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Genetic Transformation Protocols Using Zygotic Embryos as Explants: An Overview

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Plant Embryo Culture

Part of the book series: Methods in Molecular Biology ((MIMB,volume 710))

Abstract

Genetic transformation of plants is an innovative research tool which has practical significance for the development of new and improved genotypes or cultivars. However, stable introduction of genes of interest into nuclear genomes depends on several factors such as the choice of target tissue, the method of DNA delivery in the target tissue, and the appropriate method to select the transformed plants. Mature or immature zygotic embryos have been a popular choice as explant or target tissue for genetic transformation in both angiosperms and gymnosperms. As a result, considerable protocols have emerged in the literature which have been optimized for various plant species in terms of transformation methods and selection procedures for transformed plants. This article summarizes the recent advances in plant transformation using zygotic embryos as explants.

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References

  1. James C (2008) Global status of commercialized biotech/GM crops: 2008. ISAAA Brief No. 39. ISAAA, Ithaca

    Google Scholar 

  2. Chassy BM, Parrot WA, Roush R (2005) Crop biotechnology and the future of food: a scientific assessment. In: CAST commentary, Ames, Oct 2005

    Google Scholar 

  3. Fink GR (2005) A transforming principle. Cell 120:153–154

    Article  PubMed  CAS  Google Scholar 

  4. Vasil IK (2007) A short history of plant biotechnology. Phytochem Rev 7:387–394

    Article  Google Scholar 

  5. Sharma KK, Mathur PB, Thorpe TA (2005) Genetic transformation technology: status and problems. In Vitro Cell Dev Biol Plant 41:102–112

    Article  CAS  Google Scholar 

  6. Birch RG (1997) Plant transformation: problems and strategies for practical application. Annu Rev Plant Physiol Plant Mol Biol 48:297–326

    Article  PubMed  CAS  Google Scholar 

  7. Vain P (2006) Thirty years of plant transformation. Plant Biotechnol J 5:221–229

    Article  Google Scholar 

  8. Sticklen MB, Oraby HF (2005) Shoot apical meristem: a sustainable explant for genetic transformation of cereal crops. In Vitro Cell Dev Biol Plant 41:187–200

    Article  CAS  Google Scholar 

  9. Jones HD, Sparks CA (2008) Stable transformation of plants. Methods Mol Biol 513:111–130

    Article  Google Scholar 

  10. Jube S, Borthakur D, Jube S, Borthakur D (2009) Agrobacterium-mediated transformation protocol for the tree-legume Leucaena leucocephala using immature zygotic embryos. Plant Cell Tissue Organ Cult 96:325–333

    Article  PubMed  Google Scholar 

  11. Trick HN, Finer JJ (1997) SAAT: sonication-assisted Agrobacterium-mediated transformation. Transgenic Res 6:329–336

    Article  CAS  Google Scholar 

  12. Herschbach C, Kopriva S (2002) Transgenic trees as tools in tree and plant physiology. Trees 16:250–261

    Article  CAS  Google Scholar 

  13. Sundar IK, Sakthivel N (2008) Advances in selectable marker genes for plant transformation. J Plant Physiol 165:1698–1716

    Article  PubMed  CAS  Google Scholar 

  14. Aulinger IE, Peter SO, Schmid JE, Stamp P (2003) Gametic embryos of maize as a target for biolistic transformation: comparison to immature zygotic embryos. Plant Cell Rep 21:585–591

    PubMed  CAS  Google Scholar 

  15. Frame BR, Zhang H, Cocciolone SM, Sidorenko LV, Dietrich CR, Pegg SE, Zhen S, Schnable PS, Wang K (2000) Production of transgenic maize from bombarded type II callus: effect of gold particle size and callus morphology on transformation efficiency. In Vitro Cell Dev Biol Plant 36:21–29

    Article  Google Scholar 

  16. Brettschneider R, Lorz HBD (1997) Efficient transformation of scutellar tissue of immature maize embryos. Theor Appl Genet 94:737–748

    Article  CAS  Google Scholar 

  17. O’Kennedy MM, Burger JT, Berger DK (2001) Transformation of elite white maize using the particle inflow gun and detailed analysis of a low-copy integration event. Plant Cell Rep 20:721–730

    Article  Google Scholar 

  18. Wu H, Doherty A, Jones HD (2009) Agrobacterium-mediated transformation of bread and durum wheat using freshly isolated immature embryos. Methods Mol Biol 478:93–103

    Article  PubMed  Google Scholar 

  19. Ritala A, Aspegren K, Kurtén U, Salmenkallio-Marttila M, Mannonen L, Hannus R, Kauppinen V, Teeri TH, Enari TM (1994) Fertile transgenic barley by particle bombardment of immature embryos. Plant Mol Biol 24:317–325

    Article  PubMed  CAS  Google Scholar 

  20. Wan Y, Lemaux PG (1994) Generation of large numbers of independently transformed fertile barley plants. Plant Physiol 104:37–48

    PubMed  CAS  Google Scholar 

  21. O’Kennedy MM, Burger JT, Botha FC (2004) Pearl millet transformation system using the positive selectable marker gene phosphomannose isomerase. Plant Cell Rep 22:684–690

    Article  PubMed  Google Scholar 

  22. Yan B, Serinivasa MS, Collins RGB, Dinkins RD (2000) Agrobacterium tumefaciens mediated transformation of soybean using immature zygotic cotyledon explants. Plant Cell Rep 19:1090–1097

    Article  CAS  Google Scholar 

  23. Sato S, Newell C, Kolacz K, Tredo L, Finer L, Hinchee M (1993) Stable transformation via particle bombardment in two different soybean regeneration systems. Plant Cell Rep 12:408–413

    CAS  Google Scholar 

  24. Hunolda R, Burrus M, Bronnera R, Duretc J-P, Hahne G (1995) Transient gene expression in sunflower (Helianthus annuus) following microprojectile bombardment. Plant Sci 105:95–109

    Article  Google Scholar 

  25. Cai W, Gonsalves C, Tennant P, Fermin G, Souza M, Sarindu N, Zhu F, Gonsalves D (1999) A protocol for efficient transformation and regeneration of Carica papaya L. In Vitro Cell Dev Biol Plant 35:61–69

    CAS  Google Scholar 

  26. Fitch MMM, Manshardt RM, Gonsalves D, Slightom JL, Santord JC (1990) Stable transformation of papaya via microprojectile bombardment. Plant Cell Rep 9:189–194

    CAS  Google Scholar 

  27. Kloti A, Iglesias VA, Wfinn J, Burkhardt PK, Datta SK, Potrykus I (1993) Gene transfer by electroporation into intact scutellum cells of wheat embryos. Plant Cell Rep 12:671–675

    Article  Google Scholar 

  28. Tu S, Sangwan RS, Raghavan V, Verma DPS, Sangwan-Norreel BS (2005) Transformation of pollen embryo-derived explants by Agrobacterium tumefaciens in Hyoscyamus niger. Plant Cell Tissue Organ Cult 81:139–148

    Article  Google Scholar 

  29. Cruz-Hernández A, Witjaksono RE, Litz M, Lim G (1998) Agrobacterium tumefaciens – mediated transformation of embryogenic avocado cultures and regeneration of somatic embryos. Plant Cell Rep 17:497–503

    Article  Google Scholar 

  30. Kost B, Leduc N, Sautter C, Potrykus I, Neuhaus G (1996) Transient marker-gene expression during zygotic in-vitro embryogenesis of Brassica juncea (Indian mustard) following particle bombardment. Planta 198:211–220

    Article  CAS  Google Scholar 

  31. Rochange E, Serrano L, Marque C, Teulitres Z, Boudet AM (1995) DNA delivery into Eucalyptus globulus zygotic embryos through biolistics: optimization of the biological and physical parameters of bombardment for two different particle guns. Plant Cell Rep 14:674–678

    Article  Google Scholar 

  32. McKently AH, Moore GA, Doostdar H, Niedz RP (1995) Agrobacterium-mediated transformation of peanut (Arachis hypogaea L.) embryo axes and the development of transgenic plants. Plant Cell Rep 14:699–703

    Article  CAS  Google Scholar 

  33. Sangwan RS, Bourgeois Y, Sangwan-Norreel BS (1991) Genetic transformation of Arabidopsis thaliana zygotic embryos and identification of critical parameters influencing transformation efficiency. Mol Gen Genet 230:475–485

    Article  PubMed  CAS  Google Scholar 

  34. Leelavathi S, Sunnichan VG, Kumria R, Vijaykanth GP, Bhatnagar RK, Reddy VS (2004) A simple and rapid Agrobacterium-mediated transformation protocol for cotton (Gossypium hirsutum L.): embryogenic calli as a source to generate large numbers of transgenic plants. Plant Cell Rep 22:465–470

    Article  PubMed  CAS  Google Scholar 

  35. Taniguchi T, Kurita M, Itahana N, Kondo T (2004) Somatic embryogenesis and plant regeneration from immature zygotic embryos of Hinoki Cypress (Chamaecyparis obtusa Sieb. et Zucc.). Plant Cell Rep 23:26–31

    Article  PubMed  CAS  Google Scholar 

  36. Charity JA, Holland L, Donaldson SS, Grace L, Walter C (2002) Agrobacterium-mediated transformation of Pinus radiata organogenic tissue using vacuum-infiltration. Plant Cell Tissue Organ Cult 70:51–60

    Article  CAS  Google Scholar 

  37. Tang W, Sederoff R, Whetten R (2001) Regeneration of transgenic loblolly pine from zygotic embryos transformed with Agrobacterium tumefaciens. Planta 213:981–989

    Article  PubMed  CAS  Google Scholar 

  38. Lin X, Zhang W, Takechi K, Takio S, Ono K, Takano H (2005) Stable genetic transformation of Larix gmelinii L. by particle bombardment of zygotic embryos. Plant Cell Rep 24:418–425

    Article  PubMed  CAS  Google Scholar 

  39. Shyamkumar B, Anjaneyulu C, Giri CC (2007) Genetic transformation of Terminalia chebula Retz. and detection of tannin in transformed tissue. Curr Sci 92:3–10

    Google Scholar 

  40. Parasharami VA, Naik VB, von Arnold S, Nadgauda RS, Clapham DH (2006) Stable transformation of mature zygotic embryos and regeneration of transgenic plants of chir pine (Pinus roxbughii Sarg.). Plant Cell Rep 24:708–714

    Article  PubMed  CAS  Google Scholar 

  41. Tang W, Newton RJ (2005) Transgenic Christmas trees regenerated from Agrobacterium tumefaciens mediated transformation of zygotic embryos using the green fluorescence protein as a reporter. Mol Breed 16:235–246

    Article  CAS  Google Scholar 

  42. Clapham D, Demel P, Elfstrand M, Koop H-U, Sabala I, von Arnold S (2000) Gene transfer by particle bombardment to embryogenic cultures of Picea abies and the production of transgenic plantlets. Scand J For Res 15:151–160

    Article  Google Scholar 

  43. Stasolla C, Kong L, Yeung EC, Thorpe TA (2002) Maturation of somatic embryos in conifers: morphogenesis, physiology, biochemistry, and molecular biology. In Vitro Cell Dev Biol Plant 38:93–105

    Article  CAS  Google Scholar 

  44. Ishida Y, Saito H, Ohta S, Hiei Y, Komari T, Kumashiro T (1996) High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Biotechnology 14:745–750

    Article  CAS  Google Scholar 

  45. Zhu H, Muthukrishnan S, Krishnaveni S, Wilde G, Jeoung JM, Liang GH (1998) Biolistic transformation of sorghum using a rice chitinase gene. J Genet Breed 52:243–252

    CAS  Google Scholar 

  46. Zhao ZY, Gu WN, Cai TS, Tagliani L, Hondred D, Bond D, Schroeder S, Rudert M, Pierce D (2001) High throughput genetic transformation mediated by Agrobacterium tumefaciens in maize. Mol Breed 8:323–333

    Article  CAS  Google Scholar 

  47. Bohorova N, Frutos R, Royer M, Estanol P, Pacheco M, Rascon Q, McLean S, Hoisington D (2001) Novel synthetic Bacillus thuringiensis cry1B gene and the cry1B-cry1Ab translational fusion confer resistance to southwestern corn borer, sugarcane borer and fall armyworm in transgenic tropical maize. Theor Appl Genet 103:817–826

    Article  CAS  Google Scholar 

  48. Brettschneider R, Becker D, Lorz H (1997) Efficient transformation of scutellar tissue of immature maize embryos. Theor Appl Genet 94:737–748

    Article  CAS  Google Scholar 

  49. Geest AHM, Petolino JF (1998) Expression of a modified green fluorescent protein gene in transgenic maize plants and progeny. Plant Cell Rep 17:760–764

    Article  Google Scholar 

  50. Howe AR, Gasser CS, Brown SM, Padgette SR, Hart J, Parker GB, Fromm ME, Armstrong CL (2002) Glyphosate as a selective agent for the production of fertile transgenic maize (Zea mays L.) plants. Mol Breed 10:153–164

    Article  CAS  Google Scholar 

  51. Hueros G, Gomez E, Cheikh N, Edwards J, Weldon M, Salamini F, Thompson RD (1999) Identification of a promoter sequence from the BETL1 gene cluster able to confer transfer-cell-specific expression in transgenic maize. Plant Physiol 121:1143–1152

    Article  PubMed  CAS  Google Scholar 

  52. Rasco-Gaunt S, Liu D, Li CP, Doherty A, Hagemann K, Riley A, Thompson T, Brunkan C, Mitchell M, Lowe K (2003) Characterization of the expression of a novel constitutive maize promoter in transgenic wheat and maize. Plant Cell Rep 21:569–576

    PubMed  CAS  Google Scholar 

  53. Sangtong V, Moran DL, Chikwamba R, Wang K, Woodman Clikeman W, Long MJ, Lee M, Scott MP (2002) Expression and inheritance of the wheat Glu-1DX5 gene in transgenic maize. Theor Appl Genet 105:937–945

    Article  PubMed  CAS  Google Scholar 

  54. Songstad DD, Armstrong CL, Petersen WL, Hairston B, Hinchee MAW (1996) Production of transgenic maize plants and progeny by bombardment of Hi-II immature embryos. In Vitro Cell Dev Biol Plant 32:179–183

    Article  Google Scholar 

  55. Wright M, Dawson J, Dunder E, Suttie J, Reed J, Kramer C, Chang Y, Novitzky R, Wang H, Artim L (2001) Efficient biolistic transformation of maize (Zea mays L.) and wheat (Triticum aestivum L.) using the phosphomannose isomerase gene, pmi, as the selectable marker. Plant Cell Rep 20:429–436

    Article  CAS  Google Scholar 

  56. Khanna HK, Daggard GE (2003) Agrobacterium tumefaciens-mediated transformation of wheat using a superbinary vector and a polyamine-supplemented regeneration medium. Plant Cell Rep 21:429–436

    PubMed  CAS  Google Scholar 

  57. Takumi S, Shimada T (1995) Effects of three promoters on stable integration of the herbicide resistance gene in wheat culture cells through particle bombardment. Bull RIAR Ishikawa Agr Coll 4:9–16

    Google Scholar 

  58. Ahmad A, Maqbool S, Riazuddin S, Sticklen MB (2002) Expression of synthetic Cry1Ab and Cry1Ac genes in Basmati rice (Oryza sativa L.) variety 370 via Agrobacterium-mediated transformation for the control of the European corn borer (Ostrinia nubilalis). Dev Biol 38:213–220

    Article  CAS  Google Scholar 

  59. Pons MJ, Marfa V, Mele E, Messeguer J (2000) Regeneration and genetic transformation of Spanish rice cultivars using mature embryos. Euphytica 114:117–122

    Article  Google Scholar 

  60. Baruah WJ, Harwood WA, Lonsdale DA, Harvey A, Hull R, Snape JW (1999) Luciferase as a reporter gene for transformation studies in rice (Oryza sativa L.). Plant Cell Rep 18:715–720

    Article  Google Scholar 

  61. Hiruki C, Kakuta H, Hashidoko Y, Ge Z, Figueiredo G, Mizutani J (1993) Biolistic delivery of foreign DNA or genomic transcripts of plant virus full-length cDNA clones into monocotyledonous and dicotyledonous plant tissues. Proc Jpn Acad Ser B Phys Biol Sci 69:244–247

    Article  CAS  Google Scholar 

  62. Perret SJ, Valentine J, Leggett JM, Morris P (2003) Integration, expression and inheritance of transgenes in hexaploid oat (Avena sativa L.). J Plant Physiol 160:931–934

    Article  PubMed  CAS  Google Scholar 

  63. Kuai B, Perret S, Wan SM, Dalton SJ, Bettany AJE, Morris P (2001) Transformation of oat and inheritance of bar gene expression. Plant Cell Tissue Organ Cult 66:79–88

    Article  CAS  Google Scholar 

  64. Somers DA, Rines HW, Gu W, Kaeppler HF, Bushnell WR (1992) Fertile, transgenic oat plants. Biotechnology 10:1589–1594

    Article  CAS  Google Scholar 

  65. Torbert KA, Rines HW, Somers DA (1998) Transformation of oat using mature embryo-derived tissue cultures. Crop Sci 38:226–231

    Article  Google Scholar 

  66. Fang YD, Akula C, Altpeter F (2002) Agrobacterium-mediated barley (Hordeum vulgare L.) transformation using green fluorescent protein as a visual marker and sequence analysis of the T-DNA barley genomic DNA junctions. J Plant Physiol 159:1131–1138

    Article  CAS  Google Scholar 

  67. Horvath H, Rostoks N, Brueggeman R, Steffenson B, von Wettstein D, Kleinhofs A (2003) Genetically engineered stem rust ­resistance in barley using the Rpg1 gene. Proc Natl Acad Sci USA 100:364–369

    Article  PubMed  CAS  Google Scholar 

  68. Bregitzer P, Tonks D (2003) Inheritance and expression of transgenes in barley. Crop Sci 43:4–12

    Article  CAS  Google Scholar 

  69. Choi HW, Lemaux PG, Cho MJ (2003) Long-term stability of transgene expression driven by barley endosperm-specific hordein promoters in transgenic barley. Plant Cell Rep 21:1108–1120

    Article  PubMed  CAS  Google Scholar 

  70. Harwood WA, Ross SM, Bulley SM, Travella S, Busch B, Harden J, Snape JW (2002) Use of the firefly luciferase gene in a barley (Hordeum vulgare) transformation system. Plant Cell Rep 21:320–326

    Article  CAS  Google Scholar 

  71. Koprek T, Hansch R, Nerlich A, Mendel RR, Schulze J (1996) Fertile transgenic barley of different cultivars obtained by adjustment of bombardment conditions to tissue response. Plant Sci 119:79–91

    Article  CAS  Google Scholar 

  72. Manoharan M, Dahleen LS (2002) Genetic transformation of the commercial barley (Hordeum vulgare L.) cultivar Conlon by particle bombardment of callus. Plant Cell Rep 21:76–80

    Article  CAS  Google Scholar 

  73. Zhang Y, Darlington H, Jones HD, Halford NG, Napier JA, Davey MR, Lazzeri PA, Shewry PR (2003) Expression of the gamma-zein protein of maize in seeds of transgenic barley: effects on grain composition and properties. Theor Appl Genet 106:1139–1146

    PubMed  CAS  Google Scholar 

  74. Abumhadi N, Trifonova A, Takumi S, Nakamura C, Todorovska E, Getov L, Christov N, Atanassov A (2001) Development of the particle inflow gun and optimizing the particle bombardment method for efficient genetic transformation in mature embryos of cereals. Biotechnol Biotech 15:87–96

    Google Scholar 

  75. Gurel F, Gozu Kirmizi N (2000) Optimization of gene transfer into barley (Hordeum vulgare L.) mature embryos by tissue electroporation. Plant Cell Rep 19:787–791

    Article  CAS  Google Scholar 

  76. Zhao ZY, Cai TS, Miller M, Wang N, Pang H, Rudert M, Schroeder S, Hondred D, Seltzer J, Pierce D (2000) Agrobacterium-mediated sorghum transformation. Plant Mol Biol 44:789–798

    Article  PubMed  CAS  Google Scholar 

  77. Jeoung JM, Krishnaveni S, Muthukrishnan S, Trick HN, Liang GH (2002) Optimization of sorghum transformation parameters using genes for green fluorescent protein and beta-glucuronidase as visual markers. Hereditas 137:20–28

    Article  PubMed  CAS  Google Scholar 

  78. 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

    Article  CAS  Google Scholar 

  79. Goldman JJ, Hanna WW, Fleming G, Ozias-Akins P (2003) Fertile transgenic pearl millet plants recovered through microprojectile bombardment and phosphinothricin selection and apical meristem-, inflorescence-, and immature embryo-derived embryogenic tissues. Plant Cell Rep 21:999–1009

    Article  PubMed  CAS  Google Scholar 

  80. Lambe P, Dinant M, Deltour R (2000) Transgenic pearl millet (Pennisetum glaucum): I. Transgenic crops. Biotechnol Agric For 46:84–108

    CAS  Google Scholar 

  81. McKently AH, Moore GA, Doodstdar H, Niedz RP (1995) Agrobacterium-mediated transformation of peanut (Arachis hypogaea L.) embryo axes and the development of transgenic plants. Plant Cell Rep 14:699–703

    Article  CAS  Google Scholar 

  82. Ducrocq C, Sangwan RS, Sangwan-Norreel BS (1994) Production of Agrobacterium-mediated transgenic fertile plants by direct somatic embryogenesis from immature zygotic embryos of Datura innoxia. Plant Mol Biol 25:995–1009

    Article  PubMed  CAS  Google Scholar 

  83. Clapham D, Damel P, Elfstrand M, Koop H-U, Sabala I, von Arnold S (2000) Gene transfer by particle bombardment to embryogenic cultures of Picea abies and the production of transgenic plantlets. Scand J For Res 15:151–161

    Article  Google Scholar 

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Authors and Affiliations

  1. Department of Plant Science, University of Manitoba, Winnipeg, MB, Canada

    Muhammad Tahir, Ejaz A. Waraich & Claudio Stasolla

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  1. Muhammad Tahir
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  2. Ejaz A. Waraich
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  3. Claudio Stasolla
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Correspondence to Muhammad Tahir .

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Editors and Affiliations

  1. Dept. Biological Sciences, University of Calgary, University Dr. NW., 2500, Calgary, T2N 1N4, Alberta, Canada

    Trevor A. Thorpe

  2. , Department of Biological Sciences, University of Calgary, University Dr. NW 2500, Calgary, T2N 1N4, Alberta, Canada

    Edward C. Yeung

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Tahir, M., Waraich, E.A., Stasolla, C. (2011). Genetic Transformation Protocols Using Zygotic Embryos as Explants: An Overview. In: Thorpe, T., Yeung, E. (eds) Plant Embryo Culture. Methods in Molecular Biology, vol 710. Humana Press. https://doi.org/10.1007/978-1-61737-988-8_21

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  • DOI: https://doi.org/10.1007/978-1-61737-988-8_21

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