Transformation Protocol for Micro-Tom,

Plant Cell Physiol. 47(3): 426–431 (2006)

doi:10.1093/pcp/pci251, available online at http://m.wendangku.net/doc/4519931a650e52ea55189858.html JSPP ? 2006

426

Short Communication

A Highly Efficient Transformation Protocol for Micro-Tom, a Model Cultivar for Tomato Functional Genomics

Hyeon-Jin Sun 1, Sayaka Uchii 1, Shin Watanabe and Hiroshi Ezura *

Gene Research Center, Graduate School of Life and Environmental Sciences, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki, 305-8572 Japan

We report a highly efficient protocol for the Agrobac-terium -mediated genetic transformation of a miniature dwarf tomato (Lycopersicon esculentum ), Micro-Tom, a model cul tivar for tomato functional genomics. Cotyl edon exp l ants of tomato inocu l ated with Agrobacterium tumefaciens (Rhizobium radiobacter ) C58C1Rif R harboring the binary vector pIG121Hm generated a mass of chimeric non-transgenic and transgenic adventitious buds. Repeated shoot el ongation from the mass of adventitious buds on se l ection media resu l ted in the production of mu l tip l e transgenic pl ants that originated from independent trans-formation events. The transformation efficiency exceeded 40% of the explants. This protocol could become a power-ful tool for functional genomics in tomato.

Keywords : Agrobacterium tumefaciens (Rhi z

obium radio-bacter ) — Functional genomics — Lycopersicon esculentum L.— Micro-Tom — Transformation.

Abbreviations: AS, acetosyringone; EMS, ethyl methane-sulfonate; EST, expressed sequence tag; GUS, β-glucuronidase; MS,Murashige and Skoog.

With the successful conclusion of the Arabidopsis and rice genome projects, researchers in plant science recognize that genomic information is a powerful tool for both basic studies and technological developments. A specific feature of tomato (Lycopersicon esculentum L.), i.e. fruit development, is impos-sible to study fully in Arabidopsis or rice. In addition, tomato is an economically important food worldwide. For these reasons,tomato has become the next target for plant genomics studies.However, compared with Arabidopsis and rice, tomato has a relatively large genome (approximately 950Mb), which makes it difficult for a single institution to undertake the genome project. Therefore, tomato genome initiatives are currently in progress, involving the action of an international consortium called the ‘International Solanaceae Genome Project’ (SOL).Among other accomplishments, SOL has been generating information on genome resources related to tomato, such as the

results obtained by genome sequencing and expressed sequence tag (EST) sequencing (Fei et al. 2004), including sequencing of full-length cDNA and mutants induced by a variety of mutagenic treatments, such as the application of ethyl methanesulfonate (EMS; Emmanuel and Levy 2002,Menda et al. 2004). Using these resources, functional genomics have been used to study the function of genes related to tomato development (Gidoni et al. 2003, Shibata 2005, Takahashi et al.2005).

Genetic transformation is a key technology for functional genomics. To confirm the functions of genes isolated using map-based cloning of mutant alleles and omics analysis, func-tional complementation using genetic transformation is required. Previous studies on the genetic transformation of tomato have reported transformation efficiencies ranging from 6 to 37% (Hamza and Chupeau 1993, Van Roekel et al. 1993,Frary and Earle 1996, Ling et al. 1998, Vidya et al. 2000, Hu and Phillips 2001, Park et al. 2003). Nevertheless, many research groups that are using tomato as an experimental plant are having difficulty in generating transgenic tomato plants (Dr.Y . Kubo, Okayama University; Dr. A. Itai, Tottori University;Dr. K. Matsui, Yamaguchi University; and Dr. K. Aoki, Kazusa DNA Institute; personal communications at SOL and JSOL workshop), indicating that an efficient reliable protocol is required.

Micro-Tom is a miniature dwarf tomato cultivar that was originally bred for home gardening (Scott and Harbaugh 1989).This cultivar has several unique features, such as a small size that enables it to grow at a high density (1,357plants m –2), seed setting under fluorescent light and a short life cycle that allows for mature fruit to be harvested within 70–90d after sowing.These features are similar to those of Arabidopsis ; conse-quently, this tomato is considered to be a model cultivar for tomato functional genomics.

Tomato researchers in Japan have established a consor-tium for tomato genomics called Japanese SOL (JSOL). JSOL is currently establishing shared genomic resources for tomato and is participating in the international SOL activity. The genomic resources include genome sequences, macroarray pro-filing data sets using Micro-Tom (Van der Hoeven et al. 2002,

These authors contributed equally to this work.

*

Corresponding author: E-mail, ezura@gene.tsukuba.ac.jp; Fax, +81-29-853-7263.

at Shanghai Information Center for Life Sciences, CAS on July 28, 2010

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