ФИЗИОЛОГИЯ РАСТЕНИЙ, 2011, том 58, № 1, с. 102-110
ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ
УДК 581.1
DIFFERENT RATES OF CHROMOSOME ELIMINATION IN SYMMETRIC AND ASYMMETRIC SOMATIC HYBRIDIZATION BETWEEN Festuca arundinacea AND Bupleurum scorzonerifolium © 2011 Minqin Wang*, Zhenying Peng*, Le Wang, Junsheng Zhao, Jing Che, Guangmin Xia
School of Life Sciences, Shandong University, Shandong, China Received October 8, 2009
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 ^W/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.
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 ge-nomic 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 le-
* These authors contributed equally to the work.
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
thal 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 x 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-/y-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. scorzon-erifolium 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 ^W/cm2 UV irradiation for 30 s); (III) tall fescue + B. scorzonerifolium (380 p,W/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-citric 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 ^l 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 of5S rDNA spacer sequences was also used as primers (25-mers): PI (5'-GGATGGGTGACCTCCCGGGAAGTCC-3'), PII (5'-CGCTTAACTGCGGAGTTCTGATGGG-3'). PCR amplification was performed following Wang etal. [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
Table 1. Development of the fusion combinations and controls
Combinations/controls First division, days No. regenerated calli
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