TECHNICAL REPORTS: ECOLOGICAL RISK ASSESSMENT

Environmental Risk Assessment of Genetically Engineered Herbicide-Tolerant 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 , golf courses, further improve the turf grass through 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, . 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 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 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, ) 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. Plant Materials Phenotypic Characterization Unless stated otherwise, all plant materials used for the risk Th e growth and morphology (stems, leaves, seedlings, etc.) assessment study reported here have been generated by Toyama of the GM Zoysia grass were compared with those of the WT et al. (2003). Th e seeds of Zoysia grass (Zoysia japonica Steud.) cultivated under greenhouse conditions, according to previous were obtained as described previously (Bae et al., 2001; Toyama methods (Honda and Kono, 1963; Yu et al., 1974; Hong and et al., 2002, 2003). Th e Zoysia grass stolons produced were Yeam, 1985; Hwang and Choi, 1999; Kim et al., 1996; Choi vegetatively propagated in Cheju National University-approved and Yang, 2004). Table 1 lists the physicochemical properties confi ned vinyl houses as well as in a test fi eld in Nam Jeju of the soils used for the greenhouse habitat. Th e morphological County, Jeju, Korea, expressly approved for environmental risk comparisons included the plant height, the length of the blade,

208 Journal of Environmental Quality • Volume 37 • January–February 2008 width, and the angle of a plant leaf, the third-youngest leaf of each erect stem was chosen for measuring the leaf parameters to minimize the variations due to envi- ronmental factors described by Youngner (1961) and Hong and Yeam (1985), the distance between the shoot base and the lowest leaf blade, and the dry weight (up to the third leaf of the plant) after 48 h in a drying oven. Th e chlorophyll con- centration was measured using a portable chlorophyll analyzer (SPAD-502; Mi- nolta Co., Japan). For seed morphology (number, length, and width) compari- sons, the seeds were harvested from one spike, and the average weight of the seeds was measured based on those harvested from 45 individual plants. Intra-species Hybridization Potentials To investigate pollination-induced hybrid formation between the GM and the WT grasses, three test plots each containing both the GM Zoysia grass and the synchronously fl owering Fig. 1. Field testing (a) and schematic illustration (b) for cross-hybridization between genetically modifi ed (GM) and wild-type (WT) Zoysia grass at 0.5-m separation. Grass lanes, GM Zoysia WT Zoysia grass planted in a 25-cm grass; pots (25- cm diameter each), WT Zoysia grass. diam. by 20 cm deep porcelain pot were distributed within the test fi eld April, but the GM Zoysia grasses fl owered 5 to 7 d later than WT. in Nam Jeju County, Jeju. Each pot in two of the three plots Th e GM and the WT Zoysia grasses were transplanted and distrib- had three pots each of GM and WT Zoysia plants, and the uted according to a completely randomized plot design (Nakayama remaining plot contained fi ve pots each. Th e seeds harvested and Yamaguchi, 2002; Belanger et al., 2003b) and the randomized from the WT Zoysia grass were germinated and the grasses block and crossing block design (Belanger et al., 2004) or an alter- grown until three leaves appeared, then were screened for nating population combination design (Song et al., 2003). their herbicide tolerance by spraying Basta. Th e herbicide- In the alternating population combination design test, fi ve 2 tolerant lines screened were then subjected to PCR analysis blocks each (1 × 12 m ) of GM and WT Zoysia grasses were based on bar primers. Th e primers for the detection of bar distributed alternatively (Fig. 1). Figure 2 illustrates the distri- gene were 5’-GGTCTGCACCATCGTCAACC-3’ and 5’- bution patterns of pots (25-cm diam. and pot-to-pot distance ATCTCGGTGACGGGCAGGA-3’. Th e Z-A2 actin primers ?0.5 m) containing GM and WT Zoysia grasses. After allow- for the expression in Zoysia japonica were 5’-GTCAACCCTG ing growth for 10 wk under natural fi eld conditions, hybrid- TGCAGCAGTA-3’ and 5’-ATTCAGGTTGGTTGCTC- ization results were scored for WT samples in each plot as a CAC-3’. Th irty fi ve cycles of PCR were performed under the function of distance and plot design. following conditions: denaturation at 94°C for 30 s, annealing Next-nearest Neighbor (≤3 m) Cross-pollination at 61°C for 30 s, and elongation at 72°C for 45 s. Figure 3 illustrates the test for the next-nearest neighbor cross- Hybridization Potentials in Other Species fertilization showing 3-m intervals of four GM grass pots (25-cm 2 Th e GM Zoysia grass and the native weeds grew within the diam.) surrounded by one 6 by 16 m patch of WT grass. Hybrid same confi ned test fi eld during 2003–2005. In May 2004, Bas- formation within the 3-m separation was determined after 2 mo ta was sprayed both inside and within a 5-m radius outside the of growth under natural fi eld conditions. Mature WT seeds were 2 test fi eld to investigate cross-pollination between the GM grass harvested from 96 fractions of a 1 by 1 m area around a GM pot and the weeds mediated by wind. One year later, in May 2005, and dried naturally under sunlight. Fresh seeds were stored in ice hybridization in weed species was then examined on the plants box at −10°C until used. Th e seeds were dehusked mechanically having identical fl owering time by means of PCR analysis. and kept at 4°C for 1 wk. Th e seeds were sterilized in 2% sodium hypochrorite solution for 15 min and rinsed fi ve times in distilled Nearest Neighbor (0.5 m) Cross-pollination water. Th e seeds were then allowed to imbibe on wet fi lter papers During the 2-yr study (2004 through 2005) performed in both at 35°C under 4200 cd sr–1 m−2 light for 72 h, and placed at 25°C test fi elds in Cheju National University and Nam Jeju County, we under 3500 cd sr–1 m−2 light for . Th e resulting plants found that the GM and the WT Zoysia grasses fl owered in late were then sprayed with Basta to screen for hybridization.

Bae et al.: GM Zoysia Grass and Environmental Risk 209 Neighbor (3 m to 9 m) Cross-pollination Figure 4 shows a 9 by 9 m2 hexagonal test plot (initiated July 2004) enclosing the GM grass (1-m radius) surrounded by cold- and warm-season grass species at distances from 3 to 9 m. Each plot included fi ve grass species, Z. japonica, Z. sinica, Z. matrella, perennial ryegrass (Lolium perenne L.), and Kentucky bluegrass (Poa pratensis L., data not shown). GM and these grass species were trans- planted and arranged according to a plot design (Belanger et al., 2003a). Th e seeds were harvested in August 2005, dried, ger- minated, and screened for hybrid forma- tion by herbicide application. Long Distance Cross-Fertilization Potential gene fl ow from the total 936 m2 GM grass fi eld (14 × 16 m2, 16 × 40 m2, and 6 × 12 m2) to WT Zoysia grasses in the surrounding wilderness (119 Zoysia japonica and 2 Zoysia sinica sampling sites as shown in Fig. 5) within a 3-km radius was tested based on Basta screen- ing and PCR analysis. Th e majority of the sampling sites are east and northeast biased relative to the GM grass test fi eld because of the local land topography—namely high hills, woody forests, seaside, bushy valleys south, and southeast of the test fi eld. Fig. 2. Field testing (a) and schematic illustration (b) for cross hybridization between genetically modifi ed (GM) and wild-type (WT) Zoysia grass according to a randomized Unintended gene fl ow and seed propa- complete block design. Black circle, GM Zoysia grass; white circle, WT Zoysia grass. gation from the GM creeping bentgrass fi eld established in 2003 were tested in 2005. For this purpose, both herbicide screening and PCR methods were used, as described previously (see Intra-species Hy- bridization Potentials). Skin Prick Tests With informed consent, we performed a similar study with pollen extracts from GM and WT grasses on chronic allergy patients admitted at the allergy clinic of Cheju Na- tional University Hospital and on healthy volunteers over the period from October 2005 through April 2006. For the skin prick tests, twenty common inhalent aller- gens and pollen extracts of GM and WT grasses were used with positive (histamine 1 mg mL−1) and negative (0.9% NaCl) controls. Th e sensitization was defi ned when each wheal size showed more than 3 mm. One hundred twenty seven subjects (55 males, mean age of 38) were included. Among the 127 subjects, 87 individuals Fig. 3. Field testing for gene fl ow from genetically modifi ed (GM) to wild-type (WT) Zoysia grass were sensitive to inhalent allergens. within a 3-m radius. Each GM grass pot is surrounded by WT grass patches of 6 by 16 m2 area.

210 Journal of Environmental Quality • Volume 37 • January–February 2008 Results Two copies of the bar gene introduced in GM-Zoysia japonica retained its stable integration in the host plant in the T1 to

T6 generations, exhibiting a 15:1 segrega- tion ratio in accordance with Mendelian genetics, and also showing the transgenic line’s tolerance to ammonium glufosinate throughout the culture period. Th e geno- type was retained through the multiple generations in both transgenic Zoysia lines and their WT hybrids. Before subjecting the GM Zoysia japonica grass to the envi- ronmental risk assessment study reported here, we observed that the GM Zoysia grass survived application of the non-se- lective herbicide spray, whereas WT grass did not, indicating the stable inheritance of bar gene in the transgenic grass. Th e GM Zoysia grasses were cultivated in a greenhouse and periodically checked Fig. 4. Field testing and schematic illustration for cross hybridization between genetically for the herbicide tolerance at various modifi ed (GM) and wild-type (WT) Zoysia grass and it relative weed species as a function stages of growth and development. Th e of distance. (a) GM Zoysia japonica tillers (illustrated with white circle). (b) WT grass group grass lines hardened during repeated containing Zoysia japonica (Zj), Zoysia matrella (Zm), Zoysia sinica (Zs), Lolium perenne (Lp), and Poa pratensis (Pp). (c) Hexagonal arrangement illustrating a GM test area shown in (a); vegetative propagation were then used orange, 3-m radius; blue, 6-m radius; black, 9-m radius). for all the studies reported hereafter. Both the WT and GM Zoysia plants displayed between the two types of Zoysia grass was that locusts and other essentially identical germination and growth rates, and morpho- insects preferred to reside in the Basta-sprayed GM fi eld, com- logical and physiological characteristics. One interesting diff erence pared to the WT fi eld, most likely avoiding the higher amounts of selective herbicide residuals such as Pyrazosulfuron [ethyl 5-(4,6-

Fig. 5. Test for the potential gene fl ow from genetically modifi ed (GM) grass to wild-type (WT) grasses within a 3-km radius during a 2-yr period from 2003 to 2005. The GM grass fi eld is centrally located in the Wimi-Ri test fi eld in Nam Jeju County. The sampling sites shown were randomly chosen where Zoysia grasses grew. The sampling site distribution is biased in the north easterly direction from the GM grass site, whereas other directions are less favorable for grass growth due to geo-topographic factors (volcanic rocks, bushy jungles, forest, etc.).

Bae et al.: GM Zoysia Grass and Environmental Risk 211 dimethoxypyrimidin-2-ylcarbamoyl)sulfamoyl-1-methylpyrazole- able from WT grass, except for the bar-gene transgenicity of the 4-carboxylate], Alachlor (2-chloro-2′,6′-diethyl-N-methoxymethyl- former, which imbues it with tolerance to a herbicide (Basta) acetanilide), and Triclopyr (3,5,6-trichloro-2-pyridyloxyacetic acid) spray. Both GM and WT seeds showed relatively low germina- in the latter. More frequent applications (fi ve to seven times) of the tion rates, so vegetative propagation through the spreading of selective herbicide spray were required to keep the WT grass fi eld grass stolons over the soil surface became the preferred method free of weeds, whereas the non-selective herbicide (Basta), just once for the hardening and vegetative propagation of the Zoysia grass or twice was suffi cient for the GM grass fi eld. cultivars. Th e following summaries provide further details of the Th e herbicide tolerance of the bar transgenic Zoysia grass was studies including some of the subtle diff erences in grassy features stably preserved for the testing period spanning more than 2 yr. observed between the GM and the WT lines. During the same period that the Basta tolerance of the GM grass was sustained stably, they remained susceptible to non-selective Reproduction and Genetic Traits herbicides such as paraquat and glyphosate. Th us, for any reason Flowering Time if it is necessary to terminate the cultivation and spread of the During the 2-yr study conducted in 2004 and 2005, we ob- GM grass within and beyond the test fi eld, the GM plants can be served WT grasses starting to fl ower in late April, whereas the GM readily killed by applying a herbicide spray other than Basta. plants fl owered about 5 to 7 d later. Both types of grasses fully Is GM Grass Environmentally Risky? Comparative fl owered within 5 d of 10 May, formed dried pollens (i.e., inactiva- tion of anthers), and full seed formation by mid-July, consistent Characterizations of GM and WT Grasses with the fl owering times reported by Kitamura (1967). Since GM Conventional environmental risk assessments of GM crops grass began fl owering about 5 to 7 d after WT plants, the fre- have been performed in four categories, namely, (i) establishment quency of formation of intraspecifi c GM hybrids could have been of the substantial equivalence between the GM and the WT reduced. To circumvent this possibility arising from the fl owering plants, (ii) determination of pollen fl ight and potential gene fl ow, time diff erence, both GM and WT grass cultivars that fl ower at (iii) biodiversity eff ects of GM plants on unintended or non-tar- about the same time in the greenhouse were replanted in the plots get and target plants in their ecological habitats, and (iv) health of the test fi eld. However, after 2 yr this became unnecessary as risk assessments for animals including humans. We adopted the both types of grass fl owered simultaneously (see below). four-category protocol for an initial evaluation of GM Zoysia Pollination japonica before its release to agronomic habitats. Category (i) vis. substantial equivalence, is described and further discussed here Pollen formation was maximal around 10 May, and intra- along with the remaining aspects (categories ii–iv). Th e substan- species hybrid formation induced by pollination was most tial equivalence between the GM and the WT grasses has been prevalent thereafter, 15 to 18 May. We decided to introduce established on the basis of their essentially identical reproduc- WT plants having similar fl owering time into the GM grass tion rate, morphology of leaves and seeds, germination rate, and greenhouse and outdoor test fi eld on 15 May, 2004, and chemical composition. 2005. Results showed that both GM and WT grasses cross- To ascertain the grassy characteristics of the GM Zoysia plants pollinated at an average rate of 6% at close proximity. in comparison to those of WT plants, the plants were grown Morphology under identical conditions with respect to soil composition, ir- Th e morphology of Zoysia grass can be classifi ed in terms rigation, and fertilization, etc. Results from the various studies of leaf width and length, according to Kitamura’s horticultural described here indicate that GM grass displays morphological, classifi cation method (Kitamura, 1967). We examined other physiological, and genetic characteristics virtually indistinguish- appearance indices of the plants, as presented in Table 2, which shows morphological features of GM and WT Table 2. Morphological characteristics of genetically modifi ed (GM) and wild-type (WT) Zoysia grass leaves observed under fi eld growing conditions. Each value grasses. Results show that the two types of grasses indicates the mean ± standard error of fi fteen replicates. are essentially indistinguishable and any diff erences observed were statistically insignifi cant. Table 3 Plant Leaf First leaf Leaf Leaf Chlorophyll Leaf Plants height blade† height‡ width‡ angle§ contents¶ weight# compares the seed morphologies, again indicating –––––––––––––––––cm––––––––––––––––– A° g kg−1 FW g that the two types of grasses are indistinguishable. GM 20.4 ± 2.5 19.7 ± 3.5 4.0 ± 0.7 0.54 ± 0.06 24.9 ± 6.4 1.06 ± 0.17 0.20 ± 0.05 Other phenotypic traits also showed no signifi cant WT 19.9 ± 3.9 17.8 ± 3.3 3.8 ± 0.7 0.55 ± 0.08 24.5 ± 5.1 1.04 ± 0.19 0.20 ± 0.09 diff erences between the GM and WT Zoysia culti- t-test NS†† NS NS NS NS NS NS vars (Tables 2, 3, and 4). † Values measured from the third leaf. In summary, after 16 mo of cultivation in the test ‡ Length from basal zone of the shoot to fi rst leaf blade. fi eld, morphological analyses were performed and both § Angle between leaf blade axis and vertical axis. WT and GM Zoysia grass displayed a plant height of ¶ Chlorophyll contents measured from SPAD values; the values were calculated from 20 cm, leaf length of 17 to 19 cm, and leaf width of the relation curve between the UV spectrophotometer and the Chlorophyll meter 0.5 cm. Th e length of the lowest leaf blade and the (SPAD-502; MINOLTA, Japan). chlorophyll content were 4 cm and 1 g kg−1, respec- # Leaf weight; the three of fresh leaves were dried in an oven at 80°C for 3 d. tively (Table 2). After 3 mo of planting, the number †† NS, considered statistically insignifi cant at 0.05 level by t test.

212 Journal of Environmental Quality • Volume 37 • January–February 2008 Table 3. Seed characteristics of genetically modifi ed (GM) and wild-type (WT) Zoysia grass using eight morphological traits; length of fl owering culms, length of spike without rachis; length/width ratio; germination, and weight. No. of seeds Length of Length of Seed length Seed width SL/SW Frequency of Weight of Plants per spike rachis fl owering culms (SL) (SW) ratio germination 1000 seeds ––––––––––––cm–––––––––––– ––––––––mm–––––––– %g GM 49.4 ± 7.6† 4.8 ± 0.6‡ 12.1 ± 2.4‡ 3.2 ± 0.3‡ 1.5 ± 0.2‡ 2.2 ± 0.2‡ 3.7 ± 1.2 0.6 ± 0. 03§ WT 49.1 ± 7.3 4.9 ± 0.6 11.7 ± 2.7 3.1 ± 0.3 1.4 ± 0.2 2.2 ± 0.3 4.0 ± 1.0 0.6 ± 0.02 t-test NS¶ NS NS NS NS NS NS NS † Mean ± standard error of forty-fi ve replicates. ‡ Mean ± standard error of fi fteen replicates. § Mean ± standard error of fi ve replicates. ¶ NS, considered statistically insignifi cant at 0.05 level by t test. of stolons (ca. 5), its length (approx. 30.4–33.0 cm), and the leaf- GM Zoysia’s Dominance over Weeds node length (approx. 3.2–3.7 cm) were also statistically equivalent Th e Zoysia grass propagates reproductively both from seeds for the both types (Table 4). In addition, a seed’s morphological and vegetatively. Th e weight of the 1000 seeds is approx. 0.57 characterizations performed included the number of seeds per to 0.59 g. Even if wind carries the seeds over some distance, spike (ca. 49); length of fl owering culms (ca. 12 cm) and the rachis the germination rate is less than 4% under natural conditions. length (ca. 4.8 cm), seed length (ca. 3.1 mm) and width (ca. 1.5 Th us, compared to germination, Zoysia can spread itself more mm), 1000 seeds weight (ca. 0.58 g), and the rate of germination eff ectively through vegetative propagation. However, the Zoysia (approx. 3.7–4%, Table 3). Th e chemical and mineral composi- grass is not a dominant species and does not spread into weedy tions of seeds harvested from both types were also performed and areas easily. In fact, the Zoysia grass fi eld is completely domi- no signifi cant diff erences were found (data not shown). nated by the weeds within 2 to 3 yr of cohabitation. Figure 7 il- Hybridization lustrates the eff ects of dominant weeds on the GM Zoysia grass, As evident from the above result (see Pollination), no showing the dominance of the weeds over the Zoysia grass. signifi cant diff erence in pollination rates was found between the GM and the WT Zoysia grasses. We also examined the Disease Tolerance and Pathogenic Organisms pollination from GM to WT Zoysia grasses, and in the reverse Th e eff ects of GM Zoysia grass on the population of several direction. Table 5 shows that the minimum cross-pollina- pathogenic soil fungi were investigated. Table 7 lists the soil patho- tion rate was 3% and maximum was 9%, with an average of gens distributed within the soil layer and the plant segment of ap- 6%, at the nearest distance (>0 m). At a 0.5-m distance in proximately 3 cm length from the soil surface layer in the test fi eld. both randomized and completely randomized plot designs, No signifi cant diff erences in the population of the four major turf cross-pollination was approximately 1.2%, which declined to grass pathogens (Rhizoctonia spp., Pythium spp., Curvularia sp., 0.12% at a 3-m distance, and to 0% at distances greater than and Colletotrichum sp.) between WT and GM grass planted soils 3 m (Table 5). Figure 6 graphically illustrates the distance were found, with all diff erences within experimental and statistical dependence of GM-to-WT Zoysia gene fl ow. Th e best fi t for margins of error. However, the soil samples contained relatively this distance dependence from a regression analysis of the data high levels of Fusarium spp. in both WT and GM grass plots. Th e described in the results section is an exponential function as relatively dense population of this fungus is attributable to low shown in the fi gure. Similar distance dependence has been soil pH and electrical conductance (Kwon et al., 1998; Suh et al., reported for wild rice (Oryza rufi pogon) (Song et al., 2003). 2003). Fusarium spp. is a common fungal pathogen in soil, but turf grass plants are apparently unaff ected. In fact, the Fusarium Winter Dormancy spp. stimulates a plant’s growth by suppressing several co-habitat Both WT and GM Zoysia grass showed essentially identi- pathogens (Meera et al., 1993, 1994; Liu et al., 1995; Yun, 1996; cal dormancy profi les in the Nam Jeju County test fi eld, turn- Park and Yu, 2005). Th e higher density of Fusarium spp. in the ing brown, wilting by late November, and staying dormant until the next March. Table 4. Growth characteristics of genetically modifi ed (GM) and wild-type (WT) Zoysia grass using fi ve morphological traits. Each value indicates mean ± standard error of triplicates. Eff ects of GM Zoysia Grass on Coverage† Stolon No. Stolon length Leaf-node length Density§ Neighboring Weeds: Potential Weediness Plants 90DAP‡ 150DAP 90DAP 90DAP 90DAP 150DAP Gene Flow from GM Zoysia to Weeds –––––––––m2––––––––– ––––––––––––cm–––––––––––– no. cm−2 GM 0.07 ± 0.01 0.13 ± 0.06 5.2 ± 0.8 33.0 ± 7.4 3.7 ± 0.9 0.55 ± 0.04 Table 6 lists 14 co-habitant weed species WT 0.08 ± 0.01 0.13 ± 0.05 5.4 ± 1.1 30.4 ± 6.8 3.2 ± 0.7 0.57 ± 0.07 within the GM grass test plot facility. Neither t-test NS¶ NS NS NS NS NS Basta nor PCR evidence was obtained to † Coverage, about 10-cm diameter of GM and WT Zoysia grass plugged after 10 May. indicate bar-gene fl ow from the GM plant’s ‡ DAP, days after plugging. pollen to these neighboring weed species dur- § Density, number of tiller per cm2. ing the study conducted from 2003 to 2005. ¶ NS, considered statistically insignifi cant at 0.05 level by t test.

Bae et al.: GM Zoysia Grass and Environmental Risk 213 Table 5. Number of the germinated seeds tested and the hybrids Table 6. Test for the potential outcrossing between genetically identifi ed (number in parentheses) at distances from genetically modifi ed (GM)-Zoysia grass and weed plants grown within the modifi ed (GM) Zoysia grass in each plot design. Wimi-Ri test fi eld.

Distance CRD† RCBD‡ R-3§ R-9Zj¶ R-9Zs# R-9Zm†† R-9Lp‡‡ Flowering m No Scientifi c name Common name season Outcross m > 0 746(45) -§§ – – – – – 1 Spergula arvensis Corn spurry Mar.-June – 0.5 491(6) 967(12) – – – – – 2 Cerastium holosteoides Common mouse ear Mar.-June – 1 – 245(4) 72(1) 243(2) – – – 3 Stellaria media Chickweed Apr.-June – 2 – – 660(2) 145(1) – – – 4 Trigonotis peduncularis Cucumber herb Apr.-June – 3 – – 2547(3) 231(0) 83(0) 0 89(0) 5 Taraxacum offi cinate Dandelion Apr.-June – 6 – – – 209(0) 152(0) 79(0) 176(0) 6 Veronica arvensis Corn speedwell Apr.-June – 9 – – – 214(0) 104(0) 58(0) 93(0) 7 Vicia angustifolia Garden vetch Apr.-June – † CRD, completely random design. 8 Erigeron annuus Daisy fl eabane June-Sept. – ‡ RCBD, randomized complete block design. 9 Mazus pumilus Japanese mazus Apr.-Aug. – § R-3, WT Zoysia japonica within 3-m radius from GM Zoysia japonica pot 10 Youngia japonica Japanese youngia Apr.-June – (0.25-m diameter). 11 Cardamine impatiens Narrow leaf bitter cress Mar.- May – ¶ R-9Zj, WT Zoysia japonica within 9-m radius from GM Zoysia japonica 12 Gnaphalium affi ne Cudweed May-July – (1.5-m diameter). 13 Alopecurus aequalis † Orange Foxtail Apr.-June – # R-9Zs, WT Zoysia sinica within 9-m radius from GM Zoysia japonica (1.5-m 14 Poa annua † Annual Bluegrass Dec.-June – diameter). † Family of Gramineae with no hybridization from GM Zoysia grass. †† R-9Zm, WT Zoysia matrella within 9-m radius from GM Zoysia japonica (1.5-m diameter). electrophoresis (SDS-PAGE) gel, both proteins were identi- ‡‡ R-9Lp, WT Lolium perenne within 9-m radius from GM Zoysia japonica cally Western blotted at 21 to 23 kDa (Herouet et al., 2005). (1.5-m diameter). When the amino acid sequence of PAT was matched §§ Blank boxes represent no plot design data. against the known allergenic sequences using BLAST 2.2.15 GM grass soil may account for the lower levels of Curvularia sp. algorithm, SwissProt, PDB, PIR, PRF in NCBI, and Food and Colletotrichum sp., although the pathway by which the GM Allergy Research and Resource Program (FARRP)-FASTA grass soil stimulates Fusarium growth remains unknown (Table 7). Version 6, 80 or more amino acid-peptide sequences of the In fact, our study showed that other pathogenic populations were enzyme showed less than 35% homology. In addition, no suppressed by Fusarium spp. (data not shown). homology was found between the protein sequences and the eight amino acid allergen epitopes (FAO/WHO, 2001; Codex Potential Gene-induced and Allergic Hazards Alimentarius Commission, 2003; Bae, 2007). Th e pat gene is bar Gene inactivated at a pH below 4 or by heating for 30 min (Weh- Th e bar gene described earlier was originally isolated from rmann et al., 1996; ANZFA, 2001; Herouet et al., 2005). S. hygroscopicus. Its coded amino acid sequence showed an Previous animal and human studies showed that the pat gene 84% identity with the 183-amino acid polypeptide encoded posed no signifi cant health risks (Jones and Maryanski, 1991; by pat gene from S. viridochromogenes (National Center for Sjoblad et al., 1992; Schmidt, 1994; Mossinger and Dietrich, Biotechnology Information BLAST 2; Wehrmann et al., 1998; WHO, 1998; OECD, 1999; Institute of Science in 1996). On a 12% sodium dodecyl sulfate-polyacrylamide gel Society, 2003; Th omas et al., 2004; Herouet et al., 2005). Th e bar or pat gene used for the generation of GM Zoysia grass has been introduced into commercial crops such as rapeseed (Bayer CropScience), corn (Bayer CropScience), and cotton (Bayer CropScience). Th e gene introduced showed no appar- ent risks to human and animal health in terms of cytogenetic toxicity and allergenic reactions (FDA, 1992; CFSAN, 1995, 1998; FAO/WHO, 1996, 2000, 2001; Health Canada, 2001; OECD, 2001, 2002; Codex Alimentarius Commis- sion, 2003; European Commission, 2003). Allergic Reactions Kim et al. (1987) performed skin prick tests with pollen extracts of Zoysia grass and found that 5% of respiratory al- lergic patients were sensitized. Table 8 summarizes the test results. Six cases each of a wheal reaction to the WT and GM pollen extracts were found. Among the six, three subjects Fig. 6. Distance dependence for gene fl ow from the genetically modifi ed (GM) to wild-type (WT) Zoysia grass within 9-m radius in fi eld. The had respiratory allergic disorders. Th us, six subjects (4.7% of observed data can be best fi t by an exponential equation resulting the 127 test subjects) developed a positive allergic reaction from a regression analysis of the data. Bars refer to standard error.

214 Journal of Environmental Quality • Volume 37 • January–February 2008 Fig. 7. Zoysia japonica plants are overcame by dominant weed plants under natural ecological conditions. (A) Unmanaged Zoysia and weeds habitats. (B) The Zoysia after weeds were removed. (C) Weeds began to overtake Zoysia grass (1 yr without weed control). (D), (E) The same as C after 2 and 3 yr without weed control, respectively. to both WT and GM Zoysia pollens. However, no diff erence the incidence of allergic skin reactions to both GM and WT Zoysia between the two types of pollens was observed. grass was observed (Table 8). Th us, the focus of the discussion will be on the concerns about transgene fl ow from the GM Zoysia grass Discussion to compatible WT Zoysia and other weed species within and out- We fi rst reported the successful establishment of GM herbicide/ side the test fi eld in Jeju. Basta-tolerant Zoysia japonica Steud. by transforming the plant calli with the transgene bar (Toyama et al., 2003) under greenhouse Table 8. Results from the skin prick tests for common and Zoysia habitats. In the present study, we confi rmed that the transgenic grass allergens. Zoysia grass contained two copies of the bar gene. We further char- Allergen No. of patients Positive reaction acterized the phenotypic performance and the transgene introgres- % sion in the natural ecological environment of Jeju Island, Korea. Positive control (1mg/mL histamine) 127 100 All morphological and biochemical analyses suggested that the Negative control 0 0 GM Zoysia grass developed is indistinguishable from its WT plant, Dermatophagoides farinae 72 56.7 except for its Basta tolerance, under greenhouse, and fi eld habitat Dermatophagoides pteronyssinu 60 47.2 conditions (Fig. 1–6 and Tables 1–7). In addition, no diff erence in American cockroach 36 28.3 German cockroach 39 30.7 Table 7. Test for the fungal infection of genetically modifi ed (GM) and Cat and dog hair 18 14.2 wild-type (WT) Zoysia grass within the test fi eld. Horse and cattle hair 4 3.2 Fungal infection Flag 3 2.4 GM Zoysia grass WT Zoysia grass Broadleaf tree 2 1.6 Base Rhizosphere Base Rhizosphere Acicular tree 6 4.7 Fungi line† soil‡ line soil Disease Japanese cedar 5 3.9 –––––––––––––––––%––––––––––––––––– American cedar 6 4.7 Rhizoctonia spp. ND§ ND ND ND Large patch House dust-fungi 8 6.3 Pythium spp. ND ND ND ND Pythium blight Outdoor fungi 6 4.7 Curvularia sp. 2.8 3.0 4.5 8.3 Leaf blight Flowers 12 9.4 Colletotrichum sp. ND ND 1.5 ND Anthracnose Weeds 14 11.0 Fusarium spp. 75.8 19.4 22.7 11.1 Unknown Crops 10 7.9 † Shoot base. GM Zoysia grass 6 4.7 ‡ Stem’s rhizosphere soil zone. WT Zoysia grass 6 4.7 § ND, fungi not detected.

Bae et al.: GM Zoysia Grass and Environmental Risk 215 grass tillers was signifi cant (Fig. 6). Th is observa- tion is consistent with the documented cases of conventional gene fl ow and hybridization between cultivated and non-cultivated plant populations including the transgene introgression between GM bentgrass and compatible WT grasses (Reichman et al., 2006). However, at distances over 3 m the frequency of cross hybridization drops precipitously to essentially zero, as discussed below. As cited earlier, pollen-mediated introgression of herbicidal transgene cp4 epsps introduced in bentgrass has been detected within the popula- Fig. 8. Average wind velocity and directions on Jeju Island during the month of May tions of closely related grass species at up to 3.8 2005. The wind directions, maximum velocities of 6.7 m/s northerly; 5.6 m/s easterly and south easterly, 8.4 m/s southerly; 4 m/s south westerly, and 5.9 m/s km from the perimeter of the GM grass habitats westerly winds. Average monthly wind velocity was 5 m/s during the fl owering (Baack, 2006; Reichman et al., 2006). Th us, season and gene fl ow testing. when released to the natural environment, herbi- Th e GM Zoysia grass has been developed for its eventual release cide-tolerant weeds could evolve as the result of to agronomic habitats and recreational lands such as golf courses. transgene fl ow. Implications of the bentgrass case are critical for Th is work has been performed under a joint developmental agree- both ecological and societal concerns. However, it should be ment with the Jeju Provincial Government. Th e local government pointed out that the comparison of the distances of transgene is particularly interested in herbicide-tolerant grass for its potential fl ow in the present study to those of Reichman et al. (2006) environmental and economic implications for Jeju’s golf courses is not quantitatively valid, since the scale of the pollen sources and recreational parks. We anticipate that the use of GM Zoysia for between the two studies diff er markedly (we thank the referee golf courses will substantially reduce the amount and frequency of for pointing this out). weed-selective herbicide sprays performed annually. Th e volcanic However, from 121 sampling sites beyond our test area perim- island’s water supply for half a million inhabitants is underground eter up to 3-km distance (Fig. 6), we found no evidence for either springs, and concerns about potential herbicide contamination can pollen-mediated hybridization of Zoysia grass or seed dispersal, be lessened through the use of non-selective herbicide (Basta) ap- although the PCR and Basta resistance methods we used would plications to the GM grass lands. not have discriminated pollen-mediated hybrid Zoysia progeny Recently, Reichman et al. (2006) monitored the pollen or from those that grew from GM crop seeds, as pointed out by a seed transfer from a large fi eld of GM herbicide/glyphosate- referee. Th is observation contrasts with the case of GM bentgrass tolerant creeping bentgrass (Agrostis stolonifera L.) developed showing transgene fl ow at multi-kilometer distances mediated by by Scotts and Monsanto. Watrud et al. (2004) and Reichman downwind pollen and/or seed fl ights (Reichman et al., 2006), as et al. (2006) looked at cp4 epsps transgene fl ow and the escape concerns of such unintended gene fl ow was discussed previously by from large test production plots planted by Scotts in Oregon. Wipff and Fricker (2001) and Watrud et al. (2004). However, long Th e establishment of transgenic plants in wild populations distance gene fl ow is of lesser concern for GM Zoysia grass since was the result of unintended releases from Scotts fi elds. pollen/seed-mediated hybridization was not observed at distances Pollen-mediated introgression of herbicidal transgene cp4 greater than a few meters. Further studies are warranted to monitor epsps introduced in the herbicide-tolerant creeping bentrass has the dispersal of viable transgenic pollen over much greater distances been detected within populations of closely related grass spe- from the larger plots of GM Zoysia grass than those reported in this cies at up to 3.8 km from the perimeter of GM grass habitats work. Th e Zoysia japonica seeds show only a 4% germination rate (Baack, 2006; Reichman et al., 2006). When released to the after winter dormancy in its natural ecological habitats (Niwa and natural environment, transgene fl ow to related species could Takanashi, 1943; Bae, 2007), further contributing to the reduced occur, including turf grass. Th us, it is critical to consider the risk of transgene fl ow from the GM Zoysia grass. ecological and societal implications of transgenic bentgrass. Several factors can account for the lack of “long-distance” To release GM grass to agronomic habitats including golf gene fl ow from Jeju Island’s Wimi-Ri test area to the sampling courses and parks, we must address the concerns about trans- sites (Fig. 6). Th ey include inherently recalcitrant cross-pol- gene fl ow from GM Zoysia grass to other compatible grasses and lination in Zoysia japonica, low germination rate under natural weeds under the natural ecological conditions in Jeju. As a fi rst conditions, relatively small GM pollen source, land topography, step to assess the possibility of unintended environmental and and wind variations during the month of May when Zoysia ecological risks associated with transgene (bar in the present case) produces pollen in Jeju. Wind mediates cross-pollination. introgression, we performed several analyses primarily involving Th e island is known for its strong wind. Figure 8 shows wind transgene fl ow from GM Zoysia to WT Zoysia grass, as sum- directions with maximum velocities of 6.7 m/s northerly, 5.6 marized in the results section. Within short distances of 3 m or m/s easterly and south easterly, 8.4 m/s southerly, 4 m/s south less, intra-specifi c hybridization between the GM and WT Zoysia westerly, and 5.9 m/s westerly winds. Average monthly wind velocity in May 2005 was 5 m/s. Perhaps strong and multi-

216 Journal of Environmental Quality • Volume 37 • January–February 2008 directional winds may be counterproductive for pollination, as transgene. We conclude that the GM Zoysia grass developed is an both eff ective pollen fl ight and deposit are aff ected by the wind. environmentally, ecologically, and dermatologically safe grass for We examined potential transgene fl ow from GM Zoysia grass potential use in golf courses and other recreational parks on the to 14 co-habitant wild plant species within the test area. Th e Island of Jeju and possibly in other Zoysia habitats elsewhere. fourteen weed species (Table 6) with similar fl owering periods were sampled for testing the transgene introgression mediated Acknowledgments by pollen fl ight. Th ere were no incidences of cross-hybridiza- Th is work was supported in part by grants from Korea tion between the GM Zoysia and the co-habitant weed group. Ministry of Agriculture and Forestry/RDA Biogreen 21 Program Furthermore, no transgene fl ow from GM Zoysia to other grass (20050401034689 and 20050601034857), Korea Ministry of species such as perennial ryegrass, Kentucky bluegrass, tall fescue, Science and Technology/KOSEF Environmental Biotechnology and cogon grass that were co-cultivated with GM Zoysia inside National Core Research Center (R15-2003-012-010030), and the perimeter of the test area was observed. Since transgene in- City of Seogwipo Dep. of Parks and Recreation, Jeju, Korea. trogression has not been detected among the monocot and dicot grasses sampled for testing, we tentatively conclude that fl ow of References the bar gene from Zoysia japonica to other plant species is rare ANZFA. 2001. Final assessment report (inquiry-section 17)- Application A375: under ecological conditions in and around the test habitat. Food derived from glufosinate ammonium-tolerant corn line T25. 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218 Journal of Environmental Quality • Volume 37 • January–February 2008