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See discussions, stats, and author profiles for <a href= this publication at: In vitro haploid and dihaploid production via unfertilized ovule culture Article in Plant Cell Tissue and Organ Culture · March 2011 DOI: 10.1007/s11240-010-9874-6 CITATIONS READS 43 2,626 4 authors , including: K e r e M b i ra U n i v e r s i t y o f K a b i a n g a C I T AT I O N S P U B L I C AT I O N S S E E P R O F I L E Some of the authors of this publication are also working on these related projects: selection of kenyan Pumpkin landraces for vitamin A content View project Al l c o n t e n t f o l l o w i n g t h i s p a g e w a s u p l o a d e d b y K e re M b i r a o n 2 1 S e p t e m b e r 2 0 1 7. T h e u s e r h a s r e q u e s t e d e n h a n c e m e n t o f t h e d o w n l o a d e d fi l e . " id="pdf-obj-0-20" src="pdf-obj-0-20.jpg">
























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T h e u s e r h a s r e q u e s t e d e n h a n c e m e n t o f t h e d o w n l o a d e d fi l e .

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DOI 10.1007/s11240-010-9874-6

In vitro haploid and dihaploid production via unfertilized ovule


Jin-Feng Chen Li Cui Ahmed Abbas Malik Kere George Mbira

Received: 18 July 2010/Accepted: 26 October 2010 Springer Science+Business Media B.V. 2010

Abstract Haploids and doubled haploids are very important in plant breeding, enabling the time needed to produce homozygous lines to be shortened compared with conventional breeding. In the present review, emphasis is given to haploid induction through unfertilized ovule/ovary culture. Attention is given to induction of haploid plants from female gametophyte culture through analysis of factors in the processes of gynogenesis, including genotype selection, stage of ovule development, pretreatment, and culture media containing nutritional components and phytohormones. The gynogenetic approach may be of great value in discovering novel genetic recombinations. Application of double haploids in genetics and plant breeding is also highlighted. This review also identifies some existing knowledge gaps where work may increase the efficiency of this process in different plant species.



Gynogenesis Female gametophyte

J.-F. Chen (&)

L. Cui

A. A. Malik K. G. Mbira

State Key Laboratory for Crop Genetics and Germplasm Enhancement, Key Laboratory of Southern Vegetable Genetic Improvement, Nanjing Agricultural University, Nanjing, Jiangsu, People’s Republic of China e-mail:

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). Megaspores or female gametophytes of plants can be triggered in vitro to undergo sporophytic development. Production of haploid plants through gynogenesis by culturing unfertilized ovaries was first described in barley (San

Introduction and brief history

Plants with gametophytic chromosome number in their sporophyte (whether diploid or polyploid) are referred to as haploids. Haploid cells occur naturally in the gametophytic phases of higher plants, in their ovules and pollen. Female

Jin-Feng Chen and Li Cui contributed equally to this paper.

gamete cells may be manipulated to produce 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. The development of in vitro techniques for production of haploids was a major feat in the fields of biotechnology and plant breeding in the past few decades. It is documented that Blakelsee et al. (1922) pioneered this technique by first producing haploids of Datura stramonium. Thereafter, haploids were reported in many other species. Guha and Maheswari (1964) developed an anther culture technique for production of haploids through androgenesis in Datura inoxia. Haploid production by wide crossing was 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).

The process of haploid regeneration though unpollinated female gametophytes is usually described as gynogenesis. Gynogenesis has been shown to be a possible alternative source for haploid production in plants, particularly in species where androgenesis is recalcitrance or where the

Noeum 1976). Previous attempts to induce haploids through culturing unfertilized ovules failed (Yang and Zhou 1982; Lakshmi-Sita 1997; Mukhambetzhanov 1997; Germana 2006). However, successful gynogenesis is reported in many plant species, e.g., onion (Allium cepa; Bohanec et al. 1995; Luthar and Bohanec 1999), sweet potato (Ipomoea batatas; Ruth et al. 1993), tulip (Tulipa generiana; Van-Creij et al. 2000), maize (Zea mays; Tang et al. 2006), sugar beet (Beta vulgaris; Gu¨rel et al. 2000), cucumber (Ge´mes- Juha´sz et al. 2002), and wheat (Triticumdur Defs., Sibi et al. 2001). Haploids of 21 angiosperm species have been obtained from in vitro unfertilized ovule/ovary culture since 1976, in most cases using explants at uninucleate to mature embryo sac stages (Wu 2003). These pure lines can provide

Plant Cell Tiss Organ Cult REVIEW DOI 10.1007/s11240-010-9874-6 In vitro haploid and dihaploid production via unfertilized


two valuable advantages: first, direct establishment of new cultivars in selfpollinated crops, and second, development of pure lines that could be used to produce high-yielding hybrids (Shalaby 2007). The presence of one set of chromosomes facilitates isolation of recessive gene mutants (Hermsen and Ramana 1981). 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).

Genetics of double haploids

In the doubled haploid (DH) method, only two types of genotypes occur for a pair of alleles, A and a, with frequency of AA and aa, while in the diploid method, three genotypes occur with frequency of AA, Aa, and aa. Thus, if AA is a desirable genotype, the probability of obtaining this genotype is higher in the haploid method than in the diploid method. If n loci are segregating, the probability of getting the desirable genotype is (1/2) n and (1/4) n by the haploid and diploid method, respectively. Thus, the efficiency of the haploid method is obviously high when the number of genes concerned is large. Studies have been conducted to compare the DH method and other conventional breeding methods, and it was concluded that adoption of doubled haploidy does not lead to any bias of genotypes in populations, and random DHs were even found to be compatible with selected lines produced by conventional pedigree method (Winzeler et al.


Induction of gynogenesis has been tested in a significant number of economically important plants. In general, such protocols consist of different phases and can be divided into the following major components: (a) genotype selection, (b) developmental stage of ovule, (c) pretreatment, and (d) culture medium composition and phytohormones. These are the principal factors controlling induction and development of intact plants.

Genotype selection

The donor genotype is thought to play a decisive role in unfertilized ovary/ovule culture. Gynogenesis efficiency in plants is highly dependent on the variety used, the growth condition of the plants, and the quality of the donor material. Cellular and morphogenetic events during somatic embryogenesis are controlled by an array of culture conditions (medium composition and physical environment) and by genetic effects (Martı´nez et al. 2000). In onion,


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haploid induction ability is strongly influenced by donor genotype. Inbred lines and hybrid F1 tend to have higher rates of embryo production and plant regeneration compared with open pollinated populations, in which the response is generally lower (Gioffriau et al. 1997; Bohanec and Jakse 1999). This confirms genotypic-specific responses to gynogenesis. The genotype proved to be one of the most important factors affecting in vitro gynogenesis in squash. The percentage of gynogenic ovules ranged from 0% to 48.8%, depending on genotype. Cultured Yellow Bik F1 ovules of squash cultivar remained undeveloped on culture medium (Shalaby 2007). In contrast, the same medium produced the best embryogenic callus from anther cultures (Shalaby 2006). This indicates that there are differences between ovule and anther culture response within the same genotype. Similar results were reported in sweet potato (Kobayashi et al. 1993) and onion (Alan et al. 2004; Bohanec and Jakse 1999).

Stage of ovule development

The development stage of the ovules has a profound influence on gynogenesis in vitro (San Noeum and Gelebart 1986). The stage of ovule development at time of inoculation has not been studied extensively. In many crop species, such as barely, sugar beet, maize, and sunflower, several authors found that optimal gynogenesis was obtain with nearly mature embryo sacs (Hoseman and Bossoutrot 1983; San Noeum and Gelebart 1986; Lux et al. 1990). In tobacco ovule cultures, Wu and Chen (1982) reported that ovaries with young uninuclear to mature embryo sacs were responsive to gynogenesis. The induced time according to the develomental stage of the flower bud or stage of pollen development has been described. The development pattern of the female gametophyte in excised ovaries in vitro has been studied in onion, barley, rice, tobacco, Cenchrus ciliaris, and Melandrium album (Musial et al. 2005). A maturation process similar to in situ development has been found in studied species, the difference from in situ maturation being mainly a higher frequency of degenerating ovules. Histological study of cucumber embryo sac indicated that the possibility of haploid induction from megaspores or the early stages of the embryo sac can be precluded. The most responsive ovaries (ovules) had nearly mature or fully mature embryo sacs (Ge´mes-Juha´sz et al. 2002). The ovaries from various stages of stigma development responded differently in saffron. In general, ovaries with yellow stigma were the best explants for direct shoot regeneration. Later stages, i.e., explants from blooming flowers, failed to respond (Bhagyalakshmi 1999). In niger [Guizotia abyssinica (L. f.) Cass.], it was observed that only ovules collected on the day of, or 1 day before,

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anthesis were responsive to gynogenesis (Bhat and Murthy 2007). No gynogenic response was observed in ovules collected 2 or 3 days before anthesis (Bhat and Murthy 2007). Similarly, in sugar beet, flower bud collected 13 days before anthesis but with mature embryo sac was more responsive (Ferrant and Bouharmont 1994). In Eragrostis mexicana, pistils dissected 12 days before anthesis at the bi- or tricellular pollen stage showed regeneration efficiency of almost 5% (Likyelesh et al. 2006). In onion, small and large flower containing megaspore mother cells and mature embryo sacs, respectively, were less responsive than medium-sized flower having 24 nucleate embryo sacs (Musial et al. 2005). Differential gynogenic response had also been observed in numerous plant species regarding development stage of embryos, for example, grapes (Nakajima et al. 2000), pearl millet (Lakshmi et al. 2002), sugar beet (Gu¨rel et al. 2000), and cucumber (Ge´mesJuha´sz et al. 2002). Overall, it seems that the female gametophyte can be developed from a wide range of developmental stages, whereas in the male gametophyte uninuclear stage is optimal. The developmental stage of embryo sac or ovule is one of the most important factors in gynogenesis and also plays a crucial role for reprogramming a pathway from gametophytic to sporophytic.


Stress treatments are the most common factor affecting embryogenesis, with cold/heat shock and starvation treatment being commonly used. Without stress, the change from gametophytic to sporophytic phase is very difficult. Certain physical treatments (e.g., low/high temperature, dark period or starvation medium) applied to donor plants in in vitro culture may have a strong influence on embryo induction, and are also important to switch from gametophytic pathway to saprophytic development. Pretreatment can be applied at different levels of explants, such as intact flowers, isolated ovules, or inflorescence. With regard to different explants, the type, level, and duration of pretreatment are different, and the regeneration efficiencies vary as well. In onion, a total of 49 embryos were obtained from 2,660 cultured flower buds, and preconditioning of stock ovaries significantly influenced gynogenic embryogenesis (Puddephat et al. 1999). Cold treatment was found to be beneficial in durum wheat ( Sibi et al. 2001), sugar beet (Gu¨rel et al. 2000) and niger. On the other hand, Ge´mes-Juha´sz et al. (2002) demonstrated that heat

treatment at 32C during the embryo induction phase increased haploid embryo formation and plantlet regeneration from ovule cultures of cucumber (Diao et al. 2009). In the 2432C temperature range, a 1C rise in temperature had a specific effect on both embryo induction and plantlet regeneration (Ge´mes-Juha´sz et al. 2002). Similarly, ovules exposed to 32C for 4 days produced the greatest number of gynogenic ovules, followed by ovules exposed to 4C for 4 days, and produced better embryogenic response (28% and 22%, respectively) ( Shalaby 2007). Despite the cited examples of a positive influence of cold or heat treatment, in Cucurbita pepo ( Metwally et al. 1998) and niger (Bhat and Murthy 2007) no beneficial effect of cold pretreatment (at 4C) on gynogenesis was observed as compared with control; rather, it significantly decreased the embryogenic potential of unpollinated ovules. Bohanec (1998) reported that in vitro gynogenesis is generally not stimulated by shock treatment such as low or high temperatures or pregrowth on starvation medium. Ovaries/ovules are generally cultured in light, but at least in same species, e.g., saffron (Bhagyalakshmi 1999) and cucumber (Ge´mes-Juha´sz et al. 2002) , dark incubation favors gynogenesis and minimizes somatic callusing. In contrast to the above, high illumination has been reported to be beneficial for onion (Puddephat et al.


Culture medium

Culture media formulation has also contributed to the progress of gynogenic methods. The most often modified components are: (1) the source of organic nitrogen, (2) carbohydrates, and (3) growth regulators.

Generally, gynogenesis has two or many stages, and each stage may have distinct nutritional requirements. During induction, ovaries require low levels of growth regulators and to be kept in the dark or light, while for regeneration they are transferred to medium with higher growth regulator concentration and incubated in light. Culture medium is a principal factor controlling induction and development of intact plants. However, it is still difficult to draw a conclusion as to the most suitable composition for different plant species. No general recommendation can be given with regards to culture medium for in vitro gynogenesis due to differential nutritional requirements at intra- and interspecific levels (Mukhambetzhanov 1997). Gynogenesis in onion is achieved by culture of flowers on solid media. Keller and Korzun (1996) found an review of methods, after that, the medium developed by the group of Campion and collaborators are commonly applied for onion gynogenesis. However, Geoffriau et al. (1997) used B5 medium for induction and MS medium for regeneration. During


cultivation of female gametophytes, the principle of empirical selection of nutrient media, vitamins, amino acid, and growth regulators is the predominant approach. Sucrose is usually used at concentration of 23%. For corn ovaries culture, 12% sucrose was initially used (Truong-Andre and Demarly 1984). It was found that 6% sucrose promoted embryo formation and inhibited proliferation of somatic tissues in unfertilized ovary culture of wheat ( Mukhambetzhanov 1992). As the concentration of sucrose increased in summer squash, the percentage of ovules forming embryos decreased, whereas ovules cultured on medium supplemented with 90 g l -1 did not produce embryos. On the other hand, high sucrose concentration in the culture medium has been shown to be beneficial in some plant species such as carnation (Sato et al. 2000) and shallot (Sulistyaningsih et al. 2006). Several amino acids and vitamins stimulate gynogenesis. A positive effect of proline and glycine has been noted in mulberry (Thomas et al. 1999) and cucumber (Ge´mes-Juha´sz et al. 2002). Kantartzi and Roupakias (2009) found MS medium with casein was no better than SH medium without casein in cotton gynogenesis. Auxins are widely used for induction of gynogenesis, and their optimum concentrations have been reported to vary considerably from species to species. Bhagyalakshmi (1999) found that neither NAA (a-naphthaleneacetic acid) nor BA (N 6 - benzyladenine) alone supported caulogenesis in saffron. Specific ratios of NAA and BA supported caulogenesis, where an increase in NAA ( up to 26.9 mM) progressively increased the percentage response, and higher levels of BA enhanced callus formation and abnormal shoots. Thidiazuron (TDZ) is another widely used and active growth regulator in induction and regeneration media for improving gynogenic response. In cucumber, TDZ is successfully used for gynogenic induction and regeneration of embryos (Ge´mes- Juha´sz et al. 2002; Diao et al. 2009). Kielkowska and Adamus (2010) reported that medium supplement with indole-3-acetic acid (IAA) promoted embryo development (0.63%), while supplementation with 2,4-D (2 ,4-Dichlorophenoxyacetic acid) and 6-BA promoted callus development (1.34%) in culture of carrot unfertilized ovule. Addition of polyamine (spermidine and putrescine) in induction and regeneration media resulted in improved gynogenic embryos and haploid plantlets (Martı´nez et al. 2000; Ebrahimi and Zamani 2009). It can be concluded from the above findings that the culture medium composition plays a key role in induction and regeneration in plant gynogenesis.

There is no universal protocol that will result in unfertilized ovary/ovule culture in all species. Thus,


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there are differences among genotypes and species in terms of gynogenic response. The basic steps are listed in Fig. 1, in which a few of factors involving gynogenesis are list and includes the following: growth of donor plant, development stage of ovary/ovule, pretreatment, embryo or callus induction, regenerant embryo, haploid & DH and application in breeding.

The protocol and results of haploid induction through unfertilized ovule/ovary culture are summarized in Table 1.

Application of DH in genetics and plant breeding

The main advantage of doubled haploid lines is their complete homozygosis. This makes phenotypic selection for qualitative and quantitative characters much easier. Thus, DHs can improve the efficiency and speed of the usually cumbersome, time-consuming, and laborious, and sometimes rather inefficient conventional breeding methods. DH systems are widely applied in breeding ( Thomas et al. 2003), genetic mapping (Forster and Thomas 2005) , and mutation studies. Meanwhile, they can provide useful systems for transformation and fixing transformation events

cultivation of female gametophytes, the principle of empirical selection of nutrient media, vitamins, amino acid, and

Fig. 1 Basic steps and factors involved in unfertilized ovary/ovule culture protocol

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Plant Cell Tiss Organ Cult (Shim and Kasha 2003). DHs have many advantages for application in
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(Shim and Kasha 2003). DHs have many advantages for application in basic genetic research, molecular studies, and


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practical applications in plant breeding. In recent years, genetic marker technology together with new statistical methodology has aided the dissection of complex traits into locus-specific components that explain the observed variation, the so-called quantitative trait loci (QTL) (Martı´nez et al. 2002). Most economic traits are controlled by polygenes with small but cumulative effects. Although the potential of DH populations in quantitative genetics has been understood for some time, it was the advent of molecular marker maps that provided the impetus for their use in identifying loci controlling quantitative traits (Kearsey 2002). Since quantitative trait loci (QTL) effects are small and highly influenced by environmental factors, accurate phenotyping with replicated trials is essential. This is possible using the doubled haploid approach because of their true breeding nature and convenient production in large number of plants. Using DH populations, 130 quantitative traits have been mapped in nine crop species (Forster and Thomas 2005). In total, 56 DH populations were used for QTL detection (Maluszynski et al. 2003). Similarly doubled haploid plants are used for QTL mapping of various traits in several plant species, e.g., rice (Li et al. 2005; Ma et al. 2009), wheat (Dashti et al. 2007; Zhang et al. 2008), rapeseed (Zhao et al. 2008), canola ( Samizadeh et al. 2007), japonica rice (Zichao et al. 2005), and barley (Ma et al. 2004). In backcross breeding, a problem arises when the trait of interest is recessive, as it will be present only in a heterozygous condition after each backcross. The development of molecular markers combined with the doubled haploid approach provides an easier short-cut method for selection based on genotype (marker) rather than phenotype. In barley, stripe-rust-resistant lines were developed using marker-assisted backcross conversion with doubled haploid BC1 individuals (Chen et al. 1994). DH populations are commonly used in bulked sergeant analysis of barley and rapeseed, which is a popular method in marker-assisted breeding (Ardiel et al. 2002; William et al. 2002; Yi et al. 1998). A linkage map is a genetic map of a species or experimental population that shows the position of its known genes or genetic markers relative to each other in terms of recombination frequency, rather than as specific physical distance along each chromosome. Genetic map construction is relatively easy using a DH population derived from a hybrid of two homozygous parents, as the expected segregation ratio is simple, i.e., 1:1. DH populations have now been used to produce genetic maps of barley, rapeseed, rice, wheat, and pepper. DH populations played a major role in facilitating the generation of the molecular marker maps in eight crop species (Maluszynski et al. 2003). A small doubled haploid (DH) population was used to demonstrate that a dwarfing gene in barley is located

on chromosome 5H (Thomas et al. 2000). Fine mapping involves the identification of markers that are very tightly linked to a targeted gene, and required recombinant chromosome substitution or recombinant inbred lines ( Paterson et al. 1990). In barley, this was achieved through the doubled haploid method (Thomas et al. 2000). In rice, molecular markers have been found to be linked with major genes and QTLs for resistance to rice blast, bacterial blight, and sheath blight in a map produced from a DH population (Wang et al. 2001). Many commercial cultivars were originated from DH lines. In barley alone, over 100 DH lines have been released as cultivars (Thomas et al. 2003). Coupled with marker-assisted selection, DH technology is a powerful tool to speed up plant breeding.

Future prospective

Remarkable work has been done in the production of haploid and DH lines in various plant species through unfertilized ovule/ovule culture. However, much of the research work is limited to selection of responsive genotypes, explants used, formulation of suitable culture media, pretreatment, growth conditions, and chromosome doubling. In many species, unfertilized ovary/ovule culture is not sufficient to produce DH plants for breeding programs. No or very little work is currently documented regarding the genetic basis of the switch from gametophytic to sporophytic phase during

embryo production in the absence of fertilization. Thus, future research programs aimed at elucidating pathways involved in gynogenesis, such as: Molecular mechanism and Cell Biology. More research is therefore required to improve our understanding of the genes or gene action responsible for embryogenesis and the mechanism of haploid induction in embryo sac. The development of molecular markers for determining the responsive ability of gynogenesis will be used for ovary/ ovule culture. During the last decade, a great deal of attention has been given to include DH lines in plant breeding and genetics studies, i.e., QTL mapping, fine mapping, and BAC library construction for gene cloning, due to their high homozygosity and efficient production of recombinant inbred lines in short time. Due to the low frequency production of haploid and highly genotype dependency make it not very feasible for use in extensive breeding programs and genetic studies. Thus, it is necessary to explore more species and genotypes for gynogenic response and also to increase efficiency through improvement of overall protocol for gynogenesis. Use of gynogenesis for haploid plant production is mostly done in plant species having male sterility or that are unresponsive to androgenesis and microspore culture. The gynogenetic approach may provide valuable information to unravel novel genetic recombinations.



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