All about dwarfing genes in wheat

Green revolution in wheat was brought through the "Dwarfing genes". This document describes all relevant information about major dwarfing genes in wheat and the mechanism how they cause dwarfism.

1. ALL ABOUT DWARFING GENES IN WHEAT
BY:SONAM MEHTA
M.Sc. BIOTECHNOLOGY
GURU NANAK GIRLS COLLEGE
LUDHIANA
Nomenclature
Rht-B1a ( Formerly rht1): Exhibits wild type stature.
Rht-B1b ( Formerly Rht1): Exhibits semi-dwarf stature.
Rht-D1a ( Formerly rht2): Exhibits wild type stature.
Rht-D1b (Formerly Rht2) :Exhibits semi-dwarf stature.
rht8 : Exhibits wild type stature.
Rht8 : Exhibits semi-dwarf stature.
Figure .:Development of
plant height from the soil
surface to the top ligule.
plants at the 7th week in
AS(autumn sown), the
yellow line shows the site of
the top ligule in each
genotype and the two
parents.

2. Reduced height in cereals is often associated with increases in yield due to a reduced risk
of lodging, increase in partitioning of assimilates to the grain (Evans, 1993), and more
fertile florets per spikelet (Brooking and Kirby, 1981). Wheat is the most important cereal
grain crop in the world.It is the staple crop for about 35% of the human population and
also known as “king” of the cereals (Laghari et al 2010).It is a crop that has profound
social and economic importance across countries. It is the principal cereal grain crop used
for food consumption in the United States and most parts of the world. The leading
producers being China, India, Turkey, Pakistan, and Argentina; INDIA is second largest
producer of wheat in the world .World production of wheat in 2001 was 583.9 million
metric tons, occurring on 219.5 million acres. World wheat consumption in that period
was 590.6 million tons. The consumption of wheat has been increasing with the
increasing population and thus its production need to be increased. It is estimated that
over the next decade, grain production must increase by 15% to meet the global demand
and consumption of wheat as a result of a growing human population (Edgerton, 2009).
Improving and achieving yield stability remains a daunting challenge.One strategy to
meet this challenge is to increase wheat productivity by optimizing plant architecture
(defined by tillering, stature, and leaf and ear morphology). Plant architecture is of major
agronomic importance as it determines the adaptability of a plant to cultivation, harvest
index, and potential grain yield (Reinhardt and Kuhlemeier, 2002).
A decisive component of plant architecture is stature, mainly determined by stem
elongation. Wheat (Triticum aestivum L.) is an annual crop with round, hollow, and
jointed culms (stems). There are usually five elongated internodes in fully grown culms,
with each internode progressively longer towards the ear. The internode elongation,
which determines final plant height, is regulated by genes involved in brassinosteroid
(BR) and gibberellin (GA) biosynthetic or signalling pathways.Semi-dwarfing genes in
wheat made a significant contribution to the ‘Green revolution’ in the 1960s .Twenty-one
reduced height genes in wheat, Rht1 to Rht21, have been described till .Among them,
four genes, originally named Rht1, Rht3, Rht2 and Rht10, were re-designated as Rht-
B1b, Rht-B1c, Rht-D1b and Rht-D1c, respectively and these were shown to be alleles at
two loci. Semidwarfing genes led to the higher yields due to improved lodging resistance
and the resulting ability to tolerate higher rates of inorganic nitrogen-based fertilizer

3. (Gale and Youssefian, 1985). The decrease in stem stature resulted in an increase in
assimilate partitioning to developing ears, enabling greater floret survival at anthesis and
increased grain numbers per ear (Youssefian et al., 1992).-
Green revolution
The term “Green Revolution” refers to the huge increases in grain yields after the 1960s,
resulting from the introduction of new varieties of wheat and rice, particularly for use in the
developing world. This development was a major factor in maintaining per capita food supplies
worldwide in the late 20th Century despite a doubling in the world population during this time
(Evans, 1998) and was recognised by the award in 1978 of the Nobel Peace prize to Norman
Borlaug of the International Maize and Wheat Improvement Center (CIMMYT). Borlaug
developed high yielding wheat varieties suitable for growing in sub-tropical and tropical
climates. The higher grain yields were obtained in part through increased use of fertilizers and
pesticides. However, the heavier grain caused the plants to become unstable and prone to
lodging (falling over) in high winds and rain. Borlaug introduced dwarfing genes into wheat
giving the plants a stronger, shorter stem that resisted lodging.The advantages of using dwarfing
genes with high-yielding varieties was soon recognized and most commercial wheat varieties
contain such genes, in temperate as well as in sub-tropical regions. An additional benefit from
these genes has been an increase in grain yield through an improvement in the ‘harvest index’
(the proportion of plant weight in the grain). This means that a greater proportion of the
products of photosynthesis accumulates in the grains rather than in the leaves. Modern wheat
varieties have a harvest index of over 50%, with a sharp increase since the introduction of the
dwarfing genes (Evans, 1998).
 The Dwarfing Genes of wheat
The dwarfing genes in wheat are classified into two categories, GA-responsive (GAR) and GA-
insensitive (GAI), reflecting the relative magnitude of their responses to application of
exogenous GA. GA-responsive dwarfing genes show significantly enhanced growth response to
exogenous GAs (probably have mutations in GA biosynthesis pathway) while GA-insensitive
dwarfing genes show very little response to exogenous GAs (probably have mutations in GA
signaling pathway, such as Rht-D1b and Rht-B1b) . This classification is conducted at the seedling
stage, for example, based on the response of coleoptile length or the first seedling leaf

4. elongation rate to exogenous GAs .The genes associated with a semi-dwarf growth habit in
wheat are known as Reduced height (Rht) genes and many of them are dominant or semi-
dominant, indicating that they actively inhibit growth through a so-called gain-of-function
mutation. Twenty-one reduced height genes in wheat, Rht1 to Rht21, have been described.
Among them, four genes, originally named Rht1, Rht3, Rht2 and Rht10, were re-designated as
Rht-B1b, Rht-B1c, Rht-D1b and Rht-D1c, respectively, when they were shown to be alleles at two
loci.
Rht-B1b (Rht 1) and Rht-D1b (Rht 2)
The Rht-B1b (Rht1) and Rht-D1b (Rht2) semi-dwarfing genes were introduced into
commercial wheat cultivars from the Japanese variety Norin10 in the 1960s as part of
wheat improvement programs in the USA and at CIMMYT, Mexico. A reduction in plant
height improved lodging resistance and partitioning of assimilates to the developing grain
(Evans 1993).
Figure. : Rht B1b sequence in Triticum aestivum (from NCBI).

5. Figure3. : The conserved sequence of Rht-B1 ( Pearce et.al, 2011).
Figure4. : Sequence of Rht D1gene in Triticum aestivum (from NCBI).
The large increases in yield that followed the introduction of these dwarfing genes led to

6. widespread adoption of the dwarfing genes throughout the world (Gale et al. 1985).
Recently, the homoeologous genes Rht-B1b and RhtD1b were isolated from wheat (Peng
et al. 1999). They are orthologous to the Arabidopsis GAI gene, a de-repressible
modulator of gibberellic acid (GA) response (Peng et al. 1997). Both the Rht-B1b and
Rht-D1b mutations are associated with a single base-pair change leading to a TAG stop
codon shortly after the start of translation (Peng et al. 1999). These mutations reduce the
plant’s ability to respond to GA, so that exogenous application of this hormone does not
restore wild-type plant height. Hence the presence of these dwarfing genes can be
determined by testing seedlings for the lack of responsiveness to GA (Gale and Gregory
1977; Richards 1992). Although relatively easy, this test is time-consuming, not always
reliable, and does not discriminate between Rht-B1b and Rht-D1b. These limitations can
be overcome by using molecular markers for these dwarfing genes. We developed PCR-
based markers aimed at discriminating between mutant (dwarf) Rht-B1b and Rht-D1b
and their wild-type (tall) alleles.
 Rht8
The Reduced height 8 (Rht8) semi-dwarfing gene is one of the few, together with the Green
Revolution genes, to reduce stature of wheat (Triticum aestivum L.), and improve lodging
resistance, without compromising grain yield. Rht8 is widely used in dry environments such as
Mediterranean countries where it increases plant adaptability. With recent climate change, its
use could become increasingly important even in more northern latitudes. In the present study,
the characterization of Rht8 was furthered. Morphological analyses show that the semi-dwarf
phenotype of Rht8 lines is due to shorter internodal segments along the wheat culm, achieved
through reduced cell elongation. Physiological experiments show that the reduced cell
elongation is not due to defective gibberellin biosynthesis or signalling, but possibly to a reduced
sensitivity to brassinosteroids. Using a fine-resolution mapping approach and screening 3104 F2
individuals of a newly developed mapping population, the Rht8 genetic interval was reduced
from 20.5 cM to 1.29 cM. Comparative genomics with model genomes confined the Rht8
syntenic intervals to 3.3 Mb of the short arm of rice chromosome 4, and to 2 Mb of
Brachypodium distachyon chromosome 5. Rht8 appears to be of importance to South European
wheats as alternative giberellic acid (GA)-insensitive dwarfing genes do not appear to be
adapted to this environment.The very successful semi-dwarf varieties bred by CIMMYT, Mexico,

7. for distribution worldwide have been thought to carry Rht8 combined with GA-insensitive
dwarfing genes. Additional height reduction would have been obtained from pleiotropic effects
of the photoperiod-responsegene Ppd1 that is essential to the adaptability of varieties bred for
growing under short winter days in tropical and sub-tropical areas. The microsatellite analysis
showed that CIMMYT wheats lack Rht8 and carry a WMS 261 allelic variant of 165 bp that has
been associated with promoting height. This presumably has adaptive significance in partly
counteracting the effects of other dwarfing genes and preventing the plants being too short.
Most UK, German and French wheats carry an allelic variant at the WMS 261 locus with 174 bp.
This could be selected because of linkage with the recessive photoperiod-sensitive ppd1 allele
that is though to off eradaptive significance northern European wheats.The diculty in
recognising Rht8 in varieties has led to many claims concerning the distribution of the gene
worldwide and its potential benefits to breeding programmes. These claims are difficult to
substantiate. Rht8 has been reported to be present in Chinese and CIMMYT varieties (Mishra
and Kushwaha 1995) and to enhance the yield of these wheats. In reality the only
definitivestudies on the effect of Rht8 involve the use of precise geneticstocks derived from
varieties like ‘Mara’ from Italy and ‘Sava’ from Yugoslavia where both pedigree analysis and
observations on defined aneuploid stocks confirm that Rht8 must be present on the 2D
chromosome.The data from thesestudies are restricted to both a limited range of varieties and
limited array of environments. These studies suggest that Rht8 reduces height by around 8—10
cm in the UK (Worland and Law 1986; Worland et al. 1988a,b), 5 cm in mid Germany (Worland
et al. 1992) and 5—7 cm in Yugoslavia (Worland et al. 1988a, b, 1990). In all cited examples, data
were obtained on single-chromosome recombinant lines between chromosomes 2D of ‘Mara’
(Rht8) and ‘Cappelle-Desprez’ (rht8) in a homozygous ‘Cappelle-Desprez’ background. Few
significant additive genotypic effects of Rht8 were detected on other agronomic characters.
Interactions were detected between Rht8 and Ppd1 for spikelet numbers, grain size and ear
yield (Worland et al. 1988a, b). No interactive environmental effects were detected for Rht8
when similar lines were tested in England and Yugoslavia. These results suggest that at least in
Europe, within the varietal background, year and environment limitations of the trials Rht8
could be used to reduce height without adverse effect on plant yield. Recently a microsatellite
marker, WMS 261, has been identified that shows very restricted recombination with Rht8
(Korzun et al. 1998). Three main allelic variants of 165 bp, 174 bp and 192 bp have been
detected at the WMS 261 locus on the short arm of chromosome 2D. The 165-bp variant was

8. found to be diagnostic for the CIMMYT variety ‘Ciano 67’, the 174-bp variant diagnostic for
‘Cappelle Desprez’, the tall control variety in experiments determining the pleiotropiceffects of
Rht8, and the 192-bp variant diagnostic for the Italian variety ‘Mara’, the donor of Rht8. Genetic
analysis of single-chromosome recombinant lines developed in a ‘Cappelle-Desprez’ background
between 2D chromosomes of ‘Cappelle-Desprez’ and ‘Ciano67’ shows a significant height
increase of around 3—4 cm associated with the WMS 261 165-bp allele compared to the WMS
261 174-bp allele. This 3- to 4-cm increase is in addition to the5—10 cm of the WMS 261 192-bp
allele versus WMS 261 174-bp allele comparison. It is anticipated that the very close linkage of
WMS 261 to Rht8 will permit the use of the microsatellite as a marker for determining the
distribution of Rht8 in international breeding programmes and to demonstrate how Rht8 has
been transmitted from initial.crosses involving the source variety ‘Akakomugi’. In the
experiments described here over 100 varieties have been screened for allelic variants at the
WMS261 locus.The varieties were chosen to include key varieties in the pedigrees of modern
varieties that are thought to carry Rht8, varieties from diverse international breeding
programmes and check varieties that have been shown by cytological observation to carry Rht8
and are therefore able to be used to verify that recombination has not occurred between WMS
261 and Rht8 and thus verify conclusions drawn from the marker association.
Worldwide distribution of WMS 261 allelic variants
The dwarfing genes credited with playing major roles in improving wheat yields (Rht-B1b, Rht-
B1d, Rht-D1b, Rht8, Ppd1) all seem to have either originated in Japan or to have been
incorporated into early Japanese varieties before spreading into worldwide breeding
programmes. ‘Akakomugi’, the source variety for Rht8, carries the diagnostic WMS 261-192-bp
microsatellite. Of 5 other Japanese varieties tested, 2 modern varieties ‘ChikushiKomugi’ (‘Norin
121’) and ‘Fakuho-Komugi’ (‘Norin 124’)bothcarrythe Rht8 allele,as does‘Haya-Komugi’, an old
land-race and a parent of another important Japanese dwarfing source variety ‘Saitama 27’.
‘Saitama 27’ itself has not obtained the diagnostic Rht8 allele from ‘Haya-Komugi’ but carries
the WMS 261165-bp allele. The third important Japanese dwarfing gene source variety, ‘Norin
10’, carries the WMS 261174-bpallele. This indicate that even in the early decades of the
twentieth century all three major WMS 261 alleles were segregating in Japanese wheats.
 Rht12

9. Rht12 has been classified as a GA-responsive dwarfing gene , 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. , 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. Rht12, a
dominant dwarfing gene from the gamma ray-induced mutant Karcagi 522M7K of winter wheat
(here referred to as Karkagi-12), has been classified as a GA-responsive dwarf gene .Rht12
significantly decrease 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.
Additionally, the Rht12 dwarf lines showed very dark green leaves compared to tall lines. Rht12
significantly decreased plant height, by around 40%, while seedling vigour, coleoptile length and
root traits at the seedling stage were not affected adversely. Rht12 lines had significantly
increased floret fertility and grain number and achieved a higher harvest index (due to the lower
plant biomass) than the tall genotypes. However, Rht12 extended the duration of the spike
development phase, especially the duration from sowing to double ridge, and delayed anthesis
date by around 5 days. Even the dominant Vrn-B1 allele could not compensate for these effects
on phenological development, which may hamper the direct utilization of Rht12 in wheat
breeding. Another negative effect of Rht12 on yield components was that grain size was reduced
significantly. Similarly, other studies have found that Rht12 had a substantial effect on reducing
plant height without altering early vigour and significantly increased spikelet fertility, harvest
index, and lodging resistance but these were usually accompanied by delayed ear emergence
and reduced grain weight.Although Rht12 has been classified as a GA-responsive dwarfing gene
a comprehensive understanding on the response of Rht12 to exogenous GAs is lacking. Thus, the
role of Rht12, if any, in GA biosynthesis or signaling is still unclear. Moreover, it has been
recently found that the effects of the dwarfing gene Rht8, which had been considered as ‘GA-
responsive’, was possibly not due to defective GA metabolism or signaling because the wild type
and Rht8 lines responded with a very similar increase in final plant height (15% and 13%,
respectively; P,0.05) with GA3 application. It has been proposed that the effects of Rht8 are
possibly due to reduced sensitivity to brassinosteroid.
Previous research has indicated that the main disadvantage of Rht12 is the long vegetative
phase resulting in late ear emergence.

10. Origin and History of Rht-B1b , Rht-D1b and Rht8
The GA-insensitive height reducing genes Rht-B1b and Rht-D1b originated in the 1930’s
in the dwarf variety Norin 10, a derivative of the Japanese variety Daruma (Allan, 1989).
Shortly after World War II, S. C. Salmon, a wheat breeder with the USDA, visited Japan
as an advisor to the occupation army. During his visit, he received several wheat samples,
and among them was the variety Norin 10, which he sent to the USDA Small Grains
Collection Facility. In 1948, Norin 10 was obtained by Orville Vogel, a USDAARS
wheat breeder in Pullman, WA, who then crossed Norin 10 with the high-yielding variety
Brevor 14 (Allan, 1989). The Norin 10 x Brevor 14 cross was then used by Norman
Borlaug and others as part of wheat improvement programs in the United States and at
the International Maize and Wheat Improvement Center (CIMMYT) (Ellis et al, 2002).
The Japanese variety Akakomugi was the source for most of the European cultivars
carrying the Rht8 dwarfing gene (Worland et al., 1998). Rht8 is widespread in southern
and central European wheats as well as several Russian cultivars (Worland et al., 1998)
Akakomugi was first used by the Italian breeder Strampelli in the 1920’s to introduce
genes not only for semi-dwarfism (Rht8) but also, unknowingly, for early maturity (Ppd-
D1) (Worland and Law, 1986, Korzun et al., 1998).
From Italy, Rht8 made its way to Argentina before World War II and and then to Europe
and the former Soviet Union after World War II (Borojevic and Borojevic, 2005). Unlike
Rht-B1b and Rht-D1b, the Rht8 dwarfing gene from Akakomugi is sensitive to exogenous
GA.
 Wheat Norin 10
It is a semi-dwarf wheat cultivar with very large ears that was bred at an experimental station in
Iwate Prefecture, Japan. In 1935, it was registered as a numbered cultivar by Ministry of
Agriculture and Forestry .Norin 10 provided two very important genes, Rht1 and Rht2, that
resulted in reduced-height wheats, thus allowing better nutrient uptake and tillerage (when
heavily fertilised with nitrogen, tall varieties grow too high, become top-heavy, and lodge).
Genetics of Rht-B1b, Rht-D1b, and Rht8

11. Figure. : Comparison of the nucleotide sequences of the Rht-1 homeologs across the
conserved N-terminal coding region. SNPs between the Rht-1 homeologs are indicated
by lowercase letters and asterisks above the sequences .The Rht-B1b and Rht-D1b point
mutations are shown, and their positions are indicated by arrows above the sequences.
The predicted amino acid sequences of RHT-A1A, RHT-B1A, and RHT-D1A are
identical in this region.
Rht-B1 is located on chromosome 4B (Gale and Marshall, 1976), which was confirmed in both
hexaploid wheat (Rao, 1980) and durum wheat (Blanco et al., 1998).

12. Figure. :Rht-D1 is located on chromosome 4D (Gale et al., 1975).
Traditionally, selection for the Rht-B1b and Rht-D1b dwarfing genes was determined by testing
seedlings with gibberellic acid (GA). Plants with the mutant dwarfing gene show no response to
exogenous GA. Both Rht-B1b and Rht-D1b dwarfing genes are insensitive to exogenous GA. With
the introduction of marker-assisted selection, perfect markers for Rht-B1b and Rht-D1b were
developed in a doubled haploid population that was segregating for the Rht-B1b and Rht-D1b
alleles (Ellis et al., 2002).
Perfect markers detect the specific base-pair mutation responsible for the semi-dwarfing
phenotype.
The mutations involved in the Rht-B1b and Rht-D1b semi-dwarf phenotypes disrupt the GA
signaling pathway. The wild type proteins are thought to act as negative repressors of GA

13. signaling and GA acts by repressing their function (Hussain and Peng, 2003). Peng et al. (1997)
identified base substitutions in both Rht-B1b and Rht-D1b. In both cases, the mutations affects
the N-terminal region of their transcribed DELLA proteins via a substitution which produces a
stop codon shortly after the transcription start site (Peng et al., 1997), resulting in their
characteristic GA-insensitivity. DELLA proteins are a subfamily of GRAS proteins, which are
thought to act as transcriptional regulators (Pysh et al., 1999). Rht-B1b and Rht-D1b produce
stop codons in the DELLA domain, a 27 amino acid motif at the N-terminus, and in their
truncated form, these proteins are thought to act as constitutive repressors of GA-mediated
growth (Peng et al., 1997).
Gale et al. (1982) found the location of a major Rht allele on chromosome 2D with a backcross
monosomic analysis using varieties Sava (derived from Italian wheats) and Koga II. This allele
accounted for nearly all of the height difference between the two parent varieties. Another
study, by Law et al. (1981), determined that the dwarfism of Mara, an Italian variety derived
from Akakomugi, was partially caused by chromosome 2D. The causal gene was later designated
as Rht8. The microsatellite marker WMS 261 is located 0.6cM distally from Rht8 can be used as a
marker to identify lines carrying the Rht8 dwarfing gene (Korzun et al., 1998).
Rht8 has been shown to be closely linked to the photoperiod-insensitivity gene Ppd-D1 (Korzun
et al., 1998). Photoperiod-sensitive wheat varieties require long days for floral induction, while
photoperiod-insensitive varieties have the ability to flower independently of photoperiod.
Several studies have shown that varieties exhibiting photoperiod-insensitivity also exhibit earlier
heading and shorter stature than their photoperiod-sensitive counterparts (Marshall et al.,
1989; Blake et al., 2009). In addition to effects on heading date and height, Dyck et al. (2004)
found that photoperiod insensitivity had a significant effect on yield. In the current study, lines
were screened for photoperiod-insensitivity and removed to reduce confounding effects.

14. 2.12 Agronomic Traits of Rht-B1b, Rht-D1b, and Rht8
Figure : Most dwarfing mutations introduce premature stop codon
Height
The height reduction associated with Rht-B1b and Rht-D1b arises from GA insensitivity that
causes a decrease in cell elongation in juvenile leaf and stem tissue, which leads to an overall
reduction in plant height. Height reductions for cultivars carrying the Rht-B1b and Rht-D1b
dwarfing genes are similar to each other. Rht-B1b and Rht-D1b were found to reduce plant
height by 15% (Gale and Youseffian, 1985) and 24% (Allan, 1986). Another study found a
reduction of 14% and 17% for Rht-B1b and RhtD1b, respectively (Flintham et al., 1997).

15. Trethowan et al. (2001) reported an average height reduction of 36% in a population of Rht-B1b
near-isogenic lines. Blake et al. (2009) suggested that final plant height is influenced by not only
genotype but also a variety of environmental factors, such as heat, drought, and nutrient
deficiencies. Rht8 has been shown to reduce height by approximately 10% in studies from the
UK, Germany, and former Yugoslavia (Worland and Law, 1986; Worland et al., 1998).
Reductions of 3.49% (Börner et al., 1993), 7.3% (Rebetzke et al., 1999), and 12.5% (Rebetzke and
Richards, 2000) have also been reported.
Yield
Reports of the advantages and disadvantages of different Rht alleles and their standard height
counterparts have drawn varying conclusions. Increased yield potential for Rht-B1b and Rh-D1b
has been noted under high-input growing conditions (Knott, 1986; Hedden, 2003; McNeal et al.,
1972) Although Rht-B1b and Rht-D1b dwarfing genes have the potential to increase yield of
wheat grown in optimal conditions, these dwarfing genes have been associated with reductions
in yield in environments with low-inputs or abiotic stresses (Laing and Fischer, 1977; Anderson
and Smith, 1990; Richards, 1992) .The yield advantages of Rht-B1b and Rht-D1b are less obvious
in spring wheat than in winter wheat as well as in conditions of heat or drought stress (Flintham
et al., 1996). Heat and drought stress during ear initiation can reduce grain number through a
reduction in the number of competent florets and pollen viability and can reduce grain weight as
a result of shortened grain-fill period (Hoogendoorn and Gale, 1988).
Rht-B1b, Rht-D1b, and possibly Rht8, are associated with increased floret fertility which may
counteract the negative effects on yield observed with some Rht genes (Gale and Youseffian,
1985). Yield increases in semi-dwarf wheat cultivars are due, in part, to increased partitioning of
assimilates into the developing grain rather than into the stem for elongation (Flintham et al.,
1997).
Several studies suggest that there is not a significant difference between Rht-B1b and Rht-D1b in
terms of yield improvement. In Montana and Saskatchewan trials, semidwarf lines containing
Rht-B1b or Rht-D1b generally yield more than standard height lines, except in very low yielding
environments, where the standard height lines exhibited a yield advantage (Knott, 1986; McNeal
et al., 1972). Yield increases of 24% (Flintham et al., 1997) and 16% (Singh et al., 2001; Allan,
1986) have been reported for Rht-B1b and Rht-D1b. Yield increases of 21% (Chapman et al.,

16. 2007) for Rht-B1b and 30% (Blake et al., 2009) and 18% (Chapman et al., 2007) for Rht-D1b have
been reported. For cultivars carrying Rht8, yield increases of 12% (Gale etal., 1982), 9.7%
(Rebetzke and Richards, 2000), and 3.8% (Börner et al., 1993) have been reported.
 Coleoptile Length
Crop establishment is a major determinant of yield (Paulsen, 1987) and coleoptile length is an
important factor in seedling emergence and crop establishment. Reduced coleoptile length is
associated with reduced emergence and subsequent poor crop establishment (Allan, 1980). In
modern wheat cultivars, one of the most important determinants of coleoptile length is the
presence of the Rht semi-dwarfing genes.
Standard height (tall) wheats have long coleoptiles, due to normal cell elongation in the
presence of endogenous GA, and Rht-B1b and Rht-D1b semi-dwarf wheats, which are GA-
insensitive, have shortened coleoptiles (Keyes et al., 1989). Allan (1980) found that Rht-B1b and
Rht-D1b reduce coleoptile length in a similar proportion to their reduction in plant height.
Wheat cultivars carrying Rht-B1b and Rht-D1b dwarfing genes have a limited coleoptile length of
about 7.0 cm, while the coleoptiles of standard height wheats can reach up to 13.0 cm (Whan,
1976). Another study, by Trethowan etal. (2001), reported standard height average coleoptile
length at 12.4 cm and Rht-B1b semidwarf average coleoptile length at 7.8 cm.
In cultivars containing the GA-sensitive Rht8 dwarfing gene, there seems to be a negligible effect
on coleoptile length (Konzak, 1987). Additionally, Rebetzke et al. (1999) reported Rht8 semi-
dwarf coleoptiles as long as those of the standard height parent.
 Stem Solidness
The wheat stem sawfly, Cephus cinctus Norton, is a major insect pest of wheat and other cereals
across areas of western North America as well as Canada (Davis, 1955). Adults deposit eggs in
the stems and when the larva hatch, they feed on the parenchyma and vascular tissue inside of
the stem, moving down the stem until they reach near ground level, where they cut around the
inside of the stem. Then, they move down into the remaining portion of the stem to pupate and
overwinter (Hayat, 1993). Agricultural losses caused by wheat stem sawfly are estimated at $25

17. million per year (Montana State University, 1997). Currently, the main control method has been
the use of solid stemmed cultivars.
Stem solidness is caused by the development of undifferentiated parenchymous cells, or pith,
inside the stem. The thickness of the parenchymal cell walls has been reported to have a direct
relationship with larval mortality in wheats with solid stems (Roemhild, 1954). Thus far, no
published studies have examined the effect of Rht dwarfing genes on stem solidness. The
current study examines the effect of Rht genotype on stem solidness.
Effects of Exogenous GA3 Application
Gibberellins (GAs) are a major class of plant hormones that regulate plant growth and
development, from seed germination and stem elongation to fruit-set and growth . It is
important for plants to produce and maintain optimal levels of bioactive GAs to ensure
normal growth and development. Mutants with impaired GA biosynthesis or response
show typical GA-deficient phenotypes, such as dark green leaves, dwarfism and late-
flowering, while elevated exogenous GA dose or increased signaling can cause excessive
plant growth and earlier flowering . Mutants deficient in GA biosynthesis can be rescued
by exogenously applied GAs but this is not possible if the mutation is in the GA signaling
pathway .
The deployment of genes influencing plant height through the GA pathway was a major
factor in the success of the Green Revolution, which created high-yielding cultivars of
rice and wheat with shorter and sturdier culms . In contrast to the recessive, semi-dwarf
sd-1 Green Revolution allele in rice, which is a loss-of-function mutation in one of the
major GA biosynthetic genes , the reduced height Rht-B1b (Rht1) and Rht-D1b (Rht2)
Green Revolution alleles in wheat are semi-dominant gain-of-function mutations causing
impaired GA signaling and thus conferring dwarfism through constitutive repression of
cell division and elongation .
The wheat Green Revolution genes are orthologues of the Arabidopsis GA-insensitive
(gai), the rice slender1 (slr1) or the rice gai , the barley slender1 (sln1) and the maize
dwarf-8 (d8) genes. However, in addition to reducing plant stature, Rht-D1b and Rht-

18. B1b also reduce seedling vigour and coleoptile length, and may reduce crop water-use
efficiency and performance in some unfavorable environments . So, opportunities exist
for replacing Rht-B1b and Rht-D1b in wheat with alternative dwarfing genes, such as the
GA-responsive dwarfing genes (Rht4, Rht5, Rht9, Rht12, Rht13, Rht14, Rht15, Rht16 or
Rht18). These genes have been reported to reduce plant height without compromising
early plant growth . Even though there are several GA-responsive dwarfing genes in
wheat, their molecular characteristics remain obscure and the mechanisms by which they
resulted in a reduction of plant height is not well understood. The metabolic pathways of
gibberellin biosynthesis, deactivation and signaling have become relatively clear and
many of the genes involved have been identified , which lays the foundation for analysis
of GA-responsive dwarfing genes in wheat.
Generally, dwarfing genes in wheat are classified into two categories, GA-responsive
(GAR) and GA-insensitive (GAI), reflecting the relative magnitude of their responses to
application of exogenous GAs . GA-responsive dwarfing genes show significantly
enhanced growth response to exogenous GAs (probably have mutations in GA
biosynthesis pathway) while GA-insensitive dwarfing genes show very little response to
exogenous GAs (probably have mutations in GA signaling pathway, such as Rht-D1b and
Rht-B1b) . This classification has usually been conducted at the seedling stage, for
example, based on the response of coleoptile length or the first seedling leaf elongation
rate to exogenous GAs . There is less information available on the response of the GAR
dwarfing genes to exogenous GAs at later growth stages. 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. Additionally, the
Rht12 dwarf lines showed very dark green leaves compared to tall lines. Rht12
significantly decreased plant height, by around 40%, while seedling vigour, coleoptile
length and root traits at the seedling stage were not affected adversely. Rht12 lines had
significantly increased floret fertility and grain number and achieved a higher harvest
index (due to the lower plant biomass) than the tall genotypes. However, Rht12 extended
the duration of the spike development phase, especially the duration from sowing to
double ridge, and delayed anthesis date by around 5 days. . Another negative effect of

19. Rht12 on yield components was that grain size was reduced significantly. Similarly, other
studies have found that Rht12 had a substantial effect on reducing plant height without
altering early vigour and significantly increased spikelet fertility, harvest index, and
lodging resistance but these were usually accompanied by delayed ear emergence and
reduced grain weight. .Although Rht12 has been classified as a GA-responsive dwarfing
gene , a comprehensive understanding on the response of Rht12 to exogenous GAs is
lacking. Thus, the role of Rht12, if any, in GA biosynthesis or signaling is still unclear.
Moreover, it has been recently found that the effects of the dwarfing gene Rht8, which
had been considered as ‘GA-responsive’, was possibly not due to defective GA
metabolism or signaling because the wild type and Rht8 lines responded with a very
similar increase in final plant height (15% and 13%, respectively; P<0.05) with GA3
application. It has been proposed that the effects of Rht8 are possibly due to reduced
sensitivity to brassinosteroids .
DELLA Proteins
Figure8. : The amino acid sequence of DELLA protein
DELLA proteins are negative regulators of GA-induced growth. In the absence of GA, DELLA
proteins repress expression of GA response genes resulting in slow growth, whereas with GA,
there is induced phosphorylation of DELLA proteins via an unidentified kinase. The SCFSLY1
complex interacts with the GRAS domain of DELLA proteins and targets their polyubiquitination
and degradation via the ubiquitin–26S proteasome pathway (Dill et al., 2004). Small deletions of

20. the DELLA protein that interfere with degradation (Itoh et al., 2002, 2005; Liu et al., 2010), such
as a gai mutant of the Arabidopsis DELLA domain that lacks 17 amino acids near the N terminus,
cause dwarf stature (Fleck & Harberd, 2002) due to constant gene repression even in the
presence of GA. Similarly, a T-to-G substitution converts the E61 codon (GGA) to a translational
stop codon (TGA) in the Rht-D1b allele and the resultant N-terminally truncated product confers
short stature (Peng et al., 1999). Overexpression of DELLA proteins is also a powerful way to
reduce stature. Low-level gai expression caused relatively mild height reduction, whereas high-
level gai expression resulted in more severe dwarfism in Arabidopsis (Fu et al., 2001).
Figure : The GA-
DELLA signalling
mechanism. In
the absence of
GA, the DELLA
proteins are
stabilized in the
nucleus and
repress DELLA-
mediated
growth,
presumably via
modulation of
transcription of
target genes. In
the presence of
GA, GA binds to
the soluble
GID1 receptor.
In the nucleus
the GA-GID1 complex associates with the DELLA proteins, promoting a further interaction
between DELLA and the SCF
SLY1/GID2
complex. The SCF complex catalyses the polyubiquitination of

21. the DELLA protein, triggering DELLA degradation by the 26S proteasome. Destruction of the
DELLA protein derepresses the DELLA-mediated growth restraint and allows growth to occur.
DELLAs are a family of nuclear proteins that act as growth repressors throughout the life cycle of
higher plants. Derepression is mediated through the gibberellic acid (GA)-dependent
degradation of DELLAs and the key components of the GA-DELLA signalling pathway (the GA
receptor and the F-box protein involved in DELLA destruction) have recently been identified. It is
becoming increasingly clear that DELLAs promote a plant's survival by integrating its growth
responses to a wide range of endogenous and environmental signals.
Figure: DELLA proteins consist of functional GRAS and regulatory DELLAdomain at the less
conservative N-terminus of the protein .Conserved N-terminal regulatory region consists of
DELLA,LexLe and TVHYNP amino acid motif.In the C-terminus functional domains are LR1 &
LR2(Leucine rich regions);NLS(nuclear localization signal);SH2-like(src homology 2 like domains)
and VHIID,PFYRE,RVER,SAW are the amino acid motifs.
DELLAs N-termini show high homology to each other between 34 and 84 % similarity (Bolle,
2004). Mutations in these genes produce amino acid substitutions, deletions or insertions in the
N-terminal region of the translated DELLA protein (in wheat, Rht-B1b and Rht-D1b alleles
encode proteins that have deletion between DELLA and TVHYNP motifs in LExLE region). These
mutations affect binding to GA-receptor and GA allowing accumulation of mutant DELLAs and
repress growth. In the nucleus, DELLA of wild type binds to GA-receptor (e.g. GID1 known for
Arabidopsis), GA and SCF E3 ubiquitin ligase complex. Such a large complex is recognized by 26S

22. proteasome and destroyed. The disappearance of DELLA proteins stimulates GA responsive
processes such as seed germination, stem and root elongation, and fertility (Hirsh, Oldroyd,
2009). In the absence of GA, or in the case of mutation in nucleotide sequence of DELLA domen,
the ubiquitination becomes impossible. Accumulation of the mutant DELLA proteins cause
continuous 171Della mutations in plants with special emphasis on wheat growth inhibition and,
accordingly, leads to agronomically advantageous dwarfed plant height and improved straw
strength by inhibition of stem cell elongation (Dalrymple, 1986; Flintham et al., 1997; Peng et al.,
1999). As has been shown for barley embrio (Gubler et al., 2002;
http://plantcellbiology.masters.grkraj. org), DELLAs repress transcription and processing of
GAMYB gene (GA induced Amylase-beta), that is transcriptional regulator of α-amylase gene
regulatory elements called GARE (GA response elements) and induce amylase gene expression.
Figure. : Model of regulation of amylase biosynthesis by DELLA proteins
1.DELLA SCF E3 ubiquitin ligase complex degradation starts after binding with GA
2.The promoter of GAYMB gene becomes active and GAYMB transcription factor is
synthesised
3.GAYMB transcription factor activates the alpha amylase gene.

23. 4. Alpha amylase and other hydrolytic enzyme are synthesised in rough endoplasmic
reticulum and secreted by golgi body.
5.Secretory vessels go through the cell wall and alpha amylase starts the starch
degradation in endosperm.
DELLAs inhibit growth by interfering with the activity of growth-promoting transcription factors
(Harberd et al., 2009). Mutants of wheat, barley, and rice, that are affected in GA signaling,
display an altered aleurone α-amylase response. For example, dominant mutations at the
homeoallelic wheat Rht-B1a and Rht-D1a loci confer dwarfism and а reduced growth response
to GA (Börner et al., 1996; Peng et al., 1999). Severely dwarfing alleles, such as Rht-B1c, abolish
the GA response of mutant aleurone cells (Gale, Marshall, 1975; Ho et al., 1981; Börner et al.,
1996). DELLAs are conservative due to the essential role in plant cell. They help to establish GA
homeostasis by direct feedback regulation on the expression of GA biosynthetic and GA
receptor genes, and promote the expression of downstream negative components that are
putative transcription factors/regulators or ubiquitin E2/E3 enzymes (Zentella et al., 2007). In
addition, one of the putative DELLA targets, XERICO, promotes accumulation of abscisic acid
(ABA) that antagonizes GA effects. Therefore, DELLA may restrict GA-promoted processes by
modulating both GA and ABA pathways (Zentella et al., 2007).