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
Mean values for life history and fecundity traits at each location are given in Tables 2 and 3. At
the Baton Rouge location, Red c 4 exhibited greater mean number of plantsplot than non-
transgenic BG, transgenic tBG and tCP parents, but not seven of the F
2
populations entries 1, 2, 3, 6, 7, 9, 10. Red c 5 showed similar results except
for lower values when compared to F
2
population tBG × Red c 4 entry 3. Among the parents used
to make crosses in this study tCP, tBG, CP, and BG, no differences in mean number of plantsplot
were observed. Transgenic line tBG-11 was not used as a parent, but it showed a greater number
J .
Oard et
al .
Plant Science
157 2000
13 –
22
17
Table 2 Means values for plantsplot, vigor, plant height, days to first and last heading, shatter rating, and seed germination 1 and 8 weeks after harvest, Ben Hur Farm, Baton
Rouge, LA, 1997
Plant height Days from planting
Plant vigor Days from planting
Number of Entry
Seed shatter Seed germination
Seed germination 8 weeks after
rating 1–9
‡
rating 0–3
§
cm 1 week after harvest
to last heading plantsplot
†
to first heading harvest
74.5
e
37.0
bc¶
105.7
ab
2.1
abcd
3.9
c
31.9
bcde
5.5
bc
136.1
de
1. F
2
tCP× Red c 1
107.2
ab
2. F
2
tCP× 1.9
abcdef
42.0
ab
3.2
c
28.7
bcde
5.0
bcd
140.0
cd
74.7
e
Red c 2 104.0
ab
1.4
bcdefg
74.7
e
77.2
ab
157.8
abc
3. F
2
tBG× 4.0
ed
49.0
a
75.7
e
Red c 4 42.5
ab
76.0
e
100.7
b
1.9
abcde
2.3
c
37.6
abcde
5.0
bcd
4. F
2
tBG× 132.4
de
Red c 3 14.0
f
103.5
ab
0.0
g
14.8
bc
40.0
abcde
7.5
a
101.0
g
5. tBG-BSX2 92.0
a
1.6
abcdef
11.3
bc
49.6
abcd
81.7
d
6. F
2
Red c 155.9
abc
4.7
bcde
38.7
ab
1×tCP 108.3
a
2.8
ab
7. F
2
Red c 1.4
c
42.5
ab
47.1
abcde
4.0
de
160.0
ab
87.0
abcd
4×BG 2.5
abc
0.0
c
38.9
abcde
91.2
ab
42.5
ab
103.0
ab
8. Red c 4 166.9
a
4.2
cde
83.0
d
41.5
ab
101.7
ab
1.3
bcdefg
8.1
c
54.8
abcd
4.5
bcde
132.7
de
9. F
2
BG× Red c 4
108.7
a
1.1
cdefg
5.7
c
10. F
2
CP× 41.3
abcde
35.5
bc
4.5
bcde
136.6
de
84.7
cd
Red c 4 2.5
abc
0.8
c
42.4
abcde
90.2
a
11. Red c 5 105.5
ab
146.3
bcd
5.1
bcd
37.6
bc
27.0
cde
104.0
ab
0.5
efg
44.7
a
60.3
abcd
5.7
b
114.3
fg
12. tCP 92.0
a
85.5
bcd
93.7
c
0.1
g
71.8
a
85.3
a
13. tBG 4.7
bcde
26.0
de
104.7
fg
0.0
g
67.8
a
83.2
a
92.5
c
84.7
cd
106.3
fg
14. tBG-11 41.5
ab
3.5
e
101.5
ab
31.2
bcde
0.4
fg
31.3
bc
71.9
ab
5.5
bc
112.2
fg
90.0
abc
15. CP 85.2
cd
93.2
c
0.0
g
69.3
a
84.3
a
5.2
bcd
16. BG 120.1
ef
23.0
ef †
Number of plants counted 34 days after planting.
‡
Vigor rating of 1 = most vigorous, 9 = least vigorous.
§
Shatter rating: 0 = 0–10, 1 = 11–30, 2 = 31–60, 3 = \60.
¶
Means followed by the same letter are not significantly different DMRT, a = 0.05 Approximately 20 of plants from entry 6 were late maturing and did not form panicles during the course of the study.
Table 3 Mean values for seed numberpanicle, seed weightpanicle, percent seed shattering and seed germination 1 and 8 weeks after
harvest, Hope, AR, 1997
Seed germination 8 weeks Seed weight
Seed shatter Seed germination 1 week Seed number
Entry panicle g
panicle after harvest
after harvest 449.6
ab†
2.0
a
79.6
ab
1.7
e
1. F
2
tCP× 64.7
ab
Red c 1 415.0
ab
2.1
ab
2. F
2
tCP× 80.5
a
0.8
e
71.3
a
Red c 2 419.7
ab
2.2
ab
78.3
abc
4.5
e
48.0
abcd
3. F
2
tBG× Red c 4
477.9
ab
2.2
ab
4. F
2
tBG× 81.1
a
1.3
e
69.3
a
Red c 3 2.3
ab
30.4
i
5. tBG-BSX2 18.7
d
584.0
ab
30.7
def
460.1
ab
1.8
de
68.8
cde
1.8
e
6. F
2
Red c 1 38.7
bcde
× tCP
7. F
2
Red c 4 397.3
bc
2.4
ab
66.3
def
1.3
e
35.0
cde
× BG
1.5
e
81.8
a
8. Red c 4 0.4
e
264.4
c
20.0
efg
9. F
2
BG× 444.2
ab
2.4
ab
58.3
efg
0.0
e
42.7
bcde
Red c 4 412.2
ab
2.2
ab
10. F
2
CP× 69.1
bcd
0.8
e
59.3
abc
Red c 4 2.3
ab
79.1
ab
11. Red c 5 0.4
e
490.6
ab
18.6
efg
1.9
cde
55.5
g
12. tCP 48.7
b
408.3
ab
0.0
g
2.4
ab
56.6
ab
542.2
ab
67.3
a
13. tBG 6.3
fg
2.3
ab
14. tBG-11 38.7
hi
482.4
ab
63.3
a
0.0
g
2.2
ab
60.9
defg
601.6
a
24.1
cd
15. CP 6.3
fg
2.6
a
16. BG 42.6
h
565.6
ab
32.0
c
0.0
g †
Mean followed by the same letter are not significantly different DMRT, a = 0.05.
of plantsplot than BG, tBG and tCP, except for normal cultivar CP. Transgenic line tBG-BSX2
exhibited the lowest mean number of plantsplot when compared to other entries except BG. Trans-
genic line tCP as a parent showed lower mean number of plantsplot than F
2
population tCP × Red c 2, but not F
2
s from the tCP × Red c 1 cross. A greater mean number of plantsplot were
observed in F
2
populations tBG × Red c 4 and tBG × Red c 3 than the transgenic tBG parent.
No differences were found when comparing F
2
populations derived from tBG entries 3, 4 and BG entries 7, 9 parents. Similarly, no differences
were observed among F
2
populations when tCP and CP were used as parents entries 1, 2, 10. At
the Hope site, mean number of plantsplot ranged from 24.5 to 40.0, but there were no significant
differences among the entries.
Vigor ratings were taken at the Baton Rouge location and represent a visual subjective scoring
system to estimate overall ‘robustness’ of entries in the early seedling stage. It is important to note
that a high vigor rating is indicative of low plant vigor. Red c 4 and Red c 5 exhibited similar
vigor ratings to all F
2
populations. Red c 5 showed a higher vigor rating than tBG-11, but a
lower rating than transgenic line tBG-BSX2. Non- transgenic BG exhibited a higher vigor rating of
5.2 than transgenic tBG-11 with rating 3.5, but non-transgenic BG showed similar vigor to an-
other transgenic line, tBG, with a 4.7 rating. tBG- BSX2 displayed the highest vigor rating of any
other entry 7.5 and is consistent with the lowest number of plantsplot recorded in this study.
Vigor ratings were similar for CP and tCP. No differences in vigor were observed in F
2
popula- tions when either CP and tCP or BG and tBG
were used as parents entries 1, 2, 3, 4, 6, 7, 9, 10 . Vigor ratings were not recorded at the Hope site.
Plant heights for Red c 4 and Red c 5 were significantly greater at Baton Rouge, as expected,
than transgenic entries 5, 12, 13, 14 or non-trans- genic entries 15, 16 lines. Due to excessive plant
heights, severe lodging was observed in the red
rice, but not in the commercial or transgenic mate- rial. When Red c 4 was crossed to CP, a 23-cm
reduction in plant height was detected in the F
2
population entry 8 vs. 10, but plant height was similar when Red c 4 was crossed to tBG entry 8
vs. 3. Among transgenic and non-transgenic parental material, plant heights were similar, but
tBG-BSX2 was significantly shorter by 19 cm than non-transgenic BG. A significant reduction of 27
cm in plant height was found when BG was used as a female in reciprocal crosses with Red c 4
entry 7 vs. 9. Plant height was significantly shorter by 25 cm in the BG × Red c 4 F
2
s entry 9 when compared to the tBG × Red c 4 popula-
tion entry 3, but no differences were found when compared with the F
2
of tBG × Red c 3 entry 4. No differences in plant height were found in F
2
populations when either CP or tCP were used as females in crosses to red rice entries 1, 2, 10.
Significant reductions of 17 cm were observed, however, when tCP was used as a male entry 6.
Data for plant height were not recorded at the Hope location.
Days from planting to first heading at Baton Rouge varied only from zero to 2 days between
corresponding transgenic and non-transgenic lines CP, tCP and BG, tBG, tBG-11. In contrast, tCP
was later by 7 days than normal and transgenic Bengal. Red c 4 and Red c 5 differed by 1 day
compared to CP and tCP from planting to first heading, and the red rice was 5 days later than
BG, tBG, and tBG-11. When red rice biotypes were used as a male entries 1, 2, 3, 4 or as a
female entry 6 in crosses, significant reductions of 10 – 15 days from planting to first heading were
observed. This trend was not found for entries 9 or 10 that contained Red c 4 as a male parent. In
crosses with transgenic tBG and red rice entries 3, 4, days from planting to first heading were signifi-
cantly reduced by 7 – 10 days when compared to crosses with non-transgenic BG entries 7 and 9.
The same trend was found in crosses involving transgenic tCP entries 1 and 2 and non-trans-
genic CP entry 10. Days from planting to first heading were not recorded at Hope.
Transgenic and non-transgenic CP displayed similar days from planting to last heading with
tBG-BSX2 entry 5 and these entries were 8 – 11 days later than BG and tBG. Crosses of red
rice with either commercial entry 10 or trans- genic Cypress entries 1, 2 resulted in similar days
to last heading, but 20 of individuals from the Red c 1 × tCP cross entry 6 did not flower dur-
ing the entire study. Reciprocal crosses involving tCP entries 1 and 6 showed significant maternal
effects for days to last heading when Red c 1 was used as a female. In contrast, no maternal effect
was detected in reciprocal crosses with normal Bengal, transgenic Bengal and Red c 4 entries 3,
9. When BG or tBG was crossed to red rice entries 3, 4 vs. 7, 9, no differences in days to last
heading were observed. Red rice biotypes c 4 and c
5 were similar in days to last heading compared to normal and transgenic Cypress, but were signifi-
cantly later by 11 days than normal or trans- genic Bengal. Hybrid weed populations with red
rice as a parent entries 1, 2, 3, 4, 7, 9, 10 showed no differences between Red c 4 and c 5 for days
to last heading. The exception was late-maturing entry 6. Days from planting to last heading were
not recorded at the Hope site.
The extent of seed shattering was measured at both field locations. At the Baton Rouge site, seed
shatter ratings were low for all transgenic and non-transgenic lines entries 5, 12 – 16. This trend
was not observed at Hope where CP and tCP showed similar shatter percentage, but non-trans-
genic BG exhibited lower shattering than trans- genic tBG and higher shattering than tBG-BSX2.
Red c 4 and Red c 5 shared identical shatter rat- ings at Baton Rouge which were significantly
higher than all cultivars and transgenic lines. Sim- ilar results were observed at Hope except for the
high shatter percentage of tBG that was not statis- tically different from Red c 4 or Red c 5. Hybrid
populations that used red rice as a parent entries 1, 2 ,3 , 4, 6, 7, 9, 10 showed no differences in
shatter rating at Baton Rouge when compared to Red c 4 or c 5. No maternal effects on shatter
rating were found in reciprocal crosses entries 7, 9 of Red c 4 and BG. When populations of
tBG × Red c 4 entry 3 and BG × Red c 4 entry 9 were compared for seed shatter rating at Baton
Rouge, no differences were found. In contrast significantly greater shattering by 10 was
found at Hope in the tBG × Red c 4 and tBG × Red c 3 populations entries 3,4 than in the non-
transgenic
crosses BG × Red c 4
and Red c 4 × BG entries 7, 9. This result is consis-
tent with the fact that transgenic parental tBG entry 13 displayed significantly greater shattering
by 14 than the non-transgenic parental BG en-
try 16. Shatter percentage of hybrid populations Hope site with tBG as a parent entries 3,4 was
similar to those of Red c 4 and Red c 5. When shatter ratings at Baton Rouge were compared
between populations containing CP entry 10 and tCP entries 1,2, 6, no significant differences were
found. Similar results were found at the Hope site, except that the non-transgenic population contain-
ing CP entry 10 showed significantly lower shat- tering than a transgenic population containing
tCP entry 2.
Seed germination percentages 1 week after har- vest SG1 were statistically similar for transgenic
and non-transgenic lines at Baton Rouge except for CP at 31 and tBG-BSX2 at 0. At the Hope
location tBG and tBG-11 showed 31 and 35 greater SG1, respectively, than BG. Similarly, tCP
displayed greater SG1 at Hope by 25 than its non-transgenic counterpart CP. Red c 4 and c 5
exhibited low SG1 near 0 which was significantly smaller than those for all cultivars and parental
lines except CP. SG1 was not significantly differ- ent among Red c 4, Red c 5, and those crosses
with red rice parents entries 1, 2, 3, 4, 6, 7, 9, 10 for both the Baton Rouge and Hope locations.
For seed germination percentage 8 weeks after harvest SG8, no differences were found among
the cultivars and parental lines at Baton Rouge and at Hope where SG8 was unexpectedly low
when compared to SG1. When either BG or tBG was used as a parent, no differences in SG8 were
observed among corresponding populations en- tries 3, 4, 7, 9 at Baton Rouge. The same trend
was observed at Hope except for the tBG × Red c 3 population entry 4 that displayed 26 –
34
greater SG8
than the
non-transgenic populations containing BG entries 7, 9. SG8
showed no difference among populations with ei- ther CP or tCP as a parent at Baton Rouge or
Hope. Red c 4 and c 5 exhibited statistically sim- ilar SG8 when compared to all other entries at
Baton Rouge, but the same results were not found at Hope. For example, Red c 4 and c 5 exhibited
14 – 20 greater SG8 than cultivars and lines, and significantly less SG8 than hybrid populations
when red rice was used as a male entries 1, 2, 3, 4, 10, but not as a female entries 6, 7. The
exception to this trend was the BG × Red c 4 population entry 9.
The number of seedspanicle at Baton Rouge ranged from 71 to 156, but no significant differ-
ences were detected among the entries data not shown. At the Hope location all cultivars and
lines were similar in seed production per panicle. CP not unexpectedly showed greater seed fecun-
dity than Red c 4, but no differences were found when compared with Red c 5 and all crosses in-
volving red rice biotypes. As was the case for seedspanicle at Baton Rouge, no differences
among entries were found for seed weightpanicle. At the Hope site, seed weightpanicle was signifi-
cantly lower for tCP and Red c 1 × tCP than CP and tCP × Red c 1. No differences were found
among populations that used either BG, tBG, CP or tCP as a parent.
3
.
1
. Segregation of glufosinate resistance Red rice biotype c 4 plants were all pubescent
and susceptible to glufosinate as expected Table 4. Plants from tCP were all glabrous, but one out
of 60 plants was susceptible to glufosinate which suggested physical seed mixture or unstable glufos-
inate resistance in transgenic tCP. All 60 trans- genic tBG plants were glabrous, but the number of
glufosinate-susceptible plants suggested segrega- tion 1:1 R:S x
2
ratio = 2.4, P B 0.05, seed mix- ture or instability of glufosinate resistance. These
results may help explain segregation of glufosinate resistance in the F
2
populations evaluated. For example, glufosinate resistance showed one- or
two-gene Mendelian segregation in two F
2
popula- tions derived from tCP as a parent entries 1 and
2, however, a separate F
2
population derived from tCP entry 6 displayed an abnormal segrega-
tion pattern with 85 of the F
2
plants susceptible to glufosinate. A comparison of data from recipro-
cal crosses involving tCP entries 1 and 6 suggests that maternal effects can influence segregation pat-
terns for glufosinate resistance. Abnormal or un- expected segregation ratios were also observed in
two F
2
populations when tBG was used as a parent entries 3 and 4. An unusual S:R 37:23
ratio suggested that glufosinate resistance for en- try 3 was governed by recessive genes, but this
result must be verified in F
3
and later generations. The second F
2
population derived from tBG en- try 4 showed abnormal segregation patterns for
herbicide resistance and pubescent leaves that did not fit any known Mendelian ratios.
Glufosinate resistance was controlled by one or two dominant genes in a Mendelian manner for
one third of the crosses entries 1 and 2. However, the majority of the crosses 35 displayed abnor-
mal segregation for glufosinate resistance, includ- ing susceptibility as the dominant phenotype for
entries 3 and 6. Reasons for the abnormal segrega- tion may be traced back to tCP and tBG that were
probably unstable as parents Table 4. Multiple insertions of the BAR gene into CP and BG may
be another possible cause of the unusual segrega- tion ratios that were observed in this study. In
addition, tCP and tBG were previously selected from transgenic material that was evaluated by
spraying in the field with a boom nozzle and back pack sprayer unpublished data. It is possible that
the spraying method did not detect unstable lines as was done with the leaf dip method used in this
study. The leaf dip method should be used to confirm results obtained by the field spraying
method.
4. Discussion
