Abstract: Cultivated tartary buckwheat (Fagopyrum tataricum) was successfully hybridized with cultivated common buckwheat (Fagopyrum esculentum). Both diploid (2n = 16) and tetraploid (2n = 32) and hybrids were produced from interspecific crosses using ovule rescue method. The produced hybrid was confirmed using plant morphological characters, cytological characters and DNA analysis. The morphological characteristics of the hybrids and parent species are described along with estimation of genetic variation of the hybrids. Self-compatibility together with homomorphism has been introduced from F. tataricum to F. esculentum. Inheritance of flower color in the hybrids was found to be intermediate, whereas pin-style flowers and round shape seeds were found to be dominant over homomorphism and non-winged seeds. There were no obvious differences between the two species in efficiency of pollination. The meiotic observations were accorded with high seed fertility of F1 hybrids and produced normal seeds. These observations indicated that the genetic resemblance and chromosome affinity in the hybrids and parental chromosome supported the fertility of the hybrids.
INTRODUCTION
Common buckwheat, Fagopyrum esculentum (F. esculentum), is one of the main species used as a human food source in the world. Common buckwheat had several advantages such as; high protein content, short growth period and probability for fewer diseases, as well as its medical value (e.g., rutin content) for treating human diseases (Li and Zhang, 2001). Further it was described that was observed a desirable flavor and milling quality in common buckwheat (Marshall and Pomeranz, 1982; Koh et al., 1988; Choi et al., 1988; Cheng and Ni, 1992). Due to these nutritional and aesthetic values cultivation of common buckwheat (F. esculentum) as an alternative crop has been increasing day by day in Japan. However, till date productivity of common buckwheat has not yet reached to desired level with an unstable yield. Thus, buckwheat has not emerged as an economical crop; the dimorphic self incompatibility system, limited variation in its reproductive system and susceptibility to different pests and diseases being the major yield limiting factors (Campbell, 2003; Nair et al., 1999; Kreft, 1983; Guan and Adachi, 1992).
On the other hand, Fagopyrum tataricum (F. tataricum) cultivated buckwheat species grows in the Asian high-altitude mountain areas. On the other hand, F. tataricum usually called tartary buckwheat- a species which is self-compatible with homomorphic flowers and has a high yield, self-fertility and probable tolerance to frost but its seeds lack the desired flavor (Adachi, 1986; Ruszkowshi, 1980; Baniya et al., 1992).
One of the main attempts at buckwheat improvement has been interspecific hybridization to overcome the self-incompatibility problem and to introduce desirable traits such as frost tolerance from wild but related species into common buckwheat. On the other hand, though tartary buckwheat (F. tataricum) is a self-compatibility species with homomorphic flowers, high yield and probable tolerance to frost, has been hybridized with common buckwheat (Samimy et al., 1996), the hybrid did not develop beyond the vegetative stage and its seed lacked the desired flavor (Adachi, 1986; Ruszkowshi, 1980; Baniya et al., 1992).
The first successful hybridization between common buckwheat and Fagopyrum homotropicum was reported by Campbell (1995), in which the self-compatibility trait was introduced from F. homotropicum No. 1 (H1) into a common buckwheat accession B730284. Conventional breeding techniques have not been successful in hybridizing common buckwheat with tartary (Morris, 1951; Ruszkowski, 1980; Adachi et al., 1989; Samimy, 1991). So far, application of protoplast fusion has also failed because the resulting calli did not regenerate (Lachman, 1991). Ujihara et al. (1990) have successfully hybridized tetraploid wild buckwheat (Fagopyrum cymosum, 2n = 32) with tetraploid common buckwheat using ovule culture. Efforts to improve common buckwheat have been made through interspecific hybridization between common buckwheat and the related species F. cymosum (Ujihara et al., 1990; Suvoeova et al., 1994; Hirose et al., 1995; Woo et al., 1999a) and F. tataricum (Morris, 1951; Samimy, 1991; Hirose et al., 1995; Samimy et al., 1996; Chen, 1998; Wang and Campbell, 1998; Fesenko et al., 2001; Wang et al., 2002). However, limited efforts made by them showed success due to sterility of the hybrids.
Thus, the objective of this study was to overcome self-incompatibility and to introduce desirable traits such as high yield and cold tolerance into common buckwheat (F. esculentum), interspecific cross, made with tartary buckwheat (F. tataricum), a self-compatibility species with homomorphic flowers.
MATERIALS AND METHODS
Two cultivated buckwheat used for the interspecific crosses were F. tataricum cv. CT-1 (2x, 4x) and F. esculentum cv. Botansoba (2x) and cv. GreatRuby (4x) as female and male parent, respectively. These varieties were grown in a pot at the green house and crosses were made by hand pollination after flowering. Young hybridized ovules were rescued using ovule culture. This experiment was conducted from October 2005-September 2008 in Shinshu University, Japan.
Pollination: F. tataricum, which was used as female parent, was emasculated in the early morning (about 8 to 9 am) and crossed with pollen of F. esculentum (long-pin flower) by hand from 9 am to 12 pm. The crossed flowers were bagged in small plastic bags for 1-2 days after pollination.
Ovule culture: Ovaries are better enlarged 7-10 days after pollination for ovule culture. The enlarged ovaries from the crosses were collected. The ovaries were surface sterilized with 70% (v/v) ethanol for 30 sec and 2% commercial bleach containing sodium hypo chloride (NaOCl) for 10 min. Ovaries were then thoroughly washed with autoclaved distilled water several times, after which they were carefully excised under microscope and plated (1 ovule per tube) in test tubes which contained 10 mL ½ MS and MS (Sigma, UK) media (Murashige and Skoog, 1962) with the addition of different concentrations and combinations of BA, NAA, IAA, Zeatin (Nakarai chemicals Ltd., Japan) and 2 and 3% sucrose. We used two methods to regenerate to plantlets: One was developing directly into plantlets, while the other required the use of three culture media after plantlet development was completed, inducing media for callus induction, regeneration media for shoot development and rooting media for root formation. The ovule culture were maintained at 22°C±2°C in 16 h of light and an 8 h dark period.
Acclimation: Fully developed plantlets were removed from the test tube and washed in distilled water. Plantlets were then transferred to sterilized vermiculite and grown for about 2 weeks in in vitro condition. Afterward plantlets were transferred into potted soil and grown in the greenhouse.
Morphological and cytological observation: Parents and F1 hybrid plants were cultivated in the green house. The nature of the hybrids was determined by comparing their morphological and cytological characters with those of the parents. For meiotic studies, pre-anthesis flower buds were collected from the investigated plants (ten of each parent, hybrid and F2) in the early morning (8-9 am) and fixed in a freshly prepared Farmer′s solution of 3/1(v/v) ethanol and acetic acid for 24 h. Two percent acetocarmine solution was used for Pollen Mother Cell (PMC) staining for cytological studies and pollen fertility was determined by measuring the percentage of well-stained pollen grains with acetocarmine at anthesis.
DNA extraction and RAPD analysis: Genomic DNA was isolated from young and fresh leaves (0.30 g sample) using Automatic Nucleic Acids Extractor (Quick Gene-810, FUJI FILM, Japan). After template DNA was isolated from the parents and the F1 hybrids, 10 m arbitrary primer (5′ to 3′) OPK15 (CTCCTGCCAA) prepared by (Operon Technologies, CA, USA) was used for PCR (i Cycler, Bio Red, USA) amplification. Each PCR reaction included 12 μL volume containing 1x buffer (supplied by Takara, Shiga, Japan), 200 μM each of dNTPs, 0.42 μM arbitrary primer, 0.5 units of Taq DNA polymerase (Takara, Shiga, Japan) and 12 ng template DNA. The DNA PCR amplifications were performed with a BIO-RAD i Cycler as follow: one step of 3 min at 94°C, then 40 cycles of 1 min at 94°C, 2 min at 40°C, 2 min at 72°C and a final step of 5 min at 72°C. The amplification products were separated by electrophoresis (Mupid-2plus, Advancn, Japan) in 1.7% agarose gels stained by 10 μL Ethidium bromide (Nacalai Tesque Inc., Japan), then 2 μL loading buffer was added to each PCR reaction and 7 μL of each reaction was loaded on the gel. The DNA pHY marker (company by TAKARA BIO INC, Japan) in each gel was used as the check. Electrophoresis was set at 100 v for 1 h. The electrophoresed gel was photographed using UV transilluminator after staining with Ethidium bromide.
Data analysis: Data were expressed as Mean±SD. Differences of p<0.05 were considered significant. Statistical analysis was conducted using one-way ANOVA. A post hoc procedure was carried out all percentage data.
RESULTS AND DISCUSSION
The results of the production of the hybrid plants from interspecific hybridization between F. tataricum and F. esculentum, both diploid and tetraploid, through ovule culture are presented in Table 1. The percentage of emerged embryos (embryo emerged/ovule rescue) was about 11% in diploid and 16% in tetraploid. However, efficiency of hybrid recovery percentages was 3.8 and 6.1% in diploid and tetraploid, respectively. The efficiency of ovule rescue for both varieties was about 55%, while efficiency of plant recovery was poor. Similar reports were presented by Woo et al. (1999a, b), Woo and Adachi (1995), Simimy et al. (1996), Hirose et al. (1995), Ujihara et al. (1990) and Adachi (1990). Though buckwheat is an important species, unfortunately till date there is no recent data on buckwheat interspecific hybrid. The above mentioned studies concluded that the productions of interspecific hybrids are often very difficult to obtain by conventional breeding since strong pre- and post-fertilization barriers exist in the genus. The method of in vitro culture of immature hybrid embryos or ovules prior to abortion has been routinely used to circumvent these barriers. Successful ovule and embryo culture from interspecific crosses have been accomplished by Woo et al. (1999a, b), Woo and Adachi (1995), Simimy et al. (1996) and Ujihara et al. (1990). Morris (1951) obtained some immature embryos when tartary buckwheat was used as female parent, but could not obtain any plants due to premature degeneration of these embryos. Similar efforts were made by Hirose et al. (1995) who also couldn′t obtain any interspecific hybrid plants at diploid level despite culturing 30 ovules. However, they successfully regenerated 20 interspecific hybrid plants by crossing diploid F. esculentum and F. cymosum. Due to interspecific incompatibility barriers, traditional breeding technology has had difficulty establishing new cultivars evincing desirable genetic characteristics. Such challenges may be overcome by ovule rescue of the hybrid before post-zygotic abortion and recovery of the hybrid plant through in vitro culture. Tartary buckwheat possesses a cultivated self-compatibility trait and frost tolerance. Self-compatibility is one of the most important characteristics in the evaluation of Fagopyrum species. Therefore, introducing self-compatibility from F. tataricum (tartary buckwheat) into F. esculentum (common buckwheat) through interspecific hybridization has been attempted. However, conventional cross has not been successfully made between F. tataricum and F. esculentum when F. tataricum was used as the female (Woo et al., 1999a, b; Woo and Adachi, 1995; Simimy et al., 1996). In the present study 17 hybrids from 334 ovules (diploid and tetraploid) was obtained by using F. tataricum as the female the parent following ovule culture methods (Table 1). To the best of our knowledge, this is the first success case for F. tataricum and F. esculentum cross. Therefore, it was possible to produce hybrid plants through the cross combination of F. tataricum and F. esculentum by in vitro ovule culture methods. These methods can be used as a useful tool for overcoming post-zygote hybrid incompatibility in this cross combination. The hybridity was confirmed using plant morphological characters, cytological observation and RAPD analysis.
| Table 1: | Interspecific hybridization by ovule culture between F. tataricum x F. esculentum in diploid and tetraploid species |
| *Hybrid recovery (%) = (No. of hybrids/No. of ovules rescued) x100 | |
| Fig. 1: | Acclimated hybrid plant transferred in the soil pot. The bar represents 2 cm |
As shown in Fig. 1, the hybrid plantlets grew and developed leaves and branches under in vitro condition and all acclimated hybrid plants with fully developed leaves and roots in in vitro condition were transplanted into the soil pots in the green house. Successfully transplants hybrids showed normal growth and flowering.
| Table 2: | Main morphological characteristics between the parents, hybrids and F2 in diploid and tetraploid species |
| *P1 = F. tataricum, P2 = F. esculentum | |
| Fig. 2: | Comparative representation of morphological characteristics in interspecific hybrids (F. tataricumx F. esculentum), F2 and parent in the diploid (A-E) and tetraploid (F-J). The bars represent A and F = 2 cm, B, C, D, G, H and I = 1 cm and E and J = 100 cm |
Morphological observation: The morphological traits of the hybrids, F2 and parent species are presented in Table 2 and Fig. 2. The nature of the hybrids was determined by comparing their morphological characteristics, such as growth habits, flower morphology, seed and leaf, with those of the parents. The growth of the hybrids and F2 was the same or less than that of the parent species. The homomorphic flower type was found to be dominant over the pin flower type in hybrids. Segregation of homomorphism vs. pin flowers occurred in the cross between tartary and common buckwheat, both diploid and tetraploid and F1 hybrid showed either homomorphism-type flower, while F2 also showed homomorphic or pin-type flowers. The flower colors of tartary and common buckwheat are green and white respectively. The flower color of the hybrids appeared to be intermediate, like pink, but F2 flower colors were pink, white and whitish green (Fig. 2C, H). Flowers produced by the hybrid plants were the same type (homomorphic) and size as those of tartary, with white sepals similar to common buckwheat. The hybrid plant developed leaves of a different shape than those of the parents. The general leaf shape of the hybrid plant was ovate without any lobe at the base, whereas that of the parents was heart-shaped with the base of the leaf extending into two either sharp (maternal) or blunt (paternal) lobes. Generally, the flower color and leaf shape of the hybrid plants exhibited intermediate characteristics of the parents. The stem of the hybrid plant was solid, whereas the parents had hollow stems. The seed shape of tartary and common buckwheat was round and non-winged but F1 and F2 seed shape was non-winged (Fig. 2D, I). Diploid and tetraploid F1 hybrid showed normal fertility and produced F2 seed.
| Table 3: | Chromosome association at MI in interspecific hybrid (F. tataricumxF. esculentum) and parents |
| F. t = F. tataricum, F. e = F. esculentum | |
| Fig. 3: | Comparative representation of meiotic metaphase I chromosome association in interspecific hybrids (F. tataricum xF. esculentum) and parent in the diploid (A-C) and tetraploid (D-F) (x1500) |
The fertility and morphology of the diploid and tetraploid F1 and F2 plants were distinct. The diploid and tetraploid F1 and F2 plants were more fertile, taller and had darker leaves than their parents (Fig. 2E, J). These characteristics were similar to the so-called gigas features that have been observed in autopolyploid of other species (Simmonds, 1979). Evaluation of hybrid plants by studying morphology and pollen fertility was reported elsewhere (Gomathinayagam et al., 1998; Sukno et al., 1999). F1 and F2 seeds germinated normally and all F1 and F2 plants were vigorous in their vegetative growth and produced seed.
Cytological observation: The chromosome association of the hybrids and parents at metaphase-I are presented in Table 3 and Fig. 3. The meiotic behavior of parental species and hybrids was studied from PMC mostly at metaphase. Cytological studies were carried out on two ploidy-level parents and their hybrids. Both parents and hybrids show a diploid and tetraploid chromosome number of 2n = 16 and 4n = 32, with normal meiotic behavior in the parents and hybrids. In the parental species diploid and tetraploid shows a high degree of homology in chromosome pairing with 7.3 and 15.5 as the mean value of bivalent frequency and 0.38 and 0.2 the mean value for univalent and tetravalent frequency, respectively. Meiotic behavior of two ploidy-level hybrids shows that chromosomes have affinity for pairing with each other, resulting as the mean value of bivalent frequency 7.3 and 15.3 and mean value of univalent frequency little bit increasing (0.68 and 0.82) compared with the parents. Percentage of Pollen Mother Cells (PMC) in the hybrids shows no great difference from the parents, mean value of percentage about 77% in diploid and tetraploid level.
Table 4 shows the percentage of mean and standard deviation (SD) for the number of normal pollen grains per anther in F1, F2 and their parents. Pollen fertility studies were carried out individually on 10 plants on two ploidy-level parents and their hybrids and F2 plants. Percentage of pollen fertility the diploid and tetraploid parental species showed no great difference between F. tataricum and F. esculentum. Both ploidy level pollen fertility in F1 and F2 little bit decreased from the parents. F1 highest percentages of mean were 68.8 and 65.3 in diploid and tetraploid respectively. This value is not very low compared with the parents and it predicts the degree of fertile in this hybrids.
| Table 4: | Pollen fertility per anther of interspecific hybrids (F. tataricumxF. esculentum), F2 and parent species |
| Percentages are expressed as Mean±SD (min-max). a,bDifferent superscript in the same row significantly different | |
All cytological parameters indicated that meiosis proceeded normally in both parents and both hybrids and high percentage PMC and pollen fertility predict the fertility of hybrids. A similar experiment was performed in an interspecific cross between F. esculentum and F. cymosum and a hybrid plant was successfully produced (Ujihara et al., 1990). Fertile hybrids between F. homotropicum diploid and F. esculentum (Campbell, 1995; Wang and Campbell, 1998; Woo et al., 1999b) formed bivalents at meiosis. The meiotic observations were accorded with high seed fertility of F1 hybrids and produced normal seeds. These observations indicated that the genetic resemblance and chromosome affinity in the hybrids and parental chromosome support the possibility of producing fertile hybrids.
DNA characterization by RAPD analysis in hybrids and parents: Genomic DNA was isolated from the parents and the F1 hybrids were analyzed using a 10 m arbitrary primer (5′ to 3′) OPK15 (CTCCTGCCAA) prepared by Operon Technologies, CA, USA. The RAPD primer OPK15 profile that confirmed hybridity in plants obtained from cross combinations of F. tataricum x F. esculentum (profile) is presented in Fig. 4. In the RAPD-PCR analysis, the specific DNA bands amplified from the male parent (F. esculentum) were also clearly amplified in the interspecific F1 hybrids. Primer that amplified bands specific to the male parent might reveal a proper pattern of a true hybrid. These results confirmed that rescued ovule plants from the cross tartary and common buckwheat of both ploidy levels were true interspecific F1 hybrid plants. The PCR-based RAPD technique has a direct impact on plant breeding-related practices. For testing hybrid purity in buckwheat, RAPD markers were effectively used by Wang et al. (2004).
| Fig. 4: | Polymorphism of interspecific hybrids between F. tataricum and F. esculentum both diploid and tetraploid based on RAPD primer OPK15. M: Molecular marker, F. t = F. tataricum and F. e = F. esculentum |
Confirmation of F1 hybrids: Hybridity of the regenerated plants was identified through comparison of morphological traits, cytological behavior and DNA banding patterns resulting from RAPD-PCR analysis of the parent. Cultivated tartary buckwheat (F. tataricum) was successfully hybridized with cultivated common buckwheat (F. esculentum), both diploid (2n = 16) and tetraploid (2n = 32) and hybrids were produced from interspecific crosses using ovule rescue. Hybridity of plants was confirmed with their morphological features and cytological characters derived from the parent pollen. The morphological characteristics of the hybrids and parent species are described and estimation of genetic variation of the hybrids is given. Self-compatibility together with homomorphism has been introduced from F. tataricum of F. esculentum. Inheritance of flower color in the hybrids was found to be intermediate, whereas pin-style flowers and round-shape seeds were found to be dominant over homomorphism and non-winged seeds. There were no obvious differences between the two species in efficiency of pollination. In plant morphology, including leaf, stem and branching habit, the F1 hybrids generally resembled the male parent (F. esculentum). The flower type of the hybrids was homostyle (Fig. 2B, G). The cytological observations were accorded with high seed fertility of F1 hybrids and produced normal seeds (Fig. 2D, I). These observations indicated that the genetic resemblance and chromosome affinity in the hybrids and parental chromosome support the producing fertile hybrids. RAPD analysis also showed all plants were true hybrids.
In the present study, ovule culture method to produce interspecific hybrid between F. tataricumx F. esculentum the first success in these two species and may bring a major breakthrough in buckwheat breeding. Now needs to be determined are the specific factors which lead to the success of interspecific hybridization in Fagopyrum tataricumxF. esculentum in diploid and tetraploid species through ovule culture technology (phytohormones in the regeneration media or reflective of the narrow range of genotypes examined).