crops to weedy species and subsequent changes in fitness of weed populations have been a topic of
recent research. For example, interspecific F
1
hy- brids from Brassica rapa × transgenic Brassica na-
pus controlled crosses showed normal expression and Mendelian segregation of the BAR gene [7].
However, selfing of the hybrids produced no vi- able seeds, and transmission frequency of the BAR
gene was significantly reduced in one of two back- cross populations. Similar experiments using non-
transgenic material showed that B. rapa × B. napus F
1
, F
2,
and backcross generations were inter- mediate to or lower in fecundity than that of the
parents [8,9]. Transgenic B. napus × Raphanus raphanistrum wild radish F
1
hybrids and four successive generations were previously evaluated
under field conditions for seed production and BAR gene transmission [10]. Seed production was
low in F
1
populations, but increased to near wild type levels by the third generation. BAR frequency
decreased to 25 of the original by the fourth generation and was associated with reduced num-
bers of chromosomes in female parents. Hybrids produced between wild and domesticated strains
of sunflower Helianthus annus L. showed reduced seed production and dormancy for most, but not
all, of the F
1
populations that were evaluated [11]. Wild strains and domesticated cultivars of rice
have been shown to hybridize and produce viable offspring, but with varying degrees of efficiency
[12]. Cultivated rice has a companion weed, red rice Oryza sati6a, which exists in temperate and
tropical regions of the world that reduces grain yield and quality and is considered a noxious weed
in rice producing areas of the southern United States [13]. At present, red rice control is achieved
through crop rotation and paddy-water manage- ment. Frequency of hybridization between non-
transgenic cultivated rice and red rice has been evaluated in one study [14], where seeds were
collected from red rice plants found in commercial Louisiana rice fields and planted in common gar-
den experiments. Electrophoretic analysis iden- tified individuals produced from hybridization
between cultivated and red rice plants. Hybrids generally exhibited greater height and flag leaf
area than the cultivated or red rice parents. Tiller number was greater in the hybrids than in the red
rice, but not in the commercial cultivars. Overall, these results indicated that hybrid vigor did occur
for certain vegetative characteristics. Incidence of hybridization ranged from 1 for early maturing
cultivars to a high of 52 in a commercial plant- ing of the late maturing cultivar Nortai.
The BAR gene was recently transferred and evaluated in 11 different transgenic rice lines in 2
years of field-plot trials [6]. Significant differences among transgenic BAR-containing lines were ob-
served for grain yield, plant height, and date of flowering before or after treatment with Liberty.
Other work [15] showed that insertion of the nptII gene in rice was associated with reduced seed
fertility, delayed maturity, and smaller flag leaves when compared with the corresponding non-trans-
formed, protoplast-derived plants. Similar results were obtained in transgenic barley [16]. Reciprocal
crosses between two transgenic rice cultivars and a common Louisiana red rice biotype displayed sin-
gle-gene, dominant transmission and expression of the BAR gene in F
1
and F
2
generations [17]. No cytoplasmic influence on expression of the trans-
gene was detected when either the cultivated or wild strains were used as maternal parent. A total
of five quantitative trait loci QTL were detected for rice seed dormancy on four chromosomes that
explained 48 of total phenotypic variation in BC
1
F
5
lines [18]. One dormancy QTL mapped to the same location as a QTL for heading date.
The objective of this research was to investigate agronomic and fitness traits of BAR transgenic
rice-red rice hybrid populations that segregated for resistance to Liberty herbicide under field condi-
tions. We expected to gain a greater understanding of potential consequences of gene flow from herbi-
cide resistant commercial rice into red rice. To our knowledge this study was the first in the United
States to evaluate potential effects of the BAR gene on life history, fitness and seed characteristics
of red rice.
2. Materials and methods
2
.
1
. De6elopment of crop-weed hybrid populations A total of three straw hulled, awnless biotypes
1, 2, 3 and one black hulled, awned biotype 4 red rice plants were collected at a south Louisiana
commercial rice field in the summer of 1996 and used in greenhouse crosses with non-transgenic
Bengal and Cypress cultivars and Liberty resistant transgenic lines Table 1. An additional straw
hull, awnless red rice, designated biotype 5, has been previously characterized for seed dormancy
[19 – 21] and was included as a control. The trans- genic BAR lines were previously shown by labora-
tory and field tests to contain one or more stably incorporated copies of the BAR gene and express
tolerance to 1.1 kg a.i.ha field applications of Liberty herbicide [22]. F
1
seeds were planted in the greenhouse for seed increase during the winter of
1996 – 1997. F
2
seeds were harvested in the spring of 1997 and dried to 12 moisture for field plant-
ing. From each F
2
population ten seeds were tested for germination using a standard 7-day test
[21]. Only those seed lots with germination per- centages higher than 85 were planted in field
experiments.
2
.
2
. Field experiments Field experiments were planted on May 7, 1997
at the LSU Agricultural Center, Central Station- Ben Hur Farm near Baton Rouge, LA and on
May 14, 1997 at the University of Arkansas, Southwest Research and Extension Center near
Hope, Arkansas. The experimental design was a randomized complete block design with four repli-
cations. Each plot, 2 m long and 1.33 m wide, was planted using a seed drill planter with 60 seeds and
30 cm distance spacing to allow adequate growth and inspection of individual plants. Plot
sites were located outside the commercial rice pro- duction areas of the respective states. Soil prepara-
tion and irrigation management were typical for the rice agricultural systems of Louisiana and
Arkansas. Both sites received quinclorac 0.43 kg a.i.ha,
thiobencarb, 4.5
kg a.i.ha,
and propanil, 4.5 kg a.i.ha herbicides to control
weeds. At the Hope site, Permit™ halsulfuron- methyl, 70 g a.i.ha was also applied for nutsedge
Cyperus esculentus control. Except for leaf-dip scoring of herbicide resistance in F
2
populations, Liberty herbicide was not applied.
The following variables were measured at the Ben Hur site: seedling emergence counts taken at
34 days post-planting, vigor rating rating of 1 = most vigorous, 9 = least vigorous, plant height
measured from soil line to tip of flag leaf, date of first and 50 tillering, date of first, 50, and last
heading heading defined as date of panicle emer- gence from the boot. For seed shatter rating at
the Ben Hur site, four panicles per plant and four plants per plot were harvested at physiological
maturity. After collection panicles were carefully transported to a laboratory and rapped ten times
against a plastic bucket. The percentage of seeds remaining on the panicle was used to give a seed
Table 1 Description and designation of parents, F
2
families, transgenic lines, normal cultivars, and red rice biotypes evaluated in this study Description of entries
Entry Type
Designation Cypress-CPB6
a,d
× Red rice biotype 1
b,c
1 F
2
population tCP×Red c 1
F
2
population 2
tCP×Red c 2 Cypress-CPB6
a,d
× Red rice biotype 2
b,c
3 tBG×Red c 4
F
2
population Bengal-HU10
a,d
× Red rice biotype 4
b,c
tBG×Red c 3 Bengal-HU10
a,d
× Red rice biotype 3
b,c
F
2
population 4
Transgenic line 5
tBG-BXS2 Bengal-BS×2
a,d
F
2
population 6
Red c 1×tCP Red rice biotype 1
b,c
× Cypress-CPB6A
d
F
2
population 7
Red c 4×BG Red rice biotype 4
b,c
× Bengal
b,d
Red c 4 Red rice biotype 4
b,c
8 Red rice line
F
2
population 9
BG×Red c 4 Bengal
b,d
× Red rice biotype 4
b,c
CP×Red c 4 Cypress
b,d
× Red rice biotype 4
b,c
10 F
2
population 11
Red rice biotype 5
b
Red c 5 Red rice line
tCP Cypress-CPB6
a,d
Transgenic line 12
tBG 13
Bengal-HU10
a,d
Transgenic line Transgenic line
14 tBG-11
Bengal-HU11
a,d
Cypress
b,d
Normal cultivar CP
15 BG
Normal cultivar Bengal
b,d
16
a
Liberty herbicide-resistant transgenic line.
b
Non-transgenic line or cultivar.
c
Pubescent hairy leaves.
d
Glaborous smooth leaves.
shattering rating for each plot: 0 = 0 – 10, 1 = 11 – 30 , 2 = 31 – 60, 3 = \ 60. Total seed
weight, 100-seed weight, and total seed number were also determined. For seed shattering at
Hope, a four-panicle sample was placed in a 30 × 12-cm panicle envelope and rapped violently four
times against the edge of the laboratory bench. The panicles and the seeds still attached to them
were removed and weighed. The loose seed in each envelope was weighed and a ratio of loose seed
weightoriginal total panicle weight was consid- ered an estimation of shattering. Panicles were
stripped by hand into a bucket, all seed returned to the sample envelope and allowed to dry for 3
days at 50°C. The seed were then passed through a small seed lot thresher to separate the blank
florets from the seed. Both empty florets and total seed weights were obtained. A sample of 100 seeds
was weighed and the total number of seeds was calculated. A standard 100 empty floret weight
was used to calculate the number of florets that did not produce seeds.
For seed dormancy evaluation, seeds and awns if present were removed from panicles by hand.
Seed samples from individual panicles were trans- ferred to plastic containers and remained on the
lab bench at 23°C for dry after-ripening. At 1 and 8 weeks after harvest, a subsample of 15
seedspanicle was removed and tested for germina- tion and dormancy. Plastic 9-cm square Petri
dishes were prepared with three sheets of Anchor Standard brown germination paper Anchor Pa-
per, Minneapolis, MN and 8-10 ml of 0.01 Dithane or 0.005 Chlorothalonil fungicide Gro
Tech, Madison, GA diluted with deionized water. Seeds were incubated at 30°C in the dark at 100
relative humidity for 14 days.
Best management practices were used to control the seed remaining at field sites after harvest from
all three rice types: red rice, hybrid populations and white crop rice. The Ben Hur site was mowed
on November 25, 1997, the first opportunity to enter the field due to wet conditions. All plant
material with the exception of the seed samples, remained at the site. The levies remained intact to
prevent inadvertent seed burial and to allow the accumulation of winter rainfall into standing wa-
ter. At the Hope site, the remaining plant material was cut and left in the field to dry on October 14,
1997. Dry hay was placed on top of the test site and on December 10, 1997, rice stubble at the test
site was burned. The levees remained standing and the soil surface was undisturbed except for a light
cultivation operation to stimulate germination in the early spring of 1998.
The two sites were monitored after each rainfall or irrigation that was sufficient to stimulate germi-
nation. After the first recorded germination of rice andor red rice volunteers in the spring, six 1-m
square quadrants at Ben Hur and four at Hope, were established to monitor numbers of surviving
volunteers. Except for the following herbicide ap- plications, the sites remained fallow and undis-
turbed. After each new germination flush, the field was sprayed with recommended field rates of glu-
fosinate ammonium. Plots were then treated with recommended field rates of glyphosate to kill all of
the remaining living vegetation. The plots were subsequently resprayed and monitored until vol-
unteers were no longer observed. Appropriate au- thorizations
were obtained
from the
USDA-APHIS for all environmental releases field planting of the transgenic rice seed.
2
.
3
. Statistical analyses All analyses were completed on plot mean val-
ues. Analysis of variance was performed using JMP
®
[23]. The minimum model used for all analyses of variance was comprised of entries and
replications, with the entry × replication interac- tion used as the error term. Replications and the
entry × replication interaction were considered random effects. Separation of means was per-
formed using the Duncan’s Multiple Range Test.
3. Results
