УДК 581.1
Different Rates of Chromosome Elimination in Symmetric and Asymmetric Somatic Hybridization between Festuca arundinacea and Bupleurum scorzonerifolium
© 2010 г. Minqin Wang*, Zhenying Peng*, Le Wang, Junsheng Zhao, Jing Che,
Guangmin Xia
School of Life Sciences, Shandong University, Shandong, China
Received October 8, 2010

The protoplasts of tall fescue (Festuca arundinacea Schreb.) were fused with those of Bupleurum scorzonerifolium Willd. The latter were irradiated with UV at an intensity of 380 μW/cm2 for 0 s (combination I), 30 s (combination II), and 60 s (combination III) before fusion. Putative hybrid calli, leaves, and shoots were generated from the fusion products. They were recognized as somatic hybrids by a combined analysis of chromosome numbers, isozyme, RAPD, and 5S rDNA spacer sequence. The hybrid calli with morphogenetic ability and leaves/shoots differentiation had the B. scorzonerifolium phenotype, whether they were derived from symmetric fusion (UV 0 s) or asymmetric fusion (UV 30 s/60 s). Cytological tests revealed that these hybrids contained the complete set (12) of B. scorzonerifolium chromosomes and 0(4 partner tall fescue chromosomes. The tall fescue chromosomes were rapidly eliminated in combinations II and III, but gradually lost in combination I. It was noted that the green leaves and shoots were produced earlier, and the differentiation frequency was higher in combinations II and III than in combination I, which corresponded to the speed of elimination of the tall fescue chromosomes in the hybrids. Therefore, UV irradiation can indirectly promote elimination of tall fescue chromosomes and hybrid differentiation. B. scorzonerifolium can repel partner chromosomes with mechanism that differs from UV.

--------------------------------
Abbreviations: BA ( benzyladenine; MS ( Murashige and Skoog nutrient medium.
Corresponding author: Guangmin Xia. School of Life Sciences, Shandong University, Shan Da Nan Lu 27, Jinan 250100, Shandong, China; fax: (86) 531-8856-5610; e-mail: xiagm@sdu.edu.cn

*These authors contributed equally to the work.

Key words: Festuca arundinacea ( Bupleurum scorzonerifolium ( somatic hybridization ( chromosome elimination

INTRODUCTION
Wide hybridization between distantly related species remains attractive because a secondary gene pool can be extremely important for the improvement and evolution of plants. More recently, a protoplast fusion approach has been applied to broaden the genetic basis of resistance to biotic and abiotic stresses, to transfer nuclear and cytoplasmic gene(s) from different species, to combine the genomes of incompatible species, and to manipulate chromosome constitutions at ploidy levels in crop plants [1(4]. Symmetric fusion means that none of the fusion parents is subjected to treatment that can cause chromosome fragmentation or nuclear inactivation. Wild symmetric hybrids are often sterile and morphologically abnormal and may show uncontrolled chromosome exclusion and genomic instabilities [3, 5]. Therefore, during the last two decades, asymmetric somatic hybridization (also called donor(recipient fusion), based on the induction of unilateral chromosome elimination using lethal doses of X- or gamma-rays, UV irradiation, or restriction endonucleases, has been developed as a means to create morphologically normal wide hybrids [2, 6]. Irradiation of Eruca sativa protoplasts was found to enhance the plating efficiency and morphogenesis potential of the fusion products. Fusion products derived from symmetric fusion between parsley and carrot could not form hybrid colonies or embryos apart from mitotic division, whereas plants were established by asymmetric fusion. For the same combination between Lycoperscon esculentum × L. pennellii and eggplant, whole plants were obtained via asymmetric fusion instead of regenerating of leaf primordial cells via symmetric fusion [7, 8]. Donor chromosome exclusion caused directly by X-/γ-rays or UV has also been reported in many papers [9]. But there were a few studies that showed differences between radiation and phylogenetic relationships in hybrid chromosome elimination and morphogenesis.
Bupleurum scorzonerifolium Willd. (Umbelliferae) is a traditionally important Chinese herb used in the treatment of influenza, fever, malaria, and menstrual disorders in China [10]. In asymmetric somatic hybridization of Arabidopsis thaliana/B. scorzonerifolium treated with UV, we found some specific traits of B scorzonerifolium, e.g., resistance to UV irradiation and induction of its partner chromosome exclusion [11]. It is beneficial to explore the chromosome exclusion mechanisms of somatic hybridization through studying whether this indirect effect of UV will happen in monocot/B. scorzonerifolium hybrids.
Tall fescue (Festuca arundinacea Schreb.) is a deep rooted cool-season grass species widely used as a turf grass in home lawns, golf course fairways, driving ranges, and public parks. It has high tolerance to drought, heat, and water stress. It is more tolerant to saline soil conditions than many other cool-season grasses as well. It also requires minimal management, is characterized by greening early in the spring and staying green until late in the fall [12]. Although several studies have produced different combinations of somatic hybrids of tall fescue with related species [13], no study has combined tall fescue with a dicot.
The objective of this study was to create novel somatic hybrids through symmetric and asymmetric protoplast fusions between tall fescue and B. scorzonerifolium to compare the indirect action of UV-irradiation and the phylogenetic relationship of B. scorzonerifolium on the elimination of chromosomes from the monocot partner.

MATERIALS AND METHODS
Protoplast isolation, fusion, and culture. Embryo-derived calli and suspension cell cultures of tall fescue were induced from seedling-derived hypocotyls on MB2 medium (MS medium [14] containing 2 mg/l 2,4-D) at 25(C. The induction and selection of embryonic calli and suspension cell lines of B. scorzonerifolium were performed as described by Xia et al. [15]. The B. scorzonerifolium calli were subcultured on MB1 medium (MS medium containing 1 mg/l 2,4-D) for more than 16 years; they grew vigorously but lost the ability to regenerate. After 3(4 days of subculture, both suspension lines were incubated in an enzyme solution (0.6 M mannitol, 5 mM CaCl2, 1.5% cellulase Onozuka RS, and 0.3% pectolyase Y-23) for 2,0(2.5 h.
The protoplasts of recipient (tall fescue) and donor (B. scorzonerifolium) were fused using the PEG method [15] in three combinations: (I) Tall fescue + B. scorzonerifolium protoplasts; (II) Tall fescue + B. scorzonerifolium (380 μW/cm2 UV irradiation for 30 s); (III) Tall fescue + B. scorzonerifolium (380 μW/cm2 UV irradiation for 60 s). After irradiation, protoplast fusion was conducted immediately. The cultures of each untreated parental protoplasts were used as controls. Protoplast culture was performed as described by Xia and Chen [16]. The fusion products were cultured in P5 liquid medium (MS medium with 90 g/l glucose, 40 g/l sucrose, 250 mg/l D-ribose, 100 mg/l glutamine, 40 mg/l aspartic acid, 2 mg/l cysteine, 2 mg/l ascorbic acid, 500 mg/l casein hydrolysate, 1.5 mg/l NAA, and 0.25 mg/l kinetin, pH 5.8) in the dark at 25(C [15]. After the regenerated cell clusters grew into a small calli of about 1.5(2.0 mm in diameter, they were transferred onto MB1 medium for proliferation, and then onto IB medium (MS medium with 0.5 mg/l IAA and 0.5 mg/l 6-BA) for differentiation.
Analysis of isozymes and chromosome number. For electrophoresis of isozymes, the samples were ground in 0.1 M Tris(citiric acid buffer (pH 8.2) in an ice bath. The homogenate was centrifuged at 5000 g for 10 min. The supernatant was mixed with equal volume of 10% glycerol. Whereafter, 50(100 μl of each sample was loaded onto 10% polyacrylamide gel and run for 4(5 h at 4(C and 30 mA. Gels were stained for esterase and peroxidase following the procedures described by Xia and Chen [15].
For chromosome counting, calli, as well as root tips or young leaf bases of the regenerated plants, were incubated at 4(C for 24 h, then fixed at room temperature with acetic alcohol (99% methanol : acetic acid = 3 : 1), followed by washing with distilled water. The fixed samples were softened in an enzyme solution (used in protoplast isolation) for 40(60 min, washed with distilled water three times; they were kept in the water for 0.5(1.0 h. After removing the water, the samples were refixed with a small amount of acetic-alcohol for 20 min and pounded into suspensions with a rounded head glass rod. A few drops of the upper suspensions were spread on a glass slide and dried with flame. Chromosomes were stained with 5% giemsa for 30 min.
PCR analysis. DNA was extracted from the parents and putative hybrid calli or leaves using the CTAB method. Eight RAPD primers ("Operon Technology", United States) were used (OPJ12-GTCCCGTGGT; OPA8-GTGACGTAGG; OPH20-GGGAGACATC; OPA1-CAGGCCCTTC; OPF5-CCGAATTCCC; OPA19-CAAACGTCGG; OPH4-GGAAGTCGCC; and OPG10-ACAACGCGAG). And a pairs of 5S rDNA spacer sequences was also used as primers (25-mers): PI (5'-GGATGGGTGACCTCCCGGGAAGTCC-3'), PII (5'-CGCTTAACTGCGGAGTTCTGATGGG-3'). PCR amplification was performed following Wang et al. [11]. The PCR products were separated by gel electrophoresis in 1.5% (RAPD) and 2.5% (5S rDNA) agarose gels and analyzed with Syngene gel imaging system ("Syngene", United States) after staining with ethidium bromide.

RESULTS
Growth and Morphogenesis of Fusion Products

The division and differentiation of fusion products and controls are presented in table 1. The products of controls, B and T, did not divide to form visible cell lines. In combinations I(III, fusion products started to divide after 3(6 days of culture and formed visible cell lines at frequencies of 5(9% after 35(45 days of culture in P5 protoplast culture medium (fig. 1a).
Selection of putative somatic hybrids in the fusion products was based on their ability to regenerate buds and leaves [11]. Pale-yellowish calli that were 1.5(2.0 mm in diameter were transferred individually onto MB1 medium (figs. 1b(1e) and grown as independent cell lines. Compact green calli obtained from each combination (table 2) were transferred onto IB medium after 3(4 months of subculture (figs. 1f, 1g). Shoots and leaves appeared quickly in combinations II and III but slowly in combination I (figs. 1h, 1i), with successive subculture of the green calli on the IB medium. They were unable to develop further into whole plants, the same as most hybrids from distantly related parents [1, 17]. All the compact green calli and shoots/leaves are resembled the phenotypes of B. scorzonerifolium.

Chromosome Number
The chromosome numbers of most donor B. scorzonerifolium cells (over 90%) remained stable at 2n = 12 (fig. 2a) despite a long period of subculture. In the tall fescue (2n = 48), the chromosome number changed, ranging from 25 to 48 (figs. 2b, 2c). Since the chromosomes of B. scorzonerifolium are smaller than those of tall fescue, we could easily distinguish them in the hybrid cells. Chromosome numbers in the one-month-old hybrid clones, such as No 1, 11, 13, 18, and 29 from combinations I(III are shown in fig. 2. The distribution of chromosome numbers was in the range of 12(50, including a set of small B. scorzonerifolium chromosomes and some large tall fescue chromosomes. It was noted that most of the chromosome numbers from asymmetric combinations II and III ranged from 12 to 16 (figs. 2d(2f; table 2). Whereas most of the hybrid cells from symmetric combination I had more chromosomes because they contained more tall fescue chromosomes than combinations II and III at this time (figs. 2g, 2h; table 2). These tall fescue chromosomes in the hybrids from combination I were also eliminated after three months of subculture on IB medium (fig. 2i; table 2).

Isozyme Analysis
The hybrid nature of the putative hybrids was further confirmed by examining the electrophoretic patterns for esterase in combinations I(III (fig. 3; table 2). The patterns demonstrated that the product of the esterase gene from each parent was represented on the profile of the putative hybrid calli from combinations I(III. In combination I, putative hybrids 1, 7, 13, 16, 20, 29, and 31 had bands from both parents (fig. 3a; table 2). In combination II, putative hybrids 17, 18, 25, and 32 had bands from both parents and a new band was presented in the hybrid 18 (fig. 3b; table 2). In combination III, putative hybrids 8, 11, 21, and 33 had the characteristic bands of both parents and the hybrid 43 showed a new band (fig. 3c; table 2). Esterase assay confirmed the hybrid nature of them on protein level.

RAPD and 5S rDNA Spacer Sequence
Of the eight RAPD primers, three (OPG-10, OPJ-12, and OPH4) (figs. 4a(4c; table 2) could amplify polymorphic products in the parents. When amplified with primer OPG-10, hybrids 1 and 16 (combination I), 17 and 25 (combination II), 11 and 33 (combination III) had bands from both parents (fig. 4a; table 2). Amplified with primer OPJ-12, new band appeared in the hybrid 11 (combination III), and the other hybrids had bands of both parents (fig. 4b; table 2). With primer OPH4, hybrids 1 and 16 (combination I), 17 and 25 (combination II), 11 and 33 (combination III) had both parental bands (fig. 4c; table 2). These primers were used successfully to confirm the hybrids.
The results of 5S rDNA spacer analysis from partial hybrids are shown in fig. 4d and table 2. The calli and leaves/shoots of hybrids all had the fragments specific for B. scorzonerifolium and tall fescue, as well as novel band.

DISCUSION
It has been proposed that the spontaneous elimination of chromosomes from either or both parents was necessary for normal division and morphogenesis of the hybrid cells derived from remote combinations, which leads to the regeneration of asymmetric hybrids or cybrids [5]. Famelaer et al. [5] explained that the loss of recipient chromosomes was the result of a nonspecific effect of in vitro culture, but could also result from donor(recipient chromosome interactions. Sorting out of chromosome of either or both fusion parents could alleviate the incompatibility, so that their hybrids could be established after fusion. In this experiment, the hybrid leaves/shoots of tall fescue/B. scorzonerifolium generated early in combinations II and III with UV-treated B. scorzonerifolium, but developed late in combination I without UV treatment. We observed that elimination of tall fescue chromosomes was rapid in the cell lines from the asymmetric hybridization but occurred gradually during the subculture on the differentiation media from symmetric hybridization. The chromosome elimination is probability affected by the following factors, such as irradiation dosage, genotype, phylogenetic relationship between the fusion parents, ploidy level of the parents, the ratio between the donor and the recipient DNA contents, and the physiological status of the parents [3, 17]. Our results revealed that the elimination of tall fescue chromosomes with/or without UV irradiation in this study is likely due to a combination of two main factors: remote phylogenetics and the indirect influence of UV irradiation on B. scorzonerifolium. Liu et al. [3] proposed that phylogenetically distant species are possibly different from each other in chromosome behavior and severe incompatibility exists between their whole chromosome sets. Since the somatic hybrids contained nuclear and cytoplasmic genomes of phylogenetic distant species, which led to the complex interaction among nuclear(nuclear, cytoplasmic(cytoplasmic, and nuclear(cytoplasmic genomes, and this aggravated the genetic instability in the hybrid cell, and the chromosome elimination was inevitable. This complex interaction in the somatic hybrids leads to the gradually chromosome elimination until its genome reaches a relative genetic balance.
Most cell fusions demonstrated that X-, γ- and UV-irradiation are efficient means for inducing high frequencies of asymmetry [3]. These forms of radiation result in the partial or complete elimination of chromosomes or chromosome fragmentation in the donors [3]. The B. scorzonerifolium used in this study is really a special material, which has been subcultured for more than 16 years (since 1992) in our laboratory. Its chromosome number is stably maintained over a number of 12 (2n = 12) (fig. 2a).
Somatic hybrid plants were obtained from common wheat + B. scorzonerifolium [18], and Arabidopsis thaliana + B. scorzonerifolium [11] in our laboratory. In both hybridizations, the hybrids showed B. scorzonerifolium phenotypes and the chromosomes of other partner eliminated or fragmented, although the protoplasts of B. scorzonerifolium were treated with UV before fusion. Thus, the elimination of tall fescue chromosomes by hybrids involving B. scorzonerifolium as one partner is not an accidental event.
Some reports showed that UV irradiation resulted in an increased ROS generation that overwhelmed the antioxidant defense mechanisms of the target system [19]. Li and Stapleton et al. [20, 21] have reported that flavonoids effectively absorb UV in many plants. It is known that B. scorzonerifolium contains more than 70 kinds of secondary metabolites, such as flavonoids, saikosaponin, quercetin, rutin, guaiol, and others [22], which might provide some natural protection against ROS. In various plants, UV-induced flavonoid accumulation likely functions in protecting cells against damage from UV irradiation [23, 24]. In another experiment, we found that ionization and excitation induced by primary UV-irradiation led to the production of more ROS in B. scorzonerifolium than in A. thaliana. Meanwhile, it was determined that the concentrations of flavonoids were higher in B. scorzonerifolium than in A. thaliana (Wang et al., personal communication). Chatterjee and Holley [25] showed that, as a result of radiolysis of the water existing in a cellular complex irradiated with UV, new chemicals, such as some species of hydrogen radicals and solvated electrons, are produced and have a finite probability of interacting with DNA sites. Hydrogen radicals, as well as hydroxyl radicals, can react either with sugar moieties or with bases [25]. Thus, we deduced that, to protect the stable characteristics of B. scorzonerifolium DNA, secondary metabolites, such as flavonoids in B. scorzonerifolium, prevent electrons from reducing O2 into O2( or integrating with unstable radicals. Perhaps, the secondary metabolites surround the nucleus of B. scorzonerifolium and divert the electrons through the electron-transport chains to tall fescue. These reactions between water radicals and various DNA sites can lead to damage, such as base alterations, base deletions, and strand breaks of the tall fescue DNA. The elimination of tall fescue chromosomes (recipient) is unavoidable in the fusant. On the other hand, the radiation may interact with other atoms or molecules in the fusion bodies, particularly with water, to produce free radicals, which can diffuse far enough to damage different important components, including the DNA of the receptor chromosomes. This indirect effect of irradiation is important in cultured cells, in which the cytoplasm contains about 80% water [23]. Because UV irradiation may produce free radicals, it enhances the velocity of interactions, leads to chromosome breakage, and elimination of tall fescue chromosomes in the fused cells from asymmetric combinations a short time after fusion, which is favorable for hybrid morphogenesis.
The results obtained in this study demonstrated that protoplast fusion allows the production of somatic hybrids between tall fescue and B. scorzonerifolium. A significant loss of chromosomes from the receptor tall fescue was an indirect effect of UV irradiation. This confirmed the characteristics of B. scorzonerifolium in repelling exogenous genetic material, as well as resisting UV radiation. In addition, we revealed that there is a different mechanism of chromosome elimination of tall fescue in symmetric and asymmetric somatic hybrids of tall fescue/B. scorzonerifolium.
The authors thank Roberta Greenwood for her help in editing this manuscript.
This work was supported by the National Key Technology R&D Program 2007BAD59B06, National Natural Science Foundation of China no. 30870240, and Middle-Young's Scientist Encouraging Foundation by Shandong Science and Technology Committee, project no. 2005BS03023.


REFERENCES
1. Cheng A.X., Xia G.M. Somatic Hybridization between Common Wheat and Italian Ryegrass // Plant Sci. 2004. V. 166. P. 1219-1226.
2. Gleba Y.Y., Sytnik K.M. Protoplast Fusion ( Genetic Engineering in Higher Plants: Protoplast Fusion and Hybridization of Distantly Related Plant Species // Monographs on Theoretical and Applied Genetics. 1984. V. 8. P. 115-161.
3. Liu J.H., Xu X.Y., Deng X.X. Intergeneric Somatic Hybridization and Its Application to Crop Genetic Improvement // Plant Cell, Tissue Organ Cult. 2005. V. 82. P. 19-44.
4. Millam S., Paine L.A., Mackay G.R. The Integration of Protoplast Fusion-Derived Material into a Potato Breeding Programme ( a Review of Progress and Problems // Euphytica. 1995. V. 85. P. 451-455.
5. Famelaer I., Gleba Y.Y., Sidorov V.A., Kaleda V.A., Parokonny A.S., Borysyuk N.V., Cherep N.N., Negrutiu I., Jacobs M. Intergeneric Asymmetric Hybrids between Nicotiana plumbaginifolia and Nicotiana sylvestris Obtained by Gamma-Fusion // Plant Sci. 1989. V. 61. P. 105-117.
6. Sikdar S.R., Chatterjee G., Das S., Sen S.K. "Erussica", the Intergeneric Fertile Somatic Hybrid Developed through Protoplast Fusion between Eruca sativa Lam and Brassica juncea (L.) Czern. // Theor. Appl. Genet. 1990. V. 79. P. 561-567.
7. Guri A., Dunbar L.J., Sink K.C. Somatic Hybridization between Selected Lycopersicon and Solanum Species // Plant Cell Rep. 1991. V. 10. P. 76-80.
8. Liu B., Xing M., Zhang Z. H., Liu B., Xing M., Zhang Z. H., He M.Y., Hao S., Gu D.F., Zhao R., Wu X.K. Intergeneric Somatic Hybrid Plants of Nicotiana tabacum L. and Lycium barbarum L. by Protoplast Electrofusion // Acta Bot. Sinica. 1995. V. 37. P. 259-266.
9. Yue W., Xia G.M., Zhi D.Y., Chen H.M. Transfer of Salt Tolerance from Aeleuropus littoralis Sinensis to Wheat (Triticum aestivum) via Asymmetric Somatic Hybridization // Plant Sci. 2001. V. 161. P. 259-263.
10. Chang H.M., But P.P.H. Pharmacology and Applications of Chinese Material Medical. Singapore: World Scientific Publishing Co., 1987.
11. Wang M.Q., Xia G.M., Peng Z.Y. High UV-Tolerance with Introgression Hybrid Formation of Bupleurum scorzonerifolium Willd // Plant Sci. 2005. V. 168. P. 593-600.
12. Lisa L. Turfgrass Biotechnology // Plant Sci. 1996.V. 115. P. 1-8.
13. Takamizo T., Spangenberg G., Suginobu K.-I., Potrykus I. Intergeneric Somatic Hybridization in Gramineae: Somatic Hybrid Plants between Tall Fescue (Festuca arundinacea Schreb.) and Italian Ryegrass (Lolium multiflorum Lam.) // Mol. Gen. Genet. 1991. V. 231. P. 1-6.
14. Murashige T., Skoog F. A Revised Medium for Rapid Growth and Bioassays with Tobacco Tissue Cultures // Physiol. Plant. 1962. V. 15. P. 473-497.
15. Xia G.M., Chen H.M. Plant Regeneration from Intergeneric Somatic Hybridization between Triticum aestivum and Leymus chinensis (Trin) Tzvel // Plant Sci. 1996. V. 120. P. 197-203.
16. Xia G.M., Li Z.Y., Chen H.M. Direct Somatic Embryogenesis and Plant Regeneration from Protoplasts of Bupleurum scorzonerifolium Willd // Plant Cell Rep. 1992. V. 11. P. 155-158.
17. Kisaka H., Kisaka M., Kanno A., Kameya T. Production and Analysis of Plants that are Somatic Hybrids of Barley (Hordeum vulgare L.) and Carrot (Daucus carota L.) // Theor. Appl. Genet. 1997. V. 94. P. 221-226.
18. Zhou C.E., Xia G.M., Zhi D.Y., Chen Y. Genetic Characterization of Asymmetric Somatic Hybrids between Bupleurum scorzonerifolium Willd and Triticum aestivum L.: Potential Application to the Study of the Wheat Genome // Planta. 2006. V. 223, P. 714-724.
19. Farrukh A., Vaqar M.A., Hasan M. Photochemoprevention of Ultraviolet B Signaling and Photocarcinogenesis // Mutation Res. 2005. V. 571. P. 153-173.
20. Li J., Qu-Lee T.M., Raba R., Amundson R.G., Last R.L. Arabidopsis Flavonoid Mutants Are Hypersensitive to UV-B Irradiation // Plant Cell. 1993. V. 5. P. 171-179.
21. Stapleton A.E., Walbot V. Flavonoids Can Protect Maize DNA from the Induction of UV Radiation Damage // Plant Physiol. 1994. V. 105. P. 881-889.
22. Shi Q., Nie S.Q., Huang L.Q. New Progression of Chemical Component and Pharmacological Studies of Radix Bupleuri // Chin. J. Exp. Trad. Med. Form. 2002. V. 8. P. 53-56.
23. Kovács E., Keresztes Á. Effect of Gamma and UV-B/C Radiation on Plant Cells // Micron. 2002. V. 33. P. 199-210.
24. Stratmann J. Ultraviolet-B Radiation Co-opts Defense Signaling Pathways // Trends Plant Sci. 2003. V. 8. P. 526-533.
25. Chatterjee A., Holley W.R. Computer Simulation of Initial Events in the Biochemical Mechanisms of DNA Damage // Adv. Radiat. Biol. 1993. V. 17. P. 181-226.



FIGURE CAPTIONS
Fig. 1. Development of somatic hybrid clones.
a ( microcalli on P5 liquid medium; b ( microcalli on MB1 solid medium; c ( B. scorzonerifolium calli; d ( tall fescue calli; e ( putative hybrid calli on MB1 medium; f ( putative hybrid calli on IB differentiation medium (some green calli can be seen); g ( putative hybrid green calli on IB medium; h ( abnormal hybrid green shoot with root in combination II; i ( hybrid green leaves in combination III. Bar = 1 cm.

Fig. 2. Chromosomes of the parents and hybrid cell lines.
a ( chromosomes of B. scorzonerifolium calli; b and c ( chromosomes of tall fescue calli; d ( chromosomes of one-month-old calli of cell line No. 1 in combination I (containing 12 small chromosomes of B. scorzonerifolium and 34 chromosomes from tall fescue); e ( chromosomes of one-month-old calli of cell line No. 13 in combination I (containing 12 small chromosomes of B. scorzonerifolium and 16 chromosomes from tall fescue); f ( chromosomes of one-month-old calli of cell line No. 29 in combination I (containing 12 small chromosomes of B. scorzonerifolium and 11 chromosomes from tall fescue); g ( chromosomes of one-month-old calli of cell line No. 18 in combination II; h ( chromosomes of one-month-old calli of cell line No. 11 in combination III (containing 12 small chromosomes of B. scorzonerifolium and 1 chromosomes from tall fescue); i ( chromosomes of four-month-old calli of cell line No. 29 in combination I. → ( small chromosomes of B. scorzonerifolium;  ( large tall fescue chromosomes. Bar = 30 μm.

Fig. 3. Isozyme patterns of esterase in the putative hybrid cell lines and the parents.
a ( combination I; b ( combination II; c ( combination III; B ( B. scorzonerifolium; T ( tall fescue; → ( band characteristic of both parents;  ( new band.

Fig. 4. Electrophoresis patterns of PCR products using RAPD and 5S rDNA spacer sequence of regenerated clones and the parents.
a ( electrophoresis pattern of RAPD using OPG-10; b ( electrophoresis pattern of RAPD using OPJ-12; c ( electrophoresis pattern of RAPD using OPH-4; d ( 5S rDNA electrophoresis pattern using 25-mer primer pairs. T ( tall fescue; B ( B. scorzonerifolium; M ( the lambda DNA size marker cut with EcoRI + HindIII. ↑ ( specific band of B. scorzonerifolium;  ( specific band of tall fescue; ◄ ( new band.