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
been generated previously through Agrobacterium tumefaciensmediated transformation. The genetically modified (GM) Zoysia
grass survived Basta spraying and grew to maturity normally
while the wild-type (WT) grass stopped growing and died.
GM Zoysia grass will permit more efficient weed control for
various turf grass plantings such as home lawns, golf courses,
and parks. We examined the environmental/biodiversity risks
of herbicide-tolerant GM Zoysia before applying to regulatory
agencies for approval for commercial release. The GM and
WT Zoysia grass’ substantial trait equivalence, ability to crosspollinate, and gene flow in confined and unconfined test
fields were selectively analyzed for environmental/biodiversity
effects. No difference between GM and WT Zoysia grass in
substantial traits was found. To assess the potential for crosspollination and gene flow, a non-selective herbicide, Basta,
was used. Results showed that unintended cross-pollination
with and gene flow from GM Zoysia grass were not detected
in neighboring weed species examined, but were observed
in WT Zoysia grass (on average, 6% at proximity, 1.2% at a
distance of 0.5 m and 0.12% at a radius of 3 m, and 0% at
distances over 3 m). On the basis of these initial studies, we
conclude that the GM Zoysia grass generated in our laboratory
and tested in the Nam Jeju County field does not appear to
pose a significant risk when cultivated outside of test fields.
Copyright © 2008 by the American Society of Agronomy, Crop Science
Society of America, and Soil Science Society of America. All rights
reserved. No part of this periodical may be reproduced or transmitted
in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system,
without permission in writing from the publisher.
Published in J. Environ. Qual. 37:207–218 (2008).
doi:10.2134/jeq2007.0128
Received 13 Mar. 2007.
*Corresponding author ([email protected]).
© ASA, CSSA, SSSA
677 S. Segoe Rd., Madison, WI 53711 USA
T
urf grasses are commercially important species. As a perennial
monocot species, Zoysia grass is one of the most popularly
cultivated grasses for sports and recreational environments,
particularly in East Asia, because of its relatively high drought
tolerance, disease tolerance, and relatively slow growth habit. To
further improve the turf grass through plant biotechnology, the
transformation of this species (Inokuma et al., 1998; Toyama et
al., 2002; Ge et al., 2006; Li et al., 2006) has been investigated as a
prerequisite for the generation of several transgenic lines including
herbicide-tolerant grass (Toyama et al., 2003).
In a continuing effort to realize the biotechnology-based agronomic potential of turf grass, we investigated (Toyama et al., 2003)
the herbicide tolerance of Zoysia grass by introducing a bar gene that
codes for phosphinothricin N-acetyltransferase (PAT) (Thompson
et al., 1987) which catalyzes acetylation of the amino group of phosphinotricin (phosphinothricyl-L-alanyl-L-alanine). The N-acetylated
peptide can no longer inhibit the key enzyme in the nitrogen assimilation pathway, glutamine synthetase (Bayer et al., 1972). The
bar gene confers tolerance to the broad-spectrum glufosinate-based
herbicide Basta in transgenic crops. Glufosinate is not only a non-selective herbicide, but it is also quite readily biodegraded under natural conditions. Thus, 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,
Faculty of Biotechnology, Cheju National Univ., Jeju 690-756, Korea. E. Vanjildorj and
S.Y. Song, Dep. of Horticulture, Chungnam National Univ., Daejeon 305-764, Korea. S.S.
Yang and J.I. Kim, Dep. of Biotechnology (BK21 Program) and Kumho Life Science Lab.,
Chonnam National Univ., Gwangju 500-757, Korea. T.W. Kang, Applied Radiological
Science Research Inst., Cheju National Univ., Jeju 690-756, Korea. Y.J. Koh, School of
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.
J. Lee, School of Medicine, Cheju National Univ., Jeju 690-756, Korea. Y.E. Lee, Dep. of
Biotechnology, Dongguk Univ., Kyungju, Kyongbuk 780-714, Korea. K.H. Ryu, Division of
Environmental and Life Sciences, Seoul Women’s Univ., Seoul 139-774, Korea.
Abbreviations: GM, genetically modified; PAT, phosphinothricin N-acetyltransferase;
WT, wild type.
207
Table 1. Physicochemical properties of soil mixture used to grow genetically modified (GM) and wild-type (WT) Zoysia grasses. Each indicates the
mean ± standard error of three replicates.
Soil
sample
pH†
EC‡
OM§
Exchangeable cations
Available
P
K
Ca
0.1 N HCl extractable
Mg
Fe
B
Zn
Mn
Cu
GM
g kg−1
mg kg−1
——–––—cmol kg−1¶——–––—
dS m−1
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
——————––––––——mg kg−1#———––––––————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 insignificant.
in terms of minimal environmental impact. Another important
reason for our choice of the bar gene for turf grass biotechnology
application is that it enables the use of herbicide tolerance as a selectable marker for development of transgenic turf grass cultivars
having multiple genes (i.e., herbicide tolerance plus other traits
by gene pyramiding) currently in our development pipeline.
In the present study, we characterized the phenotypic performance of bar-gene transgenic Zoysia grass in the test field and
used the marker gene in preliminary assessments of the environmental/biodiversity concerns arising from GM Zoysia grass. In
view of the widely expressed concerns about the ecological and
biodiversity implications of GM crops and plants, releasing a GM
plant to agronomic habitats entails prior assessments of its risks
to the environment as well as to human and animal health. The
herbicide-tolerant GM crops that underwent such risk assessments
include creeping bentgrass (not currently commercially available
from Scotts), soybean (Monsanto and Bayer CropScience), cotton
(Monsanto, Calgene, Dow AgroSciences, and Bayer CropScience),
maize (Monsanto, Syngenta, DuPont, Bayer CropScience, and
Pioneer Hi-bred), rice (Bayer CropScience), chicory (Bejo Zaden
BV), Argentine canola (Bayer CropScience and Monsanto), Polish
canola (Bayer CropScience and Monsanto), and sugar beet (Novartis, Monsanto, and Bayer CropScience). Yaneshita et al. (1997)
studied the outcrossing or self-pollination potential of Zoysia japonica, and reported evidence of interspecific hybridization within
the genus Zoysia (Z. matrella, Z. sinica, Z. tenuifolia, and Z. macrostashya) on the basis of RFLP and morphological characterization.
In this report, we focused our attention on similar ecological and
environmental concerns arising from the release of GM Zoysia
grass to natural environment.
assessments of GM plants by the Rural Development Administration/Korea Ministry of Agriculture and Forestry.
Transformation of Zoysia japonica
The Agrobacterium-mediated transformation of Zoysia japonica
was established by our laboratory. The bar gene introduced, the
promoter used, and the selection markers and the vector chosen
have been reported in detail elsewhere (Becker, 1990, Becker et al.,
1992; Toki, 1992; Lee et al., 1998; Toyama et al., 2002, 2003).
Environmental Risk Assessments
Preparation of Plants
In T3 generation, the stolons of the herbicide-tolerant Zoysia
grass (GM Zoysia grass hereafter) were subjected to various tests.
The growth and propagation of the grass were investigated during
hardening and vegetative propagation of the stolons in one of the
isolated greenhouses. Wild-type Zoysia grass (WT) plants were
used as the control for the test. The grass stolons thus obtained
were transplanted in the confined test field. The grass plants were
transplanted in a set of porcelain pots with each pot containing
GM and WT plants separated by 25-cm radii (1 pot = 1 unit).
Genetic Stability
Materials and Methods
The GM Zoysia grasses are tolerant to the non-selective
commercial herbicide Basta (Bayer CropScience, Australia) at
a final concentration of 0.1% (w/v) glufosinate. The tolerance
to these herbicides sprayed to GM Zoysia grass was monitored
periodically throughout the T0 and T1 generations. The efficacy of herbicide spraying was assessed under optimal growth
conditions for the grasses. The growth and the herbicide action on GM Zoysia grasses and naturally occurring weed species were investigated 2 wk after Basta was applied.
Plant Materials
Phenotypic Characterization
Unless stated otherwise, all plant materials used for the risk
assessment study reported here have been generated by Toyama
et al. (2003). The seeds of Zoysia grass (Zoysia japonica Steud.)
were obtained as described previously (Bae et al., 2001; Toyama
et al., 2002, 2003). The Zoysia grass stolons produced were
vegetatively propagated in Cheju National University-approved
confined vinyl houses as well as in a test field in Nam Jeju
County, Jeju, Korea, expressly approved for environmental risk
The growth and morphology (stems, leaves, seedlings, etc.)
of the GM Zoysia grass were compared with those of the WT
cultivated under greenhouse conditions, according to previous
methods (Honda and Kono, 1963; Yu et al., 1974; Hong and
Yeam, 1985; Hwang and Choi, 1999; Kim et al., 1996; Choi
and Yang, 2004). Table 1 lists the physicochemical properties
of the soils used for the greenhouse habitat. The morphological
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 environmental 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. The chlorophyll concentration was measured using a portable
chlorophyll analyzer (SPAD-502; Minolta Co., Japan). For seed morphology
(number, length, and width) comparisons, 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
Fig. 1. Field testing (a) and schematic illustration (b) for cross-hybridization between genetically
grass and the synchronously flowering
modified (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
April, but the GM Zoysia grasses flowered 5 to 7 d later than WT.
were distributed within the test field
The GM and the WT Zoysia grasses were transplanted and distribin Nam Jeju County, Jeju. Each pot in two of the three plots
uted according to a completely randomized plot design (Nakayama
had three pots each of GM and WT Zoysia plants, and the
and Yamaguchi, 2002; Belanger et al., 2003b) and the randomized
remaining plot contained five pots each. The seeds harvested
block and crossing block design (Belanger et al., 2004) or an alterfrom the WT Zoysia grass were germinated and the grasses
nating population combination design (Song et al., 2003).
grown until three leaves appeared, then were screened for
In the alternating population combination design test, five
their herbicide tolerance by spraying Basta. The herbicideblocks
each (1 × 12 m2) of GM and WT Zoysia grasses were
tolerant lines screened were then subjected to PCR analysis
distributed alternatively (Fig. 1). Figure 2 illustrates the distribased on bar primers. The primers for the detection of bar
bution patterns of pots (25-cm diam. and pot-to-pot distance
gene were 5’-GGTCTGCACCATCGTCAACC-3’ and 5’?0.5 m) containing GM and WT Zoysia grasses. After allowATCTCGGTGACGGGCAGGA-3’. The Z-A2 actin primers
ing growth for 10 wk under natural field conditions, hybridfor the expression in Zoysia japonica were 5’-GTCAACCCTG
ization results were scored for WT samples in each plot as a
TGCAGCAGTA-3’ and 5’-ATTCAGGTTGGTTGCTCfunction of distance and plot design.
CAC-3’. Thirty five cycles of PCR were performed under the
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
The GM Zoysia grass and the native weeds grew within the
same confined test field during 2003–2005. In May 2004, Basta was sprayed both inside and within a 5-m radius outside the
test field to investigate cross-pollination between the GM grass
and the weeds mediated by wind. One year later, in May 2005,
hybridization in weed species was then examined on the plants
having identical flowering time by means of PCR analysis.
Nearest Neighbor (0.5 m) Cross-pollination
During the 2-yr study (2004 through 2005) performed in both
test fields in Cheju National University and Nam Jeju County, we
found that the GM and the WT Zoysia grasses flowered in late
Bae et al.: GM Zoysia Grass and Environmental Risk
fertilization showing 3-m intervals of four GM grass pots (25-cm
diam.) surrounded by one 6 by 16 m2 patch of WT grass. Hybrid
formation within the 3-m separation was determined after 2 mo
of growth under natural field conditions. Mature WT seeds were
harvested from 96 fractions of a 1 by 1 m2 area around a GM pot
and dried naturally under sunlight. Fresh seeds were stored in ice
box at −10°C until used. The seeds were dehusked mechanically
and kept at 4°C for 1 wk. The seeds were sterilized in 2% sodium
hypochrorite solution for 15 min and rinsed five times in distilled
water. The seeds were then allowed to imbibe on wet filter papers
at 35°C under 4200 cd sr–1 m−2 light for 72 h, and placed at 25°C
under 3500 cd sr–1 m−2 light for germination. The resulting plants
were then sprayed with Basta to screen for hybridization.
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 five 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 transplanted and arranged according to a plot
design (Belanger et al., 2003a). The seeds
were harvested in August 2005, dried, germinated, and screened for hybrid formation by herbicide application.
Long Distance Cross-Fertilization
Fig. 2. Field testing (a) and schematic illustration (b) for cross hybridization between
genetically modified (GM) and wild-type (WT) Zoysia grass according to a randomized
complete block design. Black circle, GM Zoysia grass; white circle, WT Zoysia grass.
Potential gene flow from the total
936 m2 GM grass field (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 screening and PCR analysis. The majority of the
sampling sites are east and northeast biased
relative to the GM grass test field because
of the local land topography—namely high
hills, woody forests, seaside, bushy valleys
south, and southeast of the test field.
Unintended gene flow and seed propagation from the GM creeping bentgrass
field established in 2003 were tested in
2005. For this purpose, both herbicide
screening and PCR methods were used, as
described previously (see Intra-species Hybridization Potentials).
Skin Prick Tests
Fig. 3. Field testing for gene flow from genetically modified (GM) to wild-type (WT) Zoysia grass
within a 3-m radius. Each GM grass pot is surrounded by WT grass patches of 6 by 16 m2 area.
210
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 National University Hospital and on healthy
volunteers over the period from October
2005 through April 2006. For the skin
prick tests, twenty common inhalent allergens and pollen extracts of GM and WT
grasses were used with positive (histamine
1 mg mL−1) and negative (0.9% NaCl)
controls. The sensitization was defined
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
were sensitive to inhalent allergens.
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 segregation ratio in accordance with Mendelian
genetics, and also showing the transgenic
line’s tolerance to ammonium glufosinate
throughout the culture period. The genotype was retained through the multiple
generations in both transgenic Zoysia lines
and their WT hybrids. Before subjecting
the GM Zoysia japonica grass to the environmental risk assessment study reported
here, we observed that the GM Zoysia
grass survived application of the non-selective herbicide spray, whereas WT grass
did not, indicating the stable inheritance
of bar gene in the transgenic grass.
The 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
modified (GM) and wild-type (WT) Zoysia grass and it relative weed species as a function
stages of growth and development. The
of distance. (a) GM Zoysia japonica tillers (illustrated with white circle). (b) WT grass group
containing Zoysia japonica (Zj), Zoysia matrella (Zm), Zoysia sinica (Zs), Lolium perenne (Lp),
grass lines hardened during repeated
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
between the two types of Zoysia grass was that locusts and other
the WT and GM Zoysia plants displayed
insects preferred to reside in the Basta-sprayed GM field, comessentially identical germination and growth rates, and morphopared to the WT field, most likely avoiding the higher amounts of
logical and physiological characteristics. One interesting difference
selective herbicide residuals such as Pyrazosulfuron [ethyl 5-(4,6-
Fig. 5. Test for the potential gene flow from genetically modified (GM) grass to wild-type (WT) grasses within a 3-km radius during a 2-yr period
from 2003 to 2005. The GM grass field is centrally located in the Wimi-Ri test field 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-methylpyrazole4-carboxylate], Alachlor (2-chloro-2′,6′-diethyl-N-methoxymethylacetanilide), and Triclopyr (3,5,6-trichloro-2-pyridyloxyacetic acid)
in the latter. More frequent applications (five to seven times) of the
selective herbicide spray were required to keep the WT grass field
free of weeds, whereas the non-selective herbicide (Basta), just once
or twice was sufficient for the GM grass field.
The herbicide tolerance of the bar transgenic Zoysia grass was
stably preserved for the testing period spanning more than 2 yr.
During the same period that the Basta tolerance of the GM grass
was sustained stably, they remained susceptible to non-selective
herbicides such as paraquat and glyphosate. Thus, for any reason
if it is necessary to terminate the cultivation and spread of the
GM grass within and beyond the test field, the GM plants can be
readily killed by applying a herbicide spray other than Basta.
Is GM Grass Environmentally Risky? Comparative
Characterizations of GM and WT Grasses
Conventional environmental risk assessments of GM crops
have been performed in four categories, namely, (i) establishment
of the substantial equivalence between the GM and the WT
plants, (ii) determination of pollen flight and potential gene flow,
(iii) biodiversity effects of GM plants on unintended or non-target and target plants in their ecological habitats, and (iv) health
risk assessments for animals including humans. We adopted the
four-category protocol for an initial evaluation of GM Zoysia
japonica before its release to agronomic habitats. Category (i) vis.
substantial equivalence, is described and further discussed here
along with the remaining aspects (categories ii–iv). The substantial equivalence between the GM and the WT grasses has been
established on the basis of their essentially identical reproduction rate, morphology of leaves and seeds, germination rate, and
chemical composition.
To ascertain the grassy characteristics of the GM Zoysia plants
in comparison to those of WT plants, the plants were grown
under identical conditions with respect to soil composition, irrigation, and fertilization, etc. Results from the various studies
described here indicate that GM grass displays morphological,
physiological, and genetic characteristics virtually indistinguish-
able from WT grass, except for the bar-gene transgenicity of the
former, which imbues it with tolerance to a herbicide (Basta)
spray. Both GM and WT seeds showed relatively low germination rates, so vegetative propagation through the spreading of
grass stolons over the soil surface became the preferred method
for the hardening and vegetative propagation of the Zoysia grass
cultivars. The following summaries provide further details of the
studies including some of the subtle differences in grassy features
observed between the GM and the WT lines.
Reproduction and Genetic Traits
Flowering Time
During the 2-yr study conducted in 2004 and 2005, we observed WT grasses starting to flower in late April, whereas the GM
plants flowered about 5 to 7 d later. Both types of grasses fully
flowered within 5 d of 10 May, formed dried pollens (i.e., inactivation of anthers), and full seed formation by mid-July, consistent
with the flowering times reported by Kitamura (1967). Since GM
grass began flowering about 5 to 7 d after WT plants, the frequency of formation of intraspecific GM hybrids could have been
reduced. To circumvent this possibility arising from the flowering
time difference, both GM and WT grass cultivars that flower at
about the same time in the greenhouse were replanted in the plots
of the test field. However, after 2 yr this became unnecessary as
both types of grass flowered simultaneously (see below).
Pollination
Pollen formation was maximal around 10 May, and intraspecies hybrid formation induced by pollination was most
prevalent thereafter, 15 to 18 May. We decided to introduce
WT plants having similar flowering time into the GM grass
greenhouse and outdoor test field on 15 May, 2004, and
2005. Results showed that both GM and WT grasses crosspollinated at an average rate of 6% at close proximity.
Morphology
The morphology of Zoysia grass can be classified in terms
of leaf width and length, according to Kitamura’s horticultural
classification method (Kitamura, 1967). We examined other
appearance indices of the plants, as presented in Table 2, which
shows morphological features of GM and WT
Table 2. Morphological characteristics of genetically modified (GM) and wild-type
grasses. Results show that the two types of grasses
(WT) Zoysia grass leaves observed under field growing conditions. Each value
are essentially indistinguishable and any differences
indicates the mean ± standard error of fifteen replicates.
observed were statistically insignificant. Table 3
Plant
Leaf
First leaf
Leaf
Leaf
Chlorophyll
Leaf
compares the seed morphologies, again indicating
Plants
height
blade†
height‡
width‡
angle§
contents¶
weight#
that the two types of grasses are indistinguishable.
–––––––––––––––––cm–––––––––––––––––
A°
g
g kg−1 FW
Other phenotypic traits also showed no significant
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
differences between the GM and WT Zoysia cultiWT
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
vars (Tables 2, 3, and 4).
t-test
NS††
NS
NS
NS
NS
NS
NS
In summary, after 16 mo of cultivation in the test
† Values measured from the third leaf.
field, morphological analyses were performed and both
‡ Length from basal zone of the shoot to first leaf blade.
§ 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. The 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 insignificant at 0.05 level by t test.
212
Journal of Environmental Quality • Volume 37 • January–February 2008
Table 3. Seed characteristics of genetically modified (GM) and wild-type (WT) Zoysia grass using eight morphological traits; length of flowering
culms, length of spike without rachis; length/width ratio; germination, and weight.
Plants
No. of seeds
per spike
Length of
rachis
Length of
flowering culms
––––––––––––cm––––––––––––
GM
49.4 ± 7.6†
4.8 ± 0.6‡
12.1 ± 2.4‡
WT
49.1 ± 7.3
4.9 ± 0.6
11.7 ± 2.7
t-test
NS¶
NS
NS
† Mean ± standard error of forty-five replicates.
‡ Mean ± standard error of fifteen replicates.
§ Mean ± standard error of five replicates.
¶ NS, considered statistically insignificant at 0.05 level by t test.
of stolons (ca. 5), its length (approx. 30.4–33.0 cm), and the leafnode length (approx. 3.2–3.7 cm) were also statistically equivalent
for the both types (Table 4). In addition, a seed’s morphological
characterizations performed included the number of seeds per
spike (ca. 49); length of flowering culms (ca. 12 cm) and the rachis
length (ca. 4.8 cm), seed length (ca. 3.1 mm) and width (ca. 1.5
mm), 1000 seeds weight (ca. 0.58 g), and the rate of germination
(approx. 3.7–4%, Table 3). The chemical and mineral compositions of seeds harvested from both types were also performed and
no significant differences were found (data not shown).
Hybridization
As evident from the above result (see Pollination), no
significant difference in pollination rates was found between
the GM and the WT Zoysia grasses. We also examined the
pollination from GM to WT Zoysia grasses, and in the reverse
direction. Table 5 shows that the minimum cross-pollination rate was 3% and maximum was 9%, with an average of
6%, at the nearest distance (>0 m). At a 0.5-m distance in
both randomized and completely randomized plot designs,
cross-pollination was approximately 1.2%, which declined to
0.12% at a 3-m distance, and to 0% at distances greater than
3 m (Table 5). Figure 6 graphically illustrates the distance
dependence of GM-to-WT Zoysia gene flow. The best fit for
this distance dependence from a regression analysis of the data
described in the results section is an exponential function as
shown in the figure. Similar distance dependence has been
reported for wild rice (Oryza rufipogon) (Song et al., 2003).
Winter Dormancy
Seed length
(SL)
Seed width
(SW)
––––––––mm––––––––
3.2 ± 0.3‡
1.5 ± 0.2‡
3.1 ± 0.3
1.4 ± 0.2
NS
NS
SL/SW
ratio
Frequency of
germination
Weight of
1000 seeds
2.2 ± 0.2‡
2.2 ± 0.3
NS
%
3.7 ± 1.2
4.0 ± 1.0
NS
g
0.6 ± 0. 03§
0.6 ± 0.02
NS
GM Zoysia’s Dominance over Weeds
The Zoysia grass propagates reproductively both from seeds
and vegetatively. The weight of the 1000 seeds is approx. 0.57
to 0.59 g. Even if wind carries the seeds over some distance,
the germination rate is less than 4% under natural conditions.
Thus, compared to germination, Zoysia can spread itself more
effectively through vegetative propagation. However, the Zoysia
grass is not a dominant species and does not spread into weedy
areas easily. In fact, the Zoysia grass field is completely dominated by the weeds within 2 to 3 yr of cohabitation. Figure 7 illustrates the effects of dominant weeds on the GM Zoysia grass,
showing the dominance of the weeds over the Zoysia grass.
Disease Tolerance and Pathogenic Organisms
The effects of GM Zoysia grass on the population of several
pathogenic soil fungi were investigated. Table 7 lists the soil pathogens distributed within the soil layer and the plant segment of approximately 3 cm length from the soil surface layer in the test field.
No significant differences in the population of the four major turf
grass pathogens (Rhizoctonia spp., Pythium spp., Curvularia sp.,
and Colletotrichum sp.) between WT and GM grass planted soils
were found, with all differences within experimental and statistical
margins of error. However, the soil samples contained relatively
high levels of Fusarium spp. in both WT and GM grass plots. The
relatively dense population of this fungus is attributable to low
soil pH and electrical conductance (Kwon et al., 1998; Suh et al.,
2003). Fusarium spp. is a common fungal pathogen in soil, but
turf grass plants are apparently unaffected. In fact, the Fusarium
spp. stimulates a plant’s growth by suppressing several co-habitat
pathogens (Meera et al., 1993, 1994; Liu et al., 1995; Yun, 1996;
Park and Yu, 2005). The higher density of Fusarium spp. in the
Both WT and GM Zoysia grass showed essentially identical dormancy profiles in the Nam Jeju County test field, turning brown, wilting by late November, and
staying dormant until the next March.
Table 4. Growth characteristics of genetically modified (GM) and wild-type (WT) Zoysia grass
Effects of GM Zoysia Grass on
Neighboring Weeds: Potential Weediness
Gene Flow from GM Zoysia to Weeds
Table 6 lists 14 co-habitant weed species
within the GM grass test plot facility. Neither
Basta nor PCR evidence was obtained to
indicate bar-gene flow from the GM plant’s
pollen to these neighboring weed species during the study conducted from 2003 to 2005.
Bae et al.: GM Zoysia Grass and Environmental Risk
using five morphological traits. Each value indicates mean ± standard error of triplicates.
Coverage†
Plants
90DAP‡
150DAP
Stolon No. Stolon length Leaf-node length
90DAP
90DAP
90DAP
Density§
150DAP
–––––––––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
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
t-test
NS¶
NS
NS
NS
NS
NS
† Coverage, about 10-cm diameter of GM and WT Zoysia grass plugged after 10 May.
‡ DAP, days after plugging.
§ Density, number of tiller per cm2.
¶ NS, considered statistically insignificant at 0.05 level by t test.
213
Table 5. Number of the germinated seeds tested and the hybrids
identified (number in parentheses) at distances from genetically
modified (GM) Zoysia grass in each plot design.
Distance CRD† RCBD‡
R-3§
Table 6. Test for the potential outcrossing between genetically
modified (GM)-Zoysia grass and weed plants grown within the
Wimi-Ri test field.
R-9Zj¶ R-9Zs# R-9Zm†† R-9Lp‡‡
m
m>0
746(45)
-§§
–
–
–
–
–
0.5
491(6) 967(12)
–
–
–
–
–
1
–
245(4)
72(1) 243(2)
–
–
–
2
–
–
660(2) 145(1)
–
–
–
3
–
–
2547(3) 231(0) 83(0)
0
89(0)
6
–
–
–
209(0) 152(0)
79(0)
176(0)
9
–
–
–
214(0) 104(0)
58(0)
93(0)
† CRD, completely random design.
‡ RCBD, randomized complete block design.
§ R-3, WT Zoysia japonica within 3-m radius from GM Zoysia japonica pot
(0.25-m diameter).
¶ R-9Zj, WT Zoysia japonica within 9-m radius from GM Zoysia japonica
(1.5-m diameter).
# R-9Zs, WT Zoysia sinica within 9-m radius from GM Zoysia japonica (1.5-m
diameter).
†† R-9Zm, WT Zoysia matrella within 9-m radius from GM Zoysia japonica
(1.5-m diameter).
‡‡ R-9Lp, WT Lolium perenne within 9-m radius from GM Zoysia japonica
(1.5-m diameter).
§§ Blank boxes represent no plot design data.
GM grass soil may account for the lower levels of Curvularia sp.
and Colletotrichum sp., although the pathway by which the GM
grass soil stimulates Fusarium growth remains unknown (Table 7).
In fact, our study showed that other pathogenic populations were
suppressed by Fusarium spp. (data not shown).
Potential Gene-induced and Allergic Hazards
bar Gene
The bar gene described earlier was originally isolated from
S. hygroscopicus. Its coded amino acid sequence showed an
84% identity with the 183-amino acid polypeptide encoded
by pat gene from S. viridochromogenes (National Center for
Biotechnology Information BLAST 2; Wehrmann et al.,
1996). On a 12% sodium dodecyl sulfate-polyacrylamide gel
No
Scientific name
Common name
Flowering
season Outcross
1
Spergula arvensis
Corn spurry
2
Cerastium holosteoides
Common mouse ear
Mar.-June –
Mar.-June –
3
Stellaria media
Chickweed
Apr.-June
–
4
Trigonotis peduncularis Cucumber herb
Apr.-June
–
5
Taraxacum officinate
Dandelion
Apr.-June
–
6
Veronica arvensis
Corn speedwell
Apr.-June
–
7
Vicia angustifolia
Garden vetch
Apr.-June
–
8
Erigeron annuus
Daisy fleabane
June-Sept. –
9
Mazus pumilus
Japanese mazus
Apr.-Aug.
–
10
Youngia japonica
Japanese youngia
Apr.-June
–
11
Cardamine impatiens
Narrow leaf bitter cress Mar.- May –
12
Gnaphalium affine
Cudweed
May-July
–
13
Alopecurus aequalis †
Orange Foxtail
Apr.-June
–
14
Poa annua †
Annual Bluegrass
Dec.-June –
† Family of Gramineae with no hybridization from GM Zoysia grass.
electrophoresis (SDS-PAGE) gel, both proteins were identically Western blotted at 21 to 23 kDa (Herouet et al., 2005).
When the amino acid sequence of PAT was matched
against the known allergenic sequences using BLAST 2.2.15
algorithm, SwissProt, PDB, PIR, PRF in NCBI, and Food
Allergy Research and Resource Program (FARRP)-FASTA
Version 6, 80 or more amino acid-peptide sequences of the
enzyme showed less than 35% homology. In addition, no
homology was found between the protein sequences and the
eight amino acid allergen epitopes (FAO/WHO, 2001; Codex
Alimentarius Commission, 2003; Bae, 2007). The pat gene is
inactivated at a pH below 4 or by heating for 30 min (Wehrmann et al., 1996; ANZFA, 2001; Herouet et al., 2005).
Previous animal and human studies showed that the pat gene
posed no significant health risks (Jones and Maryanski, 1991;
Sjoblad et al., 1992; Schmidt, 1994; Mossinger and Dietrich,
1998; WHO, 1998; OECD, 1999; Institute of Science in
Society, 2003; Thomas et al., 2004; Herouet et al., 2005). The
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). The gene introduced showed no apparent 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 Commission, 2003; European Commission, 2003).
Allergic Reactions
Fig. 6. Distance dependence for gene flow from the genetically modified
(GM) to wild-type (WT) Zoysia grass within 9-m radius in field. The
observed data can be best fit by an exponential equation resulting
from a regression analysis of the data. Bars refer to standard error.
214
Kim et al. (1987) performed skin prick tests with pollen
extracts of Zoysia grass and found that 5% of respiratory allergic 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
had respiratory allergic disorders. Thus, six subjects (4.7% of
the 127 test subjects) developed a positive allergic reaction
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 lawn 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 difference
between the two types of pollens was observed.
Discussion
We first reported the successful establishment of GM herbicide/
Basta-tolerant Zoysia japonica Steud. by transforming the plant calli
with the transgene bar (Toyama et al., 2003) under greenhouse
habitats. In the present study, we confirmed that the transgenic
Zoysia grass contained two copies of the bar gene. We further characterized the phenotypic performance and the transgene introgression in the natural ecological environment of Jeju Island, Korea.
All morphological and biochemical analyses suggested that the
GM Zoysia grass developed is indistinguishable from its WT plant,
except for its Basta tolerance, under greenhouse, and field habitat
conditions (Fig. 1–6 and Tables 1–7). In addition, no difference in
Table 7. Test for the fungal infection of genetically modified (GM) and
wild-type (WT) Zoysia grass within the test field.
Fungal infection
GM Zoysia grass
Fungi
WT Zoysia grass
Base Rhizosphere Base Rhizosphere
line†
soil‡
line
soil
Disease
–––––––––––––––––%–––––––––––––––––
Rhizoctonia spp.
ND§
ND
ND
ND
Large patch
Pythium spp.
ND
ND
ND
ND
Pythium blight
Curvularia sp.
2.8
3.0
4.5
8.3
Leaf blight
Colletotrichum sp.
ND
ND
1.5
ND
Anthracnose
Fusarium spp.
75.8
19.4
22.7
11.1
Unknown
† Shoot base.
‡ Stem’s rhizosphere soil zone.
§ ND, fungi not detected.
Bae et al.: GM Zoysia Grass and Environmental Risk
the incidence of allergic skin reactions to both GM and WT Zoysia
grass was observed (Table 8). Thus, the focus of the discussion will
be on the concerns about transgene flow from the GM Zoysia grass
to compatible WT Zoysia and other weed species within and outside the test field in Jeju.
Table 8. Results from the skin prick tests for common and Zoysia
grass allergens.
Allergen
No. of patients
Positive reaction
%
Positive control (1mg/mL histamine)
Negative control
Dermatophagoides farinae
127
100
0
0
72
56.7
Dermatophagoides pteronyssinu
60
47.2
American cockroach
36
28.3
German cockroach
39
30.7
Cat and dog hair
18
14.2
Horse and cattle hair
4
3.2
Flag
3
2.4
Broadleaf tree
2
1.6
Acicular tree
6
4.7
Japanese cedar
5
3.9
American cedar
6
4.7
House dust-fungi
8
6.3
Outdoor fungi
6
4.7
Flowers
12
9.4
Weeds
14
11.0
Crops
10
7.9
GM Zoysia grass
6
4.7
WT Zoysia grass
6
4.7
215
grass tillers was significant (Fig. 6). This observation is consistent with the documented cases of
conventional gene flow 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 populaFig. 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
km from the perimeter of the GM grass habitats
easterly and south easterly, 8.4 m/s southerly; 4 m/s south westerly, and 5.9 m/s
(Baack, 2006; Reichman et al., 2006). Thus,
westerly winds. Average monthly wind velocity was 5 m/s during the flowering
season and gene flow testing.
when released to the natural environment, herbicide-tolerant weeds could evolve as the result of
The GM Zoysia grass has been developed for its eventual release
Implications of the bentgrass case are critical for
transgene
fl
ow.
to agronomic habitats and recreational lands such as golf courses.
both
ecological
and societal concerns. However, it should be
This work has been performed under a joint developmental agreepointed
out
that
the comparison of the distances of transgene
ment with the Jeju Provincial Government. The local government
fl
ow
in
the
present
study to those of Reichman et al. (2006)
is particularly interested in herbicide-tolerant grass for its potential
is
not
quantitatively
valid, since the scale of the pollen sources
environmental and economic implications for Jeju’s golf courses
between
the
two
studies
differ markedly (we thank the referee
and recreational parks. We anticipate that the use of GM Zoysia for
for
pointing
this
out).
golf courses will substantially reduce the amount and frequency of
However, from 121 sampling sites beyond our test area perimweed-selective herbicide sprays performed annually. The volcanic
eter
up to 3-km distance (Fig. 6), we found no evidence for either
island’s water supply for half a million inhabitants is underground
pollen-mediated
hybridization of Zoysia grass or seed dispersal,
springs, and concerns about potential herbicide contamination can
although
the
PCR
and Basta resistance methods we used would
be lessened through the use of non-selective herbicide (Basta) apnot
have
discriminated
pollen-mediated hybrid Zoysia progeny
plications to the GM grass lands.
from
those
that
grew
from
GM crop seeds, as pointed out by a
Recently, Reichman et al. (2006) monitored the pollen or
referee.
Th
is
observation
contrasts
with the case of GM bentgrass
seed transfer from a large field of GM herbicide/glyphosateshowing
transgene
fl
ow
at
multi-kilometer
distances mediated by
tolerant creeping bentgrass (Agrostis stolonifera L.) developed
downwind
pollen
and/or
seed
fl
ights
(Reichman
et al., 2006), as
by Scotts and Monsanto. Watrud et al. (2004) and Reichman
concerns
of
such
unintended
gene
fl
ow
was
discussed
previously by
et al. (2006) looked at cp4 epsps transgene flow and the escape
Wipff
and
Fricker
(2001)
and
Watrud
et
al.
(2004).
However,
long
from large test production plots planted by Scotts in Oregon.
distance
gene
fl
ow
is
of
lesser
concern
for
GM
Zoysia
grass
since
The establishment of transgenic plants in wild populations
pollen/seed-mediated hybridization was not observed at distances
was the result of unintended releases from Scotts fields.
greater than a few meters. Further studies are warranted to monitor
Pollen-mediated introgression of herbicidal transgene cp4
the dispersal of viable transgenic pollen over much greater distances
epsps introduced in the herbicide-tolerant creeping bentrass has
from the larger plots of GM Zoysia grass than those reported in this
been detected within populations of closely related grass spework. The Zoysia japonica seeds show only a 4% germination rate
cies at up to 3.8 km from the perimeter of GM grass habitats
after winter dormancy in its natural ecological habitats (Niwa and
(Baack, 2006; Reichman et al., 2006). When released to the
Takanashi, 1943; Bae, 2007), further contributing to the reduced
natural environment, transgene flow to related species could
risk of transgene flow from the GM Zoysia grass.
occur, including turf grass. Thus, it is critical to consider the
Several factors can account for the lack of “long-distance”
ecological and societal implications of transgenic bentgrass.
gene
flow from Jeju Island’s Wimi-Ri test area to the sampling
To release GM grass to agronomic habitats including golf
sites
(Fig.
6). They include inherently recalcitrant cross-polcourses and parks, we must address the concerns about translination
in
Zoysia japonica, low germination rate under natural
gene flow from GM Zoysia grass to other compatible grasses and
conditions,
relatively small GM pollen source, land topography,
weeds under the natural ecological conditions in Jeju. As a first
and
wind
variations
during the month of May when Zoysia
step to assess the possibility of unintended environmental and
produces
pollen
in
Jeju.
Wind mediates cross-pollination.
ecological risks associated with transgene (bar in the present case)
Th
e
island
is
known
for
its
strong wind. Figure 8 shows wind
introgression, we performed several analyses primarily involving
directions
with
maximum
velocities
of 6.7 m/s northerly, 5.6
transgene flow from GM Zoysia to WT Zoysia grass, as summ/s
easterly
and
south
easterly,
8.4
m/s
southerly, 4 m/s south
marized in the results section. Within short distances of 3 m or
westerly,
and
5.9
m/s
westerly
winds.
Average
monthly wind
less, intra-specific hybridization between the GM and WT Zoysia
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
both effective pollen flight and deposit are affected by the wind.
We examined potential transgene flow from GM Zoysia grass
to 14 co-habitant wild plant species within the test area. The
fourteen weed species (Table 6) with similar flowering periods
were sampled for testing the transgene introgression mediated
by pollen flight. There were no incidences of cross-hybridization between the GM Zoysia and the co-habitant weed group.
Furthermore, no transgene flow from GM Zoysia to other grass
species such as perennial ryegrass, Kentucky bluegrass, tall fescue,
and cogon grass that were co-cultivated with GM Zoysia inside
the perimeter of the test area was observed. Since transgene introgression has not been detected among the monocot and dicot
grasses sampled for testing, we tentatively conclude that flow of
the bar gene from Zoysia japonica to other plant species is rare
under ecological conditions in and around the test habitat.
In a natural ecological environment, Zoysia grasses are readily overtaken by dominant wild plant species and they do not
survive well in the wild (Fig. 7). Since growth and propagation
of the Basta-tolerant Zoysia grass can be terminated by the use of
non-glufosinate herbicides, the risk of GM Zoysia grass spreading to unintended areas is low and controllable. Interestingly, we
observed that locusts and other insect habitants in the test field
thrived by feeding on GM Zoysia grass while avoiding the WT
Zoysia grass treated with several obligatory sprays of non-Basta
herbicides. Also, potential for horizontal gene flow (HGT) mediated by insects’ microflora and soil microorganisms is unlikely
(Bae et al., 2007). Brown et al. (2001) observed the populations
of GM canola, potato, corn, and sugarcane in their natural habitats for over 10 yr, and found that these GM crops were not more
adaptive to the environment than their WT plants. Similarly, for
the next 8 to 10 yr, we will continue to study the herbicidal activity, intra- and inter-specific transgene introgressions, and ecological effects of the GM Zoysia grass described in this report.
Finally, about 5% of pulmonary patients show positive allergic reactions to Zoysia grass’ pollen (Kim et al., 1987). We
showed similar incidences (4.7%) of allergic skin responses to
the extracts from the pollens of both GM and WT Zoysia grasses. No differences were observed between the pollens of the two
grass types, suggesting that the bar gene introduced in GM Zoysia grass does not induce production of specific allergens. This
suggestion is in agreement with preliminary reports that the bar
gene does not induce or encode for any allergen (OECD, 1999;
Codex Alimentarius Commission, 2003; Herouet et al., 2005).
Conclusions
The herbicide-tolerant GM and the WT Zoysia are substantially equivalent, except for the transgene bar’s phenotypic traits
that are conferred on the former. Transgene introgression is significant at close proximity between the GM and the WT Zoysia
grasses. However, no gene flow mediated by pollen flights was detected among the wild species within the test habitat and the WT
Zoysia grasses at 121 sampling sites multi-kilometers away from
the perimeter of the test area in Jeju. Also, the herbicide-tolerant
grass poses no specific allergic/health risks associated with the bar
Bae et al.: GM Zoysia Grass and Environmental Risk
transgene. We conclude that the GM Zoysia grass developed is an
environmentally, ecologically, and dermatologically safe grass for
potential use in golf courses and other recreational parks on the
Island of Jeju and possibly in other Zoysia habitats elsewhere.
Acknowledgments
This work was supported in part by grants from Korea
Ministry of Agriculture and Forestry/RDA Biogreen 21 Program
(20050401034689 and 20050601034857), Korea Ministry of
Science and Technology/KOSEF Environmental Biotechnology
National Core Research Center (R15-2003-012-010030), and
City of Seogwipo Dep. of Parks and Recreation, Jeju, Korea.
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Journal of Environmental Quality • Volume 37 • January–February 2008