Hybrid Calli Research Articles

Development of salt tolerant callus cultures by somatic hybridization between Oryza sativa and mangrove grass Myriostachya wightiana

Abstract Rice is one of the agronomically and nutritionally important cereal crops. It provides food for more than 50% of the world's population. However, the yield of rice crop is decreased by the salinity stress. This can be overcome by the development of salt-resistant rice varieties. In this study salt resistant callus cultures were developed by the somatic hybridization between Oryza sativa and Myriostachya wightiana. To optimize the isolation and fusion of protoplasts, the experiments were carried out with three different enzyme mixtures and different incubation periods. To know the salt tolerant ability, growth and total antioxidant activity were measured in the calli of Oryza sativa, Myriostachya wightiana, and hybrid at the different NaCl concentrations. In Oryza sativa high viable protoplasts yield was obtained by Enzyme mixture B (6.1 ± 0.2 × 106) whereas, Myriostachya wightiana gives high viable protoplasts yield by Enzyme Mixture C (3.4 ± 0.2 × 106). Optimum digestion time for the Oryza sativa was 12 h, which gave the highest number that is, 6.36 ± 0.15 × 106 protoplasts/g FW and viability were 91.2 ± 1.05%. Optimum digestion time for the Myriostachya wightiana was 24 h, which gave the highest yield that is, 3.49 ± 0.08 × 106 protoplasts/g FW and viability were 60.3 ± 1.72%. The fusion was performed with 50% (w/v) PEG-MW4000. The optimal incubation time was found as 30 and 45 min, which gave the highest number of fused viable protoplasts. A maximum number of binary fusions was achieved at 45 min incubation. The use of liquid culture medium resulted in a range of 5–20 calli per Petri dish which continued their growth. The hybrid calli show better growth in all NaCl concentrations. Hence, it was concluded that the hybrids have a better tolerance to salt stress. These results are useful in the development of salt tolerant rice varieties.

Protoplast fusion in the genus Gentiana: genomic composition and genetic stability of somatic hybrids between Gentiana kurroo Royle and G. cruciata L.

Somatic hybridization by protoplast fusion is used in breeding programs to obtain plant material that has inherited valuable traits from two different species, and in order to broaden plant genetic diversity. Somatic hybrids of the genus Gentiana could provide a useful source of new ornamental cultivars and of secondary metabolites. However, in order to evaluate its further usefulness, detailed characterization of the newly created hybrid is essential. Here, genome composition and stability of interspecific gentian somatic hybrids obtained following electrofusion of cell suspension-derived protoplasts of diploid Gentiana kurroo Royle with leaf mesophyll-derived protoplasts of tetraploid G. cruciata L. were characterized using various molecular markers and flow cytometry. AFLP and ISSR analyses revealed that all 21 hybrid plants and 3 lines of hybrid callus were genetically closer to G. cruciata than to G. kurroo. According to the results of chloroplast DNA analysis with the use of CAPS markers, all somatic hybrids inherited chloroplasts from “mesophyll” fusion partner G. cruciata. Flow cytometry revealed that polyploidization occurred, and probably it took place at the early stage of post-fusion culture. In consequence, gradual elimination of nuclear DNA, mixoploidy, and high genetic instability were observed in most hybrid plants and calli during the subsequent 4 years of in vitro culture.

Genotyping and metabolite characterization of somatic hybrids between Arabidopsis thaliana and Swertia mussotii

Symmetric and polyethylene glycol (PEG)-induced asymmetric fusions between Swertia mussotii and Arabidopsis thaliana protoplasts generated >100 putative hybrid cell lines. Of these, 65 were shown to have a hybrid origin, based on molecular markers, chromosome number, and morphology. An assay directed at the A. thaliana CACTA transposon family detected only three positive lines among the 65 hybrid calli tested. The CACTA sequence amplified from clone D14 was highly homologous with CAC2, indicating that the chromosome content of the hybrid cell lines was largely inherited from S. mussotii. Profiles of secondary metabolites identified a number of S. mussotii- or A. thaliana-specific compounds, as well as significant proportions of compounds not represented in the profile of either parent.

Genetic and biochemical characterization of somatic hybrids between Bupleurum scorzonerifolium and Gentianopsis paludosa

Ultraviolet-irradiated protoplasts of Gentianopsis paludosa were fused with those of Bupleurum scorzonerifolium and 28 independent hybrid calli were identified, five of which later differentiated into plants. A genetic analysis of these calli and regenerated plants based on chromosome number, esterase, random amplified polymorphic DNA, and 5S rDNA spacer profiling showed that the majority of their nuclear genomes were represented by the recipient biparent B. scorzonerifolium. A restriction fragment length polymorphism analysis of the plastidial genomes confirmed that DNA from both biparents was present in some of the hybrids. The secondary metabolite composition of the hybrids was analyzed by a combination of high-performance liquid chromatography and gas chromatography-mass spectrometry analysis. The content of oleanolic acid in two of the hybrid lines was substantially higher than in the donor G. paludosa, while that of swertiamarin was equal to that in G. paludosa in two of the six hybrids analyzed. A number of both G. paludosa and B. scorzonerifolium specific compounds were detected in the three hybrids analyzed by GC-MS as were several not present in either of the biparents.

Introgression of the heterologous nuclear DNAs and efficacious compositions from Swertia tetraptera Maxim. into Bupleurum scorzonerifolium Willd. via somatic hybridization

Swertia tetraptera Maxim. is an important source of secoiridoid glucosides. To produce these pharmacologically valuable compounds heterologously in somatic hybrid cell lines, S. tetraptera protoplasts were irradiated with various doses of UV light and fused with protoplasts from a long-term cell line of Bupleurum scorzonerifolium Willd. This recipient was chosen as the cell line is cytogenetically stable and fast growing; furthermore, protoplasts isolated from the cell line are readily regenerable. From a set of 86 putative hybrid calli, only two were able to regenerate viable green plants. The hybridity of the 19 of the 86 selections was revealed by a combined isozyme and RAPD analysis, supported by a karyotypic study based on genomic in situ hybridization (GISH). Clone I-3 contained 0.014% swertiamarin while the regenerants had 0.069% swertiamarin and 0.409% gentiopicroside while the III-4 plants contained only 0.015% gentiopicroside.

Asymmetric somatic hybridization between Bupleurum scorzonerifolium Willd. and Taxus chinensis var. mairei

In an attempt to transfer the genetic components needed for taxol synthesis into a more tractable plant, protoplasts of Taxus chinensis var. mairei were UV irradiated for various times prior to their fusion with protoplasts of Bupleurum scorzonerifolium. Of the 60 presumptive hybrid calli obtained, selections of the 9 most rapidly growing were recognized as hybrid clones based on a combination of their callus morphology, esterase profile, chromosome number, and RAPD genotype. The RAPD data showed that 82.4-96.8% of the hybrid genome was inherited from the recipient (B. scorzonerifolium) and 4.6-13.9% from the donor (T. chinensis), with 1.6-6.9% of the RAPD fragments being not present in either parent. None of the hybrid clones expressed a detectable quantity of taxol, but four produced more of the triterpenoid oleanolic acid than did calli of the recipient. With the use of semi-quantitative reverse transcriptase PCR, it was possible to show variation in the expression of several triterpenoid biosynthetic pathway related genes between B. scorzonerifolium and the hybrids. Increasing the concentration of oleanolic acid requires that the expression levels of the genes encoding IPP isomerase, squalene synthase, squalene epoxidase and β-amyrin synthase are increased, while at the same time, those of the genes encoding the branching enzymes cycloartenol synthase and lupeol synthase need to be reduced.

Introgression of Swertia mussotii gene into Bupleurum scorzonerifolium via somatic hybridization.

BackgroundThe wild herb Swertia mussotii is a source of the anti-hepatitis compounds swertiamarin, mangiferin and gentiopicroside. Its over-exploitation has raised the priority of producing these compounds heterologously. Somatic hybridization represents a novel approach for introgressing Swertia mussotii genes into a less endangered species.ResultsProtoplasts derived from calli of Bupleurum scorzonerifolium and S. mussotii were fused to produce 194 putative hybrid cell lines, of which three (all derived from fusions where the S. mussotii protoplasts were pre-treated for 30 s with UV light) later differentiated into green plants. The hybridity of the calli was confirmed by a combination of isozyme, RAPD and chromosomal analysis. The hybrid calli genomes were predominantly B. scorzonerifolium. GISH analysis of mitotic chromosomes confirmed that the irradiation of donor protoplasts increased the frequency of chromosome elimination and fragmentation. RFLP analysis of organellar DNA revealed that mitochondrial and chloroplast DNA of both parents coexisted and recombined in some hybrid cell lines. Some of the hybrid calli contained SmG10H from donor, and produced swertiamarin, mangiferin and certain volatile compounds characteristic of S. mussotii. The expression of SmG10H (geraniol 10-hydroxylase) was associated with the heterologous accumulation of swertiamarin.ConclusionsSomatic hybrids between B. scorzonerifolium and S. mussotii were obtained, hybrids selected all contained introgressed nuclear and cytoplasmic DNA from S. mussotii; and some produced more mangiferin than the donor itself. The introgression of SmG10H was necessary for the accumulation of swertiamarin.

Different rates of chromosome elimination in symmetric and asymmetric somatic hybridization between Festuca arundinacea and Bupleurum scorzonerifolium

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.

Ginsenoside Rb1 in asymmetric somatic hybrid calli of Daucus carota with Panax quinquefolius

American ginseng (Panax quinquefolius L.) is one of the most valuable herbs in the world. Its major active components are ginsenosides. In order to produce ginsenoside heterogeneously, somatic hybridization, a novel approach for genetic introgression, was employed in this study. Protoplasts derived from respective calli of carrot (Daucus carota var. sativus Hoffm.) and American ginseng (P. quinquefolius L.) were used as the fusion partners. Hybrid calli derived from single cell lines containing chromatin of American ginseng were confirmed by the analyses of isozyme, Random amplified polymorphic DNA (RAPD) and genomic in situ hybridization (GISH). High performance liquid chromatography (HPLC) results showed that the ginseng monomer Rb(1) was synthesized in seven of the hybrid calli identified as well as in the parent American ginseng calli but not in the parent carrot calli. Results indicated that hybrid introgression lines could produce ginsenoside Rb(1) and the ginsenoside Rb(1) biosynthesis pathway has been introgressed into carrot cells via somatic hybridization. From the point of biosafety view concerning the consumer acceptance, the potential predominance to produce ginsenosides with somatic hybridization other than with genetic transformation is discussed.

Introgression of bread wheat chromatin into tall wheatgrass via somatic hybridization

Regenerates were obtained following somatic hybridization between tall wheatgrass (Agropyron elongatum) and bread wheat (Triticum aestivum cv. Jinan177) protoplasts. Two lines (CU and XI) were self-fertile in the first (R0) and subsequent (R1 and R2) generations. The phenotype of each R1 population was uniform. All CU progeny were phenotypically similar to the tall wheatgrass parent, while XI progeny had thinner, smoother and softer leaves. Cytological analysis showed that more wheat chromatin was present in the hybrid callus than in the R1 and R2 plants, and that some intercalary translocations of wheat chromosome segments were retained in the R2 generation. AFLP profiling confirmed the presence of wheat DNA in the introgression lines. Analysis of the high molecular weight glutenin subunit content of derived seed identified three novel subunits, not present in either the wheat or the tall wheatgrass parent. Microsatellite-based profiling of the chloroplast genome of the introgression lines suggested that only chloroplast sequences from the tall wheatgrass parent were present. The specifically inherited phenomena and possible application of these hybrids are discussed.

Genotyping of somatic hybrids between Festuca arundinacea Schreb. and Triticum aestivum L.

In order to genotype hybrid genomes of distant asymmetric somatic hybrids, we synthesized hybrid calli and plants via PEG-mediated protoplast fusion between recipient tall fescue (Festuca. arundinacea Schreb.) and donor wheat (Triticum aestivum L.). Seventeen and 25 putative hybrid clones were produced from the fusion combinations I and II, each with the donor wheat protoplast treated by UV light for 30 s and 1 min, respectively. Isozyme and RAPD profiles confirmed that ten hybrid clones were obtained from combination I and 19 from combination II. Out of the 29 hybrids, 12 regenerated hybrid plants with tall fescue phenotype. Composition and methylation-variation of the nuclear and cytoplasmic genomes of some hybrids, either with or without regenerative ability, were compared by genomic in situ hybridization, restriction fragment length polymorphism, and DNA methylation-sensitive amplification polymorphism. Our results indicated that these selected hybrids all contained introgressed nuclear and cytoplasmic DNA as well as obvious methylation variations compared to both parents. However, there were no differences either in nuclear/cytoplasmic DNA or methylation degree between the regenerable and non-regenerable hybrid clones. We conclude that both regeneration complementation and genetic material balance are crucial for hybrid plant regeneration.

Production of two mandarin × trifoliate orange hybrid populations via embryo rescue with verification by SSR analysis

Intergeneric sexual hybridizations were conducted between two genera of Rutaceae, with Satsuma mandarin (Citrus unshiu Marc) and Red tangerine (C. reticulata Blanco) as maternal parents, and Poncirus trifoliata (L.) Raf as the paternal parent, in an effort to generate hybrid populations for both molecular mapping and rootstock breeding. Embryo rescue is important for citrus sexual breeding because polyembryony can interfere with hybrid embryo recovery. Immature embryos of 80, 85, 90 days after pollination (DAP) from the Satsuma mandarin (S) × trifoliate orange (P) cross, 80 and 85 DAP from the Red tangerine (R) × trifoliate orange (P) cross, were cultured on MG1.0 medium consisting of MT basal medium supplemented with 1.0 mg l−1 GA3 and 4% sucrose. The results showed that 80 DAP was the optimal time for embryo rescue of the tested crosses, as evidenced by embryos at this stage exhibiting high germination rates, 37.3% for S × P and 51.3% for R × P. Among the eight tested media, MT medium supplemented with 0.5 mg l−1 GA3 was the best one for Satsuma mandarin, and MT plus 1.0 mg l−1 GA3 for Red tangerine. A total of 85 plants were obtained from S × P, and 340 from R × P. Out of them, 44 progenies from S × P and 111 from R × P were sufficiently confirmed to be hybrids by morphological characterization and SSR analysis. In addition, two hybrid callus lines were obtained from S × P and R × P respectively.

Culture and fusion of pollen protoplasts of Brassica oleracea L. var. italica with haploid mesophyll protoplasts of B. rapa L. ssp. pekinensis

Hybrid callus was formed from the successful protoplast fusion between pollen protoplasts of Brassica oleracea var. italica and haploid mesophyll protoplasts of Brassica rapa. The pollen protoplast isolation frequency in broccoli was highly related to the ratio of trinucleate pollens in the male gametophyte population. Large quantities of pollen protoplasts with high vigor could be isolated, and the isolation frequency reached up to 90% in 6.0-7.0 mm long flower buds with about 94.7% trinucleate-stage pollens. Pollen protoplasts could be collected and purified by discontinuous gradient centrifugation. In 1% Na-alginate embedding culture, cell divisions were observed but no further development was found. The haploid mesophyll protoplasts were isolated from in vitro haploid plants of B. rapa. Results strongly showed the variability in culturability of mesophyll protoplasts from different haploid lines. Both pollen protoplasts and haploid mesophyll protoplasts retained a stable round shape in the designed prefusion solution with an osmotic pressure of 0.74 osmol/kg. Polyethylene glycol was used for the protoplast fusion, and 40% polyethylene glycol 4000 enabled the highest fusion frequency of about 20%. Some postfusion protoplasts showed cell divisions up to callus proliferation. Calli were screened by random amplified polymorphic DNA analysis for their hybrid character. Results revealed the existence of the hybrid calli. Some of the hybrid calli grew well with green color and shoot primordia. According to our knowledge, this is the first report about a hybrid formation between two haploid protoplasts. Potential comprehensive applications, as well as problems of this technique, are discussed.

First Report of Hop stunt viroid and Citrus exocortis viroid on Fig with Symptoms of Fig Mosaic Disease

AbstractReverse transcription polymerase chain reaction (RT‐PCR) was used for the detection of Hop stunt viroid (HSVd) and Citrus exocortis viroid (CEVd) in nucleic acid extracts of infected fig leaf tissues. RT‐PCR results were confirmed by cDNA sequencing. This is the first report of CEVd and HSVd infecting fig in Tunisia. Alignments and phylogenetic analysis classify the HSVd Tunisian isolates in the plum‐type group. CEVd Tunisian isolates are very closely related to the CEVd tomato hybrid callus isolate and to CEVd Gynura isolate from the USA.

Construction of a Primary RH Panel of Italian Ryegrass Genome via UV‐Induced Protoplast Fusion

Symmetric and asymmetric somatic hybrids were produced via protoplast fusion between common wheat ( TRITICUM AESTIVUM L.) cv. "Jinan 177" and Italian ryegrass ( LOLIUM MULTIFLORUM Lam.). The ryegrass without or with UV irradiation was used as a donor, providing a small amount of chromatin. In these somatic hybrids, most ryegrass chromosomes have been confirmed preferential elimination and the somatic hybrid calli and plants showed wheat-like morphology. Some of the hybrid lines were used for the analysis of distribution and heredity of donor DNA in the hybrid genome and the possibility of establishing a radiation hybrid (RH) panel of the ryegrass in the present experiment. These hybrids, subcultured for two and three years, retained the ryegrass DNA examined by RFLP and GISH analysis, respectively. Distribution of the ryegrass DNA in the wheat genomes of 20 single-cell individuals, randomly selected from hybrid cell lines produced, were analyzed by 21 ryegrass genome specific SSR markers. The average frequencies of molecular marker retention in symmetric hybrid lines (UV 0), as well as asymmetric hybrid lines from UV 30 s and 1 min were 10.88, 15.48 and 33.86, respectively. It was suggested that the UV dose increased the introgression of donor DNA into wheat genome. The ryegrass SSR fragments in most asymmetric hybrid cell lines remained stable over a period of 2 approximately 3 years. This revealed that those asymmetric somatic hybrids are suitable for the introgression of ryegrass DNA into wheat, and for RH panel and RH mapping.

고려인삼과 미국삼의 종간잡종으로부터 재분화된 식물체의 특성

The characteristics of plantlets redifferentiated from calli of F1 hybrid between Panax ginseng and Panax quinquefolius were investigated. Growth of plantlets redifferentiated from F1 hybrid was superior to the plants redifferentiated from Korean ginseng. Stem color of plantlets redifferentiated from F1 hybrid was more purple than that from Korean ginseng and leaf color of the former was also greener than that of the latter. Chunpoong, Yunpoong and Seonweon which are belonged to Korean ginseng showed same PCR band(A), while American ginseng showed different PCR band (B) in Internal Transcribed Spacer (ITS) region. F1 hybrid exhibited both A and B PCR band which belonged to Korean ginseng and American ginseng, respectively. F1 hybrid calli and plantlets redifferentiated from F1 hybrid calli showed same PCR band with that of F1 hybrid plant in ITS region. Therefore it was confirmed that piantlets redifferentiated from F1 hybrid exhibited genetic stability in ITS region.

Isolation of cytoplasts from Satsuma mandarin (Citrus unshiu Marc.) and production of alloplasmic hybrid calluses via cytoplast–protoplast fusion

Cytoplasm of Satsuma mandarin (Citrus unshiu Marc.) is known to influence seedlessness. Transfer of cytoplasm to a seedy cultivar could possibly lead to the production of seedless citrus fruits. In the present paper cytoplasts were isolated from cell suspension-derived protoplasts of Satsuma mandarin via ultra-centrifugation in a discontinuous gradient. No nucleus could be detected in the cytoplasts by DAPI (4', 6-diamidino-2-phenylindole) staining compared with normal protoplasts. The cytoplasts, with high viability and small size, did not divide during solid embedding culture. Cytoplasts of Satsuma mandarin were electrically fused with embryogenic protoplasts of Murcott tangor (C. reticulata x C. sinensis), which led to regeneration of several cell lines. Flow cytometry (FCM) indicated that the cell lines were diploids. Simple sequence repeats (SSR) and cleaved amplified polymorphism sequence (CAPS) showed that the cell lines got their nuclear DNA from the protoplast parent, whereas the cytoplast parent donated the mtDNA, confirming transfer of mtDNA from Satsuma mandarin into Murcott tangor via cytoplast-protoplast fusion though no polymorphism was detected in chloroplast DNA between the fusion partners. This is the first report on isolation and characterization of cytoplasts, together with cytoplast-protoplast fusion in Citrus, which has a potential for citrus cultivar improvement involving cytoplasm transfer via cytoplast-protoplast fusion.

Interspecific hybrids between Medicago sativa L. and annual Medicago containing Alfafa weevil resistance

Non-embryogenic protoplasts of Medicago rugosa and M. scutellata were electro-fused with iodoacetic acid-treated protoplasts of M. sativa (alfalfa). Putative somatic hybrid callus were obtained and some plants regenerated from both combinations. Hybridity of regenerants was confirmed by morphology, molecular means and cytological observations. Parental specific bands were recognized in somatic hybrids by Southern analysis. The somatic hybrids were perennial and their morphology was similar to M. sativa. Cytological observations were carried out on the somatic hybrids, their vegetative clones and self-pollinated offspring. Original somatic hybrids were aneuploids (2n=31–59), but during vegetative proliferation, their chromosome numbers reduced to 32. Those clones of hybrids formed seeds from M. sativa (+) M. rugosa by self-crossing. Chromosomal rearrangements within the parental genomes were observed in vegetative clones of hybrids and their S1 offspring by Genomic in situ Hybridization (GISH). Some of S1 offspring from M. sativa (+) M. rugosa showed better spring growth than parental M. sativa and tend to be tolerant to Alfalfa weevil. It was considered that these traits were introduced from the genome transferring M.␣rugosa chromosome to M. sativa. The cell fusion may still have a potential in transferring alien chromosomes in order to increase the genetic variation for crop breeding.

Introgression of the Haynaldia villosa genome into γ-ray-induced asymmetric somatic hybrids of wheat

To study the effect of gamma-ray treatment on donor and derived somatic hybrids, we carried out gamma-ray donor treatment experiments with a wide range of gamma-ray dosages and asymmetric somatic hybridization between protoplasts of wheat (Triticum aestivum L. Jinan 177) and protoplasts of Haynaldia villosa Schur. treated with different dosages of gamma-rays (40, 60 and 80 Gy, respectively). We first screened the putative hybrids by isozyme analysis, followed by characterization of nuclear and organellar genome composition of the hybrids. Genomic in situ hybridization on mitotic metaphases demonstrated that the donor chromosome elimination in the hybrids increased with increased gamma-ray dosage. Intergenomic chromosome recombination/translocations were observed in the hybrids from different dosages of gamma-rays. PCR amplification of 5S rDNA spacer sequences showed that only some of the regenerated hybrid clones inherited donor 5S rDNA sequences, suggesting that the donor DNA was also eliminated randomly. Restriction fragment length polymorphism analysis using mitochondrion (mt) and chloroplast (cp) gene-specific probes showed that the hybrid calli contained mt genomes of both parents and the cp genome of only one of the parents. Recombinations between parental mt as well as cp genes were found in the hybrid clones. Furthermore, development of the hybrid clones was dependent on the gamma-ray dosage used for the donor treatment. Regenerated plants were only obtained from fusion combinations of low (40 Gy) and intermediate (60 Gy) dose irradiation. The possible role and significance of gamma-rays on the introgression of small segments of donor chromosomes to the receptor is discussed.

Somatic hybrid vigor in Citrus: Direct evidence from protoplast fusion of an embryogenic callus line with a transgenic mesophyll parent expressing the GFP gene

Previous experience with citrus somatic fusion suggests that hybrid cells are generally more vigorous and have higher capacity for embryogenesis, compared to unfused cells from the embryogenic callus parent. However, this observation has never been validated due to the unavailability of an appropriate experimental design. Citrus mesophyll protoplasts never divide and regenerate into plants. Herein, mesophyll protoplasts of transgenic Valencia orange ( Citrus sinensis) which expressed the green fluorescent protein (GFP) gene were fused with protoplasts isolated from an embryogenic callus line of Shekwasha mandarin ( C. reticulata) to visually screen somatic hybrids. At the multi-cell cluster stage, the regenerated callus lines expressing GFP were morphologically identical to the callus parent regenerate. All hybrid callus lines expressing the GFP developed into embryoids, while most callus parent regenerates still remained in a callus stage. Embryoids with strong or weak GFP expression were verified to be diploid cybrid and tetraploid somatic hybrid embryoids, respectively, by flow cytometry, simple sequence repeat (SSR) and chloroplast SSR analyses. Diploid cybrid globular embryoids appeared light green in color and had a tendency to enter subsequent developmental stages earlier than the dark green tetraploids; however, GFP plants were regenerated from both sources. Thus, expression of the GFP marker gene was effectively used to visually screen somatic hybrids, and confirmed our previous observation that somatic hybrid tissue has a regeneration advantage. The intensity of GFP fluorescence and the color difference of regenerated embryoids was also an early indicator of ploidy level (based on visual GFP observation and confirmed by flow cytometry analysis), and could serve as a tool to study the regeneration mechanism of diploid cybrids via symmetric fusion in citrus and other higher plants.