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Efficient in vitro plant regeneration and generation of transgenic plants in barley (Hordeum vulgare L.) using particle bombardment

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Abstract

Plant regeneration from immature embryos of 15 Indian barley genotypes (Hordeum vulgare L.) was examined. Highest percent callus induction and number of regenerated plantlets were obtained in cultivars BL 2 (94.4 %; 12.1), RD 2668 (92.6 %; 9.1) and RD 2552 (90.8 %; 7.8). The highly responding cultivar BL 2 was selected for further development of transformation protocol. The plasmid DNA isolated from pCAMBIA1381 harbouring hptII gene as selectable marker and gusA gene as reporter was used. Particle bombardment was used for transformation of immature embryos and regeneration of transgenic plants in Indian barley genotype for the first time. Transformation experiments were carried out using different parameters and optimum conditions for DNA delivery was standardized. The transient expression of gusA gene was investigated as a preliminary test of optimum DNA delivery and for selecting the most appropriate bombardment parameters. The optimum conditions were: gold microparticles (diameter 1.0 μm) shot with 1,100 psi rupture disc pressure. The 3 cm distance between rupture disk and macrocarrier and 9 cm target tissue distance yielded high transient GUS expression. The immature embryos were bombarded twice to increase area for efficient gene delivery. Osmotic medium optimization with 0.4 M sorbitol and preculture of immature embryos for 5 days prior to bombardment resulted into efficient gene transfer in barley. Selection of transformed tissue was performed after 7 days resting step on selection medium containing 50 mg l−1 hygromycin. After two more selection steps, green shoots were rooted on MSB5 medium with 50 mg l−1 hygromycin. PCR analysis using primers specific for hptII and gusA genes and Southern blot analysis with hptII probes confirmed the stable integration of transgene in barley genome. Molecular analysis of T1 generation plantlets revealed the amplification of selectable marker hptII gene in the progeny.

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Abbreviations

GUS:

β-glucuronidase

hptII :

Hygromycin phosphotransferase

MSB5 medium:

MS salts + B5 vitamins

IEs:

Immature embryos

References

  • Altpeter F, Baisakh N, Beachy R, Bock R, Capell T, Christou P, Daniell H, Datta K, Datta SK, Dix PJ et al (2005) Particle bombardment and the genetic enhancement of crops: myths and realities. Mol Breeding 15:305–327. doi:10.1007/s11032-004-8001-y

    Article  Google Scholar 

  • Castillo AM, Egana B, Sanz JM, Cistue L (1998) Somatic embryogenesis and plant regeneration from barley cultivars grown in Spain. Plant Cell Rep 17:902–906. doi:10.1007/s002990050506

    Article  CAS  Google Scholar 

  • Chauhan M, Kothari SL (2004) Optimization of nutrient levels in the medium increases the efficiency of callus induction and plant regeneration in recalcitrant Indian barley (Hordeum vulgare L.) in vitro. In Vitro Cell Dev Biol Plant 40:520–527

    Article  CAS  Google Scholar 

  • Chernobrovkina MA, Sidorov EA, Baranov IA, Kharchenko PN, Dolgov SV (2007) The effect of the parameters of biolistic transformation of spring barley (Hordeum vulgare L.) on the level of transient expression of GFP reporter gene. Biol Bull 34:558–563. doi:10.1134/S1062359007060040

    Article  Google Scholar 

  • Dahleen LS, Manoharan M (2007) Recent advances in barley transformation. In Vitro Cell Dev Biol Plant 43:493–506. doi:10.1007/s11627-007-9068-z

    Article  CAS  Google Scholar 

  • Delporte F, Li S, Jacquemin JM (2005) Calluses initiated from thin mature embryo fragments are suitable targets for wheat transformation as assessed by long-term GUS expression studies. Plant Cell Tissue Organ Cult 80:139–149. doi:10.1007/s11240-004-9221-x

    Article  CAS  Google Scholar 

  • Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151–158. doi:10.1016/0014-4827(68)90403-5

    Article  PubMed  CAS  Google Scholar 

  • Gomez KA, Gomez AA (1984) Statistical procedures for agricultural research. Wiley, New York

    Google Scholar 

  • Harwood WA, Ross SM, Cilento P, Snape JW (2000) The effect of DNA/gold particle preparation technique, and particle bombardment device, on the transformation of barley (Hordeum vulgare). Euphytica 111:67–76. doi:10.1023/A:1003700300235

    Article  CAS  Google Scholar 

  • Hensel G, Valkov V, Middlefell-Williams J, Kumlehn J (2008) Efficient generation of transgenic barley: the way forward to modulate plant-microbe interactions. J Plant Physiol 165:71–82. doi:10.1016/j.jplph.2007.06.015

    Article  PubMed  CAS  Google Scholar 

  • Ingram HM, Power JB, Lowe KC, Davey MR (1999) Optimisation of procedures for microprojectile bombardment of microspore-derived embryos in wheat. Plant Cell Tissue Organ Cult 57:207–210. doi:10.1023/A:1006384525513

    Article  CAS  Google Scholar 

  • Jagga-Chugh S, Kachhwaha S, Sharma M, Kothari-Chajer A, Kothari SL (2012) Optimization of factors influencing microprojectile bombardment-mediated genetic transformation of seed-derived callus and regeneration of transgenic plants in Eleusine coracana (L.) Gaertn. Plant Cell Tissue Organ Cult. doi:10.1007/s11240-011-0104-7

  • Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions:beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907

    PubMed  CAS  Google Scholar 

  • Kachhwaha S, Kothari SL (1994) Plant regeneration in barley through microtillering and multiple shoot bud differentiation. Curr Sci 67:116–118

    CAS  Google Scholar 

  • Kachhwaha S, Kothari SL (1996) Plant regeneration from immature embryo explants of Hordeum spontaneum and Hordeum vulgare. Cereal Res Commun 24:27–32

    Google Scholar 

  • Kachhwaha S, Varshney A, Kothari SL (1997) Somatic embryogenesis and long term high plant regeneration from barley (Hordeum vulgare L.) using picloram. Cereal Res Commun 25:117–126

    Google Scholar 

  • Kothari SL, Chandra N (1988) Somatic embryogenesis and plant regeneration from seed callus of barley (Hordeum vulgare L.). Curr Sci 57:1351–1352

    Google Scholar 

  • Lowe BA, Shiva Prakash N, Way M, Mann MT, Spencer TM, Boddupalli RS (2009) Enhanced single copy integration events in corn via particle bombardment using low quantities of DNA. Transgenic Res 18:831–840. doi:10.1007/s11248-009-9265-0

    Article  PubMed  CAS  Google Scholar 

  • Manoharan M, Dahleen L (2002) Genetic transformation of the commercial barley (Hordeum vulgare L.) cultivar Conlon by particle bombardment of callus. Plant Cell Rep 21:76–80. doi:10.1007/s00299-002-0477-5

    Article  CAS  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497. doi:10.1111/j.1399-3054.192.tb08052.x

    Article  CAS  Google Scholar 

  • Murray F, Brettell R, Matthews P, Bishop D, Jacobsen J (2004) Comparison of Agrobacterium-mediated transformation of four barley cultivars using the GFP and GUS reporter genes. Plant Cell Rep 22:397–402. doi:10.1007/s00299-003-0704-8

    Article  PubMed  CAS  Google Scholar 

  • Patnaik D, Khurana P (2001) Wheat biotechnology: a minireview. Electron J Biotechnol 4:1–29

    Google Scholar 

  • Petrillo CP, Carneiro NP, Purcino AÁC, Carvalho CHS, Alves JD, Carneiro AA (2008) Optimization of particle bombardment parameters for the genetic transformation of Brazilian maize inbred lines. Pesq Agrop Brasileira 43:371–378. doi:10.1590/S0100-204X2008000300012

    Article  Google Scholar 

  • Rao AQ, Bakhsh A, Kiani S, Shahzad K, Shahid AA, Husnain T, Riazuddin S (2009) The myth of plant transformation. Biotechnol Adv 27:753–763

    Article  PubMed  Google Scholar 

  • Rasco Gaunt S, Riley A, Cannell M, Barcelo P, Lazzeri PA (2001) Procedures allowing the transformation of a range of European elite wheat (Triticum aestivum L.) varieties via particle bombardment. J Exp Biol 52:865–874. doi:10.1093/jexbot/52.357.865

    CAS  Google Scholar 

  • Rasco-Gaunt S, Riley A, Barcelo P, Lazzeri PA (1999) Analysis of particle bombardment parameters to optimise DNA delivery into wheat tissues. Plant Cell Rep 19:118–127. doi:10.1007/s002990050721

    Article  CAS  Google Scholar 

  • Rikiishi K, Matsuura T, Maekawa M, Noda K, Takeda K (2003) Barley lines showing prominent high green shoot regeneration in cultures derived from immature embryos. Plant Breed 122:105–111. doi:10.1046/j.1439-0523.2003.00823.x

    Article  Google Scholar 

  • Roussy I, Ahlandsberg S, Jansson C (2001) Transformation and regeneration capacities for five Nordic barley elite cultivars-evaluation of tissue culture response and transient expression. Hereditas 134:97–101. doi:10.1111/j.1601-5223.2001.00097.x

    Article  PubMed  CAS  Google Scholar 

  • Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Habour Laboratory Press, Cold Spring Habour, New York

    Google Scholar 

  • Sanford JC (2000) The development of the biolistic process. In Vitro Cell Dev Biol Plant 36:303–308

    Article  Google Scholar 

  • Shim YS, Pauls KP, Kasha KJ (2009) Transformation of isolated barley (Hordeum vulgare L.) microspores: II. Timing of pretreatment and temperatures relative to results of bombardment. Genome 52:175–190. doi:10.1139/G08-113

    Article  PubMed  CAS  Google Scholar 

  • Shou H, Frame BR, Whitham SA, Wang K (2004) Assessment of transgenic maize events produced by particle bombardment or Agrobacterium-mediated transformation. Mol Breed 13:201–208. doi:10.1023/B:MOLB.0000018767.64586.53

    Article  CAS  Google Scholar 

  • Shrawat AK, Becker D, Lörz H (2007) Agrobacterium tumefaciens-mediated genetic transformation of barley (Hordeum vulgare L.). Plant Sci 172:281–290. doi:10.1016/j.plantsci.2006.09.005

    Article  CAS  Google Scholar 

  • Sivamani E, DeLong RK, Qu R (2009) Protamine-mediated DNA coating remarkably improves bombardment transformation efficiency in plant cells. Plant Cell Rep 28:213–221. doi:10.1007/s00299-008-0636-4

    Article  PubMed  CAS  Google Scholar 

  • Sreeramanan S, Maziah M, Abdullah MP, Sariah M, Aini MN (2005) Physical and biological parameters affecting transient GUS and GFP expressions in banana via particle bombardment. Asia Pac J Mol Biol 13:35–57

    Google Scholar 

  • Tadesse Y, Sagi L, Swennen R, Jacobs M (2003) Optimisation of transformation conditions and production of transgenic sorghum (Sorghum bicolor) via microparticle bombardment. Plant Cell Tissue Organ Cult 75:1–18. doi:10.1023/A:1024664817800

    Article  CAS  Google Scholar 

  • Tingay S, McElroy D, Kalla R, Fieg S, Wang M, Thornton S, Brettell R (1997) Agrobacterium tumefaciens mediated barley transformation. Plant J 11:1369–1376. doi:10.1046/j.1365-313X.1997.11061369.x

    Article  CAS  Google Scholar 

  • Trifonova A, Madsen S, Olesen A (2001) Agrobacterium-mediated transgene delivery and integration into barley under a range of in vitro culture conditions. Plant Sci 161:871–880. doi:10.1016/S0168-9452(01)00479-4

    Article  CAS  Google Scholar 

  • Um MO, Park TI, Kim YJ, Seo HY, Kim JG, Kwon SY, Kwak SS, Yun DJ, Yun SJ (2007) Particle bombardment-mediated transformation of barley with an Arabidopsis NDPK2 cDNA. Plant Biotechnol Rep 1:71–77. doi:10.1007/s11816-007-0016-5

    Article  Google Scholar 

  • Wu H, McCormac AC, Elliott MC, Chen DF (1998) Agrobacterium-mediated stable transformation of cell suspension cultures of barley (Hordeum vulgare). Plant Cell Tissue Organ Cult 54:161–171. doi:10.1023/A:1006106916542

    Article  CAS  Google Scholar 

  • Yadav T, Kothari SL, Kachhwaha S (2011) Evaluation of regeneration potential of mature embryo derived callus in Indian cultivars of barley (Hordeum vulgare L.). J Plant Biochem Biotechnol 20:166–172. doi:10.1007/s13562-011-0042-9

    Article  Google Scholar 

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Acknowledgments

Teena Yadav thanks the Council of Scientific and Industrial Research (CSIR), HRDG, New Delhi for the award of Senior Research Fellowship and Prof. M. Shreemali, Agriculture Research Station, Durgapura, Jaipur for providing barley seeds.

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Authors and Affiliations

  1. Department of Botany, University of Rajasthan, Jaipur, 302 004, India

    Teena Yadav, Sumita Kachhwaha & S. L. Kothari

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  1. Teena Yadav
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  2. Sumita Kachhwaha
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  3. S. L. Kothari
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Correspondence to Sumita Kachhwaha.

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Yadav, T., Kachhwaha, S. & Kothari, S.L. Efficient in vitro plant regeneration and generation of transgenic plants in barley (Hordeum vulgare L.) using particle bombardment. J. Plant Biochem. Biotechnol. 22, 202–213 (2013). https://doi.org/10.1007/s13562-012-0149-7

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