Abstract
Campanula medium is an important ornamental plant known for its blue and bell-shaped flowers and a good model for studying flower colour formation and modification. The major outstanding obstacle preventing the developing of genetic modification and improvements in C. medium is a missing transformation method. For the first time, an effective and reliable in vitro regeneration and Agrobacterium-mediated transformation system has been established in this study. Different types and combinations of plant growth regulators (PGRs) were investigated for shoot regeneration by using leaf explants from three different cultivars. A high regeneration rate (50% for cv. ‘Blue double’, 58% for cv. ‘White double’ and 44% for cv. ‘Pink double’) was achieved on MS-based medium supplemented with 11.5 μM thidiazuron (TDZ) and 2.3 μM 1-naphthaleneacetic acid (NAA). In total, the Agrobacterium strains ABI, AGL1 and GV3101:pMP90, transformed with a binary plasmid harbouring NPTII and GFP gene cassettes, were evaluated for plant transformation, while various factors affecting the transformation efficiency were also identified. The highest transformation rate of about 22% was obtained from explants pre-cultured on regeneration medium and infected with AGL1. However, transgenic shoots were produced by all three Agrobacterium strains studied and confirmed by PCR and GFP fluorescence microscopy.
Key message
Campanula medium regeneration and genetic transformation methods were established while various factors affecting the transformation efficiency were identified such as pre-culture treatment of leaf explants and different Agrobacterium strains.
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Abbreviations
- BA:
-
6-Benzylaminopurine
- NAA:
-
1-Naphthaleneacetic acid
- IAA:
-
Indole-3-acetic acid
- TDZ:
-
Thidiazuron
- GFP:
-
Green fluorescent protein
References
Bosma T, Dole JM (2002) Postharvest handling of cut Campanula medium flowers. HortScience 37:954–958. https://doi.org/10.21273/HORTSCI.37.6.954
Brandt K (1992) Micropropagation of Campanula isophylla Moretti. Plant Cell Tiss Org 29:31–36. https://doi.org/10.1007/BF00036143
Brandt K (1994) Variation among and within clones in formation of roots and shoots during micropropagation of Campanula isophylla. Plant Cell Tiss Org 39:63–68. https://doi.org/10.1007/BF00037593
Dewir YH, Nurmansyah NY, da Silva JAT (2018) Thidiazuron-induced abnormalities in plant tissue cultures. Plant Cell Rep 37:1451–1470. https://doi.org/10.1007/s00299-018-2326-1
Dorokhov DB, Klocke E (1997) A rapid and economic technique for RAPD analysis of plant genomes. Genetika 33:443–450
Edwards K, Johnstone C, Thompson C (1991) A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Res 19:1349–1349. https://doi.org/10.1093/nar/19.6.1349
Frello S, Stummann BM, Serek M (2002) Shoot regeneration of Campanula carpatica Jacq. (Campanulaceae) via callus phase. Sci Hortic 93:85–90. https://doi.org/10.1016/S0304-4238(01)00312-0
Holsters M et al (1980) The functional organization of the nopaline A. tumefaciens plasmid pTiC58. Plasmid 3:212–230. https://doi.org/10.1016/0147-619X(80)90110-9
Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT (1985) A simple and general method for transferring genes into plants. Science 227:1229–1231. https://doi.org/10.1126/science.227.4691.1229
Jensen L, Hegelund JN, Olsen A, Lütken H, Müller R (2016) A natural frameshift mutation in Campanula EIL2 correlates with ethylene insensitivity in flowers. BMC Plant Biol 16:117. https://doi.org/10.1186/s12870-016-0786-4
Joung YH, Roh MS, Kamo K, Song JS (2001) Agrobacterium-mediated transformation of Campanula glomerata. Plant Cell Rep 20:289–295. https://doi.org/10.1007/s002990100341
Joung YH, Liao MS, Roh MS, Kamo K, Song JS (2002) In vitro propagation of Campanula glomerata, ‘Acaulis’ from leaf blade explants. Sci Hortic 92:137–146. https://doi.org/10.1016/S0304-4238(01)00282-5
Karimiani ZG, Serek M, Mibus H (2015) Transformation of Campanula carpatica to alter flower colour and ethylene sensitivity. J Hortic Sci Biotech 90:518–524. https://doi.org/10.1080/14620316.2015.11668708
Kato M, Shimizu H, Onozaki T, Tanikawa N, Ikeda H, Hisamatsu T, Ichimura K (2002) Role of ethylene in senescence of pollinated and unpollinated Campanula medium flowers. J Jpn Soc Hortic Sci 71:385–387. https://doi.org/10.2503/jjshs.71.385
Koncz C, Schell J (1986) The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimeric genes carried by a novel type of Agrobacterium binary vector. Mol Gen Genet 204:383–396. https://doi.org/10.1007/Bf00331014
Lazo GR, Stein PA, Ludwig RA (1991) A DNA transformation-competent Arabidopsis genomic library in Agrobacterium. Nat Biotechnol 9:963–967. https://doi.org/10.1038/nbt1091-963
Lewis P, Lynch M (1998) Campanulas—a gardener’s guide. Timber Press, Portland
Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plantarum 15:473–497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
Saez L, Aldasoro JJ (2003) A taxonomic revision of Campanula L subgenus Sicyocodon (Feer) Damboldt and subgenus Megalocalyx Damboldt (Campanulaceae) Bot J Linn Soc 141:215–241. https://doi.org/10.1046/j.1095-8339.2003.00133.x
Serek M (1991) Postharvest Characteristics of Campanula carpatica—influence of temperature programming. Gartenbauwissenschaft 56:71–74
Sivanesan L, Lee YM, Song JY, Jeong BR (2007) Adventitious shoot regeneration from leaf and petiole explants of Campanula punctata Lam. var. rubriflora Makino. Propag Ornam Plants 7:210–215. https://doi.org/10.1007/s11240-011-9983-x
Sivanesan I, Lim MY, Jeong BR (2011) Somatic embryogenesis and plant regeneration from leaf and petiole explants of Campanula punctata Lam. var. rubriflora Makino. Plant Cell Tiss Organ Cult 107:365–369. https://doi.org/10.1007/s11240-011-9983-x
Sriskandarajah S, Frello S, Serek M (2001) Induction of adventitious shoots in vitro in Campanula carpatica. Plant Cell Tiss Org 67:295–298. https://doi.org/10.1023/A:1012786818689
Sriskandarajah S, Frello S, Jorgensen K, Serek M (2004) Agrobacterium tumefaciens-mediated transformation of Campanula carpatica: factors affecting transformation and regeneration of transgenic shoots. Plant Cell Rep 23:59–63. https://doi.org/10.1007/s00299-004-0797-8
Sriskandarajah S, Mibus H, Serek M (2007) Transgenic Campanula carpatica plants with reduced ethylene sensitivity. Plant Cell Rep 26:805–813. https://doi.org/10.1007/s00299-006-0291-6
Sriskandarajah S, Mibus H, Serek M (2008) Regeneration and transformation in adult plants of Campanula species. Plant Cell Rep 27:1713–1720. https://doi.org/10.1007/s00299-008-0590-1
Tada H, Nakashima T, Kunitake H, Mori K, Tanaka M, Ishimaru K (1996) Polyacetylenes in hairy root cultures of Campanula medium L. J Plant Physiol 147:617–619. https://doi.org/10.1016/S0176-1617(96)80056-3
Acknowledgements
The authors would like to thank the lab staff and gardeners of the Section Floriculture at Leibniz University Hannover for technical assistance. Guo Li gratefully acknowledge financial support from China Scholarship Council (CSC).
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Communicated by M. I. Beruto.
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Gehl, C., Li, G. & Serek, M. An efficient protocol for Agrobacterium-mediated transformation and regeneration of Campanula medium (Canterbury bells) based on leaf disc explants. Plant Cell Tiss Organ Cult 140, 635–645 (2020). https://doi.org/10.1007/s11240-019-01758-5
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DOI: https://doi.org/10.1007/s11240-019-01758-5