Communicated by Grzegorz Żurek Volume 73 2016 P L A N T .docx

Communicated by Grzegorz Żurek
Volume 73 2016
P L A N T B R E E D I N G A N D S E E D S C I E N C E
Fardin Khazaei
1
, Majid AghaAlikhani
1
, Samad Mobasser
2
, Ali Mokhtassi-Bidgoli
1
,
Hesam Asharin
2
, Hossein Sadeghi
2
1
Department of Agronomy, Faculty of Agriculture, Tarbiat
Modares University, Tehran, Iran;
2
Seed and Plant Certification and Registration Institute,
Agricultural Research,

Education and Extension Organization (AREEO), Karaj, Iran
EVALUATION OF WHEAT (TRITICUM AESTIVUM, L.)
SEED QUALITY
OF CERTIFIED SEED AND FARM- SAVED SEED
IN THREE PROVINCES OF IRAN
ABSTRACT
The objective of this study was to study the seed quality aspects
of wheat (Triticum aestivum L.) and the
extent of weed seed contamination present in wheat seeds
produced in different regions of Iran. Four districts
(cities), each including 12 fields (six certified seed fields and
six farm-saved seed fields), were selected in
each regions (provinces). One kilogram of the wheat seed
sample was collected from each field for analysis in
the laboratory. Wheat seeding was commonly done by farm-
saved seed sourced from within the farm due to
the high costs of certified seeds purchased from outside sources,
followed by the low seed quality. The use of
a farm-saved seed resulted in a higher germination rate and a
lower mean time to germination compared with
another system. The more positive temperatures experienced by
mother plants could decrease the number of

normal seedling and seedling length vigor index. Generally
there was virtually no difference about physiologi-
cal quality between certified seed and farm- saved seed sector
that is related to lower quality of certified seed.
The certified produced seeds had the lower number of weed
seed, species and genus before and after cleaning.
The highest seed purity and 1000 seed weight was obtained
from the certified seed production system. The
need for cleaning the farm-saved seed samples before sowing is
one of the important findings of this survey.
Keywords: germination indices; seed production system; vigor;
weed seed dispersal; winnowing
INTRODUCTION
Importance of wheat (Triticum aestivum L.) as an agricultural
crop is punctuated
by the fact that it ranks second after maize in the world cereal
output and it is a staple
food for billions of people of the world. It is also the most
important winter cereal
DOI: 10.1515/plass-2016-0009
100 Fardin Khazaei et al..
grown in Iran (Zand et al. 2007). A little more than 9 million ha

of arable land in Iran
are planted with cereals, of which wheat occupies 6.6 million
ha. To grow this plant,
80-85% of the national seed requirements of the country are
derived from farm-saved
seeds, so a substantial investment has been made in agricultural
research to evaluate
the wheat farm-saved seed performance (Mobasser et al. 2012).
More than 80% of the crops in developing countries are planted
from seed stocks
of the farmers (Almekinders and Louwaars 1999). According to
the crop, the share of
the certified seed system in supplying the country's seed
requirement varies. For some
hybrid seeds, 100% of the needed seed are produced by state
companies, while for
self pollinated crops as cereals, the share of certified system is
estimated lower than
20% (Mobasser et al. 2012).
Since seed quality is a critical and basic input that affects crop
production potential,
it should reach farmers in a good quality state. Seed quality is
composed of many as-

pects where four key attributes as genetic, physical,
physiological and health quality
are clearly identified. However, seed quality can be influenced
by environmental con-
ditions and also the cultural practices used for production.
Preserving seed quality is
fundamental if the variety meet the expectation of farmers and
consumers (van Gastel
et al. 2002). Seed quality within farmer seed systems is
controlled by social norms of
reciprocity, whereas in the certified sector seed schemes, seed is
produced commer-
cially; without seed regulation, the onus is on the procedure
(i.e. the project) to main-
tain quality standards (Jones et al. 2002).
Dispersal and spread of weed seeds is an important biological
factor affecting seed
quality control and an essential element when considering crop
weed management
strategies (Michael et al. 2010). Previous studies have identified
high levels of weed
seed contamination in crop seed in Australia (Michael et al.
2010) and Ethiopia and
Syria (Bishaw 2004). In addition, Bishaw (2004) reported that

the average physical
purity of wheat seed samples collected from different districts
of Ethiopia was
98.92% and also there were significant differences in physical
purity between differ-
ent wheat growing regions in Ethiopia.
Considering seed quality, the first and the most important step
in reducing weed
infestation is the use of clean seeds (Michael et al. 2010;
Chauhan 2013). Weeds can
cause significant reductions in both productivity and seed
quality of desirable species
such as wheat (Mokhtassi-Bidgoli et al. 2013b). It has long been
thought that many
infestations of weed populations are a direct result of seeding
with contaminated crop
seed. Therefore, the objective of this study was to study the
quality aspects of wheat
seeds and the extent of weed seed contamination present in
wheat seeds produced in
different regions of Iran taking into account weather conditions,
seed sources and ag-
ronomic practices.

MATERIALS AND METHODS
A total of 144 wheat farmers were interviewed in three wheat
growing regions of
Iran. Four districts (cities), each including 12 fields (six
certified fields and six farm-
saved seed fields), were selected in each regions (provinces)
(Fig. 1). One primary
Evaluation of wheat (Triticum aestivum L.) seed quality of
certified seed and farm … 101
sample for each 500 kg of 3001-20000 kg lot size and also one
primary sample for
each 700 kg of 20001 kg and above lot size were taken and
samples were mixed and
finally one kilogram of seed sample was prepared by the divider
(ISTA hand book on
seed sampling, 2004) from each field for analysis in the
laboratory. All samples col-
lected during the survey were used to determine the number of
weed seed, genus and
species before and after cleaning. Weed seeds were separated
manually from the sam-
ples and their species and genera were identified. These weed
seeds were returned to

the samples. After that, the samples were cleaned using a
Westrup LA-LS air/screen
cleaner. The grain velocity used was 3.5 m/sec. The screen
system is consisting of
one sieve boat with 1 short scalping screen 275 × 275 mm and 2
long grading/-sand
screens 275 × 540 mm which gives a total screen area of 0.34 m
2
.
Fig. 1. Geographic distribution of wheat farms in studied areas
All samples were analyzed for seed quality (physical purity,
thousand seed
weight, germination, vigor, etc.). The tests were conducted at
the seed testing
laboratory of the Seed and Plant Certification and Registration
Institute
(SPCRI), Karaj, Iran in 2011-2012.
The meteorological data recorded during the study period in
each growing
season and geographical altitudes are given in Table 1.

Table 1
The averag0e annual maximum, minimum and mean
temperatures (°C), annual total
precipitation [mm] recorded in 2011-2012 and geographical
altitudes (m) in studied areas.
The farmers were asked to give information about their
preferences and uses of
seed sources and agronomic practices for wheat production
(Tables 2, 3 and 4).
Seed quality attributes - physical
Other seeds by number analysis
According to ISTA rules (2012), the 1000g seed sample was
evaluated for
determination of the number of other seeds.
Province Cities
Maximum
temperature
Minimum
temperature
Mean tem-
perature

Precipitation Altitude
Tehran and
Alborz
Karaj 22.4 1.3 10.6 174.1 1380
Hashtgerd&Nazarabad 21.2 1.2 10.4 181.3 1191
Varamin 19.9 6.7 13.3 148.0 918
Rey 18.6 6.5 12.5 159.0 1060
Average 20.5 3.9 11.7 165.6 1137.2
Kermanshah
Sarab Niloofar 18.5 1.9 10.1 293.0 1331
Mahidasht 18.8 2.0 10.4 344.2 1420
Kuzaran 18.8 1.7 10.2 273.3 1368
Ravansar 16.8 4.1 10.3 378.2 1362
Average 18.2 2.4 10.3 322.2 1370.2
West Azarbai-
jan
Orumiyeh 13.3 0.4 6.8 217.5 1328
Oshnavieh 13.2 0.5 6.9 327.1 1415

Naghadeh 13.9 2.3 8.0 214.0 1307
Miandoab 14.7 1.9 8.2 192.0 1300
Average 13.8 1.3 7.5 237.7 1337.5
Physical (analytical) purity test
Two replicates of 60 g from each seed sample were analyzed.
The samples
were divided into four fractions (pure seed, other seed and inert
matter). After
analysis, the percentage of each fraction (based on weight) was
calculated
(ISTA 1996).
Thousand Seed Weight (TSW)
Two replicates of 1000 seeds were separated using a seed
counter and then
the mean seed weight was calculated (Bonner 1974).
Seed quality attributes - physiological
Germination Test
Four replicates of 100 seeds from each sample were planted in

pleated paper.
Then, seeds were placed in a germination room maintained at
20°C for 8 days
for wheat according to ISTA Rules (ISTA 1996). The
germination boxes were
removed at the end of the incubation period and seedlings were
evaluated
(Bekendam and Grob 1979). Normal seedling, abnormal
seedling, dormant and
dead seeds were recorded and the average calculated based on
the final count.
Vigor tests
Germination rate
Four replicates of 100 seeds were planted from each sample and
kept at
20 ± 1°C for a maximum of 12 days in a germination room until
no further
germination took place. Normal seedlings were removed each
day at predeter-
mined size and time until all seeds that are capable to produce
normal seedlings,
germinated. An index was calculated by dividing the number of
seedlings re-

moved each day by the number of days in which they were
removed (Maguire
1962).
First seedling count
The first count was made during the germination test, and the
numbers of
normal seedlings were recorded (fourth day after planting). The
final count was
made on the eighth day and total number of normal seedlings,
number of abnor-
mal seedling, fresh seed and dead seeds was recorded (Agarwal
1986).
Seedlings shoot and root length
The seedling shoot and root lengths were assessed after the final
count. 40
normal seedlings were randomly selected from each sample and
the shoot

length was measured from the point of attachment to the
cotyledon to the tip of
the seedling. The root length was measured from the point of
attachment to the
cotyledon to the tip of the root. The average shoot or root
length was calculated
by dividing the total shoot or root lengths by the total number
of normal seed-
lings measured (Fiala 1987).
Seedling dry weight
The seedling dry weight was measured after the final count.
From each repli-
cate, ten seedlings was selected randomly and were cut free
from their cotyle-
dons and placed in envelopes and dried in an oven at 80 ± 1°C
for 24 hours. The
dried seedlings were weighed and the average seedling dry
weight was calcu-
lated.
Germination indices
The following indices were calculated: Mean time germination
(MTG) was
calculated as follows:

where ni is number of germinated seeds till i
th
day and di is number of days
from start of experiment till i
th
counting and n is total germinated seeds. Mean
daily germination (MDG) is contrast to day germination speed
(time to need for
germination a seed), MDG is ‘mean germination days’ and it is
speed germina-
tion day index and was determined as follows:
where FGP is final germination percentage and D is number of
days from
start to end of the experiment. Seedling vigor index length
(SVIL) was calcu-
lated as follows:
where LP is length of seedling and nor is number of normal
seedlings. Seed-
ling vigor index weight (SVIW) was calculated as follows:
where WDP is total weight of seedlings and nor is number of
normal seed-
lings (Ellis and Roberts 1981).

Statistical analyses
After initially testing the normal distribution of residuals, data
were subjected
to analysis of variance (ANOVA), using the GLM and Nested
procedures of
SAS (SAS Institute Inc. 2002) for a nested design. Nested
analyses were con-
ducted by nesting fields within seed production systems and
seed production
systems within districts (cities). The appropriate error term for
province F-test
was district (province). Differences between means were
determined by Dun-
can's multiple range tests at the 0.05 probability levels.
RESULTS AND DISCUSSION
Natural precipitation, temperature and farmlands management
As Table 1 shows, temperature and natural precipitation varied

among the cities.
The annual total precipitation for provinces was in the declining
order of Kerman-
shah > West Azarbaijan > Tehran and Alborz, while the average
annual tempera-
ture followed the trend West Azarbaijan < Kermanshah <
Tehran and Alborz.
In Iran, wheat seeding is commonly done by farmers saved seed
seeds
sourced from within the farm due to the high costs of certified
seeds (40% more
than farmers saved seed) purchased from outside sources,
followed by the low
seed quality (Table 2). A study by Michael et al. (2010) found
that of the 16 not
cleaned crop seed samples, nine were wheat and seven were an
alternative crop
(barley and lupin), with most samples sourced from within the
farm (94%).
Table 2
The proportion (% based on farmer reports in studied areas) of
reasons for not using the certified seeds
Three methods were used to treat the wheat seeds of farm-saved
seed section

after harvest (Table 3), with fungicide seed treatment (51%),
followed by ma-
chine cleaning (22%), and then sieving (15%). Michael et al.
(2010) reported
the share of gravity table, rotary screen and sieves considered
for the cleaning
of crop seeds were 38%, 8% and 3%, respectively.
Most farmers rely on herbicides for weed management (Table
4). The level of
weed control and price were the top two things that farmers
considered when select-
ing herbicides.
Province Cities
Maximum
temperature
Minimum
temperature
Mean tem-
perature
Precipitation Altitude
Tehran and
Alborz

Karaj 22.4 1.3 10.6 174.1 1380
Hashtgerd&Nazarabad 21.2 1.2 10.4 181.3 1191
Varamin 19.9 6.7 13.3 148.0 918
Rey 18.6 6.5 12.5 159.0 1060
Average 20.5 3.9 11.7 165.6 1137.2
Table 3
The proportion (%) of different post harvest activities on farm-
saved seed in studied areas in 2011-12
Table 4
The proportion (%) of types of weed control methods in wheat
farmlands
and seed production systems in areas studied in 2011-12.
Three main reasons of the farmers for not using of certified seed
was related to the
High price (45.8%), low seed quality (27.1%) and insufficient
supply (14.6%).
Physiological quality
There were significant differences among provinces on normal
seedling (P<0.05),

dead seed (P<0.05), abnormal seed (P<0.01), seedling shoot
length (P<0.05) and
seedling root length (P<0.01), while the effect of seed
production system was signifi-
cant only for fresh seed (Table 5). Seedling length vigor index
and both germination
rate and mean time to germination were affected significantly
by provinces and seed
production systems, respectively (Table 5). The highest and
lowest values of normal
seed, seedling shoot length, seedling root length, abnormal seed
and seedling length
vigor index of wheat were related to West Azarbaijan and
Kermanshah provinces,
respectively, while the highest and lowest dead seed values
were related to Tehran &
Alborz and Kermanshah provinces, respectively (Table 5).
Maximum and minimum
fresh seed percentages were obtained from farm-saved seed and
certified seed pro-
duction systems, respectively (Table 5). The use of a farm-
saved seed production sys-
tem resulted in a higher germination rate and a lower mean time
to germination com-

pared with certified seed system (Table 5). The reason of these
finding might is re-
lated to incorrect management at the nucleolus seed production
by the formal institu-
tions.
Province Sieving Machine cleaning
Fungicide seed
treatment
No action
Tehran and Alborz 6.2 16.7 60.4 16.7
Kermanshah 2.1 45.8 52.1 0.0
West Azarbaijan 35.4 4.2 39.6 20.9
Average 14.6 22.2 50.7 12.5
Province Hand weeding Herbicide application No control
Tehran and Alborz 4.2 82.3 13.5
Kermanshah 1.1 88.5 10.4
West Azarbaijan 0.0 93.8 6.3
Seed production system
Informal 0.7 79.2 20.1
Formal 2.8 97.2 0.0

Average 1.8 88.2 10.1
Table 5a
Physiological quality (vigour) of wheat seed collected from
certified seed and farm-saved seed sources in
2011-2012. Summary of F significance from analysis of
variance (ANOVA) of experimental factors
Table 5b
Physiological quality (vigour) of wheat seed collected from
certified seed and farm-saved seed sources in
2011-2012. Summary of F significance from analysis of
variance (ANOVA) of experimental factors
Means within a column and study factor followed by the same
letter are not significantly different according
to Duncan's multiple range test at the 0.05 probability level of
significance; n.s., * and ** are non significant,
significant at the 0.05 and 0.01 probability levels, respectively.
Provinces
Normal

seedling [%]
Abnormal
seedling [%]
Ungerminated seeds Seedling
shoot length
[cm]
Hypocotyl
length
[cm]
Seedling
root length
[cm]
Seedling
fresh weight
[cm]
Dead seed
[%]
Fresh seed
[%]
Tehran and

Alborz
95.8125ab 2.0208 b 1.6667a 0.9167a 21.6998b 11.3738a
10.3265b 1.1940a
Kermanshah 95.0417b 3.7500a 0.4792b 1.0833a 21.4017b
11.7840a 9.6194b 1.3638a
West Azar-
baijan
97.0625 a 1.7708b 0.8333ab 0.7708a 22.8085a 11.1667a
11.6463a 1.0456a
Farm-saved
seed
95.7917a 2.4583a 0.9444a 1.1250a 21.9040a 11.3461a 10.5589
a 1.0092a
Certified
seed
96.1528a 2.5694a 1.0417a 0.7222b 22.0360a 11.5368a 10.5025
a 1.3931a
Source
Provinces * ** * ns * ns ** ns
Cities ns ns * ns ns ns ns ns
Seed sys-
tems

ns ns ns ** ns ns ns ns
Provinces
Seedling dry
weight [g]
Final germi-
nation [%]
Germination
rate
Mean daily
germination
[number/day]
Mean time to
germination
[day]
Seedling
length vigor
index
Seedling
weight vigor
index

Tehran and
Alborz
0.166042a 97.8333a 61.466a 39.181a 1.75292a 2077.45b
15.8327a
Kermanshah 0.162708a 98.6458a 61.485a 42.124a 1.75729a
2031.63b 15.3531a
West Azarbai-
jan
0.169375a 98.6458a 59.578a 40.754a 1.79771a 2211.51a
16.3171a
Farm-saved
seed
0.160139a 98.1389a 61.898a 40.7665a 1.74556 b 2097.23a
15.2331a
Certified seed 0.171944a 98.6111a 59.788b 40.6060a 1.79306 a
2116.50a 16.4356a
Source
Provinces ns ns ns ns ns ** ns
Cities ns ns ns * ns ns ns
Seed systems ns ns ** ns ** ns ns

These findings are in contrast with those of Kshetri (2010) who
observed the
highest levels in seed quality attributes such as germination,
1000 seed weight
genetic purity percentage in certified seed. It was noticed that
farmers who used
own saved seeds produced seeds with less germination
percentage compared to
farmers who used certified seeds.
Both biotic and abiotic conditions experienced by mother-plants
during the
formation, development and maturation of seed affect the
phenotype of the off-
spring. In the present study, the average annual temperature was
significantly
correlated with normal seed (r=-0.66, P<0.05), seedling shoot
length (r=0.66,
P<0.05), seedling root length (r=-0.74, P<0.01) and seedling
length vigor index
(r=-0.65, P<0.05). As can be seen, the more positive
temperatures experienced
by mother plants can decrease the number of normal seed and

seedling length
vigor index. In fact, high temperature combined with drought
and high winds in
the maternal environment may negatively affected plants,
especially during the
heading, flowering and grain filling stages. The results of Sales
et al. (2013)
have indicated that stressful environmental conditions
experienced by mother
plants may act as cues signalizing unfavorable conditions for
seed germination.
Generally there was virtually no difference about physiological
quality be-
tween certified seed and farm- saved seed sector (just small
number of traits
were statistically different) that is might related to reduction of
financial support
of the government and incorrect management in formal seed
production system.
Weed seed contamination before and after cleaning
The number of weed seed, genus and species in wheat before
and after clean-
ing was affected significantly by seed production system
(P<0.01). In addition,

significant differences for the number of weed genus and
species in wheat after
cleaning were observed between provinces (P<0.05) (Table 6).
The maximum
and minimum number of weed seed, genus and species before
and after clean-
ing were related to farm-saved seed and certified seed
production systems, re-
spectively (Table 6). The highest and lowest number of weed
genus and species
after cleaning was related to Kermanshah and Tehran and
Alborz provinces,
respectively (Table 6). These results are in line with those of
Bishaw (2004)
who reported highly significant differences in seed quality for
seed samples col-
lected from different regions and seed systems for wheat and
barley crops in
Syria and Ethiopia in a way that certified seeds showed better
conditions.
As above mentioned, the number of other crop seed and other
distinguishable
variety were significantly lower in certified seed than that of
seed produced by

farm-saved seed production system. In certified seed
production, farmers re-
moved the off-types and all other crop plants during rouging
due to compulsion
of seed certification. So, it is necessary to suggest to farmers to
adopt proper
practice in additional management as in certified seed
production for maintain-
ing the physical and genetic purity of seed. As it is indicated in
Table 4, certi-
fied seed farmers have applied more weed control compared to
farm-saved seed
farmers and this can be one of the reasons for lower rate of
weed seeds in certi-
fied seed samples than farm-saved seed. The certified seeds had
the lower num-
ber of weed seed, species and genus by 50%, 8% and 8% before
cleaning and
by 17%, 45% and 45% after cleaning, respectively, compared
with the produced

seeds of the farm-saved seed section (Table 6). Furthermore,
cleaned seeds had
significantly the lower number of weed seed, genus and species
by 91%, 25%
and 74% for certified seeds and by 97%, 83% and 83% for farm-
saved seeds,
respectively, than uncleaned indicting the importance of
employing crop clean-
ing techniques.
Table 6
The number of weed seed, genus and species within wheat seed
before and after cleaning in provinces
and seed production systems tested in 2011-12. Summary of F
significance from
analysis of variance (ANOVA) of experimental factors
significance; n.s., * and ** are nonsignificant,
As it can be seen, cleaned seeds had some level of weed seed
contamination.
Michael et al. (2010) found that it is impossible to completely

remove all for-
eign weeds seeds from grain due to high costs and the purchase
of certified seed
does not guarantee weed seed–free status. On the other hand,
the differences
among the provinces regarding to seed (species) purity, and the
lower rate of
weed seed contamination in certified seed might be related to
origin of the seed
source, agricultural practices, and field's inspection by
inspectors. There were
negative significant correlations for the number of weed genus
and species be-
fore and after cleaning vs. planted area and vs. nitrogen
application (data not
shown).
As may be expected, the presence of more N may promote
higher crop
growth rates and plant spacing will be low enough to permit
additional weed
plants per area unit (Mokhtassi-Bidgoli et al. 2013a). It seems
that the larger the
Provinces

The number of weed
Seed Genus Species Seed Genus Species
Before cleaning After cleaning
Tehran and Alborz 385.3a 7.73b 8.67a 28.13a 1.44b 1.56b
Kermanshah 413.5a 10.15ab 11.54a 32.06a 3.00a 3.56a
West Azarbaijan 445.3a 11.63a 13.17a 26.00a 1.96ab 2.23ab
Farm-saved seed 552.8a 10.82a 12.24a 49.24a 2.75a 3.17a
Certified seed 276.7b 8.85b 10.01b 8.22b 1.51b 1.74b
Source
Provinces ns ns ns ns * *
Cities ns ns ns ns ns ns
Seed systems ** ** ** ** ** **
area under cultivation, the higher the economic return, the
greater the power of
the farmer and consequently the greater the control of the weed.
Analytical purity and seed loss during cleaning

There were significant differences between different seed
sources for analytical
purity (Table 7). The highest seed purity and 1000 seed weight
was obtained from
the certified seed production system (Table 7). This finding is
in line with those of
Hasan (1995) who reported significantly higher purity values
for wheat certified
seed compared to farm-saved seed source except in other crop
seed contamination.
Also this result is in contrast with Ensermu et al. (1998) that
reported no significant
difference for analytical purity and other crop seeds
contamination of seed samples
collected from different sources of wheat in Ethiopia.
Table 7
The values of seed loss weight after cleaning, seed purity and
1000 seed weight in provinces
and seed production systems tested in 2011-12. Summary of F
significance
from analysis of variance (ANOVA) of experimental factors

significance; n.s., * and ** are nonsignificant,
Based on the results the highest seed loss after cleaning was
related to Tehran
and Alborz province and farmers saved seed section (Table 7).
Since all of the produced seeds by the farmers are not supposed
to be planted
for the next season and just some part of the seed (about 250 kg
× h
-1
) is needed
for planting and also as we find the seed loss during the
cleaning in farm-saved
seed is about 15% (37.5kg) and furthermore the price of 1kg of
certified seed is
about 50% more than farmers saved seed. So providing the
certified seed for
Provinces
Seed loss weight
after cleaning
[g]

Seed purity
[%]
Weed seed
[%]
Inert matter
[%]
1000 seed
weight
[g]
Tehran and Alborz 157.86a 99.43a 0.16a 0.41a 44.30a
Kermanshah 133.33ab 99.41a 0.10a 0.50a 43.16a
West Azarbaijan 108.00b 99.51a 0.10a 0.39a 41.47a
Farm-saved seed 148.87a 99.34b 0.21a 0.45a 41.74b
Certified seed 117.26b 99.56a 0.03b 0.41a 44.22a
Source
Provinces ns ns ns ns ns
Cities ns ns ns * ns
Seed systems ** * ** ns **

farmers of farm-saved seed is not economical. Also in the other
side as we find
the certified seed did not have enough quality and if the
problems in this section
don’t be solved, we don't recommend the farmers use certified
seed and its bet-
ter the farmers in the farm-saved seed sector clean the needed
seed for planting
in the next season that can be considered as important outcome
of the research.
Detected weed seed species
Based on the results, 24 families included 73 weed genus and
124 weed spe-
cies were detected in studied areas. The highest and lowest
weed diversity was
related to dicotyledonous (97) and monocotyledonous (27)
respectively. About
90% of weed species were C3 plants (included 18
monocotyledonous and 93
dicotyledonous) and just 10% of weed species were C4
(included 9 monocotyle-

donous and 4 dicotyledonous). Annual (80 species), perennial
(38) and biennial
(6) weed had the highest diversity in respect of life cycle,
respectively.
Generally, the highest variation of weed species was related to
Poacea (24)
and Fabaceae (18) families, while the lowest weed species was
included Dipsa-
caceae, Euphorbiaceae, Primulaceae, Resedaceae and
Scrophulariaceae
(Table 8). The same results were reported by Norozzadeh et al
(2008) and
Koocheki et al. (2006).
Table 8
Weeds family detected and its number in studied traits
Koocheki et al. (2006) by Assessing species and functional
diversity and
community structure for weeds in wheat and sugar beet in Iran,
reported that the
total number of weed species were 72 and 52 species in wheat
and sugar beet,
respectively. Also he showed the Poaceae and Asteraceae had
the most diverse

amongst monocotyledonous and dicotyledonous groups in the
wheat fields
while Poaceae and Brassicaceae were the most diverse family
amongst mono-
cotyledonous and dicotyledonous groups in the sugar beet
fields.
Family Number Family Number
Poacea 24 Malvaceae 3
Fabaceae 18 Liliaceae 3
Brassicaceae 11 Rubiaceae 3
Polygonaceae 10 Lamiaceae 2
Apiaceae 6 Plantaginaceae 2
Asteraceae 6 Ranunculaceae 2
Boraginaceae 6 Rosaceae 2
Caryophillaceae 6 Dipsacaceae 1
Chenopodiaceae 5 Euphorbiaceae 1
Amaranthaceae 4 Primulaceae 1
Convolvulaceae 3 Resedaceae 1
Cuscutaceae 3 Scrophulariaceae 1

Norozzadeh et al. (2008) by evaluation of species, functional
and structural
diversity of weeds in wheat fields of three provinces in Iran
showed that the
weeds of wheat fields were belong to 26 families and 120
species. The majority
of weed species were of Asteraceae (20 species) and Poaceae
(25 species)
amongst dicotyledonous and monocotyledonous, respectively.
In another research, Poggio et al. (2004) by evaluation of
Structure of weed
communities occurring in pea and wheat crops in the Rolling
Pampa of Argen-
tina reported that the weed community in pea was more diverse
than wheat
field. 12 weed species included Polygonum aviculare, Lolium
perenne, Convol-
vulus arvensis, Secale cereale, Hordeum vulgare, Chenopodium
album, Conrin-
gia orientalis, Galium tricornatum, Avena fatua, Convolvulus
sepium, Sorghum
halepense and Panicum miliaceum which were more diverse
than the others in

the studied areas and seed production systems were analyzed.
Lolium perenne, Convolvulus arvensis, Secale cereale,
Chenopodium album,
Conringia orientalis, and Galium tricornatum were affected
significantly by the
provinces (Table 9).
Table 9a
Effect of provinces and seed production systems on weed seed
number. Summary
of F significance from analysis of variance (ANOVA) of
experimental factors
Source
Polygonum

aviculare
Lolium peren-
ne
Convolvulus
arvensis
Secale cereale
Hordeum
vulgare
Chenopodium
album
Provinces
Tehran and Al-
borz
93.44 123.32 7.25 0.33 37.73 42.04
Kermanshah 182.84 0.04 38.31 0.00 11.90 5.73
West Azarbaijan 82.04 0.77 75.92 66.48 16.94 10.25
F test ns * ** * ns *
Farm-saved seed 165.63 52.53 71.40 44.46 33.43 25.43
Certified seed 72.97 30.22 40.28 0.08 10.94 13.25
F test ns ns ns ns * ns

Table 9b
Effect of provinces and seed production systems on weed seed
number. Summary
of F significance from analysis of variance (ANOVA) of
experimental factors
While just Conringia orientalis, Galium tricornatum and
Hordeum vulgare
were affected significantly by the seed systems.
Furthermore some weed species as Hordeum spontaneum,
Hordeum disti-
cum, Secale cereal, Avenea fatua, Avenea luduviciana,
Acroptilon repens, Gly-
cegrrhiza glabra, Vicia Villosa, Lathyrus sativus, Malva
neglecta, Convolvulus

arvensis, Turgenia latifolia and Lisaea heterocarpa that are
considered as nox-
ious weed were removed very few through cleaning (data no
Shown).
Difference in hand weeding, fertilizer and fungicide application
management,
the crop rotation and also weed seed bank of the soil might are
the reason for
difference the weed seed in provinces and seed systems.
CONCLUSIONS
Generally there was virtually no difference about physiological
quality be-
tween certified seed and farm- saved seed sector that is related
to lower quality
of certified seed. This issue might occurred through more
reduction of financial
support of the government and also incorrect management of
formal seed pro-
duction sector and consequently reduction of seed quality in the
breeder seed,
foundation seed and finally certified seed systems.
Since some part of the produced seed (about 250 kg × ha
-1

) is needed for
planting and also as we find the seed loss during the cleaning in
farmers saved
seed is about 15% (37.5kg) and furthermore the price of 1kg of
certified seed is
about 50% more than farmers saved seed. So providing the
certified seed for
farm-saved seed farmers is not economical. Also in the other
side as we ob-
Source
Conringia
orientalis
Galium
tricornatum
Avena fatua
Convolvulus
sepium
Sorghum
halepense
Panicum
miliaceum
Provinces
Tehran and Alborz 0.00 0.00 0.79 3.00 5.25 10.92

Kermanshah 44.43 33.44 8.27 5.56 6.65 5.25
West Azarbaijan 4.67 1.75 22.40 22.13 14.44 9.31
F test ** ** ns ns ns ns
Farm- saved seed 25.28 21.03 18.60 15.49 12.03 10.56
Certified seed 6.79 2.43 2.38 4.97 5.71 6.43
F test * * * ns ns ns
served, the certified seed in studied areas did not have enough
quality and if the
problems in this section don’t be solved, it is recommend the
farmers don’t use
certified seed and its better the farmers in the farmers saved
seed sector clean
the needed seed for planting in the next season.
Also it was recognized, the cleaning is an inevitable practice,
which should
be conducted to reduce weeds in seed samples especially for
farmers saved seed

samples that are supposed to be planted for the next season.
Prevention is the
most important method of dealing with weeds. Once a weed has
entered an area
and become established, eradication is far more expensive and it
is likely that
greater resources will be required to control its further spread
and decrease its
impact. So, the first step in weed prevention is considered as
preventing the en-
try of weeds into fields. It is also necessary to organize the
Seed Quality Man-
agement training program effectively in farmer conditions for
strengthening
farmer seed system in the country. The need for cleaning the
farmers saved seed
samples before sowing is one of the important findings of this
survey. Thereby,
removing a large quantity of weeds and other crop seeds from
the main sample
will result in more purity in harvested seeds, less herbicide
application, less soil
and water contamination, more yield and production of healthy
food. it is sug-

gested that not only the same study in another parts of the
country but also in
another countries be conducted to get more information about
the seed systems
and making the strategies for improvement the seed systems
based on the re-
sults.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge the financial support
provided for this
survey by the Research deputy of Tarbiat Modares University
(Iran) and techni-
cal support provided Seed and Plant Certification and
Registration Institute
(Iran).
REFERENCES
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of considerable importance. Seed Res. 14:1-11
Almekinders C.J.M, Louwaars N.P. 1999. Farmers' seed
production: new approaches and practices. Intermediate
Technology Publications Ltd, London, UK.
Bekendam J, Grob R. 1979. Handbook for seedling evaluation.
ISTA, Zürich, Switzerland.

Bishaw Z. 2004. Wheat and barley seed systems in Ethiopia and
Syria [PhD dissertation]. Wageningen Uni-
versity, the Netherlands.
Bonner F.T. 1974. Seed testing. In Seeds of Woody Plants in the
United States, Agriculture Handbook No.
450. For. Service, USDA, Washington D.C.
Chauhan B.S. 2013. Strategies to manage weedy rice in Asia.
Crop Prot. 48:51-56.
Ensermu R, Mwangi W.M, Verkuijl H, Hasenna M, Alemayehu
Z. 1998. Farmers' wheat seed sources and
seed management in Chilalo Awraja, Ethiopia. CIMMYT,
Mexico and IAR, Ethiopia.
Fiala F. 1987. Handbook of vigour test methods. ISTA, Zürich,
Switzerland.
Gastel van A.J.G., Bishaw Z., Gregg B.R. 2002. Wheat seed
production. In: Curtis BC, Rajaram S, Gomez
Macpherson H, editors. Bread wheat- improvement and
production. FAO, Rome; p. 463-482.
Hasan BMA. 1995. A survey of wheat seed quality in Jordan
[MSc thesis]. University of Jordan, Amman,

Jordan.
ISTA. 1996. International Rules for Seed Testing., Zürich,
Switzerland.
ISTA. 2004. Handbook on Seed Health Testing. Zürich,
Switzerland.
ISTA. 2013. International Rules for Seed Testing. Zürich,
Switzerland.
Jones RB., Bramel P., Longley C., Remington T. 2002. The
need to look beyond the production and provision
of relief seed: experiences from southern Sudan. Disasters.
26:302-315.
Koocheki A, Nassiri Mahallati M, Tabrizi L, Azizi G, Jahan M.
2006. Assessing species and functional diver-
sity and community structure for weeds in wheat and sugar beet
in Iran. Iranian Journal of Field Crops
Research. 4(1):105-129. In Persian.
Kshetri B.T. 2010. On-farm management and quality assessment
of farmers’ saved wheat seed in the western
Terai, Nepal. Agron J N. 1:50-60.
Maguire J.D. 1962. Speed of germination - aid in selection and
evaluation for seedling emergence and vigor.
Crop Sci. 2:176-177.

Michael P.J., Owen M.J., Powles S.B. 2010. Herbicide-resistant
weed seeds contaminate grain sown in the
Western Australian grainbelt. Weed Sci. 58:466-472.
Mobasser S., Jazayeri M.R., Khazaei F., Sadeghi L. 2012.
Wheat seed contamination with seed-borne diseases
in cold climatic zone of Iran. Int J Plant Prod. 6:337-352.
Mokhtassi-Bidgoli A., AghaAlikhani M., Nassiri-Mahallati M.,
Zand E., Gonzalez-Andujar J.L., Azari A.
2013a. Agronomic performance, seed quality and nitrogen
uptake of Descurainia sophia in response to
different nitrogen rates and water regimes. Ind Crop Prod.
44:583-592.
Mokhtassi-Bidgoli A., Navarrete L., AghaAlikhani M.,
Gonzalez-Andujar JL. 2013b. Modelling the popula-
tion dynamic and management of Bromus diandrus in a non-
tillage system. Crop Prot. 43:128-133.
Norozzadeh Sh., Rashed Mohasel M.H., Nassiri Mahallati M.,
Koocheki A., AbbasPour M. 2008. Evaluation
of species, functional and structural diversity of weeds in wheat
fields of Northern, Southern and Razavi
Khorasan provinces. Iranian Journal of Field Crops Research.
2(2):471-485. In Persian.
Poggio S.L., Satorre E.H., Delafuente E.B. 2004. Structure of
weed communities occurring in pea and wheat

crops in the Rolling Pampa Argentina .Agriculture Ecosystems
and Environment 103: 225-235.
Sales N.M., Pérez-García F., Silveira F.A.O. 2013. Consistent
variation in seed germination across an envi-
ronmental gradient in a Neotropical savanna. S Afr J Bot.
87:129-133.
SAS Institute Inc. 2002. The SAS System for Windows, Release
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Cary, NC, USA.
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M., Bagherani N., Barjastehe A., Khayamia
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–emergence herbicides in Iran. Crop Prot. 26:746-752.
Good pre s e ntation for the two pape rs you chos e ! I’ve
include d s uggestions, corre ctions,
and note s in blue and re d font.
THE EFFECTS OF GLYPHOSATE ON THE CONTENT OF
MINERAL NUTRIENTS
IN SEEDS AND LEAVES and THE EFFECTS OF
GENE/GENTICALLY MODIFIED

SEED ON THE ROOT COLONIZATION AND SUDDEN
DEATH SYNDROME OF
SOYBEANS (Glycine max (L.) Me rr.)
ABSTRACT
This report reflects on two separate soybean studies that
were performed by others. The
objective of the paper is to demonstrate the various effects that
occur in soybeans in the field
using genetically modified gene with glyphosate and a
greenhouse experiment using non-
glyphosate resistant soybean crops to study the effects of
glyphosate drift on plant growth and
concentration of mineral nutrients in leaves and seeds. The
materials used include specific
research performed by others on soybeans regarding the use of
glyphosate and illustrates the
outcome of each of the findings. The first study used Roundup
Ready (RR) soybeans of
unknown cultivars grown in field conditions to determine
glyphosate effects on Fusarium solani
(Fsg) root colonization and sudden death syndrome (SDS). Data
from this study indicate that the
development of SDS on RR soybeans is influenced by genotype.
Farmers planting RR soybeans
in Fsg infested fields are encouraged to select cultivars with
resistance to SDS.
The second study was performed in greenhouse conditions to
study the effects of glyphosate drift
on plant growth and concentrations of mineral nutrients in
leaves and seeds of non-glyphosate
resistant soybean plants at both the early growth stage and at
the grain seed maturation. The

results suggested that glyphosate might interfere with uptake
and re-translocation of Calcium
(Ca), Manganese (Mn), Iron (Fe), and Magnesium (Mg), most
probably by binding and thus
immobilizing them. The decreases in seed concentration of Ca,
Mn, Fe, and Mg by glyphosate
are very specific and may affect seed quality. At grain seed
maturation, however, glyphosate did
not reduce seed concentrations of Nitrogen (N), Potassium (K),
Phosphorus (P), Zinc (Zn) and
Copper (Cu). In fact, according to the study, they increased with
the exception of Phosphorus.
The major findings indicate that the glyphosate genetically
modified soybean seeds do not
outperform conventional seeds in all cases for any given
environment for desirable traits and that
glyphosate drift impairs the plant’s ability of the non-
glyphosate resistant variety to decrease
seed yield and affect the generative organs whereby, the seed
showed decreased concentrations
of Ca, Mn, Fe, and Mg which raises concern in terms of seed
quality (How seed quality was
measured?). The desirable traits are to increase yield, resistance
to herbicide (in the case of the
transgene study) without adverse affects, and improve the
nutritiona l value of food. However, in
order to obtain and sustain these desirable traits via genetic
engineering, there is an input cost
associated which may not be desirable and may offset any
desired gains in the long term. By
placing glyphosate gene into the soybean and spraying the
soybean crop with glyphosate raises
the question whether this would have the same effect as the
spray drift experiment performed on
the non-resistant glyphosate relative to nutrients or does the

inserted gene “protect” the plant
from the decreases in mineral nutrients and decrease in seed
yield. These types of studies need to
be performed to address such questions.
2
INTRODUCTION
Most commercial transgenic varieties of corn, cotton,
soybeans, and canola/rapeseed contain
only two traits herbicide and insect resistance. Genetically
Engineered (GE) seeds, or Genetically
Modified (GM) also called transgenic seeds are those seeds that
have been genetically modified
by adding specific genes to enhance a desired trait such as
herbicide or insect tolerance through
molecular biology techniques. Currently, two herbicide resistant
cropping systems are common
for soybean, corn, canola/rapeseed, and cotton: Roundup Ready
(active agent, glyphosate) and
Liberty Link (active agent, glufosinate).
This paper will be limited to the effects of only one trait; the
herbicide, glyphosate and will look
at performance and results established based on the findings.
This paper will not address the
processes of mutation and natural selection — as in cross-
pollination or grafting — but rather the
use of biotechnology to alter DNA to attain desired traits in
field studies and the effects of non-

glyphosate resistant variety of soybean on mineral nutrients.
Glyphosate is a non-selective herbicide registered for use on
many food and non-food field crops
as well as non-crop areas where total vegetation control is
desired (1). When applied at lower
rates, glyphosate also is a plant growth regulator (1).
Glyphosate is usually formulated as an
isopropylamine salt (2). At the time these studies were
performed, the Roundup Ready
formulation contained this salt as various glyphosate formulas
later entered the market;
formulations were modified (3). Some plants have been
genetically engineered to be resistant to
glyphosate and soybeans are an example of such a plant.
Glyphosate acts by enzyme inhibition in the shikimic acid
pathway (4). The target of the
herbicide is the enzyme 5-enolpyruvylshikimate-3-phosphate
synthetase (EPSPS) (4). Inhibition
of EPSP disrupts plant synthesis of aromatic compounds such as
vitamin K, ubiquinones, and the
amino acids tryptophan, tyrosine, and phenylalanine (4).
Glyphosate is absorbed across the plant
surface and is translocated throughout the plant (4). The
herbicide tends to accumulate in plant
regions with actively dividing cells (4). Plants exposed to
glyphosate display stunted growth,
loss of green coloration, leaf wrinkling or malformation, and
tissue death. Death of the plant may
take several days to weeks (4-20 days) to occur (4). These
amino acids are essential for crucial
plant processes such as protein synthesis, cell wall formation,
defense against pathogens and
insects, production of hormones, and production of compounds
required in energy transduction

such as plastoquinone (Duke 1988). Plastoquinone refers to any
of a group of quinones that are
involved in electron transport in chloroplasts during
photosynthesis (5). A gene for glyphosate-
tolerant EPSPS was isolated from the soil bacterium
Agrobacterium sp. strain CP4 and was
introduced into the soybean genome by means of the particle
acceleration method (Barry et al.
1992; Padgette et al., 1996a). Upon glyphosate treatment,
soybean expressing glyphosate-
tolerant EPSPS remains unaffected because of the continued
action of the introduced EPSPS that
meets the plant’s need for aromatic amino acids.
The chemical composition of seeds is basically determined by
genetic factors and varies among
different species and seed parts. However, it is influenced by
environmental and cultural
practices (Copeland & McDonald, 2001). Because of genetic
factors, the chemical composition
of seeds varies widely among species and even among varieties.
3
Every seed is genetically unique producing a population that
respond best to changing or
different environments because they are a product of sexual
recombination. Therefore, seeds
provide evolutionary advantages. However, a very important
thing to note is that in addition to
the manipulation of genes through crossing and selection, plant
breeders have been able to
manipulate the chemical composition of many domesticated

crops and increase their value as
food, fiber, or raw material. The effects of glyphosate through
genetic engineering of plants and
selective breeding in addition to environmental conditions add
much complexity to the desired
outcomes.
Thus, the reproductive background of the soybean along with an
explanation of maturity
grouping and growth stages would be beneficial in the
understanding of the conventional
reproductive and vegetative process, whereby, the transgenic
process on the other hand is
demonstrated in the objectives of one of the following studies.
These will be important for the
analysis of the studies as well as concluding the results.
Re productive Background on Soybe an (Glycine max (L.) Me
rr.), Maturity Groups and
Growth Stage s
Soybean is considered a self-pollinated species, propagated
commercially by seed. Artificial
hybridization is used for cultivar breeding. The soybean flower
stigma is receptive to pollen
approximately 24 hours before anthesis and remains receptive
48 hours after anthesis. The
anthers mature in the bud and directly pollinate the stigma of
the same flower. As a result,
soybeans exhibit a high percentage of self-fertilization, and
cross pollination is usually less than
one percent (Caviness, 1966). A soybean plant can produce as
many as 400 pods, with two to
twenty pods at a single node. Each pod contains one to five
seeds. Neither the seedpod, nor the
seed, has morphological characteristics that would encourage

animal transportation (6).
Because the first soybean study uses maturity groupings, a
description identifying them is useful.
Varieties grown in the United States are divided into 13
maturity groups (MG) (Wiatrak, et al.,
year?). The earlier varieties bloom when days are long and
nights are short, while the later-
maturing varieties bloom under relatively shorter days and
longer nights. Earlier and later
maturing groups develop differently and knowing the growth
habit of different maturity groups
(MG) can help with the crop management (Wiatrak et.al., year).
Most indeterminate varieties are adapted to maturity group IV
and earlier. These varieties have
overlapping vegetative and reproductive growth stages (Wiatrak
et.al., year). Terminal growth
bud on the main stem continues to grow after the first bloom
and most of the pods are on the
main (Wiatrak et.al., year). Flowers and pods develop at
different times and rates depending on
node locations (Wiatrak et.al., year). Nodes with the earliest
flowers located near the bottom of
stem; therefore, an indeterminate plant may contain pods with
developing seed at lower nodes
while upper nodes contain only small pods or flowers (Wiatrak
et.al., year).
Most varieties with a determinate growth habit (maturity group
V and later) have distinct
vegetative and reproductive growth stages (Wiatrak et.al., year).
Number of nodes and plant
height of the main stem are terminated at the full bloom stage
(R2) (Wiatrak et.al., year).
However, branch growth continues after first bloom and the

number of pods often greater on
branches than the main stem (Wiatrak et.al., year). See Table 1,
Vegetative and Reproductive
4
Stages for the definition of the various stages and Figure 1,
Illustrated Soybean Stages to
understand the reproductive and vegetative stages. In addition
to maturity growth, these stages
illustrate where they occur. This is helpful when discussion of
the timing of glyphosate
application is performed. The early reproductive period (R1 to
R5.5) is sensitive to altered source
strength and crop growth rate (Palle Pedersen, 2003-2007).
Pederson also indicated that yield is
mostly determined by seed number. Neither of the two studies
correlated this.
Table 1. Ve ge tative & Reproductive Stages of De velopme nt
of Soybe ans
Ve ge tative Stage s Re productive Stage s
VE (emergence)
VC (cotyledon stage)
V1 (first trifoliolate)
V2 (second trifoliolate)
V3 (third trifoliolate)
V(n) (nth trifoliolate)
V6 (flowering will soon start)
R1 (beginning bloom, first flower)
R2 (full bloom, flower in top 2 nodes)

R3 (beginning pod, 3/16" pod in top 4 nodes)
R4 (full pod, 3/4" pod in top 4 nodes)
R5 (1/8" seed in top 4 nodes)
R6 (full size seed in top 4 nodes)
R7 (beginning maturity, one mature pod)
R8 (full maturity, 95% of pods on the plant are mature)
Source: Nort h Dakot a St at e Universit y
Figure 1, Illus trate d Soybe an Stage s
Ve ge tative Stage s Re
productive Stage s
* V-Stage s * R- Stage s
*VE,VC,V1,V2,V3, Vn * R1,
R2, R3, …R8
* Starts at Flowe ring
Source: Iowa St at e Universit y, Dept of Agronomy
5
MATERIALS AND METHODS
Because soybean crops rate as the highest transgenic crop
in the world, two soybean
studies have been selected. See Table 2, Global Area of
Transgenic Crops 1996-2002 by Crop.
Observations from the table illustrate that soybeans are the most

genetically modified crop by
acreage followed by corn as the second largest by acreage for
genetic modification of crop. The
important factor to mention is that most of these genetically
modified crops that are planted in
the fields are rotated by another genetically modified crop
followed by yet another genetically
modified crop. Hence, the undesirable trait of herbicide
resistance and the growth of weeds that
no longer respond to the chemical, glyphosate. See Table 3,
Changes in the Pounds of
Glyphosate Applied per Acre per Crop Year on Corn, Cotton,
and Soybeans: 1996-2007.
Table (this is Figure ) 2, Global Are a of Trans ge nic Crops
1996-2002
(Source: ht t p://cls.casa.colost ate.edu/t
ransgeniccrops/references.html#James2002)
Table 3, Change s in the Pounds of Glyphos ate Applie d pe
r Acre pe r Crop Ye ar on Corn,
Cotton, and Soybe ans : 1996-2007
Crop and Period Glyphosate Rate
in 1996
Total Increase (Pounds
active ingredient per
Acre)
Percent
Change

Average Annual
% Change in
Period Noted
Corn (1996-2005) 0.68 0.27 39% 4.3%
Cotton (1996-2007) 0.63 1.26 199.8% 18.2%
Soybean (1996-2006) 0.69 0.67 97.6% 9.8%
Not e: All use dat a is from t he USDA Nat ional Agricult ural
St at ist ics Service (NASS) annual surveys of pest icide use,
and t ake
int o account bot h changes in t he one-t ime rat e of
application and t he average number of applicat ions per crop
year. Corn was last
surveyed by NASS in 2005, Cot t on in 2007, and Soybeans in
2006. (Source: www.organic-cent er.org)
The above information is us e ful, but it is not Mate rials &
Me thods. It can be move d to the
“Introduction” s e ction.
http://cls.casa.colostate.edu/transgeniccrops/references.html#Ja
mes2002
http://www.organic-center.org/
6
Firs t Soybe an Study - Mate rials and Me thods are lis ted
from actual s tudy
From the 1st soybean study, the objective was to evaluate
the effects of glyphosate on root

colonization by Fusarium solani f. sp. glycines (Fsg) and
development of sudden death syndrome
(SDS) on Round Up Ready (RR) soybean. Ten Roundup Ready
(RR) soybean cultivars from
maturity group (MG) II to VI were used. There were two
cultivars per maturity group. The
cultivars were provided by Monsanto without disclosing their
identity. Each cultivar was
previously characterized for SDS resistance (by the companies)
and was identified as either
partially resistant or susceptible to SDS. The cultivars in each
maturity group included one SDS
resistant and one SDS susceptible cultivar. Five RR cultivar
pairs that contrasted for SDS
resistance from maturity groups (MG) II to VI were evaluated
with and without glyphosate
application. The Fsg root infection severity (IS), colony
forming units per gram of root (CFU),
SDS leaf scorch disease index (DX), and grain yield were
determined. Each maturity group was
considered a separate experiment. The cultivar pairs within each
experiment were evaluated at
two locations per year for two years. All fields were selected
based on a history of uniform SDS
leaf symptoms expression. All plots were established using
conventional tilling. All plots were
treated with a pre-emergence herbicide combination of 0.84 kg
(active ingredient) a.i. ha_1
trifluralin [2,6-dinitro-N ,N-dipropyl-4-(trifluoromethyl)-
benzamine] and 0.07 kg a.i. ha_1
imaziquin (2-[4,5-dihydro-4-methyl-4-(1-methylethyl)-5-oxo-
1H-imida zol-2-yl]-3-
quinolinecarboxylic). Treatments consisted of glyphosate-
sprayed and a non-sprayed control. At
the R6 growth stage, subplots were rated for SDS disease
incidence (DI) and disease severity

(DS). Disease incidence was the percent of plants in the subplot
with visible SDS leaf symptoms.
Disease severity (percentage leaf surface chlorotic/necrotic)
was rated on a scale of 1 to 9 as
described: (1 =0–10%/1–5%, 2= 10–20%/6–10%, 3=20–
40%/10–20%, 4=40–60%/20–40%,
5=>60%/=40%, 6=up to 33% premature defoliation, 7 = up to
66% premature defoliation,
8=>66% premature defoliation, and 9 = premature death of
plant). Disease index (DX, expressed
as %) was then calculated as (DI _ DS)/9, with a possible range
of 0 (no disease) to 100 (death of
all plants). Eight taproots were harvested from each subplot at
R6 growth stage. Four roots were
used for IS and four for CFU determination. For IS
determination, taproots were washed and
surface sterilized in 0.5% (v/v) sodium hypochlorite. Each
taproot was cut into several 1-
cmsegments. Ten root segments were randomly selected and
placed on a Petri plate containing a
selective medium composed of 20 g agar, 20 g sucrose, 1 g
KH2PO4, 0.5 gMgSO4·7H2O, 1 g
KNO3 and 1 L distille d water; autoclaved at 110°C for 40 min
and cooled to 42°C before 0.3 g
streptomycin sulfate, 0.1 g neomycin, 0.1 g chlortetracycline,
0.05 g rifampicin, and 0.23 g
pentachloronitrobenze ne (PCNB) (Terrachlor, Uniroyal
Chemical Co., Vaugntuk, CT) were
added. Infection severity was determined as the percentage of
root segments yielding slow-
growing, blue fungus (Njiti et al., 1997). A total of 40 root
segments from 4 taproots per subplot
were used in IS determination. For determination of CFU, the
four taproots from each subplot
were treated as one sample. The roots were washed, surface-
sterilized in 0.5% (v/v) sodium

hypochlorite , oven dried at 28°C for 48 h, ground using a
Wiley cutting mill (Thomas Scientific,
Swedesboro, NJ) to pass through a 40-mesh screen, weighed,
and suspended in 2 L of distilled
water. One mL of the suspension was transferred onto each of
five Petri plates containing the
selective medium described above. Plates were inoculated and
incubated at room temperature for
14 d. The number of slow-growing, appressed, and blue fungal
colonies (Rupe et al., 1996) was
counted as CFUs. The CFU per gram of root tissue was
calculated (Luo et al., 2001). At the R8
developmental stage, the middle two rows for each plot were
harvested with a two-row combine.
7
The seeds were cleaned without losing broken seeds. Yield was
calculated for each plot at 13 g
kg _1 seed moisture content. Data were collected for root IS in
all test environments, CFU in 10
environments (2 per MG) in 1998, disease index in 4
environments for MG II and III; 3
environments for MG V and VI and 0 environment for MG IV,
and grain yield in 4 environments
in MG II, III, IV, and V; and 3 environments in MG VI. In this
study, environment was
considered a random factor and cultivar and glyphosate were
considered fixed factors. To avoid
violation of the assumptions for analysis of variance, the CFU
values were subjected to
logarithmic transformation using the formula log10(CFU + 1).
The values of DX and IS were

subjected to square root transformation using the formulae
√(DX + 0.5) and √(IS + 0.5),
respectively. Transformed data were subjected to analysis of
variance (SAS Inst., 1985).
Analysis of variance was conducted for combined environments
to test possible interaction of
treatments with environments.
Se cond Soybe an Study - Mate rial and Me thods are lis ted
from actual s tudy
For the second soybean study: the objective was to study the
effects of glyphosate drift on
plant growth and concentrations of mineral nutrients in leaves
and seeds of non-glyphosate
resistant soybeans. It was demonstrated that glyphosate reduced
seed and leaf concentrations of
calcium, manganese, magnesium, and iron in non-glyphosate
resistant soybean. The non-
glyphosate resistant soybeans (Cultivar: Nova) were grown
under greenhouse conditions
equipped with an evaporative cooling system (24-28; 21-24°C
day/night) under natural daylight
during the summer season. The soil was transported from
Turkey where soil Zn deficiency has
been well documented (Cakmak et al., 1999). The
concentrations of DTPA-extractable Zn, Fe,
Mn, and Cu were 0.10, 5.6, 7.2, and 1.7, respectively measured
according to the method
described by Lindsay and Norvell, 1978. Soil was not
inoculated with N-fixing bacteria. Eight
seeds were sown per pot and after emergence the seedlings were
thinned to four per pot. Plants
were watered daily with deionized water. Two separate pot
experiments were conducted in a
randomized block design with 4 (in the first experiment) or 6

(in the second experiment)
replications. In the first experiment, plants were grown for 3
weeks under greenhouse conditions,
and foliar glyphosate applications were made at 13 days after
planting. At harvest, the shoot apex
together with the youngest expanded leaves was harvested to
represent the young plant parts and
the remaining leaves as old plant part. The leaf samples were
analyzed for mineral nutrient
concentrations as described below. In the second experiment,
plants were grown until seed
maturation in the plastic pots containing 2.8 kg loamy clay soil.
In addition to the basal fertilizer
treatment, 100mg N as Ca(NO3)2 and 50mg P and 63mg K as
KH2PO4 per kg soil was applied
into soil at the R1 growth stage. Glyphosate applications during
the second experiment were
made at the V4, V6 and early R1 stages. Plants were harvested
at the grain maturation stage, and
the grains harvested were used for determination of grain yield
and for analysis of mineral
nutrient concentrations. In all foliar applications, the
isopropylamine salt of glyphosate
(Roundup Ultra Herbicide, Monsanto Ltd., Adana, Turkey) with
an active ingredient (a.i.) 480 g
L−1 was used. In the first experiment, when plants were 13 days
old, foliar applications of
glyphosate was initiated and realized three times with two days
intervals. The doses applied for
simulating foliar glyphosate drift were 0.06, 0.2, and 0.6% of
the recommended application rate
(i.e. 1.44 kg ha−1 a.i. glyphosate applied with 200 L of water
ha−1: 31.55mM glyphosate as
active ingredient), which are equivalent to 19, 63 and 189µM
glyphosate in the spray solution,

8
respectively. These concentrations have been selected based on
the preliminary tests to
determine the different sub lethal doses of glyphosate and the
selected doses were similar to
those used previously by De Maria et al. (2006) and Eker et al.
(2006). In the experiment done
until seed maturation, the simulated drift rates of the glyphosate
applied at the V4, V6 and early
R1 stages and were 0, 0.3, 0.6, 0.9 and 1.2% of the
recommended application rate which are
equivalent to 0, 95, 189, 284 and 379µM glyphosate in the spray
solution, respectively.
Glyphosate solutions were freshly prepared before the foliar
treatments. Applications were made
onto the leaves using a plastic hand-sprayer until most of the
leaves became wet but not allowing
any surface runoff. Changes in levels of chlorophyll (SPAD
value) were measured on the same
area of the youngest expanded leaves before harvest using a
chlorophyll meter (Minolta SPAD-
502, Japan). For the measurement of mineral nutrient
concentrations, the dried and ground leaf
and seed samples were digested in 2 ml 35% H2O2 and 4 ml
65% HNO3 by using a microwave
digestion system (MarsExpress; CEM Corp., Matthews, NC,
USA). The analysis of P, K, Mg,
Ca, Mn, Fe, Zn and Cu was performed by using inductively
coupled plasma optical emission
spectrometry (ICP-OES) (Vista-Pro Axial; Varian Pty Ltd.,
Mulgrave, Australia). Total nitrogen
concentrations of the samples were measured by an automated N

analyzer (TruSpec CN, LECO
Corp., Michigan, USA). All treatments were performed in four
and six independent replications
in the first and second experiment, respectively. Differences
among the means and treatments
were compared by the least significant difference (LSD) test at
P = 0.05 probability level.
Shikimic acid accumulation was determined in the youngest
leaves at the growing points and
oldest trifoliate leaves using a modified method of Cromartie
and Polge (2002). About 500mg (±
25 mg) of fresh leaf tissues was extracted in 5 ml of 0.25N HCl
in an ice cold mortar and
centrifuged at 15,000×g for 15min at +4 °C. The resulting
supernatant was diluted with 0.25N
HCl at a ratio of 1:10 (v:v) and directly used in the colorimetric
assay. Aliquots of 200µL of 1:10
diluted samples were mixed with 400µL of reaction solution
consisting of 0.25% periodic acid
and 0.25% Na meta-periodate. All samples were incubated at
room temperature for 1 h to
oxidize the shikimic acid. Finally 400µL of quenching solution
consisting of 0.6M NaOH and
0.22M Na2SO3 was added to yield a yellow color chromophore
that correlates directly with the
amount of shikimic acid in the samples. Concentration of
shikimic acid was determined by
measuring the absorbance at 380nm using a standard curve of 0–
200µM shikimic acid (S5375;
Sigma, St. Louis, MO, USA) and presented on a fresh weight
basis.
RESULTS

Firs t Soybe an Study Re s ults lis ted are adapte d from actual
s tudy
There was significant (P ≤ 0.05) variation among
environments in MG III, IV, V, and VI for
IS; MG II and VI for CFU; MG II, III, and VI for DX. There
were no significant Glyphosate X
Environment interactions for any disease measure within any
maturity groups. Across cultivars
within each maturity group, there was no significant difference
between glyphosate sprayed and
non-sprayed subplots for all disease measures. There was no
Glyphosate X Cultivar interaction
in any disease measure within any maturity group. For each
disease measure, significant
variation between cultivars was only observed in some maturity
groups. There was significant
variation between cultivars for IS in MG IV and VI, for CFU in
MG VI, and for DX in MG III
9
and VI. The SDS-resistant cultivar generally had less disease
(as measured by IS, CFU, and DX)
than the SDS-susceptible cultivar except for IS in MG III, V,
and VI. The application of
glyphosate did not reduce yield in this study. There was
significant Glyphosate X Cultivar
interaction for yield in MG VI. In MG VI, the SDS-susceptible
cultivar sprayed with glyphosate
had significantly higher grain yield than the non-sprayed.
In this study root colonization by Fsg and SDS leaf symptoms

did not significantly increase
following the application of glyphosate. Data from this study
indicate that the development of
SDS on RR soybean is influenced by the genotype of the
cultivar. Farmers planting RR soybean
in Fsg-infested fields are encouraged to select cultivars that
have been shown to be resistant to
SDS. See Figure 2, Bar graphs of glyphosate cultivar interaction
means for yield.
The study was performed by Victor N. Njiti, Oval Myers Jr.,
Daniel Schroeder, and David A.
Lightfoot and published in Agron. J. 95:1140-1145 (2003).
Fig. 2. Bar graphs of Glyphos ate X Cultivar inte raction me
ans for yie ld. Clust ers of bars for MG II
t o V are averaged over four environment s and MG VI is
averaged over t hree environments.
Source: Agronomy Journal, Vol. 95, Sept -Oct 2003
Se cond Soybe an Study Re s ults lis ted are adapte d from
actual s tudy
The effect of foliar-applied glyphosate on the shoot growth
and leaf concentrations of
mineral nutrients was first studied in 21-dayold plants by
analyzing young and old leaves
separately. The first reaction of plants to increasing glyphosate
application up to 0.6% of the
recommended dose was the development of chlorosis on the
youngest leaves. As expected,
chlorophyll concentrations (SPAD values) showed a clear
decrease by glyphosate. Increasing
application of glyphosate significantly reduced dry weight of

young leaves, but did not affect the
dry weight of the old leaves. The dry weight ratio of older parts
to younger parts of plants was
nearly 2 at the nil glyphosate treatment and increased to about
10 at the highest glyphosate
treatment. Increasing rates of glyphosate caused marked
increases in shikimate concentrations of
Yi
el
d/
M
g/
H
a
Maturity Group (Mg) Sprayed_S Non-Spray_S Sprayed_R
Non-Spray_R
10
young parts of plants; but, remained less effective on the
shikimate concentrations in the old
parts of plants. Among the mineral nutrients analyzed, the leaf
concentrations of K, P, Cu and Zn
were not affected, or even increased significantly in case of P
and Cu in young leaves by
glyphosate. Phosphorus concentrations of young leaves showed
significant increases by
increasing glyphosate rate that might be related to accumulation

of P-containing glyphosate in
young parts of plants. In the case of Fe concentrations of leaves,
there were inconsistent changes
by glyphosate applications. But, at the highest glyphosate rate,
leaf concentrations of Fe showed
clear declining tendency when compared to other glyphosate
treatments. In contrast to other
mineral nutrients analyzed, the leaf concentrations of Ca, Mn
and Mg were reduced greatly by
glyphosate, particularly Ca and Mn in young leaves. For
example, in young leaves, the
concentration of Ca was reduced from 14.9 to 9.0mgkg−1 and
Mn was reduced from 182 to
29mgkg−1. In the second experiment, plants were grown until
grain maturation, and glyphosate
was sprayed three times at the V4, V6 and early R1 stages.
Distinct decreases in shoot biomass
production and seed yield by glyphosate were found at the
higher glyphosate rates (e.g., 0.9 and
1.2% of the recommended dose) which also resulted in
development of chlorosis on young
leaves. When compared to the shoot dry weight, seed yield was
more clearly declined by
glyphosate. At the 1.2% glyphosate application, seed yield was
declined by nearly 5-fold when
compared to the plants without glyphosate application; but, this
decrease was only 2.8-fold for
shoot dryweight, indicating higher sensitivity of generative
organs to glyphosate. Increases in
glyphosate application resulted in significant changes in seed
concentrations of all measured
elements with exception of P. At the highest level of the
glyphosate application, the seed
concentrations of N, K, Zn and Cu were significantly increased
when compared to the control

plants without glyphosate treatment. By contrast, the seed
concentrations of Ca, Mn, Fe and Mg
were significantly reduced by the highest rates of glyphosate,
particularly in the case of Ca and
Mn (Table 4). To verify such clear effects of glyphosate on seed
concentrations of Mn and Ca
and partly on Fe and Mg, this experiment was repeated by using
0, 0.3 and 1.2% glyphosate
doses, and the results obtained.
The effects of glyphosate drift on plant growth and
concentrations of mineral nutrients in leaves
and seeds of non-glyphosate resistant soybeans demonstrated in
Table 4, of the foliar-applied
glyphosate on the shoot growth and leaf concentrations of
mineral nutrients in 21-day old plants
by analyzing young and old leaves separately. The first reaction
of plants to increasing
glyphosate application up to 0.6% of the recommended dose was
the development of chlorosis
on the youngest leaves. The chlorophyll concentrations (SPAD
values) showed a clear decrease
by glyphosate. Increasing application of glyphosate
significantly reduced dry weight of young
leaves, but did not affect the dry weight of the old leaves (Table
4). The dry weight ratio of older
parts to younger parts of plants was nearly 2 at the nil
glyphosate treatment and increased to
about 10 at the highest glyphosate treatment (Table 4).
Increasing rates of glyphosate caused
marked increases in shikimate concentrations of young parts of
plants; but, remained less
effective on the Shikimate concentrations in the old parts of
plants (Table 4).

11
Table 4. Effe ct of incre as ing glyphos ate rate s on SPAD
value s (chlorophyll) and dry matte r
production of young and old le ave s of 21-day-old s oybe an
plants grown unde r gre e nhouse
conditions . T he values represent means of four independent
replicat ions.
Glyphosate rate(% of
recommended)a
SPAD
value
Dry Matter Production Shikimate
Young leaves (mg
plantµ 1)
Old leaves (mg
plantµ 1)
Young leaves
(µ mol g−1 FW)
Old leaves
(µ mol g−1 FW)
a Glyphosat e was applied t o foliage at t he concent rations of
0.06% (19_M glyphosate), 0.2% (63_M glyphosat e) and 0.6%
(189_M glyphosat e) of t he recommended applicat ion rate for

weed cont rol.
Among the mineral nutrients analyzed, the leaf concentrations
of K, P, Cu and Zn were not
affected, or even increased significantly in case of P and Cu in
young leaves by glyphosate
(Table 5). Phosphorus concentrations of young leaves showed
significant increases by increasing
glyphosate rate that might be related to accumulation of P-
containing glyphosate in young parts
of plants (Table 5). In the case of Fe concentrations of leaves,
there were inconsistent changes by
glyphosate applications. But, at the highest glyphosate rate, leaf
concentrations of Fe showed a
clear declining tendency when compared to other glyphosate
treatments (Table 5). In contrast to
other mineral nutrients analyzed, the leaf concentrations of Ca,
Mn and Mg were reduced greatly
by glyphosate, particularly Ca and Mn in young leaves (Table
5). For example, in young leaves,
the concentration of Ca was reduced from 14.9 to 9.0mgkgµ1
and Mn was reduced from 182 to
129mgkgµ1. In the second experiment, plants were grown until
grain maturation, and glyphosate
was sprayed three times at the V4, V6 and early R1 stages.
Distinct decreases in shoot biomass
production and seed yield by glyphosate were found at the
higher glyphosate rates (e.g., 0.9 and
1.2% of the recommended dose) which also resulted in
development of chlorosis on young
leaves (Fig. 2; Table 3). When compared to the shoot dry
weight, seed yield was more clearly
declined by glyphosate. At the 1.2% glyphosate application,
seed yield was declined by nearly 5-
fold when compared to the plants without glyphosate
application; but, this decrease was only

2.8-fold for shoot dry weight, indicating higher sensitivity of
generative organs to glyphosate.
Increases in glyphosate application resulted in significant
changes in seed concentrations of all
measured elements with exception of P (Table 5). At the highest
level of the glyphosate
application, the seed concentrations of N, K, Zn and Cu were
significantly increased when
compared to the control plants without glyphosate treatment. By
contrast, the seed concentrations
of Ca, Mn, Fe and Mg were significantly reduced by the highest
rates of glyphosate, particularly
in the case of Ca and Mn (Table 5). To verify such clear effects
of glyphosate on seed
concentrations of Mn and Ca and partly on Fe and Mg, this
experiment was repeated by using 0,
0.3 and 1.2% glyphosate doses, and the results obtained
confirmed the results given in Table 5
(no data shown).
0 3 4.4 183 346 1.6 2.4
0.06 31.9 103 353 1.4 2.2
0.2 23.0 120 373 9.3 2.2
0.6 12.0 40 440 34.6 6.3
LSD0.05 1.6 33 NS 3.2 1.7
12

Table 5. Effe ct of incre as ing glyphos ate rate s on the
concentrations of mine ral nutrie nts in
young and old le ave s of 21-day-old s oybe an plants grown
unde r gre e nhous e conditions . T he
values represent means of four independent replicat ions.
Glyphosate rate (%
of recommended)a
P
(gkg−1
DW)
K (g
kg−1
DW)
Ca (g kg−1
DW)
Mg (g kg−1
DW)
Fe (mg kg−1
DW)
Mn (mg kg−1
DW)
Zn (mg kg−1
DW)
Cu (mg kg−1
DW)
Young leaves

0 4.5 33.7 14.9 6.4 45 182 81 5.5
0.06 5.0 28.6 12.5 6.1 59 152 78 5.8
0.2 5.2 30.9 11.2 5.6 53 148 77 6.1
0.6 6.0 28.3 9.0 4.6 42 129 89 7.2
LSD0.05 0.9 NS 1.0 0.5 14 25 NS 0.8
Old leaves
0 2.3 26.3 22.3 8.8 49 232 93 5.5
0.06 2.2 24.0 18.4 7.2 54 160 78 4.8
0.2 2.3 25.6 19.2 7.6 51 190 84 4.9
0.6 2.3 25.2 15.6 5.8 40 121 65 4.1
LSD0.05 NS NS NS 2.7 NS 75 NS NS
0.06% (19µM glyphosat e), 0.2% (63µM glyphosat e) and 0.6%
(189µM glyphosat e) of t he recommended applicat ion rat e for
weed cont rol.
Reproductive organs are known to be more sensitive to
glyphosate than the vegetative tissues.
The reported decreases in seed concentration of Fe, Mn, Ca and
Mg by glyphosate raise concerns
in terms of seed quality. Seed reserves of mineral nutrients play
an important role in seed
viability and seedling vigor and establishment, particularly
under adverse soil conditions (Welch,
1999). It is of great importance to examine the concentrations of
mineral nutrients in grains
harvested in cropping systems with frequent glyphosate
applications. Preharvest application of
glyphosate to stimulate grain maturation is a common practice
in many cropping systems. In
most cases, this practice is, however, associated with poor seed
germination and reduced
seedling vigor (Bennett and Shaw, 2000; Baig et al., 2003).
Such adverse effects of pre-harvest
applied glyphosate on seed germination and seedling growth

might also be related to the
reduction or physiological inactivation of mineral nutrients in
seeds, such as Ca and Mn. For
better understanding and characterization of the adverse effects
of glyphosate on plant growth,
future studies should also focus on differentiation between
bound and unbound divalent cations
in the glyphosate-treated plants. Currently, experiments are on-
going to test the role of
glyphosate on physiological availability of divalent cations by
measuring the activity of enzymes
which are dependent on those cations.
The study was performed by Ismail Cakmak, Atilla Yazici,
Yusuf Tutus, Levent Ozturk Sabanci
University, Faculty of Engineering and Natural Sciences, 34956
Istanbul, Turkey. See Table 6,
Effect of increasing glyphosate rates on grain and shoot matter
production of soybean plants, and
Figure 3, Effect of increasing glyphosate rates on growth of
soybean plants.
13
Table 6. Effe ct of incre as ing glyphos ate rate s on grain and
s hoot dry matte r production of

s oybe an plants grown until grain maturation s tage unde r gre
e nhouse conditions. T he values
represent means of six independent replicat ions.
Glyphosat e rat e (% of recommended)a Seed yield (g plant -1)
Shoot yield (g plant -1)
0 12.5 19.3
0.3 13.4 18.7
0.6 12.1 20.0
0.9 9.2 14.6
1.2 2.3 6.8
LSD0.05 1.9 2.6
0.3% (95µM glyphosat e), 0.6% (189µM glyphosat e), 0.9%
(284µM
glyphosat e) and 1.2% (379µM glyphosat e) of t he
recommended applicat ion rat e for weed cont rol.
Fig. 3. Effe ct of incre as ing glyphos ate rate s on growth of s
oybe an plants unde r gre e nhouse
conditions . T he values on t he pot s represent % of t he
recommended applicat ion rat e for weed cont rol corresponding
t o 0, 95,
189, 284 and 379µM glyphosat e.
DISCUSSION
Herbicide resistance, insect resistance, yield, vigor, viability
and nutrition are some of the
reasons that transgenic seeds are being adopted. The reason
Roundup Ready soybeans were
created was to reduce the use of pesticide and not to increase

yield. As a result, there is more
pesticide use on soybeans since RR soybeans have been on the
market.
Glyphosate use increased more than six-fold between 1992 and
2002, to become the most used
herbicide in the United States (Gianessi and Reigner, 2006).
Most of this increase is due to the
adoption of Genetically Resistant Crops (GRCs). (Antonio L.
Cerdeira and Stephen O. Duke,
2006). The most striking finding is that GE crops have been
responsible for an increase of 383
million pounds of herbicide use in the U.S. over the first 13
years of commercial use of GE crops
1996-2008 (Benbrook 2009). The average rate of herbicides
applied to conventional soybean
acres dropped from 1.19 pounds of active ingredient per acre in
1996 to 0.49 pounds in 2008.
(Figure 4.0). The steady reduction in the rate of application of
conventional soybean herbicides
accounts for roughly one-half of the difference in herbicide use
on GE versus conventional
soybean acres. The increase in the total pounds of herbicides
applied to HT soybean acres, from
14
0.89 pounds in 1996 to 1.65 pounds in 2008, accounts for the
other one-half of the difference
(Benbrook 2009).
From another study performed by Benbrook (2001), states the
following about RR soybean
herbicide use:

• Slightly more pounds of herbicides are applied on the average
acre of Roundup-Ready
(RR) soybeans compared to the average acre planted to
conventional soybean varieties.
• Fewer herbicide active ingredients are applied on the average
acre of RR soybeans
relative to the average conventional acre.
• Average per acre pounds of herbicide applied on RR soybeans
exceeds by 2- to 10-fold
herbicide use on the approximate 30% of soybean acres where
farmers depend largely on
low-dose imidazolinone and sulfonylurea herbicides.
• Herbicide use on RR soybean acres is gradually rising as a
result of weed shifts, late-
season weed escapes leading to a buildup in weed seedbanks,
and the loss of
susceptibility to glyphosate in some weed species (Hartzler,
1999; HRAC, 2001).
However, Benbrook also stated the obvious that farmers have
embraced the technology because
it greatly simplifies soybean weed management and provides
additional degrees of freedom in
managing weeds (Gianessi and Carpenter, 2000; ERS, 1999).
Figure 4.0. He rbicide Us e Rate on All RR and Conve ntional
Soybe an Acre s
A study performed at the University of Missouri, herbicide-

tolerant soybeans appear to suffer
what is referred to as "yield drag." (7,8). It explains that in
some areas and on some farms the
tendency of Roundup Ready soybean varieties to yield less than
their comparable, conventional
counterparts although, variable, but overall, they appear to
average yields that are five to ten
percent lower per acre.
15
In addition, they stated that they experienced impaired root
development, nodulation, and that
nitrogen fixation is likely account for the yield drag (7, 8).
Drought conditions worsen the effects.
The bacterium that facilitates nodulation and nitrogen fixation
in the root zone is apparently
sensitive to both Roundup and drought (7, 8). University of
Missouri scientists reported problems
with germination of Roundup Ready soybeans in the 2001 crop
year (8). Experiments conducted
in 1997 through 2000 at two Missouri locations revealed that
Roundup Ready soybeans
receiving glyphosate at recommended rates had significantly
higher incidence of Fusarium on
roots within one week of application compared with" soybeans
that did not receive glyphosate,
reported Pat Donald, MU plant pathologist, and Robert Kremer,
an MU soil scientist and USDA
Agricultural Research Service microbiologist (8). It has also
reported increased levels of cracked
soy beans at harvest due to dry weather - work has already
established that RR soybeans are

especially vulnerable in drought compared to conventional
varieties (7, 8). This was followed by
poor germination from many soy seed bags when planting took
place in the spring. University of
Arkansas scientists have shown that root development,
nodulation and nitrogen fixation are
impaired in some RR soybean varieties and that the effects are
worse under conditions of
drought stress or in relatively infertile fields (9).
In reference to the Reproductive Soybean Maturity section
mentioned earlier, Wiatrak et.al.,
indicated that adequate soil moisture from bloom through pod
fill is important for good yields,
farmers should consider planting varieties from more than one
maturity group to reduce the risks
of drought damage during pod fill.
These findings support the first soybean study in this paper
where they evaluated the effects of
glyphosate on root colonization by Fsg and development of SDS
on RR soybeans and it was
recommended that farmers planting RR soybeans in Fsg infested
fields are encouraged to select
cultivars that are resistant to SDS. Table7, RR Soybean Yield
Compared to Conv Varieties.
Table 7. Roundup Re ady (RR) Soybe an Yie ld Compare d to
Conve ntional (Conv) Varie tie s
in Eight State s , 1998 [bus hels pe r acre ]
States Trial
Mean
Top Five
Varieties

Top Variety
Conv RR Conv RR Conv RR
Illinois 58 60 65 65 67 67
Iowa 61 57 64 60 66 60
Michigan 66 64 74 69 78 70
Minnesota 66 61 73 67 74 69
Nebraska 58 51 65 58 66 60
Ohio 60 58 67 63 69 65
South Dakota 49 44 54 50 56 51
Wisconsin 71 69 85 82 87 84
Source: ‘P erformance of T ransgenic Soybeans – Nort hern
U.S.’, E.S. Oplinger, M.J. Mart inka, K.A. Schmit z.
16
From a nutritional standpoint, an abstract of a study (Lappe, et.
al.) published in the Journal of
Medicinal Foods (Vol. 1, No. 4) claims that Roundup Ready
soybeans may have reduced levels
of isoflavones. The authors further claim the implications are
meaningful since isoflavones are
potentially beneficial nutritional components in soybeans. Dr.
Clare Hasler, Executive Director

of the Functional Foods Health Program at the University of
Illinois Urbana-Champaign, and a
member of the Journal of Medicinal Foods' editorial review
board, expressed concern about the
Lappe paper. Dr. Hasler stated, "Concluding that the results of
the Lappe paper are biologica lly
relevant would be inappropriate and misleading since the
scientific literature clearly suggests that
isoflavone amounts in soybeans can vary as much as 300
percent or more." A publication by the
University of Illinois shows that numerous environmental
factors, such as weather during the
growing season, and even the slope of the field where the
soybeans are grown, can lead to
variability in the isoflavone content of soybeans. "Variability is
due to the varietal component
and the environmental component," said Dr. Don Bullock,
Associate Professor of Biometry, at
the University of Illinois at Urbana-Champaign. "Right now, the
environmental effect is far
greater than the varietal effect."
These publications and comments support the findings in the
second soybean study in this paper
where the reported decreases in seed concentration of Fe, Ca,
Mn and Mg by glyphosate raised
concerns in terms of seed quality in that seed reserves of
mineral nutrients play a important role
in seed viability, and seedling vigor and establishment,
particularly under adverse soil
conditions. Therefore, environment is a significant variable in
addition to soil.
However, in the cases presented here, there are additional
reasons that may need to be
considered in order to grant the crop grower the full economic

benefits that come with such
guarantees such as herbicide resistance, increased yields,
nutrient composition. The costs of
weed control, the equipment needed (including maintenance) to
collect seeds, costs of
conventional breeding to obtain yields without the added costs
of additional herbicide when
added with the high cost of the transgenic seeds. Based on the
numbers in the above tables, it
seems that some growers may even come out ahead with the use
of selecting particular cultivars
for their intended purpose based on their soil, and
environmental conditions. The overwhelming
evidence suggests that there needs to be additional research that
performs tight studies that
mimic identical situations. For example, studies suggest that
there exists different genes used
throughout the years (improvements made over prior years for
RR crops, i.e. stacked genes),
climate is always variable, application of herbicide/insecticide
timing is variable (before, during
and after harvest), not to mention soil conditions and timing of
planting, use of various maturity
groups, and as one of the publications mentioned, even consider
the slope of the field where
soybeans are grown.
It is very well known that the environment plays a significant
role in determining additional
stress levels that add a variable of complexity to growing any
type of crop. The objective of this
paper is to demonstrate the effects that occur with using a
glyphosate resistant soybean crops
with genetic engineering and using glyphosate on non-resistant
soybean crops with desirable
traits such as increasing yield, resistance to herbicide (in the

case of the transgenic soybean study
without adverse affects), and improve the nutritional value of
food. The outcome of these results
leads to the conclusive evidence that there is more complexity
as all the variables are difficult to
test at any one time for consistent results and more rigorous
scientific studies are needed.
17
CONCLUSION
Trials are typically conducted in different regions of a state and
cover different maturity groups
at different stages of growth. Soybean varieties are often sold
and studied within maturity
groups. In general, the longer the maturity period for a variety,
the higher the expected yields. In
the second soybean study it was difficult to compare maturity
groups to the first soybean study as
they used the vegetative or reproductive stages. It would have
been helpful to tie these together
to observe those stages within the maturity groups for
concentrations of mineral nutrients. It is
inappropriate to compare the yields harvested of a long season
variety in contrast to a short
season one as the first study also had overlapping maturity
groups.
In some of the states where these trials have been carried out
used a variety of tillage and
planting systems. The first soybean study used conventional
tillage when establishing their plot

and it was treated with a pre-emergent herbicide combination.
The study also had various soil
conditions, silty, clay, loam combination and fine-loamy,
mixed, superactive. In the second
study, the soil was transported from the Eskisehir region in
Central Anatolia, Turkey where Zn
deficiency is well documented.
In summary, there is not one “cookie cutter” transgenic seed
that will work for everyone in every
situation. After all, Mother Nature breeds seeds that are best
adapted to the environment. The
seeds have survived this long. Everything else is just a gamble.
It is extremely complicated to understand the biological
evolution and the diversity of life and
bring in manipulating a few genes in seeds. Seeds are life.
The heritable characteristics, which can be biochemical and
anatomical, largely determine what
capabilities an organism will have, how it will behave, and how
likely it is to survive and
reproduce. This is not well known with transgenic seeds.
The variation of organisms within a species increases the
chance of survival of the species, and
how the great diversity of species on Earth increases the chance
of survival of life in the event of
major global changes. How can any transgenic seeds do that?
This would mean that science can
predict the future and “make” seeds accordingly. Is this ideal of
Genetically Engineered Seeds?
As the tables and the content of information within them as
presented in this paper indicate that
the jury is still out. We just do not know the consequences of

what we have done so far. To
march forward without additional scientific data is simply not
science. The charts below
represent a simple summary.
Be ne fits of Trans ge nics
Crop/
Cultivar
Studies
Growth
Conditions
Germination Vigor Viability Persistence Longevity Less
tilling
Less
soil
erosion
Less
pesticide
use
Selective
Breeding/
Cultivar
just as good
Soybean1/
Unknown

Field Variable Variable Variable Variable Unknown Yes No
Yes
Soybean2/
Nova
Greenhouse Variable Variable Variable No Unknown N/A No
Yes
18
Ris ks of Trans ge nics
Crop/
Cultivar
Studies
More herbicide
/insecticide use
More weeds or weed
resistance
Unwanted gene flow Irreparable changes in species diversity
and
genetic diversity within a species
Soybean1/
Unknown
Yes Yes Undetermined Unknown
Soybean2/

Nova
Yes Yes Undetermined Unknown
Good discussion and conclusions! Generally, the “Conclusion”
section does not include tables.
Your references are a bit confusing. Most science journals do
not number their sources in
References or, more often, Literature Cited. When a format is
set for the Literature Cited,
remain true to the format. Your references look as though your
borrowed them from different
sources and kept their formats (which are not the same). But
you should determine which style
you will use for the paper and remain true to one format. Most
are listed in alphabetical order by
lead author last name – not numbered.
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20
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