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Efficient Agrobacterium-mediated genetic transformation of oilseed mustard [Brassica juncea (L.) Czern.] using leaf piece explants

Abstract

Leaf piece explants of five Brassica juncea (L.) Czern. cultivars were transformed with an Agrobacterium tumefaciens strain EHA105 harboring the plasmid pCAMBIA1301, which contains the β-glucuronidase (uidA) and hygromycin phosphotransferase (hpt) genes under the control of cauliflower mosaic virus 35S (CaMV35S) promoter. Transgenic plants were regenerated on Murashige and Skoog (MS) medium fortified with 8.87 µM 6-benzylaminopurine, 0.22 µM 2,4-dichlorophenoxyacetic acid, and 20 µM silver nitrate in the presence of 30 mg/l hygromycin. When co-culture took place in the presence of 100 µM acetosyringone, the efficiency of stable transformation was found to be approximately 19% in the T 0 generation, with the transgenic plants and their progeny showing constitutive GUS expression in different plant organs. Southern blot hybridization of uidA and hpt genes confirmed transgene integration within the genome of transformed plants of each cultivar. Inheritance of hpt gene for single copy T-DNA inserts showed a 3:1 pattern of Mendelian segregation in progeny plants through germination of T 1 seeds on MS medium containing 30 mg/l hygromycin. The protocol described here reports superior transformation efficiency over previously published protocols and should contribute to enhanced biotechnology applications in B. juncea.

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References

  • Akasaka-Kennedy Y.; Yoshida H.; Takahata Y. Efficient plant regeneration from leaves of rapeseed (Brasica napus L.): the influence of AgNO3 and genotype. Plant Cell Rep. 24: 649–654; 2005. doi:10.1007/s00299-005-0010-8.

    PubMed  Article  CAS  Google Scholar 

  • Barfield D. G.; Pua E. C. Gene transfer in plants of Brassica juncea using Agrobacterium tumifaciens-mediated transformation. Plant Cell Rep. 10: 308–314; 1991.

    Article  CAS  Google Scholar 

  • Cardoza V.; Stewart C. N. Agrobacterium-mediated transformation of canola. In: CurtisI. S. (ed) Transgenic Crops of the World-Essential Protocols, Chapter 28. Kluwer Academic, Netherland; 2004: 379–387 pp

    Google Scholar 

  • Chi G. L.; Barfield D. G.; Sim G. E.; Pua E. C. Effect of AgNO3 and aminoethoxyvinyleglycine on in vitro shoot and root organogenesis from seedling explants of recalcitrant Brassica genotypes. Plant Cell Rep. 9: 195–198; 1990. doi:10.1007/BF00232178.

    Article  CAS  Google Scholar 

  • Comai L.; Facciotti D.; Hiatt W. R.; Thompson G.; Rose R. E.; Stalker D. M. Expression in plants of a mutant aroA gene from Salmonella typhimurium confers tolerance to glyphosate. Nature 317: 741–744; 1985. doi:10.1038/317741a0.

    Article  CAS  Google Scholar 

  • Das B.; Goswami L.; Ray S.; Ghosh S.; Bhattacharyya S.; Das S.; Majumder A. L. Agrobacterium-mediated transformation of Brassica juncea with cyanobacterial (Synechocystis PCC6803) delta-6 desaturase gene leads to production of gamma-linolenic acid. Plant Cell Tiss. Org. Cult. 86: 219–231; 2006. doi:10.1007/s11240-006-9111-5.

    Article  CAS  Google Scholar 

  • Dutta I.; Majumder P.; Saha P.; Ray K.; Das S. Constitutive and phloem specific expression of Allium sativum leaf agglutinin (ASAL) to engineer aphid (Lipaphis erysimi) resistance in transgenic Indian mustard (Brassica juncea). Plant Sci. 169: 996–1007; 2005. doi:10.1016/j.plantsci.2005.05.016.

    Article  CAS  Google Scholar 

  • Gong H.; Pua E. C. Identification and expression of genes associated with shoot regeneration from leaf disc explants of mustard (Brassica juncea) in vitro. Plant Sci. 167: 1191–1201; 2004. doi:10.1016/j.plantsci.2004.04.032.

    Article  CAS  Google Scholar 

  • Grover A.; Pental D. Breeding objectives and requirements for producing transgenics for major field crops of India. Curr. Sci. 84: 310–320; 2003.

    Google Scholar 

  • Hachey J. E.; Sharma K. K.; Moloney M. M. Efficient shoot regeneration of Brassica campestris using coyledon explants cultured in-vitro. Plant Cell Rep. 9: 549–554; 1991. doi:10.1007/BF00232329.

    Article  CAS  Google Scholar 

  • Hajdukiewicz P.; Svab Z.; Maliga P. The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol. Biol. 25: 989–994; 1994. doi:10.1007/BF00014672.

    PubMed  Article  CAS  Google Scholar 

  • Hong H.; Datla N.; Darwin W. R.; Patrick S. C.; Samuel L. M.; Xiao Q. High-level production of γ-linolenic acid in Brassica juncea using a Δ6-desaturase from Pythium irregulare. Plant Physiol. 129: 354–362; 2002. doi:10.1104/pp.001495.

    PubMed  Article  CAS  Google Scholar 

  • Hood E. E.; Helmer G. L.; Fraley R. T.; Chilton M. The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA. J. Bacteriol. 168: 1291–1301; 1986.

    PubMed  CAS  Google Scholar 

  • Horsch R. B.; Fry J. E.; Hoffmann N. L.; Eichholtz D.; Rogers S. G.; Frayley R. T. A simple and general method for transferring genes into plants. Science 227: 1229–1231; 1985. doi:10.1126/science.227.4691.1229.

    Article  CAS  Google Scholar 

  • Jagannath A.; Arumugam N.; Gupta V.; Pradhan A.; Burma P. K.; Pental D. Development of transgenic barstar lines and identification of male sterile (barnase)/restorer (barstar) combination for heterosis breeding in Indian oilseed mustard (Brassica juncea). Curr. Sci. 82: 46–52; 2002.

    CAS  Google Scholar 

  • Kanrar S.; Venkateshwari J.; Kirti P. B.; Chopra V. L. Transgenic Indian mustard (Brassica juncea) with resistance to mustard aphid (Lipaphis erysimi Kalt.). Plant Cell Rep. 20: 976–981; 2002. doi:10.1007/s00299-001-0422-z.

    Article  CAS  Google Scholar 

  • Kellar W. A.; Armstrong K. C. Embryogenesis and plant regeneration in Brassica napus anther cultures. Can. J. Bot. 55: 1383–1388; 1977.

    Article  Google Scholar 

  • Klimaszewska K.; Keller K. High frequency plant regeneration from thin cell layer explants of Brassica napus. Plant Cell Tiss. Org. Cult. 24: 183–197; 1985. doi:10.1007/BF00040193.

    Article  Google Scholar 

  • Kumar, P. R. Rapeseed mustard research in India: 21st century strategies. 10th International Rapeseed Congress, Canberra, Australia. 1999.

  • Mehra S.; Pareek A.; Bandyopadhyay P.; Sharma P.; Burma P. K.; Pental D. Development of transgenics in Indian oilseed mustard (Brassica juncea) resistant to herbicide phosphinothricin. Curr. Sci. 78: 1358–1364; 2000.

    CAS  Google Scholar 

  • Miguel C. M.; Oliveira M. M. Transgenic almond (Prunus dulcis Mill.) plants obtained by Agrobacterium-mediated transformation of leaf explants. Plant Cell Rep. 18: 387–393; 1999. doi:10.1007/s002990050591.

    Article  CAS  Google Scholar 

  • Murashige T.; Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15: 473–497; 1962. doi:10.1111/j.1399-3054.1962.tb08052.x.

    Article  CAS  Google Scholar 

  • Pental D.; Pradhan A. K.; Sodhi S. Y.; Mukhopadhyay A. Variations among Brassica juncea cultivars for regeneration from hypocotyl explants and optimization of conditions for Agrobacterium-mediated genetic transformation. Plant Cell Rep. 12: 462–467; 1993. doi:10.1007/BF00234713.

    Article  CAS  Google Scholar 

  • Pradhan A. K.; Sodhi Y. S.; Mukhopadhay A.; Pental D. Heterosis breeding in Indian mustard (Brassica juncea L. Czern and Coss): analysis of component characters contributing to heterosis for yield. Euphytica 69: 219–229; 1993. doi:10.1007/BF00022368.

    Article  Google Scholar 

  • Pua E. C.; Chi G. L. De novo shoot morphogenesis and plant growth of mustard (Brassica juncea) in vitro in relation to ethylene. Physiol. Plant 88: 467–474; 1993. doi:10.1111/j.1399-3054.1993.tb01361.x.

    Article  CAS  Google Scholar 

  • Radke S. E.; Turner J. C.; Facciotti D. Transformation and regeneration of Brassica rapa using Agrobacterium tumefaciens. Plant Cell Rep. 11: 499–505; 1992. doi:10.1007/BF00236265.

    Article  Google Scholar 

  • Sandal I.; Saini U.; Lacroix B.; Bhattacharya A.; Ahuja S. P.; Citovsky V. Agrobacterium-mediated genetic transformation of tea leaf explants: effects of counteracting bactericidity of leaf polyphenols without loss of bacterial virulence. Plant Cell Rep. 26: 169–176; 2007. doi:10.1007/s00299-006-0211-9.

    PubMed  Article  CAS  Google Scholar 

  • Sivaraman I.; Amurugam N.; Sodhi S. Y.; Gupta V.; Mukhopadhyay A.; Pradhan K. A.; Burma K. P.; Pental D. Development of high oleic and low linoleic acid transgenics in a zero erucic acid Brassica juncea L. (Indian mustard) line by antisense suppression of the fad2 gene. Mol. Breed. 13: 365–375; 2004. doi:10.1023/B:MOLB.0000034092.47934.d6.

    Article  CAS  Google Scholar 

  • Song G. Q.; Sink K. C. Agrobacterium tumifaciens-mediated transformation of blueberry (Vaccinium corymbosumi L.). Plant Cell Rep. 23: 475–484; 2004. doi:10.1007/s00299-004-0842-7.

    PubMed  Article  CAS  Google Scholar 

  • Spangenberg G.; Koop H. U.; Litcher R.; Schweiger H. G. Microculture of single protoplasts of Brassica napus. Physiol. Plant 66: 1–8; 1986. doi:10.1111/j.1399-3054.1986.tb01223.x.

    Article  CAS  Google Scholar 

  • Stringam G. R. Regeneration in stem explants of haploid rapeseed (Brassica napus L.). Plant Sci. Ltrs. 9: 115–119; 1977. doi:10.1016/0304-4211(77)90088-8.

    Article  CAS  Google Scholar 

  • Van Wordragen M. F.; De Jong J.; Schornagel J. J.; Dons H. J. M. Rapid screening for host-bacterium interactions in Agrobacterium-mediated gene transfer to Chrysanthemum, by using the gus-intron gene. Plant Sci. 81: 207–214; 1992. doi:10.1016/0168-9452(92)90044-M.

    Article  Google Scholar 

  • Vervliet G.; Holsters M.; Teuchy H.; Van Montagu M.; Schell J. Characterization of different plaque-forming and defective temperate phages in Agrobacterium strains. J. Gen. Virol. 26: 33–48; 1975. doi:10.1099/0022-1317-26-1-33.

    PubMed  Article  CAS  Google Scholar 

  • Visser R. G. F.; Jacobsen E.; Hesseling-Meinders A.; Schans M. J.; Witholt B.; Feenstra W. J. Transformation of homozygous diploid potato with an Agrobacterium tumefaciens binary vector system by adventitious shoot regeneration on leaf and stem segments. Plant Mol. Biol. 12: 329–337; 1989. doi:10.1007/BF00043210.

    Article  CAS  Google Scholar 

  • Xu J. H.; Davey M. R.; Cocking E. C. Plant regeneration from root protoplasts of Brassica. Plant Sci. Ltrs. 24: 117–121; 1982. doi:10.1016/0304-4211(82)90016-5.

    Article  Google Scholar 

  • Yang M. Z.; Jia S. R.; Pua E. C. High frequency of plant regeneration from hypocotyl explants of Brassica carinata A. Br. Plant Cell Tiss. Org. Cult. 24: 79–82; 1991. doi:10.1007/BF00039734.

    Article  Google Scholar 

  • Zhang H. X.; Hodson J. N.; Williams J. P.; Blumwald E. Engineering salt-tolerant Brassica plants: Characterization of yield and seed oil quality in transgenic plants with increased vacuolar sodium accumulation. Proc. Natl. Acad. Sci. U S A 98: 12832–12836; 2001. doi:10.1073/pnas.231476498.

    PubMed  Article  CAS  Google Scholar 

  • Zhu Y. L.; Elizabeth A. H.; Pilon-Smits Tarun A. S.; Weber S. U.; Jouanin L.; Terry N. Cadmium tolerance and accumulation in Indian mustard is enhanced by overexpressing γ-glutamylcysteine synthase. Plant Physiol. 121: 1169–1177; 1999. doi:10.1104/pp.121.4.1169.

    PubMed  Article  CAS  Google Scholar 

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Acknowledgements

Authors are thankful to Prof. Barbara Hohn, FMI, Basel, Switzerland for providing the plasmid pCAMBIA1301 and the Agrobacterium strain EHA105. We are grateful to the Council of Scientific and Industrial Research, Government of India, New Delhi, India for providing fellowship to ID and PS. We are also thankful to Behrampur Pulse and Oilseed Research Station, West Bengal, India for providing seeds of B. juncea cultivars. The support of Bose Institute, Kolkata, India is greatly acknowledged. For back-up service, Mr. Arup Kumar Dey is sincerely acknowledged. We also gratefully acknowledge the reviewers for corrections and suggestions to our manuscript.

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Correspondence to Prasenjit Saha.

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Dutta, I., Saha, P. & Das, S. Efficient Agrobacterium-mediated genetic transformation of oilseed mustard [Brassica juncea (L.) Czern.] using leaf piece explants. In Vitro Cell.Dev.Biol.-Plant 44, 401–411 (2008). https://doi.org/10.1007/s11627-008-9150-1

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  • DOI: https://doi.org/10.1007/s11627-008-9150-1

Keywords

  • Agrobacterium-mediated transformation
  • Brassica juncea
  • Leaf explants