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Progress and prospects for interspecific hybridization in buckwheat and the genus Fagopyrum
Biotechnology Advances 31 (2013) 1768–1775
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Research review paper
Progress and prospects for interspecific hybridization in buckwheat and the genus Fagopyrum Nóra Mendler-Drienyovszki ⁎, Andrew J. Cal 1, Judit Dobránszki Research Institute of Nyíregyháza, Research Institutes and Study Farm, Centre for Agricultural and Applied Economic Sciences, University of Debrecen, Nyíregyháza, P.O. Box 12, H-4400, Hungary
a r t i c l e
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Article history: Received 27 May 2013 Received in revised form 9 September 2013 Accepted 11 September 2013 Available online 20 September 2013 Keywords: Embryo rescue Heterostylic incompatibility Flower type Fagopyrum homotropicum Sporophytic incompatibility
a b s t r a c t Cultivated buckwheat, such as common (Fagopyrum esculentum Moench.) and tartary (Fagopyrum tataricum (L.) Gaertn.) buckwheat, is one of the most versatile crops for forage and food and has several benefits for human health. Interspecific hybridization between Fagopyrum species is of great importance to improvement of buckwheat. Hybridization would allow the transfer of agronomical beneficial characteristics from wild Fagopyrum species, including self-pollination and increased fertility, frost tolerance, and higher content of beneficial compounds. However, conventional breeding methods are only partially applicable because of the selfincompatibility and incompatibility barriers between different species. Present review summarizes the morphology of self-incompatibility, the genetic and cellular basis of incompatibility between different Fagopyrum species. In many interspecific crosses hybrid embryos are aborted after successful pollination due to post-zygotic incompatibility. The use of in vitro embryo rescue after interspecific hybridization has been successful in circumventing breeding barriers between Fagopyrum species. Methods applied successfully for the construction of interspecific hybrids are discussed in detail. © 2013 Elsevier Inc. All rights reserved.
Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . 2. Genetics of incompatibility . . . . . . . . . . . . . . . . . . 3. Interspecific hybridization and its benefits for cultivated buckwheat 4. Methods for overcoming the species barrier . . . . . . . . . . . 5. Future prospects of buckwheat breeding . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction Buckwheat (Fagopyrum species) has been a popular grain and forage crop for centuries, with a traditional range of cultivation stretching from eastern Europe to Japan and with the origin of domestication thought to be eastern Tibetan plateau, bordering the Chinese province of Yunnan (Gondola and Papp, 2010; Ohnishi, 1998a,b). A member of the Polygonaceae family, buckwheat falls outside the Poaceae and is technically a pseudo-cereal. Accordingly, buckwheat is free from gluten, a common human allergen found in wheat, barley, and rye products (Hoffman, 1975; Rewers, 2004). Buckwheat grain is high in nutrients, with protein content between ~20 and 25%. The major storage protein in buckwheat seeds is the 13S globulin (Aubrecht and Biacs, 1999; ⁎ Corresponding author. E-mail address: [email protected] (N. Mendler-Drienyovszki). 1 Present address: 2001 Fleet St Baltimore, MD 21231, USA. 0734-9750/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.biotechadv.2013.09.004
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Christa and Soral-Śmietana, 2008; Li and Zhang, 2001; Sangma and Chrungoo, 2010); the seeds also contain 8S vicilin as a minor storage protein (Radović et al., 1999). Buckwheat seeds are a source of rutin, B1 (thiamine), B2 (riboflavin), and B6 vitamins (pyridoxine) (Bonafaccia et al., 2003; Fabjan et al., 2003); they contain lysine, methionine, threonine, arginine (Christa and Soral-Śmietana, 2008), important microelements such as Zn, Cu, Mn, and Se (Stibilj et al., 2004), and macroelements such as K, Na, Ca, and Mg (Christa and Soral-Śmietana, 2008; Ikeda, 2002; Wei et al., 2003). The starch of buckwheat includes numerous compounds that have been identified to have beneficial effects on human health, including lowering both blood lipid and sugar levels (He et al., 1995; Kawa et al., 2003; Obendorf et al., 1993). Buckwheat belongs to the genus Fagopyrum (family Polygonaceae), which consists of two mono-phyletic groups: the cymosum and urophyllum (Sangma and Chrungoo, 2010). Both cultivated species, Fagopyrum esculentum Moench. (common buckwheat) and Fagopyrum tataricum (L.) Gaertn. (tartary buckwheat), cluster in the cymosum
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clade (Fig. 1). However, F. esculentum is more closely related to Fagopyrum homotropicum Ohnishi, while F. tataricum clusters with Fagopyrum cymosum, indicating that the modern crops were domesticated separately from two closely related species (Ohnishi and Matsuoka, 1996; Yasui and Ohnishi, 1998). While both cultivated species F. esculentum and F. tataricum are diploid, their progenitor species, F. homotropicum and F. cymosum, exist as both diploids and tetraploids (Campbell, 1976; Ohsako, 2000). Tetraploid F. homotropicum is the result of hybridization between F. homotropicum (diploid) × F. esculentum ssp. ancestrale (Tomiyoshi et al., 2012). The origins of the two cultivated Fagopyrum species and their wild relatives that are targets for hybridization are shown in Table 1. The prominence of buckwheat as a grain crop has fallen over the last century as cereals have risen to prominence. Relative to major cereals, buckwheat yield is unstable and is often plagued by low seed set (Woo et al., 2004), which can be attributed to both environmental complications such as high temperature and water stress, and intrinsic biological factors, e.g. self-incompatibility caused by heterostylism, defective reproductive organs, failure of fertilization, and embryo abortion (Adachi, 1990; Guan and Adachi, 1992; Hirose et al., 1994; Kreft, 1983; Marshall, 1969; Marshall and Pomeranz, 1982; Obendorf and Slawinska, 1993; Woo et al., 1995, 2010). Under optimal environmental conditions and agrotechnical factors (sowing date, row-width, fertilizers, etc.), buckwheat yield is highly dependent on the action of pollinators, specifically bees (Ruszowszki, 1980). Common buckwheat (F. esculentum Moench.) has been cultivated in all parts of the world because it survives and grows also under adverse environmental conditions (Niroula et al., 2006), matures rapidly, and is susceptible to few diseases (Yasui et al., 2004). It has short growing season (Asaduzzaman et al., 2009), the growing intensity is vigorous and indeterminate. The common buckwheat has been used for honey production and useful as a green manure crop in the low-productivity land (Campbell, 1997). It has desirable flavor and milling quality (Cheng and Ni, 1992; Marshall and Pomeranz, 1982). The reliance on pollinators is due to self-incompatibility in F. esculentum. The lack of compatibility is sporophytic, partly due to floral structure. Buckwheat
I. ’cymosum’ group
Fagopyrum esculentum Moench. Fagopyrum homotropicum Ohnishi Fagopyrum tataricum L. Fagopyrum giganteum Krot. Fagopyrum cymosum L. II. ’urophylleum’ group Fagopyrum macrocarpum Ohsako&Ohnishi Fagopyrum pleioramosum Ohnishi
Fagopyrum capillatum Ohnishi Fagopyrum gracilipes (Hemsl.) Dammer Fagopyrum gilessii Hemsl. Fig. 1. The suspected relationships of 10 Fagopyrum species including the cultivated species based on Sharma and Jana (2002), Rout and Chrungoo (2007), Gondola and Papp (2010) and Sangma and Chrungoo (2010).
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flowers are dimorphic; ‘pin’ type flowers have long pistols and short stamens, while ‘thrum’ type flowers have short pistols and long stamens (Adachi et al., 1983; Cawoy et al., 2009; Kreft, 1986; Nagatomo, 1961) (Fig. 2). Individuals bear only one flower type and, in addition to structural barriers, biochemical barriers are believed to exist as self pollen often fails to germinate and is unable to grow functional pollen tubes (Adachi, 1990; Nettancourt, 1977). Successful fertilization can arise only between plants with different floral structures.
2. Genetics of incompatibility Flower type ratio segregates consistent with single locus inheritance and ‘thrum’ flowers are dominant to ‘pin’ (Garber and Quisenberry, 1927a). Although floral architecture is linked with incompatibility type, incompatibility is mainly due to early pollen tube abortion, not floral structure (Nettancourt, 1977), and is inherited sporophytically (Garber and Quisenberry, 1927b; Lewis and Jones, 1992). It is unknown how many gene products comprise the self-compatibility locus (S-locus). Recently, Yasui et al. (2012) identified two transcripts that are linked to the S-locus and expressed exclusively in the style of thrum flowers. Through homology analysis of 610 kb of genomic DNA surrounding these transcripts they identified only one other gene, which is expressed in both thrum and pin styles. It is possible that several gene fragments in the region produce functional transcripts. The transfer of the self-pollinating mechanism to the cross-pollinated common buckwheat would be of particular importance, since this would reduce the dependence on insect pollination (Garber and Quisenberry, 1927b; Kovalenko and Laptev, 1979; Kovalenko and Shumny, 2004; Woo et al., 2006) and allow for increased rate of seed set which might result in higher yield. The discovery of a wild, self-compatible species with homomorphic flowers, F. homotropicum, by Ohmi Ohnishi (1995) in South-western China has provided a new germplasm source for buckwheat breeders. F. homotropicum has 19 accessions which have been characterized morphologically (Ohnishi and Asano, 1999). Campbell (1995) and Woo et al. (1999) made crosses between populations of F. esculentum and F. homotropicum and found that the homomorph, self-compatible style was controlled by a single locus (Sh). They determined the dominance relations of the between the three haplotypes determining flower structure as follows: S N Sh N s. Therefore the flower type of F1 population from crosses between F. esculentum ‘pin’ style (ss) and F. homotropicum (ShSh) is homomorph (sSh). Genetic incompatibility was determined to be sporophytic by Dahlgren (1922). According to Wang et al. (2005a) the homomorph flower type and the self-compatibility can be controlled by more complex genetic interactions, and they posited a 2-gene model in F. homotropicum species and in its progenies. They found that the use of self-incompatible F. esculentum ‘pin’ type in interspecific hybridization with homostyly type is more desirable for plant breeding than ‘thrum’ type (Ss), as the ‘thrum’ plants carry both ‘pin’ and self-compatible alleles which makes the selection of progenies of homostyly from ‘pin’ difficult. Recently, much progress has been made to identify the molecular basis of heterostylistic incompatibility of buckwheat. Ohnishi and Ohata (1987) produced the first genetic map in buckwheat with the use of morphological and isozyme markers. Maps based on DNAbased polymorphisms have also been constructed, including an AFLP map based on a segregation of an interspecific F. esculentum × F. homotropicum cross (Yasui et al., 2004) that was used to identify markers linked to the self-incompatibility locus and the locus responsible for brittle pedicels in wild buckwheat (Matsui et al., 2004). Additionally, a genetic map based on 87 RAPD and STS markers has been deployed to map variants in four seed storage protein subunits in a cross between common and wild buckwheat (Pan and Chen, 2010). Finally, an intra-specific map based on a cross between two varieties of F. esculentum has been created using microsatellite and AFLP markers, and
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Table 1 Chromosome numbers, desirable and undesirable traits, origin and progenitors, and phylogenic relationships of Fagopyrum esculentum, Fagopyrum tataricum and Fagopyrum homotropicum (diploid) might be introgressed by interspecific hybridization to cultivated buckwheat, based on Campbell (1997), Woo et al. (2001), Rout and Chrungoo (2007) and Gondola and Papp (2010). Species
Chromosome Desirable traits number
F. esculentum — cultivated in the 2n = 16 temperate Europe, Japan (Indo-Myanmar)
Resistance to seed shattering, good flour quality, easy dehulling
F. tataricum — cultivated in the Himalaya
2n = 16
F. homotropicum
2n = 16
High seed set, high rutin content of seeds, self-pollination, frost tolerance, vigorous plant, resistance to seed shattering Self-pollination, high seed set, frost tolerance
several of these markers also work in F. tataricum (Konishi and Ohnishi, 2006). Although there is no current genome project in Fagopyrum, several studies have taken advantage of genomic techniques to character the molecular basis of buckwheat variation. Yasui et al. (2008) constructed a BAC library of 142,000 large insert clones, representing an estimated 7–8-fold coverage of the F. esculentum genome. They used previously identified markers to the S locus to identify linked BAC clones and create an STS marker for the dwarf locus. Hara et al. (2011) sequenced cDNA clones to conduct a homology-based search to locate candidate genes co-segregating with QTLs controlling photoperiod sensitivity, and next
A
Undesirable Origin and progenitors traits
Putative ancestors of cultivated Fagopyrum species
Wild relatives
Crosspollination, yield instability
Western and Central Himalaya, East Himalaya (one accession)
F. cymosum (wild ancestor: F. homotropicum)
F. homotropicum F. cymosum
Western and Central Himalaya, Japan (one accession)
F. cymosum
F. giganteum F. cymosum
Seed shattering
Eastern Tibet and Yunnan and Sichuan Provinces of China
F. cymosum
generation sequencing has been used to study floral transcript diversity between F. esculentum and F. tataricum (Logacheva et al., 2011). In an impressive convergence of new sequencing technology and a decade's accumulation of genetic resources in buckwheat, Yasui et al. (2012) identified a novel gene that may control short-styled floral morphology. They sequenced the styler transcriptome from genetically similar longand short-styled sibs, identifying four transcripts exclusively expressed in short styles. Using a long-styler mutant found on a chimeric shortstyler plant, they were able to identify the S-ELF3 transcript as deleted in the long-styler chimeric branch. The exclusivity of S-ELF3 expression to short-styler plants was consistent in self-incompatible accessions of
B
C
anther stigma
stamen
style
Fig. 2. Heteromorph and homomorph flower types of Fagopyrum. Structure of ‘pin’ type flowers (A) with long styles and short stamens; structure of ‘thrum’ type flowers (B) with short styles and long stamens. Homomorph flower structure of F1 hybrids between F. esculentum × F. homotropicum with the same length of styles and stamens (C).
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F. cymosum and the distant Fagopyrum urophyllum. As sequencing costs continue to plummet, an acceleration of molecular research in buckwheat through the utilization of genomic resources appears to be on the horizon. 3. Interspecific hybridization and its benefits for cultivated buckwheat Interspecific hybridization in the genus Fagopyrum goes back to a long time. The aim of these crossings was the transfer of favorable properties from the wild species to the cultivated ones, sometimes from one cultivated species to another one. In the course of the latter variation, the good nutritional traits of common buckwheat have been attempted to transfer to tartary buckwheat. Self-pollination, higher fertility rates, frost tolerance, vigor, and high rutin content are desirable traits of tartary buckwheat which breeders have wanted to transfer to common buckwheat (Woo et al., 1999). While interspecific hybridization with common buckwheat's progenitor species, F. homotropicum, can be a useful technique for transferring traits such as self-compatibility and cold tolerance, it can also re-introduce undesirable characteristics, such as seed shattering, which have been eliminated through the domestication process. Campbell (1995) and Woo and Adachi (1994, 1997) were the first ones who reported successful interspecific hybridization between F. homotropicum and F. esculentum at diploid level, in which the progenies were fertile. Inbreeding depression could be detected in the homostylic inbred lines of F. esculentum (Mukasa et al., 2010), but inbreeding depression has not been observed in selfcompatible buckwheat breeding lines created through interspecific hybridization (Matsui et al., 2003). After the transfer of desirable traits from F. homotropicum into the common buckwheat, such as self-pollination and frost resistance (Table 1), backcrosses must be made to F. esculentum in order to eliminate seed shattering and other undesirable traits (Wang et al., 2005b; Woo et al., 2001). Interspecific hybridization can also be applied to improve quality (bitterness) and agronomic characteristics (ease of dehulling) of buckwheat seeds (Campbell, 1995). Campbell (1995) carried out further back-crosses to F. esculentum to transfer the desirable traits (self-pollinating system, frost tolerance) of F. homotropicum to F. esculentum. Woo et al. (1999) reported successful hybridization between F. homotropicum, F. esculentum, and F tataricum. They conclude that the ‘thrum’ plants of F. esculentum could be successfully used to increase the production efficiency of the hybrids. After interspecific crossings between F. homotropicum lines, selfincompatible F. esculentum ‘pin’ and self-pollinating homostylous breeding lines more than 50% of F2 hybrids had lower rutin content but two F2 hybrids had 9.38-fold and 34.92-fold higher rutin content compared to parents (Wang et al., 2004). This result indicated the possibility of selection for high rutin content using recurrent selection on each generation provided that the population size was high enough. Wang et al. (2005c) reported the first successful interspecific hybridization between Fagopyrum species with different ploidy levels. Tetraploid F. homotropicum and diploid F. esculentum ‘pin’ plants were used for crossings. However, for culturing of hybrid embryos in vitro embryo rescue was necessary. All the flower types of F1 hybrid plants were homostylic and the morphology of these plants was similar to F. homotropicum, including the seed shattering habit. The F1 hybrids were triploid and had 24 chromosomes according to their cytogenetic analysis. Fertility and morphology of F2 generation differed depending on ploidy. F2 hybrids were diploid, aneuploidy and hexaploid. Diploid plants with homostylic flower type and hexaploids with ‘pin’ flower type were self-fertile while diploids with ‘pin’ type flower, aneuploids and hexaploids with homostylic flower type were partially fertile (Wang et al., 2005c). Seed shattering is the main undesirable trait in the wild buckwheat relative that impedes use of interspecific hybrids; abscission of the pedicel in F. homotropicum causes the seeds to drop to the ground, rendering them unharvestable. In all studies examining shattering in
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interspecific hybrids between common buckwheat and F. homotropicum, shattering exhibited dominance in the F1 (Campbell, 1995; Wang and Campbell, 1998; Wang et al., 2005b). In some crosses, genetic linkage is observed between flower type and shattering habit, though the linkage is not perfect and segregants have been identified in large populations (Wang et al., 2005b). Based on interspecific crosses between diploid F. homotropicum and F. esculentum, Wang et al. (2005b) found evidence for multiple segregation ratios for the shattering character in hybrids with various segregation ratios, for which they proposed a 3-gene-model with recessive epistasis, where variation in the shattering genes in different accessions of common buckwheat used was responsible for the multiplicity of segregation ratios. They found three types of segregation in F2 population (3:1, 9:7, 27:37) and four types of segregation in F3 population (1:0, 3:1, 9:7, 27:37) regarding shattering to nonshattering. According to Wang et al. (2005b) there are genetic correlations between seed shattering and self-compatible homomorphic flower type and between non-shattering and self-incompatible ‘pin’ flowers (Fesenko et al., 1998). This linkage can be overcome by use of a large segregating population (Wang et al., 2005b). Chen et al. (2004) studied the big-achene group of Fagopyrum, which contains the F. esculentum subsp. ancestrale Ohnishi, F. tataricum, F. homotropicum and other four Fagopyrum species. They reported that there are close relationships between these species in the big achene group (Chen, 1999) and F. homotropicum is similar in morphology to the F. esculentum, so it can be crossed with each other easily. Contrary to previous studies, they found that crossability was high between both F. esculentum and F. homotropicum (28%), and between F. esculentum and F. tataricum (37%). According to the descendant it was concluded that achene dormancy of F. homotropicum was controlled by the sporophyte genotypes. There have also been successful attempts to hybride common buckwheat to diploid accessions of its progenitor, F. homotropicum (Campbell, 1995; Woo and Adachi, 1994), and the use of thrum-type flowers of F. esculentum was found to increase compatibility in these hybridizations (Woo et al., 1999). 4. Methods for overcoming the species barrier Although the molecular mechanism of incompatibility remain unresolved, chemical treatments have been found which can overcome it, such as actinomycin, a RNA synthesis inhibitor, and cycloheximide, a protein synthesis inhibitor (Asher and Drewlow, 1970; Ferrari and Wallace, 1976a, 1976b; Franklin et al., 1992; MiljušĐukić et al., 2010; Sarker et al., 1988). Phosphatase inhibitors, such as ocadaic acid and cantharidin, have also been successful circumventing the self-incompatibility of buckwheat; when applied on isolated pistils, self-pollen tube inhibition is relieved (Miljuš-Đukić et al., 2003). Another group of inhibitors, calcium antagonists (verapamil, jonophore) were successfully used for cessation of self-incompatibility in buckwheat, implicating Ca2+ signaling in the SI response of buckwheat (Franklin-Tong et al., 1997; Miljuš-Đukić et al., 2003). Conventional breeding methods have not been successful in producing interspecific hybridization of buckwheat species (Ruszowszki, 1980; Samimy et al., 1996; Woo et al., 2006). In many cases the hybrid embryo aborted due to incompatibility (Woo et al., 2006). Based on the observations of Guan and Adachi (1992) there are two types of sterility in the abnormal embryo sac. One type is the lack of fertilization, i.e. the pollen tube to germinate (Ruszowszki, 1980) and failure of the pollen tube to target and penetrate the micropyle and ovary (Samimy, 1991; Woo et al., 1999). The second type, abortive sterility, is due to the degradation of the endosperm after fertilization (Shaikh et al., 2002a). Postfertilization barriers, such as abortive sterility, can be overcome through the use of tissue culture methods, such as embryo rescue (Table 2). At high summer temperature Woo et al. (2010) observed abnormal embryo sac 3 days after pollination, which led to incomplete development, failure of fertilization, or abortion of the embryo. They observed
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that the occurrence of abnormality of either type was higher in tetraploids (90–100%) than in diploids (50–77%). According to Guan and Adachi (1992) the stress of high temperature indicates ultrastructural changes of organelles, abortion of embryo sac and endosperm. Initial attempts to cross common and tartary buckwheat with conventional breeding were unsuccessful. The barriers to hybridization of these species appear to be post-zygotic. Though pollen from 10 species within Fagopyrum all had germination and initial pollen tube growth on F. esculentum styles, and F. esculentum pollen was able to penetrate the micropyle of F. tataricum (Hirose et al., 1994). According to Woo et al. (2008) there are correlation between pistils and pollens and the growth of pollen tube is different during the interspecific hybridization making a barrier for these crosses. Five categories were determined in accordance with the degree of compatibility (from high compatibility to incompatibility). In the first, highly compatible category, the pollen tube elongation and its penetration into the style occur 6–24 h after pollination; in the second category, slightly compatible, pollen tube elongation and penetration are delayed for 1–6 h. The final three categories are comprised by three different types of incompatibility: inhibition of pollen tube at the stigma (type I), inhibition of pollen tube at the style (type II), and inhibition of pollen tube at the stylodium (type III). Microscopic examination of hybrid F. esculentum × F. tataricum embryos found numerous cytological defects, including synergid degradation, organellar aggregation and deformation, and defective embryo sac development (Shaikh et al., 2002b). The use of embryo culture from a cross of common buckwheat pollen on tartary buckwheat styles was able to overcome the post-zygotic barrier, but the resulting hybrids were sterile (Samimy et al., 1996). Alternate methods of embryo rescue eventually succeeded in developing a fertile hybrid between these species (Wagatsuma and Un-no, 1995). After embryo rescue from interspecific crosses between F. esculentum and F. tataricum the ovules were observed with light and transmission electron microscopy (Shaikh et al., 2002b). The embryos were rescued at 1 to 5 days after pollination. According to their observation several abnormalities can be detected at 1 day after pollination. The cytoplasm of synergids were degenerated and showed ultrastructural abnormalities.
Several cell organelles were aggregated. The mitochondria were irregularly shaped, and the mitochondrial cristae disappeared. The active cytoplasm of both egg and central cells indicated the normal function of hybrid embryo sac for fertilization. If the fertilization was unsuccessful – like in Shaikh et al. (2002b) studies – the pollen tube may not have entered into the synergid. This results in cross incompatibility between F. esculentum and F. tataricum. They also reported non-zygotic development in the cross combinations between F. esculentum and F. tataricum at 5 days post-pollination. In this case the synergids deformed and degenerated, the endosperm showed abnormal phenomenon, and ribosome density in the cytoplasm of endosperm decreased. The changes in ribosome density caused lowered protein synthesis. The fusion of nuclear was completed but cell division was halted, and the zygote did not develop further. The third studied barrier of the interspecific hybridization was the shrunken or small embryo sac in the hybrid ovules. Two breeding lines of F. homotropicum and three lines of F. esculentum were used for crossings by Wang et al. (2005c), and all F1 hybrids plant obtained by using in vitro embryo rescue method were self-compatible with homostylic flowers, demonstrating the dominance of self-compatibility. Evaluation of F2 and F3 population indicated that two genes with complementary interaction were controlling the self-compatibility. Woo et al. (2008) determined the abnormalities of different hybrid embryos and classified them in five categories: undivided zygote, degenerated and collapsed endosperm, degenerated embryo and endosperm, collapsed embryo and endosperm, floating embryo without suspensor. The introduction of in vitro embryo rescue (Table 2, Fig. 3) in the 1990s has been tremendously successful in overcoming the barrier of embryo abortion that made the production of Fagopyrum interspecific hybrids impossible with conventional breeding techniques (Ruszowszki, 1980; Samimy et al., 1996; Woo et al., 2006). In the embryo rescue technique, hybridized and enlarged ovules are removed from the pistol after pollination, however, the time between hybridization and ovary excision has varied considerably among studies. Woo et al. (2006) removed ovaries 3–5 days post pollination while several studies (Asaduzzaman et al., 2009; Niroula et al., 2006; Wang and Campbell, 1998) waited 7–10 days
Table 2 Successful interspecific hybridization of Fagopyrum species using embryo rescue. Ploidy level
No. of crosses
No. of ovules rescue
No. of embryos emerged
Hybrid recovery (%)
Fertility of progenies (%) F1
F. esculentum × F. tataricum Diploid 88 Diploid 104 Diploid 275 Tetraploid 344
No data No data 68.8 ± 1.07 65.3 ± 1.76
References
F2
3 2 156 178
0 0 16 28
0 0 3.8 6.1
111 2 16 9
13 2 5 6
3 50 2.7 4.1
No data No data No data No data
Wang and Campbell (1998) Wang and Campbell (1998) Niroula et al. (2006) Niroula et al. (2006)
22 16 19 40 24.8 21.1 19
No data No data No data No data No data No data No data
Wang and Campbell (1998) Wang and Campbell (1998) Wang and Campbell (1998) Wang and Campbell (1998) Wang et al. (2002) Wang et al. (2002) Wang et al. (2005c)
4 29 40
No data No data No data
Wang and Campbell (1998) Wang and Campbell (1998) Wang et al. (2005c)
17.9 17.1 3
No data No data No data
Wang et al. (2002) Wang et al. (2002) Asaduzzaman et al. (2009)
51.6 ± 18.77 54.4 ± 17.63
Niroula et al. (2006) Niroula et al. (2006) Asaduzzaman et al. (2009) Asaduzzaman et al. (2009)
F. tataricum × F. esculentum
Diploid Diploid
150 98
F. esculentum × F. homotropicum
Diploid Diploid × tetraploid Diploid × tetraploid
1115
71 90 26 10 145 194 194
5 4
F. homotropicum × F. esculentum
Tetraploid × diploid F. tataricum × F. homotropicum Diploid 79 Tetraploid 41 Diploid 100
10 7 10
4 2 4
28 7 12
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A
B
C
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The desirable traits of buckwheat were transferred by using in vitro ovule culture method (Asaduzzaman et al., 2009). The effect of growth regulators was described using direct and indirect regeneration protocols, but they did not discuss these two types of regeneration further. The crossings were made between F. tataricum and F. esculentum, and after fertilization the embryos were rescued and put for direct regeneration onto half-strength MS and whole MS media (Murashige and Skoog, 1962) with different plant growth regulators. Two types of hybrid plant regeneration methods were used. One is the direct method, when the ovules were cultured on a medium containing NAA, IAA, BA and zeatin. The hybrids were tested in different ploidy level (diploid, tetraploid) by Asaduzzaman et al. (2009) and they conclude that the plant growth regulator content of the medium is an important factor for the direct regeneration of hybrid buckwheat plants (Table 4). The second method is the indirect hybrid plant regeneration method wherein the embryos were rescued from the buckwheat (diploid or tetraploid) hybrids for callus induction prior to placing them onto plant regeneration medium (Table 4).
5. Future prospects of buckwheat breeding
D
Fig. 3. Production of fertile F1 hybrids between F. esculentum × F. homotropicum using embryo rescue method. Enlarged ovule removed 8 days after pollination and placed onto medium (A). In vitro developing 2-week-old hybrid embryo (B). In vitro culture of regenerated hybrid plantlet (C). Acclimated hybrid plants in pots (D).
As a result of embryo and ovary rescue interspecific hybrids of Fagopyrum species have been produced successfully for some time; however, several back crosses are necessary to eliminate disadvantageous wild traits before these hybrids can be utilized for agriculture. Pollination rate and hereby the yield stability in cultivated varieties could be improved by transfer of self-compatibility from F. homotropicum because the insect-dependence decreases in a self-pollinated buckwheat variety. Further advancement can be expected at a greater pace as molecular markers and other biotechnologies are applied in buckwheat breeding. Systematic characterization of the buckwheat genome and DNA polymorphism in the germplasm of domesticated and wild Fagopyrum species will allow for the identification of agronomically valuable traits, such as frost tolerance, resistance to shattering, and high rutin content, and determining phylogenic relationships within the genus. Genome mapping and marker assisted selection (MAS) are efficient tools in crop improvement and buckwheat breeding would be buttressed by the identification of molecular variations in Fagopyrum species.
Table 3 Media used for embryo rescue. Medium composition
before the removal, and Campbell (1995) did not remove the ovaries until 18–20 days post-pollination. After excision, ovaries are surfaced sterilized first with 70% ethanol for 30 s, the with 2% sodium hypochloride for time minutes. Following several rinses of distilled water, the ovaries are plated in test tubes. The embryo culture is maintained at 22 ± 2 °C in 16 h of light and 8 h dark period (Asaduzzaman et al., 2009). Fully developed plantlets are removed from the test tubes, washed with distilled water, and transferred to sterile vermiculite. After two weeks, plantlets were transplanted into pots and grown in a greenhouse. Wang and Campbell (1999) compared four different methods of buckwheat embryo culture: test tube with solid medium (one embryo on 10 ml medium gelled by 0.7% agar), Petri dish with tissue paper soaked by media (10 ml liquid medium in the Petri dish with 2 layers of tissue paper), beaker with 10 ml liquid media, and beaker with 10 ml tap water. Interspecific hybrids between F. esculentum and F. homotropicum were used for all treatments. They found the Petri dish method, first reported in their paper, to be the simplest and most rapid of methods tested. The media used for embryo rescue are summarized in Table 3.
F. esculentum × F. homotropicum 1/2 MS 1/2 MS + 10 mg l−1 L-tyrozine + 10 mg l−1 L-arginine + 2 mg l−1 zeatin MS + 2 mg l−1 zeatin + 10 mg l−1 L-tyrozine + 10 mg l−1 L-arginine + 3% sucrose F. esculentum × F. tataricum MS + 2 mg l−1 zeatin 1/2 MS MS + BA + NAA + IAA + zeatin + 3% sucrose MS + 3% sucrose + 0.2 mg l−1 IAA + 2 mg l−1 BA MS basal salt mixture (1/2 NH4NO3 + 1/2 KNO3) + MS vitamins + 5% sucrose + 2 mg l−1 casein hydrolysate + 0.2 mg l−1 IAA + 0.5 mg l−1 GA3 + 1.0 mg l −1 zeatin F. tataricum × F. homotropicum 1/2 MS + 10 mg l−1 L-tyrozine + 10 mg l−1 L-arginine + 2 mg l−1 zeatin
References Wang and Campbell (1998) Wang et al. (2005c)
Wang and Campbell (1998) Asaduzzaman et al. (2009) Niroula et al. (2006) Samimy et al. (1996)
Wang and Campbell (1998)
MS: Murashige and Skoog (1962), BA: benzyladenine, IAA: indole acetic acid, NAA: naftil acetic acid.
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Table 4 Type of regeneration methods applied for embryo rescue. Type of the method
Medium CIa
PRa MS + zeatin 2 mg l−1 + 3% sucrose
Direct
−1
Indirect
a
1/2 MS + BA 1 mg l−1 + NAA 0.2 mg l−1 + 3% sucrose 1/2 MS + BA 1 mg l−1 + NAA 0.2 mg l−1 + 3% sucrose
1/2 MS + BA 1 mg l + NAA 0.1 mg l−1 + 3% sucrose MS + 3% sucrose + 0.2 mg l−1 IAA + 2 mgL−1 BA MS + zeatin 2 mg l−1 + IAA 0.2 mg l −1 + 3% sucrose MS + zeatin 2 mg l−1 + IAA 0.2 mg l −1 + 3% sucrose
Ploidy level of parents
Embryos emerged (%) [no. of hybrids]
No. of shoots per callus
References
Diploid
25 [3]
–
Tetraploid
40 [5]
–
40
–
Diploid
[2]
4.5
Tetraploid
[2]
4.5
Asaduzzaman et al. (2009) Asaduzzaman et al. (2009) Niroula et al. (2006) Asaduzzaman et al. (2009) Asaduzzaman et al. (2009)
PR = plant regeneration, CI = callus induction, 1/2 MS: half-strength Murashige and Skoog medium (1962).
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Research review paper
Progress and prospects for interspecific hybridization in buckwheat and the genus Fagopyrum Nóra Mendler-Drienyovszki ⁎, Andrew J. Cal 1, Judit Dobránszki Research Institute of Nyíregyháza, Research Institutes and Study Farm, Centre for Agricultural and Applied Economic Sciences, University of Debrecen, Nyíregyháza, P.O. Box 12, H-4400, Hungary
a r t i c l e
i n f o
Article history: Received 27 May 2013 Received in revised form 9 September 2013 Accepted 11 September 2013 Available online 20 September 2013 Keywords: Embryo rescue Heterostylic incompatibility Flower type Fagopyrum homotropicum Sporophytic incompatibility
a b s t r a c t Cultivated buckwheat, such as common (Fagopyrum esculentum Moench.) and tartary (Fagopyrum tataricum (L.) Gaertn.) buckwheat, is one of the most versatile crops for forage and food and has several benefits for human health. Interspecific hybridization between Fagopyrum species is of great importance to improvement of buckwheat. Hybridization would allow the transfer of agronomical beneficial characteristics from wild Fagopyrum species, including self-pollination and increased fertility, frost tolerance, and higher content of beneficial compounds. However, conventional breeding methods are only partially applicable because of the selfincompatibility and incompatibility barriers between different species. Present review summarizes the morphology of self-incompatibility, the genetic and cellular basis of incompatibility between different Fagopyrum species. In many interspecific crosses hybrid embryos are aborted after successful pollination due to post-zygotic incompatibility. The use of in vitro embryo rescue after interspecific hybridization has been successful in circumventing breeding barriers between Fagopyrum species. Methods applied successfully for the construction of interspecific hybrids are discussed in detail. © 2013 Elsevier Inc. All rights reserved.
Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . 2. Genetics of incompatibility . . . . . . . . . . . . . . . . . . 3. Interspecific hybridization and its benefits for cultivated buckwheat 4. Methods for overcoming the species barrier . . . . . . . . . . . 5. Future prospects of buckwheat breeding . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction Buckwheat (Fagopyrum species) has been a popular grain and forage crop for centuries, with a traditional range of cultivation stretching from eastern Europe to Japan and with the origin of domestication thought to be eastern Tibetan plateau, bordering the Chinese province of Yunnan (Gondola and Papp, 2010; Ohnishi, 1998a,b). A member of the Polygonaceae family, buckwheat falls outside the Poaceae and is technically a pseudo-cereal. Accordingly, buckwheat is free from gluten, a common human allergen found in wheat, barley, and rye products (Hoffman, 1975; Rewers, 2004). Buckwheat grain is high in nutrients, with protein content between ~20 and 25%. The major storage protein in buckwheat seeds is the 13S globulin (Aubrecht and Biacs, 1999; ⁎ Corresponding author. E-mail address: [email protected] (N. Mendler-Drienyovszki). 1 Present address: 2001 Fleet St Baltimore, MD 21231, USA. 0734-9750/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.biotechadv.2013.09.004
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Christa and Soral-Śmietana, 2008; Li and Zhang, 2001; Sangma and Chrungoo, 2010); the seeds also contain 8S vicilin as a minor storage protein (Radović et al., 1999). Buckwheat seeds are a source of rutin, B1 (thiamine), B2 (riboflavin), and B6 vitamins (pyridoxine) (Bonafaccia et al., 2003; Fabjan et al., 2003); they contain lysine, methionine, threonine, arginine (Christa and Soral-Śmietana, 2008), important microelements such as Zn, Cu, Mn, and Se (Stibilj et al., 2004), and macroelements such as K, Na, Ca, and Mg (Christa and Soral-Śmietana, 2008; Ikeda, 2002; Wei et al., 2003). The starch of buckwheat includes numerous compounds that have been identified to have beneficial effects on human health, including lowering both blood lipid and sugar levels (He et al., 1995; Kawa et al., 2003; Obendorf et al., 1993). Buckwheat belongs to the genus Fagopyrum (family Polygonaceae), which consists of two mono-phyletic groups: the cymosum and urophyllum (Sangma and Chrungoo, 2010). Both cultivated species, Fagopyrum esculentum Moench. (common buckwheat) and Fagopyrum tataricum (L.) Gaertn. (tartary buckwheat), cluster in the cymosum
N. Mendler-Drienyovszki et al. / Biotechnology Advances 31 (2013) 1768–1775
clade (Fig. 1). However, F. esculentum is more closely related to Fagopyrum homotropicum Ohnishi, while F. tataricum clusters with Fagopyrum cymosum, indicating that the modern crops were domesticated separately from two closely related species (Ohnishi and Matsuoka, 1996; Yasui and Ohnishi, 1998). While both cultivated species F. esculentum and F. tataricum are diploid, their progenitor species, F. homotropicum and F. cymosum, exist as both diploids and tetraploids (Campbell, 1976; Ohsako, 2000). Tetraploid F. homotropicum is the result of hybridization between F. homotropicum (diploid) × F. esculentum ssp. ancestrale (Tomiyoshi et al., 2012). The origins of the two cultivated Fagopyrum species and their wild relatives that are targets for hybridization are shown in Table 1. The prominence of buckwheat as a grain crop has fallen over the last century as cereals have risen to prominence. Relative to major cereals, buckwheat yield is unstable and is often plagued by low seed set (Woo et al., 2004), which can be attributed to both environmental complications such as high temperature and water stress, and intrinsic biological factors, e.g. self-incompatibility caused by heterostylism, defective reproductive organs, failure of fertilization, and embryo abortion (Adachi, 1990; Guan and Adachi, 1992; Hirose et al., 1994; Kreft, 1983; Marshall, 1969; Marshall and Pomeranz, 1982; Obendorf and Slawinska, 1993; Woo et al., 1995, 2010). Under optimal environmental conditions and agrotechnical factors (sowing date, row-width, fertilizers, etc.), buckwheat yield is highly dependent on the action of pollinators, specifically bees (Ruszowszki, 1980). Common buckwheat (F. esculentum Moench.) has been cultivated in all parts of the world because it survives and grows also under adverse environmental conditions (Niroula et al., 2006), matures rapidly, and is susceptible to few diseases (Yasui et al., 2004). It has short growing season (Asaduzzaman et al., 2009), the growing intensity is vigorous and indeterminate. The common buckwheat has been used for honey production and useful as a green manure crop in the low-productivity land (Campbell, 1997). It has desirable flavor and milling quality (Cheng and Ni, 1992; Marshall and Pomeranz, 1982). The reliance on pollinators is due to self-incompatibility in F. esculentum. The lack of compatibility is sporophytic, partly due to floral structure. Buckwheat
I. ’cymosum’ group
Fagopyrum esculentum Moench. Fagopyrum homotropicum Ohnishi Fagopyrum tataricum L. Fagopyrum giganteum Krot. Fagopyrum cymosum L. II. ’urophylleum’ group Fagopyrum macrocarpum Ohsako&Ohnishi Fagopyrum pleioramosum Ohnishi
Fagopyrum capillatum Ohnishi Fagopyrum gracilipes (Hemsl.) Dammer Fagopyrum gilessii Hemsl. Fig. 1. The suspected relationships of 10 Fagopyrum species including the cultivated species based on Sharma and Jana (2002), Rout and Chrungoo (2007), Gondola and Papp (2010) and Sangma and Chrungoo (2010).
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flowers are dimorphic; ‘pin’ type flowers have long pistols and short stamens, while ‘thrum’ type flowers have short pistols and long stamens (Adachi et al., 1983; Cawoy et al., 2009; Kreft, 1986; Nagatomo, 1961) (Fig. 2). Individuals bear only one flower type and, in addition to structural barriers, biochemical barriers are believed to exist as self pollen often fails to germinate and is unable to grow functional pollen tubes (Adachi, 1990; Nettancourt, 1977). Successful fertilization can arise only between plants with different floral structures.
2. Genetics of incompatibility Flower type ratio segregates consistent with single locus inheritance and ‘thrum’ flowers are dominant to ‘pin’ (Garber and Quisenberry, 1927a). Although floral architecture is linked with incompatibility type, incompatibility is mainly due to early pollen tube abortion, not floral structure (Nettancourt, 1977), and is inherited sporophytically (Garber and Quisenberry, 1927b; Lewis and Jones, 1992). It is unknown how many gene products comprise the self-compatibility locus (S-locus). Recently, Yasui et al. (2012) identified two transcripts that are linked to the S-locus and expressed exclusively in the style of thrum flowers. Through homology analysis of 610 kb of genomic DNA surrounding these transcripts they identified only one other gene, which is expressed in both thrum and pin styles. It is possible that several gene fragments in the region produce functional transcripts. The transfer of the self-pollinating mechanism to the cross-pollinated common buckwheat would be of particular importance, since this would reduce the dependence on insect pollination (Garber and Quisenberry, 1927b; Kovalenko and Laptev, 1979; Kovalenko and Shumny, 2004; Woo et al., 2006) and allow for increased rate of seed set which might result in higher yield. The discovery of a wild, self-compatible species with homomorphic flowers, F. homotropicum, by Ohmi Ohnishi (1995) in South-western China has provided a new germplasm source for buckwheat breeders. F. homotropicum has 19 accessions which have been characterized morphologically (Ohnishi and Asano, 1999). Campbell (1995) and Woo et al. (1999) made crosses between populations of F. esculentum and F. homotropicum and found that the homomorph, self-compatible style was controlled by a single locus (Sh). They determined the dominance relations of the between the three haplotypes determining flower structure as follows: S N Sh N s. Therefore the flower type of F1 population from crosses between F. esculentum ‘pin’ style (ss) and F. homotropicum (ShSh) is homomorph (sSh). Genetic incompatibility was determined to be sporophytic by Dahlgren (1922). According to Wang et al. (2005a) the homomorph flower type and the self-compatibility can be controlled by more complex genetic interactions, and they posited a 2-gene model in F. homotropicum species and in its progenies. They found that the use of self-incompatible F. esculentum ‘pin’ type in interspecific hybridization with homostyly type is more desirable for plant breeding than ‘thrum’ type (Ss), as the ‘thrum’ plants carry both ‘pin’ and self-compatible alleles which makes the selection of progenies of homostyly from ‘pin’ difficult. Recently, much progress has been made to identify the molecular basis of heterostylistic incompatibility of buckwheat. Ohnishi and Ohata (1987) produced the first genetic map in buckwheat with the use of morphological and isozyme markers. Maps based on DNAbased polymorphisms have also been constructed, including an AFLP map based on a segregation of an interspecific F. esculentum × F. homotropicum cross (Yasui et al., 2004) that was used to identify markers linked to the self-incompatibility locus and the locus responsible for brittle pedicels in wild buckwheat (Matsui et al., 2004). Additionally, a genetic map based on 87 RAPD and STS markers has been deployed to map variants in four seed storage protein subunits in a cross between common and wild buckwheat (Pan and Chen, 2010). Finally, an intra-specific map based on a cross between two varieties of F. esculentum has been created using microsatellite and AFLP markers, and
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Table 1 Chromosome numbers, desirable and undesirable traits, origin and progenitors, and phylogenic relationships of Fagopyrum esculentum, Fagopyrum tataricum and Fagopyrum homotropicum (diploid) might be introgressed by interspecific hybridization to cultivated buckwheat, based on Campbell (1997), Woo et al. (2001), Rout and Chrungoo (2007) and Gondola and Papp (2010). Species
Chromosome Desirable traits number
F. esculentum — cultivated in the 2n = 16 temperate Europe, Japan (Indo-Myanmar)
Resistance to seed shattering, good flour quality, easy dehulling
F. tataricum — cultivated in the Himalaya
2n = 16
F. homotropicum
2n = 16
High seed set, high rutin content of seeds, self-pollination, frost tolerance, vigorous plant, resistance to seed shattering Self-pollination, high seed set, frost tolerance
several of these markers also work in F. tataricum (Konishi and Ohnishi, 2006). Although there is no current genome project in Fagopyrum, several studies have taken advantage of genomic techniques to character the molecular basis of buckwheat variation. Yasui et al. (2008) constructed a BAC library of 142,000 large insert clones, representing an estimated 7–8-fold coverage of the F. esculentum genome. They used previously identified markers to the S locus to identify linked BAC clones and create an STS marker for the dwarf locus. Hara et al. (2011) sequenced cDNA clones to conduct a homology-based search to locate candidate genes co-segregating with QTLs controlling photoperiod sensitivity, and next
A
Undesirable Origin and progenitors traits
Putative ancestors of cultivated Fagopyrum species
Wild relatives
Crosspollination, yield instability
Western and Central Himalaya, East Himalaya (one accession)
F. cymosum (wild ancestor: F. homotropicum)
F. homotropicum F. cymosum
Western and Central Himalaya, Japan (one accession)
F. cymosum
F. giganteum F. cymosum
Seed shattering
Eastern Tibet and Yunnan and Sichuan Provinces of China
F. cymosum
generation sequencing has been used to study floral transcript diversity between F. esculentum and F. tataricum (Logacheva et al., 2011). In an impressive convergence of new sequencing technology and a decade's accumulation of genetic resources in buckwheat, Yasui et al. (2012) identified a novel gene that may control short-styled floral morphology. They sequenced the styler transcriptome from genetically similar longand short-styled sibs, identifying four transcripts exclusively expressed in short styles. Using a long-styler mutant found on a chimeric shortstyler plant, they were able to identify the S-ELF3 transcript as deleted in the long-styler chimeric branch. The exclusivity of S-ELF3 expression to short-styler plants was consistent in self-incompatible accessions of
B
C
anther stigma
stamen
style
Fig. 2. Heteromorph and homomorph flower types of Fagopyrum. Structure of ‘pin’ type flowers (A) with long styles and short stamens; structure of ‘thrum’ type flowers (B) with short styles and long stamens. Homomorph flower structure of F1 hybrids between F. esculentum × F. homotropicum with the same length of styles and stamens (C).
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F. cymosum and the distant Fagopyrum urophyllum. As sequencing costs continue to plummet, an acceleration of molecular research in buckwheat through the utilization of genomic resources appears to be on the horizon. 3. Interspecific hybridization and its benefits for cultivated buckwheat Interspecific hybridization in the genus Fagopyrum goes back to a long time. The aim of these crossings was the transfer of favorable properties from the wild species to the cultivated ones, sometimes from one cultivated species to another one. In the course of the latter variation, the good nutritional traits of common buckwheat have been attempted to transfer to tartary buckwheat. Self-pollination, higher fertility rates, frost tolerance, vigor, and high rutin content are desirable traits of tartary buckwheat which breeders have wanted to transfer to common buckwheat (Woo et al., 1999). While interspecific hybridization with common buckwheat's progenitor species, F. homotropicum, can be a useful technique for transferring traits such as self-compatibility and cold tolerance, it can also re-introduce undesirable characteristics, such as seed shattering, which have been eliminated through the domestication process. Campbell (1995) and Woo and Adachi (1994, 1997) were the first ones who reported successful interspecific hybridization between F. homotropicum and F. esculentum at diploid level, in which the progenies were fertile. Inbreeding depression could be detected in the homostylic inbred lines of F. esculentum (Mukasa et al., 2010), but inbreeding depression has not been observed in selfcompatible buckwheat breeding lines created through interspecific hybridization (Matsui et al., 2003). After the transfer of desirable traits from F. homotropicum into the common buckwheat, such as self-pollination and frost resistance (Table 1), backcrosses must be made to F. esculentum in order to eliminate seed shattering and other undesirable traits (Wang et al., 2005b; Woo et al., 2001). Interspecific hybridization can also be applied to improve quality (bitterness) and agronomic characteristics (ease of dehulling) of buckwheat seeds (Campbell, 1995). Campbell (1995) carried out further back-crosses to F. esculentum to transfer the desirable traits (self-pollinating system, frost tolerance) of F. homotropicum to F. esculentum. Woo et al. (1999) reported successful hybridization between F. homotropicum, F. esculentum, and F tataricum. They conclude that the ‘thrum’ plants of F. esculentum could be successfully used to increase the production efficiency of the hybrids. After interspecific crossings between F. homotropicum lines, selfincompatible F. esculentum ‘pin’ and self-pollinating homostylous breeding lines more than 50% of F2 hybrids had lower rutin content but two F2 hybrids had 9.38-fold and 34.92-fold higher rutin content compared to parents (Wang et al., 2004). This result indicated the possibility of selection for high rutin content using recurrent selection on each generation provided that the population size was high enough. Wang et al. (2005c) reported the first successful interspecific hybridization between Fagopyrum species with different ploidy levels. Tetraploid F. homotropicum and diploid F. esculentum ‘pin’ plants were used for crossings. However, for culturing of hybrid embryos in vitro embryo rescue was necessary. All the flower types of F1 hybrid plants were homostylic and the morphology of these plants was similar to F. homotropicum, including the seed shattering habit. The F1 hybrids were triploid and had 24 chromosomes according to their cytogenetic analysis. Fertility and morphology of F2 generation differed depending on ploidy. F2 hybrids were diploid, aneuploidy and hexaploid. Diploid plants with homostylic flower type and hexaploids with ‘pin’ flower type were self-fertile while diploids with ‘pin’ type flower, aneuploids and hexaploids with homostylic flower type were partially fertile (Wang et al., 2005c). Seed shattering is the main undesirable trait in the wild buckwheat relative that impedes use of interspecific hybrids; abscission of the pedicel in F. homotropicum causes the seeds to drop to the ground, rendering them unharvestable. In all studies examining shattering in
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interspecific hybrids between common buckwheat and F. homotropicum, shattering exhibited dominance in the F1 (Campbell, 1995; Wang and Campbell, 1998; Wang et al., 2005b). In some crosses, genetic linkage is observed between flower type and shattering habit, though the linkage is not perfect and segregants have been identified in large populations (Wang et al., 2005b). Based on interspecific crosses between diploid F. homotropicum and F. esculentum, Wang et al. (2005b) found evidence for multiple segregation ratios for the shattering character in hybrids with various segregation ratios, for which they proposed a 3-gene-model with recessive epistasis, where variation in the shattering genes in different accessions of common buckwheat used was responsible for the multiplicity of segregation ratios. They found three types of segregation in F2 population (3:1, 9:7, 27:37) and four types of segregation in F3 population (1:0, 3:1, 9:7, 27:37) regarding shattering to nonshattering. According to Wang et al. (2005b) there are genetic correlations between seed shattering and self-compatible homomorphic flower type and between non-shattering and self-incompatible ‘pin’ flowers (Fesenko et al., 1998). This linkage can be overcome by use of a large segregating population (Wang et al., 2005b). Chen et al. (2004) studied the big-achene group of Fagopyrum, which contains the F. esculentum subsp. ancestrale Ohnishi, F. tataricum, F. homotropicum and other four Fagopyrum species. They reported that there are close relationships between these species in the big achene group (Chen, 1999) and F. homotropicum is similar in morphology to the F. esculentum, so it can be crossed with each other easily. Contrary to previous studies, they found that crossability was high between both F. esculentum and F. homotropicum (28%), and between F. esculentum and F. tataricum (37%). According to the descendant it was concluded that achene dormancy of F. homotropicum was controlled by the sporophyte genotypes. There have also been successful attempts to hybride common buckwheat to diploid accessions of its progenitor, F. homotropicum (Campbell, 1995; Woo and Adachi, 1994), and the use of thrum-type flowers of F. esculentum was found to increase compatibility in these hybridizations (Woo et al., 1999). 4. Methods for overcoming the species barrier Although the molecular mechanism of incompatibility remain unresolved, chemical treatments have been found which can overcome it, such as actinomycin, a RNA synthesis inhibitor, and cycloheximide, a protein synthesis inhibitor (Asher and Drewlow, 1970; Ferrari and Wallace, 1976a, 1976b; Franklin et al., 1992; MiljušĐukić et al., 2010; Sarker et al., 1988). Phosphatase inhibitors, such as ocadaic acid and cantharidin, have also been successful circumventing the self-incompatibility of buckwheat; when applied on isolated pistils, self-pollen tube inhibition is relieved (Miljuš-Đukić et al., 2003). Another group of inhibitors, calcium antagonists (verapamil, jonophore) were successfully used for cessation of self-incompatibility in buckwheat, implicating Ca2+ signaling in the SI response of buckwheat (Franklin-Tong et al., 1997; Miljuš-Đukić et al., 2003). Conventional breeding methods have not been successful in producing interspecific hybridization of buckwheat species (Ruszowszki, 1980; Samimy et al., 1996; Woo et al., 2006). In many cases the hybrid embryo aborted due to incompatibility (Woo et al., 2006). Based on the observations of Guan and Adachi (1992) there are two types of sterility in the abnormal embryo sac. One type is the lack of fertilization, i.e. the pollen tube to germinate (Ruszowszki, 1980) and failure of the pollen tube to target and penetrate the micropyle and ovary (Samimy, 1991; Woo et al., 1999). The second type, abortive sterility, is due to the degradation of the endosperm after fertilization (Shaikh et al., 2002a). Postfertilization barriers, such as abortive sterility, can be overcome through the use of tissue culture methods, such as embryo rescue (Table 2). At high summer temperature Woo et al. (2010) observed abnormal embryo sac 3 days after pollination, which led to incomplete development, failure of fertilization, or abortion of the embryo. They observed
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that the occurrence of abnormality of either type was higher in tetraploids (90–100%) than in diploids (50–77%). According to Guan and Adachi (1992) the stress of high temperature indicates ultrastructural changes of organelles, abortion of embryo sac and endosperm. Initial attempts to cross common and tartary buckwheat with conventional breeding were unsuccessful. The barriers to hybridization of these species appear to be post-zygotic. Though pollen from 10 species within Fagopyrum all had germination and initial pollen tube growth on F. esculentum styles, and F. esculentum pollen was able to penetrate the micropyle of F. tataricum (Hirose et al., 1994). According to Woo et al. (2008) there are correlation between pistils and pollens and the growth of pollen tube is different during the interspecific hybridization making a barrier for these crosses. Five categories were determined in accordance with the degree of compatibility (from high compatibility to incompatibility). In the first, highly compatible category, the pollen tube elongation and its penetration into the style occur 6–24 h after pollination; in the second category, slightly compatible, pollen tube elongation and penetration are delayed for 1–6 h. The final three categories are comprised by three different types of incompatibility: inhibition of pollen tube at the stigma (type I), inhibition of pollen tube at the style (type II), and inhibition of pollen tube at the stylodium (type III). Microscopic examination of hybrid F. esculentum × F. tataricum embryos found numerous cytological defects, including synergid degradation, organellar aggregation and deformation, and defective embryo sac development (Shaikh et al., 2002b). The use of embryo culture from a cross of common buckwheat pollen on tartary buckwheat styles was able to overcome the post-zygotic barrier, but the resulting hybrids were sterile (Samimy et al., 1996). Alternate methods of embryo rescue eventually succeeded in developing a fertile hybrid between these species (Wagatsuma and Un-no, 1995). After embryo rescue from interspecific crosses between F. esculentum and F. tataricum the ovules were observed with light and transmission electron microscopy (Shaikh et al., 2002b). The embryos were rescued at 1 to 5 days after pollination. According to their observation several abnormalities can be detected at 1 day after pollination. The cytoplasm of synergids were degenerated and showed ultrastructural abnormalities.
Several cell organelles were aggregated. The mitochondria were irregularly shaped, and the mitochondrial cristae disappeared. The active cytoplasm of both egg and central cells indicated the normal function of hybrid embryo sac for fertilization. If the fertilization was unsuccessful – like in Shaikh et al. (2002b) studies – the pollen tube may not have entered into the synergid. This results in cross incompatibility between F. esculentum and F. tataricum. They also reported non-zygotic development in the cross combinations between F. esculentum and F. tataricum at 5 days post-pollination. In this case the synergids deformed and degenerated, the endosperm showed abnormal phenomenon, and ribosome density in the cytoplasm of endosperm decreased. The changes in ribosome density caused lowered protein synthesis. The fusion of nuclear was completed but cell division was halted, and the zygote did not develop further. The third studied barrier of the interspecific hybridization was the shrunken or small embryo sac in the hybrid ovules. Two breeding lines of F. homotropicum and three lines of F. esculentum were used for crossings by Wang et al. (2005c), and all F1 hybrids plant obtained by using in vitro embryo rescue method were self-compatible with homostylic flowers, demonstrating the dominance of self-compatibility. Evaluation of F2 and F3 population indicated that two genes with complementary interaction were controlling the self-compatibility. Woo et al. (2008) determined the abnormalities of different hybrid embryos and classified them in five categories: undivided zygote, degenerated and collapsed endosperm, degenerated embryo and endosperm, collapsed embryo and endosperm, floating embryo without suspensor. The introduction of in vitro embryo rescue (Table 2, Fig. 3) in the 1990s has been tremendously successful in overcoming the barrier of embryo abortion that made the production of Fagopyrum interspecific hybrids impossible with conventional breeding techniques (Ruszowszki, 1980; Samimy et al., 1996; Woo et al., 2006). In the embryo rescue technique, hybridized and enlarged ovules are removed from the pistol after pollination, however, the time between hybridization and ovary excision has varied considerably among studies. Woo et al. (2006) removed ovaries 3–5 days post pollination while several studies (Asaduzzaman et al., 2009; Niroula et al., 2006; Wang and Campbell, 1998) waited 7–10 days
Table 2 Successful interspecific hybridization of Fagopyrum species using embryo rescue. Ploidy level
No. of crosses
No. of ovules rescue
No. of embryos emerged
Hybrid recovery (%)
Fertility of progenies (%) F1
F. esculentum × F. tataricum Diploid 88 Diploid 104 Diploid 275 Tetraploid 344
No data No data 68.8 ± 1.07 65.3 ± 1.76
References
F2
3 2 156 178
0 0 16 28
0 0 3.8 6.1
111 2 16 9
13 2 5 6
3 50 2.7 4.1
No data No data No data No data
Wang and Campbell (1998) Wang and Campbell (1998) Niroula et al. (2006) Niroula et al. (2006)
22 16 19 40 24.8 21.1 19
No data No data No data No data No data No data No data
Wang and Campbell (1998) Wang and Campbell (1998) Wang and Campbell (1998) Wang and Campbell (1998) Wang et al. (2002) Wang et al. (2002) Wang et al. (2005c)
4 29 40
No data No data No data
Wang and Campbell (1998) Wang and Campbell (1998) Wang et al. (2005c)
17.9 17.1 3
No data No data No data
Wang et al. (2002) Wang et al. (2002) Asaduzzaman et al. (2009)
51.6 ± 18.77 54.4 ± 17.63
Niroula et al. (2006) Niroula et al. (2006) Asaduzzaman et al. (2009) Asaduzzaman et al. (2009)
F. tataricum × F. esculentum
Diploid Diploid
150 98
F. esculentum × F. homotropicum
Diploid Diploid × tetraploid Diploid × tetraploid
1115
71 90 26 10 145 194 194
5 4
F. homotropicum × F. esculentum
Tetraploid × diploid F. tataricum × F. homotropicum Diploid 79 Tetraploid 41 Diploid 100
10 7 10
4 2 4
28 7 12
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A
B
C
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The desirable traits of buckwheat were transferred by using in vitro ovule culture method (Asaduzzaman et al., 2009). The effect of growth regulators was described using direct and indirect regeneration protocols, but they did not discuss these two types of regeneration further. The crossings were made between F. tataricum and F. esculentum, and after fertilization the embryos were rescued and put for direct regeneration onto half-strength MS and whole MS media (Murashige and Skoog, 1962) with different plant growth regulators. Two types of hybrid plant regeneration methods were used. One is the direct method, when the ovules were cultured on a medium containing NAA, IAA, BA and zeatin. The hybrids were tested in different ploidy level (diploid, tetraploid) by Asaduzzaman et al. (2009) and they conclude that the plant growth regulator content of the medium is an important factor for the direct regeneration of hybrid buckwheat plants (Table 4). The second method is the indirect hybrid plant regeneration method wherein the embryos were rescued from the buckwheat (diploid or tetraploid) hybrids for callus induction prior to placing them onto plant regeneration medium (Table 4).
5. Future prospects of buckwheat breeding
D
Fig. 3. Production of fertile F1 hybrids between F. esculentum × F. homotropicum using embryo rescue method. Enlarged ovule removed 8 days after pollination and placed onto medium (A). In vitro developing 2-week-old hybrid embryo (B). In vitro culture of regenerated hybrid plantlet (C). Acclimated hybrid plants in pots (D).
As a result of embryo and ovary rescue interspecific hybrids of Fagopyrum species have been produced successfully for some time; however, several back crosses are necessary to eliminate disadvantageous wild traits before these hybrids can be utilized for agriculture. Pollination rate and hereby the yield stability in cultivated varieties could be improved by transfer of self-compatibility from F. homotropicum because the insect-dependence decreases in a self-pollinated buckwheat variety. Further advancement can be expected at a greater pace as molecular markers and other biotechnologies are applied in buckwheat breeding. Systematic characterization of the buckwheat genome and DNA polymorphism in the germplasm of domesticated and wild Fagopyrum species will allow for the identification of agronomically valuable traits, such as frost tolerance, resistance to shattering, and high rutin content, and determining phylogenic relationships within the genus. Genome mapping and marker assisted selection (MAS) are efficient tools in crop improvement and buckwheat breeding would be buttressed by the identification of molecular variations in Fagopyrum species.
Table 3 Media used for embryo rescue. Medium composition
before the removal, and Campbell (1995) did not remove the ovaries until 18–20 days post-pollination. After excision, ovaries are surfaced sterilized first with 70% ethanol for 30 s, the with 2% sodium hypochloride for time minutes. Following several rinses of distilled water, the ovaries are plated in test tubes. The embryo culture is maintained at 22 ± 2 °C in 16 h of light and 8 h dark period (Asaduzzaman et al., 2009). Fully developed plantlets are removed from the test tubes, washed with distilled water, and transferred to sterile vermiculite. After two weeks, plantlets were transplanted into pots and grown in a greenhouse. Wang and Campbell (1999) compared four different methods of buckwheat embryo culture: test tube with solid medium (one embryo on 10 ml medium gelled by 0.7% agar), Petri dish with tissue paper soaked by media (10 ml liquid medium in the Petri dish with 2 layers of tissue paper), beaker with 10 ml liquid media, and beaker with 10 ml tap water. Interspecific hybrids between F. esculentum and F. homotropicum were used for all treatments. They found the Petri dish method, first reported in their paper, to be the simplest and most rapid of methods tested. The media used for embryo rescue are summarized in Table 3.
F. esculentum × F. homotropicum 1/2 MS 1/2 MS + 10 mg l−1 L-tyrozine + 10 mg l−1 L-arginine + 2 mg l−1 zeatin MS + 2 mg l−1 zeatin + 10 mg l−1 L-tyrozine + 10 mg l−1 L-arginine + 3% sucrose F. esculentum × F. tataricum MS + 2 mg l−1 zeatin 1/2 MS MS + BA + NAA + IAA + zeatin + 3% sucrose MS + 3% sucrose + 0.2 mg l−1 IAA + 2 mg l−1 BA MS basal salt mixture (1/2 NH4NO3 + 1/2 KNO3) + MS vitamins + 5% sucrose + 2 mg l−1 casein hydrolysate + 0.2 mg l−1 IAA + 0.5 mg l−1 GA3 + 1.0 mg l −1 zeatin F. tataricum × F. homotropicum 1/2 MS + 10 mg l−1 L-tyrozine + 10 mg l−1 L-arginine + 2 mg l−1 zeatin
References Wang and Campbell (1998) Wang et al. (2005c)
Wang and Campbell (1998) Asaduzzaman et al. (2009) Niroula et al. (2006) Samimy et al. (1996)
Wang and Campbell (1998)
MS: Murashige and Skoog (1962), BA: benzyladenine, IAA: indole acetic acid, NAA: naftil acetic acid.
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Table 4 Type of regeneration methods applied for embryo rescue. Type of the method
Medium CIa
PRa MS + zeatin 2 mg l−1 + 3% sucrose
Direct
−1
Indirect
a
1/2 MS + BA 1 mg l−1 + NAA 0.2 mg l−1 + 3% sucrose 1/2 MS + BA 1 mg l−1 + NAA 0.2 mg l−1 + 3% sucrose
1/2 MS + BA 1 mg l + NAA 0.1 mg l−1 + 3% sucrose MS + 3% sucrose + 0.2 mg l−1 IAA + 2 mgL−1 BA MS + zeatin 2 mg l−1 + IAA 0.2 mg l −1 + 3% sucrose MS + zeatin 2 mg l−1 + IAA 0.2 mg l −1 + 3% sucrose
Ploidy level of parents
Embryos emerged (%) [no. of hybrids]
No. of shoots per callus
References
Diploid
25 [3]
–
Tetraploid
40 [5]
–
40
–
Diploid
[2]
4.5
Tetraploid
[2]
4.5
Asaduzzaman et al. (2009) Asaduzzaman et al. (2009) Niroula et al. (2006) Asaduzzaman et al. (2009) Asaduzzaman et al. (2009)
PR = plant regeneration, CI = callus induction, 1/2 MS: half-strength Murashige and Skoog medium (1962).
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