30

REVIEW OF LITERATURE

31

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.

32

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.

33

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

34

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

35

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

36

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

37

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

38

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

39

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

40

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

41

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

42

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

43

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)

44

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

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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

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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.