Annals of Botany 89: 491-497, 2002
© 2002 Annals of Botany Company
Green Fluorescent Protein as a Visual Marker in Somatic Hybridization
1Departamento de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias, Apartado Oficial 46113, Moncada (Valencia), Spain
* For correspondence. Fax 34 96 139 0240, e-mail lnavarro{at}ivia.es
Received: 4 June 2001; Returned for revision: 10 October 2001; Accepted: 14 December 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSLITERATURE CITED |
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Using a transgenic citrus plant expressing Green Fluorescent Protein (GFP) as a parent in somatic fusion experiments, we investigated the suitability of GFP as an in vivo marker to follow the processes of protoplast fusion, regeneration and selection of hybrid plants. A high level of GFP expression was detected in transgenic citrus protoplasts, hybrid callus, embryos and plants. It is demonstrated that GFP can be used for the continuous monitoring of the fusion process, localization of hybrid colonies and callus, and selection of somatic hybrid embryos and plants.
Key words: Protoplast fusion, genetic transformation, green fluorescent protein, GFP, woody plant, vital marker, Citrus reticulata, Citrus sinensis, Poncirus trifoliata, Agrobacterium tumefaciens.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSLITERATURE CITED |
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Somatic hybridization by means of protoplast fusion enables some of the problems associated with traditional breeding to be overcome, including bypassing of sexual barriers among species and genera, thus providing an efficient biotechnological alternative for crop improvement (Bajaj, 1994). Somatic hybrids have been produced for a few species from economically important families, including Solanaceae, Rutaceae, Brassicaceae, Fabaceae and Poaceae (Johnson and Veilleux, 2001). However, in most cases, recovery of whole plants from fused protoplasts has been unsuccessful; this is mainly due to difficulties in regenerating the hybrid products.
Several strategies have been attempted for the selection of somatic hybrid tissue (Gleba and Shlumukov, 1990), including genetic and/or physiological complementation, parental cell inactivation, mechanical selection (based on morphologically different protoplasts or differential staining of parent cells), enriched culture of mechanically or flow cytometry-selected hybrid cells and transgene-based selection. However, application of these strategies has been limited due to difficulties with the mutant generation in complementation or antibiotic-based protoplast selection, or the effectiveness of low-density cell culture. Utilization of stable non-destructive markers would facilitate the development and/or improvement of protoplast fusion and regeneration of somatic hybrids.
In recent years, the green fluorescent protein (GFP) from Aequorea victoria has become one of the leading and most studied reporter proteins in biochemistry, cell biology and biotechnology (Plautz et al., 1996; Leffel et al., 1997; Misteli and Spector, 1997; Tsien, 1998). GFP has been used as an in vivo fluorescent marker both in prokaryotic (Dhandayuthapani et al., 1995; Valdivia et al., 1996) and eukaryotic systems (Tsien, 1998). GFP emits stable and distinctive green fluorescence when expressed by living cells, without addition of any cofactors or subtracts but oxygen (Chalfie et al., 1994). For this reason, GFP is being used extensively as a reporter in plant biology studies (Tian et al., 1997; Chytilova et al., 1999; Elliott et al., 1999; Molinier et al., 2000) and as a scorable marker in plant genetic transformation (Ghorbel et al., 1999; Escobar et al., 2000).
A transgenic citrus plant expressing the gfp gene has been used as the parental material in somatic hybridization experiments to study the possible advantages of the constitutive presence of an in vivo marker during the processes of protoplast fusion and somatic hybrid regeneration. The fact that there is a well established somatic hybridization system for citrus (Grosser et al., 2000; Olivares-Fuster et al., 2000) makes it a suitable model for this purpose.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSLITERATURE CITED |
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Plant materials
Non-embryogenic protoplasts were obtained from fully expanded leaves of a citrange Carrizo [Citrus sinensis (L.) Osbeck x Poncirus trifoliata (L.) Raf.] transgenic plant previously obtained in our laboratory (Ghorbel et al., 1999) by Agrobacterium tumefaciens transformation with the binary plasmid pBin19-sgfp containing the NOSpro-nptII-NOSter and 35Spro-sgfp-35Ster gene cassettes (Chiu et al., 1996). The plant is not chimeric and expresses constitutively the gfp gene (Ghorbel et al., 1999). It was propagated clonally and maintained in a glasshouse. C. reticulata Blanco nucellar-derived callus was used as a source of embryogenic protoplasts as suspension cultures maintained in liquid hormone-free medium (Murashige and Skoog, 1962) supplemented with 25 g l–1 sucrose and 0·5 g l–1 malt extract, and a 2-week subculture cycle with moderate shaking, 25 °C and a 16 h daylight period.
Protoplast isolation, fusion and culture
Protoplasts from embryogenic cells and leaves were isolated according to Grosser and Gmitter (1990) using the enzyme solution described by Grosser and Chandler (1987). Evaluation of GFP as a protoplast viability reporter was carried out by comparing direct cell viability based on GFP-presence with the viability of transgenic protoplasts submitted to the FDA (fluorescein diacetate) test (Nadel, 1989).
Protoplasts were chemically fused according to Grosser and Gmitter (1990). Briefly, cell suspensions adjusted to 106 protoplasts ml–1 from each parent were mixed, and approx. 50 µl of the mixture was placed onto a 60 x 15 mm dish. Two drops of 40 % PEG (polyethylene glycol) were added and, after 10 mins, two more drops of a high pH/high calcium solution were added to complete membrane fusion. Protoplasts were washed carefully with 0·6 M BH3 (Grosser and Gmitter, 1990), a Murashige and Tucker (1969) based protoplast culture medium. Protoplasts were cultured in approx. 0·5 ml of the same medium on sealed plates and incubated in the dark at 28 °C.
Plant regeneration
During the following 6 weeks, osmotic reduction in culture dishes was gradually achieved according to Grosser and Gmitter (1990), and small calli were transferred onto solidified basal medium (BM) consisting of Murashige and Tucker (1969) salts supplemented with 50 g l–1 sucrose, 0·5 g l–1 malt extract and 8 g l–1 agar. Spontaneous embryogenesis occurred, and somatic embryos were transferred to 1500 medium (BM medium plus 1·5 g l–1 malt extract) for enlargement (Vardi and Galun, 1988). BM medium supplemented with 1 mg l–1 gibberellic acid and 0·02 mg l–1 l naphthalene acetic acid was used for shoot induction, and solid hormone-free BM medium was used for shoot elongation and rooting. Rooted plants with three or four fully expanded leaves were transferred to a peat substratum and acclimatized in a glasshouse.
Somatic hybrid identification
For ploidy analysis, young leaves were collected from all in vitro-regenerated plants and analysed in a Partec Ploidy Analyzer flow cytometer (PA) (Partec®, Münster, Germany) as described previously (Olivares-Fuster et al., 2000).
Genetic identification of regenerated plants was performed by PCR amplification of DNA microsatellite regions. The previously described TAA15 microsatellite locus (Kijas et al., 1997) was selected for the genetic identification of hybrids on the basis of previous analyses, which had established that the locus was polymorphic for the two parents used in this study. Genomic DNA was extracted from both parents and regenerated plant leaves according to Dellaporta et al. (1983). PCR thermocycling conditions were as described in Kijas et al. (1997). PCR amplification products were analysed by electrophoresis on 3 % agarose mini-gels and stained with ethidium bromide. ‘PCR 100 bp low ladder’ (Sigma, St Louis, MO, USA) was used as a molecular weight standard.
GFP detection and photography
Protoplasts and colonies were examined under a Leica DMRB microscope equipped with either a Leica GFP filter cube that blocked red autofluorescence from chlorophyll, or a Leica I3 filter cube that did not block it, and an HBO 50 W high-pressure mercury bulb as the light source. Photographs were taken on Kodak Ektachrome 1600 film for fluorescence and Kodak Ektachrome 160T film for white light photography.
Callus and differentiated plant tissue were examined under a Leica MZ12 stereomicroscope equipped with a Leica Fluorescence module comprising a 480/40 nm exciter filter, a 505 nm LP dichromatic beam splitter and a 510 LP barrier filter. Again the light source was an HBO 50 W high-pressure mercury bulb. Photographs were taken on Kodak Ektachrome 400 film.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSLITERATURE CITED |
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Citrus somatic hybridization is a well-established technology based on the capacity of nucellar-derived callus cultures and their protoplasts to regenerate whole plants via somatic embryogenesis under specific culture conditions (Grosser et al., 2000). Leaf-derived protoplasts cannot regenerate under these conditions. Thus, the citrus somatic hybridization system uses the regeneration capabilities of only one parent as the selectable marker. These characteristics make citrus an appropriate model to investigate the possibilities of using gfp as an in vivo marker in somatic fusions, because we can infer that GFP-positive tissue with regeneration capabilities will be hybrid.
GFP lights up the somatic hybridization process
Protoplast isolation was readily accomplished from both embryogenic callus and leaf tissues. Protoplast isolation yields from transgenic citrus leaves were similar to those routinely obtained from non-transgenic plants (data not shown). Investigating the suitability of GFP as a cell viability reporter for transgenic citrus protoplasts, we found that viability estimation based on the presence of GFP (86·3 ± 2·9) was significantly higher than that based on the FDA test (78·1 ± 3·9). Viability over-estimation based on the presence of GFP could be related both to the additional cell death that occurred during the FDA test and to the higher stability of GFP; some cells which are no longer able to retain FDA due to membrane damage may still show GFP fluorescence. GFP, when available, could be used to estimate protoplast viability based on the fact that no cell manipulation is required as is the case in conventional viability tests.
Only spherical protoplasts showed GFP presence when transgenic protoplasts were observed with a filter which blocked chlorophyll fluorescence (Fig. 1A and B). Candidate viable transgenic protoplasts observed under blue light and a wide wavelength filter appeared spherical and partially yellow as result of the combination of both red (from chlorophyll) and green (from GFP) fluorescence, while candidate non-viable protoplasts appeared red due to chlorophyll autofluorescence (Fig. 1A, C, F and H), thus allowing direct estimation of protoplast viability.
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Different patterns of cell adhesion were recognizable after chemically induced fusion between callus and leaf protoplasts (Fig. 1D), and protoplast adhesion identified by morphological traits (chloroplasts in leaf-derived protoplasts) was confirmed by the presence of GFP (Fig. 1E). GFP accumulation in one parent did not affect heterokaryon formation. When non-transgenic parents are used in chemically induced protoplast fusion, the heterokaryon formation rate is approx. 6 % (Olivares-Fuster et al., 2000), and similar values were obtained in this study. Heterokaryon cells were easily identified based on morphological traits, or GFP or chlorophyll fluorescence (Fig. 1F–H).
Even when morphological traits of the parents allow visual confirmation of heterokaryon formation, as is the case for citrus, once protoplasts divide and the cell wall has formed, regeneration is usually a blind process. The presence of GFP allowed visual and direct identification of hybrid tissues even after cell wall regeneration and cell division (Fig. 2A and B). gfp-expressing hybrid tissues were easily detected on the basis of green fluorescence, facilitating in vivo identification of hybrid colonies and embryos from the culture mixture by exposing the plates to blue light (Fig. 2C–F). The presence of GFP did not hamper the somatic embryogenesis process and, as in other citrus protoplast fusion experiments, eight somatic embryos were obtained, on average, per culture plate. After regeneration, stable gfp expression was also found at the plant level (data not shown). In this study six plants were regenerated and acclimatized in a glasshouse, all of them constitutively expressing the gfp gene.
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Genetic analysis of regenerated plants was performed by PCR amplification of a specific citrus DNA microsatellite region (locus TAA15; Kijas et al., 1997). Figure 3A shows the complementary DNA pattern obtained for two of the regenerated plants, as well as the pattern for both parents, C. reticulata and Carrizo citrange. In our study, all GFP-positive regenerated plants were confirmed as somatic hybrids after microsatellite analysis. Flow cytometry analysis of GFP-positive regenerated plants confirmed their allotetraploid origin. Figure 3B shows the histogram obtained after cytometric analysis of one of the GFP-positive regenerated plants. All GFP-positive plants were found to be tetraploid after flow cytometry analysis.
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High levels of GFP expression do not affect hybrid cell division and somatic embryogenesis
A high level of GFP expression was always detected in transgenic citrus protoplasts, hybrid calli, embryos and plants. Ghorbel et al. (1999) also showed consistent GFP expression during regeneration of whole plants from GFP-transgenic citrus cells. Using arabidopsis or tobacco, previous authors have reported difficulties in obtaining whole plants from the brightest transformants using different versions of gfp (Chiu et al., 1996; Haselof et al., 1997; Rouwendal et al., 1997). It was suggested that high levels of constitutively expressed GFP were toxic or interfered with the regeneration of transgenic shoots. No toxic effects were found during transgenic citrus plant regeneration (Ghorbel et al., 1999; Fleming et al., 2000) or transgenic somatic hybrid regeneration in this work. Therefore, the toxic effects cannot be attributed to the synthetic gfp version used under the constitutive 35S promoter from the Cauliflower Mosaic Virus (CaMV). It is possible that citrus cells are less sensitive to GFP than arabidopsis or tobacco cells, or that the 35SCaMV promoter yields lower GFP expression in citrus cells than in those of other species. These observations are supported by the results of Rouwendal et al. (1997) who found that potato transformation was very inefficient and most of the resulting transformants grew poorly, whereas tobacco transformants regenerated normally using the same gfp construct.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSLITERATURE CITED |
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This study is the first to show the potential of green fluorescent protein as a somatic hybridization marker. In citrus, GFP could be useful in the regeneration of hybrids between citrus and related species where somatic hybridization has been unsuccessful to date, for cybrid synthesis or in gametosomatic fusion. Moreover, GFP-based selection is a positive process since it does not involve the death of non-desirable cells as is the case for antibiotic resistance-based selection (Vazquez-Tello et al., 1996), thus minimizing the risk of cell death and the release of toxic compounds, which could affect the viability and regeneration capacity of the hybrids and thus invalidate results.
These results suggest that GFP could be used to either improve or establish a somatic hybridization protocol in other plant systems. When non-morphologically distinct protoplasts are used in cell fusion experiments, availability of different colour variants of this marker (blue, cyan or yellow) (Haselof, 1999) should allow in vivo evaluation of fusion parameters over heterokaryon formation and heterokaryon survival rates. Furthermore, use of colour variant versions of the protein, or sub-cellular protein localization signals (cytoplasmic, nuclear, organellar) could provide additional advantages for in vivo monitoring of the protoplast fusion process and of the hybrid or cybrid regeneration pathways in other plant species.
Somatic fusion efficiency should be directly related to the number of hybrid plants regenerated. Although there is a correlation between heterokaryon formation rate and the number of somatic hybrid plants obtained, the regeneration process involves so many steps that in many cases the correlation is weak. Adjusting culture conditions to ensure successful hybrid regeneration is a time- and resource-consuming process, and is sometimes a major barrier for the establishment of somatic hybridization procedures because the global effect of different conditions cannot be scored until plants have regenerated and been analysed genetically. The presence of GFP allows identification of hybrid tissue in vivo at all times, thereby facilitating direct study of specific culture conditions such as osmoticum pressure, carbon source, growth factors, culture density, light period and temperature throughout the whole hybrid regeneration process.
Confirmation of the hybrid nature of regenerated plants is a critical step when designing a somatic hybridization procedure because the number of regenerated hybrids should be the factor which distinguishes between the fusion, culture and regeneration conditions being tested. However, analysis of regenerated plants involves studying molecular markers such as isoenzymes, RAPDs or RFLPs, which usually requires the availability of tissue from glasshouse-acclimatized plants. In our study, the presence of GFP allowed early (colonies, embryos, shoots) and direct (in vivo and visual) identification of hybrid tissue, including hybrid plants, thus saving time and resources in the final step of the somatic hybridization process.
Use of this transgenic marker should be restricted to the process of setting up somatic hybridization conditions, which could later be used with non-transgenic parents.
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ACKNOWLEDGEMENTS |
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We thank Dr R. Ghorbel for selection of the transgenic plant; Dr Carmen Brisa (Department of Biology, Universitat de València) for advice and technical facilities; Drs C. R. Arias, E. Garay and R. Aznar (Department of Microbiology, Universitat de València) for technical facilities; R. Lluch (Leica, Spain) for technical support; and Dr V. Moreno (IBMCP, Universitat Politècnica de València-CSIC) for comments on this manuscript. This research was supported by a grant from the Instituto Nacional de Investigaciones Agrarias (SC97–102) and from CICYT-European Union (1FD97–0822). O.O.-F. received a fellowship from the Instituto Nacional de Investigaciones Agrarias.
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LITERATURE CITED |
|---|
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-
Bajaj YPS. 1994. Plant protoplast and genetic engineering. In: Bajaj YPS, ed. Biotechnology in agriculture and forestry, Vol. 29. Berlin, Heidelberg, New York: Springer.
Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC. 1994. Green fluorescent protein as a marker for gene expression. Science 263: 802–805.
Chiu W, Niwa Y, Zeng W, Hirano T. 1996. Engineered GFP as a vital reporter in plants. Current Biology 6: 325–330.[CrossRef][Web of Science][Medline]
Chytilova E, Macas J, Galbraith DW. 1999. Green fluorescent protein targeted to the nucleus, a transgenic phenotype useful on studies in plant biology. Annals of Botany 83: 645–654.
Dellaporta SI, Wood J, Hicks JB. 1983. A plant DNA minipreparation: version II. Plant Molecular Biology Reports 1: 19–21.
Dhandayuthapani S, Via LE, Thomas CA, Horowitz PM, Deretic P, Deretic V. 1995. Green fluorescent protein as a marker for gene expression and cell biology of mycobacterial interactions with macrophages. Molecular Microbiology 17: 901–912.[CrossRef][Web of Science][Medline]
Elliott AR, Campbell JA, Dugdale B, Brettell RIS, Grof CPL. 1999. Green-fluorescent protein facilitates rapid in vivo detection of genetically transformed plant cells. Plant Cell Reports 18: 707–714.[CrossRef]
Escobar MA, Park J, Polito VS, Leslie CA, Uratsu SL, McGranahan GH, Dandekar AM. 2000. Using GFP as scorable marker in walnut somatic embryo transformation. Annals of Botany 85: 831–835.
Fleming GH, Olivares-Fuster O, Fatta del Bosco S, Grosser JW. 2000. An alternative method for the genetic transformation of sweet orange. In Vitro-Plant 36: 450–455.
Ghorbel R, Juarez J, Navarro L, Peña L. 1999. Green fluorescent protein as a valuable marker for efficient transformation and improved regeneration of recalcitrant woody plants. Theoretical and Applied Genetics 99: 350–358.[CrossRef]
Gleba YY, Shlumukov LR. 1990. Selection of somatic hybrids. In: Dix PJ, ed. Plant cell line selection. New York: VCH
Grosser JW, Chandler JL. 1987. Aseptic isolation of leaf protoplasts from Citrus, Poncirus, Citrus x Poncirus hybrids and Severinia for use in somatic hybridization experiments. Scientia Horticulturaee 31: 253–257.
Grosser JW, Gmitter FG Jr. 1990. Protoplast fusion and citrus improvement. Plant Breeding Reviews 8: 339–374.
Grosser JW, Ollitrault P, Olivares-Fuster O. 2000. Somatic hybridization in citrus: an effective tool to facilitate variety improvement. In Vitro-Plant 36: 434–449.
Haseloff J. 1999. GFP variants for multispectral imaging of living cells. Methods in Cell Biology 58: 139–151.[Web of Science][Medline]
Haseloff J, Siemering KR, Prasher DC, Hodge S. 1997. Removal of a cryptic intron and subcellular localisation of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. Proceedings of the Natural Academy of Sciences of the USA 91: 12501–12504.
Johnson AAT, Veilleux RE. 2001. Somatic hybridization and applications in plant breeding. Plant Breeding Reviews 20: 167–225.
Kijas JMH, Thomas MR, Fowler JCS, Roose ML. 1997. Integration of trinucleotide microsatellites into a linkage map of Citrus. Theoretical and Applied Genetics 94: 701–706.[CrossRef]
Leffel SM, Mabon SA, Stewart CN Jr. 1997. Applications of green fluorescent protein in plants. BioTechniques 23: 912–918.[Web of Science][Medline]
Misteli T, Spector DL. 1997. Applications of the green fluorescent protein in cell biology and biotechnology. Nature Biotechnology 15: 961–964.[CrossRef][Web of Science][Medline]
Molinier J, Himber C, Hahne G. 2000. Use of green fluorescent protein for detection of transformed shoots and homozygous offspring. Plant Cell Reports 19: 219–223.
Murashige T, Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15: 473–497.[CrossRef]
Murashige T, Tucker DPH. 1969. Growth factor requirement of citrus tissue culture. Proceedings of the 1st International Citrus Symposium 3: 1155–1161.
Nadel BL. 1989. Use of fluorescein diacetate in Citrus tissue cultures for determination of cell viability and selection of mutants. Scientia Horticulturaee 39: 15–21.
Olivares-Fuster O, Asins MJ, Duran-Vila N, Navarro L. 2000. Cryopreserved callus, a source of protoplasts for citrus improvement. Journal of Horticultural Science and Biotechnology 75: 635–640.
Plautz JD, Day RN, Dailey GM, Welsh SB, Hall JC, Halpain S, Kay SA. 1996. Green fluorescent protein and its derivatives as versatile markers for gene expression in living Drosophila melanogaster, plant and mammalian cells. Gene 173: 83–87.[CrossRef][Web of Science][Medline]
Rouwendal GJA, Mendes O, Wolbert EHJ, Douwe de Boer A. 1997. Enhanced expression of tobacco of the gene encoding green fluorescent protein by modification of its codon usage. Plant Molecular Biology 33: 989–999.[CrossRef][Web of Science][Medline]
Tian L, Seguin A, Charest PJ. 1997. Expression of the green fluorescent protein in conifer tissues. Plant Cell Reports 16: 267–271.[CrossRef]
Tsien RY. 1998. The green fluorescent protein. Annual Reviews of Biochemistry 67: 509–544.
Valdivia RH, Hromockyj AE, Monack D, Ramakrishnan L, Falkow S. 1996. Applications for green fluorescent protein (GFP) in the study of host–pathogen interactions. Gene 173: 47–52.[CrossRef][Web of Science][Medline]
Vardi A, Galun E. 1988. Recent advances in protoplast culture of horticultural crops: Citrus. Scientia Horticulturaee 37: 217–230.[CrossRef]
Vazquez-Tello A, Yang LJ, Hidaka M, Uozumi T. 1996. Inherited chilling tolerance in somatic hybrids of transgenic Hibiscus rosa-sinensis x transgenic Lavatera thuringiaca selected by double-antibiotic resistance. Plant Cell Reports 15: 506–511.[CrossRef]
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