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REVIEW OF LITERATURE
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2. REVIEW OF LITERATURE
Since the beginning of agricultural production, there has been a continuous effort
to grow more and better quality food to feed ever increasing populations. Both improved
cultural practices and improved crop plants have allowed us to divert more human
resources to non-agricultural activities while still increasing agricultural production.
Malthusian population predictions continue to alarm agricultural researchers, especially
plant breeders, to seek new technologies that will continue to allow us to produce more
and better food by fewer people on less land. Both improvement of existing cultivars and
development of new high-yielding cultivars are common goals for breeders of all the
crops. In vitro haploid and double haploid production is among the new technologies that
show great promise towards the goal of increasing yields by making similar germplasm
available for many crop plant breeding programs.
Haploids occur spontaneously at a low frequency or they can be induced by several
methods. A variety of methods used to obtain these haploids and DHs are in vivo modified
pollination methods and in vitro culture of immature gametophytes. In vivo modified
pollination method comprises chromosome elimination subsequent to wide hybridization
developed by Kasha and Kao (1970) and pollination with irradiated pollen or pollen from
a triploid plant. In vitro tissue culture methods consist of culturing the immature
gametophytes resulting in gametic embryogenesis. Gametic embryogenesis is one of the
different routes of embryogenesis present in the plant kingdom, and it consists in the
capacity of male (microspore or immature pollen grain) or female (egg cell) gametophytes
to irreversibly switch from their gametophytic pathway of development towards a
sporophytic one. Each gametic cell possesses a unique genome where every gene is
present as a single copy. Exploitation of this unique genetic unit and the totipotency of the
plant cell is the basis of anther/pollen or ovary/ovule culture for the production of haploid
plants.
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A haploid plant derived from a diploid species is more appropriately termed a
monoploid since it has only one set of chromosomes (i.e. one genome only) (Fehr, 1993;
Quisenberry and Reitz, 1967). However, 2n refers to somatic chromosome number; while,
‘x’ represents the basic chromosome number (i.e. the chromosome number in one genome
of a specific monoploid species) (Folling and Olesen, 2002). The totipotent nature of the
haploid cell is being efficiently and effectively explored in different facets of modern
biological and agricultural research disciplines. Haploids can improve the efficiency and
the speed of the usually cumbersome, time-consuming, laborious and sometimes rather
inefficient conventional breeding methods. Haplo-diploidization through gametic
embryogenesis allows single-step development of complete homozygous lines from
heterozygous parents. In a conventional breeding program, a pure line is developed after
several generations of selfing which is not possible in male sterile plants and self
incompatible plants. In very simple terms, a doubled haploid (DH) is a genotype produced
when haploid cells undergo the process of chromosome duplication (Chawla, 2002). The
DH technology platform offers a rapid mode of truly homozygous line production that
help to expedite crop breeding programs where homogeneity is an absolutely essential
parameter for rapid crop development. The DH plants produced from
monohaploids/allohaploids represent pure bred lines. Since homozygous plants are
produced in a single generation, the time period necessary for cultivar development could
be efficiently reduced by 3-4 years. The selection efficiency can also substantially
improve DH production because the phenotype of the plant is not masked by the
dominance effects. The heritable traits encoded by recessive gene(s) could be efficiently
detected and a small population of DH plants is necessary while screening for desirable
recombinants (Snape et al., 1986). To be useful, however, it is important to note that an
efficient and reliable method of haploid and DH production will be essential.
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The haploidy technology has now been adapted in different plant breeding
programs across all the major continents as the most commonly used approach for rapid
crop development for transferring genes of interest, chromosomal segments or even
complete chromosomes by means of distant hybridization (Ceoloni and Jauhar, 2006;
Baenziger and DePauw, 2009; Touraev et al., 2009). As an example, maize-induced
chromosome elimination offers a very useful approach for rapid haploid plant production
in bread wheat and durum wheat (Basu et al., 2011). Fairly recently, pearl millet
[Pennisetum glaucum (L.) R.Br.] and Tripsacum spp. pollen sources also served an
identical role in haploid production in maize (Touraev et al., 2009).
Haploid plants and haploid-derived homozygous lines are useful in several
domains of basic research in the realms of classical plant genetics and cytogenetics,
modern molecular genetics including induced mutagenesis, site-directed mutagenesis,
genetic transformation research, genome mapping and assessing distant genome
relationships, gene dosage effects, analysis of linkages, mechanisms of the genetic control
of chromosomal pairing and in the conventional plant breeding studies (Chawla, 2002;
Cuthbert et al., 2008; Touraev et al., 2009). Integration of the haploidy technology with
other available biotechnological tools such as Marker Assisted Selection (MAS) and
transgene technologies could also effectively expedite the crop improvement programs
running all across the globe (Folling and Olesen, 2002).
2.1 Haploids in mapping and genetic analysis
By efficiently utilizing DH populations, Quantitative Trait Loci (QTLs) associated
with yield and yield components have been successfully identified allowing marker-
assisted breeding approaches to be employed in several major wheat improvement
programs (Touraev et al., 2009). The haploidy technique has played an important role in
practical plant breeding as can be seen in widely grown DH cultivars in all the major
continents where some of them have earned the recognition of dominant cultivars. The
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DH populations, which are similar in genetics to Recombinant Inbred Lines (RILs)
generated by single seed descent approach, have been applied for mapping QTLs for
several desirable characters (Munoz-Amatriain et al., 2008). Wheat cultivars developed
from DH technology have been released for cultivation and have now turned out as
dominant cultivars in several countries across the globe (Baenziger and DePauw, 2009;
Touraev et al., 2009).
Haploid technology is particularly effective in accelerating breeding when
combined with other biotechnologies, such as MAS (Forster et al., 2007b; Tuvesson et al.,
2007; Werner et al., 2007). SSR-, SCAR-, CAPS-, RAPD-, ISSR-, AFLP-,
retrotransposon-based markers and SNP, and also isoenzymes and protein profiles, are
among the most used markers by breeders (De Vienne, 2003). Haploid procedures have
proved to be very beneficial in developing molecular maps and in analysing quantitative
trait loci. Low-copy number (RFLP) markers, detected using of Southern analysis, are
excellent tools for generating linkage maps. Further improvement can be achieved by the
conversion of RFLPs to more practical PCR-based markers (Larson et al., 1996). One of
the latest developments is the simple sequence repeats (SSR) method, also known as
micro-satellites, providing a new class of PCR-based DNA markers (Liu et al., 1996). Up
to now, successful genetic mapping was reported for such species as barley (Bezant et al.,
1996; Graner et al., 1996; Graner and Tekauz, 1996; Graner et al., 1991; Heun et al., 1991;
Larson et al., 1996; Laurie et al., 1995; Weyen et al., 1996), Brassica (Bohuon et al.,
1996; Dion et al., 1995; Lydiate et al., 1993; Ramsay et al., 1996; Romagosa et al., 1996;
Thormann et al., 1996), maize (Bentolila et al., 1992; Murigneaux et al., 1993a,b), pepper
(Lefebvre and Palloix, 1996) and rice ( Yu et al., 1996; Zhang et al., 1996). The doubled
haploid method is also considered to be superior to the conventional quantitative trait
analysis (Chebotar and Chalyk, 1996). DH lines allow for the estimation of linkages,
especially in case of additive genes (Surma et al., 1991). One of the examples of
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quantitative trait analysis is the estimation of the gene number for rice plant height,
panicle length, number of productive panicles per plant and 1000 grain weight in three
crosses (Liu et al., 1994). Another example is the analysis of QTL controlling of flowering
time and plant height in spring barley (Bezant et al., 1996). The genetic analysis of broad
spectrum resistance was also reported for pepper (Dogimont et al., 1996) and rice (Zhang
et al., 1996).
2.2 Haploids in induction and isolation of mutants
After mutagenic treatment of callus from leaf veins of haploid Nicotiana sylvestris
with ethyl methanesulfonate or ethyleneimin haploid as well as homozygous diploid
mutants could be selected (Malepszy et al., 1977). The haploid androgenic system is also
applicable for the isolation or induction of mutants, e.g. those showing high levels of oleic
acid (Turner and Faccioti, 1990; Haung et al., 1991) or of chlorsulphuron and
imidazolonone herbicide tolerance (Swanson et al., 1988, 1989) in Brassica napus. The
haploid petunia is an excellent system for gene tagging and for studying of transposable
elements (Renckens et al., 1996). Nicotiana plumbaginifolia haploid and diploid
protoplasts appeared to be a good system to induce salt and drought tolerant mutants
(Sumaryati et al., 1992).
2.3 Haploids in transformation and transgenics
Haploid microspores, protoplasts and explants can be used for transformation
procedures. It is easier to assess gene expression in haploid than in diploid plants.
Bombardment of haploid microspores resulted in homozygous transgenic and fertile
barley plants (Lutticke et al., 1995). One of the examples of direct transformation is
polyethylene glycol transformation of rice protoplasts derived from microspore suspension
(Chair et al., 1996). In this experiment transient activity of three serial genes was analysed
and for the progeny of three diploid transgenic plants the integration of DNA was
demonstrated. Herbicide-resistant Indica rice plants from IRRI breeding line IR72 after
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PEG-mediated transformation of protoplasts were obtained (Datta et al., 1992). From
protoplasts, isolated from microspore derived suspension culture, haploid transformed
plants were regenerated after electroporation method (Sukhapinda et al., 1993). Haploid
embryo segment transformation in Brassica napus using an octopine-producing strain of
Agrobacterium tumefaciens was achieved (Swanson and Erickson, 1989).
Although in vitro culture of gametes is more or less a standard tool for plant
breeders in many crops, particularly Brassicaceae and cereals, this has yet to be achieved
in fruit crop breeding since the deployment of gametic embryogenesis in fruit crops
improvement is still hampered by low frequencies of embryo induction, albinism, plant
regeneration, plant survival and the genotype-dependent response (Germana, 2006). Since
1970s, extensive research has been carried out to obtain haploids for fruit tree breeding
through gametic embryogenesis (Chen, 1986; Ochatt and Zhang, 1996). However, as
reviewed by Ochatt and Zhang (1996), this has not always given satisfactory results. A
better understanding of the gametic embryogenesis process, the improvement of currently
available techniques and the development of new technologies could make haploid
production a powerful fruit crop breeding tool in the future, enabling in these genotypes
the effective exploitation of the potential of gamete biotechnology (Germana, 2006). With
fruit crops, characterized by a long reproductive cycle, a high degree of heterozygosity,
large size, and sometimes, self-incompatibility, there is no way to obtain haploidization
through conventional methods.
The development of in vitro techniques for the production of haploids was a major
feat in the fields of biotechnology and plant breeding in the past few decades. It is well
documented that Blakeslee et al. (1922) pioneered this technique by first noticing natural
haploid of Datura stramonium. Thereafter, haploids were reported in many other species.
Guha and Maheshwari (1964) developed an anther culture technique for the production of
haploids through androgenesis in Datura inoxia. Haploid production by wide crossing was
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reported in barley (Kasha and Kao, 1970) and tobacco (Burk et al., 1979). Tobacco,
rapeseed and barley are the most responsive species for doubled haploid production.
Doubled haploid methodologies have now been applied to over 250 plant species
(Maluszynski et al., 2003). In 1974, Kasha reported spontaneously developed haploids in
over 100 angiosperm species generally in very low numbers and with low viability.
Androgenesis and gynogenesis are currently the most commonly used haploidization
techniques. While the former was developed during 1960s – 1970s, the latter is now being
increasingly applied to numerous new species.
2.4 In vitro haploid production via unfertilized ovule/ovary culture
Gynogenesis has become one of the available options in species with which other
methods have failed or have been prone to mutation induction in the process of
regeneration. For example, early attempts to obtain doubled haploid lines in beetroot (Beta
vulgaris) involved androgenesis which later was replaced with gynogenesis (Van Geyt and
Jacobs, 1986). Gynogenesis is the only best option in species where androgenesis is a
recalcitrance or the level of albino regenerated plants is high (reaching in most cases
100%), or due to male sterility and dioecious nature of plants (Thomas et al., 2000; Bhat
and Murthy, 2007). It, therefore, plays a limited role in cereals, although it was induced in
certain genotypes of wheat (Zhu et al., 1981; Matzk, 1991; Comeau et al., 1992; Matzk et
al., 1995), barley (Gaj and Gaj, 1996; Gaj, 1998) and rice (Zhou and Yang, 1981).
Gynogenic haploids can be induced from isolated ovules, ovaries and even by
culturing of flower buds (Keller, 1990; Bohanec et al., 1995; Jakse et al., 1996; Musial et
al. 2005). Several reviews (Maheshwari & Rangaswamy, 1965; Rangan, 1982) have
described initial efforts on ovary and ovule culture. At first, ovary culture was used to gain
a better understanding of several aspects of fruits physiology, such as morphogenesis and
physiological and biochemical changes. With ovary culture, these could be studied under
controlled environmental and nutritional conditions. Ovaries from several species have
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been grown in vitro with variable success. Difficulty in growing very young or minute
embryos led to attempts to culture ovules. Although mechanisms redirecting female
gametophytic cells into sporophytic development have still not been discovered,
substantial progress has been made in optimizing induction procedures, at least for
economically important species.
The first in vitro induced haploid plants of gynogenic origin were achieved by
culturing unfertilized ovaries in barley by San Noeum in 1976. Several earlier attempts to
induce haploids by culturing unfertilized ovules failed (Yang and Zhou, 1982). Despite
other successful examples in a number of plant species, it should be mentioned that
culturing unfertilized female ovules has often ended in failure in inducing either un-
organized or organized haploid tissues. During cultivation, ovaries and ovules often only
increase their size by cell proliferation of somatic tissue around the female gametophyte
but the embryo sac elements did not show morphogenetic activity (Mukhambetzhanov,
1997). Some failures and early attempts are reviewed by Lakshmi-Sita (1997),
Mukhambetzhanov (1997) and, more recently, for fruit crops by Germana (2006)
although, in general, failures to induce haploid plants are often not published. The
occasionally held belief that female gametophytes, given appropriate stimulation, can be
easily induced to form multi-cellular structures and subsequent embryo formation is
therefore incorrect. In fact, several decades after the first discoveries, haploid induction by
culturing un-pollinated ovules is still more or less problematic and limited to a relatively
small number of plant species.
Gynogenesis has been the most successful method used for production of haploid
plants in many species (Lux et al., 1990; Hansen et al., 1995; Alan et al., 2003).
Successful gynogenesis is reported in many plant species such as maize (Zea mays; Tang
et al., 2006), wheat (Triticum dur Defs.; Sibi et al., 2001), onion (Allium cepa; Muren,
1989; Bohanec et al., 1995; Luthar and Bohanec, 1999; Alan et al., 2007; Ebrahami and
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Zamani, 2009), sugar beet (Beta vulgaris; Van Geyt et al., 1987; Gurel et al., 2000), sweet
potato (Ipomea batatas; Ruth et al., 1993), tulip (Tulipa generiana; Van-Creij et al.,
2000), cucumber (Cucumis sativus; Gemes-Juhasz et al., 2002; Diao et al., 2009), apple
(Zhang and Lespinasse, 1988), gerbera (Sitbon, 1981; Meynet and Sibi, 1984; Miyoshi
and Asakura, 1996; Tosca et al., 1995, 1999), carnation (Sato et al., 2000), tef (Gugsa et
al., 2006), squash (Cucurbita pepo; Shalaby, 2007), mandarin orange (Froelicher et al.,
2007), niger (Guizotia abyssinica; Bhat and Murthy, 2008), sunflower (Yang et al., 1986),
Egyptian henbane (Chand and Basu, 1998), Easter lily (Ramsay et al., 2003) and mulberry
(Thomas et al., 1999). Attempts in cotton have also been reported recently (Kantartzi and
Roupakias, 2009).
2.5 Factors affecting in vitro gynogenesis
Various factors are indeed playing a vital role for gametic embryogenesis in vitro.
Genotype, organ used in culture, the stage of development of the embryo sac, media
components and culture conditions, pretreatments given to the explants were extensively
reviewed by Yang and Zhou (1982), San Noeum and Gelebart (1986) and Keller and
Korzun (1996b). Megaspores or female gametophytes of plants can be triggered in vitro to
undergo sporophytic development. For majority of species triggering factors promoting
haploid embryogenesis are not apparent, but media constituents such as phytohormones
and carbohydrates evidently have some role in reprogramming.
2.5.1 Genotype of the plant
Genotype of the donor plant is one of the most important factors for the induction
of gynogenic plants. Genotypic differences in response were demonstrated in Hordeum
vulgare (San Noeum, 1979), Oryza sativa (Zhou and Yang, 1981; Zhou et al., 1986),
Gerbera jamesonii (Sitbon, 1981), Triticum aestivum (Zhu et al., 1981), Helianthus annus
(Gelebart and San, 1987; Badea et al., 1989), Allium cepa (Campion et al., 1992; Muren,
1989; Keller, 1990) and Beta vulgaris (Van Geyt et al., 1987; Lux et al., 1990). Since each
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genotype shows a different response, a specific protocol must be followed for maximal
efficiency. Close to one hundred onion genotypes/accessions were subjected to a standard
procedure and revealed a high level of genetic variability in gynogenic response (varying
from 0 to 10 green plants regenerated per hundred cultured flowers). Most of the
regenerated plants appeared to be haploid and artificial chromosome doubling was
required to produce the DHs (Grzebelus and Adamus, 2004). Genotypic variation is a
serious problem for the overall success of haploid plant production from unfertilized
ovules of sugar beet (Doctrinal et al., 1989; Lux et al., 1990; Hansen et al., 1995; Gurel et
al., 2000). Genes responsible for the initiation of apomictic embryo development from
unfertilized egg cells (parthenogenesis) may also play a role in gynogenesis (Wedzony et
al., 2009). This potentially can open up new perspectives for in vitro gynogenesis.
2.5.2 Developmental stage of embryo sac
It is not easy to observe directly the embryo sacs at the time of inoculation, so an
indirect judgement is more feasible. It is necessary to correlate development with an easily
observable morphological feature. In case of cereals, the stage of pollen development is
often used as a test. In practice, however, most common flower characteristics used are:
anther colour (cereals), position of ovule in the ovary (gerbera), position of style related to
anthers and corolla (lettuce, sunflower), shape and height of the ovule (sugar beet), shape
of the ovary and silk emergence (maize). Haploids of most species have been obtained
from in vitro gynogenesis using explants at uninucleate to mature embryo sac stages (Wu,
2003). With the exception of mulberry, barley and maize, other species (onion, sugar
beet, squash, sunflower, gerbera, Hyosciamus muticus and Melandrium album) have been
reported to be optimally inoculated at earlier flower developmental stages (Bohanec,
2009a). In sugar beet, flower buds collected 1–3 days before anthesis possessed a mature
embryo sac (Ferrant and Bouharmont, 1994). A sugar beet embryo sac is capable of being
fertilized 5 days before anther dehiscence, but Van Geyt et al. (1987) reported a
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degeneration of young spherical ovules after a few days in culture. In sunflower, flowers
possess a young but completely developed embryo sac 2–3 days before anthesis (Yang et
al., 1986). Uninucleate to four nucleate embryo sacs, corresponding to late uninucleate or
early-binucleate stage of pollen were found in rice (Zhou et al., 1986) and were reported
as superior to earlier or mature stages. Similar results were obtained in onion (Musial et al.
2005), in which the smallest flowers predominantly (containing megaspore mother cells)
and largest (containing mature embryo sacs) were less responsive than medium size
flowers, containing 2–4 nucleate embryo sacs. In niger, it was observed that only ovules
collected on the day of, or 1 day before, anthesis were responsive to gynogenesis (Bhat
and Murthy, 2007).
2.5.3 Culture conditions
For gynogenesis, both physical and chemical properties of the culture medium are
very critical. In onion the medium developed by the group of Campion and collaborators
named BDS (Campion and Alloni, 1990; Campion et al., 1992, 1995) is commonly
applied. However, Geoffriau et al. (1997) used B5 medium for induction and MS for
regeneration. Researchers from Lubliana University (Bohanec and Jakse, 1999; Jakse et
al., 2003; Jakse and Bohanec, 2003) and from the Agricultural University of Krakow
(Michalik et al., 2000a,b; Nowak, 2000; Adamus et al., 2001; Grzebelus and Adamus,
2004) contributed vastly to recent progress. Martinez et al. (2000) reported positive effects
of polyamines (putrescine and spermidine) in case of in vitro gynogenesis in onion. A
novel approach was described by Martinez (2003) where polyamines are components of
the induction medium and where embryos were subcultured before transfer onto the
regeneration medium.
Addition of charcoal increased frequency of embryo formation (D’Halluin and
Keimer, 1986; Gurel et al., 2000), but inclusion of AgNO3 decreased or completely
inhibited their formation (Gurel et al., 2000) in beet. Physical properties of the culture
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medium used for anti-mitotic treatment were also important during chromosome doubling.
Higher rates (29.1%) were seen when haploid shoots were immersed in liquid medium
versus culturing on agarose- or agar-solidified medium, 20.7% and 15.4%, respectively
(Gurel et al., 2000). Culturing ovaries in the dark for two weeks before transferring to
light/dark conditions produced significantly more diploid shoots from ovary-derived callus
than those kept continuously in the dark (Gurel and Gurel, 1998). Galatowitsch and Smith
(1990) reported that adventitious shoot regeneration originated mostly from diploid cells
of the ovary tissue, unless they were not spontaneously doubled haploids, as also reported
by others (D’Halluin and Keimer, 1986; Wang et al., 1991). The ovary was also used for
haploid plant production, if callus from diploid cells of ovary tissue was removed and
ovules transferred to auxin-free medium with 0.5% charcoal (Van Geyt et al., 1987).
Callus from the nucellus tissues accounts for most of the callus obtained, unless activated
charcoal has been added to the embryo induction medium. Inclusion of activated charcoal
in the medium prevents callus formation from the mother tissue, which hampers the
development of haploid plantlets (Speckmann et al., 1986; Van Geyt et al., 1987).
Rongbai et al. (1998, 1999) reported callus mediated haploid plant regeneration
from rice ovaries in N6 medium supplemented with 2.0 mg/l NAA + 1.0 mg/l BA, 0.6 –
0.8% DMSO along with 5% sucrose. In Durum wheat (Triticum durum Desf.) ovaries
cultured on half-strength MS with 2 mg/l 2,4-D, 1 mg/l kinetin & 12% sucrose gave rise to
callus-mediated regeneration with all the regenerates being haploids (Mdarhri-Alaoui et
al., 1998). Ovaries cultured on a modified medium with 2 mg/l 2,4-D, 0.5 mg/l kinetin,
6% maltose induced callus mediated regeneration and predominantly haploid regeneration
(Sibi et al., 2001). Maize (Zea mays L.) ovaries cultured on MS medium supplemented
with 3 mg/l 2,4-D and 12% sucrose produced mixoploid plants (Truong-Andre and
Demarly, 1984). Gynogenesis in flax (Linum usitatissimum L.) was reported by Obert et
al. (2004) and subsequently by Bartosova et al. (2005, 2006). Callogenesis was induced
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from cold and warm pretreated female gametes. Ovaries were isolated from flax flower
buds 24 h before anthesis. Induction was achieved after treatment on N6 (Chu, 1978)
medium with NAA and BAP (each at a concentration of 1 mg/l). Formed calli were
transferred to MS medium supplemented with 1 mg/l 2,4-D. Regeneration via
organogenesis was performed on MS medium supplemented with 0.5 mg/l TDZ and 0.5
mg/l TDZ + 0.5 mg/l BAP. Shoots were finally rooted on MS medium containing 20%
sucrose or on MS medium with 10% sucrose and 0.1% of activated charcoal (Obert et al.,
2004).
2.5.4 Type of explants for culture
Gynogenesis in onion is achieved via the culture of flower buds, ovary and ovules
(Campion and Alloni, 1990; Campion et al., 1992; Keller, 1990) on solid media.
Gynogenesis induced from isolated ovules appeared to be successful for sugar beet
(Bossoutrot and Hosemans, 1985; Goska, 1985; Doctrinal et al., 1989; Galatowitsch and
Smith, 1990; Lux et al., 1990; Ragot and Steen, 1992; Gurel et al., 2000; Wremerth-Weich
and Levall, 2003), red beet (Baransky, 1996) and fodder beet (Kikindonov, 2003). In rice,
gynogenic development was reported to be most efficient when entire florets (with intact
pistils, stamens and glumes attached to a piece of receptacle) were cultured as a unit, while
dissected pistils proved unresponsive (Zhou and Yang, 1981). Gynogenic haploids of a
female clone of mulberry (Morus alba L.) were obtained by in vitro culture of
unpollinated ovaries from in vitro developed inflorescences (Dennis et al., 1999). In
cucumber and melon, ovary culture was reported to produce haploids and double haploids
(Ficcadenti et al., 1999; Gemes-Juhasz et al., 2002; Diao et al., 2009; Malik et al., 2011)
whereas in squash (Cucurbita pepo) Metwally et al. (1998) and Shalaby (2007) proved
significance of ovule culture in inducing gynogenesis. Zhang and Lespinasse (1988)
reported in apple the induction of gynogenesis through in vitro culture of unpollinated
ovaries and ovules, without plant regeneration. In tef (Eragrostis tef) Gugsa et al. (2006)
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have shown recently that gynogenesis can be induced from unpollinated inflorescence
explants which was efficient for three genotypes.
2.5.5 Pretreatments of the explants before culture
In onion, Puddephat et al. (1999) failed to show substantial improvement of the
efficiency by plant stress pre-treatment prior to ovary culture. In beet, the basic protocol
was developed in late 1980s (Bossoutrot and Hosemans, 1985; Goska, 1985; Doctrinal et
al., 1989). Cold treatment of inflorescences at 8ºC for 1 week, combined with relatively
high temperature (30ºC) of the induction phase can be regarded as the latest improvement
of the procedure (Wremerth-Weich and Levall, 2003). Cold pretreatment of flower buds
induces haploid embryos (Gurel et al., 2000), although previous studies reported no effect
(D’Halluin and Keimer, 1986). In flax (Linum usitatissimum L.) callus was induced from
cold (8°C for 72 h) and warm (32°C for 8 h) pretreated female gametes in ovary cultures
of 3 flax genotypes. Callus thus obtained was subsequently regenerated into plantlets
(Obert et al., 2004). In niger, cold treatment of the ovules was not effective for
gynogenesis induction (Bhat and Murthy, 2007).
2.6 Origin of haploids
Gynogenic origin of regenerated plants in onion was confirmed by embryological
studies (Musial et al., 2001, 2005). Most haploid plants from unfertilized ovules of sugar
beet were from direct embryogenesis. As shown by studying the histology of developing
embryos, Ferrant and Bouharmont (1994) reported that viable gynogenetic embryos
originated only from the egg cell. In other species, besides egg cells, synergids and
antipodals are also capable of undergoing embryogenesis or callus formation
(Mukhambetzhanov, 1997; Bohanec, 2009b). In tomato, an attempt for the induction of
gynogenesis by means of unfertilized ovary culture was reported (Bal & Abak, 2003).
According to this report, the ovary cultured on MS medium with various growth
45
regulators allowed gynogenic development of ovules. Histological analysis revealed mass
of cells in the embryo sacs which resembled globular embryos.
2.7 Gynogenesis in cucurbitaceae
Cucumber (Cucumis sativus L.) ovaries cultured on CBM medium supplemented
with 0.02 mg/l TDZ + 40 g/l sucrose shown to induce 18.4% haploids resulting in 7.1% of
plant regenerants (Gemes-Juhasz et al., 2002). Whereas Diao et al. (2009) reported
embryo formation frequency of 72.7% resulting in 9.0% plant regeneration with 51.5%
spontaneous double haploids on MS medium. Although ovary culture has been studied to
produce haploids and doubled haploids in cucumber, the low frequency of gynogenesis
and regeneration rates has limited the practical use of this technique in breeding programs.
Similarly, in melons, 63.3% embryos formed with plantlet regeneration occurred at 22.5%
(Malik et al., 2011). In squash (Cucurbita pepo), 2,4-D at 1.0 and 5.0 mg/l induced
gynogenesis at the rate of 11.5% (Metwally et al., 1998). Most plantlets per 100 cultured
ovules resulted from ovules without cold treatment compared to 4°C for 2, 4 and 8 days
(Metwally et al., 1998). Shalaby (2007) reported ovules cultured on MS medium
supplemented with KN and 2,4-D at 1 mg/l and 3% sucrose induced 48.8% embryogenic
response and 15 plants per 25 ovules cultured in a Petri dish. Among the plants
regenerated, 65% were haploids and remaining double haploids (Shalaby, 2007).
2.8 Use of irradiated pollen for in vitro haploid production
It is well known that treatment by ionizing rays may cause the breakage of
chromosomes, thus creating chromosome aberrations. The irradiation of gametes has been
used extensively in pollination experiments. Pollination of female flowers with irradiated
pollen can be considered as an alternate technique for the induction of gynogenic plants
when other methods have been unsuccessful. This technique was used firstly with embryo
culture of different species of Nicotiana (Pandey and Phung, 1982). Irradiated pollen can
germinate on the stigma, grow within the style and reach the embryo sac, but cannot
46
fertilize the egg-cell and the polar nuclei (Cuny et al., 1992). Genetically inactive but
germinable pollen can be used to stimulate the division of the egg cell, and thus induce
parthenogenetic haploid production (Stairs and Mergen, 1964; Savaskan and Toker, 1991;
Todorova et al., 2004). In this method, generally endosperm will not be produced because
of which the embryo, even if induced, will not grow to maturity and generally degenerate
during the development. In order to rescue the embryos, they have to be harvested and
cultured on to a suitable nutrient medium for assisting their maturity. Since only haploid
cell is involved in the formation of the embryo, the resulting plantlet would also be
haploid unless endoduplicated.
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., 1976). Successful chemical treatments include application 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 brassinolide to emasculated
stigmas of Arabidopsis, Brassica and Tradescantia (Kitani, 1994). Similarly
parthenogenesis was induced by pollen from a triploid plant followed by in vitro embryo
culture (Oiyama and Kobayashi, 1993; Germana and Chiancone, 2001).
It was pioneered by Winton and Einspahr (1968) who irradiated Populus alba
pollen with 100 radians of gamma irradiation and pollinated female flowers of P.
tremuloides. One slow growing plant from the cross was produced with haploid
chromosome number. Soon after, this method was then successfully used to obtain
haploids in wheat (Snape et al., 1983), barley (Powell et al., 1983), Petunia hybrida
(Raquin, 1985), muskmelon (Sauton and Dumas de Vaulx, 1987), cabbage (Dore, 1989)
and rose (Meynet et al., 1994). Induced parthenogenesis in carrot was studied previously
by Rode and Dumas de Vaulx (1987). They pollinated male sterile flowers with irradiated
carrot pollen, and culture of immature seeds resulted in two haploid plants. Dore and
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Marie (1993) produced gynogenetic plants of onion after crossing with irradiated pollen.
However, it should not be ignored that various factors such as genotype, environmental
elements, embryo yield and irradiation dose have extremely large effect on the success of
irradiated pollen technique (Sari et al., 1992; Ficcadenti et al., 1995; Dore et al., 1995).
2.8.1 Genotype
Haploid production is greatly influenced by the genotype in parthenogenesis
induced by irradiated pollen (Hougas et al., 1964; Chase, 1969; Rowe, 1974; Yang and
Zhou, 1982; Zhang and Lespinasse, 1991). In capsicum, the genetic background of the
female parent was essential in generating polyembryonic seeds with haploid embryos
(Campos and Morgan, 1960). High frequencies of haploids were obtained by utilizing
specific maternal genotypes in black cottonwood (Stettler et al., 1969). Different
Nicotiana species are known to possess specific genes capable of inducing the
development of parthenogenic haploids and diploids (Pandey, 1983; Pandey and Phung,
1982). In kiwifruit, certain pollinators appeared more effective in inducing haploidy than
others that induced parthenogenic diploids in the same recipient genotype (Pandey et al.,
1990). In Cucumis melo, strong influence of the genotype on haploid production was
observed (Ficcadenti et al., 1995; Sauton, 1988). The vigour and physiological state of the
parents were also found to be important for the haploid response in melon (Sauton, 1988;
Cuny et al., 1993).
2.8.2 Types of radiation used for gynogenesis
Pollen irradiation (X-rays and γ-rays) and subsequent pollination of female flowers
is the most widely used technique to induce in situ parthenogenetic haploid plants.
Various authors reported the use of X-rays for parthenogenetic haploids and double
haploids. Individual flowers were irradiated with X-rays in Capsicum frutescens (Campos
and Morgan, 1960). Sauton and Dumas de Vaulx (1987), Kato et al. (1993) have used soft
X-rays for parthenogenesis in melon. Sato et al. (2000) produced doubled haploids in
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carnation, Sugiyama et al. (2002) in watermelon and recently Yahata et al. (2010) induced
haploids in citrus.
Gamma rays are commonly used in haploid programs because of their simple
application, good penetration, reproducibility, high mutation frequency, and low disposal
(lethal) problems (Chahal and Gosal, 2002). This technique was used firstly with embryo
culture of different species of Nicotiana (Pandey and Phung, 1982). Pollen of black
cottonwood was collected from flower buds and irradiated directly with γ-rays (Stettler,
1968). Cucumis melo (Sauton and Dumas de Vaulx, 1987), onion (Dore and Marie, 1993),
kiwifruit (Pandey et al., 1990), apple (Lecuyer et al., 1991; Zhang and Lespinasse, 1991)
and rose (Meynet et al., 1994) are some of the examples in which the γ-rays have been
successfully used for haploid induction.
2.8.3 Dose of irradiation
Values of LD50 (dose to inhibit germination in 50% pollen) have been reported
between 35 Krad and 550 Krad (350 Gy and 5500 Gy) depending on the species of plant.
Doses which do not prevent germination may, however, greatly depress pollen tube
growth, often resulting in short tubes with burst tips (Casarett, 1968). After hydration, the
pollen grain produces an outgrowth from an aperture or thin area in the wall. This is the
site of tip growth that results in the production of a pollen tube, which will ultimately
convey the sperm cells to the embryo sac (Lord & Russell, 2002). This indicates that
germination and ultimate survival of pollen have different responses to radiobiological
injury (Casarett, 1968). Irradiation doses should not be so high as to inhibit pollen tube
germination but high enough to disturb normal fertilization and to avoid the development
of diploid hybrid embryos (Dore et al., 1995).
The doses applied vary greatly from one species to another: 5-20 Gy in barley
(Powell et al., 1983), 200-1200 Gy in cabbage (Dore, 1989), and the effects induced differ
greatly. In melon, no influence on pollen germination was found with gamma ray doses of
49
500 – 3,600 Gy (Cuny and Roudot, 1991). In Petunia, Raquin (1985) used the doses of
60-100 kR (0.6-1.0 kGy). Cucumber was treated with the dose 0.3-1.0 kGy (Sauton,
1989), 0.3 kGy (Przyborowski and Niemirowicz-Szczytt, 1994), 0.3, 0.45 and 0.6 kGy of
gamma rays (Caglar and Abak, 1996) 100 Gy (Faris et al., 1999; Lotfi et al., 1999), 150
Gy (Xie et al., 2005), and 500 Gy (Claveria et al., 2005; Dolcet-Sanjuan et al., 2006).
Embryos and haploid plants were also obtained from lower irradiation doses (25 and 50
Gy) in summer squash (Kurtar et al., 2002) and in pumpkin (50 and 100 Gy, Kurtar et al.,
2009) and in winter squash (50 and 100 Gy, Kurtar and Balkaya, 2010). On the other
hand, haploid embryo induction was obtained at relatively higher doses (200–300 Gy) in
watermelon (Gursoz et al., 1991; Sari et al., 1994). Todorova et al. (2004) showed γ-
radiation of 600 and 900 Gy induced 11 haploid plants from 5 genotypes in sunflower.
These results may be attributed to the radio-resistance of pollen and also to
biologic efficiency of irradiation. A linear relationship between radio-resistance and pollen
size, which is also a function of the amount of DNA in the nucleus has been reported
(Brewbaker and Emery, 1962; Alison and Casareft, 1968; Shridhar, 1992; Jain et al.,
1996). Pollens of winter squash are one of the largest pollen (as in squash and pumpkin) in
vegetables (average width 180 µm). Melon, watermelon, and cucumber pollen are smaller
than winter squash (average 50, 60 and 65 µm, respectively) (Sensoy et al., 2003).
Moreover, melon, watermelon and cucumber have 3 apertures, whereas winter squash has
12 apertures. Hence, winter squash pollens are more sensitive to dehydration and rapid
loss of their viability as reported in squash (Nepi and Pacini, 1993).
2.9 Parthenogenesis in cucurbits
The irradiated pollen technique is an effective method for the induction of haploid
embryos in Cucurbits. Induction of in situ haploid embryos and obtaining in vitro haploid
plants have been achieved using irradiated pollen technique in cucumber (Truong-Andre
1988; Niemirowicz-Szczytt and Dumas de Vaulx 1989; Przyborowski and Niemirowicz-
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Szczytt, 1994; Caglar and Abak, 1999; Faris et al., 1999; Faris and Niemirowicz-Szczytt,
1999; Lotfi et al., 1999; Claveria et al., 2005; Dolcet-Sanjuan et al., 2006), melon (Sauton
and Dumas de Vaulx, 1987; Sauton, 1988; Cuny et al., 1992; Maestro-Tejada 1992; Sari et
al., 1992; Abak et al., 1996; Lotfi et al., 2003; Lim and Earle, 2008, 2009; Ari et al.,
2010), watermelon (Gursoz et al., 1991; Sari et al., 1994, 1999; Jaskani et al., 2005b),
snake cucumber (Yanmaz et al., 1999; Taner et al., 2000), squash (Kurtar et al., 2002),
pumpkin (Kurtar et al., 2009) and in winter squash (Kurtar and Balkaya, 2010).
2.10 Parthenogenesis in fruit trees
Parthenogenesis induced in vivo by irradiated pollen, followed by in vitro culture
of embryos, can be an alternative method of obtaining haploids in fruit crops. Two
cultivars of apple, Lodi and Erovan, were used as female parents for pollination with
irradiated pollen. Pollen was irradiated by gamma rays at doses 500 to 1500 Gy. Ovules
from young fruits (1-4 weeks after pollination) were cultured on MS medium
supplemented with NAA, BA and GA. The same technique has been successfully applied
to other apple cultivars also with different gamma-rays (Zhang et al., 1987; Zhang and
Lespinasse, 1991; Zhang et al., 1992; De Witte and Keulemans, 1994; Hofer and
Lespinasse, 1996), in Pyrus communis L. (Bouvier et al., 1993), Prunus avium L. (Hofer
and Grafe, 2003), Actinidia deliciosa (A. Chev) (Pandey et al., 1990; Chalak and Legave,
1997). In Citrus natsudaidai haploid seedlings were first obtained by the application of
gamma rays (Karasawa, 1971). One haploid embryo was obtained in an immature seed
from a diploid (Clementine mandarin) x diploid (Pearl tangelo) cross (Esen and Soost,
1972). Nine haploid plants and two embryogenic callus lines were obtained in Citrus
clementina after in situ parthenogenesis induced by pollen irradiated at 300, 600 and 900
Gy (Ollitrault et al., 1996). Froelicher et al. (2007) produced five haploid plantlets from
three mandarin genotypes by pollinating with pollen of Meyer lemon (C. meyeri Y.
Tanaka) irradiated at 150 and 300 Gy of γ-rays. Recently, Aleza et al. (2009) reported
51
induction of 270 fruits producing 1744 seeds, out of which only 51 embryos were cultured
after pollination of irradiated pollen with γ-ray at 500 Gy in citrus. Only 13 embryos
responded and 8 haploid plants produced directly and 4 embryos gave rise to callus which
subsequently produced 12 haploids (Aleza et al., 2009). In European plum Prunus
domestica L. 200 Gy γ-ray dose induced formation of 2n endosperm and abnormal
embryo development (only heart-shaped embryos) which did not develop further (Peixe et
al., 2000).
Parthenogenetic tri-haploids were induced in hexaploid kiwifruit by irradiated
pollen. The best results were obtained with a dosage of 500-1500 Gy and the genotype of
the pollen parent greatly influenced the ability to obtain both seedlings and tri-haploids
(Chalak and Legave, 1997). Spontaneous doubling was also observed. Pandey et al.
(1990) and Chalak and Legave (1997) induced parthenogenesis in kiwifruit (Actinidia
deliciosa).
Besides of many advantages there are also drawbacks in the application of
irradiated pollen technique as detecting and excising haploid embryos (Lotfi et al., 2003)
and doubling of the haploid chromosome to obtain fertile plants (Lim and Earle, 2008).
Moreover the availability of radiation source might become a limiting factor as the
radiation facility is very expensive and majority of the laboratories will not afford. Thus,
in vitro culture of unpollinated ovaries and ovules and use of irradiated pollen for
induction of gynogenic haploids have been extensively and successfully carried out in
various plant species due to the positive gynogenetic responses that can be obtained by
using wider range of developmental and physiological stages of the embryo sac and
various cultural conditions than is possible with androgenesis, which is more restricted to
the stage of anthers that can be cultured. More varied approaches can thus be applied to
the generation of haploids from the female tissues than from the male.
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2.11 Chromosome doubling procedures for the production of doubled haploids
Female gamete cells may be manipulated to produce haploid embryos in contrast
to normal fertilization of ovules by pollen grains. Induced or spontaneous chromosome
doubling can generate completely homozygous doubled haploid plants (Jain et al., 1996).
Complete homozygous genotypes are precisely repeatable and hence have increased
heritability of quantitative characters. This enhances selection efficiency of desired traits.
A characteristic of gynogenic haploid induction is a low percentage of spontaneous
chromosome doubling resulting in the majority of regenerants being haploid. Data giving
high proportions of diploid regenerants are often preliminary and based on a low number
of haploids, or in some cases not proven to be homozygous. The situation is similar in
gynogenic induction induced by pollination treatments also resulting predominantly in
haploid regenerants. Approaches to chromosome doubling have been reviewed by Kasha
and Maluszynski (2003). Only a few other observations should be added. Chromosome
doubling is achieved by using colchicine or other antimitotic agents like oryzalin,
aminoprophosmethyl (APM) etc. to obtain fertile plants (Lim and Earle, 2008, 2009;
Yetisir and Sari, 2003). Large numbers of treated individuals are often needed to obtain
reproducible results. Bohanec (2009a) quoted diploidization treatments in onion embryos
were repeatable when the experimental unit was high (400–500 embryos per treatment,
Jakse et al., 2003). Another observation is that the optimal treatment should be a
compromise between the efficiency in chromosome doubling and the mortality of treated
tissues. The latter data are often not given.
Beet doubled haploids are used in breeding programs (Zakhariev and Kikindonov,
1997), including hybrid breeding (Kikindonov and Kikindonov, 2001). Production of
completely homozygous, doubled haploids was achieved through chromosome doubling
by treating haploids with anti-mitotic agents, primarily by including colchicine in the
culture medium, either during multiplication (Bossoutrot and Hosemans, 1985; Ragot and
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Steen, 1992; Gurel et al., 2000, 2003) or directly at the ovule culture stage (Hansen et al.,
1994). Several other agents, including APM, pronamide, trifluralin and oryzalin, were also
tested (Hansen et al., 1998, 2000); APM was the most effective. In a comparative study,
trifluralin was as effective as colchicine, although at much lower concentrations (Gurel et
al., 2000) and thus was strongly recommended because it is much cheaper and reported to
be less toxic (Zhao et al., 1996).
In onion, colchicine, oryzaline, trifluraline and APM were tested and eventually a
medium containing 50 µM APM applied for 24 hours was found to be the most effective
(Grzebelus and Adamus, 2004). Various other methods can be used to apply colchicine in
in vitro and in in vivo growth conditions like adding colchicine to the growth media in in
vitro culture, immersing roots, plants and single node cuttings into colchicine solution,
application of colchicine to lateral buds by medicine dropper and immersing shoot tips of
in vivo grown plants (Yetisir and Sari, 2003). Besides the rate of in vitro chromosome
duplication is low in haploid melons it was reported that shoot tip immersion into
colchicine solution in cantaloupe melon was the most efficient method (Koksal et al.,
2002; Yetisir and Sari, 2003). Durum wheat haploid plants were produced after distant
hybridization with maize pollen. After three weeks of growth all haploid plants were
colchicine treated as a root-treatment procedure (Mujeeb-Kazi and Riera-Lizarazu, 1996).
Successful chromosome doubling was reported to be achieved from the seed setting on the
colchicine-treated polyhaploid plants in producing haploid onion cultivars like ‘Sefid-e-
Kurdistan’ and ‘Sefide-Neishabour’ (Touraev et al., 2009).
Chemicals used to induce chromosome doubling (spindle inhibitors or anti-
microtubule agents) target the whole meristematic domain, resulting in a large proportion
of mixoploid plants. An alternative approach to doubling the chromosomes of haploid
plants can be based on spontaneous chromosome doubling during adventitious in vitro
regeneration, instead of chemical treatments. Such treatment of diploid tissues often leads
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to increased ploidy. For instance in hop, Skof et al. (2007) induced up to 58.6%
tetraploids. In this case, doubled regenerants are probably regenerated from a single
doubled cell and, as such, often do not possess mixoploid tissues. This approach has
already been attempted in haploid onion plants (Alan et al., 2007), in which regeneration
from flower buds resulted in 60.7% of spontaneously doubled plants. The low frequency
of mixoploidy, low mortality and simultaneous chromosome doubling and clonal
multiplication of breeding lines are why an in vitro adventitious regeneration approach
used for chromosome doubling deserves further attention.