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Integration of maize nuclear and mitochondrial DNA into the wheat genome through somatic hybridization
Plant Science 165 (2003) 1001 /1008 www.elsevier.com/locate/plantsci
Integration of maize nuclear and mitochondrial DNA into the wheat genome through somatic hybridization Chunhui Xu, Guangmin Xia *, Daying Zhi, Fengning Xiang, Huimin Chen School of Life Sciences, Shandong University, Jinan 250100 Shandong, China Received 24 February 2003; received in revised form 19 June 2003; accepted 19 June 2003
Abstract Protoplasts were isolated from cultured cells of wheat and maize and fused using PEG. Calli and green plants were regenerated following irradiation of the maize, and some tested positive for hybridity using morphological, isozymic and various DNA-based marker systems. Genomic in situ hybridization (GISH) of selected maize-carrying regenerants showed that some maize chromatin was dispersed throughout the wheat nuclear genome. Cytogenomic analysis, using RFLP and SSR, demonstrated the presence of both wheat and maize loci in the mitochondrial genome, so that, in the hybrid individuals, either distinct mitochondria from each parent coexist and/or recombinant mitochondria have been generated. With respect to the chloroplast genome, there was no evidence of the presence of any introgression from maize. # 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Wheat; Maize; Asymmetric somatic hybridization; Nuclear genome; Cytogenome; Integration
1. Introduction The two cereals, wheat and maize, are among the most important crop species. Attempts to combine their genomes via sexual hybridization have failed, because in hybrid embryos, maize chromosomes are eliminated at an early developmental stage, so that the chromosomes present in the mature embryo are all of wheat origin [1,2]. No evidence that these haploid plants express any maize genes has been produced, and plant morphology is uniformly wheat-like [2]. However, there is some evidence that high copy repetitive sequences can be successfully transferred from maize to wheat by crossing [3,4]. Since hybrid embryos can be rescued in tissue culture, pollinating wheat with maize has been widely used as a means for constructing haploid wheat plants, whose chromosome complement can later be doubled to generate doubled haploid (DH) progenies.
* Corresponding author. Tel.: /86-531-856-4525; fax: /86-531856-5610. E-mail address: [email protected] (G. Xia).
Somatic hybridization provides plant breeders with the possibility of increasing genetic variability and overcoming sexual cross-incompatibility of plants [5,6]. Recently, symmetric and asymmetric somatic hybridization between remotely related species has been widely exploited [7 /9]. In a recent report, Szarka et al. [10] have shown that this approach can be successful in creating introgression materials between maize and wheat. In these experiments, a number of regenerants were produced after an extended period in culture; all had a maize-like phenotype, without any obvious morphological traits being expressed from the wheat parent, and all were sterile. In the present study, we have combined the genomes of wheat and maize via asymmetric somatic hybridization, in an attempt to generate novel material for the breeding, potentially, of both parental species. In addition, somatic hybrids from different combinations should provide relevant materials both to understand the mechanism of maize chromosome elimination in the zygote, and to study interactions between the wheat nucleus and the maize cytoplasm, and vice versa.
0168-9452/03/$ - see front matter # 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0168-9452(03)00287-5
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2. Materials and methods 2.1. Materials Two independent cell cultures of common wheat (Triticum aestivum L. cv. Jinan177, 2n/42), and one of maize (Zea mays L. cv. Qi319, 2n/20) were used. Embryogenic calli of both wheat and maize were induced from excised immature embryos on MB2 medium (MB medium [11] containing 1 /2 mg/l 2,4-D) at 25 8C. Yellow, granular calli were subcultured and small granular calli were selected for suspension [12]. The wheat embryogenic calli (named 176, with 30 /38 chromosomes [13]) were sub-cultured on MB2 media for 3 years, and the suspension cells (named Cha9, with 21/ 29 chrmosomes [14]) were sub-cultured on MB2 liquid media for 8 years. Maize Qi319 calli (with 22 /30 chromosomes) were sub-cultured for 2 years on MB2 solid media. For the fusion experiments, 176 and Qi319 calli were used after 6 /7 days of sub-culture and Cha9 suspension was used after 3/4 days of sub-culture.
3. Methods 3.1. Protoplast isolation, fusion and culture For 176 and Qi319, yellow and fresh calli of wheat line 176 and maize line Qi319 were cut into small pieces, and were incubated in enzyme solution (0.6 M mannitol, 5 mM CaCl2, 1.5% cellulase Onozyka RS and 0.3% pectolyase Y-23) for 3 and 4 h, respectively. Wheat suspension line Cha9 was incubated with the enzyme solution for 4 h directly until large amounts of protoplasts was isolated. For asymmetric hybridization, monolayer protoplasts of maize line Qi319 were put onto 3 cm petri dishes and irradiated by UV at an intensity of 380 mW/cm2 for 30 s or 1 min, and mixed with wheat protoplasts. Seven combinations (including controls) were tried in our experiments, a: Qi319 (UV 30 s); b: Cha9 (/) 176; c: Cha9 (/) Qi319 (UV 30 s); d: 176 (/) Qi319 (UV 30 s); e: Cha9 (/) 176 (/) Qi319; f: Cha9 (/) 176 (/) Qi319 (UV 30 s); g: Cha9 (/) 176 (/) Qi319 (UV 1 min). Cell fusion was undertaken as following: protoplast mixture was precipitated for 30 min, then four drops of PEG solution (0.11 M glucose, 0.09 M Ca(NO3)2, 40% (w/v) PEG 6000) were added to the border of the protoplast mixture and incubated for 15/20 min. Add four drops of 0.27 M Ca(NO3)2 for two times, incubate for 10 min each time. Replace the solution with washing buffer for two times, incubate for 10 min each time. Replace the washing buffer with P5 liquid medium [11], incubate for 10 min. Refresh the P5 medium and culture the fusion products in the dark at 25 8C.
After the regenerated calli grew to about 2 mm in diameter, they were transferred onto MB2 medium. When the calli were large enough, they were transferred onto IB medium (MB medium added with 0.5 mg/l IAA and 0.5 mg/l 6-BA) for plant regeneration. The plants were cultured on S medium (B5 medium added with 5 mg/l NAA, 1 mg/l IBA and 0.1 mg/l 6-BA) and SD medium (B5 medium added with 5 mg/l NAA, 1 mg/l IBA, 0.1 mg/l 6-BA and 1 mg/l chlorocholine chloride) for rooting and strengthening. Plants with strong roots were planted into soil. 3.2. Isozyme analysis Plant materials (calli or leaves) were ground in 0.1 M Tris /HCl (pH 8.3) using a ratio of 1:2 (weight of plant material to volume of buffer) and were centrifuged at 12 000 rpm for 10 min. The supernatant was electrophoresed with PAGE in 3% spacer gel-10% separating gel. For peroxidase isozyme analysis, the gel was stained with 100 ml staining buffer containing 20 ml 2% benzidine, 70.4 mg vitamin C, 20 ml 0.6% H2O2 and 60 ml distilled water. For esterase analysis, the gel was stained with 90 ml phosphate buffer (pH 6.4) containing 90 mg fast blue B salt, 6 ml 2% alph-Naphthyl acetate, 3 ml 2% beta-Naphthyl acetate (both dissolved in a little acetone and 80% alcohol) [15]. 3.3. PCR analysis DNA was extracted using a CTAB method. Each 20 ml PCR solution contained 2 ml 10/PCR buffer, 1.5 mM MgCl2, 100 mM of each dNTP, 50 ng template DNA, and 1 U r-TAQ (Takara, P.R.China). For RAPD analysis, 20 ng RAPD primer (OPERON, USA) was included; for 5S rDNA spacer sequence analysis, each primer (sequences as reported by Zhou et al. [16]) was present at 1 mM. The RAPD PCR program was: 94 8C 5 min; 94 8C 10 s, 36 8C 30 s, 72 8C 1 min, 45 cycles; 72 8C 7 min, while the specific PCR program was 94 8C 5 min; 94 8C 1 min, 60 8C 1 min 20 s, 72 8C 2 min, 35 cycles; 72 8C 7 min. PCR products were electrophoresed in 1.5% (RAPDs) and 2.5% (5S rDNA) agarose gels. Gels were stained with 0.5 mg/ml ethdium bromide and analyzed with Syngene gel imaging system (Syngene, USA). For SSR analysis, four pairs of chloroplast SSR primers WCt6, WCt7, WCt11 and WCt13 [17] were used. The program was: 94 8C 5 min; 94 8C 1 min, 55 8C 1 min 15 s, 72 8C 2 min, 35 cycles; 72 8C 7 min. The PCR products were run out on 4.0 /6.0% PAGE containing 7 mol/l urea and visualized as following: gel was put into 10% glacial acetic acid, gently rotated for 40 min, and washed with double distilled water for three times (5, 5 and 10 min, respectively), then put into staining buffer (1 g AgNO3 and 1.5 ml formaldehyde in 1 l double
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distilled water), rotated gently for 30 min; washed with double distilled water for 5 s, rotated gently in developer (1.5 ml formaldehyde and 200 ml 10 mg/ml sodium thiosulphate in 1 l pre-cooled 3% (w/v) Na2CO3) until the bands became clear, then put into 10% glacial acetic acid and rotated for 3 /5 min, washed with double distilled water for two times, 2 min each time. The gel was photographed after dried in room temperature. 3.4. RFLP analysis Total genomic DNA was restricted with Hin dIII. Each 400 ml restriction solution contained 10 mM Tris / HCl (pH 7.5), 10 mM MgCl2, 1 mM dithiothreitol, 50 mM NaCl, 75 U Hin dIII (Takara, P.R. China) and 20 mg total genomic DNA. The reaction was performed at 37 8C overnight. The restriction products were electrophoresed in 0.8 /1.0% agarose at 4 8C overnight. The DNA was transferred onto Hybond nylon membrane (Amersham-Phamarcia, UK) [18]. The 3.94 kb Bam HIEco RI cox1 fragment from pBN6601 and the 2.7 kb Hin dIII atp6 fragment were used as probes for cytogenome analysis [19]. Probe labeling and blot analysis was performed with ECL Random Labelling and Detection System (Version II), Amersham-Phamarcia, following the manufacturers’ instructions.
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Table 1 Calli regeneration and plant differentiation of the combinations/ controls Combinations/controls
Calli regeneration
Plant differentiation
Qi319 (UV 30 s) Cha9 (/) 176 176 (/) Qi319 (UV 30 s) Cha9 (/) Qi319 (UV 30 s) Cha9 (/) 176 (/) Qi319 Cha9 (/) 176 (/) Qi319 (UV 30 s) Cha9 (/) 176 (/) Qi319 (UV 1 min)
None None None
None None None
one clone/three dishes, slow growing 25 clones/three dishes, fast growing 10 clones/three dishes, fast growing 30 clones/six dishes, fast growing
None Albinos Green plants from eight clones None
essential for the regeneration and normal growing of calli and plants; the plants regenerated from this combination must therefore be hybrids of Cha9, 176 and Qi319 (UV 30 s). Interestingly, calli derived from the same combination, but where Qi319 was not treated with UV, regenerated only albino plants (Fig. 1A); while for the combination Cha9 (/) 176 (/) Qi319 (UV 1 min), calli were produced, but no plants could be regenerated. 4.2. Morphology
3.5. Chromosome preparation for GISH The in situ hybridization procedure followed Zhou et al. [14]. Maize genomic probe was prepared by autoclaving total DNA of maize Qi319 for 5 min. Images were generated on a Nikon Eclipse E600 fluorescence microscope and captured with a Nikon Coolpix 990 digital camera.
The morphology of the hybrids regenerated from Cha9 (/) 176 (/) Qi319 (UV 30 s) resembled that of wheat (Fig. 1B), although some regenerants possessed branched spikes (Fig. 1C). This kind of mutation often happens in the tissue culture of maize [20], but had not been reported in the tissue culture of wheat. 4.3. Isozyme analysis
4. Results 4.1. Calli and plant regeneration Calli and plant regeneration of the seven combinations/controls are shown in Table 1. As shown in Table 1, the optimum combination was Cha9 (/) 176 (/) Qi319 (UV 30 s). Ten callus clones were formed, which grew fast on MB2 medium, and green plants were regenerated frequently from eight clones. Many seedlings flowered, but all were sterile. Control Qi319 (UV 30 s) did not regenerate into calli; for combination Cha9 (/) Qi319 (UV 30 s), although a few calli regenerated, the frequency was very low and the calli grew much slower than those from Cha9 (/) 176 (/) Qi319 (UV 30 s), and no plants were regenerated. In the combination 176 (/) Qi319 (UV 30 s), no calli were formed. Thus, in the combination Cha9 (/) 176 (/) Qi319 (UV 30 s), both Cha9 and 176 were
Peroxidase and esterase were used to analyse the hybrid plants. The peroxidase profiles are shown in Fig. 2. The profile of all calli included bands characteristic of Cha9 and 176; those of clones 1, 3, 4, 6, 7, 10 had a band that co-migrated with one from Qi319, not present in the profile of either Cha9 or 176; and those of clones 1, 4, 6, 7 included bands not present in any parental profile (Fig. 2A). Among the leaf-derived peroxidase profiles, all clones contained bands characteristic of both Jinan177 and Qi319, while clones 1, 5, 7 also contained new bands (Fig. 2B). This evidence supports the hybridity status of the materials. Esterase profiles were similarly supportive (data not shown). 4.4. RAPD analysis Seven of the nineteen primers used produced evidence for hybridity. Inspection of callus DNA RAPDs using OPF-05 (Fig. 3A) shows that all the clones contained
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Fig. 1. Morphology of the regenerated plants. (A) Albino plantlet regenerated from a callus derived from the fusion combination Cha9 (/) 176 (/) Qi319. (B) Hybrid plant with normal morphology, regenerated from combination Cha9 (/) 176 (/) Qi319 (UV 30 s). (C) Hybrid plant with branching spikes regenerated from combination Cha9 (/) 176 (/) Qi319 (UV30 s). 0/: Three spikes on one tiller.
specific sequences present in Cha9 and 176; clones 1, 2, 3, 6, 7, 10 contained specific sequences from Qi319; while clones 1, 2, 3, 5, 6, 7 contained new sequences not present in either parent. For leaf DNA RAPDs generated with primer OPH-08, all clones showed specific bands from Jinan177 and new bands, while clones 1, 2, 3, 6, 7 contained products from Qi319 (Fig. 3B). 4.5. 5S rDNA spacer sequence analysis The result of 5S rDNA spacer sequence analysis of leaf DNA is shown in Fig. 4. The regenerated plants all had the fragments specific for wheat Jinan177 and one or more sequence not present in either parent. The new bands may result from the recombination of the parental genome.
4.6. RFLP analysis The hybridization patterns of DNA from calli with cox1 and atp6 probes are shown in Fig. 5. For cox1 , the analyzed clones all contained fragments derived from each of the three parents, along with some new fragments (Fig. 5A). For the atp6 probe, the RFLP profiles of the analyzed clones all resemble those of Cha9 and 176, although clones 3 and 5 contain new fragments (Fig. 5B). These results suggest that the mitochondrial genome of the hybrids is mixed. This may result from either coexistence of intact wheat and maize mitochondria, or the formation of some hybrid mitochondria as a result of a recombination mechanism. The majority of the mitochrondrial genome, however, appears to be wheat-based.
Fig. 2. Peroxidase analysis of the calli and plants regenerated from somatic hybridization combination Cha9 (/) 176 (/) Qi319 (UV 30 s) (A) Calli (B) Plants. Cha9, 176: the two lines of wheat Jinan177 Qi319: calli or leaves of maize Qi319 177: leaves of wheat Jinan177. -: Characteristic bands of wheat. : Characteristic bands of maize. 0/: New bands.
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Fig. 3. RAPD analysis results of calli and plants regenerated from somatic hybridization combination Cha9 (/) 176 (/) Qi319 (UV 30 s). (A) Result of calli with primer OPF-5; (B) result of plants with primer OPH-8. M: lDNA/Hin dIII/Eco RI molecular marker. 1, 2, 3, 5, 6, 7, 10: Number of the regenerated clones. Cha9, 176: the two lines of wheat Jinan177. Qi319: calli or leaves of maize Qi319. 177: leaves of wheat Jinan177. -: Characteristic bands of wheat. : Characteristic bands of maize. 0/: New bands.
Fig. 4. 5 s rDNA spacer sequence analysis of the plants regenerated from somatic hybridization combination Cha9 (/) 176 (/) Qi319 (UV 30 s). M: lDNA/Hin dIII/Eco RI molecular marker. 1, 2, 3, 5, 6, 7: Number of the regenerated clones. 177: leaves of wheat Jinan177. Qi319: leaves of maize Qi319. -: Characteristic band of wheat. 0/: New bands.
4.7. SSR analysis The SSR analysis results with WCt6 and WCt7 are shown in Fig. 6. The profiles of the clones were all identical to that from Cha9 and 176, with no evidence for the presence of maize DNA in the chloroplast. Similar results were obtained with WCt11 and WCt13. These results suggest that the chloroplast genome of the hybrids was inherited from Cha9 and 176.
Fig. 6. SSR analysis result of the calli regenerated from somatic hybridization combination Cha9 (/) 176 (/) Qi319 (UV 30 s). (Left) primer WCt6 (Right) primer WCt7. M: molecular marker. 1, 2, 3, 5, 6, 7, 10: Number of the regenerated clones. Cha9, 176: the two lines of wheat Jinan177. Qi319: calli of maize Qi319 -: Characteristic bands of wheat :Characteristic bands of maize
4.8. GISH analysis In situ hybridization results showed that hybrid chromosome number ranged between 32 and 44, mostly in the range of 36/42. Chromosomes from the root tips
Fig. 5. RFLP results of the calli regenerated from the somatic hybridization combination Cha9 (/) 176 (/) Qi319 (UV 30 s). (A) Result with cox1 probe; (B) result with atp6 probe M: lDNA/Hin dIII/Eco RI molecular marker. 1, 2, 3, 4, 5: Number of the regenerated clones. Cha9, 176: the two lines of wheat Jinan177 Qi319: calli of maize Qi319. -: Characteristic bands of wheat. : Characteristic bands of maize. 0/: New bands.
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Fig. 7. GISH results on root tip cells of the regenerated plants, wheat Jinan177 and maize Qi319. Total genomic DNA of Qi319 was labeled as probe. (A) clone 1 regenerated from combination Cha9 (/) 176 (/) Qi319 (UV 30 s). (Left) prophase cell; (Right) metaphase cell. (B) whet Jian177 (C): maize Qi319. 0/: Hybridization sites on the chromosomes. Bar: 5 mm.
of clone 1 regenerating plants, wheat Jinan 177 and maize Qi319 are shown in Fig. 7. From Fig. 7A, it is clear that many hybridization sites are dispersed throughout the chromatin/chromosomes. Other analyzed hybrid clones showed the same results. This shows that multiple short chromatin lengths of maize have been introgressed into the wheat genome.
5. Discussion DH progenies of wheat are useful in breeding and many DH populations have been used for genetic mapping. However, 2 /5% of the wheat DH plants derived from wheat /maize crosses displayed morphological variation, which is incompatible with their theoretical homogeneity [1]. Chen et al. [3] have reported that a highly repeated sequence of maize was transferred into the DH progenies by crossing wheat with maize, thus proving at the molecular level that maize sequences
can be transferred into DH progenies of wheat through fertilization, although at a very low frequency. Szarka et al. [10] have recently reported obtaining a small number of hybrid plants of maize and wheat through symmetric somatic hybridization, but, in contrast to our results, the hybrid plants were phenotypically more similar to maize than to wheat, and showed dispersion of wheat chromatin across the maize genome. The contrasting results may due to the different materials and methods usedsuspension cells of maize and mesophyllous cells of wheat via symmetric somatic hybridization, versus cultured cells of maize and wheat via asymmetric somatic hybridization. Our results suggest that in asymmetric somatic hybridization derived hybrids, although greatly resembling wheat in phenotype, maize DNA is transferred into wheat at a significantly higher frequency, and that the possibility of transferring functional DNA sequence could be increased. Thus in principle, it may not be impossible to obtain hybrids of wheat and maize that
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resemble wheat and possess some agronomic traits of maize. Of particular significance is the possibility of obtaining transfer of non-nuclear maize sequences, which is not possible in sexually derived hybrids. Some important traits, notably the C4/C3 character, thus may be transferable into wheat by this path. The phenomenon of complementary regeneration of hybrid plants is not uncommon [11,14,21,22]. This refers to the situation where the protoplasts of both parents are either unable to regenerate, or have low regeneration ability, while the hybrids have good regeneration ability and can form normal plants. In our early studies, hybrid plants were obtained from the fusion of protoplasts derived from suspension cells of wheat Jinan 177 with those from different calli or suspension cultures of grasses. In the present report, the combinations Cha9 (/) Qi319 (UV 30 s) and 176 (/) Qi319 (UV 30 s) could either not form calli, or formed calli that had no ability to differentiate. Only the combination Cha9 (/) 176 (/) Qi319 (UV 30 s) was able to generate green plants through complementation of their genomes. Szarka et al. [10] have suggested that a prolonged in vitro culture period improves the viability and regeneration potential, possibly because this favors the situation where many chromosomes are lost. This may be the case in our experiments as well, since the material used had been in culture for some years, and the two types of parent wheat cultures only had 30 /38 and 21/29 chromosomes. Both Cha9 and 176 were derived from Jinan 177, but it seemed that both were required for successful regeneration of plants. Thus we suggest that Cha9 and 176 are non-identical genetically, and act in a complementary fashion in the fusion process. This method of mixing two types of wheat cultures as fusion recipient has been used in some of our previous studies to obtain hybrid plants [13,23]. Apart from combination Cha9 (/) 176 (/) Qi319 (UV 30 s), we also tried combinations Cha9 (/) 176 (/) Qi319 (symmetric fusion) and Cha9 (/) 176 (/) Qi319 (UV 1 min). As described, Cha9 (/) 176 (/) Qi319 (UV 1 min) only formed calli, while Cha9 (/) 176 (/) Qi319 regenerated albino plantlets. For Cha9 (/) 176 (/) Qi319 (UV 1 min), the reason for the failure may be that the maize DNA was so seriously damaged that regenerable hybrids could not be formed. Also, too many fragments of maize DNA inserting into wheat may have seriously interfered with the expression of wheat genes (including genome imbalance and insertional inactivation) and thus influenced the regeneration ability of hybrid calli. For Cha9 (/) 176 (/) Qi319, perhaps the maize insertions were too few to allow the regeneration of normal plants. Previous studies on the cytogenomic composition of somatic hybrids have shown different scenarios. For example: among the mitochondria, some can be rearranged while others can be identical to one (or both) of the parents; chloroplasts can be derived entirely from
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only one parent [24]; mitochondria can recombine, chloroplast derive from one parent [25]; mitochondria can be identical to one of the parents, coexist, or recombine, with the chloroplasts identical to one parent [26]; Spangenberg et al. [19] studied the relationship between cytogenome constitution and the dose of X-ray and found that, with an increasing of the dose of radiation, the cytogenome became increasingly more like that of the recipient. In our study, the mitochondrial genome of the hybrids were the result of the coexistence or the recombination of the parents; while in contrast, the chloroplast genome of the hybrids were the same as that of the wheat parents. This is only partially consistent with the situation found in somatic hybrids of wheat and Haynaldia villosa , where mitochondria coexisted or recombined, but the chloroplasts segregated and recombined randomly [14].
Acknowledgements This study was supported by National Natural Science Foundation of China, No. 30070397, Transcentury Training Program Foundation for the Talents by the Ministry of Education in China, and National 863 High Technology Research and Development Project No. 2001AA241032. We are grateful to Dr Robert Koebner (John Innes Centre, UK) for language correction. Thanks Dr. Suiyun Chen and Jing Wang for their help in SSR and GISH analysis.
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Integration of maize nuclear and mitochondrial DNA into the wheat genome through somatic hybridization Chunhui Xu, Guangmin Xia *, Daying Zhi, Fengning Xiang, Huimin Chen School of Life Sciences, Shandong University, Jinan 250100 Shandong, China Received 24 February 2003; received in revised form 19 June 2003; accepted 19 June 2003
Abstract Protoplasts were isolated from cultured cells of wheat and maize and fused using PEG. Calli and green plants were regenerated following irradiation of the maize, and some tested positive for hybridity using morphological, isozymic and various DNA-based marker systems. Genomic in situ hybridization (GISH) of selected maize-carrying regenerants showed that some maize chromatin was dispersed throughout the wheat nuclear genome. Cytogenomic analysis, using RFLP and SSR, demonstrated the presence of both wheat and maize loci in the mitochondrial genome, so that, in the hybrid individuals, either distinct mitochondria from each parent coexist and/or recombinant mitochondria have been generated. With respect to the chloroplast genome, there was no evidence of the presence of any introgression from maize. # 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Wheat; Maize; Asymmetric somatic hybridization; Nuclear genome; Cytogenome; Integration
1. Introduction The two cereals, wheat and maize, are among the most important crop species. Attempts to combine their genomes via sexual hybridization have failed, because in hybrid embryos, maize chromosomes are eliminated at an early developmental stage, so that the chromosomes present in the mature embryo are all of wheat origin [1,2]. No evidence that these haploid plants express any maize genes has been produced, and plant morphology is uniformly wheat-like [2]. However, there is some evidence that high copy repetitive sequences can be successfully transferred from maize to wheat by crossing [3,4]. Since hybrid embryos can be rescued in tissue culture, pollinating wheat with maize has been widely used as a means for constructing haploid wheat plants, whose chromosome complement can later be doubled to generate doubled haploid (DH) progenies.
* Corresponding author. Tel.: /86-531-856-4525; fax: /86-531856-5610. E-mail address: [email protected] (G. Xia).
Somatic hybridization provides plant breeders with the possibility of increasing genetic variability and overcoming sexual cross-incompatibility of plants [5,6]. Recently, symmetric and asymmetric somatic hybridization between remotely related species has been widely exploited [7 /9]. In a recent report, Szarka et al. [10] have shown that this approach can be successful in creating introgression materials between maize and wheat. In these experiments, a number of regenerants were produced after an extended period in culture; all had a maize-like phenotype, without any obvious morphological traits being expressed from the wheat parent, and all were sterile. In the present study, we have combined the genomes of wheat and maize via asymmetric somatic hybridization, in an attempt to generate novel material for the breeding, potentially, of both parental species. In addition, somatic hybrids from different combinations should provide relevant materials both to understand the mechanism of maize chromosome elimination in the zygote, and to study interactions between the wheat nucleus and the maize cytoplasm, and vice versa.
0168-9452/03/$ - see front matter # 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0168-9452(03)00287-5
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2. Materials and methods 2.1. Materials Two independent cell cultures of common wheat (Triticum aestivum L. cv. Jinan177, 2n/42), and one of maize (Zea mays L. cv. Qi319, 2n/20) were used. Embryogenic calli of both wheat and maize were induced from excised immature embryos on MB2 medium (MB medium [11] containing 1 /2 mg/l 2,4-D) at 25 8C. Yellow, granular calli were subcultured and small granular calli were selected for suspension [12]. The wheat embryogenic calli (named 176, with 30 /38 chromosomes [13]) were sub-cultured on MB2 media for 3 years, and the suspension cells (named Cha9, with 21/ 29 chrmosomes [14]) were sub-cultured on MB2 liquid media for 8 years. Maize Qi319 calli (with 22 /30 chromosomes) were sub-cultured for 2 years on MB2 solid media. For the fusion experiments, 176 and Qi319 calli were used after 6 /7 days of sub-culture and Cha9 suspension was used after 3/4 days of sub-culture.
3. Methods 3.1. Protoplast isolation, fusion and culture For 176 and Qi319, yellow and fresh calli of wheat line 176 and maize line Qi319 were cut into small pieces, and were incubated in enzyme solution (0.6 M mannitol, 5 mM CaCl2, 1.5% cellulase Onozyka RS and 0.3% pectolyase Y-23) for 3 and 4 h, respectively. Wheat suspension line Cha9 was incubated with the enzyme solution for 4 h directly until large amounts of protoplasts was isolated. For asymmetric hybridization, monolayer protoplasts of maize line Qi319 were put onto 3 cm petri dishes and irradiated by UV at an intensity of 380 mW/cm2 for 30 s or 1 min, and mixed with wheat protoplasts. Seven combinations (including controls) were tried in our experiments, a: Qi319 (UV 30 s); b: Cha9 (/) 176; c: Cha9 (/) Qi319 (UV 30 s); d: 176 (/) Qi319 (UV 30 s); e: Cha9 (/) 176 (/) Qi319; f: Cha9 (/) 176 (/) Qi319 (UV 30 s); g: Cha9 (/) 176 (/) Qi319 (UV 1 min). Cell fusion was undertaken as following: protoplast mixture was precipitated for 30 min, then four drops of PEG solution (0.11 M glucose, 0.09 M Ca(NO3)2, 40% (w/v) PEG 6000) were added to the border of the protoplast mixture and incubated for 15/20 min. Add four drops of 0.27 M Ca(NO3)2 for two times, incubate for 10 min each time. Replace the solution with washing buffer for two times, incubate for 10 min each time. Replace the washing buffer with P5 liquid medium [11], incubate for 10 min. Refresh the P5 medium and culture the fusion products in the dark at 25 8C.
After the regenerated calli grew to about 2 mm in diameter, they were transferred onto MB2 medium. When the calli were large enough, they were transferred onto IB medium (MB medium added with 0.5 mg/l IAA and 0.5 mg/l 6-BA) for plant regeneration. The plants were cultured on S medium (B5 medium added with 5 mg/l NAA, 1 mg/l IBA and 0.1 mg/l 6-BA) and SD medium (B5 medium added with 5 mg/l NAA, 1 mg/l IBA, 0.1 mg/l 6-BA and 1 mg/l chlorocholine chloride) for rooting and strengthening. Plants with strong roots were planted into soil. 3.2. Isozyme analysis Plant materials (calli or leaves) were ground in 0.1 M Tris /HCl (pH 8.3) using a ratio of 1:2 (weight of plant material to volume of buffer) and were centrifuged at 12 000 rpm for 10 min. The supernatant was electrophoresed with PAGE in 3% spacer gel-10% separating gel. For peroxidase isozyme analysis, the gel was stained with 100 ml staining buffer containing 20 ml 2% benzidine, 70.4 mg vitamin C, 20 ml 0.6% H2O2 and 60 ml distilled water. For esterase analysis, the gel was stained with 90 ml phosphate buffer (pH 6.4) containing 90 mg fast blue B salt, 6 ml 2% alph-Naphthyl acetate, 3 ml 2% beta-Naphthyl acetate (both dissolved in a little acetone and 80% alcohol) [15]. 3.3. PCR analysis DNA was extracted using a CTAB method. Each 20 ml PCR solution contained 2 ml 10/PCR buffer, 1.5 mM MgCl2, 100 mM of each dNTP, 50 ng template DNA, and 1 U r-TAQ (Takara, P.R.China). For RAPD analysis, 20 ng RAPD primer (OPERON, USA) was included; for 5S rDNA spacer sequence analysis, each primer (sequences as reported by Zhou et al. [16]) was present at 1 mM. The RAPD PCR program was: 94 8C 5 min; 94 8C 10 s, 36 8C 30 s, 72 8C 1 min, 45 cycles; 72 8C 7 min, while the specific PCR program was 94 8C 5 min; 94 8C 1 min, 60 8C 1 min 20 s, 72 8C 2 min, 35 cycles; 72 8C 7 min. PCR products were electrophoresed in 1.5% (RAPDs) and 2.5% (5S rDNA) agarose gels. Gels were stained with 0.5 mg/ml ethdium bromide and analyzed with Syngene gel imaging system (Syngene, USA). For SSR analysis, four pairs of chloroplast SSR primers WCt6, WCt7, WCt11 and WCt13 [17] were used. The program was: 94 8C 5 min; 94 8C 1 min, 55 8C 1 min 15 s, 72 8C 2 min, 35 cycles; 72 8C 7 min. The PCR products were run out on 4.0 /6.0% PAGE containing 7 mol/l urea and visualized as following: gel was put into 10% glacial acetic acid, gently rotated for 40 min, and washed with double distilled water for three times (5, 5 and 10 min, respectively), then put into staining buffer (1 g AgNO3 and 1.5 ml formaldehyde in 1 l double
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distilled water), rotated gently for 30 min; washed with double distilled water for 5 s, rotated gently in developer (1.5 ml formaldehyde and 200 ml 10 mg/ml sodium thiosulphate in 1 l pre-cooled 3% (w/v) Na2CO3) until the bands became clear, then put into 10% glacial acetic acid and rotated for 3 /5 min, washed with double distilled water for two times, 2 min each time. The gel was photographed after dried in room temperature. 3.4. RFLP analysis Total genomic DNA was restricted with Hin dIII. Each 400 ml restriction solution contained 10 mM Tris / HCl (pH 7.5), 10 mM MgCl2, 1 mM dithiothreitol, 50 mM NaCl, 75 U Hin dIII (Takara, P.R. China) and 20 mg total genomic DNA. The reaction was performed at 37 8C overnight. The restriction products were electrophoresed in 0.8 /1.0% agarose at 4 8C overnight. The DNA was transferred onto Hybond nylon membrane (Amersham-Phamarcia, UK) [18]. The 3.94 kb Bam HIEco RI cox1 fragment from pBN6601 and the 2.7 kb Hin dIII atp6 fragment were used as probes for cytogenome analysis [19]. Probe labeling and blot analysis was performed with ECL Random Labelling and Detection System (Version II), Amersham-Phamarcia, following the manufacturers’ instructions.
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Table 1 Calli regeneration and plant differentiation of the combinations/ controls Combinations/controls
Calli regeneration
Plant differentiation
Qi319 (UV 30 s) Cha9 (/) 176 176 (/) Qi319 (UV 30 s) Cha9 (/) Qi319 (UV 30 s) Cha9 (/) 176 (/) Qi319 Cha9 (/) 176 (/) Qi319 (UV 30 s) Cha9 (/) 176 (/) Qi319 (UV 1 min)
None None None
None None None
one clone/three dishes, slow growing 25 clones/three dishes, fast growing 10 clones/three dishes, fast growing 30 clones/six dishes, fast growing
None Albinos Green plants from eight clones None
essential for the regeneration and normal growing of calli and plants; the plants regenerated from this combination must therefore be hybrids of Cha9, 176 and Qi319 (UV 30 s). Interestingly, calli derived from the same combination, but where Qi319 was not treated with UV, regenerated only albino plants (Fig. 1A); while for the combination Cha9 (/) 176 (/) Qi319 (UV 1 min), calli were produced, but no plants could be regenerated. 4.2. Morphology
3.5. Chromosome preparation for GISH The in situ hybridization procedure followed Zhou et al. [14]. Maize genomic probe was prepared by autoclaving total DNA of maize Qi319 for 5 min. Images were generated on a Nikon Eclipse E600 fluorescence microscope and captured with a Nikon Coolpix 990 digital camera.
The morphology of the hybrids regenerated from Cha9 (/) 176 (/) Qi319 (UV 30 s) resembled that of wheat (Fig. 1B), although some regenerants possessed branched spikes (Fig. 1C). This kind of mutation often happens in the tissue culture of maize [20], but had not been reported in the tissue culture of wheat. 4.3. Isozyme analysis
4. Results 4.1. Calli and plant regeneration Calli and plant regeneration of the seven combinations/controls are shown in Table 1. As shown in Table 1, the optimum combination was Cha9 (/) 176 (/) Qi319 (UV 30 s). Ten callus clones were formed, which grew fast on MB2 medium, and green plants were regenerated frequently from eight clones. Many seedlings flowered, but all were sterile. Control Qi319 (UV 30 s) did not regenerate into calli; for combination Cha9 (/) Qi319 (UV 30 s), although a few calli regenerated, the frequency was very low and the calli grew much slower than those from Cha9 (/) 176 (/) Qi319 (UV 30 s), and no plants were regenerated. In the combination 176 (/) Qi319 (UV 30 s), no calli were formed. Thus, in the combination Cha9 (/) 176 (/) Qi319 (UV 30 s), both Cha9 and 176 were
Peroxidase and esterase were used to analyse the hybrid plants. The peroxidase profiles are shown in Fig. 2. The profile of all calli included bands characteristic of Cha9 and 176; those of clones 1, 3, 4, 6, 7, 10 had a band that co-migrated with one from Qi319, not present in the profile of either Cha9 or 176; and those of clones 1, 4, 6, 7 included bands not present in any parental profile (Fig. 2A). Among the leaf-derived peroxidase profiles, all clones contained bands characteristic of both Jinan177 and Qi319, while clones 1, 5, 7 also contained new bands (Fig. 2B). This evidence supports the hybridity status of the materials. Esterase profiles were similarly supportive (data not shown). 4.4. RAPD analysis Seven of the nineteen primers used produced evidence for hybridity. Inspection of callus DNA RAPDs using OPF-05 (Fig. 3A) shows that all the clones contained
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Fig. 1. Morphology of the regenerated plants. (A) Albino plantlet regenerated from a callus derived from the fusion combination Cha9 (/) 176 (/) Qi319. (B) Hybrid plant with normal morphology, regenerated from combination Cha9 (/) 176 (/) Qi319 (UV 30 s). (C) Hybrid plant with branching spikes regenerated from combination Cha9 (/) 176 (/) Qi319 (UV30 s). 0/: Three spikes on one tiller.
specific sequences present in Cha9 and 176; clones 1, 2, 3, 6, 7, 10 contained specific sequences from Qi319; while clones 1, 2, 3, 5, 6, 7 contained new sequences not present in either parent. For leaf DNA RAPDs generated with primer OPH-08, all clones showed specific bands from Jinan177 and new bands, while clones 1, 2, 3, 6, 7 contained products from Qi319 (Fig. 3B). 4.5. 5S rDNA spacer sequence analysis The result of 5S rDNA spacer sequence analysis of leaf DNA is shown in Fig. 4. The regenerated plants all had the fragments specific for wheat Jinan177 and one or more sequence not present in either parent. The new bands may result from the recombination of the parental genome.
4.6. RFLP analysis The hybridization patterns of DNA from calli with cox1 and atp6 probes are shown in Fig. 5. For cox1 , the analyzed clones all contained fragments derived from each of the three parents, along with some new fragments (Fig. 5A). For the atp6 probe, the RFLP profiles of the analyzed clones all resemble those of Cha9 and 176, although clones 3 and 5 contain new fragments (Fig. 5B). These results suggest that the mitochondrial genome of the hybrids is mixed. This may result from either coexistence of intact wheat and maize mitochondria, or the formation of some hybrid mitochondria as a result of a recombination mechanism. The majority of the mitochrondrial genome, however, appears to be wheat-based.
Fig. 2. Peroxidase analysis of the calli and plants regenerated from somatic hybridization combination Cha9 (/) 176 (/) Qi319 (UV 30 s) (A) Calli (B) Plants. Cha9, 176: the two lines of wheat Jinan177 Qi319: calli or leaves of maize Qi319 177: leaves of wheat Jinan177. -: Characteristic bands of wheat. : Characteristic bands of maize. 0/: New bands.
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Fig. 3. RAPD analysis results of calli and plants regenerated from somatic hybridization combination Cha9 (/) 176 (/) Qi319 (UV 30 s). (A) Result of calli with primer OPF-5; (B) result of plants with primer OPH-8. M: lDNA/Hin dIII/Eco RI molecular marker. 1, 2, 3, 5, 6, 7, 10: Number of the regenerated clones. Cha9, 176: the two lines of wheat Jinan177. Qi319: calli or leaves of maize Qi319. 177: leaves of wheat Jinan177. -: Characteristic bands of wheat. : Characteristic bands of maize. 0/: New bands.
Fig. 4. 5 s rDNA spacer sequence analysis of the plants regenerated from somatic hybridization combination Cha9 (/) 176 (/) Qi319 (UV 30 s). M: lDNA/Hin dIII/Eco RI molecular marker. 1, 2, 3, 5, 6, 7: Number of the regenerated clones. 177: leaves of wheat Jinan177. Qi319: leaves of maize Qi319. -: Characteristic band of wheat. 0/: New bands.
4.7. SSR analysis The SSR analysis results with WCt6 and WCt7 are shown in Fig. 6. The profiles of the clones were all identical to that from Cha9 and 176, with no evidence for the presence of maize DNA in the chloroplast. Similar results were obtained with WCt11 and WCt13. These results suggest that the chloroplast genome of the hybrids was inherited from Cha9 and 176.
Fig. 6. SSR analysis result of the calli regenerated from somatic hybridization combination Cha9 (/) 176 (/) Qi319 (UV 30 s). (Left) primer WCt6 (Right) primer WCt7. M: molecular marker. 1, 2, 3, 5, 6, 7, 10: Number of the regenerated clones. Cha9, 176: the two lines of wheat Jinan177. Qi319: calli of maize Qi319 -: Characteristic bands of wheat :Characteristic bands of maize
4.8. GISH analysis In situ hybridization results showed that hybrid chromosome number ranged between 32 and 44, mostly in the range of 36/42. Chromosomes from the root tips
Fig. 5. RFLP results of the calli regenerated from the somatic hybridization combination Cha9 (/) 176 (/) Qi319 (UV 30 s). (A) Result with cox1 probe; (B) result with atp6 probe M: lDNA/Hin dIII/Eco RI molecular marker. 1, 2, 3, 4, 5: Number of the regenerated clones. Cha9, 176: the two lines of wheat Jinan177 Qi319: calli of maize Qi319. -: Characteristic bands of wheat. : Characteristic bands of maize. 0/: New bands.
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Fig. 7. GISH results on root tip cells of the regenerated plants, wheat Jinan177 and maize Qi319. Total genomic DNA of Qi319 was labeled as probe. (A) clone 1 regenerated from combination Cha9 (/) 176 (/) Qi319 (UV 30 s). (Left) prophase cell; (Right) metaphase cell. (B) whet Jian177 (C): maize Qi319. 0/: Hybridization sites on the chromosomes. Bar: 5 mm.
of clone 1 regenerating plants, wheat Jinan 177 and maize Qi319 are shown in Fig. 7. From Fig. 7A, it is clear that many hybridization sites are dispersed throughout the chromatin/chromosomes. Other analyzed hybrid clones showed the same results. This shows that multiple short chromatin lengths of maize have been introgressed into the wheat genome.
5. Discussion DH progenies of wheat are useful in breeding and many DH populations have been used for genetic mapping. However, 2 /5% of the wheat DH plants derived from wheat /maize crosses displayed morphological variation, which is incompatible with their theoretical homogeneity [1]. Chen et al. [3] have reported that a highly repeated sequence of maize was transferred into the DH progenies by crossing wheat with maize, thus proving at the molecular level that maize sequences
can be transferred into DH progenies of wheat through fertilization, although at a very low frequency. Szarka et al. [10] have recently reported obtaining a small number of hybrid plants of maize and wheat through symmetric somatic hybridization, but, in contrast to our results, the hybrid plants were phenotypically more similar to maize than to wheat, and showed dispersion of wheat chromatin across the maize genome. The contrasting results may due to the different materials and methods usedsuspension cells of maize and mesophyllous cells of wheat via symmetric somatic hybridization, versus cultured cells of maize and wheat via asymmetric somatic hybridization. Our results suggest that in asymmetric somatic hybridization derived hybrids, although greatly resembling wheat in phenotype, maize DNA is transferred into wheat at a significantly higher frequency, and that the possibility of transferring functional DNA sequence could be increased. Thus in principle, it may not be impossible to obtain hybrids of wheat and maize that
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resemble wheat and possess some agronomic traits of maize. Of particular significance is the possibility of obtaining transfer of non-nuclear maize sequences, which is not possible in sexually derived hybrids. Some important traits, notably the C4/C3 character, thus may be transferable into wheat by this path. The phenomenon of complementary regeneration of hybrid plants is not uncommon [11,14,21,22]. This refers to the situation where the protoplasts of both parents are either unable to regenerate, or have low regeneration ability, while the hybrids have good regeneration ability and can form normal plants. In our early studies, hybrid plants were obtained from the fusion of protoplasts derived from suspension cells of wheat Jinan 177 with those from different calli or suspension cultures of grasses. In the present report, the combinations Cha9 (/) Qi319 (UV 30 s) and 176 (/) Qi319 (UV 30 s) could either not form calli, or formed calli that had no ability to differentiate. Only the combination Cha9 (/) 176 (/) Qi319 (UV 30 s) was able to generate green plants through complementation of their genomes. Szarka et al. [10] have suggested that a prolonged in vitro culture period improves the viability and regeneration potential, possibly because this favors the situation where many chromosomes are lost. This may be the case in our experiments as well, since the material used had been in culture for some years, and the two types of parent wheat cultures only had 30 /38 and 21/29 chromosomes. Both Cha9 and 176 were derived from Jinan 177, but it seemed that both were required for successful regeneration of plants. Thus we suggest that Cha9 and 176 are non-identical genetically, and act in a complementary fashion in the fusion process. This method of mixing two types of wheat cultures as fusion recipient has been used in some of our previous studies to obtain hybrid plants [13,23]. Apart from combination Cha9 (/) 176 (/) Qi319 (UV 30 s), we also tried combinations Cha9 (/) 176 (/) Qi319 (symmetric fusion) and Cha9 (/) 176 (/) Qi319 (UV 1 min). As described, Cha9 (/) 176 (/) Qi319 (UV 1 min) only formed calli, while Cha9 (/) 176 (/) Qi319 regenerated albino plantlets. For Cha9 (/) 176 (/) Qi319 (UV 1 min), the reason for the failure may be that the maize DNA was so seriously damaged that regenerable hybrids could not be formed. Also, too many fragments of maize DNA inserting into wheat may have seriously interfered with the expression of wheat genes (including genome imbalance and insertional inactivation) and thus influenced the regeneration ability of hybrid calli. For Cha9 (/) 176 (/) Qi319, perhaps the maize insertions were too few to allow the regeneration of normal plants. Previous studies on the cytogenomic composition of somatic hybrids have shown different scenarios. For example: among the mitochondria, some can be rearranged while others can be identical to one (or both) of the parents; chloroplasts can be derived entirely from
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only one parent [24]; mitochondria can recombine, chloroplast derive from one parent [25]; mitochondria can be identical to one of the parents, coexist, or recombine, with the chloroplasts identical to one parent [26]; Spangenberg et al. [19] studied the relationship between cytogenome constitution and the dose of X-ray and found that, with an increasing of the dose of radiation, the cytogenome became increasingly more like that of the recipient. In our study, the mitochondrial genome of the hybrids were the result of the coexistence or the recombination of the parents; while in contrast, the chloroplast genome of the hybrids were the same as that of the wheat parents. This is only partially consistent with the situation found in somatic hybrids of wheat and Haynaldia villosa , where mitochondria coexisted or recombined, but the chloroplasts segregated and recombined randomly [14].
Acknowledgements This study was supported by National Natural Science Foundation of China, No. 30070397, Transcentury Training Program Foundation for the Talents by the Ministry of Education in China, and National 863 High Technology Research and Development Project No. 2001AA241032. We are grateful to Dr Robert Koebner (John Innes Centre, UK) for language correction. Thanks Dr. Suiyun Chen and Jing Wang for their help in SSR and GISH analysis.
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