Review TRENDS in Plant Science Vol.12 No.

The resurgence of haploids in higher

plants
Brian P. Forster1, Erwin Heberle-Bors2, Ken J. Kasha3 and Alisher Touraev2
1
SCRI, Genetics Programme, Invergowrie, Dundee, DD2 5DA, UK
2
Max F. Perutz Laboratories, Department of Plant Molecular Biology, Vienna University, Dr. Bohrgasse 9, A-1030 Vienna, Austria
3
Department of Plant Agriculture, University of Guelph, Guelph, Ontario, N1G 2W1, Canada

The life cycle of plants proceeds via alternating
generations of sporophytes and gametophytes. The
dominant and most obvious life form of higher plants
is the free-living sporophyte. The sporophyte is the
product of fertilization of male and female gametes
and contains a set of chromosomes from each parent;
its genomic constitution is 2n. Chromosome reduction
at meiosis means cells of the gametophytes carry half
the sporophytic complement of chromosomes (n). Plant
haploid research began with the discovery that sporo-
phytes can be produced in higher plants carrying the
gametic chromosome number (n instead of 2n) and that
their chromosome number can subsequently be doubled
up by colchicine treatment. Recent technological inno-
vations, greater understanding of underlying control
mechanisms and an expansion of end-user applications
has brought about a resurgence of interest in haploids in
higher plants.
The discovery of haploids
Dorothy Bergner was the first to describe the natural
occurrence of sporophytic haploids in the weed species
Datura stramonium in 1922 [1]. This was quickly followed
by similar reports in tobacco (Nicotiana tabacum) [2],
wheat (Triticum aestivum) [3] and subsequently in several
other species [4,5]. Unfortunately it took another 40 years
to develop protocols to produce haploid plants in the
laboratory: the breakthrough came with anther culture
in Datura [6,7] followed by chromosome elimination, sub-
sequent to wide hybridization [8]. A rapid expansion in
research ensued with further development of chromosome
doubling techniques (n ! 2n) that converted sterile hap-
loids (H) into fertile, homozygous doubled haploid (DH)
plants [9]. A milestone was set with the release of the first
DH crop plant, the cultivar Maris Haplona of rapeseed
(Brassica napus) in the early 1970s [10] and Mingo in
barley (Hordeum vulgare) in 1980 [11], although DH lines
in maize (Zea mays) have previously been successful for
commercial production using spontaneously occurring
haploids [12]. The surge in interest gave rise to the First
International Symposium: ‘Haploids in Higher Plants’
which took place in 1974 [13]. At that time there was a
great deal of optimism about the opportunities that hap-
loids might bring, particularly DHs, because the DHs were
Corresponding author: Touraev, A. (alisher.touraev@univie.ac.at).
Available online 12 July 2007.
www.sciencedirect.com 1360-1385/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.2007.06.007
extremely valuable to geneticists and plant breeders
wishing to exploit true breeding lines. But, in his opening
address on the status of haploid research, Sir Ralph Riley
[5] had these sobering words ‘I believe it is quite likely that
haploid research will contribute cultivars to agriculture in
several crops in the future. However, the more extreme
claims of the enthusiasts for haploid breeding must be
treated with proper caution. . .’. This advice of caution
proved to be wise. As with Datura anther culture, devel-
oping similar techniques for DH production in other
species proved troublesome and time consuming and
research slumped into an empirical, technical phase. As
a consequence there was little take up by researchers and
plant breeders who were more interested in the end pro-
duct – DH lines – than in developing enabling technologies.
However, recent scientific and technological innovations,
greater understanding of underlying mechanisms and an
expansion of end-user applications has brought about a
resurgence of interest in haploids in higher plants. Nowa-
days, haploids and doubled haploids have been reported in
more than 200 plant species belonging to almost all
families of plant kingdom, from Aconitum carmichaeli to
Zingiber officinale [14], using various spontaneous and
induced (in vitro) methods (Figure 1).
Haploids from wide species hybridization and
chromosome elimination
Haploid embryos can be produced in plants after
pollination by distantly related species [8,15,16]; the pro-
cess is best documented in cereals. The mechanisms
involved are still being elucidated, but in most cases
normal double fertilization takes place to form a hybrid
zygote and endosperm. Subsequent cell division in the
zygote results in the elimination of paternal chromosomes
leaving a haploid embryo. The rapidly dividing endosperm
also suffers chromosomal elimination and usually aborts
early in seed development; as a consequence the haploid
embryo must be rescued by in vitro culture. The first and
most widely used method of this type is the ‘bulbosum’
method in which a high frequency of haploids of cultivated
barley, Hordeum vulgare can be obtained after crossing
with the related species Hordeum bulbosum [8]. The
method was quickly adopted and improved by researchers
and barley breeders worldwide [17]. It has advantages in
that the methods involved (emasculation, pollination,
embryo culture) are familiar to breeders, cross combi-
nations can be manipulated to maximize haploid embryo
 Review TRENDS in Plant Science Vol.12 No.8 369

Figure 1. Methods of plant haploid production. Haploids can be induced either spontaneously or by various in vitro methods using female and male parts of the plant.
Spontaneous haploids can be observed via semigamy, polyembryony, chromosome elimination, gynogenesis and androgenesis at extremely low frequencies.
Alternatively, haploids can be induced via in vitro cultured anthers or microspores (androgenesis), ovules or ovaries (gynogenesis) and by distant hybridization using
irradiated pollen or pollen from another species. Androgenesis – male parthenogenesis in which the embryo contains only paternal chromosomes owing to the failure of
the egg nucleus to participate in fertilization or the regeneration of whole plants from sexual male cell culture: anthers or isolated immature pollen. Gynogenesis –
spontaneous or induced female parthenogenesis in which the embryo contains only maternal chromosomes owing to the failure of the sperm cell to fuse with the egg
nucleus. Interspecific crossing – development of a haploid embryo by fertilizing an ovule with pollen of another species and the subsequent elimination of the
chromosomes of the pollen. Pollination with irradiated pollen – development of haploid embryo by fertilizing an ovule with irradiated (inactive) pollen that nevertheless is
capable of introducing cellular divisions in the ovule and in the development of the embryo. Semigamy – an abnormal type of fertilization whereby either reduced or
unreduced male and female gametes participate in embryo formation but fertilization does not occur. Polyembryony – the production of two or more embryos in one seed,
owing either to the existence and fertilization of more than one embryonic sac or to the origination of embryos outside of the embryonic sac.

production by eliminating genotypic dependency on the
maternal side, and there is no seedling mortality caused
by albinism (a major problem in microspore-derived hap-
loids of cereals). However, there are disadvantages: flower-
ing times of both parents need to be matched (seasonal
limitations) and additional chemicals treatments, such as
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colchicine, are required to double up the chromosome
complement of haploid plants (n ! 2n). In species other
than cereals, inefficient chromosome doubling is con-
sidered to be the main bottleneck of this technology. Wide
hybridization is also effective in doubled haploid pro-
duction in wheat [18,19]. Several species, for example,
370 Review TRENDS in Plant Science
H. bulbosum, teosinte, sorghum and pearl millet have been
tested as pollinators, but currently the most popular pollen
donor is maize (Zea mays), which is also effective in indu-
cing haploid embryos in triticale (xTriticosecale) [20], rye
(Secale cereale) [21], and oat (Avena sativa) [22]. A similar
system operates in potato (Solanum tuberosum) [23]. Cul-
tivated potato, S. tuberosum, is tetraploid (2n = 4x) and the
term di-haploids is used to described haploid plants with
the gametic chromosome constitution. Matrilineal potato
di-haploids are produced by inter-ploidy crosses of tetra-
ploid S. tuberosum with the diploid relative Solanum
phureja. In these crosses, both sperm nuclei can fuse with
the central cell of the ovule to form a functional endosperm
that initiates and supports the parthenogenic development
of the unfertilized egg [23]. The frequency of seed contain-
ing DH embryos is low, but these can be easily differen-
tiated from hybrid seed by exploiting a colour marker gene
carried by the pollinator line. In maize (Z. mays) inbred
‘inducer’ lines are available [24] that can produce 10%
haploid progeny, but the mechanisms involved are poorly
understood and the genetics is complex [24]. As in the
potato system, a seed colour marker is used to select for
haploid embryos.
Haploids via gynogenesis
In gynogenesis, haploid cells of the female gametophyte
(usually the unfertilized egg cell) are stimulated to develop
into an embryo in an induced process similar to partheno-
genesis. Gynogenesis is used in onion (Allium cepa) and
sugarbeet (Beta vulgaris) and some trees.
In onion (A. cepa), gynogenesis takes place in flower bud
or ovary culture. No particular stress pre-treatment is
applied although the growth temperature of the donor
plant before flowering is crucial [25]. Culturing involves
a two step process: an induction medium containing hor-
mones and a regeneration medium with reduced or without
hormones. All plants regenerated are haploid and require
chromosome doubling treatments. As in the biennial onion,
flowering takes place in the first year; the use of DHs in
onion breeding not only produces recombinant inbreds
but saves time. The method used in sugarbeet (B. vulgaris)
is similar although here a cold treatment of inflorescences
(8 8C for one week) combined with high temperatures
(30 8C) during the induction phase improve the response
[26]. Doubled haploids in beets have been used in breeding,
including the production of F1 hybrids using DH parents
[27].
Gynogenesis can also be induced by using irradiated
pollen [28]. This has been achieved in trees by irradiating
the pollen of a related species, which is then used to
pollinate the desired parent to induce gynogenetic haploid
production. Currently gynogenesis is the least favoured
technique because of the low efficiency, but the value of
doubled haploids in species that do not respond to more
efficient techniques makes the method worthwhile.
Haploids from isolated and in vitro cultured anthers
Anther culture is often the method of choice for doubled
haploid production in crop plants [29]. Good aseptic tech-
niques are required, but methods are generally simple and
applicable to a wide range of crops [14]. In general, haploid
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Vol.12 No.8
plants are regenerated in vitro from the microspores
contained in the anther and require chromosome doubling
treatments. A few species, such as barley, regenerate a
large number of DHs as a result of induced chromosome
doubling during early cell division of microspores [30].
Although the application of anther culture is widespread,
the processes involved are poorly understood. Investi-
gations have been hampered by the presence of the spor-
ophytic anther wall that prevents direct access to the
microspores contained within. This has become an import-
ant issue because although many species respond to anther
culture, responsive genotypes can be a limiting factor and
there is a demand to study, understand and manipulate
microspore embryogenesis to develop genotype-indepen-
dent methods. There is also a need to ensure direct embry-
ogenesis to eliminate an intermediary callus phase that
can promote gametoclonal variation among regenerants.
‘Shed microspore’ culture is a simple modification of
anther culture in which anthers are stimulated to dehisce
and release their microspores, usually into a liquid med-
ium of high osmolality. The method is successful in several
species, including barley (H. vulgare) [31], wheat (T. aes-
tivum) [32], tobacco (N. tabacum) [33] and pepper (Capsi-
cum annum) [34]. The large number of species in which
anther culture has been used successfully to produce hap-
loids is ample evidence of the benefits it offers. Its single
biggest advantage is its simplicity. Large-scale anther
culture operations can overcome the disadvantage of low
per-anther yield. However, the presence of extraneous
tissues (e.g. the anther wall) makes this a messy system
for cell biology and other precise studies.
Haploids from isolated and in vitro cultured
microspores
Isolated microspore culture has several advantages over
anther culture (Figure 2). By removing the anther wall,
confounding effects of maternal sporophytic tissues are
avoided. More importantly, the isolated single celled
microspores can be manipulated and studied using cell
biology, microbiology and functional genomics techniques
[35]. High-density cultures of synchronized microspores
can be set up containing >1000 embryos per ml. Devel-
opment in culture is independent of the sporophytic,
diploid anther wall tissues and, thus, the media com-
ponents and culture treatments have direct access to the
microspores. In a rich medium with high sucrose, isolated
microspores develop into mature pollen that are fertile,
whereas various stress treatments induce microspores or
immature pollen grains to convert into embryogenic micro-
spores or pollen grains that after transfer to rich medium
develop into embryos [35]. Under optimal conditions more
microspores can be induced to convert and, thus, far more
embryos are produced than in anther cultures. This
advantage makes microspore cultures a good experimental
system for studying embryogenesis because large numbers
of synchronous embryos in different stages can be collected.
Although a late-comer compared with anther culture,
the potential of microspore culture has been realized early
on, and efficient procedures have been developed to pro-
duce DHs in microspore cultures of tobacco, rapeseed,
pepper, wheat, barley and rice [14,35].
 Review TRENDS in Plant Science Vol.12 No.8 371

Figure 2. Developmental pathways of tobacco microspores in vivo and in vitro. Tobacco microspores develop in vivo into mature bi-cellular pollen (middle of the panel).
Isolated microspores can be cultured in vitro until the formation of mature, fertile pollen in a rich medium with high sucrose concentration (right panel). However,
microspores subjected to starvation at high temperatures form embryogenic cells that develop into haploid embryos and plants when cultured in a rich medium at normal
temperatures (left panel). The haploid plants obtained can be diploidized by treatment with colchicines or other anti-mitotic drugs.

Microspore culture has provided a wealth of information
about the mechanisms underlying microspore embryogen-
esis. For example, studies of microspore cultures led to the
identification of the stress in various forms that converts
microspores from the gametophytic to the sporophytic path-
way, or from a cell with limited developmental potential into
a totipotent or embryonic stem cell [36]. Several genes
have been isolated and characterized as being involved in
the reprogramming of microspores from the gametophytic to
the sporophytic phase, making microspore embryogenesis
an interesting system in which to study cellular reprogram-
ming, phase change and totipotency in higher plants
[37–39]. Chromatin changes and other aspects of whole-
sale genome reorganization, autophagy and/or apoptosis
involved in the degradation of gametophytic structures
[37,38], genetic similarities to cellular reprogramming in
other organisms, including animals (‘stemness’ genes), are
exciting new perspectives for future research.
Doubled haploids in crop improvement
Sherret S. Chase pioneered the use of haploids in breeding:
in 1946 he exploited a spontaneous parthenogenesis
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system to produce the first maize doubled haploid inbreds
[40]. Selected DH lines were used to produce commercial
hybrids [41]. DH cultivars are now a feature in many crop
species, for barley (H. vulgare) it is estimated that 50% of
contemporary cultivars in Europe are produced via a
doubled haploid system. In vegetable crops, DHs are used
prominently as parents for F1 hybrid seed production. A
table of contemporary DH cultivars in various crops can be
found on the COST Action 851 website (http://www.scri.
sari.ac.uk/assoc/COST851/default.htm). There is currently
much interest in expanding DH for F1 hybrid production to
high value crops such as medicinal and aromatic plants.
Because breeding has been neglected in these species, DH
for F1 production has the potential to make significant
advances in providing high, stable and predictive yields of
the raw biochemicals processed by pharmaceutical and
neutraceutical industries [42]. An exciting and somewhat
surprising development has been the production of fertile
DH lines in species such as rye (S. cereale) [43] and forage
grasses, Festuca and Lolium [44], that suffer from inbreed-
ing depression (i.e. they cannot easily produce fertile
homozygous lines by self pollination). Therefore, novel
372 Review TRENDS in Plant Science
homozygous DHs provide new opportunities for genetic
studies and plant breeding in these species.
Backcross conversion is a standard plant breeding
method for improving an elite line defective in a particular
trait. The elite line is crossed with a donor line possessing
the trait to be introgressed, the F1 hybrid is then back-
crossed to the elite line and subsequent backcrosses clear
most of the donor genome except the portion carrying the
desired trait. The process is effective but it is time-
consuming, requiring several rounds of crossing and
recombination. Doubled haploidy combined with mar-
ker-assisted selection provides a short cut. In Figure 3,
doubled haploidy is shown at the first backcross gener-
ation, but earlier or later generation intervention is
possible depending on the efficiency of DH production;
the selection of the desired genotype is essentially a num-
bers game and, therefore, the more efficient the DH sys-
tem, the earlier it can be applied. The method has been
used successfully to introgress stripe rust resistance in
barley (H. vulgare) [45].
Reversible male sterility and doubled haploid production
are two valuable technologies required for F1 hybrid breed-
ing. Recently an F1 hybrid seed technology was developed
based on metabolic engineering of glutamine in developing
tobacco anthers and pollen [46]. Tobacco GS1 with single or
double point mutations were fused to the tapetum-specific
TA29 or the microspore-specific NTM19 promoter and
transformed into tobacco, resulting in male sterility owing
to pollen abortion close to the first pollen mitosis. A non-
segregating population of homozygous DH male-sterile
plants was generated through microspore embryogenesis.
Fertility restoration was achieved by spraying plants with
Figure 3. Scheme for combining doubled haploidy with marker-assisted selection for accelerated backcross (BC) conversion, adapted from Ref. [48].
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Vol.12 No.8
glutamine or by pollination with pollen matured in vitro in
glutamine-containing medium. This innovative breeding
technology is the first example of a molecularly designed
breeding technology that combines reversible male sterility
and DH production.
DH plants also play an integral part in reverse breeding,
a recently developed technology [47]. This technology
involves the recovery of DHs from microspores of plants
in which no or limited recombination has occurred (by
suppressing or preventing recombination, e.g. by gene
knockout of key meiotic genes). The resulting recombinant
inbred populations can be screened via molecular markers
to identify those with complementary combinations of
chromosomes to allow an original heterozygous parent of
the DH to be reconstructed by hybridizing the two indi-
viduals. Consequently, different parents with different
chromosome constitutions can be identified to reconstruct
existing F1 hybrids.
Doubled haploids in genetics
DHs have been a key feature in establishing chromosomes
maps in a range of species, notably barley (H. vulgare), rice
(Oryza sativa), rapeseed (B. napus) and wheat (T. aesti-
vum) [48]; they have also provided the vast majority of
mapped genetic markers, >90% in some species. The
advent of high-throughput genotyping means that genetic
maps can be developed within weeks of producing and
extracting DNA from a segregating DH population. In
out-pollinating species, genetic analysis can be simplified
by using at least one DH parent in the initial cross to
produce a segregating population – this has been success-
ful in vegetables such as the Brassica oleracea complex,
 Review TRENDS in Plant Science
namely cabbage, cauliflower, broccoli and Brussels sprouts
[49], and the fodder grass Lolium perenne [50].
Marker maps provide a platform for trait mapping,
which is of particular interest to plant breeders. In respon-
sive crop species, such as barley, the process of producing a
segregating DH population from an F1 hybrid to find
marker–trait associations has become routine [51]. DHs
are also ideal in establishing marker–trait associations in
bulked segregant analysis (BSA), a technique that com-
pares individuals from two extremes of a population distri-
bution for a given trait, for example, disease resistance and
susceptibility [52]. As with quantitative trait mapping,
accuracy in BSA is dependent upon robust phenotyping;
because DHs can be repeatedly tested, reliable data, in-
cluding field data from trials over several seasons, can be
obtained. DNA from the selected DHs can then be geno-
typed progressively with a range of marker systems. BSA
and DH analysis has been successful in establishing mar-
ker-assisted selection for several breeding traits, mainly
disease and pest resistance but also quality traits [52].
DHs currently play an important role in genomics. A
popular method of identifying genes controlling a trait is to
trawl through expressed sequence tags (ESTs) and to map
their chromosome position relative to the trait in question.
Candidates that co-locate can be physically mapped using
BAC libraries, and their association verified. DHs play a
vital role in integrating genetic and physical maps, thereby
providing precision in targeting candidate genes [53,54].
Induced mutation provides another method of linking
genes to phenotypes. Large-scale TILLING [55–57] pro-
jects or mutation grids [58] have been developed in several
species for forward and reverse genetics. It is desirable that
mutant populations are derived from an inbred or a homo-
zygous line (e.g. a DH line) to avoid the detection of false
positives owing to inherent variation in the starting
material. Doubled haploidy is also useful in rapid isolation
and purification of selected mutants in subsequent gener-
ations in a manner that is similar to the backcross con-
version method shown in Figure 3. Seeds are the most
common targets for mutation treatments; but microspores
offer an interesting alternative. By targeting the mutation
treatment at single gametic cells and then inducing embry-
ogenesis and DH plant production, it is possible to create a
population of homozygous mutant lines directly [59]. The
masking effects of chimeras or heterozygosity are pre-
cluded, enabling the expression and identification of both
recessive and dominant traits in the haploid cells, tissues
and plants. The development of improved DH protocols
now makes this a viable option for many species. However,
the disadvantage is that homozygous lethal mutations
would be eliminated (in other populations these can be
maintained as heterozygotes).
Other applications of doubled haploids
As in mutagenesis, the totipotent microspore is a prime
target for transformation: it is an easily available and
accessible single-cell target that can now be manipulated
in a large number of plant species [35]. In addition, the
transgenes can be studied in both haploid and DH plants.
Although the primary target is the uni-cellular micro-
spore, cells or explants at all stages from microspore
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Vol.12 No.8 373
embryogenesis and regeneration have been used as
recipients for gene delivery; the choice being determined
by regeneration potential after treatment. Many transform-
ation techniques have been tried, including microinjection,
electroporation, particle bombardment and Agrobacterium
tumefaciens-mediated transformation [35].
Particle bombardment of barley embryogenic
microspores has resulted in transgenic doubled haploids
that were homozygous for the transgene [60]; similar
results have been reported in B. napus [61]. However,
transformation frequency needs to be improved because
the best frequency reported was extremely low, with one
transgenic being recovered among 200 000 bombarded
microspores (Y.S. Shim, PhD Thesis, University of Guelph,
2006). A more efficient transformation of barley micro-
spore-derived multi-cellular structures using A. tumefa-
ciens has been reported [62], demonstrating the feasibility
of this technology for other cereals where microspore
embryogenesis is amenable. However, chromosome dou-
bling at this later developmental stage results in chimeric
plants that require a more careful analysis and selection.
In tobacco, particle bombardment has been directed at
embryogenic and non-embryogenic microspores and imma-
ture pollen [63,64]. The original method involved bombard-
ing embryogenic microspores after a stress pre-treatment,
and then visually selecting haploid embryos using the vital
GUS stain IMAGINEGREEN, which were doubled using
colchicine [63]. A more recently developed method involves
bombarding and stress inducing microspores immediately
after isolation, and then visually selecting transgenic
embryos for GFP and RFP expression (T. Resch and A.
Touraev, unpublished). Bombarded microspores are also
allowed to develop into mature pollen in vitro. Matured
pollen from this novel male germ line transformation
system has been used for in vivo pollination and transfor-
mants can be selected from the resulting seed and seed-
lings [64].
In summary, the resurgence of interest in haploid
research is apparent from: (i) the large number of hap-
loid-derived crop cultivars in a wide range of species; (ii)
the availability of reliable tissue culture protocols; (iii) the
many applications in gamete and embryo biology; (iv)
genetics (mapping, gene discovery and identification);
and (v) application in mutation and transformation stu-
dies. The potential in these areas is now being realized
with real and tangible results. In the past, doubled hap-
loidy has been restrained by technological problems but
many hurdles have been overcome empirically and have
resulted in major steps forward in the study of embryogen-
esis. We can expect to identify many interesting genes
involved in microspore reprogramming and embryogen-
esis. This should lead to a better understanding of these
processes and more efficient protocols for the production of
haploid and doubled haploid plants.
Acknowledgements
We thank Tatiana Resch and Kristina Belogradova for the artwork.
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Does history count? by K. Anderson
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Environmentalism out of the Industrial Revolution by C. Macleod

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