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Production and genetic characterization of somatic hybrids between leaf mustard (Brassica juncea) and broccoli (Brassica oleracea)

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Abstract

Inter-specific somatic hybrids of leaf mustard (Brassica juncea) and broccoli (Brassica oleracea) were established to introduce cytoplasmic male sterility (CMS) and Verticillium dahliae Kleb. resistance from broccoli to leaf mustard. Protoplasts isolated from hypocotyls and cotyledons of inbred lines of leaf mustard and broccoli were fused using 40% (w/v) polyethelene glycol and then cultured in modified k8p medium supplemented with 0.2 mg/l 2,4-dichlorophenoxi-acetic acid, 0.5 mg/l 6-benzyladenine (BA), 0.1 mg/l naphthaleneacetic acid (NAA), and 0.1 mg/l kinetin (Kin), and 0.4 M mannitol as osmoticum. At the eight- to ten-cell stage, divided cells were transferred to Kao’s basal medium supplemented with 0.3 M sucrose as carbon source and 0.1% agarose, 2 mg/l BA, 2 mg/l zeatin (ZEA), 1 mg/l NAA, and 0.5 mg/l Kin for callus proliferation. After 35 d, when small calli reached 2–3 mm in diameter, calli were transferred to regeneration medium containing 5 mg/l ZEA and 2 mg/l indole-3-acetic acid. The chromosome numbers of putative somatic hybrids varied from 46 to 54. A total of ten plants showed a 0.5-kb, CMS-specific band, while two regenerated plants showed a missing band similar to that of leaf mustard by polymerase chain reaction amplification using Ogura CMS-specific primers. Hybrid state was confirmed by random amplified polymorphic DNA analysis. Regenerated plants had normal petals and stamens, but only two plants produced pollen and set seed.

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References

  • Akagi H.; Taguchi T.; Fujimura T. Stable inheritance and expression of the CMS traits introduced by asymmetric protoplast fusion. Theor. Appl. Genet. 91: 563–567; 1995.

    Article  Google Scholar 

  • Arumugam N.; Mukhopadhyay A.; Gupta N.; Sodhi Y. S.; Verma J. K.; Pental D.; Pradhan A. K. Somatic cell hybridization of ‘oxy’ CMS Brassica juncea (AABB) with B. oleracea (CC) for correction of chlorosis and transfer of novel organelle combinations to allotetraploed Brassicas. Theor. Appl. Genet. 100: 1043–1049; 2000.

    Article  Google Scholar 

  • Barsby T. L.; Yarrow S. A.; Shepard J. F. A rapid and efficient alternative procedure for the regeneration of plants from hypocotyl protopalsts of Brassica napus. Plant Cell Rep. 5: 101–103; 1986.

    Google Scholar 

  • Benabdelmouna A.; Guéritaine G.; Abirached-Darmency M.; Darmency H. Genome discrimination in progeny of interspecific hybrids between Brassica napus and Raphanus raphanistrum. Genome 46: 469–472; 2003.

    Article  PubMed  CAS  Google Scholar 

  • Bhat R. G.; Subbarao K. V. Reaction of broccoli to isolates of Verticillium dahliae from various hosts. Plant Disease 85: 141–146; 2001.

    Article  Google Scholar 

  • Borgato L.; Conicella C.; Pisani F.; Furini A. Production and characterization of arboreous and fertile Solanum melongena + Solanum marginatum somatic hybrid plants. Planta 226: 961–969; 2007.

    Article  PubMed  CAS  Google Scholar 

  • Brodelius P.; Nilsson K. Entrapment of plant cells in different matrixes: A Comparative Study. FEBS Lett. 122: 312; 1980.

    Article  CAS  Google Scholar 

  • Cardi T.; Earle E. D. Production of new CMS Brassica oleracea by transfer of ‘Anand’ cytoplasm from B. rapa through protoplast fusion. Theor. Appl. Genet. 94: 204–212; 1997.

    Article  Google Scholar 

  • Chatterjee G.; Sikdar S. R.; Das S.; Sen S. K. Intergeneric somatic hybrid production through protoplast fusion between Brassica juncea and Diplotaxis murlis. Theor. Appl. Genet. 76: 915–922; 1988.

    Article  Google Scholar 

  • Cheigh H. S.; Park K. Y. Biochemical, microbiological and nutritional aspects of Kimchi (Korean fermented vegetable products). Crit. Rev. Food Sci. Nutr. 34: 175–177; 1994.

    Article  PubMed  CAS  Google Scholar 

  • Chen F. H.; Wang H.; Li Z. Y. Production and genetic analysis of partial hybrids in intertribal crosses between Brassica species (B. rapa; B. napus) and Capsella bursa pastoris. Plant Cell Rep. 26: 1791–1800; 2007.

    Article  PubMed  CAS  Google Scholar 

  • Chen L. P.; Zhang M. F.; Li C. S.; Yutaka H. Production of inter specific somatic hybrids between tuber mustard (Brassica juncea) and red cabbage (Brassica oleracea). Plant Cell Tiss. Org. Cult. 80: 305–311; 2005.

    Article  Google Scholar 

  • Cheng A. X.; Xia G. M.; Zhi D. Y.; Chen H. M. Intermediate fertile Triticum aestivum (+) Agropyron elongatum somatic hybrids are generated by low doses of UV irradiation. Cell Res. 14: 86–91; 2004.

    Article  PubMed  CAS  Google Scholar 

  • Cho Y. S.; Park S. K. Studies on the chemical components of Korea Brassica juncea Cosson. Sunchon Natl. Univ. Bull. 9: 169–172; 1990.

    Google Scholar 

  • Delpierre N.; Boccon-Gibod J. An extensive hairy root production precedes shoot regeneration in protoplast-derived calli of cauliflower (Brassica oleracea var. botrytis). Plant Cell Rep. 11: 351–354; 1992.

    Article  Google Scholar 

  • Doyle J. J.; Doyle J. I. Isolation of plant DNA from fresh tissue. Focus 12: 13–15; 1990.

    Google Scholar 

  • Du X. Z.; Ge X. H.; Yao X. C.; Zhao Z. G.; Li Z. Y. Production and cytogenetic characterization of intertribal somatic hybrids between Brassica napus and Isatis indigotica and backcrossing progenies. Plant Cell Rep. 28: 1105–1113; 2009.

    Article  PubMed  CAS  Google Scholar 

  • Dudits D.; Maroy E.; Praznovszky T.; Olah Z.; Gyorgyey J.; Cella R. Transfer of resistance traits from carrot into tobacco by asymmetric somatic hybridization: regeneration of fertile plants. Proc. Natl. Acad. Sci. USA 84: 8434–8438; 1987.

    Article  PubMed  CAS  Google Scholar 

  • Forsberg J.; Dixelius C.; Lagercrantz U.; Glimelius K. UV dose-dependent DNA elimination in asymmetric somatic hybrids between Brassica napus and Arabidopsis thaliana. Plant Sci. 131: 65–76; 1998.

    Article  CAS  Google Scholar 

  • Glimelius K. Somatic hybridization. In: Gómez-Campo C. (ed) Biology of Brassica coenospecies. Elsevier, Amsterdam, pp 107–148; 1999.

    Chapter  Google Scholar 

  • Glimelius K.; Mats D.; Hugo F. F. Selection and enrichment of plant protoplast heterokaryons of Brassicaceae by flow sorting. Plant Sci. 45: 133–141; 1986.

    Article  CAS  Google Scholar 

  • Hu Q.; Andersen S. B.; Dixelius C.; Hansen L. N. Production of fertile intergeneric somatic hybrids between Brassica napus and Sinapis arvensis for the enrichment of the rapeseed gene pool. Plant Cell Rep. 21: 147–152; 2002a.

    Article  CAS  Google Scholar 

  • Hu Q.; Hansen L. N.; Laursen J.; Dixelius C.; Andersen S. B. Intergeneric hybrids between Brassica napus and Orychophragmus violaceus containing traits of agronomic importance for oilseed rape breeding. Theor. Appl. Genet. 105: 834–840; 2002b.

    Article  PubMed  CAS  Google Scholar 

  • Ji Y. F.; Pertuze R.; Chetelat R. T. Genomic differentiation by GISH in inter-specific and inter-generic hybrids of tomato and related nightshades. Chromosome Res. 12: 107–116; 2004.

    Article  PubMed  CAS  Google Scholar 

  • Jourdan P. S.; Earle E. D.; Mutschler M. A. Atrazine resistant cauliflower obtained by somatic hybridization between Brassica oleracea and ATR-B.napus. Theor. Appl. Genet. 78: 271–279; 1989.

    Article  Google Scholar 

  • Kao K. N.; Michcharyluk M. R. Nutritional requirements for growth of Vicia hajastana cells and protoplast at very low population density in liquid media. Planta 126: 105–110; 1975.

    Article  CAS  Google Scholar 

  • Kim B. K.; Park K. Y.; Kim B. K. Antimutagenic and anticancer effects of leaf mustard and leaf mustard Kimchi. Journal of Food Sci. Nut. 12: 84–88; 2007.

    Article  Google Scholar 

  • Kirti P. B.; Baldev A.; Gaikwad K.; Chopra V. L. Introgression of a gene restoring fertility to CMS (Trachystoma) Brassica juncea and the genetics of restoration. Plant Breed. 116: 259–262; 1997.

    Article  Google Scholar 

  • Kirti P. B.; Narasimhulu S. B.; Prakash S.; Chopra V. L. Production and characterization of inter-generic somatic hybrids of Trachystoma ballii and Brassica juncea. Plant Cell Rep. 11: 90–92; 1992.

    Google Scholar 

  • Kirti P. B.; Prakash S.; Chopra V. L. Inter-specific hybridization between Brassica juncea and B. spinecens through protoplast fusion. Plant Cell Rep. 9: 639–642; 1991.

    Article  Google Scholar 

  • Kisaka H.; Kisaka M.; Kanno A.; Kameya T. Intergeneric somatic hybridization of rice (Oryza sativa L.) and barley (Hordeum vulgare L.) by protoplast fusion. Plant Cell Rep. 17: 362–367; 1998.

    Article  CAS  Google Scholar 

  • Koike S. T.; Subbarao K. V.; Davis R. M.; Gordon T. R.; Hubbard J. C. Verticillium wilt of cauliflower in California. Plant Dis. 78: 1116–1121; 1994.

    Article  Google Scholar 

  • Lee I. H.; Park J. I.; Jeong O. S.; Jung H. J.; Jung G. H.; Nou I. S. Genetic relationship based on RAPD analysis of Yeosu Dolsan leaf mustard (Brassica juncea). Journal of Life Science 20: 66–70; 2010.

    CAS  Google Scholar 

  • Leitch A. R.; Schwarzacher T.; Jackson D.; Leitch I. J. Microscopy handbook No. 27 In situ hybridization: a practical guide. Bios Scientific, Oxford; 1994.

    Google Scholar 

  • Leung A. Y.; Steven F. Encyclopedia of common natural ingredients used in food, drugs; and cosmetics. 2nd ed. Wiley, New York; 1995.

    Google Scholar 

  • Li M. Y. Recognition and prevention of Brassica fusarium wilt. China Vegetable 2: 60; 2004.

    Google Scholar 

  • Li M. Y.; Ma Y. J.; Yao J. L. Be wary of the spread of the fusarium wilt of Brassica vegetable in China. China Plant Protection 1: 21–22; 2006.

    Google Scholar 

  • Li M. Y.; Zhang T. T.; Li X. H.; Yan H. Fusarium wilts in crucifer and identification of pathogen. Plant Protection 29: 44–45; 2003.

    Google Scholar 

  • Lian Y. J.; Lim H. T. Plant regeneration of B. juncea through plant tissue and protoplast culture. J. Plant Biotechnol. 3: 27–31; 2001.

    Google Scholar 

  • Lim H. S.; Park K. O.; Nishizawa N.; Bae S. O.; Choi M. R. Cytotoxicity of extracts from Dolsan leaf mustard Kimchi treated with lactic acid bacteria on lung and gastric cancer cells. Biotechnol. Bioprocess Eng. 13: 174–181; 2008.

    Article  CAS  Google Scholar 

  • Liu P. Y. Chinese mustard. In: Liu P. Y. (ed) Origin; Evolution; Classification and Distribution of Chinese Mustard. China Agriculture Press, Beijing, pp 1–26; 1996.

    Google Scholar 

  • Lörz H.; Larkin P. I.; Thomson I.; Scowcroft W. R. Improvement protoplast culture and agarose media. Plant Cell Tiss. Org. Cult. 2: 217–226; 1982.

    Article  Google Scholar 

  • Menke U.; Schilde-Rentschler L.; Ruoss B.; Zanke C.; Hemleben V.; Ninnemann H. Somatic hybrids between the cultivated potato Solanum tuberosum L. and the 1EBN wild species Solanum pinnatisectum Dun.: morphological and molecular characterization. Theor. Appl. Genet. 92: 617–626; 1996.

    Article  Google Scholar 

  • Menzel L.; Morgan A.; Brown S.; Maliga P. Fusion-mediated combination of Ogura-type cytoplasmic male sterility with Brassica napus plastids using X-irradiated CMS protoplasts. Plant Cell Rep. 6: 98–101; 1987.

    Google Scholar 

  • Murashige T.; Skoog F. A revised medium for rapid grown and bio-assays with tobacco tissue cultures. Physiol Plant 15: 473–497; 1962.

    Article  CAS  Google Scholar 

  • Ren J. P.; Dickson M. H.; Earle E. D. Improved resistance to bacterial soft rot by protoplast fusion between Brassica rapa and B. oleracea. Theor. Appl. Genet. 100: 810–819; 2000.

    Article  Google Scholar 

  • Schoenmaker H. C.; Nobel E. M.; Koornneef M. Use of leaky nitrate reductase-deficient mutants of tomato (Lycopersicon esculentum Mill.) for selection of somatic hybrid cell lines with wild type potato (Solanum tuberosum L.). Plant Cell Tiss. Org. Cult. 31: 151–154; 1992.

    Article  Google Scholar 

  • Sigareva M. A.; Earle E. D. Direct transfer of a cold-tolerant Ogura male sterile cytoplasm into cabbage (Brassica oleracea ssp. capitata) via protoplast fusion. Theor. Appl. Genet. 94: 213–220; 1997.

    Article  Google Scholar 

  • Sjödin C.; Glimelius K. Transfer resistance against Phoma lingam to Brassica napus by asymmetric somatic hybridization combined with toxin selection. Theor. Appl. Genet. 78: 513–520; 1989.

    Article  Google Scholar 

  • Snowdon R. J.; Köhler W.; Friedt W.; Köhler A. Genomic in situ hybridization in Brassica amphidiploids and interspecific hybrids. Theor. Appl. Genet. 95: 1320–1324; 1997.

    Article  CAS  Google Scholar 

  • Tu Y. Q.; Sun J.; Liu Y.; Ge X. H.; Zhao Z. G.; Yao X. C.; Li Z. Y. Production and characterization of intertribal somatic hybrids of Raphanus sativus and Brassica rapa with dye and medicinal plant Isatis indigotica. Plant Cell Rep. 27: 873–883; 2008.

    Article  PubMed  CAS  Google Scholar 

  • Vamling K.; Glimelius K. Regeneration of plants from protoplasts of oilseed Brassica crops. Bajaj YPS (eds). Biotechnology in agriculture and forestry 10: 385–417. Plant protoplasts and genetic engineering. Springer-Verlag Berlin, 1990.

  • Vatsya B.; Bhaskaran S. Plant regeneration from cotyledonary protoplast of cauliflower (Brassica oleracea L. botrytis L.). Protoplasma 113: 161–163; 1982.

    Article  Google Scholar 

  • Waara S.; Glimelius K. The potential of somatic hybridization in crop breeding. Euphytica 85: 217–233; 1995.

    Article  Google Scholar 

  • Walters T. W.; Earle E. D. Organellar segregation, rearrangement and recombination in protoplast fusion-derived Brassica oleracea calli. Theor. Appl. Genet. 85: 761–769; 1993.

    Article  Google Scholar 

  • Walters T. W.; Mutschler M. A.; Earle E. D. Protoplast fusion-derived Ogura male sterile cauliflower with cold tolerance. Plant Cell Rep 10: 624–628; 1992.

    Article  Google Scholar 

  • Wang M. Q.; Zhao J. S.; Peng Z. Y.; Guo W.; Wang Y.; Wang L.; Xia G. M. Chromosomes are eliminated in the symmetric fusion between Arabidopsis thaliana L. and Bupleurum scorzonerifolium Willd. Plant Cell Tiss. Org. Cult. 92: 121–130; 2008.

    Article  Google Scholar 

  • Wang Y. P.; Snowdon R. J.; Rudloff E.; Wehling P.; Friedt W.; Sonntag K. Cytogenetic characterization and fae1 gene variation in progenies from asymmetric somatic hybrids between Brassica napus and Crambe abyssinica. Genome 47: 724–731; 2004.

    Article  PubMed  CAS  Google Scholar 

  • Wang Y. P.; Zhao X. X.; Sonntag K.; Wehling P.; Snowdon R. J. Behaviour of Sinapis alba chromosome in a Brassica napus background revealed by genomic in situ hybridization. Chromosome Res. 13: 819–826; 2005.

    Article  PubMed  CAS  Google Scholar 

  • Xu C. H.; Xia G. M.; Zhi D. Y.; Xiang F. N.; Chen H. M. Integration of maize nuclear and mitochondrial DNA into the wheat genome through somatic hybridization. Plant Sci. 165: 1001–1008; 2003.

    Article  CAS  Google Scholar 

  • Xu Y. S.; Clark M. S.; Pehu E. Use of RAPD markers to screen somatic hybrids between Solanum tuberosum and S. brevidens. Plant Cell Rep. 12: 107–109; 1993.

    Google Scholar 

  • Yamagishi H.; Glimelius K. Somatic hybrids between Arabidopsis thaliana and cytoplasmic male-sterile radish (Raphanus sativus). Plant Cell Rep. 22: 52–58; 2003.

    Article  PubMed  CAS  Google Scholar 

  • Yoo E. J.; Choi M. R.; Lim H. S. The relationship between ACE inhibitory activity and degradations of sulfur containing materials in Dolsan leaf mustard juice. Biotechnol. Bioprocess Eng. 9: 400–404; 2004.

    Article  CAS  Google Scholar 

  • Zhao Z. G.; Hu T. T.; Ge X. H.; Du X. Z.; Ding L.; Li Z. Y. Production and characterization of intergeneric somatic hybrids between Brassica nupus and Orychophragmus violaceus and their backcrossing progenies. Plant Cell Rep. 27: 1611–1621; 2008.

    Article  PubMed  CAS  Google Scholar 

  • Zhong X. B.; Hans J. J.; Zabel P. Preparation of tomato meiotic pachytene and mitotic metaphase chromosomes suitable for fluorescence in situ hybridization (FISH). Chromosome Res. 4: 24–28; 1996.

    Article  PubMed  CAS  Google Scholar 

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  1. College of Life Science, Linyi University, Shandong, Linyi, 276005, China

    Yu-Ji Lian, Guang-Zhe Lin & Xiao-Mei Zhao

  2. College of Biomedical Science, Kangwon National University, Chunchon, 200-701, South Korea

    Hak-Tae Lim

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  1. Yu-Ji Lian
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  2. Guang-Zhe Lin
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Lian, YJ., Lin, GZ., Zhao, XM. et al. Production and genetic characterization of somatic hybrids between leaf mustard (Brassica juncea) and broccoli (Brassica oleracea). In Vitro Cell.Dev.Biol.-Plant 47, 289–296 (2011). https://doi.org/10.1007/s11627-011-9355-6

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