TECHNICAL REPORTS: ECOLOGICAL RISK ASSESSMENT Environmental Risk Assessment of Genetically Engineered Herbicide-Tolerant Zoysia japonica T. W. Bae Cheju National University E. Vanjildorj and S. Y. Song Chungnam National University S. Nishiguchi Cheju National University S. S. Yang Chonnam National University I. J. Song, T. Chandrasekhar and T. W. Kang Cheju National University J. I. Kim Chonnam National University Y. J. Koh Sunchon National University S. Y. Park Cheju Halla College J. Lee Cheju National University Y.-E. Lee Dongguk University K. H. Ryu Seoul Women’s University K. Z. Riu, P.-S. Song,* and H. Y. Lee Cheju National University Herbicide-tolerant Zoysia grass (Zoysia japonica Steud.) has urf grasses are commercially important species. As a perennial been generated previously through Agrobacterium tumefaciens- Tmonocot species, Zoysia grass is one of the most popularly mediated transformation. Th e genetically modifi ed (GM) Zoysia cultivated grasses for sports and recreational environments, grass survived Basta spraying and grew to maturity normally while the wild-type (WT) grass stopped growing and died. particularly in East Asia, because of its relatively high drought GM Zoysia grass will permit more effi cient weed control for tolerance, disease tolerance, and relatively slow growth habit. To various turf grass plantings such as home lawns, golf courses, further improve the turf grass through plant biotechnology, the and parks. We examined the environmental/biodiversity risks transformation of this species (Inokuma et al., 1998; Toyama et of herbicide-tolerant GM Zoysia before applying to regulatory al., 2002; Ge et al., 2006; Li et al., 2006) has been investigated as a agencies for approval for commercial release. Th e GM and WT Zoysia grass’ substantial trait equivalence, ability to cross- prerequisite for the generation of several transgenic lines including pollinate, and gene fl ow in confi ned and unconfi ned test herbicide-tolerant grass (Toyama et al., 2003). fi elds were selectively analyzed for environmental/biodiversity In a continuing eff ort to realize the biotechnology-based agro- eff ects. No diff erence between GM and WT Zoysia grass in nomic potential of turf grass, we investigated (Toyama et al., 2003) substantial traits was found. To assess the potential for cross- the herbicide tolerance of Zoysia grass by introducing a bar gene that pollination and gene fl ow, a non-selective herbicide, Basta, was used. Results showed that unintended cross-pollination codes for phosphinothricin N-acetyltransferase (PAT) (Th ompson with and gene fl ow from GM Zoysia grass were not detected et al., 1987) which catalyzes acetylation of the amino group of phos- in neighboring weed species examined, but were observed phinotricin (phosphinothricyl-L-alanyl-L-alanine). Th e N-acetylated in WT Zoysia grass (on average, 6% at proximity, 1.2% at a peptide can no longer inhibit the key enzyme in the nitrogen as- distance of 0.5 m and 0.12% at a radius of 3 m, and 0% at similation pathway, glutamine synthetase (Bayer et al., 1972). Th e distances over 3 m). On the basis of these initial studies, we conclude that the GM Zoysia grass generated in our laboratory bar gene confers tolerance to the broad-spectrum glufosinate-based and tested in the Nam Jeju County fi eld does not appear to herbicide Basta in transgenic crops. Glufosinate is not only a non-se- pose a signifi cant risk when cultivated outside of test fi elds. lective herbicide, but it is also quite readily biodegraded under natu- ral conditions. Th us, we consider Basta as the herbicide of choice T.W. Bae, S. Nishiguchi, I.J. Song, T. Chandrasekhar, K.Z. Riu, P.-S. Song, and H.Y. Lee, Copyright © 2008 by the American Society of Agronomy, Crop Science Faculty of Biotechnology, Cheju National Univ., Jeju 690-756, Korea. E. Vanjildorj and Society of America, and Soil Science Society of America. All rights S.Y. Song, Dep. of Horticulture, Chungnam National Univ., Daejeon 305-764, Korea. S.S. reserved. No part of this periodical may be reproduced or transmitted Yang and J.I. Kim, Dep. of Biotechnology (BK21 Program) and Kumho Life Science Lab., in any form or by any means, electronic or mechanical, including pho- Chonnam National Univ., Gwangju 500-757, Korea. T.W. Kang, Applied Radiological tocopying, recording, or any information storage and retrieval system, Science Research Inst., Cheju National Univ., Jeju 690-756, Korea. Y.J. Koh, School of without permission in writing from the publisher. Environmental and Agricultural Science, Sunchon National Univ., Sunchon 540-742, Korea. S.Y. Park, Dep. of Clinical Pathology, Cheju Halla College, Jeju 690-708, Korea. Published in J. Environ. Qual. 37:207–218 (2008). J. Lee, School of Medicine, Cheju National Univ., Jeju 690-756, Korea. Y.E. Lee, Dep. of doi:10.2134/jeq2007.0128 Biotechnology, Dongguk Univ., Kyungju, Kyongbuk 780-714, Korea. K.H. Ryu, Division of Received 13 Mar. 2007. Environmental and Life Sciences, Seoul Women’s Univ., Seoul 139-774, Korea. *Corresponding author ([email protected]). © ASA, CSSA, SSSA Abbreviations: GM, genetically modifi ed; PAT, phosphinothricin N-acetyltransferase; 677 S. Segoe Rd., Madison, WI 53711 USA WT, wild type. 207 Table 1. Physicochemical properties of soil mixture used to grow genetically modifi ed (GM) and wild-type (WT) Zoysia grasses. Each indicates the mean ± standard error of three replicates. Soil Available Exchangeable cations 0.1 N HCl extractable sample pH† EC‡ OM§ P KCaMgFeBZnMnCu dS m−1 g kg−1 mg kg−1 ——–––—cmol kg−1¶——–––— ——————––––––——mg kg−1#———––––––————- GM 4.86 ± 0.17 0.032 ± 0.00 46.9 ± 6.8 13.5 ± 2.57 0.78 ± 0.13 0.45 ± 0.14 0.36 ± 0.13 19.1 ± 2.08 0.85 ± 0.13 1.58 ± 0.32 27.8 ± 1.39 0.83 ± 0.07 WT 4.97 ± 0.06 0.036 ± 0.00 40.8 ± 0.6 14.8 ± 2.34 1.00 ± 0.40 0.55 ± 0.07 0.43 ± 0.05 21.5 ± 1.20 0.65 ± 0.21 1.69 ± 0.23 29.0 ± 0.98 1.29 ± 0.66 t-test NS†† NS NS NS NS NS NS NS NS NS NS NS † pH of soil: water (1:5). ‡ EC, electrical conductivity. § OM, organic matter. ¶ cmol kg-1, centimols of positive charge per kilogram of soil. # mg kg-1, cation concentration. †† NS, statistically insignifi cant. in terms of minimal environmental impact. Another important assessments of GM plants by the Rural Development Adminis- reason for our choice of the bar gene for turf grass biotechnology tration/Korea Ministry of Agriculture and Forestry. application is that it enables the use of herbicide tolerance as a se- lectable marker for development of transgenic turf grass cultivars Transformation of Zoysia japonica having multiple genes (i.e., herbicide tolerance plus other traits Th e Agrobacterium-mediated transformation of Zoysia japonica by gene pyramiding) currently in our development pipeline. was established by our laboratory. Th e bar gene introduced, the In the present study, we characterized the phenotypic perfor- promoter used, and the selection markers and the vector chosen mance of bar-gene transgenic Zoysia grass in the test fi eld and have been reported in detail elsewhere (Becker, 1990, Becker et al., used the marker gene in preliminary assessments of the environ- 1992; Toki, 1992; Lee et al., 1998; Toyama et al., 2002, 2003). mental/biodiversity concerns arising from GM Zoysia grass. In view of the widely expressed concerns about the ecological and Environmental Risk Assessments biodiversity implications of GM crops and plants, releasing a GM Preparation of Plants plant to agronomic habitats entails prior assessments of its risks In T generation, the stolons of the herbicide-tolerant Zoysia to the environment as well as to human and animal health. Th e 3 grass (GM Zoysia grass hereafter) were subjected to various tests. herbicide-tolerant GM crops that underwent such risk assessments Th e growth and propagation of the grass were investigated during include creeping bentgrass (not currently commercially available hardening and vegetative propagation of the stolons in one of the from Scotts), soybean (Monsanto and Bayer CropScience), cotton isolated greenhouses. Wild-type Zoysia grass (WT) plants were (Monsanto, Calgene, Dow AgroSciences, and Bayer CropScience), used as the control for the test. Th e grass stolons thus obtained maize (Monsanto, Syngenta, DuPont, Bayer CropScience, and were transplanted in the confi ned test fi eld. Th e grass plants were Pioneer Hi-bred), rice (Bayer CropScience), chicory (Bejo Zaden transplanted in a set of porcelain pots with each pot containing BV), Argentine canola (Bayer CropScience and Monsanto), Polish GM and WT plants separated by 25-cm radii (1 pot = 1 unit). canola (Bayer CropScience and Monsanto), and sugar beet (No- vartis, Monsanto, and Bayer CropScience). Yaneshita et al. (1997) Genetic Stability studied the outcrossing or self-pollination potential of Zoysia ja- Th e GM Zoysia grasses are tolerant to the non-selective ponica, and reported evidence of interspecifi c hybridization within commercial herbicide Basta (Bayer CropScience, Australia) at the genus Zoysia (Z. matrella, Z. sinica, Z. tenuifolia, and Z. macro- a fi nal concentration of 0.1% (w/v) glufosinate. Th e tolerance stashya) on the basis of RFLP and morphological characterization. to these herbicides sprayed to GM Zoysia grass was monitored In this report, we focused our attention on similar ecological and periodically throughout the T0 and T1 generations. Th e effi - environmental concerns arising from the release of GM Zoysia cacy of herbicide spraying was assessed under optimal growth grass to natural environment. conditions for the grasses. Th e growth and the herbicide ac- tion on GM Zoysia grasses and naturally occurring weed spe- Materials and Methods cies were investigated 2 wk after Basta was applied.