Basso Marcos Fernando, da Cunha Bárbara Andrade Dias Brito, Ribeiro Ana Paula, Martins Polyana Kelly, de Souza Wagner Rodrigo, de Oliveira Nelson Geraldo, Nakayama Thiago Jonas, Augusto das Chagas Noqueli Casari Raphael, Santiago Thais Ribeiro, Vinecky Felipe, Cançado Letícia Jungmann, de Sousa Carlos Antônio Ferreira, de Oliveira Patricia Abrão, de Souza Silvana Aparecida Creste Dias, Cançado Geraldo Magela de Almeida, Kobayashi Adilson Kenji, Molinari Hugo Bruno Correa
Genetics and Biotechnology Laboratory, National Center for Agroenergy Research (CNPAE), Brazilian Agricultural Research Corporation (EMBRAPA), Brasília, Distrito Federal, Brazil.
Agronomic Institute of Campinas (IAC), Ribeirão Preto, São Paulo, Brazil.
Curr Protoc Plant Biol. 2017 Sep;2(3):221-239. doi: 10.1002/cppb.20055.
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.
甘蔗（甘蔗属）是禾本科的一种单子叶半多年生C4草本植物。其积累高含量蔗糖和生物量的能力使其成为制糖和生物燃料生产最重要的作物之一。由于甘蔗复杂的多倍体和非整倍体基因组，传统的甘蔗育种方法存在一些局限性。然而，通过生物技术工程进行改良目前是引入经济重要性状最有前景的替代方法。在这项工作中，我们提出了一种改进的方案，用于利用未成熟顶茎来源的胚性愈伤组织培养物，通过根癌农杆菌介导对商业甘蔗杂交种进行转化。将携带二元载体的预处理根癌农杆菌与愈伤组织培养物进行转化，该二元载体编码感兴趣基因的表达盒和双丙氨膦抗性基因（bar赋予对草铵膦除草剂的抗性）。该方案已成功用于转化商业甘蔗品种SP80 - 3280，突出表现为：（i）降低了顽拗性和氧化；（ii）胚性愈伤组织产量高；（iii）选择效果改善；（iv）转化植株的芽再生和生根。总之，这些改进产生了2.2%的转化效率。该方案为通过基因工程改良甘蔗的常规程序提供了可靠工具。© 2017约翰威立父子公司版权所有
