GA-Responsive Dwarfing Gene Rht12 Affects the Developmental and Agronomic Traits in Common Bread Wheat

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DOI: 10.1371/journal.pone.0062285 · Source: PubMed
Abstract
Opportunities exist for replacing reduced height (Rht) genes Rht-B1b and Rht-D1b with alternative dwarfing genes, such as the gibberellin-responsive gene Rht12, for bread wheat improvement. However, a comprehensive understanding of the effects and mode of action of Rht12 is lacking. In the present study, the effects of Rht12 were characterized by analyzing its effects on seeding vigour, seedling roots, leaf and stem morphology, spike development and carbohydrate assimilation and distribution. This was carried out in the four genotypes of F2:3 lines derived from a cross between Ningchun45 and Karcagi (12) in two experiments of autumn sowing and spring sowing. Rht12 significantly decreased stem length (43%∼48% for peduncle) and leaf length (25%∼30% for flag leaf) while the thickness of the internode walls and width of the leaves were increased. Though the final plant stature was shortened (40%) by Rht12, the seedling vigour, especially coleoptile length and root traits at the seedling stage, were not affected adversely. Rht12 elongated the duration of the spike development phase, improved the proportion of spike dry weight at anthesis and significantly increased floret fertility (14%) in the autumn sowing experiment. However, Rht12 delayed anthesis date by around 5 days and even the dominant Vrn-B1 allele could not compensate this negative effect. Additionally, grain size was reduced with the ability to support spike development after anthesis decreased in Rht12 lines. Finally, grain yield was similar between the dwarf and tall lines in the autumn sowing experiment. Thus, Rht12 could substantially reduce plant height without altering seeding vigour and significantly increase spikelet fertility in the favourable autumn sowing environment. The successful utilization of Rht12 in breeding programs will require careful selection since it might delay ear emergence. Nonetheless, the potential exists for wheat improvement by using Rht12.

Figures

GA-Responsive Dwarfing Gene
Rht12
Affects the
Developmental and Agronomic Traits in Common Bread
Wheat
Liang Chen
1
, Andrew L. Phillips
2
, Anthony G. Condon
3
, Martin A. J. Parry
2
, Yin-Gang Hu
1,4
*
1State Key Laboratory of Crop Stress Biology for Arid Areas and College of Agronomy, Northwest A&F University, Yangling, Shaanxi, China, 2Department of Plant Biology
and Crop Science, Rothamsted Research, Harpenden, Herts, United Kingdom, 3CSIRO Plant Industry, Canberra, Australia, 4Institute of Water Saving Agriculture in Arid
Regions of China, Northwest A&F University, Yangling, Shaanxi, China
Abstract
Opportunities exist for replacing reduced height (Rht) genes Rht-B1b and Rht-D1b with alternative dwarfing genes, such as
the gibberellin-responsive gene Rht12, for bread wheat improvement. However, a comprehensive understanding of the
effects and mode of action of Rht12 is lacking. In the present study, the effects of Rht12 were characterized by analyzing its
effects on seeding vigour, seedling roots, leaf and stem morphology, spike development and carbohydrate assimilation and
distribution. This was carried out in the four genotypes of F
2:3
lines derived from a cross between Ningchun45 and Karcagi
(12) in two experiments of autumn sowing and spring sowing. Rht12 significantly decreased stem length (43%,48% for
peduncle) and leaf length (25%,30% for flag leaf) while the thickness of the internode walls and width of the leaves were
increased. Though the final plant stature was shortened (40%) by Rht12, the seedling vigour, especially coleoptile length
and root traits at the seedling stage, were not affected adversely. Rht12 elongated the duration of the spike development
phase, improved the proportion of spike dry weight at anthesis and significantly increased floret fertility (14%) in the
autumn sowing experiment. However, Rht12 delayed anthesis date by around 5 days and even the dominant Vrn-B1 allele
could not compensate this negative effect. Additionally, grain size was reduced with the ability to support spike
development after anthesis decreased in Rht12 lines. Finally, grain yield was similar between the dwarf and tall lines in the
autumn sowing experiment. Thus, Rht12 could substantially reduce plant height without altering seeding vigour and
significantly increase spikelet fertility in the favourable autumn sowing environment. The successful utilization of Rht12 in
breeding programs will require careful selection since it might delay ear emergence. Nonetheless, the potential exists for
wheat improvement by using Rht12.
Citation: Chen L, Phillips AL, Condon AG, Parry MAJ, Hu Y-G (2013) GA-Responsive Dwarfing Gene Rht12 Affects the Developmental and Agronomic Traits in
Common Bread Wheat. PLoS ONE 8(4): e62285. doi:10.1371/journal.pone.0062285
Editor: Miguel A. Blazquez, Instituto de Biologı
´a Molecular y Celular de Plantas, Spain
Received February 23, 2013; Accepted March 19, 2013; Published April 26, 2013
Copyright: ß2013 Chen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the sub-project of the 863 Program (2011AA100504, 2013AA102902) of the Ministry of Science and Technology, the key
project of Chinese Universities Scientific Fund, Northwest A&F University (ZD2012002) and the China 111 Project (B12007), P. R. China, as well as the ACIAR Project
(CIM/2005/111) of Australia. ALP and MAJP are supported by the Biotechnology and Biological Sciences Research Council (BBSRC) of the UK under the 20:20
WheatHInstitute Strategic Programme. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: huyingang@nwsuaf.edu.cn
Introduction
The introduction of semi-dwarfing genes into wheat was a major
event in breeding high yielding varieties during the Green
Revolution [1]. The greater grain yields were associated with
improved lodging resistance and the resulting ability to tolerate
higher rates of chemical fertilizers [2], and also with increased
harvest index [3]. The gibberellin acid (GA) insensitive semi-
dwarfing genes Rht-B1b (Rht1) and Rht-D1b (Rht2) are widely used
to reduce plant height and increase grain yield in wheat breeding
programs. Rht-B1b and Rht-D1b reduce stem internode length and
therefore overall plant height by decreasing the sensitivity of
vegetative and reproductive tissues to endogenous GA [4,5].
However, reduced stature also contributes to reduced seedling
vigour and coleoptile length and may reduce crop water-use
efficiency [6–9], and performance in unfavourable environments
[5,10,11]. Longer coleoptiles may permit crops to be sown at the
optimal time to increase biomass and yield [12] while deep sowing
of short coleoptile Rht-B1b and Rht-D1b alleles commonly results in
fewer, later emerging seedlings with low relative growth rates, leaf
area and biomass [13], and ultimately lower final biomass, fewer
spikes and yield [13,14]. But, in irrigated and fertilised environ-
ments, the height reduction associated with Rht-B1b and Rht-D1b
may not be sufficient. Excessive height and severe lodging can
occur in semi-dwarf varieties carrying these alleles [15,16].
Further, genetic reductions in plant height of the Rht-B1b+Rht-
D1b doubled-dwarfs usually produce much less biomass and result
in slow development of seedling leaf area though they significantly
reduced lodging and have a greater harvest index [7,10,17]. A
range of dwarfing genes is needed at our disposal to achieve the
appropriate height reduction for different environments [7]. The
modest height-reducing gene Rht8 may be suitable to reduce final
plant height without compromising early plant growth. Rht8 has
been used in breeding programs in different environments [18,19]
as it has no effect on coleoptile length or seedling vigour [5,20] and
PLOS ONE | www.plosone.org 1 April 2013 | Volume 8 | Issue 4 | e62285
there is potential for developing shorter Rht8+Rht-B1b/D1b sesqui-
dwarfs with reduced lodging susceptibility and without limiting
crop establishment [21]. Preliminary evidence indicates the
potential of several other GA-responsive dwarfing genes, including
Rht4,Rht5,Rht9,Rht12,Rht13 and Rht14, to reduce plant height
without affecting seedling vigour [22]. It is currently not clear
whether these alternative dwarfing genes can be used to improve
wheat yield and lodging tolerance [23].
The life cycle of wheat can be divided into phases based on the
main organs being differentiated [24]. The pre-anthesis late
reproductive phase (from terminal spikelet initiation to anthesis) of
stem elongation is particularly important for yield [25,26] because
the number of fertile florets at anthesis will be determined during
this phase. Wheat yield can be improved through increasing the
duration of the late reproductive phase by manipulation of spike
developmental rates [27,28]. The developmental phases prior to
anthesis may be sensitive to the environment (photoperiod or
temperature) that they encounter or the concentrations of different
endogenous hormones (gibberellin, auxin or cytokinin) associated
with development [29–32]. Indeed, several dwarfing genes are
associated with lesions in GA biosynthesis or signalling [33,34].
The possibility exists that the duration of spike development
phases can be optimised using dwarfing genes, as done with genes
affecting photoperiod or vernalization requirement [26,35,36].
Previous reports have shown that GA-insensitive dwarfing genes
have no effect on the initiation of leaf and spikelet primordia at the
shoot apex nor on the number of leaves and internodes. The
primary effect of these dwarf genes on growth is to reduce the rate
of leaf expansion, stem elongation and vegetative dry matter
accumulation [37,38]. However, the effects of GA-responsive
dwarfing genes on plant growth and spike development have not
been investigated and the potential of optimising the duration of
late reproductive phase to improve ear fertility using GA-
responsive dwarfing genes is unknown. Additionally, winter wheat
requires several weeks at low temperature to flower. This process,
vernalization, is controlled by three major genes, Vrn-A1,Vrn-B1
and Vrn-D1 which locate to the long arm of chromosomes 5A, 5B,
and 5D, respectively [39,40]. The spring habit alleles at these loci
are dominant while recessive alleles at all three loci determine
winter growth habit [41,42]. The dominant Vrn-A1 allele provides
complete insensitivity to vernalization (achieving the double ridge
stage without low temperature) whereas the dominant Vrn-B1 and
Vrn-D1 alleles each provide a reduced vernalization requirement
compared to winter alleles [43]. Thus, these genes can be used to
modify the flowering time in wheat.
Rht12, a dominant GA-responsive dwarfing gene from the
gamma ray induced mutant Karcagi 522M7K of winter wheat
[44], has been shown to be located distally in the long arm of
chromosome 5A, approximately 5.4 cM from locus Xwms291
[45,46]. Rht12 has been described as having few negative effects on
yield components and, with a reduction of height by around 46%
[44], it has a stronger effect on height than either Rht-B1b or Rht-
D1b. Worland et al. [2] reported that Rht12 reduced height
without altering ear size and significantly increased spikelet
fertility, but this was always accompanied by delayed ear
emergence. There was no significant difference in early root
growth and root architecture between Rht12 dwarf lines and the
control lines, but the effects on total root length were still unclear
due to the different background or experimental methodology
[47]. Recently, it was found that Rht12 increased grain yield,
harvest index and lodging resistance while reducing grain weight
[21]. However, it is still difficult to evaluate the effects of Rht12
comprehensively, which would include effects on vegetative
organs, spike development or other agronomic traits. Moreover,
Rht12 has not been used in wheat breeding, and its full potential
remains uncertain.
The objectives of this work were to analyse the effect of Rht12
on leaf and stem morphology, phenological development up to
anthesis and on agronomic traits and yield components. Due to
the late ear emergence associated with the Rht12 allele, the
dominant Vrn-B1 gene was introduced from a tall, spring-habit
parent to analyse the effect of Rht12 on spike development.
Materials and Methods
General Description
The experiments were carried out during the two growing
seasons of 2010–2011 and 2011–2012 in the experimental field of
the Institute of Water Saving Agriculture in Arid Regions of
China, Northwest A&F University, Yangling, Shaanxi, China
(34u179N, 108u049E, at an elevation of 506 m). To avoid water
stress, supplemental irrigation was provided as needed. Weeds
were manually removed where necessary, and fungicides and
insecticides were applied to prevent diseases and insect damage.
Weather data were recorded at an automated weather station at
the site.
Plant Material
A cross was made using Ningchun 45 as the female and Karcagi
(12) as pollen donor in May, 2009. Wheat seeds of Karcagi (12)
(Triticum aestivum L.), a gamma ray-induced mutant carrying the
dominant GA-responsive dwarfing gene Rht12 and strong winter
habit as detected with the recessive loci for all three Vrn-1 genes,
were generously provided by the Australian Winter Cereal
Collection. Ningchun 45 (Triticum aestivum L.), is a tall Chinese
spring wheat cultivar widely used in the semi-arid areas of
Northwest Spring Wheat Region of China, which carries the
dominant vernalization gene Vrn-B1 and lacks any known
dwarfing genes as detected by molecular markers.
The F
2
population was sown as spaced plants in the field in
October, 2010. The individuals of the F
2
population were
numbered, the plant height and other agronomic traits of each
individual were recorded, and the presence or absence of the loci
for the dwarfing gene Rht12 and vernalization gene Vrn-B1 in each
numbered individual was determined using the corresponding
molecular markers (see below for details). The individuals with the
four kinds of homozygous genotypes of Rht12Rht12Vrn-B1Vrn-B1
(abbreviated as RRBB), Rht12Rht12vrn-B1vrn-B1 (RRbb),
rht12rht12Vrn-B1Vrn-B1 (rrBB) and rht12rht12vrn-B1vrn-B1 (rrbb)
were then selected and used to develop the F
2:3
lines for further
analysis.
The two parents and 57 F
2:3
homozygous lines were used in
experiments to evaluate the effects of dwarfing gene Rht12 in the
2011–2012 growing season. There were two sowing dates:
October 6, 2011 (Autumn Sowing, AS) and February 6, 2012
(Spring Sowing, SS). Among those F
2:3
homozygous lines, 15, 13,
17 and 12 lines had the genotypes Rht12Rht12Vrn-B1Vrn-B1,
Rht12Rht12vrn-B1vrn-B1,rht12rht12Vrn-B1Vrn-B1, and
rht12rht12vrn-B1vrn-B1, respectively, as identified in the F
2
population. The lines and parents were sown in plots of four
rows 2 m long and 25 cm apart, with seeds spaced 5 cm apart
within rows. The parents and the 28 dwarf and 29 tall F
2:3
lines
were randomly arranged to avoid competitive effects with two
replications.
Genotyping of Rht12 and Vrn-B1
The molecular markers WMS291 (Xgwm291) and Intr1-B-F/
Intr1-B-R3/Intr1-B-R4 were used to determine the genotypes of
Rht12 Affects the Agronomic Traits in Wheat
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individuals for the presence or absence of the dwarfing gene Rht12
[46] and the vernalization gene Vrn-B1 [48] in the individual
plants of the F
2
population, respectively.
Genomic DNA from young leaves was isolated from the two
parents and each F
2
individual using the CTAB method [49].
DNA concentrations were measured using a spectrophotometer
and normalized to 50 ng/mL. The PCR protocols for the three
markers were as described [46,48]. The PCR profiles were: an
initial denaturation at 95uC for 2 min was followed by 40 cycles at
94uC for 20 s, 55uC, 63uC and 58uC (for the three markers,
respectively) for 30 s, 72uC for 90 s and a final extension of 72uC
for 5 min. PCR products for SSR marker Xgwm291 were
visualized on a 8% polyacrylamide gel (19:1 of Acrylamide to
bis ratio) in 16TBE buffer at 250V for 1.5 h, then stained by silver
buffer using a modified procedure described previously [50]. PCR
products for Intr1-B-F and Intr1-B-R3 or R4 were separated on
1% agarose gels with 16TAE buffer at 115 V for 40 min, stained
in an ethidium bromide buffer for 15 min, and then visualized by
Gel Doc XR (BioRad Laboratories, Inc.).
Coleoptile Length and Seedling Root Traits
Coleoptile length from the seed to the tip of the coleoptile was
measured with a ruler after germination in a darkened growth
chamber at 20uC after 200uCd using the method described [5,51].
Seedling root growth characteristics were assessed using a ‘Cigar’
method as follows. Five grains of each line were arranged in a line
(3 cm from the upper edge) on a 32629 cm germination paper
(Anchor, USA), and then rolled as a cigar with the seeds at the top.
Then the cigar was stood vertically in a tank (50635630 cm)
containing Hoagland solution (10 cm depth) at 20uC until
200uCd. The number of seminal roots, maximum root length,
and total root length were measured. Root and shoot dry mass
were also investigated after drying at 60uC for 72 h. Seedling
vigour was evaluated as the area of the first two leaves, coleoptile
length and the seedling root length.
Spike Development and Fertility
Spike differentiation was investigated on three randomly
selected plants from each field plot. Beginning from the three
leaf stage (Z12), plants were sampled every 3 days and the main
shoot was dissected to determine the timing of the double ridge
formation (DR) and the terminal spikelet initiation (TS) in the
apical meristem, as described by [52] using a digital Stereo
Microscope (Nikon, SMZ1500). The timing of other stages of spike
differentiation was also recorded successively. Pictures were taken
using a digital camera linked to the microscope. The timing of
heading (Z55) and anthesis (Z65) was visually determined when
50% of the plants per plot had reached these stages.
At anthesis, five plants in the central row of each plot were
harvested and the number of fertile florets in the main shoot spike
was counted before drying. Florets were considered fertile when
the stigmatic branches were spread wide, with either pollen grains
present on them or with green anthers [53].
Carbohydrate Assimilation and Distribution
After investigation of fertility, the five plants harvested at
anthesis were partitioned to investigate the dry weight of the main
shoot (PDM), dry weight of the main shoot spike (SDM), dry
weight of tiller spikes (SDT) and the total dry matter excluding
roots (TDP). The partitioning of total dry matter to reproductive
organs (dry weight of total spike including main shoot and tiller
spikes) and the spike to stem ratio in main shoots were then
calculated. The number of green leaves on the main shoot was
counted from the flag leaf down and included fractions of leaves
that were partially yellow [54].
Also from these samples, the dry weights of flag leaf, the second
leaf, the third leaf, the peduncle, the fifth internode, the fourth
internode, the basal three internodes on the main shoot stems and
leaves including leaf sheaths were determined. Samples were also
collected at the 14th, 18th, 22nd and 30th day after anthesis as
described above to analyse the dynamic changes of the dry weight
of spike, stem and leaf of the main shoot between different
genotypes. All samples were oven-dried at 60uC for 72 h and
weighed separately [26,55].
Plant Height, Leaf and Internode Character
Plant height was determined at maturity as the distance from
the soil surface to the top of the ear (awns excluded) of ten plants
for each plot. Seedling height was measured as the distance from
the soil surface to the ligule of the last fully emerged leaf of ten
plants for each plot and was used to analyze the growth rate of
different lines (capacity to produce biomass) weekly from Z12 until
heading. At the three leaf stage (Z13) [54], five plants per plot were
randomly selected and tagged, and the number of leaves emerging
on the main shoot was noted two or three times a week until
anthesis [56]. The length and width of each leaf were measured
when the leaf was fully elongated.
All of the genotypes have six internodes, the first internode of
more than 1 cm was defined as internode1, although internodes
shorter than 1 cm were occasionally observed in some individuals
[57]. Subsequent internodes up the stem were numbered as 2, 3, 4,
5, and 6 (peduncle), respectively. The lengths of internodes (mm)
were measured from the mid-point of their subtending nodes.
Stem diameter (mm) was measured at the middle of each
internode using digital calipers (TESA Etalon). Internodes were
then cut at their centre point and digital calipers were used to
measure the stem wall thickness (mm). Two measurements of stem
wall thickness were taken on opposite sides of the stem from which
a mean value was calculated [57]. Characters of each internode
were measured when they fully developed.
Lodging was scored at maturity from the fraction of the area
affected and the severity of lodging in those areas noted, on a scale
of 0 for a standing crop to 90 for a crop flat on the ground [54].
Yield Components and Yield
The main shoot ears of ten plants of each line at maturity were
measured for assessment of spike length, spikelets per spike, grains
per spike, total grains in the uppermost three spikelets and in the
bottom three spikelets of the spike. The fertile shoots per plant
were also measured in the same plants.
Due to the frequent sampling prior to harvest and the limited
number of plants in each plot (seeds from individual F
2
plants were
limited), only 25 to 40 plants remained in each plot at harvest.
Plants were hand-cut at ground level and the number of plants
harvested in each plot was recorded. The total above-ground dry
biomass of each line was determined before threshing, and then
the average biomass per plant, average yield per plant, harvest
index (calculated as the ratio of grain to total above-ground
biomass) and 1000-grain weight were determined.
Data Analysis
For each parameter measured, the mean value for each line (15
RRBB, 13 RRbb, 17 rrBB and 12 rrbb lines) was calculated and
statistical evaluation of the data was carried out by ANOVA
analysis with multiple comparisons (LSD test at the 0.05 level)
using the statistical package SPSS18.0. Association analysis was
also made with SPSS18.0. Whenever thermal time was used to
Rht12 Affects the Agronomic Traits in Wheat
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measure developmental progress, 0uC was chosen as base
temperature.
Results
The Pleiotropic Effects of Rht12 in F
2
Progeny
Among the F
2
segregating population, 228 plants were
genotyped using the SSR markers linked with Rht12 and Vrn-B1,
respectively. Individuals homozygous at those two loci were
selected and classified into four groups. Thus the F
2
individuals
composing 15 with RRBB (Rht12Rht12Vrn-B1Vrn-B1), 13 with
RRbb (Rht12Rht12vrn-B1vrn-B1), 17 with rrBB (rht12rht12Vrn-
B1Vrn-B1) and 12 with rrbb (rht12rht12vrn-B1vrn-B1) were identi-
fied.
All progenies segregated into two distinct height classes, due to
the strong effects of Rht12, and the homozygous recessive rht12
progeny could be clearly identified by their tall stature. The
primary and pleiotropic effects of Rht12 are shown in Table 1.
Plant height was significantly decreased from an average of
106 cm in the rr genotypes to 76 cm in the RR genotypes;
a reduction of 28% in plant height was thus observed. The
individuals with heterozygous genotype (Rr) were approximately
4 cm taller than RR (data not shown). The spike length, number
of spikelets per spike and 1000-grain weight were also reduced
after introducing the dwarfing gene Rht12. Whereas, the grain
number per spike, the effective spike number, and the fertility of
the top three spikelets were increased in the homozygous dwarf
plants (Table 1). Moreover, the ear emergence time and flowering
time of the homozygous dwarf individuals were significantly
delayed, for flowering by an average of 4 days (Table 1), but with
some plants by as much as 10 days compared with the tall plants.
Seedling Vigour and Seedling Root Characters
In the F
2:3
lines, no significant difference was observed for the
length or width of the first leaf between the tall and dwarf groups,
while the length of the second leaf of the dwarf group was shorter
than that of the tall group (with a 1.6 cm reduction) (Table 2). The
total area of the first two leaves in the dwarf group were less than
that of the tall group by 1.1 cm
2
(9%) and 0.55 cm
2
(11%) in the
AS and SS experiments, respectively (Table 2). Although the area
of individual leaves at seedling stage of the dwarf group was
slightly reduced, the emergence of new leaves was advanced 3 days
earlier than the tall group (data not shown), which may benefit
seedling growth. However, genotype rrBB achieved the largest leaf
area while RRbb had the smallest leaf area in both the AS and SS
experiments (Table 2), indicating that the combination of RR and
bb may affect seedling leaf growth. Additionally, no significant
difference in coleoptile length was observed between the dwarf and
tall groups (Table 3), suggesting that Rht12 may confer a semi-
dwarf height in wheat, while allowing no reduction in coleoptile
length.
Based on the ‘Cigar’ test for the root characters at the seedling
stage, the total number of seminal roots was significantly decreased
in the dwarf group (2 roots fewer) (Fig. 1), while the total root
length in the dwarf group was reduced by 6 cm (9%) compared to
that of the tall group (Table 3). The maximum root length of the
dwarf group was 3 cm (15%) longer than that of the tall group,
which may be beneficial for water acquisition in deeper soil in dry
environments, assuming this seedling trait is expressed in adult
plants.
The difference between the total root length of the tall and
dwarf genotypes was mainly determined by the difference in total
number of roots and not by the root elongation rate. The root dry
mass was decreased by 0.7 mg (11%) in the dwarf lines. While, the
dry mass ratio of root to shoot of the dwarf lines (0.49) was
significantly greater than that of the tall lines (0.44). It suggested
that Rht12 did not greatly affect the seedling root growth although
it significantly reduced the total number of roots. There was no
significant difference in root architecture between BB and bb
genotypes.
Spike Development, Spike Dry Weight at Anthesis and
Floret Fertility
Spike development was significantly delayed and the duration of
the spike development phase was elongated in the Rht12 dwarf
lines compared to that of tall lines under both AS and SS
experiments (Table 4). In the AS experiment, spike development
of the dwarf lines with Rht12 either with Vrn-B1 or vrn-B1 was
delayed by 16 days to reach the double ridge stage, which meant
that an additional thermal time of 45uCd was needed compared to
the tall lines with the winter growth habit. The effect of the
dominant vernalization gene Vrn-B1 was masked in the dwarf
lines, while in the tall lines without Rht12, the lines with Vrn-B1
reached double ridge stage about 60 days (30uCd) earlier than
those with vrn-B1. However, the anthesis date of the dwarf lines
was about 5 days (110uCd) later than that of the tall lines (Table 4
and Fig. 2). In the SS experiment, the RRBB lines required an
additional 9 days (140uCd) to reach the double ridge stage
compared to the rrBB lines and needed a further 6 days (135uCd)
Table 1. Effects of Rht12 on plant height, spike traits and yield components in the F
2
progeny.
Genotype/
variety
Plant height
(cm)
Spike length
(cm)
No. of
spikelets
spike
21
No. of grains
in the
top three
spikelets
No. of grains
in the
basal three
spikelets
Grain number
spike
21
1000-grain
weight (g)
Effective
Tiller
number
SW-AN* (uC
d)
RRBB 75.566.97b 14.260.89b 20.561.01ab 5.560.23a 4.260.35a 42.464.65a 37.663.41b 12.462.94a 216.0 (1585)a
RRbb 76.467.43b 13.860.90b 19.961.12b 5.460.25a 3.960.40a 41.664.87a 36.362.31b 12.063.21a 216.3 (1592)a
rrBB 105.264.62a 15.160.82a 21.160.74a 4.460.21b 4.260.29a 39.664.29b 45.663.75a 8.762.68b 212.0(1479)b
Rrbb 107.264.61a 14.960.76ab 20.661.04ab 4.160.14b 4.360.24a 38.563.85b 44.563.29a 8.662.56b 212.5 (1500)b
Karcagi 73.062.48 13.760.66 20.060.77 6.260.15 3.960.23 43.663.29 33.961.39 12.761.43 221.0 (1633)
Nchun45 103.061.94 15.460.58 21.660.70 5.060.19 4.960.19 44.564.11 43.262.40 9.061.16 211.7 (1471)
*Data are the duration days (d) with calculated thermal time (uC d) in parenthesis; SW: sowing date; AN: anthesis date. Karcagi and Nchun45 represent the two parents
Karcagi (12) and Ningchun45, respectively. Data are means 6SD (standard deviation) of each genotype. Data of the two parents were not considered in the statistical
significance testing. Different letters within columns indicate statistically significant differences (P,0.05).
doi:10.1371/journal.pone.0062285.t001
Rht12 Affects the Agronomic Traits in Wheat
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Table 2. Seedling vigour of different groups of the F
2:3
lines in the autumn-sown (AS) and spring-sown (SS) experiments.
Experiment Genotype/variety
Length of the 1
st
leaf
(cm)
Width of the 1
st
leaf
(cm)
Area of the 1
st
leaf
(cm
2
)
Length of the 2
nd
leaf
(cm)
Width of the 2
nd
leaf
(cm)
Area of the 2
nd
leaf
(cm
2
)
Total area of two leaves
(cm
2
)
AS RRBB 10.160.31a 0.560.04a 4.060.58a 13.060.70b 0.660.04a 6.260.81b 10.261.36b
RRbb 10.060.22a 0.560.04a 4.060.47a 12.860.72b 0.660.04a 6.160.83b 10.161.23b
rrBB 10.560.54a 0.560.04a 4.260.66a 15.061.44 a 0.660.04a 7.261.24a 11.461.61a
rrbb 10.260.52a 0.560.03a 4.160.59a 14.461.32a 0.660.03a 6.961.18a 11.061.32ab
Karcagi 9.060.19 0.560.04 3.660.43 11.060.35 0.660.04 5.360.54 8.960.89
Nchun45 12.360.39 0.560.04 4.960.54 15.960.70 0.660.04 7.660.77 12.561.36
SS RRBB 8.560.85a 0.360.04a 2.060.97a 10.460.58ab 0.360.04a 2.560.89a 4.560.93a
RRbb 8.260.87a 0.360.04a 2.061.05a 9.960. 47b 0.360.04a 2.460.72a 4.460.90a
rrBB 9.060.82a 0.360.04a 2.260.91a 11.661.15a 0.360.04a 2.861.28a 5.061.57a
rrbb 8.760.80a 0.360.03a 2.16090a 11.360.87a 0.360.03a 2.760.94a 4.861.14a
Karcagi 7.160.31 0.360.04 1.760.43 8.360.35 0.360.04 2.060.58 3.760.89
Nchun45 9.560.35 0.360.04 2.360.58 12.260.58 0.460.04 3.960.77 6.261.01
All data are means 6SD of each genotype. Data of the two parents were not considered in the statistical significance testing. Different letters within columns indicate statistically significant differences (P,0.05).
doi:10.1371/journal.pone.0062285.t002
Table 3. Effects of Rht12 on coleoptile length and root traits of different groups of the F
2:3
lines at the seedling stage.
Genotype Coleoptile length (cm)
Number of seminal
roots
Maximum root length*
(cm) Total root length (cm) Root dry mass (mg) Shoot dry mass (mg)
Root to shoot Ratio of dry
mass
RRBB 4.8760.56a 3.060.08b 23.2561.90a 57.4663.29b 5.5760.35b 11.3261.20b 0.4960.01a
RRbb 4.9660.52a 3.060.14b 22.9761.62a 57.1462.74b 5.5160.29b 11.1561.26b 0.4960.02a
rrBB 5.2060.49a 5.160.16a 20.4261.44b 63.3964.33a 6.2860.37a 14.2961.81a 0.4460.02b
rrbb 5.1360.60a 5.060.21a 20.6561.52b 63.2063.15 a 6.2660.24a 14.3361.66a 0.4460.01b
Karcagi(12) 4.160.22 3.060.08 22.4760.93 49.6762.52 4.6460.23 10.4360.97 0.4460.01
Nchun45 6.260.29 5.360.08 24.2861.98 68.4264.80 7.2360.50 14.5862.05 0.5060.03
*Mean value of the longest root length in each set of lines. Data are means 6SD of each genotype. Data of the two parents were not considered in the statistical significance testing. Different letters within columns indicate
statistically significant differences (P,0.05).
doi:10.1371/journal.pone.0062285.t003
Rht12 Affects the Agronomic Traits in Wheat
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to reach anthesis compared to the tall lines. Whereas, rrBB lines
reached double ridge stage about 5 days (60uCd) faster than rrbb
lines (Table 4).
In both AS and SS experiments, there was no significant
difference between RRBB and RRbb in spike development,
suggesting that the dwarfing Rht12 allele might have an epistatic
effect on Vrn-B1. The tall spring lines (rrBB) developed faster than
the tall winter lines (rrbb) before terminal spikelet stage while they
had no difference on flowing time (Table 4). This indicated that
genotypes having dominant Vrn-B1 needed less time to undergo
vernalization in tall lines. However, the effects of Vrn-B1 on plant
development were not strong enough to promote early flowering
in either tall or dwarf genotypes.
In the AS experiment, spike lengths in RRBB and RRbb groups
were significantly decreased (7%) compared with rrBB, while there
was no significant difference observed with rrbb (Table 5). The
number of spikelets per spike in RRbb was significantly reduced
(3%) compared to that in rrBB and rrbb groups, while there was
no significant difference between RRBB and rrBB/rrbb. The rrBB
lines showed the largest number of spikelets per spike while RRbb
had the smallest (Table 5). The dominant Vrn-B1 allele likely plays
a major role in determining spikelet number in this population and
the combination of rr and BB produces more spikelets per spike.
This phenomenon was also observed in the SS experiment. But,
total florets per spike of RRBB, RRbb and rrbb were significantly
decreased compared with that of rrBB in both experiments. This
indicates that the BB allele apparently has an effect on initiation of
florets in tall plants.
In the AS experiment, Rht12 significantly increased floret
fertility (14%) and produced more fertile florets (10%) compared
with the tall genotypes (Table 5). Correlation analysis showed that
the elongated duration of the late reproduction phase pre-anthesis
(thermal time accumulated) associated with Rht12 was highly
correlated with the increased number of fertile florets, and the
duration of TS-AN explained almost 87% (R
2
= 0.865) of variation
of the ear fertility. However, possibly due to slow development of
the vegetative phase (SW-DR) and early reproductive phase (DR-
TS), which shortened the period of time available under
favourable conditions prior to flowering (TS-AN), the dwarf lines
produced fewer (23%) fertile florets per spike than that of the tall
lines in the SS experiment. The decreased duration of late
reproduction phase pre-anthesis (thermal time accumulated) was
highly correlated with the reduced number of fertile florets
(R
2
= 0.908) in the SS experiment. There were more fertile florets
in the top three spikelets of dwarf plants in both experiments
(Table 5), suggesting that Rht12 had a stronger production
Figure 1. Seminal root morphology of the dwarf and tall lines at the seedling stage determined by the Cigar method. Panel A shows
the dwarf lines and B shows the tall lines. The tall lines always have two more seminal roots than the dwarf lines (P,0.05).
doi:10.1371/journal.pone.0062285.g001
Rht12 Affects the Agronomic Traits in Wheat
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potential in the top spikelets. There was no significant difference
between BB and bb in floret fertility.
Dry matter partitioning to the spikes during the late re-
production phase is particularly important for yield because during
this phase the number of fertile florets at anthesis is determined
[26]. In the AS experiment, the dry weights of the main shoot
spikes at anthesis were similar among the four genotypes (Table 6).
Whereas, the dry weight of the tiller spikes per plant in dwarf
groups were higher (9%) than that of the tall groups, which might
result from the increased number of tiller spikes in dwarf plants
rather than bigger tiller spikes. Because of the reduction in plant
stature, the dry weight of the main shoot (SDM) and the dry
weight of the above-ground biomass (TDP) of the dwarf lines at
anthesis were significantly decreased. While, the ratios of main
shoot spike dry weight (SDM) to main shoot dry weight (PDM)
and the total spike dry weight (SDM+SDT) to plant dry weight
(TDP) of the dwarf lines was significantly increased compared to
that of the tall lines (Table 6). This indicates that Rht12 could
increase the partitioning of dry matter to spikes during the late
reproductive phase, potentially advantageous in achieving a greater
availability of assimilates to support floret survival in developing
spikes and a greater mass per carpel at anthesis, resulting in the
greater production of fertile florets. But, in the SS experiment,
SDM, SDT, and SDM/PDM of the dwarf genotypes at anthesis
were all significantly lower than that of the tall genotypes, while
(SDM+SDT)/TDP was not different between the dwarf and tall
groups, and a fertility reduction was observed in the dwarf plants.
It seemed that the environment effects of sowing date were greater
on dwarf lines than on tall lines, and the differences between the
two sowing date experiments were more evident in dwarf lines
though all lines produced less total dry matter in the SS
experiment than in the AS experiment.
Furthermore, in the AS experiment, spikelet dry weight and
number of fertile florets per spikelet of dwarf lines increased by,
respectively, 2% and 13% compared with tall lines. Conversely, in
the SS experiment, spikelet dry weight and number of fertile florets
per spikelet of tall lines were larger than that of dwarf lines by 28%
and 27%, respectively (Table 5 and Table 6). Despite these
contrasting responses, in both experiments spikelet dry weight was
highly correlated with the number of fertile florets per spikelet at
anthesis with R
2
of 0.81 and 0.99 respectively, in the AS and SS
experiment.
Figure 2. Spike development of the tall (rrBB, A1–A3) and dwarf (RRBB, B1–B3) genotypes in the AS experiment. A1 and B1 show spike
development at the early double ridge stage of tall genotypes and at metaphase of single ridge stage of the dwarf genotypes at 630uCd (75d after
sowing), respectively; A2 and B2 show spike development at terminal spikelet initiation stage of the tall genotypes and at anaphase of double ridge
stage of the dwarf genotypes at 770uCd (165d after sowing), respectively; A3 and B3 show the floret morphology at yellow anther stage/anthesis of
the tall genotypes and at green anther stage of the dwarf genotypes at 1430uCd (209d after sowing), respectively. Scale bars = 100 mm.
doi:10.1371/journal.pone.0062285.g002
Rht12 Affects the Agronomic Traits in Wheat
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Plant Height and Associated Traits
Plant height was greatly reduced by the Rht12 dwarfing gene. In
the AS experiment, the plant height of dwarf lines carrying Rht12
was reduced by 45 cm (37%) through reducing the length of
internodes, and the peduncle length was reduced by 20 cm (43%)
in dwarf lines compared with that of the tall lines (Figs 3A, 3B and
Table S1). In the SS experiment, plant height was reduced by
50 cm (40%) in the dwarf lines compared with that of the tall lines
with the reduction of peduncle length by 23 cm (48%). The
pattern of reduced internode length compared with the tall
genotypes was thus similar in both the AS and SS experiments
(Fig. 3C and Table S1).
The culm elongated faster in tall lines than in dwarf lines from
seedling stage (Fig. 4), with the tall lines reaching jointing stage
earlier than the dwarf lines and producing longer internodes and
more biomass; this difference was sustained to maturity in both
experiments. The shortest individual internode was more often
observed in RRbb lines in both AS and SS experiments. However,
there was no significant difference in the length of internodes
between BB and bb lines (Table S1). Although no significant
difference was observed in diameter of the internodes between
dwarf and tall lines, the thickness of the internode walls of the
dwarf lines was significantly greater than that of tall lines in both
experiments (Table S2). In particular, the wall thickness of the first
Table 4. Duration days (d) and thermal time (uC d) of different pre-anthesis developmental phases of different groups of the F
2:3
lines in the autumn-sown (AS) and spring-sown (SS) experiments.
Experiment Genotype/variety SW-AN SW-DR DR-TS TS-AN
Total leaf
number
AS RRBB 214.0(1555.8)a 153.0(702.2)a 24.0(160.1)a 37.0(693.5)a 14.1a
RRbb 215.5(1584.6)a 154.0(705.0)a 24.5(168.3)a 37.0(711.3)a 14.2a
rrBB 209.0(1437.7)b 73.0(629.0)b 94.0(144.3)b 42.0(664.4)b 12.4c
rrbb 210.5(1468.4)b 137.0(658.2)d 33.0(136.2)a 40.5(674.0)b 13.6b
Karcagi 220.0(1661.6) 162.0(747.1) 22.0(194.0) 36.0(720.5) 16.0
Nchun45 210.0(1458.9) 65.0(615.7) 102.0(156.1) 48.0(687.1) 11.0
SS RRBB 114.0(1367.5)a 74.0(566.0)a 12.0(217.2)a 28.0(543.3)b 11.0a
RRbb 115.0(1385.1)a 75.0(582.1)a 12.0(234.0)a 28.0(566.0)b 10.9a
rrBB 108.0(1231.6)b 65.0(428.0)c 11.0(184.4)b 32.0(619.2)a 9.2c
rrbb 109.0(1266.6)b 69.0(485.3)b 10.0(181.0)b 30.0(600.3)a 10.1b
Karcagi 121.0(1471.5) 79.0(650.4) 13.0(249.0) 29.0(572.1) 12.0
Nchun45 106.0(1192.8) 64.0(413.5) 11.0(191.2) 31.0(588.1) 9.0
Data are the duration days (d) with calculated thermal time (uC d) in parenthesis; SW: sowing date, DR: double ridge formation date, TS: terminal spikelet initiation date,
AN: anthesis date. All data are means of each genotype. Statistical analysis was carried out using the thermal time. Data of the two parents were not considered in the
statistical significance testing. Different letters within columns indicate statistically significant differences (P,0.05).
doi:10.1371/journal.pone.0062285.t004
Table 5. Effects of Rht12 on the spike characters and fertility of different groups of the F
2:3
lines in the autumn-sown (AS) and
spring-sown (SS) experiments.
Experiment
Genotype/
variety
Spike length
(cm)
Spikelets
spike
21
Florets initiated
spike
21
Fertile florets
spike
21
Fertile florets in
the top three
spikelet Fertility*
AS RRBB 13.661.05b 20.260.89ab 197.0616.42b 48.863.60a 6.160.19a 0.2560.01a
RRbb 13.560.71b 19.960.79b 194.2614.78b 49.363.52a 6.360.32a 0.2560.01a
rrBB 14.660.99a 20.761.15a 202.9617.03a 45.263.77b 4.260.33b 0.2260.01b
rrbb 14.1a60.73a 20.461.07a 198.6615.28b 44.164.02b 4.360.17b 0.2260.01b
Karcagi 12.460.54 18.760.66 169.9613.71 46.763.82 7.360.19 0.2760.01
Nchun45 14.761.01 21.561.39 204.5618.05 50.164.76 5.260.08 0.2460.01
SS RRBB 14.460.97ab 20.861.20b 201.8619.83c 39.862.01b 4.860.12a 0.2060.01b
RRbb 14.061.19b 20.461.26b 198.5616.87c 37.761.98b 4.460.18ab 0.1960.01b
rrBB 15.360.78a 21.661.11a 215.0620.86a 51.062.60a 4.160.08b 0.2460.02a
rrbb 15.060.83a 21.461.04a 210.0618.46b 49.961.97a 4.360.10ab 0.2460.01a
Karcagi 12.760.46 19.260.62 181.3615.57 35.361.28 4.360.08 0.1960.01
Nchun45 15.360.77 21.961.32 220.2616.46 55.463.37 4.160.19 0.2560.02
*Fertility is estimated as the ratio of fertility florets to total floret per spike of the main shoot spike. All data are means 6SD of each genotype. Data of the two parents
were not considered in the statistical significance testing. Different letters within columns indicate statistically significant differences (P,0.05).
doi:10.1371/journal.pone.0062285.t005
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and second internodes of the dwarf lines was increased by
0.25 mm (32%) and 0.22 mm (34%) in the AS experiment and
0.26 mm (28%) and 0.25 mm (31%) in the SS experiment,
respectively. These shorter and thicker internodes might confer
a greater resistance to lodging in the dwarf plants [57]. Due to the
occurrence of storms at anthesis stage and at the middle grain
filling stage, lodging occurred twice in the AS experiment; serious
lodging was only observed in the tall lines (average lodging score is
39, Table S2), while all of the dwarf lines kept an upright posture.
There was no significant difference observed in lodging between
BB and bb lines.
There was no significant difference in the number of green
leaves (average of 3.6) on main stem at anthesis between the
four genotypes in both experiments. Whereas, the leaf length of
dwarf lines with Rht12 was significantly decreased (Table S3). In
the AS experiment, the leaves of the dwarf lines were
Table 6. Dry weight of spikes and the whole plant at anthesis of different groups of the F
2:3
lines in the autumn-sown (AS) and
spring-sown (SS) experiments.
Experiment Genotype/variety SDM (g) SDT (g) PDM (g) TDP (g) SDM/PDM (SDM+SDT)/TDP
AS RRBB 0.7160.05a 5.1760.54a 3.1660.32b 25.2861.95b 0.2260.01a 0.2360.02a
RRbb 0.7060.04a 5.2060.58a 3.0560.38b 26.1461.88b 0.2360.02a 0.2260.01a
rrBB 0.7160.06a 4.8360.62b 3.9560.46a 37.0963.10a 0.1860.01b 0.1560.03b
rrbb 0.7160.04a 4.7260.68b 3.7760.41a 38.7862.85a 0.1960.01b 0.1460.02b
Karcagi 0.6460.03 4.6260.36 2.9360.35 22.5561.55 0.2260.01 0.2360.01
Nchun45 0.8860.05 4.1760.30 4.1860.35 33.6862.30 0.2160.02 0.1560.01
SS RRBB 0.6460.04b 1.3060.25b 3.4560.30b 10.2461.85b 0.1960.01b 0.1960.01a
RRbb 0.6260.03b 1.3060.19b 3.3860.35b 10.6261.56b 0.1860.01b 0.1860.01a
rrBB 0.8560.06a 1.9060.30a 4.1060.54a 15.4162.27a 0.2160.02a 0.1860.02a
rrbb 0.8360.06a 1.8060.25a 3.9560.50a 14.8862.58a 0.2160.02a 0.1860.01a
Karcagi 0.4660.03 0.5360.12 2.7460.26 7.1161.13 0.1760.01 0.1460.01
Nchun45 0.9160.03 2.0060.18 4.7960.45 17.3161.58 0.1960.01 0.1760.01
Note: SDM is spike dry weight of the main shoot, SDT is spike dry weight of the tillers, PDM is the dry weight of the main shoot, TDP is the total dry weight of one plant.
Data are means 6SD of each genotype. Data of the two parents were not considered in the statistical significance testing. Different letters within columns indicate
statistically significant differences (P,0.05).
doi:10.1371/journal.pone.0062285.t006
Figure 3. Culm morphology of the four genotypes in the AS and SS experiments. A: the mature plant morphology of the four genotypes
and the two parents in the AS experiment. B: schematic representation of internode elongation patterns of the four genotypes and the two parents
in AS experiment. C: Schematic representation of internode elongation patterns of the four genotypes in the SS experiment.
doi:10.1371/journal.pone.0062285.g003
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significantly shorter than those of the tall lines by as much as
6 cm (30%), and their widths were greater (10%,25%) than
those of the tall lines. Thus the total area of the uppermost
three leaves on the main stem of the dwarf lines was only
slightly (3%) less than that of the tall lines (Table S3). This may
enable the dwarf lines to have a similar ability to produce
sufficient photosynthate for the development of the kernels.
Additionally, in both sowings, RRBB lines had significantly
larger leaf area (4% and 6%, respectively) than RRbb whereas
there was no effect of Vrn-B1 in tall lines, suggesting that the
dominant Vrn-B1 could promote leaf growth in dwarf lines.
Dry Matter Accumulation and Distribution of the Main
Shoot after Anthesis
Dynamic changes in the dry weight of different organs in the
main shoot after anthesis were different between the dwarf and tall
genotypes. In the AS experiment, the dry weight of the flag leaf,
the second last leaf, the fourth internode and the fifth internode
increased until the 14
th
day (18
th
day for peduncle) after anthesis in
the dwarf lines, while the dry weight of the leaves and internodes
were all increased until the 18
th
day after anthesis, except the dry
weight of the second last leaf, in the tall lines. The dry weight of
the third last leaf decreased after anthesis in both dwarf and tall
lines (Fig. 5 and Table S4). However, assimilates stored in leaves
and stems of dwarf lines were transferred to spikes earlier than in
the tall plants (Fig. 5). Similar results were also observed in the SS
experiment on the dynamic change of dry weight of the leaves and
stems (Fig. 6 and Table S5). In both AS and SS experiments, the
maximum dry weights of the flag leaf were larger than that of the
peduncle or the fifth internode in the dwarf plants while the
opposite was observed in the tall plants. This indicated that Rht12
affected internode growth more than leaf expansion. Finally, the
total dry weight of the main shoot (excluding the spike) decreased
by 0.67 g (30%) from its maximum to that measured at 30 days
after anthesis in dwarf lines while it decreased by 1.37 g (35%) in
tall lines in AS experiment; in the SS experiment the dry weight
decreased by 0.87 g (33%) and 1.61 g (39%) in dwarf and tall
lines, respectively. This suggests that the capability to support
grain-filling (translocate shoot dry matter to the ear) was greatly
reduced for dwarf lines, a conclusion supported by the observed
greater gain in spike dry weight of tall lines (Figs. 5C and 6C).
Yield and Yield Components
Seed set and grain numbers were significantly increased in
Rht12 dwarf lines compared to that in tall lines in the AS
experiment, while fewer grains per spike were produced in dwarf
lines under the conditions encountered in the SS experiment due
to lower fertility (Table 7). For both sowings, there were more
grains in the top three spikelets due to their higher floret fertility in
dwarf lines than in tall lines (Tables 5, 7). This suggested that
Rht12 could increase grain numbers at the top of spikes and the top
three spikelets in dwarf lines had stronger production potential
than in tall plants. There was no significant difference between BB
and bb lines for grain numbers.
A significant reduction in 1000-grain weight was found in Rht12
dwarf lines compared to that in tall lines in both AS and SS
experiments, possibly due to a combination of the 5-day delay in
time to anthesis and the reduced plant biomass associated with
Rht12. Whereas, the increase in grain numbers and in the number
of fertile ears per plant of the dwarf lines resulted in there being no
significant difference in plant yield between the dwarf and tall lines
(Table 7). Additionally, as reported above, biomass per plant was
decreased significantly in the dwarf genotypes, resulting in a net
increase in harvest index. This indicates that, if total biomass could
be maintained, Rht12 might have the potential to decrease plant
stature while increasing the grain yield. In the SS experiment, due
to the lower fertility and less productive tillers of the dwarf lines,
the grain yield of dwarf lines was much lower (32%) than that of
Figure 4. Development of plant height from the soil surface to the top ligule. A: AS experiment, the final height is achieved at week 22. B:
SS experiment, the final height is achieved at week 16. C: plants at the 7
th
week in AS experiment, the yellow line shows the site of the top ligule in
each genotype and the two parents.
doi:10.1371/journal.pone.0062285.g004
Rht12 Affects the Agronomic Traits in Wheat
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Figure 5. Dynamic changes in dry weight of different organs in the main shoot after anthesis in the AS experiment. A: dwarf lines; B:
tall lines; C: spike dry weight of the dwarf and tall lines. The main culm comprises 6 internodes, the sixth internode is the peduncle. The leaves are
numbered from the flag leaf down on the main stem. RR: dwarf lines; rr: tall lines.
doi:10.1371/journal.pone.0062285.g005
Figure 6. Dynamic changes in dry weight of different organs in the main shoot in the SS experiment. A: dwarf lines; B: tall lines; C: spike
dry weight of the dwarf and tall lines. The main culm comprises 6 elongated internodes, the sixth internode is the peduncle. The leaves are numbered
from the flag leaf down on the main stem. RR: dwarf alleles; rr: tall alleles.
doi:10.1371/journal.pone.0062285.g006
Rht12 Affects the Agronomic Traits in Wheat
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the tall plants (Table 7). However, again due to the much-reduced
biomass, the harvest index of the dwarf plants was higher than in
the tall plants. This suggested that the dwarf genotypes could
achieve a higher harvest index more easily than the tall genotypes
in different environments. There was no significant difference
between BB and bb lines on plant biomass, 1000-grain weight or
grain yield.
Discussion
This study is part of a series of experiments carried out to
investigate and obtain a better understanding of the effects of the
dwarfing gene Rht12 on physiological attributes of the wheat crop.
To improve the winter habit and the late ear emergence of Rht12
plants [2], the dominant vernalization gene Vrn-B1 was introduced
to compensate for these effects. Contrasting homozygous lines with
or without Rht12 and Vrn-B1 genes (RRBB, RRbb, rrBB and rrbb)
were selected in a F
2
segregating population and assessed as F
2:3
lines to evaluate the effects of the dwarfing gene Rht12. Although
the homozygous F
2:3
lines do not share a common background as
with near-isogenic lines (NILs), single chromosome recombinant
inbred lines (RILs) or doubled haploid (DH) lines, the large
number of lines studied allowed the effects of other loci to be
reduced or neutralized, which coupled with the strong phenotypic
effect of Rht12, gave good resolution between genotypes. A similar
strategy could be used to assess the effects of other dwarfing genes
with strong effects on plant height (such as Rht4,Rht5,Rht13 and
Rht18) to evaluate their potential use in crop breeding for
improving agronomic performance or to determine their effects
in different target environments.
To better understand the effects of Rht12, the F
2:3
lines were
sown at two sowing dates to provide winter and spring wheat
growing environments, which was important for determining the
spike development traits associated with Rht12. In the AS
experiment, all plants should have received adequate vernaliza-
tion, as around 6 weeks below 5uCd are considered sufficient to
complete vernalization of most wheat cultivars [58]. However,
RRbb and even the RRBB group showed a longer duration to
double-ridge and flowered later than the rrbb group. Moreover, in
the SS experiment, rrBB and even rrbb reached the double ridge
stage earlier and showed a higher ear fertility (than in the AS
experiment) than RRBB, which still had a significantly elongated
duration to double ridge and showed poor ear fertility. Therefore,
the effects of Rht12 on reduction in the rate of spike development
and elongation of the duration of spike development phase, as well
as its epistatic effects on Vrn-B1 were revealed in the two sowing
date experiments. This finding is different than that of the GA-
insensitive Rht dwarf genes which had no effect on the duration of
phenological development and reduced only the rates of vegetative
and reproductive development [37,38]. The effects of Rht12
measured in the two experiments are thus very similar except for
ear fertility and effective tiller number. The difference in ear
fertility and effective tiller number between sowings could be the
result of the elongated duration of plant development associated
with Rht12: due to the slower growth rate and the strong winter
growth habit caused by the strong effect of Rht12, also interacting
with the higher temperature after the initiation of terminal
spikelet, the dwarf plants had a shorter period of time under
favourable conditions to develop fertile florets prior to flower
[2,26], and finally produced fewer competent florets than tall
plants in the SS experiment. Additionally, the delayed elongation
stage and the shorter time available to initiate new tillers and
develop tiller spikes also decreased the number of tillers with fertile
spikes after floret initiation stage in the dwarf lines. It is suggested
that while floret development and effective tiller development were
more sensitive to temperature than other traits in dwarf lines, it is
feasible to evaluate other pleiotropic effects of Rht12 using these
two sowing date experiments under winter and spring growth
conditions.
The reduction of plant height by Rht12 was 47 cm (,40%) in
this study. A similar result was reported by Rebetzke et al. [21]
who found that Rht12 reduced plant height by around 44 cm
(,45%), which was stronger than the dwarfing genes most widely
used in commercial varieties, such as Rht1 (,20%), Rht2 (,20%)
or Rht8 (,7%) [21]. The reduction of plant height caused by these
dwarf or semi-dwarf genes might result from reduced cell
elongation rather than cell division [4,34,55]. Additionally, it
was observed that while the diameter of the internodes was not
Table 7. Effects of Rht12 on yield components and harvest index of different groups of the F
2:3
lines in the autumn-sown (AS) and
spring-sown (SS) experiments.
Experiment
Genotype/
variety
Grain number
spike
21
1000-grain
weight (g)
Number of efficient
spikes plant
21
Plant yield
*
(g) Plant biomass
*
(g) Harvest index
AS RRBB 45.863.95a 31.963.37b 15.461.24a 12.462.32a 35.666.86b 0.3560.02a
RRbb 44.663.46a 32.563.17b 14.861.73a 12.862.31a 35.366.13b 0.3660.03a
rrBB 41.663.63b 43.264.58a 12.662.19b 13.262.72a 42.668.29a 0.3160.03b
rrbb 40.863.64b 43.064.47a 13.461.42b 13.162.08a 42.566.79a 0.3160.04b
Karcagi 43.063.99 29.562.75 15.262.32 12.162.13 33.667.67 0.3660.02
Nchun45 45.464.34 50.363.02 10.461.28 13.562.01 40.868.50 0.3360.03
SS RRBB 36.362.59b 30.063.14b 2.660.50b 3.760.46b 11.862.56b 0.3260.01a
RRbb 34.162.88b 30.563.10b 2.260.36b 3.660.54b 12.562.63b 0.3060.02a
rrBB 47.464.21a 36.763.71a 4.060.87a 5.761.24a 20.965.15a 0.2860.03b
rrbb 46.763.29a 35.263.53a 3.560.62a 5.161.32a 19.163.83a 0.2660.02b
Karcagi 31.061.78 28.063.25 1.960.23 1.960.46 8.460.89 0.2360.01
Nchun45 50.164.26 37.263.16 4.360.27 6.361.59 21.562.13 0.2960.02
*These data are the mean values of 25,40 plants. All data are means 6SD of each genotype. Data of the two parents were not considered in the statistical significance
testing. Different letters within columns indicate statistically significant differences (P,0.05).
doi:10.1371/journal.pone.0062285.t007
Rht12 Affects the Agronomic Traits in Wheat
PLOS ONE | www.plosone.org 12 April 2013 | Volume 8 | Issue 4 | e62285
affected by Rht12, the width of the stem wall was increased
significantly in dwarf plants, which probably benefits lodging
resistance [38]. Indeed, along with reduced height, thicker stem
walls may contribute to the better lodging resistance of the dwarf
plants in this study, and confirmed that dwarf genes had been an
important factor in lodging resistance of commercial varieties
[15,17].
During the vegetative phase, the crop initiates leaves until the
point of floral initiation, which is generally indicated by the
formation of the first double ridge in the apex [26]. Rht12 lines
developed more leaves than the equivalent tall genotypes due to
the longer duration to double ridge. In contrast, previous reports
have shown that GA-insensitive dwarfing genes (Rht1,Rht2 and
Rht3) do not affect the timing of plant developmental events, nor
the final numbers of leaves and internodes [3,37,38,43]. This
suggests that the two classes of dwarfing genes may act on plant
growth and development via different mechanisms. While, similar
with that in the GA-insensitive dwarf genotypes, the number of
elongated internodes in Rht12 lines did not change compared with
the tall lines, indicating that Rht12 affects the number of leaves but
not the number of internodes that elongate. Despite initiating
more leaves, there was no significant difference either in the
number of green leaves at anthesis or the area of the three upper
leaves at grain-filling stage between the dwarf and tall groups,
which is important for radiation interception and biomass
production in Rht12 lines.
It is argued that the pre-anthesis late reproductive phase (TS-
AN) coinciding with the period of rapid stem elongation, is
particularly important for yield [25,26] because the number of
fertile florets at anthesis is determined during this phase [21,59].
Moreover, it has been found that the greater number of fertile
florets per spike at anthesis in dwarf lines than in tall lines is due to
reduced degeneration of floret primordia in the late reproductive
phase pre-anthesis rather than differences in the maximum
number of floret primordia initiated [60,61]. Lengthening the
duration of TS-AN in wheat would arguably improve yield
potential by increasing the number of fertile florets at anthesis
[62,63]. In the AS experiment, Rht12 lines had a longer TS-AN
duration (uCd), which correlated with a significant increase in
floret survival, although the total number of florets initiated was
similar to the tall lines. The duration of TS-AN was also highly
correlated with the number of fertile florets in the SS experiment,
although in this case the dwarf lines actually had fewer fertile
florets than the tall lines. Allocating a greater proportion of dry
mass to the ear may also result in more fertile florets at anthesis
[26,64], but this does not imply that ears of dwarf genotypes are
always heavier than those of tall genotypes, because total shoot dry
matter has also to be considered [37]. With reduced competition
due to a much shorter stem, Rht12 could increase the partitioning
of dry matter to spikes during the late reproductive phase, leading
to a greater mass per carpel at anthesis, which in turn could result
in an improved proportion of fertilized florets and finally produce
more grains per spikelet and per ear.
Although the Rht12 dwarf lines had a slower growth rate during
stem elongation, the seeding vigour was similar to tall lines. The
coleoptile length of dwarf plants was only about 3 mm (5%)
shorter than that of the tall plants. Moreover, the length and width
of the first leaves were not significantly different between the four
genotypes. It has previously been observed that Rht12 had no
effect on seeding vigour and coleoptile length [21,65], which offers
the opportunity to reduce plant stature without compromising on
early growth and crop establishment [20,22]. In contrast, Rht-B1b
or Rht-D1b are associated with shorter coleoptiles and smaller
seedling leaf area and have a poor emergence when sown deeply
[13].
It has been reported that Rht12 had no negative effects on root
dry mass [66]. In this study, a reduction in total number of seminal
roots and a significant reduction in root dry mass (11%) were
observed in Rht12 dwarf lines at the seedling stage. These different
findings may be due to the effect of either different genetic
backgrounds or different growing environments, or both. In
contrast, a longer maximum root length and a higher dry mass
ratio of root to shoot were achieved in Rht12 dwarf lines. Thus,
Rht12 had both negative and positive effects on root growth
attributes. Further work should be conducted to explore the
possibility of enhancing aspects of root growth such as root length
using Rht12 to enable crops to grow in drier environments.
The increase in ear fertility is observed as one the most
important advantages of almost all the major height-reducing
genes [3,21,37,55]. Kernels per spike, especially the fertility of the
top three spikelets were significantly increased in Rht12 dwarf lines
in the AS experiment. Consistent with that, Rht12 significantly
increased ear fertility in different varietal backgrounds, which
results in the increase of grain numbers per spike [2]. Whereas,
spike length, spikelet number per spike and (particularly) grain size
were all reduced in the Rht12 dwarf lines in this study as the lowest
spike length and spikelet number per spike were observed in RRbb
genotype while the highest in rrBB lines in both AS and SS
experiments. Introduction of the dominant vernalization gene can
help to compensate some of the effects of Rht12 on yield
parameters. Similar reductions in spikelet numbers have pre-
viously been correlated with genes other than height-reducing,
particularly with Ppd1, where an average of two fewer spikelets per
spike were produced [67].
The reduction in leaf length in lines containing dwarfing or
semi-dwarfing Rht-1 alleles (i.e. Rht-B1b = Rht1,Rht-D1b = Rht2
and Rhtb1c = Rht3) is mainly due to shortening of cell length while
the number of cells is not modified, and it has been suggested that
Rht-1 alleles may likewise limit cell length in the pericarp to reduce
potential grain size [4]. However, Miralles et al. [55] found that
Rht-1 alleles did not modify cell length nor width in the pericarp,
but reduced the total number of cells in that tissue, presumably by
affecting cell division. It was also observed that the smaller average
grain size conferred by Rht alleles were not only a consequence of
a higher grain number produced in distal florets, but were also due
to smaller grains at certain positions within the spike [3,59].
Additionally, it has been suggested that reduced grain size with Rht
alleles was associated with a competitive response to the increased
spikelet fertility under limiting photosynthate availability, rather
than a primary effect of the dwarfing genes [59]. Furthermore,
flowering time can also influence grain size, because delayed
flowering shortens the period of time available with favourable
conditions prior to harvest [2]. In this study, the delay of 5–6 days
in flowering time associated with Rht12 could have affected the
development and size of grains. Also, the small size of vegetative
organs (especially the flag leaf and peduncle) of Rht12 lines may
have limited the assimilates available for producing large grains.
Thus, the reduction in grain size of different dwarf genes may be
through different modes of action from each other [6,21,55].
The pleiotropic effects of Rht12 on several yield components
were evaluated in the present study. Rht12 increased the number
of grains per spike and reduced grain size, resulting in an overall
reduction in spike yield. Whereas, whole plant yields are
dependent on both ear yields and the number of fertile ears
produced per plant. Due to the increased number of tillers in
dwarf plants (in AS experiment), the final yield per plant was not
significantly different between the tall and dwarf plants. Although
Rht12 Affects the Agronomic Traits in Wheat
PLOS ONE | www.plosone.org 13 April 2013 | Volume 8 | Issue 4 | e62285
the plant biomass of dwarf plants was reduced, the harvest index of
Rht12 dwarf plants increased significantly in both AS and SS
experiments. The greater harvest index and grain number for
Rht12 alleles was consistent with the effects of other dwarfing genes
that reduced competition between growing florets and elongating
stems [21]. Rht12 has potential for increasing harvest index and
total biomass in autumn sowing environment without compro-
mising on establishment and early growth of seedlings, but it
should be noted that in this study Rht12 dwarf lines had lower
yields in the spring sowing environment.
Rht12 has been classified as a GA-responsive dwarfing gene [2],
but its role, if any, in GA biosynthesis or signalling remains
unknown. Rht12 is located on chromosome 5AL, linked to
Xgwm291 at a distance of 5.4 cM. Thus, high resolution mapping
should be initiated for eventual map-based cloning of Rht12.To
date none of the dwarfing genes are cloned except a few GA-
insensitive dwarf genes [33]. More information on these genes is
needed for a better understanding of how the dwarf genes act on
plant growth and what their roles are in GA biosynthesis or
signalling. However, it was found that the effects of the dwarf gene
Rht8 which had been considered as ‘GA-sensitive’ was possibly not
due to the defective gibberellin biosynthesis or signalling, but
possibly to a reduced sensitivity to brassinosteroids [34]. Similarly,
there may be more complex relations between Rht genes and GAs
uncovered by future research.
Previous research has indicated that the main disadvantage of
Rht12 is the long vegetative phase resulting in late ear emergence
[2]. In this study, even the dominant Vrn-B1, which accelerates
flowering, could not compensate for the negative effect of Rht12 on
flowering time. RRBB and RRbb had a similar duration of SW-
DR while rrBB had shorter SW-DR duration than rrbb,
suggesting that Rht12 might be partially epistatic to Vrn-B1.
Therefore, development-promoting genes need to be combined
with Rht12 to compensate for the delay in ear emergence in order
to exploit the potential of Rht12 in breeding programs. In this
study, one dwarf line with earlier ear emergence time (2 days
earlier than other dwarf lines) was observed, indicating that
selection for early ear emergence may be possible among Rht12
progeny. We have established other crosses for this purpose and
also for further analysing the potential of Rht12 for crop
improvement.
Supporting Information
Table S1 Plant height and internode length of different
groups of the F
2:3
lines in the autumn-sown (AS) and
spring-sown (SS) experiments. All data are means 6SD of
each genotype. Data of the two parents were not considered in the
statistical significance testing. Different letters within columns
indicate statistically significant differences (P,0.05).
(DOC)
Table S2 Diameter and wall thickness of the internodes
of different groups of the F
2:3
lines in the autumn-sown
(AS) and spring-sown (SS) experiments. Note: DI is
internode diameter at the mid-point; WT is wall thickness. The
sixth internode is the peduncle. All data are means 6SD of each
genotype. Data of the two parents were not considered in the
statistical significance testing. Different letters within columns
indicate statistically significant differences (P,0.05).
(DOC)
Table S3 The mean length, width and area of the top
three leaves of different groups of the F
2:3
lines in the
autumn-sown (AS) and spring-sown (SS) experiments at
grain-filling stage. *, The second and third leaves are from the
flag leaf down on the main shoot. All data are means 6SD of each
genotype. Data of the two parents were not considered in the
statistical significance testing. Different letters within columns
indicate statistically significant differences (P,0.05).
(DOC)
Table S4 Dry weight (mg) of different organs in main
shoot after anthesis of different groups of the F
2:3
lines
in the autumn-sown (AS) experiment. *, The main culm
comprises 6 internodes, the sixth internode is the peduncle. The
leaves are numbered from the flag leaf down on the main stem.
RR: dwarf alleles; rr: tall alleles. DDW is the difference between
maximum dry weight and minimum dry weight, with its
proportion to the maximum dry weight in the parenthesis. Values
are given as the mean 6SD.
(DOC)
Table S5 Dry weight (mg) of different organs in main
shoot after anthesis of different groups of the F
2:3
lines
in spring-sown (SS) experiment. *, The main culm comprises
6 internodes, the sixth internode is the peduncle. The leaves are
numbered from the flag leaf down on the main stem. RR: dwarf
alleles; rr: tall alleles. DDW is the difference between maximum
dry weight and minimum dry weight, with its proportion to the
maximum dry weight in the parenthesis. Values are given as the
mean 6SD.
(DOC)
Acknowledgments
We are grateful to Peter Hedden, Stephen Thomas and Yidan Li at
Rothamsted Research for their helpful discussion. We also thank OPTI-
CHINA (an EU/CAAS funded project) which aims to link the crop
improvement research activities carried out by European and Chinese
researchers by providing LC the opportunity of training at Rothamsted
Research, UK.
Author Contributions
Conceived and designed the experiments: YGH AGC LC. Performed the
experiments: LC YGH AGC. Analyzed the data: LC YGH ALP.
Contributed reagents/materials/analysis tools: YGH LC AGC. Wrote
the paper: LC YGH ALP MAJP.
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Rht12 Affects the Agronomic Traits in Wheat
PLOS ONE | www.plosone.org 15 April 2013 | Volume 8 | Issue 4 | e62285
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Rht12 Affects the Agronomic Traits in Wheat
PLOS ONE | www.plosone.org 16 April 2013 | Volume 8 | Issue 4 | e62285
    • "We confirm the detrimental effect of Rht12 on mean grain weight [17,46], but additionally demonstrate that this is often in excess of that predicted by the effect of crop height estimated from the relationship with Rht-X1x alleles. Despite this 'extra' detrimental effect of Rht12, oth- ers [46] have still explained the small grains from Rht12 as being a result of inadequate postanthesis assimilate supply from the very short plants, rather than propose direct effects of GA deficiency. We show that specific weight, the packing density of grain, can be negatively and closely related to reductions in crop height as manipulated by Rht-X1x alleles. "
    [Show abstract] [Hide abstract] ABSTRACT: The effects of dwarfing alleles (reduced height, Rht) in near isogenic lines on wheat grain quality are characterised in field experiments and related to effects on crop height, grain yield and GA-sensitivity. Alleles included those that conferred GA-insensitivity (Rht-B1b, Rht-B1c, Rht-D1b, Rht-D1c) as well as those that retained GA-sensitivity (rht(tall), Rht8, Rht8 + Ppd-D1a, Rht12). Full characterisation was facilitated by including factors with which the effects of Rht alleles are known to interact for grain yield (i.e. system, [conventional or organic]; tillage intensity [plough-based, minimum or zero]; nitrogen fertilizer level [0-450 kg N/ha]; and genetic backgrounds varying in height [cvs Maris Huntsman, Maris Widgeon, and Mercia]. Allele effects on mean grain weight and grain specific weight were positively associated with final crop height: dwarfing reduced these quality criteria irrespective of crop management or GA-sensitivity. In all but two experiments the effects of dwarfing alleles on grain nitrogen and sulphur concentrations were closely and negatively related to effects on grain yield, e.g. a quadratic relationship between grain yield and crop height manipulated by the GA-insensitive alleles was mirrored by quadratic relationships for nitrogen and sulphur concentrations: the highest yields and most dilute concentrations occurred around 80cm. In one of the two exceptional experiments the GA-insensitive Rht-B1b and Rht-B1c significantly (P
    Full-text · Article · May 2016
    • "These genes not only reduce plant height but also decrease cell size to reduce leaf size during early growth (Miralles et al., 1998). The use of alternative gibberellin-sensitive dwarfing genes, such as Rht8 and Rht12, has been shown to reduce plant height and also improve the expression of early vigour (Botwright et al., 2005; Chen et al., 2013; Rebetzke et al., 2012). The populations used in this study, contained both GA-sensitive alternate dwarfing genes, Rht8, Rht12 and Rht13, and traditional GA-insensitive dwarfing genes, Rht-B1b (Rht1), Rht-D1b (Rht2). "
    [Show abstract] [Hide abstract] ABSTRACT: Increases in wheat performance under drought have been demonstrated in selection for factors contributing to greater water-use efficiency. Crop growth models have indicated the potential for significant increases in wheat yields across a wide range of wheat growing environments when both greater early vigour and higher integrated transpiration efficiency (TE) are selected together. However, greater early vigour comes with the possible trade-off of decreasing TE due to the production of larger, thinner leaves. A number of topcross-derived populations were specially developed for greater early vigour across a range of elite commercial backgrounds. These populations were assessed across multiple field environments and shown to vary significantly for early leaf area development (mean plant leaf area of 17-54cm2 at 3.5 leaf stage) and carbon isotope discrimination (20.8-22.4%), and exceeded the range for both traits across tested commercial varieties and parental genotypes. We demonstrate that it is possible to combine greater early vigour and greater integrated TE (measured as lower carbon isotope discrimination (CID)), and that increased integrated TE is associated, in part, with increases in photosynthetic activity. We also show that alternate GA-sensitive dwarfing alleles allow greater expression of early vigour when compared to traditional GA-insensitive dwarfing alleles. The potential exists for development of high water use efficiency wheat varieties combining alleles for greater early vigour and integrated transpiration efficiency.
    Article · Nov 2015
    • "At higher nitrogen levels, plant height and fresh weight of productive tillers increased. Plant height appears to be the major contributor to lodging tolerance (Chen et al., 2013; Kuczyńska et al., 2013; Ren et al., 2014 ). In previous study, lodging severity in shortstem varieties was very low and no significant variation was observed for yield and lodging scores while plant height was strongly correlated with lodging scores at all growth stages (Navabi et al., 2006). "
    [Show abstract] [Hide abstract] ABSTRACT: Field experiments were performed in Kroměříž, Czech Republic in 2012–2014 and evaluated for spring barley comprising treatments which different sowing rates and nitrogen fertilization rates. The objective of this study was to determine parameters of stand structure in early (BBCH 39) growth stages of barley that would enable evaluation of lodging risk. Lodging was significantly affected by both year and nitrogen fertilization rate. Increased nitrogen fertilization rates demonstrably increased lodging. Higher fertilization rates manifested in increased tiller biomass weight, greater plant height, and smaller roots. Similarly, the protein content of harvested grain was strongly affected by year and nitrogen application rate. Higher fertilization rates also resulted in grain with higher protein content. Sowing density did not affect either lodging level or protein content. Yields were affected only by year. Increased tiller biomass weight and increased plant height during early growth stages are significant indicators of lodging risk. Correlation between lodging and tiller biomass and plant height was higher than 0.5 and statistically significant (P = 0.000).
    Article · Aug 2015
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