sugarcane. Transgenic Research, 20, 503–512. doi: 10.1007/s11248‐010‐9432‐3.
Key References
  Anderson & Birch (2012). See above.
  Discusses several critical points and key parameters to improve sugarcane tissue culture, transformation efficiency, and shoot regeneration.
  Dong et al. (2014). See above.
  Describes an optimized Agrobacterium‐mediated sugarcane transformation method using heat shock at 45°C and vacuum‐infiltration of embryogenic callus during inoculation, desiccation during the co‐cultivation stage, and the use of neomycin phosphotransferase II (nptII) or phosphomannoseisomerase (PMI) selectable markers genes and geneticin or mannose as selection agents.
  Wu & Altpeter (2015). See above.
  Describes a protocol for transformation of commercial sugarcane genotypes by hypervirulent Agrobacterium tumefaciens strain AGL1 using embryogeninc callus induced from immature top stalks. Geneticin and paromomicin were used as selective agents in callus phase and shoot regeneration, respectively.
PDF or HTML at Wiley Online Library