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
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Abstract

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

     
 
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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
     
 
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Materials

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

  Materials
  • 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
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Figures

Videos

Literature Cited

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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.
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