Plant Biotechnology Journal (2010) 8, pp. 377–424 doi: 10.1111/j.1467-7652.2009.00498.

Review article
Haploids in flowering plants: origins and exploitation
Jim M. Dunwell*
School of Biological Sciences, University of Reading, Whiteknights, Reading, UK

Received 12 October 2009; Summary

revised 9 December 2009;
accepted 12 December 2009.
*Correspondence (fax 0118 378 8160;
e-mail J.Dunwell@reading.ac.uk)
Keywords: plant breeding,
homozygous, anther culture, pollen,
microspore.
The first haploid angiosperm, a dwarf form of cotton with half the normal chromosome
complement, was discovered in 1920, and in the ninety years since then such plants
have been identified in many other species. They can occur either spontaneously or can
be induced by modified pollination methods in vivo, or by in vitro culture of immature
male or female gametophytes. Haploids represent an immediate, one-stage route to
homozygous diploids and thence to F1 hybrid production. The commercial exploitation
of heterosis in such F1 hybrids leads to the development of hybrid seed companies and
subsequently to the GM revolution in agriculture. This review describes the range of
techniques available for the isolation or induction of haploids and discusses their value
in a range of areas, from fundamental research on mutant isolation and transformation,
through to applied aspects of quantitative genetics and plant breeding. It will also focus
on how molecular methods have been used recently to explore some of the underlying
aspects of this fascinating developmental phenomenon.

will draw parallels with some of the related processes

Introduction
Most organisms contain the genetic contributions of both
parents. However, there are significant exceptions to this
rule. Specifically, it is possible to isolate haploid organisms,
including plants, that contain the same number of chro-
mosomes in their somatic cells as do the normal gametes
of the species. The term ‘haploid sporophyte’ is generally
used to designate such sporophytes having the gametic
chromosome number (Palmer and Keller, 2005a). This
review describes the range of techniques available for the
isolation or induction of haploid plants and discusses their
value in a range of areas, from fundamental research on
mutant isolation and transformation, through to applied
aspects of quantitative genetics and plant breeding. It will
also focus on how molecular methods have been used
recently to explore some of the underlying aspects of this
fascinating developmental phenomenon.
Evolutionary context
Before considering the details of the gametophyte to
sporophyte induction process in higher plants, this review
occurring elsewhere in the plant kingdom.
Facultative apogamy
The induction of sporophytes from microspores (see
below) has a directly analogous phenomenon within the
lower plant kingdom; it is the apogamous production of
sporophytes from gametophytes of ferns and mosses. This
parallel has been drawn previously but it is worth noting
the similarities of these two distantly related processes. It
is well over a century ago that Farlow (1874) described
the outgrowth of a shoot from a prothallus of the fern
Pteris cretica. Such facultative apogamy has been
described subsequently in several other fern species (see
review by Sheffield and Bell, 1987), and it is from some of
these experimental studies that the most intriguing
evidence has derived.
Probably, the most extensively studied system is in Pteri-
dium aquilinum gametophytes, where Whittier and
Steeves (1960) found that high concentrations of sucrose,
glucose, fructose and maltose led to an increased
frequency of apogamous outgrowths. The effects of the

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Journal compilation ª 2010 Blackwell Publishing Ltd 377
378 Jim M. Dunwell
application of various carbohydrates (Roberts-Oehlschlager
et al., 1990) will be discussed in the following sections. In a
related study, Hirsch (1975) showed a definite nutritional
role of sucrose in determining the balance of gametophytic
to sporophytic development in the fern Microgramma
vaccinifolia. She concluded that increased carbohydrate
supply (4% sucrose) led to increased production of sporo-
phytes and that starvation conditions favoured gameto-
phyte development. This concept of starvation will also be
considered in more detail in relation to theories of micro-
spore embryo development.
Identification of haploids
Haploids of higher plants can be distinguished from their
diploid equivalents in many ways. Most obviously, they
are smaller in appearance, partly because of their smaller
cell size; in general terms, cell volume in plants in directly
related to their ploidy level. The haploid status of a plant
can be confirmed using any of a variety of methods.
These include direct measurement of the chromosome
number using conventional cytological techniques and
measurement of the DNA content using flow cytometry
(Bohanec, 2003), and indirect methods based on guard
cell and plastid dimensions (Lee and Hecht, 1975; Qin
and Rotino, 1995; Yuan et al., 2009). At a genetic level,
although the detection of homozygosity in doubled hap-
loids (DHs) using isozyme-based techniques (Liu and
Douches, 1993) is still practised (Bouvier et al., 2002;
Grafe, 2003; Toppino et al., 2008), these have now lar-
gely been replaced by methods based on DNA markers
(Verdoodt et al., 1998; Chani et al., 2000; Eimert et al.,
2003; Belicuas et al., 2007; Diao et al., 2009). A variety
of morphological and other methods used for high-
throughput screening for haploids will be described in
the following sections.
In vivo occurrence of haploid plants
Methods for haploid production have been reviewed in sev-
eral extensive volumes and reviews (Magoon and Khanna,
1963; Kasha, 1974; Maluszynski et al., 2003a,b; Palmer
et al. 2005; Xu et al., 2007; Touraev et al., 2009). In
summary, efforts have been made to obtain haploids from
several hundred species, although efficient and reproduc-
ible DH protocols are available only for less than thirty.
Additionally, although both general and specific informa-
tion is given in these and other crop-specific reviews
(Braniste et al., 1984; Zhang et al., 1990; Andersen, 2005;
Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 377–424
Germanà, 2006, 2007), these publications usually focus
on the in vitro induction techniques described below in
the second section of this review. They do not assess in
similar detail the more general occurrence of haploids
as rare events in a range of different breeding systems
(Lacadena, 1974).
Spontaneous haploids
According to Harland (1955), the distinction for isolating
the first haploid flowering plants belongs to Miss O.S.
Atteck at the Cotton Research Station, Trinidad, almost a
century ago. She discovered that a well-known variant
of a type of Sea Island Cotton known as ‘Man Cotton’
(Harland, 1920, 1936) was the probable haploid compo-
nent of the relatively numerous pairs of twins (see below)
found in that variety (Burd, 1924). As recorded by Chase
(1969), the first haploid angiosperm for which cytological
proof was obtained was found in 1921 by Dorothy
Bergner in Datura stramonium (Blakeslee et al., 1922). The
two haploid individuals were among a number of plants
of abnormal appearance produced in an attempt to
induce chromosomal irregularities by the application of
cold as a stimulus. The second definitive haploid was
reported in Nicotiana tabacum (Clausen and Mann, 1924),
with the first confirmed haploid from a cereal species
reported by Gaines and Aase (1926) from Triticum
compactum var. humboldtii, a winter variety of the Pacific
Northwest. In the following years, occasional haploids
were reported in a range of species (Table 1). The specific
example of maize is discussed in more detail later.
By Kimber and Riley (1963) were able to record the
occurrence of haploids from at least 71 species, represent-
ing 39 genera in 16 families of angiosperms, and that
number has grown substantially in the decades since, with
examples from Agropyron (Sakamoto, 1964), alfalfa (Bing-
ham, 1969), peach (Hesse, 1971; Toyama, 1974), Citrus
(Karasawa, 1971; Yahata et al., 2005) and Trillium (Uchi-
no, 1973). The exact source of haploids in these species,
in terms of their embryological origin, is often unknown.
Similar examination of the published literature for gymno-
sperms shows several examples of spontaneous haploids
(Stettler et al., 1969; Piesch and Stettler, 1971; Stettler and
Bawa, 1971; Isakov et al., 1981) that include a unique hap-
loid form of Thuja plicata called Thuja gigantea ‘gracilis’
Beissn (Pohlheim, 1968; Simak et al., 1974) (see below).
At a cellular, rather than whole plant level, induction
of meiosis has been reported in somatic cells. For exam-
ple, spontaneous reduction in the number of somatic
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 Haploids in flowering plants 379

Table 1 Additional selected examples of spontaneous haploids and many genetic investigations have been published.
These will be summarized here.
Species Reference(s)
Individual and specific mention must be given to maize,
Tobacco Chipman and Goodspeed, 1927; Ruttle, 1928; Kostoff, the crop in which haploids have had the greatest commer-
1929; Goodspeed and Avery, 1929; McCray, 1932; cial significance (see below). According to Randolph (1932),
Povolochko, 1937; Ivanov, 1938; Bolsunov, 1939;
haploidy in maize was first reported by L.J. Stadler, and him-
Prakken, 1943
Crepis Hollingshead, 1928; Gerassimova, 1936 self, in unpublished articles presented before Section O of a
Matthiola Lesley and Frost, 1928 meeting of the American Association for the Advancement
Oenothera Gates, 1929; Gates and Goodwin, 1930; Davis and of Science held in Des Moines, Iowa in 1929. Following the
Kulkarni, 1930; Catcheside, 1932; Harte, 1973
work of Chase (1949, 1952, 1963, 1964), maize lines with
Tomato Lindstrom, 1929, 1941; Lindstrom and Koos, 1931;
Humphrey, 1934; Newcomer, 1941; Rick, 1945;
higher yields of haploids became widely exploited (see sec-
Kirillova and Bogdanova, 1978 tion on breeding below). These include ‘Stock 6’ (Coe,
Rice Morinaga and Fukushima, 1931, 1934; Ramiah et al., 1959a,b; Coe and Sarkar, 1964) and similar derivatives
1933, 1935
(Chalyk, 1994, 1999a,b, 2000a,b; Chalyk and Chebotar,
Pharbitis U, 1932; Katayama, 1935a
2000; Liu and Song, 2000a; Chalyk and Rotarenco, 2001;
Portulacea Okura, 1933
Brassica Morinaga and Fukushima, 1933; Komatsu, 1936; Eder and Chalyk, 2002; Zhang et al., 2008d). Recent molec-
Ramanujam, 1941; Mizushima, 1944; Kuriyama and ular investigations have shown this type of maternal haploid
Watanabe, 1955; Olsson and Hagberg, 1955; occurrence in maize to be controlled a major locus with
Fukushima and Iwasa, 1964; Prakash, 1973
incomplete penetrance (Barret et al., 2008).
Wheat Chizaki, 1933, 1934; Smith, 1946
Barley Johansen, 1934; Tometorp, 1939 An additional haploid-inducing gene in maize is the
Cotton Skovsted, 1935; Grüneberg, 1936 indeterminate gametophyte (ig) gene located on chromo-
Rye Müntzing, 1937a,b, 1938; Levan, 1942; Mostafa et al., some 3 (Kermicle, 1969, 1971; Pollacsek, 1992; Kindiger
1993
and Hamann, 1993) and encoding an LOB domain protein
Millet Gill et al., 1937; Powell et al., 1975
Potato Lamm, 1938; Bains and Howard, 1950; Hougas and
(Evans, 2007). This gene in maize is unique among
Peloquin, 1957; Rieman et al., 1959; Hougas et al., mutants that act in the haploid gametophytes; mutants
1964), oat (Nordenskiöld, 1939; Nishiyama, 1961 are viable and have an increased number of nuclei before
Oat Nordenskiöld, 1939; Nishiyama, 1961 cellularization of the embryo sac. Additionally, ig1 restricts
Prunus Pratassenja, 1939
the embryogenic potential of cells that lack one of the
Capsicum Nishiyama, 1940; Morgan and Rappleye, 1954
Lily Cooper, 1943 two parental genomes, so that mutant embryo sacs pro-
Sorghum Brown, 1943 duce haploid progeny, of both maternal and paternal ori-
Guayule Bergner, 1944; Stebbins and Kodani, 1944 gin, at a higher rate than wild-type embryos (Kermicle,
Orchid Hagerup, 1944, 1947; Kondo, 1970
1969). This gene has been exploited for the production of
Asparagus Randall and Rick, 1945
Maize Chase, 1949
paternal haploids with maternal cytoplasm (Kindiger and
Castorbean Poole and Hadley, 1954 Hamann, 1993). The long history of haploidy in maize,
Poplar Tralau, 1957; Kopecky, 1960 including the first identification of lines with increased fre-
Poppy Saito, 1958
quencies of haploids, has been recently reviewed in con-
Coffee Vishveshwara, 1960; Sreenivasan et al., 1982
siderable detail by Chang and Coe (2009).
Strawberry Islam, 1961
Jute Jacob and Sen, 1961 A gene with related affects to ig is the haploid initiator
Coconut Ninan and Raveendranath, 1965 gene (hap) in barley (Hagberg and Hagberg, 1980, 1981;
Oil palm Dunwell et al. (unpublished) Mogensen, 1982). The function of this gene is to prevent
fertilization of the egg cell, while not affecting fertilization
of the polar nuclei and development of the endosperm.
chromosomes was described in the root meristem of Ha- The haploid embryos do not require in vitro culture as
plopappus gracilis by Ames and Mitra (1966). seed development is relatively normal.
The related phenomenon of semigamy refers to an unu-
sual form of facultative apomixis in which fusion of male
Genetic control of haploid induction
and female nuclei does not occur but instead they both
From an early date, great interest developed in any possi- divide independently, giving rise to a chimaeric embryo
ble evidence for a genetic control of haploid induction, with both male and female components. Semigamous

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380 Jim M. Dunwell
plants produce elevated frequencies of maternal haploids,
paternal haploids, chimaeric maternal ⁄ paternal haploids
and trichimaeric progenies with zygotic as well as parental
haploid sectors. A defined mutant of cotton (G. barba-
dense L.) causing high frequencies of semigamy was iso-
lated by Turcotte and Feaster (1963, 1969) who showed
that it was caused by the allele Se. Related results in cot-
ton have been reported by Chaudhari (1978, 1979), Jia
et al. (1989) and Hodnett (2006). The semigamy gene was
also exploited in an interesting study of seedling lethality
in cotton by Stelly and Rooney (1989) who used virescent
(v,) and glandless mutant (gl2gh) markers to facilitate
identification of parental origins of haploids and sectors of
chimaeric haploids. Semigamy has also been recorded in
Coix aquatica (Rao and Narayana, 1980) and cocoa
(Lanaud, 1988).
Hybridization
In both native and cultivated plants, haploids have been
observed among the progeny of a great diversity of
crosses between either random or selected parents within
the same or different species.
Intraspecific hybridization
In some species, haploids have been generated from dip-
loid material (e.g. Haplopappus: Jackson and Jordan, 1975),
and in other cases, diploids have been produced from tet-
raploids. Examples of the latter occur in Parthenium (Gerstel
and Mishanec, 1950), Sorghum (Duara and Stebbins,
1952), Sisymbrium (Khoshoo, 1957) and alfalfa (Bingham
and Binek, 1969; Bingham, 1971). In a similar study,
Katayama (1935b) found haploid plants in progeny of the
allopolyploid Aegilotriticum.
Some of these include crosses between parents with
different ploidy levels. For example, a haploid of Potentilla
argentea was identified in the progeny of a
diploid · tetraploid cross (Asker, 1983). Similarly, a haploid
Citrus plant was generated from an interploidy cross
between diploids and triploids (Oiyama and Kobayashi,
1993; Germanà and Chiancone, 2001), and haploids of
sugar beet (Cistué et al., 1985) were identified by crossing
diploid male-sterile plants with green hypocotyls and
tetraploid fodder beets homozygous for red hypocotyls
(Bosemark, 1971).
Wide hybridization
Use of wide crosses between, rather than within, a species
is one of the most effective methods for haploid induction
Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 377–424
and has been used successfully in many species (We˛dzony
et al., 2009). These examples include early results from
interspecific hybridization in tobacco (Kostoff, 1930,
1934), a technique subsequently confirmed in that genus
by Apparao and Varma (1972) and Burk et al. (1979).
Similar results were reported in Portulacea grandiflora by
Okura (1933) who stated ‘Two haploids were found
among 168 F2 interspecific-hybrids (Magenta No. 22 ·
Orange No. 35) and in the following year one haploid was
obtained from l00 F3 plants. Their parents were normal
morphologically and cytologically. These haploids are of
about half the stature of P. grandiflora with all organs
proportionally smaller.’
A related example within this category involves the use
of diploid Solanum tuberosum L. as maternal parents and
selected S. phureja clones as prickle pollinators (Peloquin
et al., 1996). In one such cross, from about 2 million seeds
screened more than 500 monohaploid plants were
obtained (Uijtewaal et al., 1987).
In an example of intergeneric hybridization, Nakajima
(1935) reported the pollination of T. turgidum (2n = 28)
with the pollen of a single plant of Secale cereale and the
production of 182 kernels. They were sown and only two
individuals were raised; one of them was a F1 hybrid and
the other a haploid plant of T. turgidum. Other recent
relevant examples in this category include haploid genera-
tion in Oenothera (Haustein, 1961), sorghum (Magoon
et al., 1961), strawberry (Barrientos and Bringhurst, 1973),
Elymus (Lu, 1992), chicory (Doré et al., 1996), pear (Inoue
et al., 2004) and Brassica napa (Tu et al., 2009). Alien
cytoplasm from Aegilops species has also been used in
wheat (Kihara and Tsunewaki, 1962; Hsam and Zeller,
1993), and a haploid radish plant was obtained spontane-
ously from an R. sativus-B. oleracea monosomic addition
line (Kaneko, 1980).
Chromosome elimination
In some of these cases, it is likely that the haploids were
induced by a process of selective chromosome elimination
that follows certain interspecific pollinations. This phenom-
enon was first discovered in barley (Kasha and Kao, 1970)
with crosses between Hordeum vulgare and H. bulbosum,
and the identification of H. vulgare haploids (Houben and
Pickering, 2009). It is now used routinely in wheat
(Barclay, 1975; Suenaga, 1994; Moradi et al., 2009) and
other cereal breeding programmes; haploids are induced
in these species following pollination with maize (Sidhu
et al., 2006) or other distantly related cereal species
(Pratap et al., 2005; Komeda et al., 2007). Cytological
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 Haploids in flowering plants 381

aspects of the chromosome elimination process have been
well described (Mochida et al., 2004; Gernand et al.,
2005). The frequency of haploids from these crosses
depends on a range of genetic (Bitsch et al., 2000; Garcı́a-
Llamas et al., 2004) and experimental variables, including
the light intensity during the early stages of embryo devel-
opment (Campbell et al., 2001).
In addition to the possibility of generating chromosome
addition lines (Kynast et al., 2001) and partial hybrids
(Riera-Lizarazu et al., 1996), there is an intriguing possibil-
ity of residual maize DNA being present in haploids or
doubled haploids of wheat (or other species) produced
after such chromosome elimination. To date, there has
been limited investigation of this possibility, and the results
are inconsistent. Some authors provide evidence for the
transfer of maize-specific DNA (Chen et al., 1998, 1999,
2000), whereas no evidence of introgression was reported
in two other studies (Brazauskas et al., 2004; Fischer,
2004). However, in a related recent study, a Stock-6-
derived, haploid-inducing line CAUHOI with high kernel oil
This occurrence of so-called twin seedlings (Nezhevenko
and Shumnyi, 1970), and their potential as a source of
haploids in angiosperms, has been known for many years
in a wide range of species (Table 2).
A similar approach is possible with conifers (Clare and
Johnstone, 1931; Nishimura, 1960; Dogra, 1967). For
example, Illies (1964) found ten haploid seedlings in the
course of an extensive cytological study covering 435
progenies in Picea abies Karst. The haploids showed
various abnormalities. Five were members of a pair of
twins; the other five germinated radicle first and all were
unusually small.
The number of seed examined in these studies and
the haploid frequencies vary greatly. One of the largest
was the screen in tobacco in which 104 000 germinated
Table 2 Selected examples of haploids derived from twin
seedlings
Species Reference(s)

content (KOC), was used as the pollinator to produce

Wheat Namikawa and Kawakami, 1934; Kihara, 1936; Yamamoto,
maternal haploids from a maize hybrid with low KOC. 1936; Kasparayan, 1939; Wilson and Ross, 1961; Kihara and
CAUHOI is homozygous for the dominant marker gene Tsunewaki, 1963
R1-nj (Li et al., 2009) (see above). Simple sequence repeat Cotton Harland, 1936; Webber, 1938, 1940; Skovsted, 1939; Silow and
Stephens, 1944; Endrizzi, 1959; Blank and Allison, 1963;
(SSR) analysis showed that 43.18% of the haploids carried
Owings et al., 1964; Lee, 1970
segments from CAUHOI, and a small proportion (average Poa Engelbert, 1941; Nielsen, 1946
1.84%) of the genome of CAUHOI was introgressed into Dactylis Müntzing, 1943
haploids. Haploid kernels with high KOC had a higher fre- Maize Sharman, 1942; Pešev et al., 1976
Pepper Christensen and Bamford, 1943
quency of segment introgression from CAUHOI (2.92%)
Asparagus Randall and Rick, 1945; Marks, 1973; Uno et al., 2002
than that in haploid kernels with low KOC (1.79%), show- Sesbania Haque, 1946
ing that the marker gene R1-nj and high-oil genes from Oak, ash Johnsson, 1946
CAUHOI were expressed during the development of some Robinia Kanezawa, 1948

haploid embryos, and confirmed that the DNA introgres- Abies Kanezawa, 1949
Tobacco Cameron, 1949; De Nettancourt and Stokes, 1960
sion from the inducer parent occurred during maternal
Bromus Nielsen, 1951; Wilton et al., 1963
haploid induction. Sorghum Kidd, 1952
Squash Hayase, 1954
Sugar beet Fischer, 1956, 1962; Kruse, 1961
Parthenogenesis and polyembryony Melilotus Jaranowski, 1961
Rice Choi and Chung, 1961; Sharma and Virmani, 1990; Yang, 2008
In this process, the egg cell within the embryo sac usually Coconut Whitehead and Chapman, 1962
develops into an embryo without involvement of the Hieracium Skalinska, 1968
sperm nucleus. Usually, this process occurs at low fre- Lupin Kazimierska and Kazimierski, 1969, 1970
Cocoa Dublin, 1972, 1973a; Sounigo et al., 2003
quency. Such rare haploid plants among many normal dip-
Soybean Kenworthy et al., 1973; Ahmad et al., 1975, 1977
loid seed offspring (Kendall, 1934) are difficult to detect
Coffee Dublin and Parvais, 1976; Lashermes et al., 1994
unless specific genetic markers in the pollinators are used Flax Bellini, 1985; Secor and Russell, 1988
for selection, as exemplified for maize (Bordes et al., Blueberry Dweikat and Lyrene, 1990
1997) and apple (Lespinasse et al., 1983). However, Date palm Veerasamy and Arekal, 1993
Onion Specht et al., 2001
female haploids in seeds of angiosperms are frequent
Almond Martı́nez-Gómez and Gradziel, 2004; Martı́nez-Gómez
among seeds germinating with two or more seedlings et al., 2002
(Clayton and Yawney, 1972).

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382 Jim M. Dunwell

seeds gave rise to nine pairs of twins from which 10 Pollen treatments
seedlings died (De Nettancourt and Stokes, 1960).
Numerous attempts have been made to increase the fre-
Among the survivors, one individual of a surviving twin
quency of haploids by treating the pollen, prior to pollina-
pair proved to be haploid and came from a seed of the
tion, with various physical or chemical agents. For
dark tobacco variety Ky 151. Similarly, in C. annuum,
example, induction of female-derived haploid embryos fol-
haploids occur frequently as members of twin and trip-
lowing pollination with irradiated pollen has been used
let seedlings arising from polyembryonic seeds; three
successfully in many species (Table 3).
triplet and 291 twin seedlings were obtained from
In a specific variation of pollen irradiation, Chat et al.
78 005 germinated seeds (Christensen and Bamford,
(2003) reported, for the first time, the complete
1943) and a later combined count indicated 1619 multi-
uncoupling of the transmission of organelle and nuclear
ple seedlings from among 300 683 germinated seeds
genomes in Actinidia deliciosa (kiwifruit), a plant species
(Morgan and Rappleye, 1954). Frequencies of twin seed-
known for its paternal mode of chloroplast inheritance.
lings among such Capsicum cultivars varied from 0.06%
After pollen irradiation, transmission of nuclear genes
to 0.65%, with the differences being statistically signifi-
from the pollen parent to the progeny was inhibited, but
cant. Homozygous lines having high, intermediate and
transmission of the chloroplast genome was not. This
low frequencies of polyembryony were isolated by col-
demonstrates that plastids can be discharged from the
chicine treatment of haploid sporophytes, with the high
pollen tube into the egg with little or no concomitant
line producing 2.85% multiple seedlings in comparison
transmission of paternal nuclear genes. In androgenetic
with 0.65% for the parental cultivar ‘Goliath’. Eighty-
plants, however, haploid embryos are produced that
five per cent of all twin seedlings produced by this
originate from the development of a male gamete within
derived line were of the unattached n–2n type com-
the embryo sac of the female parent, resulting in
pared with 34% of this type for the parental line (Mor-
uniparental paternal inheritance of the nuclear genome,
gan and Rappleye, 1954). In a related study,
whereas the organelle genomes are still contributed by
characteristic and distinctive frequencies of these individ-
the female parent.
ual lines demonstrated a definite effect of the female
Such androgenetic plants (formerly described as
genotype on polyembryony (Morgan and Rappleye,
patrogenic—Collins and Kempton, 1916; Bartlett, 1916),
1954; Campos and Morgan, 1960).
Another species in which a series of detailed studies
of polyembryony has been conducted is flax (Linum usi- Table 3 Maternal haploids after pollen irradiation
tatissimum) (Wricke, 1954; Thompson, 1977; Green and Species Reference(s)
Salisbury, 1983; Bellini, 1985; Levieil and Huyghe, 1985;
Murray, 1985; Rowland and Weerasena, 1986; Huyghe, Wheat Katayama, 1934
Petunia Rick, 1943
1987; Secor and Russell, 1988). For example, Kappert
Capsicum Morgan and Rappleye, 1951, 1954; Campos and Morgan, 1960
(1933, 1950) showed that the frequency of polyembry-
Maize Morgan, 1976; Morgan and Rappleye, 1951
ony in this species is controlled by recessive genes. The Tobacco Dulieu, 1964
effect of these genes was seen in two ways, a maternal Apple Zhang et al., 1988; Zhang and Lespinasse, 1991;
effect and a zygotic one. The maternal effect was the De Witte and Keulemans, 1994
Pear Bouvier et al., 1993; Inoue et al., 2004
more obvious, and strains were selected that produced
Lily Morgan and Rappleye, 1951
high or low frequencies of polyembryony. The influence Onion Doré and Marie, 1993
of the maternal effect was such that the frequency of Rose Meynet et al., 1994; Lim et al., 2005
polyembryonic seed could not be increased above that Blackberry Naess et al., 1998
Melon Sauton and Dumas de Vaulx, 1987; Lotfi et al., 2003;
found on selfing, even when pollination was with pollen
Lim and Earle, 2008
from male parents known to have a higher frequency Cucumber Przyborowski and Nlemirowicz-Szgytt, 1994; Çaglar and Abak,
of polyembryony when selfed (Kappert, 1950). The fre- 1999; Faris and Niemirowicz-Szczytt, 1999; Faris et al., 1999;
quency of haploid–diploid twins (Murray, 1980, 1985) Claveria et al., 2005

was higher in self-pollinated than in cross-pollinated Squash Kurtar et al., 2002

Sunflower Todorova et al., 1997, 2004; Drumeva et al., 2005
material (Kappert, 1950). This method has also been
Kiwifruit Chalak and Legave, 1997
used for applied purposes (Plessers, 1963; Rajhathy, Citrus Froelicher et al., 2007
1976).

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although considerably rarer than apomictic plants and to a
lesser extent than gynogenetic plants (i.e. haploid plants
deriving from the development of the female gamete),
have been reported several times since the early twentieth
century (e.g. Clausen and Lammerts, 1929; Kehr, 1951;
Burk, 1962; Singh and Cornu, 1976). They are known to
occur spontaneously, at a low frequency, in various angio-
sperm genera belonging to both mono- and dicotyledons,
for example, Capsicum (Campos and Morgan, 1958),
Nicotiana (Burk, 1962), Petunia (Singh and Cornu, 1976),
Zea (Yarnell and Hills, 1959; Chase, 1963) and Brassica
(Chen and Heneen, 1989). In the 1960s, Goodsell (1961)
and Chase (1963) demonstrated that maize androgenetic
haploids had a maternal organellar genome, together with
a haploid paternal nuclear genome. In the 1980s, the
same conclusion was reached for Nicotiana and Petunia by
using molecular markers (Pelletier et al., 1987; Raquin
et al., 1989; Horlow et al., 1993).
Paternal apomixis was also recently reported in the
endangered Mediterranean cypress, Cupressus dupreziana
(Pichot et al., 2008). This species acts as a surrogate
mother for the development of all-paternal embryos from
pollen grains. C. dupreziana production of Cupressus sem-
pervirens haploid or diploid seedlings from C. sempervirens
pollen was also demonstrated. The haploid progeny was
derived from the embryogenic development of haploid
gametes, but the origin of the diploid progeny remained
unknown. Recently, Nava et al. (2009) reported the onto-
genic origin of the diploid C. sempervirens progeny. They
analysed the heterozygosity of 63 diploid all-paternal
C. sempervirens seedlings using highly variable co-domi-
nant nuclear SSR markers. The bi-parental inheritance of
the markers was checked in C. sempervirens controlled
crosses. A high level of polymorphism was observed
among the diploid all-paternal trees. All but three individu-
als exhibited single-band profiles as expected for homo-
zygotes, which may originate from natural diploidization
of a C. sempervirens haploid embryo or from the fusion of
two male gametes produced by the same C. sempervirens
microgametophyte.
As an alternative to irradiation of pollen, heat-treated
pollen has been used successfully for haploid induction in
aspen (Winton and Einspahr, 1968) and maize (Mathur
et al., 1980). Successful chemical treatments include appli-
cation of pollen with toluidine blue in trees (Al-Yasiri and
Rodgers, 1971; Illies, 1974), treatment of maize silks with
maleic hydrazide (Deanon, 1957) and application of bras-
sinolide to emasculated stigmas of Arabidopsis, Brassica
and Tradescantia (Kitani, 1994).
ª 2010 The Author
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Haploids in flowering plants 383
Haploid induction following a simple delay in the timing
of pollination was first developed in wheat (Kihara, 1940).
This procedure, which simply involves pollinating a few
days after the first receptivity of the stigma, was subse-
quently developed in many species including maize (Rotar-
enco and Mihailov, 2007).
Methods of screening for haploids
Apart from the identification of plants with unusual char-
acteristics, often those being small and weak in appear-
ance, there are several other methods that have been
used in directed screening for haploids (also see the previ-
ous section). These include screening for small seeds in
maize (Tyrnov and Zavalishina, 1972), cocoa (Dublin,
1973b) and citrus (Harusaki et al., 1996; Toolapong et al.,
1996; Kita et al., 2001), X-ray screening of seed to iden-
tify small embryos in pine (Toda and Satô, 1967), spruce
(Simak et al., 1968), melon (Sauton et al., 1989) and
apple (Bouvier et al., 1992), and selection of small seed-
lings in sugar beet (Maletskaya et al., 2009).
Differential buoyancy had been used previously in
date palm to identify seeds with different sugar content
(Nada, 1955), and similar techniques were subsequently
adopted for haploid isolation. For example, floatation in
a solution of hydrochloric acid (Aalders, 1958) or on cul-
ture medium (Lotfi and Salehi, 2008) was successful in
cucumber. In the future, phenotyping methods based on
optical projection tomography (Lee et al., 2006), diffrac-
tion enhanced imaging (Young et al., 2007) or miniatur-
ized magnetic resonance imaging (Koizumi et al., 2008)
may be valuable.
In addition to the use of physical traits, various
genetic markers have been employed. The most exten-
sive examples are those from maize, in which a range
of embryo colour markers have been adopted in large-
scale screening for use in commercial programmes (see
below). A potential alternative method in maize is sepa-
ration of haploids on the basis of differential oil content
(Rotarenco et al., 2007). Examples from other species
include use of a seedling leaf marker in kale (Thompson,
1956) and potato (Peloquin and Hougas, 1959), virus
sensitivity in tobacco (De Nettancourt and Stokes, 1960),
a seedling marker in tomato (Ecochard et al., 1969),
a hypocotyl colour marker in sugar beet (Bosemark,
1971), an anthocyanin ‘axil spot’ marker in cocoa (Dub-
lin, 1973c), a dominant red colour marker in pear (Bou-
vier et al., 1993), and the use of virescent and glandless
mutant markers to identify haploids induced by the
384 Jim M. Dunwell
semigamy gene in cotton (Stelly and Rooney, 1989).
Another notable example of the use of a seedling mar-
ker is that in asparagus where the frequency of parthe-
nogenesis among single seedlings was approximately 1
seedling in 2400. The marker gene technique increased
the efficiency of cytological examination from approxi-
mately 1% or 2% to 90% (Bassett et al., 1971).
A particularly sophisticated method for screening is that
developed by Hamza et al. (1993) for the isolation of
spontaneous haploid tomato plants from glasshouse
grown seedlings obtained from crosses involving a trans-
genic parental line in which a counter-selectable chimaeric
gene has been introduced. Transgenic seeds transformed
with the aux2 gene, a gene of Agrobacterium rhizogenes
that transforms naphthalene acetamide (NAM) into naph-
thalene acetic acid (NAA), did not develop roots in the
presence of NAM, whereas wild-type tomato seeds devel-
oped a normal rooting system in its presence. Transgenic
plants homozygous for aux2 (cv. ‘UC82b’) were used to
pollinate male-sterile (ms322) tomato plants (cv. ‘Ape-
dice’). Using NAM as a toxic substrate to kill heterozygous
diploid plants carrying aux2, the authors selected for three
maternal haploid plants resulting from the development of
the female nucleus without fertilization. Although, mater-
nal haploid selection using the aux2 marker was less effi-
cient than the visual screening of haploid plants displaying
recessive morphological markers of the female parent, it
did provide evidence for the feasibility of haploid selection
in species for which no morphological markers are avail-
able. A similar transgenic approach using nitrite reductase
silencing was developed for tobacco by Vaucheret et al.
(1995).
In vitro induction of haploids
‘We ought to be able to devise methods of growing tissue
cultures of plants and be in a position to regenerate a
whole plant from any single cell’ (Blakeslee, 1939).
In addition to the diverse approaches taken to identify
spontaneous haploids, many and various methods have
been developed for the in vitro induction of haploids from
either male or female gametophytes. In addition to the
general reviews mentioned earlier, there are other publica-
tions related to haploid induction in specific plant groups
or species. These include reviews on conifers (Huhtinen,
1972), spices and vegetables (Juhász et al., 2006) and fruit
trees (Germanà, 2006). The following sections will first
describe the range of methods for induction from male
and female cells.
Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 377–424
Anther and microspore culture
As Haldane (1932) stated ‘Clearly a higher plant species is
at the mercy of its pollen grains’, and although this motto
is undoubtedly true in evolutionary terms, it is only rela-
tively recently that we have been able to disrupt, almost
at will, this critical phase of plant development. The basic
pathway of pollen differentiation will be described in the
following sections, prior to a review of the various path-
ways induced during the process of in vitro induction.
Normal pollen development
Asymmetric cell division and establishment of
polarity. Development of a mature male gametophyte
depends on the asymmetric first pollen grain mitosis
(PGM) in the haploid microspore and the subsequent dif-
ferentiation of two distinct cells, a large vegetative cell
and a smaller generative cell, which later divides either
before or after anthesis to produce two sperm cells
(Figure 1). However, the asymmetry of the first PGM, pre-
sumably a consequence of existing polarity in the micro-
spore, is not invariable, and there are many examples of
occasional errors in this process. These will be described in
the following section.
The well-established cytological (Figure 2), ultrastructural
(Figure 3) and histological descriptions of normal gameto-
phyte development have recently been complemented
with novel optical techniques that include a detailed study
of mitochondrial (Matsushima et al., 2008) and plastid
(Tang et al., 2009) dynamics.
Transcript profiling during normal development. Until
recently, little was known about the patterns of gene
expression that exist in the developing anther and pollen
grain, but the availability of global transcript profiling has
now provided a wealth of interesting detail (Wilson and
Zhang, 2009). For example, analysis of the rice anther
transcriptome (Hayashi et al., 2009; Huang et al., 2009)
includes evidence for large-scale antisense transcription, a
phenomenon for which there is becoming extensive evi-
dence in other tissues (Poole et al., 2008). Similarly,
expression profiling has been conducted on mature pollen
of species including maize (Ma et al., 2008), rice (Hobo
et al., 2008; Mihara et al., 2008; Suwabe et al., 2008),
soybean (Haerizadeh et al., 2009), tomato (Frank et al.,
2009) and the dimorphic sperm cells in Plumbago zeyla-
nica (Gou et al., 2009).
Particularly, fascinating recent studies include reports
that small RNAs are present and functional in pollen
ª 2010 The Author

(a) Nicotiana tabacum
1st PGM
2nd PGM
(b) Hordeum vulgare
1st PGM
2nd PGM
Figure 1 Diagram of pollen development. Pattern of cell division in
tobacco Nicotiana tabacum (a) and barley Hordeum vulgare (b). In
both species, the first PGM occurs in a nucleus displaced to a position
adjacent to the pollen wall. The generative cell migrates to the interior
of the vegetative cell. In tobacco, the pollen is shed in a bicellular
form, with the second PGM occurring in the pollen tube. In barley,
the second PGM occurs prior to anthesis and pollen germination.
Scale bar is 20 l.
(Fujioka et al., 2008; Chambers and Shuai, 2009; Grant-
Downton et al., 2009; Slotkin et al., 2009) and that epige-
netic differences (Johnson and Bender, 2009) exist
between the two components of the pollen grain, the
vegetative and generative cells (Ribeiro et al., 2009; Schoft
et al., 2009). Such information should be considered in
the light of evidence that a dynamic reciprocal regulatory
circuit controls gametophytic development (Johnston
et al., 2008; Johnston and Gruissem, 2009).
Further molecular genetic understanding of pollen
development will be aided by the recent analysis of a
collection of Ds insertional mutants associated with
ª 2010 The Author
Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 377–424
Haploids in flowering plants 385
defects in male gametophyte development and function in
Arabidopsis (Boavida et al., 2009).
It is to be expected that this range of molecular, cellular
and genetic techniques will soon be applied to some of
the variant forms of development that either occur in vivo
or that are induced during in vitro culture.
Abnormal pollen development in vivo
Various forms of abnormal types of pollen development
in vivo had been described many years before the first
evidence of induced cell division in pollen in culture. These
abnormalities include both delays in development and ⁄ or
extra divisions in the component nuclei and cells of the
gametophyte. These will be discussed in turn in the
following paragraphs.
Smaller less developed pollen, often with symmetric rather
than asymmetric first pollen grain mitosis. It has been rec-
ognized for several decades that anthers of many, if not
most, species contain, as well as the major population of
starch filled mature pollen, a low frequency of small, often
starch-free grains (Figure 4). This so-called dimorphism
(Liang, 1985) is found in its most extreme form in species
such as Tripogandra amplexicaulis (Mattsson, 1982) where
one type of anther contains only small grains not capable
of participating in fertilization. Nutritional starvation
because of breakdown of the anther filament has been
implicated in this phenomenon. In some such cases, the
nuclei of the small grains were observed to be undergoing
or to have undergone supernumerary mitosis(es). The rele-
vance of these observations to the behaviour of immature
pollen grains in cultured anthers (see below) grew out of
the initial finding that the protein and RNA contents of
embryogenic tobacco microspores were typically at a low
level one-sixth that of nonembryogenic grains (Bhojwani
et al., 1973). In other words, the normal synthetic pro-
cesses of the pollen had been inhibited in those grains
destined for sporophytic development. For a long period
thereafter, it was not clear whether such grains pre-
existed in anthers (Figure 5) or were induced by the cul-
ture process. Various aspects of this debate have been
rehearsed over several years (Roberts-Oehlschlager and
Dunwell, 1991) with perhaps the most detailed
recent description of the arguments being that provided
by Heberle-Bors and Reinert (1981).
Various cytological abnormalities are often associated
with these dimorphic grains. The two most common
abnormalities are, first, grains in which the first PGM does
not lead to two different nuclei and cells but rather to an
386 Jim M. Dunwell

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

(j) (k) (l)

Figure 2 Pollen development in maize, Zea mays. All samples are stained with acetocarmine. (a) Tetrad of spores after meiosis. (b) Individual
microspore after dissolution of the callose around the tetrad. (c) Partially vacuolated microspore with thickening wall and with single pore visible.
(d) Fully vacuolated microspore with nucleus adjacent to the wall, and diametrically opposite the pore. Nucleolus is evident in the nucleus.
(e) Microspore with nucleus entering prophase of the first PGM. (f) Metaphase (n = 10) of first PGM. (g) Equatorial view of first PGM. (h) Anaphase
of first PGM. (i) Bicellular pollen grain with small generative cell separated by hemispherical wall from vegetative cell, the nucleus of which has
migrated across the grain to a position adjacent to the pore. (j) Pollen grain with partly regressed vacuole of vegetative cell. Nucleolus evident in
vegetative nucleus. (k) Pollen grain during period of rapid biosynthetic activity with minimal vacuole. (l) Mature pollen grain with vegetative
nucleus and one of the two sperm cells, products of the second PGM. Scale bar is 20 l.

equal division, after which a cell wall may or may not
form between the nuclei. Frequently, however, the spindle
lies in a central position and is oriented parallel to the long
axis of the microspore. In this position, the metaphase
spindle is symmetrical and spindle fibres are equally visible
at the two poles. Anaphase movement is also symmetrical
(e.g. Allium, Brumfield, 1941). The telophase nuclei result-
ing from the division are equal in size, and a cell plate is
formed equidistant between the two nuclei, dividing the
microspore into equal halves. This observation provided
some of the first evidence that asymmetry of the resultant
cells results from the position of the spindle with respect
to the wall of the microspore.
Additional divisions. Anomalous division of the vegetative
cell of pollen was first described in petaloid anthers of
Hyacinthus orientalis, in the nineteenth century (Němec,
1898). This phenomenon was discussed subsequently by
De Mol (1921), Stow (1930), Geitler (1935), Poddubnaja-
Arnoldi (1936) and Naithani (1937). Similar observation
were reported following starvation in tomato plants,
Lycopersicon esculentum, (Howlett, 1936) and by heat

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Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 377–424
 Haploids in flowering plants 387

(a) (b)

(c) (d)

(e) (f)

Figure 3 Pollen development in Datura innoxia. Transmission electron micrographs of microspores in vivo. (a) Unicellular microspore with nucleus
adjacent to the intine, prior to the first PGM. (b) Mitosis of nucleus showing chromosomes attached to microtubules, with mitotic spindle perpen-
dicular to the pollen wall. (c) Formation of small generative cell separated by hemispherical phragmoplast from large vegetative cell. Both cells
have distinct nucleoli at this stage. (d) Generative cell wall is complete. (e) More mature pollen grain with spherical generative cell now migrated
into the interior of the cytoplasm of the vegetative cell. The large vegetative nucleus has a prominent nucleolus during this stage of rapid synthesis
of storage compounds. The chromatin with the generative nucleus is undergoing condensation. (f) Initial stage of germination of the pollen grain
showing generative cell with condensed chromatin and adjacent vegetative nucleus, amoeboid in shape with indistinct nuclear structures. Scale
bars are 10 l.

treatment (8–38 C) in Tradescantia paludosa (Sax, 1935,
1937). Additional divisions in the products of a symmetric
PGM in dwarf (dimorphic) pollen in Amaryllis were
described by Upcott (1939) and in a particular clone of
Tradescantia bracteata by La Cour (1949). Darlington and
Thomas (1941) found pollen grains of Sorghum purpureo-
sericeum with extra divisions of the vegetative nuclei, but
in this case, these divisions were influenced by the
presence of heterochromatic B chromosomes supernumer-
ary to the basic set. The similarity of these naturally occur-
ring modes of development to those induced by the
culture process will be evident when in vitro observations
are described in the following paragraphs.
An interesting observation relevant to this discussion
was made recently in Arabidopsis, where it was reported
that inhibition of expression of retinoblastoma protein,

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Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 377–424
388 Jim M. Dunwell

(b)
(a)

(c)

Figure 4 Dimorphic pollen in vivo. (a,b) Scanning electron micrographs of freeze-fractured barley anthers showing arrangement of normal ovoid
grains embedded in the tapetum around the interior circumference, and abnormal small spherical grains free in the loculus of the anther. Scale
bars are 100 l. (c) Pollen of Papaver somniferum stained with acetocarmine and potassium iodide. Normal grains show orange staining of
protein ⁄ nucleic acids and dark blue ⁄ black staining of starch grains. Abnormal small grains have reduced staining. Scale bar is 50 l.

a conserved inhibitor of cell proliferation, caused the lim-
ited hyperproliferation of the vegetative and to a lesser
degree of the sperm cells (Chen et al., 2009). This obser-
vation may allow novel approaches to the objective of
inducing division of the pollen cells.
Microspore embryogenesis
During the 1960s, a major breakthrough was achieved by
the discovery that immature pollen grains (microspores) of
the solanaceous species Datura innoxia could develop into
haploid embryos if anthers were cultured under specific
conditions in vitro (Guha and Maheshwari, 1964). This
remarkable discovery, an accidental consequence of a
study of meiosis (Guha-Mukherjee, 1999), was subse-
quently extended to many other species, principally to
members of the Solanaceae (Dunwell and Sunderland,
1973) (Figures 5, 6 and 7b), Brassicaceae and Poaceae
(Figures 7a and 8). Interestingly, much of the early pio-
neering work on this subject was conducted in China
(Chu et al., 1976), during the final period of the Cultural
Revolution (Anon, 1976a,b).
The various developmental, physiological and environ-
mental factors that influence the diversion of the
normal pattern of gametophytic development towards a

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Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 377–424

Figure 5 Genetic segregation from tobacco anthers in vitro.
Microspore-derived plantlets developing from an anther of a Nicotiana
tabacum plant heterozygous Su ⁄ su (light green) for the semidominant
sulphur nuclear mutation. The haploid plantlets are segregating
50 : 50 as green (su) or yellow (Su). Scale bar is 1 mm.
sporophytic one (Seguı́-Simarro and Nuez, 2008a) were
the subject of many early studies on model species (Dun-
well, 1976; Dunwell and Thurling, 1985) that have now
been complemented by results from a greater range of
species (We˛dzony et al., 2009). Examples of recent specific
studies are provided in Table 4. Useful recent general
reviews include those provided by Srivastava and Chaturv-
edi (2008), Pratap et al. (2009) and Touraev et al. (2009).
Cytological and ultrastructural (Figures 7 and 9) studies
have shown that in general terms, the pathways along
which pollen is diverted to its sporophytic existence com-
prise the A pathway where the embryo results principally
from repeated division of the vegetative cell (Figure 7b
and 9a,b); the B pathway which follows from division in a
microspore that has undergone an equal division
(Figure 7b) and the C pathway in which an embryo devel-
ops after the fusion of generative and vegetative cells. As
described in examples in vivo (Sax, 1935, 1937; La Cour,
1949) (see above), polarity of the first pollen division may
be disturbed such that the mitotic spindle develops parallel
to the microspore wall instead of in the more usual per-
pendicular plane. The cell plate does not then curve
around one of the daughter nuclei but develops straight
across the spore, partitioning it equally or unequally,
according to the position of the nucleus in relation to the
long axis of the spore.
The theory that incomplete generative cell wall
formation is a necessary prerequisite for later embryogenic
division was first proposed in tobacco by Vazart (1971)
ª 2010 The Author
Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 377–424
Haploids in flowering plants 389
and by Dunwell and Sunderland (1976c) who found by
ultrastructural observation in Datura innoxia that anoma-
lous orientation of the mitotic spindle during the first
PGM often leads to such failure of wall formation (Fig-
ure 9c). Application of various media components have
also been shown to induce symmetrical mitoses. An exam-
ple of this is the use of lithium treatment of tobacco mi-
crospores (Zonia and Tupý, 1995) and heat treatment in
eggplant (Bal et al., 2009).
Genotype
After the initial experiments on this subject in rice (Oryza
sativa), it became obvious that the response of anthers in
culture is determined to a large extent by the genotype of
the donor plants. The best evidence has been obtained in
the cereals, barley (Hordeum vulgare) and wheat (Triticum
aestivum). Two of the basic components of yield, the
percentage of anthers producing microspore derivatives,
and the number of regenerants produced per anther,
appear to be determined independently. However, it has
not yet been possible to relate these in vitro responses to
any specific genetic locus.
There is an equally important role of genotype if
microspores are cultured in isolation, rather than in the
confines of the anther. Recent results in this area include
those on Brassica oleracea (Wei et al., 2008) and oat
(Sidhu and Davies, 2009).
Preculture environment
This variable was investigated first in tobacco (Nicotiana
tabacum) (Dunwell, 1976), where it was found that both
photoperiod and light intensity affected the yield of micro-
spore embryos and plantlets. Subsequent studies on barley
(Foroughi-Wehr and Mix, 1976), oilseed rape (Brassica
napus ssp. oleifera) (Keller and Stringham, 1978; Dunwell
and Cornish, 1985), turnip (Brassica campestris) (Keller
et al., 1983) and wheat (Lazar et al., 1984) showed that the
temperature under which the donor plants are grown can
also markedly affect the culture response. However, it is not
possible to make general recommendations about optimal
growth conditions as they seem to vary between species.
As well as physical variables, two early reports on
tobacco indicated that the nitrogen status of plants greatly
affects the yield of microspore embryos (Sunderland,
1978; Tsay, 1981, 1982). In the first study, ‘nitrogen-
starved’ plants gave better results than those supplied
with fertilizer, whereas in the second study, five nitrogen
treatments were compared, revealing an optimum
nitrogen concentration of 15 mM in the culture solution.
390 Jim M. Dunwell

(a) (b)

(c) (d)

Figure 6 Microspore embryogenesis in Solanaceae. (a–c) Scanning electron micrographs of microspore-derived embryos of Nicotiana tabacum. (d)
Multicellular microspore-derived embryo of Solanum tuberosum stained with acetocarmine. Scale bars are 20 l.

However, both studies were conducted on plants grown
in a glasshouse, and as there is a slight effect of nitrogen
status on flowering date, interpretation of the results is to
some extent confounded by the possible effect of differ-
ent conditions during flowering.
The benefit of various starvation and stress techniques
has also been reported recently in chickpea (Cicer arieti-
num L.) (Grewal et al., 2009). To induce microspore
division in anthers, the following stress treatments were
used: (i) flower clusters were treated at 4 C for 4 d, (ii)
anthers were subjected to electric shock treatment of
three exponentially decaying pulses of 50–400 V with
25 lF capacitance and 25 X resistance, (iii) anthers were
centrifuged at 168–1509 g for 2–15 min, and finally (iv)
anthers were cultured for 4 d in high-osmotic pressure
(563 mmol) liquid medium (see below). A similar study on
genotypes of pea, grass pea and M. truncatula has shown
that submission of flower buds to a cold period prior to
anther excision or microspore isolation, modifying the
osmotic pressure of the medium during initial culture and
electroporation of isolated microspores were the three
major individual stress agents to have an impact on the
efficiency of androgenetic proliferation and subsequent
differentiation from the microspores (Ochatt et al., 2009).
Stage of pollen development
This is one of the most crucial aspects of the culture pro-
cess as small differences in developmental age produce

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Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 377–424
 Haploids in flowering plants 391

(a) (b)

Figure 7 Cell division during embryogenesis in vitro. Microspore of barley (a) showing simultaneous mitoses of four nuclei and multicellular micro-
spore of potato (b) after probable symmetrical division of large vegetative cell (cf Figure 10c) products and with several smaller generative cell
products. Both microspores stained with acetocarmine. Scale bar is 10 l.

(a) (b)

Figure 8 Anther culture of barley. (a,b) Scanning electron micrographs of microspore-derived embryos of barley emerging from anthers cultured
on solid medium. Scale bars are 100 l.

great differences in yield. For example, in tobacco, a dif-
ference of 2 mm in corolla length may lead to a fourfold
difference in the yield of pollen plantlets (Dunwell, 1976).
The exact stage of pollen development which is most
readily diverted to a sporophytic pathway seems to vary
with species. In tobacco, it is the time of the first PGM, in
some species including Nicotiana sylvestris it is later, while
in others, notably the cereals, it is much earlier when the
microspore is still uninucleate. Exact determination of pol-
len stage requires a cytological analysis but for practical
large-scale programmes many investigators prefer to rely
on a simply measured, although less reliable, external mor-
phological indicator, such as corolla length.
Flower ⁄ anther pretreatment
In many species, it has been found that yields may be
increased substantially by pretreatment of the anther after
its excision from the plant and prior to its incubation on cul-
ture medium. Initial evidence on this topic came from mem-
bers of the Solanaceae. For example, the effect on the
embryogenic potential of tobacco anthers of a preculture
period in a water-saturated atmosphere was examined (Dun-
well, 1981). It was found that maintaining excised anthers in
such an atmosphere for 2 or 3 d at 23 C increased the num-
ber of anthers subsequently producing pollen plants fourfold
and doubled the number in which embryos were induced.
This increase in frequency was negatively correlated with the

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392 Jim M. Dunwell

Table 4 Recent advances in anther culture on a water ⁄ agar medium—indeed water may be beneficial
at the start of culture—continued embryo survival is stimu-
Species Reference(s)
lated by addition of a dilute mineral salt mixture and a
Anemone coronaria Laura et al., 2006; Laura and Allavena, 2007 carbon source (usually sucrose). The salt mixtures recom-
Cucumis sativus L. Song et al., 2007 mended are half strength Murashige and Skoog (1962) for
Tomato Bal and Abak, 2007; Seguı́-Simarro and Nuez, 2007 the Solanaceae and the N6 mixture for cereals.
Musa balbisiana Bakry et al., 2008
In those species (e.g. Gramineae, Cruciferae) in which
Capsicum annuum L. Kim et al., 2008; Nowaczyk et al., 2009
Phlox Razdan et al., 2008
mature pollen is shed in the tricellular condition, high
Triticale _ et al., 2008, 2009
Zur sucrose levels (6%–17%) (Dunwell and Thurling, 1985)
Rice Bagheri et al., 2009; Cha-um et al., 2009 are beneficial, whereas for those in which mature pollen is
Durum wheat Cistué et al., 2009; Plamenov et al., 2009
bicellular (e.g. Solanaceae) 2%–5% is usually optimal.
Loquat Li et al., 2008
Short-term treatment with high osmotica have recently
Cacti Garcia et al., 2009
Brassica campestris Wang et al., 2009a been recommended in chickpea (Grewal et al., 2009).
Coffee Silva et al., 2009 Specific modification of the carbon source include use of
Salix viminalis Wojciechowicz and Kikowska, 2009 include widespread evidence of the benefit of the disac-
Physalis Escobar-Guzmán et al., 2009
charide maltose in species including potato (Batty and
Solanum sessiliflorum Romani et al., 2009
Caraway Smýkalová et al., 2009 Dunwell, 1989), barley (Scott et al., 1995), lupin (Skrzypek
et al., 2008), pepper (Kim et al., 2008) and oat (Sidhu and
Davies, 2009).
rate of anther senescence in culture. It is suggested that Most members of the Solanaceae do not require an
some component of the usual culture medium is toxic to auxin addition, in contrast to members of the Gramineae
anthers during the early period of culture. and Cruciferae where 2,4-D is the auxin usually supplied.
This approach was been extended to many other spe- Originally, agar-based media were used for anther culture
cies, most importantly the cereals such as barley where a but subsequently liquid media were recommended and it
28-day treatment of the spikes at 4 C leads to a much became known that agar contains compounds inhibitory
increased yield of microspore calluses. In potato (Solanum to pollen embryo production. A further advantage of
tuberosum) a 2-day treatment at 6 C is recommended liquid culture is that it allows the release of microspore
(Wenzel et al., 1983). The optimum temperature and derivatives into the medium, thereby reducing possible
duration of pretreatment vary with species and it has been competition effects between developing embryos. A disad-
found that results also depend upon the type of explant vantage, in that sinking of these embryos may lead to
(tillers, inflorescences, whole buds or isolated anthers) and inhibitory anaerobic conditions, may be overcome by addi-
specific size of storage vessel used (Dunwell, 1981). tion of Ficoll, first described by (Kao, 1981) and now used
Recent data include results from Triticale in which it has widely (Marchand et al., 2008; Cistué et al., 2009). As an
been shown that anther preculture conditions could gen- alternative, agarose rather than agar has been used as the
erate oxidative stress and change the cellular metabolic solidifying agent.
activity, which could subsequently be reflected in the cell An associated area of research concerns methods to
viability and the efficiency of microspore embryogenesis reduce the amount of tissue browning, a phenomenon
_ et al., 2009).
(Zur caused by the presence of oxidized phenols. Application
A particularly successful form of pretreatment involves of anti-oxidants is often beneficial. With certain species,
the use of mannitol, a sugar alcohol usually considered as addition of activated charcoal is beneficial either in an
a nonmetabolizable carbon source. This method was agar-solidified medium or in a two-phase system in which
devised for barley (Roberts-Oehlschlager and Dunwell, anthers are floated on liquid medium overlying an
1990) and has since been confirmed in that crop (Muñoz- agar-solidified medium containing charcoal. Charcoal
Amatriaı́n et al., 2006) and extended to many others alters the medium in a number of ways by adsorbing
(Labbani et al., 2007). naphthaleneacetic acid (NAA) and cytokinins, thiamine
HCl and nicotinic acid, and iron chelates.
Nutrient composition Indeed, iron is one of the most critical components of
Although a low yield of microspore-derived embryos can the medium. The most detailed evidence is available for
be obtained in some species such as tobacco by culture tobacco wherein initiation of embryogenic division is iron

ª 2010 The Author

Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 377–424
 Haploids in flowering plants 393

(a) (b)

(c)

Figure 9 Microspore embryogenesis in tobacco. Transmission electron micrographs of Nicotiana tabacum microspores cultured in vitro. (a) Spore
showing induced symmetrical division of vegetative cell, and generative cell still attached to interior of spore wall. Axis of this division is perpendic-
ular to the axis of the first PGM. (b) Spore showing induced symmetrical division of vegetative cell, and generative cell still attached to interior of
spore wall. Axis of this division is parallel to the axis of the first PGM. (c) Binucleate spore after symmetrical division of unicellular spore. The cell
wall separating the two nuclei is incomplete, providing opportunity for subsequent nuclear fusion and generation of diploid embryo. Scale bars
are 10 l.

independent (Havranek and Vagera, 1979); at 0–30 lM
FeNaEDTA only globular embryos form whereas at
40–150 lM whole plantlets are produced (Vagera et al.,
1979). Addition of iron also induces senescence of the
anther wall (Vagera and Havranek, 1983), an important
factor in the determination of pollen plant production in
many species (Mii, 1980). Among other metal salts, the
effect of ZnSO4 concentration on barley microspore
embryogenesis has also been investigated. All the results
indicate that the beneficial effect of Zn2+ is exerted mainly
during the culture phase by increasing the number of
embryos and leading to an increased number of green
plants. However, it has no effect on percentage of regener-
ation or green plants (Echavarri et al., 2008). It is interesting
to note that zinc is a potent regulator of programmed cell
death (PCD) in animals and has recently also been impli-
cated as a factor inducing somatic embryogenesis of Nor-
way spruce (Picea abies) (Helmersson et al., 2008).
Addition of silver nitrate has also been recommended
for anther culture of barley, a species with many
recalcitrant cultivars and in which albinism is also a
problem (Jacquard et al., 2009b).

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Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 377–424
394 Jim M. Dunwell
Culture environment
The usual incubation temperature is between 25 C and
30 C, although temperatures outside this range have
been used, at least during the early period of culture. For
example, a temperature of 35 C for 1–2 days at the start
of culture is necessary to induce high yields of embryos
from many Brassica species, e.g. broccoli (Brassica oleracea
cultivar italica) (Keller and Armstrong, 1983), Brassica hirta
(Klimaszewska and Keller, 1983), winter cultivars of oil-
seed rape (Dunwell et al., 1985). In wheat, a temperature
of 28–30 C was reported as suitable (Ouyang et al.,
1983). However, higher incubation temperatures may
possibly lead to increased yields of albino regenerants in
cereals (see below).
Anther orientation
Since the original study of this topic in tobacco (Sunder-
land and Dunwell, 1972), there have been only three fur-
ther related investigations. In the first (Sopory and
Maheshwari, 1976), anthers of Datura innoxia were placed
on solidified medium in one of four orientations. Any sub-
mersion of the anther in the medium completely inhibited
the response and optimal results were obtained when the
anthers were laid flat with the filament side in contact
with the medium. In a very similar study on tobacco
(Misoo et al., 1981), the highest and most rapid response
was achieved if anthers were cultured with the basal half
submerged in the medium.
Yang and Zhou (1979) compared three inoculation
methods for rice. First, excised spikelets were incubated
with the pedicel inserted into the medium; secondly, spik-
elets were placed horizontally with the anthers in contact
with the medium; and thirdly, excised anthers were placed
in contact with the medium. Some abnormal pollen divi-
sions occurred in anthers incubated in the first two meth-
ods, but only the third method gave high yields of pollen
calluses. Similar results were reported in barley by
Shannon et al. (1985).
Culture atmosphere
One often neglected aspect of in vitro culture of anthers
and pollen is the culture atmosphere. It was found in early
experiments on tobacco that a small volume (0–5 mL per
anther) almost completely suppressed embryo induction
(Dunwell, 1979). Removal of specific components of the
culture atmosphere (ethylene, carbon dioxide, oxygen)
influenced the response of the anthers but did not pro-
duce a satisfactory explanation of the inhibition of pollen
embryogenesis by the small culture atmosphere volume.
Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 377–424
Until the complete spectrum of gases is analysed through-
out the culture period, thus enabling manipulations of
their individual levels to be attempted, the induction of
pollen embryogenesis will remain dependent upon the for-
tuitous combination of atmospheric components.
Cocultivation
The density of the culture (i.e. number of anthers per unit
volume of medium) has been found to be of great impor-
tance in anther culture of many cereal species. This effect
is related to the marked advantage of using medium pre-
conditioned with other reproductive tissues or organs, par-
ticularly ovaries (Broughton, 2008; Cistué et al., 2009).
Similarly, an improved results with isolated microspore cul-
ture of pepper (Capsicum annuum L.) using co-culture
with ovary tissues of pepper or wheat has been recently
reported (Lantos et al., 2009). The exact chemical basis for
this effect is not known, although there is good evidence
for a stimulatory role of arabinogalactans (Letarte et al.,
2006).
Molecular investigations
Since the first description of microspore-derived embryos,
there has been a search for the molecular basis underlying
this developmental switch. Initially, these investigations
were limited to histological investigations such as that of
Bhojwani et al. (1973) who found that the protein and
RNA contents of embryogenic tobacco microspores were
about one-sixth that of nonembryogenic grains. This sug-
gested that the normal biosynthetic processes of the pol-
len had been inhibited in those grains induced into
sporophytic development.
The advent of more sophisticated analytical techniques
has produced a diversity of investigations on the develop-
ment of cellular and histological markers of embryogenesis
(Hosp et al., 2007). For example, in olive (Olea europaea
L.), anti-RNA and pectin-specific monoclonal antibodies
have been used to characterize embryogenic microspores
(Solı́s et al., 2008). This type of study has also now been
complemented by a range of molecular investigations
(Chan and Pauls, 2007; Tsuwamoto et al., 2007; Seguı́-
Simarro and Nuez, 2008a; Lee et al., 2009). These
approaches in B. napus include the use of gene expression
profiling, a technique that has revealed the expression of
several embryogenesis-related transcription factor genes
like BABY BOOM ERF ⁄ AP2 (BBM) (Boutilier et al., 2002),
LEC1 and LEC2, during the first 2–3 d of microspore cul-
ture (Malik et al., 2008). Indeed, ectopic expression studies
have shown that BBM expression is sufficient to induce
ª 2010 The Author

spontaneous somatic embryo formation in both Arabidop-
sis and B. napus. Similar studies on pretreatments in barley
microspore embryogenesis stimulates expression of plant
defence genes including germin-like proteins (Jacquard
et al., 2009a), an important group of functionally diverse
proteins involved in the response of plants to a range of
biotic and abiotic stress (Dunwell et al., 2008).
In a related recent study designed to combine molecular
methods with cellular localization in barley, RT-PCR
together with in situ hybridization on sections (ISH) and
whole-mount in situ hybridization (WISH) were used to
analyse the expression of the early-culture abundant gene
(ECA1), which is expressed during microspore embryogen-
esis, and a polygalacturonase gene (HvPG1), a late pollen
gene expressed during gametogenesis only after micro-
spore division (Pulido et al., 2009). Both ECA1 and HvPG1
genes were transcriptionally active after stress pretreat-
ment in the same populations of microspore-derived struc-
tures, those comprising the sporophytically induced ones.
ECA1 transcripts were also detected after 3 days’ culture.
These results suggest the possibility of using ECA1 gene
expression as a marker for the induction of microspore
embryogenesis and the earliest stages of this process.
Additionally, they demonstrate that WISH is a suitable
technique for studying gene expression in embryogenic
microspore populations and, because different structures
can be examined individually, is an appropriate comple-
ment to transcriptomic profile analyses in the study of
early microspore embryogenesis.
A recent study that is particularly relevant to the issue
of pollen cell differentiation concerns the function of
Arabidopsis NEDD1, a WD40 repeat protein related to the
animal NEDD1 ⁄ GCP-WD protein, which interacts with the
c-tubulin complex (Zeng et al., 2009). A T-DNA insertional
allele of the single NEDD1 gene was isolated and main-
tained in heterozygous sporophytes, and NEDD1’s function
in cell division was analysed in haploid microspores pro-
duced by the heterozygote. Approximately half of the
dividing microspores exhibited aberrant microtubule (MT)
organization, where spindles were no longer restricted to
the cell periphery and became abnormally elongated. After
mitosis, MTs aggregated between reforming nuclei but
failed to appear in a bipolar configuration. Consequently,
the defective microspores did not form a continuous cell
plate, and two identical nuclei were produced with no dif-
ferentiation into generative and vegetative cells (Bal et al.,
2009).
In terms of evolutionary significance, probably the
most interesting recent advance is the direct induction of
ª 2010 The Author
Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 377–424
Haploids in flowering plants 395
zygotic-like embryogenesis in microspores of B. napus
(Joosen et al., 2007; Supena et al., 2008). Although
regeneration from microspores of this species has been
known for many years, recent modifications in culture
conditions involving more precision in the application of
high temperature, have provided a process most analo-
gous to zygotic embryogenesis. These findings represent a
major breakthrough and will greatly facilitate the study of
plant embryogenesis in an isolated system.
A related recent investigation of microspore embryogen-
esis in the same species involved applications of buthio-
nine sulfoximine (BSO), an inhibitor of GSH (reduced
glutathione), which switches the cellular glutathione pool
towards the oxidized form GSSG (Stasolla et al., 2008).
This compound positively influences embryo quality by
improving the structure of the shoot apical meristem and
promoting embryo maturation, both of which improve the
postembryonic performance of the embryos. To investigate
the mechanisms underlying this improvement in embryo
quality, the transcript profiles of developing microspore-
derived embryos cultured in the absence (control) or
presence of BSO were analysed using a 15 000-element
B. napus oligo microarray. BSO affected the transcription
and activities of key enzymes involved in ascorbate metab-
olism, which resulted in major fluctuations in cellular
ascorbate levels. It also activated many genes controlling
meristem formation and function; these included ZWILLE,
SHOOTMERISTEMLESS and ARGONAUTE.
Also relevant to oilseed rape is the recent announce-
ment by The National Research Council of Canada (NRC)
in Saskatoon, Saskatchewan and the Centre for BioSys-
tems Genomics (CBSG) in the Netherlands that they intend
to collaborate on a research project to better understand
the signalling pathways that are involved in the formation
of haploid embryos—research that will develop new and
improved varieties of oilseed rape (canola) in a shorter time-
to-market (http://www.nrc-cnrc.gc.ca/eng/news/nrc/2009/
07/23/crop-production.html).
However, despite this continuous range of improve-
ments, much of it is still devoted to a rather narrow range
of species and there is still no method that can be univer-
sally recommended with a new species of interest; much
progress still depends on long and tedious comparisons of
media and environmental conditions.
Ovary and ovule culture
An alternative means of haploid induction is by ovary or
ovule culture, whereby the female megaspore, rather than
the male microspore, is induced to develop into an entire
396 Jim M. Dunwell
plant. This technique was developed first in barley (San
Noeum, 1976) and has now been extended to a number
of other species, most if not all of which have also been
successful in anther culture. General aspects of the ovary
culture technique have been reviewed extensively (Yang
and Zhou, 1982; Dunwell, 1986; Bohanec, 2009). Specific
crops used for this technique include sugar beet (Van Geyt
et al., 1987), apple (Zhang and Lespinasse, 1988), gerbera
(Tosca et al., 1999), onion (Muren, 1989; Alan et al.,
2007; Ebrahimi and Zamani, 2009) and carnation (Sato
et al., 2000); more recent examples include tef (Gugsa
et al., 2006), maize (Tang et al., 2006); squash (Cucurbita
pepo L.) (Shalaby, 2007), mandarin orange (Froelicher
et al., 2007), niger (Guizotia abyssinica) (Bhat and Murthy,
2008) and cucumber (Diao et al., 2009). Attempts in
cotton have also been reported recently (Kantartzi and
Roupakias, 2009).
Products of culture
Initially, it was expected that the plants derived from the
various forms of haploid cell would be haploid in composi-
tion and normal in phenotype. This assumption was soon
proved to be incorrect. The presence of induced genetic
or epigenetic variation is a particular issue in those tech-
niques that involve a prolonged period of callus culture
rather than direct embryogenesis.
Albinos
One of the major problems to be overcome before the
anther or microspore culture technique can be widely
applied in cereal breeding programmes is the occurrence
of large numbers of albinos amongst the regenerants
(Caredda et al., 2000; Torp and Andersen, 2009). The
exact cause(s) remain obscure. Although albino plants
remain vegetative and the subsequent inheritance of albi-
nism cannot be investigated, it has been known for many
years that the frequency of albino regenerants has a
strong genetic component. This phenomenon has been
investigated most recently by Muñoz-Amatriaı́n et al.
(a) (b) (c)
Figure 10 Ploidy series of Datura innoxia. Leaves taken from plants at similar stage of growth. (a) Haploid from anther culture. (b) Diploid from
anther culture. (c) Diploid from seed. (d) Triploid from anther culture. (e) Tetraploid from anther culture. (f) Hexaploid from anther culture. In
addition to differences in overall leaf size, note difference in leaf shape from oblique in haploid, through rounded ⁄ truncate in diploids and
triploids to cordate in tetraploid and hexaploid. Scale bar is 1 cm.
Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 377–424
(2009) who used microarray methods with barley microsp-
ores to identify specific genes linked to albinism.
The frequency of albinos is also determined by specific
components of the culture or preculture medium (Labbani
et al., 2007; Soriano et al., 2008; Jacquard et al., 2009b;
Niroula and Bimb, 2009). Also their frequency varies with
pollen stage at inoculation. In this context, it is known
that the plastid genomes in albinos show substantial
changes (Dunford and Walden, 1991). However, until the
status of the genome during normal in vivo development
is established it will remain unclear whether the changes
identified in regenerants pre-exist or are induced by the
culture process.
Ploidy variants and gametoclonal variation
It has been known for many years that contrary to initial
expectations, the plants derived from the various in vitro
techniques are not all haploid and that a range of ploidies,
including ploidy chimaeras (Yamamoto and Tominaga,
2004), can be generated. A good example of this is provided
by an analysis of microspore-derived plants regenerated
from cultured anthers of Datura innoxia. These plants show
ploidies from haploid to hexaploid (Figure 10). It is known
that some of the nonhaploid plants probably originate from
nonhaploid microspores (Collins et al., 1974), whilst the
triploid plants originate from a process of endoreduplication
occurring during the early divisions of the microspores in
culture (Sunderland et al., 1974). Of particular significance
in the origin of nonhaploids is the high frequency of incom-
plete cell wall formation occurring between the vegetative
and generative nuclei (Dunwell and Sunderland, 1974a,b,
1975, 1976a,b,c), or at a later stage of division of vegetative
and ⁄ or generative cell products. These processes have been
studied more recently by González-Melendi et al. (2005)
and Seguı́-Simarro and Nuez (2008b).
Plants with a nonhaploid chromosome complement may
have intrinsic practical value. For example triploid plants
of papaya derived via anther culture have been shown to
combine short stature with a high yield of large seedless
(d) (e) (f)
ª 2010 The Author

fruits and may have great potential for exploitation in both
breeding and commercial fruit production (Rimberia et al.,
2007).
A series of discussions of the opportunities for analysis
of gene expression in various ploidies using next genera-
tion sequencing have been published recently (Peng et al.,
2008; Zhang et al., 2008a, 2009b). Similarly, a proteomic
analysis has revealed differences between haploid and
diploid embryos of cork oak (Gómez et al., 2009).
Aside from the gross differences in ploidy, there is
extensive evidence for more subtle genetic changes in
regenerants (Suenaga and Nakajima, 1993). It is possible,
for example, that the occurrence of albinism (see above) is
merely one aspect of rearrangements to the nuclear and
organellar genomes that occur in pollen or its derivatives,
either before or during culture. Evidence of various genetic
and molecular changes in microspore regenerants has
accumulated over several decades. Some of this evidence,
such as that in barley (Powell et al., 1984, 1986), is based
solely on analyses of whole plant characters. However,
there is also evidence from tobacco (Dhillon et al., 1983)
and N. sylvestris (De Paepe et al., 1982) that the regener-
ants may differ in nuclear DNA quantity and quality from
donor plants. There is also genetic evidence from tobacco
for specific cytoplasmic changes (Matzinger and Burk,
1984). It is not known how this variation is related to the
general area of somaclonal variation and whether it will
prove advantageous or disadvantageous to plant breeders.
A more detailed analysis of the molecular basis of such
variation has since been quantified by using a range of
markers in pepper (Juhász et al., 2001) and including SSRs
in barley (Bednarek et al., 2007). Anther culture of rice
has also been shown to activate a new class of Miniature
inverted-repeat transposable elements (MITEs) called mini-
ature Ping (mPing) (Kikuchi et al., 2003).
As donor plants in most breeding programmes (see
below) are of the F1 or F2 generation, such gametoclonal
variation may be present but not identified amongst the
genetically diverse segregants.
The most recent review of environmentally induced and
developmentally regulated genomic variation, including
that occurring in haploid regenerants, is that of Li (2009).
Comparison of efficiency
Various studies have examined the relative efficiency of
haploid production from both conventional and in vitro
techniques in barley (Huang et al., 1984; Powell et al.,
1984, 1986), wheat (Guzy-Wróbelska and Szarejko,
ª 2010 The Author
Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 377–424
Haploids in flowering plants 397
2003), apple (Zhang, 1988; Lespinasse et al., 1999; Höfer
et al., 2008), triticale (Thiemt and Oettler, 2008) and flax
(Obert et al., 2009).
Chromosome doubling
The earliest examples of chromosome doubling occurred
by selfing and decapitation in tomato (Lindstrom, 1929,
1941 Lindstrom and Koos, 1931). After the isolation of
the antimitotic chemical colchicine in the 1930s, this com-
pound became the usual method of choice (Newcomer,
1941; Toole and Bamford, 1947; Singsit, 1991; Liu and
Song, 2000b; Lanaud, 2006a,b; Lim and Earle, 2008,
2009). Interestingly, colchicine has also been used to gen-
erate haploids in several species including sugar beet
(Levan, 1945), sorghum (Sanders and Franzke, 1962;
Simantel and Ross, 1964) and maize (Hu et al., 1991).
There are various comparative studies of the effects of
antimitotic agents on development and genome doubling.
For example, chromosome doubling of haploid embryos of
(Quercus suber) was evaluated by flow cytometry mea-
surements and it was shown that nuclear DNA duplication
and embryo survival was most efficiently induced with
oryzalin 0.01 mM for 48 h (Pintos et al., 2007). A recent
comparative study in B. napus found that mean rate of
DH plants from trifluralin treatment was 85.7%, from col-
chicine 74.1% and 66.5% in the case of oryzalin, while
only 42.3% in the untreated control variant. Additional in
vivo application of colchicine at the plantlet stage did not
significantly increase the mean rate of DH plants (55.6%)
(Klı́ma et al., 2008). Other related studies include those on
gynogenic onion (Grzebelus and Adamus, 2004) and of
the use of nitrous oxide gas as a doubling agent (Kato,
2002; Kitamura et al., 2009).
The wide range of these techniques for chromosome
doubling have been reviewed by Kasha (2005) and some
are the subject of patent applications (see below).
Exploitation
Haploids have been exploited in a wide range of
theoretical and practical aspects of plant biology
and genetics, and some of these will be summarized in
the following paragraphs.
Horticultural interest
Haploids may have intrinsic value because of their overall
reduction in size compared with diploids. For example, the
398 Jim M. Dunwell
Figure 11 Haploid Pelargonium ‘Kleine Liebling’. Scale bar is 5 cm.
well-known cultivar of pelargonium ‘Kleine Liebling’ (and
its variants) (Figure 11) is in fact a spontaneous haploid
(Daker, 1966, 1967; Li, 2005). According to Daker, 1967,
‘It is not known whether Kleine Liebling arose as a haploid
seedling, or as a bud sport, but it appears likely to have
originated in Germany in 1925, while the variegated form,
a chlorophyll mutant, was raised by Miller of Los Altos,
USA in 1956.’ Several colour variants of this cultivar are
available from Fibrex Nursery Ltd, Honeybourne Road, Peb-
worth, Stratford-upon-Avon, Warwickshire, CV37 8XP,
England (http://www.fibrex.co.uk/).
Also of much interest is Pohlheim’s (1968) report of a
haploid form of Thuja plicata called Thuja gigantea ‘graci-
lis’ Beissn. This was first described in 1896 (Beissner, L.
cited by Pohlheim, 1968) although it was not recognized
as a haploid at that time. Beissner had observed several
branches of the gracilis form to revert to the normal form;
Pohlheim verified this observation and found the reverted
tissue to be diploid.
Recently, it has been reported that haploids can be gen-
erated from in vitro flowers of various tetraploid gentian
hybrids, and these may have future commercial value
(Morgan et al., 2009).
Mutant isolation
At a genomic level, haploids may be considered as
being mutants in their own right (Blakeslee et al.,
1922), and recently the genetic and phenotypic conse-
quences of ploidy differences have been described in
detail in maize (Riddle et al., 2006, 2009). Haploids also
have value in allowing the isolation of mutations, which
may be masked in a diploid (Maluszynski and Kasha,
Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 377–424
2002; Jambhulkar, 2007; Szarejko and Forster, 2007).
This approach was confirmed using haploid tobacco
from which lines with high photosynthetic activity were
selected (Medrano and Primo-Millo, 1985) and in isola-
tion of disease resistance in melons (Kuzuya et al.,
2003). Also dwarf potato mutants deficient in gibberellin
biosynthesis were isolated from anther culture–derived
lines of potato (Valkonen et al., 1999), and a giant
embryo mutant derived from an anther culture of rice
has been recently described (Park et al., 2009).
Recent examples in maize anther culture include in vitro
microspore selection with oxidative stress stimulators
(Ambrus et al., 2006; Darkó et al., 2009). To produce DH
maize plants tolerant of oxidative stress, in vitro micro-
spore selection was carried with reactive oxygen species
(ROS) progenitors such as paraquat, menadione, tert-buty-
lhydroperoxide (t-BHP) and methionine combined with
riboflavin. Similar studies have been conducted in wheat
in an attempt to isolate lines resistant to aluminium (Bakos
et al., 2008). Other examples include mutagenesis of
microspores of Brassica species to induce fatty acid
modifications (Ferrie et al., 2008), the isolation of embryo-
genic from nonembryogenic lines in oilseed rape (Malik
et al., 2008) and the production of TILLING populations in
Brassica oleracea (Himelblau et al., 2009).
The genetic advantage of selection in haploid cells is
also the basis of their proposed use as a bioassay system
for monitoring possible mutagenic effects of herbicides
(Pohlheim et al., 1977) or environmental pollutants
(Christianson and Chiscon, 1978).
Transformation
Haploids also have value in transformation programmes. If
haploids are transformed directly, then true-breeding dip-
loid transgenics can be produced in one step following
doubling of chromosomes. Recent examples include those
from rice (Chen et al., 2006), oilseed rape (Cegielska-Taras
et al., 2008; Abdollahi et al., 2009) and barley (Obert
et al., 2008; Shim et al., 2009). Transformation of haploid
callus of poplar has also been reported (Deutsch, 2004;
Deutsch et al., 2004). Other specialized examples involving
selection among transgenics include efficient screening of
tobacco plants expressing an anti-human immunodefi-
ciency virus monoclonal antibody (Floss et al., 2009),
barley plants expressing recombinant collagen (Eskelin
et al., 2009) and oilseed rape with enhanced resistance to
the clubroot pathogen (Plasmodiophora brassicae) (Reiss
et al., 2009) or pollen beetle (Åhman et al., 2009).
ª 2010 The Author
 Haploids in flowering plants 399

A similar recent study involves the transformation of (a)

anther culture–derived Festulolium plants (Lolium perenne
L. · Festuca pratensis Huds.) by Agrobacterium tumefac-
iens (Guo et al., 2009). In another related example, a new
L. temulentum line, NFLT12 (Reg. No. GS-7, PI 655941),
was developed using anther culture on an F2 population
derived from a cross of two parental L. temulentum lines
(Wang et al., 2009b). Haploid and DH plants were
obtained and their tissue culture responsiveness and sensi-
tivity to genetic transformation were tested. NFLT12 is
highly responsive to tissue culture, is readily transformable
and is expected to provide a resource for those using
L. temulentum as a model species for functional genomics
studies.
An interesting novel approach of potential value in
transformation was recently described by Chugh et al.
(2009) who demonstrated in triticale that various synthetic
peptides were able efficiently to transduce the GUS
enzyme in its functional form into unicellular microspores
at a maximum frequency of 31%.

Haploids and plant breeding

‘They (haploids) cannot yet be produced to order. When

this can be done it will lead to great speeding up of plant
breeding.’ Harland (1955).
(b)
The most important use of haploids is based on the fact
that marked improvements in the economics of plant
breeding can be achieved via DH production, as selection
and other procedural efficiencies can be markedly
improved by using true-breeding (homozygous) progenies
(Nei, 1963; Melchers, 1972; Kihara, 1979; Choo, 1981;
Hermsen and Ramanna, 1981; Snape, 1989; Jauhar et al.,
2009; Travadon et al., 2009; Wan et al., 2009). With DH
production systems, homozygosity is achieved in one gen-
eration (Figure 12). Thus, the breeder can eliminate the
numerous cycles of inbreeding necessary to achieve practi-
cal levels of homozygosity by conventional methods. Figure 12 Microspore-derived plants of barley. (a) Microspore-derived
Indeed, absolute homozygosity for all traits is not achiev- haploid (left) and seed-derived diploid (right) plants of barley. Scale
bar is 10 cm. Haploid plant is infertile and therefore showing contin-
able by conventional breeding methods. Consequently, an
ued vegetative growth and tillering, in the absence of monocarpic
efficient DH technology would enable breeders to reduce senescence. (b) Field trial of progeny from microspore-derived doubled
the time and the cost of cultivar development relative to haploid barley plants.
conventional breeding practices (Thomas et al., 2003;
Forster and Thomas, 2005; Forster et al., 2007).
The advantage of haploid breeding is displayed in crops based on the use of DH lines. However, owing to inbreed-
with a variety of genome complements (Ortiz et al., 2009) ing depression, these lines cannot be used directly but
and breeding systems (Kruse, 1980). According to Herm- only as parental inbred lines for the production of hybrid
sen and Ramanna (1981), ‘Just as in self-pollinators, the varieties. When inbred lines are being developed via
application of haploidy in cross-pollinated diploid crops is haploids, all barriers to repeated selfing, which are

ª 2010 The Author

Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 377–424
400 Jim M. Dunwell
characteristics of natural cross-pollinators are bypassed,
e.g. dioecy, self incompatibility and long juvenile periods.
The time saving is particularly apparent in biennial crops
and in crops with a long juvenile period. Only via haploidy
can inbred lines be developed in these crops.’
It is often stated that the first DH cultivar in a crop plant
was the cultivar Maris Haplona of rapeseed (Brassica na-
pus) in the early 1970s (Thompson, 1972) and Mingo in
barley (Hordeum vulgare) in 1980 (Ho and Jones, 1980),
although DH lines derived from spontaneously occurring
haploids have previously been successfully exploited for
commercial production in maize (Chase, 1951). In fact,
the first DH cultivar was the tomato ‘Marglobe’ produced
about 50 years previously by Morrison (1932). The signifi-
cance of this often overlooked breakthrough in plant
breeding was discussed by Cook (1936) (see also in the
context of patent protection). As well as having value in
their own right as potential new varieties in inbreeding
crops, homozygous plants are required to generate F1
hybrid plants, crosses between selected homozygous
males and females. These F1 plants often exhibit so-called
hybrid vigour (heterosis) (Hochholdinger and Hoecker,
2007), a characteristic and often dramatic increase in yield
compared with either parent and first described by Shull
(1908). The first explanation of these results came in 1917
from Donald Jones who produced a Mendelian explana-
tion of the advantage of hybrids (Jones, 1917).
This genetic phenomenon is the foundation of the
hybrid corn companies which grew in importance (and
commercial value) in the second half of the 20th century.
For example, Pioneer-HiBred seed company was founded
in 1926 following the discovery of the value of hybrids. In
1933, only about 1% of corn grown in the USA was
hybrid, a value that has now increased to 95% because of
the greatly increased yields achieved; during the period
from 1920 to c. 2000, the yield of hybrid corn rose from
about 25 bushels per acre to 140 (Crow, 1998; Duvick,
2001).
The role of haploids in this history of maize breeding is
very significant (Smith et al., 2008; Chang and Coe,
2009). As described earlier, the first reports of their occur-
rence in maize were made concurrently by L. F. Randolph
and L. J. Stadler (Stadler, 1931, 1940) in unpublished arti-
cles read in 1929. However, according to East (1930), Pro-
fessor R.A. Emerson had for a number of years previously
been endeavouring to isolate monoploids parthenogeneti-
cally with the hope that some diploid seed could be
obtained from them. The application of DH maize was
subsequently commercialized after the development of
Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 377–424
simple procedures utilizing selected haploid-inducing lines
(Sarkar and Coe, 1966, 1971; Sarkar et al., 1972; Khoklov
et al., 1975; Khvatova, 1976; Lashermes and Beckert,
1988), often aided by colour markers (see above) (Green-
blatt and Bock, 1967). According to Geiger (2009), the
most effective inducer (average rate about 8%) is line
RWS, derived from a cross between the Russian inducer
synthetic KEMS (Shatskaya et al., 1994) and the French
inducer line WS14 (Lashermes and Beckert, 1988). A num-
ber of theoretical studies that discuss the most favourable
generation from which maize haploids should be extracted
have recently been published (Bordes et al., 2006; Gallais
and Bordes, 2007; Longin et al., 2007; Wegenast et al.,
2008, 2009; Bernardo, 2009; Gallais, 2009; Mayor and
Bernardo, 2009).
There is considerably less information about the global
benefit of haploids in breeding crops other than maize. As
mentioned earlier, much of the early adoption of anther
culture technology in plant breeding was conducted in
China (Anon, 1976a) on species including egg plant
(Anon, 1976b), rice (Anon, 1976c) and wheat (Chu et al.,
1976). More recently, in a review of the application of
haploids in wheat, it is stated that some DH wheat culti-
vars have become dominant in specific regions of the
world (Jauhar et al., 2009). Thus, in Canada in 2007,
three of the five most widely grown cultivars in all grades
of the Canada Western Red Spring (CWRS) wheat class
were DH cultivars and ‘Lillian’ accounted for about 15%
of the CWRS acreage. Similarly, ‘AC Andrew’ is an anther
culture–derived DH line that accounted for 99% of the
Canada Western Soft White Spring acreage. Recent
assessments of the value of DH barley varieties have been
provided by Gomez-Pando et al. (2009a,b).
A recent specific example of the utilization of haploids
in oilseed rape was in a study designed to improve the
S. sclerotiorum resistance of ‘Hui5200’, an elite ‘Polima’
CMS restorer line, by introgression and fixation of resis-
tance alleles from the partially resistant cultivar ‘NingRS-1’
via phenotypic selection, marker-assisted background
selection and microspore culture (Yu et al., 2009). After
three cycles of selection and consideration of economic
traits and genetic distance analysis, four resistant DH
restorer lines with elite economic traits were finally
selected. The obtained resistant restorer lines have been
used to produce commercial F1 hybrids.
Probably, the most complete list of haploid-derived vari-
eties is that provided by the COST 851 programme, a
European Union funded research network entitled
‘Gametic cells and molecular breeding for crop improve-
ª 2010 The Author

ment’ that ran from 2001 to 2006 (http://www.scri.ac.uk/
assoc/COST851/Default.htm). The final report is available at
http://cost.esf.org/typo3conf/ext/bzb_securelink/pushFile.
php?cuid=253&file=fileadmin/domain_files/ABFS/Action_851/
progress_report/progress_report-851.pdf. The list of DH
varieties (http://www.scri.ac.uk/assoc/COST851/Default.
htm) shows a total of almost 300 such varieties from
asparagus, barley, Brassica, eggplant, melon, pepper, rape-
seed, rice, swede, tobacco, triticale and wheat. In this con-
text, it is relevant to note that the current status of DH
methods in medicinal crops has been reviewed recently by
Ferrie (2009) (see also details in patent applications).
A notable absence from the list of haploid-derived varie-
ties is any example from woody species (Andersen, 2005),
a group in which their theoretical advantage would be
greatest. According to Palmer and Keller (2005b), ‘The
wide application of haploid technology to the improve-
ment of woody species would be a valuable addition to
other biotechnological techniques aimed at improvement
of these species.’ This general lack of progress with woody
species (Stettler and Howe, 1966) is due mainly to the pres-
ent emphasis on methods involving an in vitro phase, and
the problems associated with the general intransigence of
woody species to growth under such conditions. Similarly,
Vanwynsberghe et al. (2005) stated ‘The use of homozy-
gous androgenic genotypes from ‘‘Braeburn’’ in apple
breeding programmes is currently not a realistic approach,
partly because of the low efficiency of anther culture, but
mainly because of the reduced vigour and severe sterility of
the androgenic genotypes produced.’ However, there have
been significant recent advances with palms that represent
new approaches to haploid isolation. In coconut, the first
microspore-derived plants have been reported (Perera
et al., 2008a,b, 2009), and in oil palm a high-throughput
screening process involving flow cytometry has successfully
identified haploids and doubled haploids among anoma-
lous seedlings (Dunwell et al., unpublished). Progress has
also been made in coffee (Lashermes et al., 2006).
There are many commercial and noncommercial sites
that promote the virtues of haploids, particularly in maize.
Commercial examples include those from Pioneer Hi-Bred
(http://www.pioneer.com/CMRoot/Pioneer/research/pipeline/
brochures/DblHapBrch17.pdf) and Great Lakes Hybrids
(http://www.greatlakeshybrids.com/performance/research-
information/doubled-haploid-breeding-technology/), while
an example from CIMMYT is available at http://www.
cimmyt.org/english/wps/news/2008/may/doubledHaploids.
htm. It should also be stressed that for many years, both
the Food and Agriculture Organisation of the United
ª 2010 The Author
Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 377–424
Haploids in flowering plants 401
Nations (FAO) and the International Atomic Energy Agency
(IAEA) have supported and coordinated research that
focuses on the development of more efficient DH produc-
tion methods and their application in plant breeding in
developing countries.
In addition to their exploitation in practical breeding
programmes, DH lines have widespread application in fun-
damental quantitative genetics (Pink et al., 2008) and SNP
discovery (Trick et al., 2009). Specific recent examples of
the utilization of DH lines in QTL mapping in cereals
include studies of laccase enzymes in maize (Andersen
et al., 2009); Fusarium head blight resistance in barley
(Marchand et al., 2008); arsenic accumulation (Zhang
et al., 2008b), agronomic traits (Lapitan et al., 2009), stem
height (Ma et al., 2009), and cooking quality (Govindaraj
et al., 2009) in rice; and agronomic traits (Chu et al.,
2008), plant height (Zhang et al., 2008c), heading date
(Zhang et al., 2009c), flour colour (Zhang et al., 2009d),
phosphorus use efficiency (Su et al., 2009), zinc deficiency
(Genc et al., 2009) and Septori tritici blotch resistance
(Raman et al., 2009) in wheat.
Examples in oilseed rape include selection of lines for
phytosterol content (Amar et al., 2008), erucic acid con-
tent (Nath et al., 2009), plant height (Cheng et al., 2009)
and general yield traits (Shi et al., 2009). In tobacco, QTLs
have been identified for the effect of a chromosome seg-
ment marked by the Php gene (for resistance to Phytoph-
thora nicotianae) on reproduction of tobacco cyst
nematodes (Johnson et al., 2009).
In a related study, Potokina et al. (2008) reported the
genome-wide occurrence of limited pleiotropy of cis-regu-
latory mutations in barley using Affymetrix analysis of
22 840 genes in a population of 139 DH lines derived
from a cross between the cultivars Steptoe and Morex.
An interesting application of haploids in the breeding of
the cultivated marine brown alga Undaria pinnatifida is pro-
vided by the results of Shan and Pang (2009) who conducted
a genetic analysis of sporophytic offspring originating from
mono-crossing of male and female gametophyte clones.
Cytogenetic analysis
Cytogenetic studies of the haploids of polyploid species
(Jauhar, 2006; Birchler and Veitia, 2007; Birchler et al.,
2007) have contributed to our knowledge of the pairing
behaviour between the various constituent genomes of
these species; however, in some instances, the nature of
the association between the homoeologous chromosomes
and their authenticity have been questioned. For example,
402 Jim M. Dunwell

the cultivated cotton (Gossypium hirsutum, 2n = 4x = 52) titution. The frequency of dyads at the tetrad stage of pol-
(Mursal, 1978) is an allotetraploid composed of the A and len development (2.6%) suggested that unreduced
D subgenomes that are closely similar to the A genome. gametes were preferentially selected in microspore culture.
Similar analysis has been conducted in castor oil plant These authors discuss the potential for using microspore
(Narain and Singh, 1968) and in oilseed rape by Nicolas culture of unreduced gametes in such hybrids to map
et al. (2007, 2009) who investigated the genetic regula- Brassica centromeres through half-tetrad analysis.
tion of meiotic cross-overs between related genomes in The simplified assembly provided by a haploid genome
various haploids and hybrids. Haploids of sugar beet were is the basis of several genome sequencing programmes.
also used in the development of basic cytogenetic proto- For example, it was reported in January 2007 that the
cols (De Jong and De Bock, 1978). Department of Energy’s Joint Genome Institute would
In a related study, it was suggested that the radish gen- sequence the peach genome in 2008. This will involve an
ome (n = 9) might comprise three pairs of homoeologous 8· coverage of the peach haploid cultivar ‘Lovell’, the
chromosomes, with the remaining three chromosomes same cultivar used in the physical map. Also, in January
carrying the homologous region(s) that results in a triva-
lent formation (Kaneko et al., 2003). Table 5 Granted US patents
A slightly different approach is the study on obtaining
Number Date Species Subject Inventor(s)
primary trisomics by the isolated microspore culture of
autotetraploid Chinese cabbage (Shen et al., 2006). This 4835339 1986 Tomato Anther culture* Evans et al.
material was used subsequently to determine the n + 1 4840906 1989 Barley Anther culture Hunter
5049503 1991 Brassica Anther culture Swanson et al.
gamete transmission rate and to locate the gene control-
5066594 1991 Maize Transformation DeBonte et al.
ling 2n gamete formation on the corresponding chromo- 5066830 1991 Capsicum Anther culture Morrison et al.
some (Zhang et al., 2009a). 5272072 1993 Barley Transformation Kaneko and Ito
5306864 1994 Maize Anther culture Petolino
5322789 1994 Maize Anther culture Genovesi et al.
Linkage maps and genome sequencing 5445961 1995 Maize Anther culture Genovesi et al.
5492827 1996 Cucumber Ovule culture Dirks
A high-resolution transcript linkage map of barley was cre- 5547866 1996 Taxus Secondary products Durzan et al.
ated using a single DH mapping population, only 3¢-end 5602310 1997 Maize Anther culture Petolino
5629183 1997 Tobacco Transformation Saunders et al.
expressed sequence tags (ESTs) and only PCR-based
5639951 1997 Maize ig gene Bosemark et al.
assays. Cultivar ‘Haruna Nijo’ and an ancestral wild-form
5749169 1998 Maize ig gene Briggs
accession ‘H602’ were used as EST donors and crossing 5770788 1998 Maize Anther culture Jia
parents of the mapping population (Sato et al., 2009). Of 5840557 1998 Tobacco Transformation Heberle-Bors et al.
the 10 366 primer sets developed from a nonredundant 5900375 1999 Brassica Anther culture Simmonds et al.
5929300 1999 Tobacco Transformation Burke et al.
set of 3’EST sequences, 7700 sets generated useful ampli-
6200808 2001 Brassica Anther culture Simmonds et al.
cons and 3975 (52%) showed polymorphisms between 6316694 2001 Brassica Transformation Dormann et al.
the mapping parents. Of these, 2890 (28% of the total) 6362393 2002 Wheat Anther culture Konzak et al.
were mapped by single nucleotide polymorphisms (1717), 6407314 2002 Tobacco Microspore-specific Oldenhof
promoter
cleaved amplified polymorphic sequence (933) and INDELs
6764854 2004 Rice Anther culture Konzak et al.
(240). This study involved an estimated 9% of the genes
6812028 2004 Wheat Anther culture Kasha et al.
of barley. A linkage map for oat has also been generated 6861576 2005 Brassica Microspore-specific Drouaud et al.
from a DH population (Tanhuanpää et al., 2008). promoter
In an analysis of the products of male meiosis in micro- 7135615 2006 Maize Chromosome doubling Kato
7148402 2006 Arabidopsis Embryo-specific gene Niu and Chua
spore-derived progeny from a Brassica napus x Brassica
7151170 2006 Brassica Embryo-specific gene Boutilier et al.
carinata interspecific hybrid, it was shown by genotyping 7297838 2007 Flax Transformation Chen and Dribnecki
at 102 SSR marker loci and nuclear DNA contents pro- 7439416 2008 Maize ig gene Evans
vided strong evidence that 26 of the 28 progeny (93%) 7442549 2008 Orchid Parthenogenesis Ichihashi
7502113 2009 Maize Nondestructive sampling Deppermann et al.
were derived from unreduced (2n) gametes (Nelson et al.,
7572635 2009 Maize Transformation Armstrong et al.
2009). This high level of marker heterozygosity, and
parallel spindles at Anaphase II in the hybrid, indicated *In these tables, the term ‘anther culture’ may include techniques for
that unreduced gametes were formed by first division res- microspore culture.

ª 2010 The Author

Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 377–424
 Haploids in flowering plants 403

2008 Washington State University, in collaboration with
the University of Washington and the French National
Institute for Agricultural Research (INRA) initiated the
sequencing of a DH line from the apple cultivar Golden
Delicious. This marks the start of an international commu-
nity initiative to generate a reference genome for apple.
Similarly, is was reported in 2008 that as part of the
construction of a coffee physical map and sequencing of
the coffee genome, two Coffea canephora BAC libraries
(Eco RI and Hind III) had been constructed from a DH
C. canephora genotype that was mapped previously so
that the physical map can be anchored to the mole-
cular map (http://www.coffeegenome.org/communications/
report/Minutes_ICGN_meeting_Campinas_Sept08.pdf). Fin-
ally, it has recently been reported that a haploid clementine
line induced by the technique of in situ gynogenesis induced
by irradiated pollen has been selected by the International
Citrus Genomic Consortium as the reference line for
sequencing (Aleza et al., 2009), and that the Pacific North-
west Pear Research Bureau has funded the initial stages of
sequencing of a pear DH line.
In an interesting prediction of future trends in plant
breeding, Yu (2009) includes efficient protocols for
haploid induction among a list of technologies that will
be required to exploit the potential of the genomics
revolution.
Patents
The application of intellectual property (IP) protection and
patenting system to haploid plants has been reviewed
recently (Dunwell, 2009). Possibly, the first detailed consid-
eration of the potential of such IP protection in the con-
text of haploid plants was that published by Cook (1936).
He was speculating on the production of a DH cultivar of
tomato ‘Marglobe’ by Morrison (1932) and wrote percep-
tively: ‘Thus there is a possibility that Mr. Morrison’s
pioneer breeding work with haploid tomatoes will some

Table 6 U.S. patent applications

Number Date Species Subject Inventor(s)

20020104128 2002 Wheat Anther culture Konzak et al.

20020151057 2002 Maize Anther culture Zheng et al.
20040210959 2004 Flax Transformation Armstrong et al.
20040226059 2004 Wheat Anther culture Kasha et al.
20050262595 2005 Arabidopsis Embryo-specific gene Niu et al.
20050071898 2005 Arabidopsis Embryo-specific gene Zuo et al.
20050198711 2005 Maize ig gene Evans
20050289663 2005 Orchid Parthenogenesis Ichihashi
20050289673 2005 Maize Transformation Armstrong al.
20060179498 2006 Brassica, etc. Reverse breeding Dirks et al.
20060185033 2006 Maize Transformation Zhao et al.
20060260005 2006 Brassica Transformation Chen and Celio
20070107077 2007 Maize Transformation Chen and Tulsieram
20070204366 2007 Maize Nondestructive sampling Deppermann et al.
20070292951 2007 Brassica Carbon source Ilic-Grubor et al.
20080072339 2008 Calibrachoa Anther culture Stover et al.
20080072343 2008 Maize Haploid-inducing lines Wang et al.
20080098496 2008 Vegetables Reverse breeding Van Dun et al.
20080134353 2008 Arabidopsis Embryogenesis Dirks et al.
20080189801 2008 Maize Haploid-inducing lines Williams et al.
20080216191 2008 Maize Chromosome doubling Barton et al.
20080216198 2008 Maize Transformation Zhao et al.
20080293059 2008 Barley Haploid variation Bednarek et al.
20080299658 2008 Maize Transformation Zhao et al.
20090025288 2009 Maize Nondestructive sampling Deppermann et al.
20090064361 2009 Maize, etc. Gene mapping Butruille et al.
20090070891 2009 Maize, etc. Transgene breeding Foley et al.
20090100538 2009 Apiaceae Anther culture Ferrie et al.
20090105077 2009 Maize Chromosome doubling Bhatti et al.
20090151025 2009 Maize ig gene Evans
20090210959 2009 Saponaria Anther culture Ferrie et al.

ª 2010 The Author

Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 377–424
404 Jim M. Dunwell

Table 7 Non-USA patent documents Table 7 Continued

Number Date Species Inventor(s) Number Date Species Inventor(s)

World Patent System CN 101385441 2009 Zantedeschia Zhang et al.

WO 86 ⁄ 00495 1986 Wheat Picard CN 101385442 2009 Zantedeschia Zhang et al.
WO 92 ⁄ 14828 1992 Barley Tallberg et al. CN 101377481 2009 Maize Chen et al.
WO 94 ⁄ 01999 1994 Barley Holm et al. CN 101401550 2009 Eggplant Lian et al.
WO 95 ⁄ 26628 1995 Barley Salmenkallio-Marttila et al. CN 101433183 2009 Brassica Liu
WO 02 ⁄ 01940 2002 Wheat Jensen et al. CN 101433184 2009 Brassica Liu
WO 2004 ⁄ 032607 2004 Orchid Miyoshi CN 101449657 2009 Cauliflower Sun et al.
WO 2004 ⁄ 042066 2004 Tobacco Touraev et al. CN 101473789 2009 Mustard Wan et al.
WO 2005 ⁄ 004586 2005 Maize Bordes et al. Japanese patents
WO 2005 ⁄ 084420 2005 Wheat Shen JP59205922 1984 NA Samueru et al.
WO 2006 ⁄ 116876 2006 Saponaria Ferrie et al. JP1067130 1989 Rice Negishi et al.
WO 2006 ⁄ 125310 2006 Apiaceae Ferrie et al. 01-168294 1989 Alkaloids Takahashi et al.
WO 2006 ⁄ 128707 2006 Tomato Dirks et al. 02-002382 1990 Alkaloids Takahashi et al.
WO 2007 ⁄ 038075 2007 Maize Barton et al. 02-231022 1990 Cucumber Dirks
WO 2008 ⁄ 114000 2008 Oil palm Nelson et al. 02-242622 1990 Stevia Murakami et al.
WO 2009 ⁄ 029771 2009 Maize, etc. Butruille et al. 02-255093 1990 Alkaloids Takahashi et al.
WO 2009 ⁄ 059777 2009 Cucumber Crienen et al. JP3280817 1991 Cucumber Fujishita et al.
European patents 03-155721 1991 Gramineae Takahara
0 127 313 1989 Wheat Sherba et al. JP4112730 1992 Brassica Hamaoka et al.
0 171 310 1990 Wheat Picard JP5219849 1993 Melon Kato et al.
Chinese patents JP6237657 1994 Melon Kato et al.
CN 1080114 1994 Seaweed Chaoyuan et al. 06-098648 1994 Arabidopsis Kitani
CN 1212828 1999 Wheat Weng 07-143829 1995 Maize Bosemark et al.
CN 2394719 2000 Pollen sorting Guo et al. JP8126444 1996 Carnation Sato et al.
CN 1331914 2002 Tobacco He et al. JP9172893 1997 Cyclamen Kitaura et al.
CN 1418537 2003 Capsicum Li et al. 09-084478 1997 Maize Kato et al.
CN 1418950 2003 Capsicum Gu et al. JP11318249 1999 NA Kato et al.
CN 1421124 2003 Brassica Sun et al. 2004-049163 2004 Orchid Ichihashi et al.
CN 1421125 2003 Brassica Sun et al. Korean patents
CN 1473462 2004 Camelina Zhang et al. KR 20050089235 2005 Capsicum Park et al.
CN 1484945 2004 Radish Mei KR 20060117080 2006 Transformation Sung
CN 1543780 2004 Radish Li KR 100736150 2007 Capsicum Kim et al.
CN 1552198 2004 Cauliflower Sun et al. Canadian patents
CN 1555678 2004 Rice Xu et al. CA 1236700 1988 Wheat Sherba et al.
CN 1586116 2005 Oilseed rape Zhang CA 2019989 1990 Maize Mitchell et al.
CN 1653885 2005 Pumpkin Wang et al. CA 2444797 2002 Maize Bidney et al.
CN 1659955 2005 Muskmelon Wang et al. Russian patents
CN 1723767 2006 NA Mei SU 1497212 1989 Cedar Ruguzov et al.
CN 1737122 2006 Cucumber Wei SU 1520096 1989 Barley Rodin et al.
CN 1784952 2006 Cabbage Zhang SU 921138 1996 Maize Tyrnov et al.
CN 1799332 2006 Eriobotrya Li RU 2150823 2000 Flax Poljakov
CN 1836496 2006 Chilli pepper Zhang Ukrainian patents
CN 101011028 2007 Chrysanthemum Wang UA 20078 2007 Brassica Klymchuk et al.
CN 101049081 2007 Cruciferae Chen UA 21988 2007 Wheat Ihnatova et al.
CN 101135642 2008 Brassica Hou et al. UA 82013 2008 F1 breeding Fedorovych et al.
CN 101228841 2008 Wheat Zhao et al. Bulgarian patent
CN 101243776 2008 Brassica Huang et al. BG 51816 1993 Tobacco Kintja et al.
CN 101248753 2008 Brassica Ma et al. Roumanian patent
CN 101250516 2008 Brassica Shi et al. RO 113602 1998 Flax Popescu et al.
CN 101283674 2008 Radish Gong et al. Moldovan patent
CN 101292626 2008 Brassica Fu et al. MD 56 2009 Tomato Saltanovici et al.
CN 101317548 2008 Cucumber Chen et al. Spanish patent
CN 101343619 2009 Radish Zhang et al. ES 2180385 2003 Cork oak de la Vega et al.
CN 101356276 2009 Brassica Chen French patent
CN 101371650 2009 Capsicum Yong et al. FR 2896952 2007 Brassica Leroux et al.

ª 2010 The Author

Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 377–424

day be cited as the beginning of ‘‘haploid plant breeding
stations’’ where this interesting technique will be utilized
to the limit of its possibilities. Instead of growing acres of
seedlings and throwing all but a handful away, there may
come a time when our future Burbanks -will grow acres
of haploids to get a few dozen seeds-each one of these
representing the beginning of a unique pure line, which
will form the basis of later breeding experiments.’ He con-
tinued ‘At the moment we do not know what the result
might be of cross pollinating haploids,… When such an
experiment is tried it might have considerable importance
in settling certain moot points concerning the causes of
hybrid vigour, and it might be very important in future
breeding programs. Other ‘‘perhaps’’ uses of haploids
might be suggested, so purely speculative, however, as
hardly to be suitable subjects for plant patents—basic or
otherwise.’
This prediction has been fulfilled to some extent, albeit
gradually, over the subsequent 77 years. Specifically, the
strong commercial interest in methods for the production
and exploitation of haploid plants is exemplified by the
extensive number of granted patents and patent applica-
tions from the USA (Tables 5 and 6) and elsewhere
(Table 7). Further details and discussion of some of these
examples are provided in Dunwell (2009).
Conclusion
This review has summarized a long and fascinating history
of haploids in higher plants. Their occurrence developed
gradually from being a sporadic and random process in
vivo to one which could be predicted, especially with the
discovery of efficient in vitro methods for the induction of
sporophytes from gametophytic cells. Their value has also
increased in many areas of fundamental plant science, but
most particularly in the context of plant breeding and their
place in the route to homozygous lines and thence to pure
cultivars and F1 hybrids. This process of exploitation can
be expected to continue in the race to improve the effi-
ciency and yield of crop plants at a time of increasing
challenge to maintain food security (Royal Society, 2009).
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