Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci.
DOI 10.1007/s40011-017-0870-z
REVIEW
Doubled Haploidy Techniques in Wheat (Triticum aestivum L.):
An Overview
Madhu Patial1 • Dharam Pal1 • Anjana Thakur2 • Ram Swaroop Bana3
Sunny Patial2
Received: 5 April 2016 / Revised: 10 March 2017 / Accepted: 25 April 2017
 The National Academy of Sciences, India 2017
Abstract Wheat crop has a critical role in current food
system and also in the future global food security. Global
wheat demand in 2010 reached 666 million metric tons
(MMT). If the demand growth rate remains constant, it has
been predicted that the global wheat consumption would
surpass 880 MMT by 2050. Fulfilling this demand needs
new and more efficient wheat breeding methodologies.
Conventional breeding has led to the development of
number of varieties, but with the changing climatic regime
accompanied with fast and continuous changing nature of
biotic and abiotic stresses there is an urgent need to fasten
the breeding methods. Hence, biotechnological tool like DH
becomes an important weapon. The production of haploid
plants from hybrids, followed by chromosome doubling
will provide wheat breeder with a mean to accelerate the
development of true breeding lines. Doubled haploid (DH)
populations have lot of applications in plant breeding like
cultivar and germplasm development, transferring traits
from wild types, studying components of quantitative
genetics and whole genome mapping. Among different DH
production techniques, anther culture and Hordeum bulbo-
sum have stronger genotypic specificity whereby, wide
hybridization comes up with a solution. Amongst various
wide hybridization techniques, DH production via Imperata
& Madhu Patial
mcaquarian@gmail.com
1
ICAR-Indian Agricultural Research Institute, Regional
Station, Tutikandi Centre, Shimla, Himachal Pradesh 04,
India
2
Chaudhary Sarvan Kumar Himachal Pradesh Krishi
Vishvavidyalya, Palampur, Himachal Pradesh 176061, India
3
ICAR-Indian Agricultural Research Institute, New Delhi
110012, India
•
cylindrica has been found to be the most economical and
efficient. The genotypic nonspecific production lacks
somaclonal variation and albino plants development
alongwith having higher regeneration rate coupled with
lower cost. Thus, integration of I. cylindrica mediated DH
system with conventional breeding will be instrumental for
future wheat breeding programmes.
Keywords Anther culture
Haploid

Imperata cylindrica
Maize mediated DH

Triticum aestivum
Introduction
Wheat is one of the ‘‘big three’’ staple crops alongwith rice
and maize which together make three-fourth of the world
grain production. Wheat alone occupies 25% of the world’s
cultivated area [1] and provides more than 20% of the
protein and calories for the world population [2]. Over the
last 50 years, breeding programmes worldwide have
achieved significant genetic gains in wheat yield, leading to
an increase in wheat yields of 0.7 t/ha/decade [3]. But, the
demand of more than 880 million metric ton has been
predicted by 2050 [4]. This necessity is deterred by too low
recent rates of yield growth which is aggravated by con-
tinuous shrinking and deteriorating production environ-
ment and natural resources alongwith continuous changing
nature of biotic and abiotic stresses. No doubt, to feed the
world in near future, the foreseeable plant breeding will
play a primary role, what will change is the tools that will
support/complement it. Thus, there is an urgent need to
develop new and more efficient breeding methodologies to
complement existing techniques, which will drive faster
yield gains in wheat.
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A varietal development programme in wheat requires
selfing until a condition of complete homozygosity is
attained, so as to ensure genetic purity and uniformity.
Wheat homozygous lines are conventionally being bred
by pedigree selection or by single seed descent technique.
However, eliminating heterozygosity is a time consuming
task (at least six growing season) using these traditional
breeding methods. To reduce this time, plant breeders are
integrating new plant biotechnological methods with tra-
ditional breeding techniques. In this aspect, one of the
advanced breeding technologies being employed to sup-
port the development of new wheat varieties and accel-
erate the genetic progress is doubled haploidy [5]. This
technology significantly reduces the varietal development
time (up to 2–3 years) alongwith generating 100%
homozygosity.
A ‘doubled haploid (DH)’ is a genotype formed when
haploid (n) cells successfully undergo either spontaneous
or artificial induced chromosome doubling. Haploids are
sporophytes that contain gametic chromosome numbers
(n). The haploids from einkorn, emmer, and dinkel wheat
possess n = x = 7, n = 2x = 14, and n = 3x = 21
chromosomes with genomic constitution of A, AB, and
ABD, respectively [6–8]. Spontaneous development of
haploid plants was first described by Blakeslee et al. [9] in
Datura stramonium. But, spontaneous haploid production
was not the solution (due to very low frequency) for its
commercial application. With the achievement of in vitro
culture of Datura anther by Guha and Maheshwari [10], the
potential of haploid for plant breeding arose. Breeding
wheat using DH technique is of great interest to geneticists
and breeders because of their complete homozygosity and
the short duration (1–2 years) of their production cycle and
the need for smaller population sizes [11]. Selection also
becomes more efficient, since the testing of homozygotic
lines in the field experiments gives a more realistic picture
of agronomic performance [12].
The doubled haploidy system has been widely used in
wheat breeding programs and numerous new wheat culti-
vars of DH origin were released in Europe, USA, Canada,
Brazil and China [13, 14]. DH populations are frequently
used in phenotypic and genetic research including con-
struction of molecular maps, localization of loci responsi-
ble for qualitative and quantitative traits (QTLs), and in
genomics and other molecular studies [15, 16]. DHs are
also frequently used in plant genome mapping [17] and
microspore derived embryos can be the target for genetic
transformation [18]. Therefore, DH lines are considered as
an attractive tool for both plant breeders and geneticists
because many of the problems associated with the assess-
ment of segregating populations can be overcome by their
usage. Moreover, DH technique saves at least three to four
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M. Patial et al.
generations of self-pollination for the fixation of homozy-
gous pure lines [19].
Traditional Versus DH Methods
The traditional method of stabilizing the genes and
developing a pure line requires self-pollinating plants for
eight or nine generations. On the other hand, the doubled-
haploid method is much faster and can produce a new,
genetically stable pure line in 1 year (Fig. 1).
Double haploid breeding not only helps in accelerating
conventional plant breeding programmes and make early
release of cultivars with superior and desirable traits but
also it has greater utility in other research aspects of plant
breeding, genetics and genetic engineering. Major differ-
ence between conventional and DH breeding are briefly
highlighted in Table 1.
DHs are important constituent of germplasm. These
also help in complementing backcross breeding by
transferring genes of interest between wild relatives thus
breaking genetic barriers. On the other hand, unique
complete homozygous nature of DHs, less time require-
ment to produce a large number of DHs, absence of
heterozygosity, efficiency over conventional systems and
absence of gametoclonal variation in DHs make them
very valuable material for genetic and molecular stud-
ies. The goals of the traditional method and the DH
method are almost same. However, the DH method
reaches those goals much faster.
DH Induction Techniques in Wheat
DH technology enables gene stacking and enhances ‘‘for-
ward breeding’’ by allowing hybrids to be bred with new
traits without locking up the germplasm. Although signif-
icance of DHs has been recognized for long time but with
the advent of biotechnology it has received renewed
emphasis. Researchers have applied different methods for
production of DHs in wheat [20–24] which includes anther
culture, ovule culture, chromosome elimination following
wide hybridization, haploid inducer gene/s and chemical
treatments. Zhang et al. [25] used meiotic restitution genes
to synthesize DH (SynDH) in wheat. An inducer line with
gene(s) for meiotic restitution and an alien species that can
be crossed without embryo rescue are needed. It is a three-
step procedure involving hybridization to induce recom-
bination, interspecific hybridization to extract haploids, and
spontaneous chromosome doubling by selfing the inter-
specific F1, with no special equipment or treatments
involved in DH production. Maheshwari et al. [26] and
Ravi and Chan [27] in Arabidopsis have proposed a
Doubled Haploidy Techniques in Wheat (Triticum aestivum L.): An Overview
Fig. 1 Schematic description of
comparison of conventional and
doubled haploidy technique
(embryo culture)
Table 1 Major difference between conventional and DH breeding
S. no. Particulars
1 Time required for cultivar development
2 Fixation of heterosis
3 100% homozygosity attainable
4 Expenditure
5 Recessive mutants identification
technology-driven approach which involves a centromere-
mediated genome elimination procedure for the develop-
ment of DH. In this method, haploids are produced from
the seed of plants by manipulating centromere-specific
histone CENH3. When the cenh3 mutant expressing
abnormal CENH3 is crossed to the wild type, chromo-
somes from the mutant are eliminated and eventually
haploids are produced. This method could be applied to
any plant because the CENH3 is universal in eukaryotes.
This approach has been undertaken in different crops but
success in wheat has not been reported [28] till date. All
these methods have varying rate of accuracy and efficacy.
Among all the DH induction techniques, anther culture
[29, 30] and wide hybridization of wheat with barley [31],
maize [32] and Imperata cylindrica [20] have been widely
used in breeding programmes in wheat and appear to be
the most effective for DH production due to their high
efficacy.
Haploidy breeding Conventional breeding
2–3 years 7–8 years or more
Possible Not possible
Yes No
More Less
Very easy Difficult
Anther Culture
In vitro androgenesis in wheat is being employed effi-
ciently in many countries of the world for the development
of DH lines. The first success in regeneration of wheat
plants through anther culture was achieved in the early
1970s [33–36]. The early culture methods primarily relied
upon the natural occurrence of embryogenic microspores
within certain genotypes, hence were inefficient, yielding
only a fraction of the plants. But, with the development of
the first haploid plants from anthers of Datura inoxia [10]
this technique has been speeded up. Anther and microspore
culture has the greatest potential, because it can be a key
for efficient DH breeding system on a large-scale as more
number of DH plants per floret can be obtained. The
technique uses the concept of in situ embryogenic nature of
pollen grains. For triggering embryogenesis and blocking
of gametophytic pathway, stress application forms a crucial
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 M. Patial et al.

step in this technique. The followings are the basic steps technique for haploid breeding, selection, various genetic
involved in anther culture in wheat: and physiological studies [41].
No doubt, the anther culture technique is being used in
• Collection of the flower buds at the onset of flowering
many cereal breeding programmes as more cost-effective
(at the mid–late uni-nucleate stage of development).
technique [19, 42]; however, the application of anther
Plants at boot stage are suitable for spike collection.
culture method in wheat breeding had several disadvan-
• The selected flower buds are transferred to the laminar
tages, among which the genotypic specificity is an impor-
airflow and surface sterile in closed buds. The flower
tant one. Many wheat genotypes, as well as F1 and F2
buds are surface sterilized by immersion in 70%
breeding combinations are incapable of morphogenesis in
ethanol for 10 s followed immediately by 10 min in
anther culture, which makes this method too expensive to
2% sodium hypochlorite. They are washed three times
be used for routine purposes. The haploid production
with sterile distilled water. Finally, the buds are
response varies not only among species but also within a
transferred to a sterile petridish.
species, with few genotypes having great response while
• The anthers with a pair of sterilized forceps are
others being recalcitrant. Almouslem et al. [43] Garcia-
removed and the anthers with the filaments are placed
Llamas et al. [44] reported hexaploid wheat genotypes to
to another petridish. The filaments are cut gently.
be more responsive compared to durum wheat genotypes
During excision of anthers, special care should be taken
and Sharma et al. [45] reported more responsiveness of
to ensure that they are not injured in any way. Damaged
winter genotypes than spring genotypes. Therefore, the
anthers should be discarded as they often form callus
technique is used in anther culture responsive genotypes
tissue from the damaged parts other than the pollen.
only. But, since the anther culture ability is a heritable trait
• Anthers are placed on agar solidified basal MS or White
and can be transferred into agriculturally desirable material
or Nitsch and Nitsch medium.
by crossing so responsiveness of hybrid combinations can
• The cultures are kept initially in dark. After 3–4 weeks,
be created [46] but again, it will increases the cost and time
the anthers normally undergo pollen embryogenesis
period of DH production. Other factor is albinism which
and haploid plantlets appear from the cultured anther.
remains a major hurdle to obtain haploids and DHs in
In some cases, anther may undergo proliferation to
wheat [47]. This lack of chlorophyll results from plastid
form callus tissue which can be induced to differentiate
deficiency in albino plants due to nuclear genes interacting
into haploid plants. At this stage, the cultures are
with pathways in plastid development [48]. The QTL for
incubated at 24–28 C in a 14 h daylight regime at
green plantlet in barley [49], rye [50], triticale [51, 52] and
about 2000 lux.
wheat [53] has been identified. Table 2 compiles the
• Approximately 50 mm tall plantlets are freed from agar
researchers who have induced haploids via anther culture
by gently washing with running tap water and then
in wheat crop.
transferred to small pot containing autoclaved potting
compost. Each plantlet is covered with a glass beaker to
prevent desiccation and is maintain in a well-lit humid Table 2 Anther culture technique used by different workers for
induction of haploids in wheat
green-house. After one week, the glass beakers are
removed and the plantlets are transferred to larger pots Authors Year Maximum number References
of green plant
to mature and finally flower.
regenerants/100
anthers

Marsolais et al. 1986 11.19 [54]

Progress in Anther Culture Technique of DH Lashermes et al. 1991 22.40 [55]
Production in Wheat Lashermes 1992 18.80 [56]
Alvarenz 1992 36.70 [57]
A number of DH wheat varieties (Florin, Jinghua 1, Yun- Trottier et al. 1993 46.70 [58]
hua 1, McKenzie etc.) have been developed by the anther Masojc et al. 1993 6.20 [59]
culture method [37]. In wheat, the first success via anther Alvarez et al. 1994 49.00 [60]
culture was achieved in China [38, 39] where two cultivars
Sadasivaiah et al. 1999 4.73 [61]
Jinghua No. 1 and No. 764 were selected in a breeding
Kim et al. 2005 22.00 [62]
program using DH lines. This method was also used in
Khiabani et al. 2008 24.13 [63]
France and Hungary, where two cultivars Florin [37] and
EL-Hennawy et al. 2011 10.00 [19]
Gk Delibab [40] were released in 1985 and 1995. Studies
Lantos et al. 2013 28.67 [64]
concerning the wheat anther culture technique produced
Castillo et al. 2015 58.00 [65]
significant results and it is now possible to use this

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Doubled Haploidy Techniques in Wheat (Triticum aestivum L.): An Overview
Factors Affecting DH Development Following
Anther Culture
Apart from genotypic factors [66, 67] the development of
wheat DH it is also affected by the following factors.
• Environmental conditions [45, 68]: Researchers had
highlighted the higher regeneration response of the
plants grown under controlled environmental condi-
tions (e.g., phytotron) than the field grown plants [69].
• Physiological status of the donor plants [70]: The
frequency of androgenesis is usually higher in anthers
harvested at the beginning of the flowering period and
declines with plant age [71]. Also, it has been observed
that the anthers from primary tillers in most cereals
(except rice) were more responsive than those from
lateral tillers.
• The developmental stage of the microspore at the time
of culturing strongly affects the efficiency of haploid
induction via influencing the microspore’s totipotency
during in vitro androgenesis. In wheat, spikes contain-
ing anthers with pollen at the mid–late uninucleate
stage of development have been reported to be more
effective [72].
• Pre-treatment of the donor material [70]: Pre-treatment
is needed to arrest the microspores in their gameto-
phytic pathway. Pre-treatment of cultured anthers/
pollen grains—like application of certain physical
(temperature shock, centrifugation, c irradiation) and
chemical (auxins) treatments to cultured anthers or
pollen grains prior to standard culture room conditions,
has been proven advantageous for in vitro androgen-
esis. For example, cold-temperature pretreatment at
4 C for 5 weeks was reported to be effective to
improve embryogenesis induction and green plant
regeneration in otherwise recalcitrant durum wheat
genotypes [73]. Mannitol pretreatment is also effective
in improving the efficiency of anther-culture in durum
wheat [73, 74].
• Chemical composition of the culture medium including
plant growth regulators [75, 76] and other amendments:
The reprogramming of explants from gametophytic to
sporophytic pathway, for example, depends upon the
type and concentration of carbohydrates and plant
growth regulators [77]. The requirement of culture
medium is species and genotype specific. Hence, there
is no single culture medium that would be suitable for
haploid induction systems in various crops. For exam-
ple Grauda et al. [78] noted an increased DH produc-
tion efficiency in wheat through the utilization of
androgenic microspore culture induction medium with
copper, which has been effective in reducing albino
plants and increasing green plant regenerants.
Pretreatment of wheat spikes with 0.4 M mannitol at
4 C followed by embryoid induction and regeneration
in a medium fortified with ascorbic acid was found to
produces highest number of green plants [79].
• Incubation conditions including temperature, light and
duration of the induction [80]: Donor plant temperature
and light conditions influencing haploid induction in
wheat has been reported by many researchers [81–83].
For anther and microspore culture as well as for embryo
culture in wheat 9 maize and wheat 9 I. cylindrica
system, initial incubation of cultures in dark has found
to be the best for haploid regeneration [20].
Limitations of Anther Culture
Though considerable progress has been made in the field of
wheat anther culture since 1973, use of anther culture to its
wider application in wheat breeding is limited by:
• its strong genotype specificity [43, 44],
• low frequency of haploids [47],
• unpredictable genetic alterations due to gametoclonal
variation [47] and,
• high rate of albinism in regenerants [47].
But, despite these limitations, the anther culture technique
has been successfully applied in many wheat breeding
programs and there have been continuous efforts for
increasing the efficiency of this DH production technique.
Although earlier workers obtained low frequencies (0.7%)
of green plants [34], the frequency of microspore embryo-
genesis in common wheat has been improved considerably
[84]. Liu et al. [72] has developed an efficient DH
production system using microspores which includes
multiple modified steps (e.g., pre-treatment of spikes with
inducer, microspore isolation, complex culture media
system), and therefore is rather complex. Also, Grauda
et al. [85] have reported increased DH production
efficiency through the utilization of AMC (modified MS)
induction medium with added copper. Hence, incorporat-
ing different modifications anther culture technique can be
used for efficiently production of DH wheat plants.
DH Production via Wide Hybridization
The technique of DH production via wide hybridization has
opened new possibilities of integrating DH system in wheat
breeding programs [20, 24, 86–88]. Non-responsive geno-
types for callus induction and plantlet formation in the
anther culture method proved to be good parental material
in this technique [89]. To produce DH plants via inter-
123

specific/inter-generic hybridization, wheat spiklets are
crossed with another closely related species that stimulates
the beginnings of cell differentiation. However, the dif-
ference between the two crossed species is sufficiently
wide that the genome of the other species does not become
involved, thus rendering the development of haploids.
Haploid embryos in formed caryopses lacked endosperm
which can be successfully obtained by in vivo treatments
with plant growth regulators on pollinated spikes [23, 90].
These haploid embryos are cultured under laboratory
conditions. The chromosomes are stimulated chemically
for duplicating them, developing into a plant that bears two
identical haploid genomes. This double haploid individual
can then be selfed. This technique is expensive, but to a
breeder the time saved justifies the cost.
Several inter-specific/inter-generic hybridization meth-
ods are used for haploid production in wheat. These
methods include wheat 9 I. cylindrica [20], wheat 9 -
maize [87], wheat 9 pearl millet [91], wheat 9 tripsacum
[92], wheat 9 teosinte [93], wheat 9 barley [31] and
wheat 9 job’s tears [94]. Such inter-generic crosses have
been found to be effective for the production of DH plants
in wheat. All these methods include embryo rescue tech-
nique and haploids are formed due to chromosomes elim-
ination of the pollen parent during embryo development.
Hordeum bulbosum Mediated DH Technique
In Hordeum bulbosum mediated DH technique, the H.
bulbosum chromosomes are eliminated during embryo
development from a hybrid zygote [31]. The underlying
mechanism of this technique is uniparental chromosome
elimination during early development stages of a hybrid
embryo leading to the formation of haploid embryo. In this
system, the presences of incompatible genes (Kr1 and Kr2)
situated on the 5A and 5B chromosomes expresses in the
style of most of the wheat genotypes and inhibit H. bul-
bosum pollen tube growth, thereby reducing the cross-
ability between wheat and H. bulbosum [95]. For this
reason, at present, this method is restricted to crossable
wheat genotypes only.
Wheat 3 Pearl Millet and Wheat 3 Sorghum
System of DH Induction
Crossing of wheat with sorghum [96] and pear millet [91]
resulted in successful fertilization and elimination of
paternal chromosomes from hybrid zygote suggesting their
potential for DH production in wheat. High frequencies of
wheat DH were obtained when crossing was done followed
by 2, 4-D treatment in both the species. However, crosses
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M. Patial et al.
with sorghum showed a strong genotypic response.
Therefore, among all methods, wheat 9 maize and
wheat 9 I. cylindrica are the best for DH production in
wheat [24, 97].
Maize Mediated DH System
In maize mediated DH system, the intergeneric crosses
between wheat and maize followed by elimination of the
genome of maize has been considered an efficient method
for the induction of haploid zygotic embryos [98]. This
system was reported by Zenkteler and Nitzsche [99] for the
first time. The production of haploid wheat plants through
wheat 9 maize hybridization was reported by Laurie and
Bennett [87] through spikelet culture and Comeau et al.
[100] through ovule culture. Laurie and Bennett [87]
developed the first in vitro method to rescue the haploid
wheat embryos from the wheat 9 maize crosses. Later,
Laurie and Bennett [101] showed that when maize pollen
pollinates the wheat floret, wheat egg is fertilized and
zygote is formed. Initially, the resultant zygote has haploid
number (n) of wheat and maize chromosomes but, since the
karyotypes of wheat and maize are different so the zygotes
are unstable. Centromere of maize chromosomes fails to
attach to spindle fibers. Hence, maize chromosomes are
unable to move to spindle poles during cell division. This
non-attachment of chromosomes is due to loss of cen-
tromeric activity of maize chromosomes. As a result, maize
chromosomes are rapidly lost and after a few cell divisions
haploid embryo having 21 wheat chromosomes are formed
and no endosperm is formed as a result of wheat 9 maize
cross. This system is genotypic non-dependent and more
efficient and simple than wheat 9 H. bulbosum [88] or
anther culture [61, 102]. The maize pollen is insensitive to
the incompatible genes (Kr1 and Kr2) situated on the 5A
and 5B wheat chromosomes [88], tissue culture associated
variations and albinism in regenerants [81]. Moreover, the
sexual route of DH production systems in wheat is free
from tissue culture associated variations and the problem of
albinism in regenerants.
In wheat 9 maize system, the genotypic effect on hap-
loid embryo production is still controversial phenomenon
in literature. Suenaga and Nakajima [103] reported that
maize genotypes influence the success of wheat 9 maize
crossing system. Laurie and Reymondie [104] observed
that in crosses of winter and spring wheat with F1 hybrid of
maize, significant differences for haploid embryo produc-
tion were present between groups but absent within groups.
Suenaga and Nakajima [103] studied the effects of maize
pollen on haploid wheat embryo production and observed
differences in haploid embryo production by different
maize genotypes on single wheat genotype. Suenaga [88]
Doubled Haploidy Techniques in Wheat (Triticum aestivum L.): An Overview
studied the effect of 55 maize varieties and lines by pol-
linating Fukuho-Komigi as female parent and their study
revealed that considerable genotypic differences with
embryo formation rate ranging from 16 to 36%. Verma
et al. [105] quantified the effect of maize genotypes
through line (wheat) 9 tester (maize) analysis and found
that striking differences of maize genotypes on frequency
of haploid embryos formation and plantlets regeneration.
They also concluded that the genotypic influence of maize
genotype was greater than that of wheat. But, the genotypic
specificity of the wheat 9 maize mediated haploid pro-
duction system is not up to the magnitude, as reported for
other haploid production systems including anther culture
[89, 102] and wheat 9 H. bulbosum [106]. Hence, haploid
induction rate in wheat via maize mediated system can be
enhanced by selecting of more responsive maize
genotypes.
Most of the investigators except Oury et al. [107]
reported that wheat 9 maize system is superior over
androgenesis. Kisana et al. [89] and Sadasivaiah et al. [61]
showed that wheat 9 maize is quite fast, easiest and 2–4
times superior in terms of overall plants regeneration.
Sadasivaiah et al. [61] obtained 6.29 green haploid plants
out of 100 florets pollinated as compared to 1.64 from 100
cultured anthers. The apparent higher efficacy for green
haploid plant regeneration through this technique is due to
the higher rate of embryo formation, higher germination
rate of polyhaploid embryo and non existence of albinism.
Therefore, based on studies conducted by different work-
ers, it can be concluded that maize mediated DH produc-
tion technique is more efficient than anther culture. The
low level of genotype specificity, absence of albinism and
ease of application, make this technique more efficient than
anther culture for the production of polyhaploids in com-
mon wheat.
Factors Affecting DH Production and Studies
in Maize Mediated Technique of DH Production
Haploid production efficiency in maize mediated system is
affected by the proportion of pollinated florets which
develop haploid embryos (yield). Yields of haploid
embryos have been reported to be as high as 53% [108] and
as low as 1% [103] depending upon a wide range of
variables. Factors that affect the yield of haploid embryos
include genotypic differences between individual wheat
and maize lines [91, 109], the timing and use of exogenous
growth substances to stimulate ovule development [103]
and environmental factors (especially temperature) during
and following pollination. Haploid production efficiency is
also influenced by the proportion of haploid embryos which
germinate and develop into plantlets (germination
percentage). Other factor which influences the germination
success of haploid embryos is the timing of embryo rescue
[110–112]. Haploid embryos rescued from 2 to 3 weeks
after pollination with maize gives greater haploid produc-
tion efficiency [89, 104, 108]. Several types of solid media
have been successfully used for germination of haploid
embryos, including Norstog II [100] orchid agar [87], half
strength MS [103, 113] and B5 [109, 114] and hence
composition and proportion of nutritional and hormonal
factors in the germination medium also affect the success
of DH development.
Pratap et al. [115] compared four maize genotypes by
crossing them with seven Triticale 9 wheat F1s for the
haploid induction parameters and reported considerable
variation for these parameters among the maize genotypes.
Niroula et al. [21] reported that the efficiency of polyploidy
induction in wheat through wheat 9 maize system is also
determined by wheat genotypes under study. They have also
reported that the higher number of seed set always not yiel-
ded higher number of culturable embryos and the higher
number of culturable size of embryo is one of the crucial
factors that the determine the germination and post germi-
nation efficiency. Moradi et al. [116] reported that the per-
centage of haploid regeneration and DH production is
dependent on the wheat genotypes, but it is independent of
maize genotypes. Dhiman et al. [117] compared seven maize
genotypes for their efficiency in haploid induction parame-
ters in the elite spring wheat cross F1s and reported that every
maize genotype behave differently in the haploid induction
parameters. Inagaki and Mujeeb-Kazi [118] compared the
frequencies of polyhaploid production in three hexaploid
wheat varieties; each pollinated with maize, pearl millet and
sorghum pollen and found that all the wheat varieties pro-
duced embryos in crosses with maize lines. However,
embryo formation in wheat lines crosses with pearl millet
and sorghum was highly genotype specific. Maize mediated
polyhaploid production was inferred to be more stable than
those mediated by pearl millet and sorghum lines.
The chromosome elimination approach following
wheat 9 maize system has been successfully utilized for
the production of DHs from the winter 9 spring wheat
hybrids [45, 119]. Wang et al. [97] studied the frequency of
fertilization and embryo formation in wheat 9 maize
crosses. Hybrid embryos and endosperms obtained from
wheat 9 maize hybridization were karyotypically unsta-
ble and were characterized by rapid elimination of the
maize chromosomes to produce haploid wheat embryos.
Hence, the reduced genotypic specificity, absence of
albinism and ease of application make the wheat 9 maize
hybridization technique more efficient than the anther
culture and the Bulbosum technique for the production of
haploids in common wheat. This system has been widely
modified and used for DH production in wheat in many
123
 M. Patial et al.

Table 3 Different wide hybridization techniques used by researchers for production of wheat DHs
Haploid induction Method Authors Year Maximun number of Maximum haploid References
haploid embryo plant regenerated
rescued of a cross of a cross

Wheat 9 maize and Matzk and Mahn 1994 – 450 [123]

wheat 9 pearl millet crosses
Wheat 9 sorghum Ohkawa et al. 1992 – 38 [96]
Hordeum bulbosum mediated DH Jie and Snape 1987 27 – [124]
Bozorgipour and Snape 1991 4 1 [125]
Inagaki and Tahir 1990 55 21 [109]
Khan et al. 2014 33 – [126]
Maize mediated DH system Laurie and Bennet 1988 47 31 [87]
Inagaki and Tahir 1990 113 67 [109]
Laurie and Reymondie 1991 311 191 [104]
Lefebvre and Devaux 1996 1703 610 [127]
Wedzony et al. 1998 – 76 [128]
Mihailescu and Giura 1998 2220 1821 [129]
Niroula et al. 2007 53 26 [21]
Khan and Ahmed 2011 23 – [130]
Hussain et al. 2012 15 – [131]
Bhattacharya et al. 2015 27 – [23]
– Not reported

parts of the world by different researchers (Table 3).
Accordingly, Inagaki and Tahir [120], Sun et al. [121] and
Kasha et al. [122] advocated the use of this technique for
breeding purpose by raising a large number of wheat
haploids.
Imperata cylindrica Mediated DH Technique
Chaudhary et al. [20] has identified another system of wide
hybridization which performs superior over maize- mediated
technique. This system uses I. cylindrica (cogon grass) as a
pollen source and has been found to be more efficient alter-
native pollen source for DH production in wheat. I. cylindrica
is a winter season plant and coincides well for flowering with
that of wheat and triticale under natural conditions. I. cylin-
drica is genotype nonspecific for hybridization with any
variety of wheat, triticale or their derivatives and its pollen is
readily available in abundance during the wheat hybridization
period. This wild weedy perennial grass doesn’t require
repeated sowings and is available under natural conditions in
almost all parts of the world.
Cytological investigation of the wheat 9 I. cylindrica
mediated chromosome elimination system has shown that
there is no endosperm formation and the elimination of
chromosomes of I. cylindrica takes place in the first
zygotic division thus allowing the production of embryo-
carrying seeds [132]. Chaudhary [133] has reported that
combination of I. cylindrica- mediated DH production and
molecular cytogenetic techniques like GISH and FISH can
accelerate the alien introgression mediated wheat breeding
programmes in various farming systems.
Wheat 9 I. cylindrica system of DH breeding following
chromosome elimination approach has number of advan-
tages over the existing wheat 9 maize system as
following.
• Since, I. cylindrica is a winter season plant its flowering
coincides well with wheat under natural conditions and
there is no need to artificially raise the pollen parent as
desired in case of maize. Also, the pollens of I.
cylindrica are available in abundance during the wheat
hybridization period.
• Like maize, I. cylindrica is genotypic non-specific and
hence superior to anther culture system.
• I. cylindrica performs significantly better than maize
for all the haploid induction parameters in wheat and
triticale and can produce more DH populations in
comparison to the maize-mediated system in the same
time period.
Hence, I. cylindrica mediated system of DH breeding has
been proved to be very efficient and economically viable
approach for acceleration of wheat breeding endeavours
with enhanced precision and efficiency. This innovation
has globally opened new vistas for commercial utilization
of the new system for large scale production of DHs of
wheat to be used directly as improved varieties or gene
mapping populations.

123
Doubled Haploidy Techniques in Wheat (Triticum aestivum L.): An Overview

Progress in Imperata cylindrica Mediated highlighted the importance of I. cylindrica mediated

Technique of DH Production in Wheat
A comparison of efficacy of wheat embryo development by
wheat 9 maize and wheat 9 I. cylindrica systems by dif-
ferent researchers [20, 24, 115] concluded the superiority
of former system than latter. Hence, higher rate of embryo
development, absence of albinism and higher regeneration
rate from embryo makes this wheat 9 I. cylindrica cross-
ing system efficient and easy to use than other techniques
used for DH production in wheat (Table 4).
Pratap et al. [115] tested different Graminae genera and
reported that among all, haploid plant production via I.
cylindrica produced more embryos and haploids over
others. Chaudhary [134] reported that I. cylindrica per-
formed significantly better than maize for all the haploid
induction parameters in wheat, triticale and their deriva-
tives. Chaudhary et al. [135] reported that I. cylindrica
performed better than maize in all the crosses of win-
ter 9 winter wheat, spring 9 spring wheat, win-
ter 9 spring wheat derivatives, whereas it exhibited par
excellence in triticale and wheat 9 rye derivatives.
Kishore et al. [136] used maize and I. cylindrica for
haploid induction in spring and winter wheat cross with
Himalayan rye derivatives and observed that I. cylindrica
produced more haploid embryos whereas maize did not
produce any haploid embryo. Chaudhary et al. [137]
reported that I. cylindrica performed better than maize and
leads to high precision genetic upgradation in wheat.
Badiyal et al. [138] had highlighted the potential of I.
cylindrica mediated approach for haploid induction in
Triticale 9 wheat cross. Mahato and Chaudhary [24] have
also highlighted the relative efficiency of I. cylindrica over
maize for haploid induction in T. durum. In this technique,
the chromosomes of I. cylindrica are completely elimi-
nated from the nuclei in the first cell division due to lack of
functional kinetochores [132]. Patial et al. [139, 140] also
chromosome elimination technique for getting high fre-
quency of haploid plants in wheat. Mahto and Chaudhary
[24] reported I. cylindrica to be significantly better pollen
source for haploid induction in durum wheat over maize in
terms of pseudoseed formation, embryo formation, haploid
regeneration and haploid formation efficiency.
Although a lot of technical refinements have been made
in the past, in order to use this system in practical
breeding programmes, further strategic research on
enhancement of embryo formation, germination and green
plant regenerations are urgently needed. These compo-
nents, in future, can be improved by manipulating suit-
able auxin sources and various environmental factors such
as light and temperature regimes. This technique could
thus complement conventional breeding programmes and
accelerate the release of new varieties in developed
countries, as well as in developing countries where rapid
varietal development is critical for sustainable wheat
production systems. Therefore, the integration of DH
technology via wheat 9 I cylindrica system into wheat
genetics and breeding program is extremely useful to
reduce the breeding cycle, develop new cultivars and
improve our understanding of agronomically important
genetic traits.
Advantage of Wide Crosses for DH Induction
in Comparison to Anther Culture
The superiority of wide crosses over other techniques of
DH production includes:
• higher efficacy (2–3 times more efficient for green plant
production than anther culture),
• simplicity,
• less genotype dependent response,

Table 4 Use of I. cylindrica mediated chromosome elimination technique by some researchers for development of wheat haploids
Haploid induction Method Author Year Maximum seed Maximum embryo Maximum References
formation formation regeneration
frequency (%) frequency (%) frequency (%)

Imperata cylindrica mediated DH Chaudhary et al. 2005 84.5 47.7 75.0 [20]
Pratap et al. 2005 – 25.48 34.17 [115]
Chaudhary et al. 2008 84.5 47.7 75.0 [134]
Rather et al. 2013 77.45 41.00 17.15 [141]
Badiyal et al. 2014 50.23 33.15 71.25 [138]
Patial et al. 2015 95.97 – – [140]
Celiktas et al. 2015 31.1 15.5 11.5 [142]
Chaudhary et al. 2015 57.70 9.58 0.057 [143]
– not reported

123
 M. Patial et al.

• less gametoclonal variation, and • In wheat, DH lines have been used for identifying and
• less time consuming. mapping QTLs associated with resistance to foliar
disease of wheat, important agronomic traits, variation
in grain yield in response to varying nitrogen applica-
tion [150], grain quality [151], common bunt resistance
Uses of Double Haploids in Basic and Applied
and plant height [152], leaf rust (LR) resistance, stem
Plant Breeding Research
rust resistance [153], stripe rust resistance [154] and
adult plant yellow rust and stem rust resistance.
The production of haploids followed by induced chromo-
Recently, QTL controlling Ergot sclerotia size in
some doubling is widely being used in advanced breeding
hexaploid wheat has been identified by using a DH
programs of several agricultural species. In wheat number
mapping population [155].
of varieties has been developed by one or the other method
• Due to complete homozygous nature, DH population
of haploid production in different parts of the world
are ideal for genome mapping.
(Table 5). The DH-derived wheat cultivars currently
• DH and MAS technologies have been successfully
occupy significant acreage in some of the countries. For
employed to improve host plant resistance in wheat. For
example, twenty- five wheat cultivars accounted for more
example wheat DH has been produced which are
than one-third of the Canadian wheat acreage, with Lillian
resistant to fusarium head blight (Fusarium gramin-
and AC Andrew being the most widely grown wheat cul-
earum) [156], stem rust (Puccinia graminis f. sp. tritici)
tivars in Canada [144, 145]. Also, a DH-derived wheat
[157], and stripe rust (Puccinia striiformis f. sp. tritici)
cultivar Glossa grew in 16% of the total wheat area
[158].
(300,000 ha) just in 5 years after its release in Romania
• Haploid embryos can be used for engineering of DH
[146]. Designated lab like Heartland Plant Innovations
homozygous for the transgene. A drought tolerant and
(HPI)—a public private partnership– has established a DH
stable transgenic haploid wheat plants using the barley
laboratory at Manhattan, Kansas (USA) for use by public
gene HVA1was bred using explants from anther
and private wheat breeders [147]. DHs have several uses as
culture-derived haploid embryos of the commercial
following.
wheat cultivar CPAN1676 through Agrobatcerium-
• Double haploid technology reduces the varietal devel- mediated genetic transformation [159]. The transgenic
opment time from 12–13 to 6–7 years by decreasing the plants were chromosome doubled with colchicine to
selection cycles for obtaining desired homozygosity. produce DHs that did not show transgene silencing
• DH lines can be directly released as variety or can until the T4 generation. Furthermore, these DH plants
make a valuable genetic stock. had faster seed germination and seedling establishment
• DH lines derived from hybrid crosses can be used to and show better drought tolerance that non-transgenic
extract lines with yield potential similar to hybrids DH plants.
[148]. For example, the development of hybrids DH • In some plants of horticultural importance where
lines originating from heterotic F1 crosses in wheat, vegetative propagation take too much time for com-
when evaluated together with their respective parental mercialization, anther culture can be used.
lines and control, performed well and few transgressed
significantly the higher yielding parent and the control
cultivar [19].
Conclusion
• DH lines are the best material for quantitative
genetic studies due to their complete homozygous
The DH technology, undoubtedly, is the powerful mean to
nature.
modernize wheat breeding for deriving completely
• In landraces, in vitro DH technology can be used to
homozygous lines for hybrid development and their
overcome the loss of variability which occurs at later
deployment. But, its implementation requires new skills on
generations in outbreeding species due to increased
DH line production. Proper training of the manpower in
homozygosity [149]. DH technologies can unlock the
various aspect of DH production (haploid induction, hap-
genetic diversity from highly heterogeneous landrace
loid identification, chromosome doubling, and DH line
populations thereby broadening of the cultigens gene
recovery) is the first and crucial point for the success of DH
pool.
production. Secondly, the haploid plants obtained are often
• DH lines are used to study the components of
weak and vulnerable to various environmental stresses; the
quantitative genetics including gene linkage, number
technique for the same has to be standardized. Thirdly,
of genes governing the trait, interactions, additive
integration of the DH technology with other available
variances and location of genes on chromosomes.

123
Doubled Haploidy Techniques in Wheat (Triticum aestivum L.): An Overview
Table 5 Wheat DH varieties produced in different countries by using different haploid induction techniques
Haploid production method Varieties Country
Anther culture Hua Pei 1, Lung Hua 1, China
Jinghua 1, Yunhua 1,
Yunhua 2
Kharoba Morocco Dwivedi et al.
Florin France
McKenzie Canada
Wheat 9 Maize Glosa, Faur F, Litera, Miranda, Romania Saulescu et al.
Gruia
BRS 328 Brazil
Emerson, AAC Elevate, AAC Canada
Connery, Snowstar, Sunrise,
Bhishaj, Lillian etc.
Wheat 9 Imperata Him Pratham India
cylindrica
biotechnological tools like marker assisted selection,
induced mutagenesis and transgenic development will
enhance the genetic gains and breeding efficiency, and
ultimately leads to the fast development of hybrids. No
doubt lot of efforts in DH production via different method
has been done but still its wide use is limited due to
decreased number of DH plants production. The supple-
mentation of DH technology into wheat genetics and
breeding program is extremely useful to reduce the
breeding cycle, develop new cultivars and improve our
understanding of agronomically important genetic traits.
Hence, integration and joint efforts of industries,
researchers from a range of discipline and breeders are
needed to develop the best strategies for deploying DH
system for practical use in wheat breeding.
Acknowledgements The authors are thankful to Dr. K.V. Prabhu, JD
(R), ICAR-IARI, New Delhi and Dr. H.K. Chaudhary, Head, CSK,
Himachal Pradesh Krishi Vishvavidyalaya, Palampur for providing
necessary help for initiation of I. cylindrica mediated DH work at
ICAR-IARI, Regional Station, Shimla. Funding was provided by
Indian Council of Agricultural Research.
Compliance with Ethical Standards
Conflict of interest The authors do not have any conflict of interest
to declare.
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