American Journal of Plant Sciences, 2012, 3, 368-372 http://dx.doi.org/10.4236/ajps.2012.33044 Published Online March 2012 (http://www.SciRP.org/journal/ajps) The Mutated Acetolactate Synthase Gene from Rice as a Non-Antibiotic Selection Marker for Transformation of Bamboo Cells Shinjiro Ogita * , Nanaka Kikuchi, Taiji Nomura, Yasuo Kato Laboratory of Plant and Cell Engineering, Department of Biotechnology, Toyama Prefectural University, Toyama, Japan. Email: * ogita@pu-toyama.ac.jp Received October 12 th , 2011; revised November 26 th , 2011; accepted December 27 th , 2011 ABSTRACT Previously, we developed a particle bombardment-mediated transformation protocol in Phyllostachys nigra bamboo by expressing hygromycin phosphotransferase gene (HPT) and neomycin phosphotransferase II gene (NPT II). Although these marker genes could introduce to several tissue cultured organs (e.g. leaves, buds, and calli) of Phyllostachs bam- boo species, some organs showed a high susceptibility and/or a low selectivity to hygromycin and kanamycin. In this report, therefore, we describe advantages and technical details for generating stable transgenic bamboo cells using the particle bombardment method with the mutated-acetolactate synthase gene (mALS) from rice (W548L/S627IOsALS) as a non-antibiotic selection marker. A facile and efficient transformation was achieved with the mALS gene and enhanced fluorescent protein gene (mCherry). Approximately 490 and 1400 mCherry-expressing cells/dish/shot in average were observed in both P. bambusoides and P. nigra under fluorescent stereo-microscope. Stable transgenic bamboo cell lines were generated in a selection medium supplemented with 0.1 μM of bispyribac-sodium (BS) as ALS inhibitor. The in- tegration of mALS gene was identified by in vivo ALS enzyme assay and a PCR-restriction fragment length polymer- phism (RFLP) based detection procedures. Keywords: Bamboo; Mutated Acetolactate Synthase Gene; Particle Bombardment; Suspension Culture 1. Introduction Acetolactate synthase (ALS: EC 2.2.1.6) is the common enzyme in the biosynthetic pathway of the branched- chain amino acids leucine, isoleucine, and valine. A mutated- ALS (mALS) gene was isolated from pyrimidinyl car- boxy herbicide (Pyriminobac, pyrithiobac-sodium and bis- piribac-sodium)-resistant somaclonal variation rice cells and found to have a single amino acid change from serine to isoleucine in a conserved region [1]. Recently, it was developed that the mALS gene can be used as a new se- lectable marker to produce transgenic plants such as Ara- bidopsis [2], rice [3-5], soybean [6], and wheat [7]. These contributions allow us to overcome many difficulties for the selection of transformants of the target plants. It is very important contribution to improve a new vector system/plural vector choices as powerful tools for understanding physiological/ molecular biological events and/or engineering complex biosynthetic pasway(s) of target plant cells by introducing multiple genes. At pre- sent, however, little information is available concerning the transformation of bamboo plants. In order to inves- tigate/improve biosynthetic pathway(s) of bamboo plants, it is essential to develop an efficient transformation pro- tocol for a model cell culture system. Previously, we developed a cell culture system [8] and a particle bombardment-mediated transformation proto- col in Phyllostachys nigra bamboo by expressing hygro- mycin phosphotransferase gene (HPT) and neomycin phosphotransferase II gene (NPT II) [9]. Although this system has been currently used for a regular transfor- mation technique of bamboo cells in our researches, there is scope for improvement such as a high toxicity and/or a low selectivity of antibiotics in some organs (e.g. leaves and buds) or cell lines as shown in Figure 1(A). In this report, therefore, we described advantages and technical details for generating stable transgenic bamboo cells using the particle bombardment method with mALS gene as a non-antibiotic selection marker. 2. Materials and Methods 2.1. Bamboo Cell Cultures Callus cultures of two bamboo species, both P. bam- * Corresponding author. Copyright © 2012 SciRes. AJPS