Agrobacterium-mediated transformation of European chestnut somatic embryos with a Castanea sativa (Mill.) endochitinase gene

Author

• E. Corredoira 
• , M. C. San José
• , A. M. Vieitez
• , I. Allona
• , C. Aragoncillo
• , A. Ballester

Abstract

Chestnut blight, caused by Cryphonectria parasitica, is a severe disease that has devastated chestnut stands in North America and Europe. Genes encoding hydrolytic enzymes such as chitinases, which can degrade fungal cell wall components, are attractive candidates for improving disease resistance. This report describes a reliable and efficient protocol for the Agrobacterium-mediated transformation of somatic embryos of European chestnut with the endogenous CsCh3 gene that codes for chitinase. The transformation efficiency, determined on the basis of the fluorescence of surviving explants, was genotype-dependent. Although somatic embryos of all three lines evaluated were transformed, the best results were obtained with somatic embryos derived from line CI-9 (20 %). The addition of silver thiosulphate (20 or 40 μM) improved the transformation efficiency of somatic embryos derived from lines CI-3 and CI-9, although the differences were not significant. A total of 88 independent transformed lines were obtained. The presence of transgenes was confirmed by green fluorescent protein (GFP) expression, PCR and Southern blot analysis. Transgenic lines were maintained by secondary embryogenesis or cryopreservation following vitrification procedures. Maturation and germination of transformed somatic embryos yielded transgenic plants. Fluorescence indicating overexpression of the transgenes was observed in somatic embryos and also in shoots and leaves. No phenotypic differences were found relative to control plants, suggesting a lack of any cytotoxic effects of the GFP.

References

1. Allona I, Collada C, Casado R, Paz-Ares J, Aragoncillo C (1996) Bacterial expression of an active class Ib chitinase from Castanea sativa cotyledons. Plant Mol Biol 32:1171–1176CrossRefPubMed
2. Álvarez R, Ordás RJ (2007) Improved genetic transformation protocol for cork oak (Quercus suber L.). Plant Cell, Tissue Organ Cult 91:45–52CrossRef
3. Anagnostakis SL (1995) The pathogens and pests of chestnuts. In: Andrews JH, Tommerup I (eds) Advances in botanical research (Vol 21). Academic Press, New York, pp 125–145
4. Anagnostakis SL (2001) The effect of multiple importations of pests and pathogens on a native tree. Biol Invasions 3:245–254CrossRef
5. Andrade GM, Nairn CJ, Le HT, Merkle SA (2009) Sexually mature transgenic American chestnut trees via embryogenic suspension-based transformation. Plant Cell Rep 28:1385–1397CrossRefPubMed
6. Barakat A, DiLoreto DS, Zhang Y, Smith C, Baier K, Powell WA, Wheeler N, Sederoff R, Carlson JE (2009) Comparison of the transcriptomes of American chestnut (Castanea dentata) and Chinese chestnut (Castanea mollissima) in response to the chestnut blight infection. BMC Plant Biol 9:51CrossRefPubMedPubMedCentral
7. Bezirganoglu I, Hwang S-Y, Fang TJ, Shaw J-F (2013) Transgenic lines of melon (Cucumis melo L. var. makuwa cv. ‘Silver Light’) expressing antifungal protein and chitinase genes exhibit enhanced resistance to fungal pathogens. Plant Cell, Tissue Organ Cult 112:227–237CrossRef
8. Ceasar SA, Ignacimuthu S (2012) Genetic engineering of crop plants for fungal resistance: role of antifugal genes. Biotechnol Lett 34:995–1002CrossRefPubMed
9. Collada C, Casado R, Fraile A, Aragoncillo C (1992) Basic endochitinases are major proteins in Castanea sativacotyledons. Plant Physiol 100:778–783CrossRefPubMedPubMedCentral
10. Corrado G, Arciello S, Fanti P, Fiandra L, Garonna A, Digilio MC, Lorito M, Giordana B, Pennacchio F, Rao R (2008) The Chitinase A from the baculovirus AcMNPV enhances resistance to both fungi and herbivorous pests in tobacco. Transgenic Res 17:557–571CrossRefPubMed
11. Corredoira E, Ballester A, Vieitez AM (2003) Proliferation, maturation and germination of Castanea sativa Mill. somatic embryos originated from leaf explants. Ann Bot 92:129–136CrossRefPubMedPubMedCentral
12. Corredoira E, Montenegro D, San José MC, Vieitez AM, Ballester A (2004) Agrobacterium-mediated transformation of European chestnut embryogenic cultures. Plant Cell Rep 23:311–318CrossRefPubMed
13. Corredoira E, Ballester A, Vieitez FJ, Vieitez AM (2006) Somatic embryogenesis in chestnut. In: Mujib A, Samaj J (eds) Plant cell monographs, Vol. 2, somatic embryogenesis. Springer, Berlin, pp 177–199
14. Corredoira E, San José MC, Vieitez AM, Ballester A (2007) Improving genetic transformation of European chestnut and cryopreservation of transgenic lines. Plant Cell, Tissue Organ Cult 91:281–288CrossRef
15. Corredoira E, Valladares S, Vieitez AM, Ballester A (2008) Improved germination of somatic embryos and plant recovery of European chestnut. In Vitro Cell Dev Biol Plant 44:307–315CrossRef
16. Corredoira E, Valladares S, Allona I, Aragoncillo C, Vieitez AM, Ballester A (2012) Genetic transformation of European chestnut somatic embryos with a native thaumatin-like protein (CsTL1) gene isolated from Castanea sativa seeds. Tree Physiol 32:1389–1402CrossRefPubMed
17. Corredoira E, Valladares S, Vieitez AM, Ballester A (2015) Chestnut, European (Castanea sativa). In: Wang K (ed) Methods in molecular biology Agrobacterium protocols. Springer, New York, pp 163–176
18. Dan Y (2008) Biological functions of antioxidants in plant transformation. In Vitro Cell Dev Biol Plant 44:149–161CrossRef
19. De la Riva G, Gónzalez-Cabrera J, Vázquez-Padron R, Ayra-Pardo C (1998) Agrobacterium tumefaciens: a natural tool for plant transformation. Electron J Biotechnol 1:1–15
20. Dhekney SA, Li ZT, Gray DJ (2011) Grapevines engineered to express cisgenic Vitis vinifera thaumatin-like protein exhibit fungal disease resistance. In Vitro Cell Dev Biol-Plant 47:458–466CrossRef
21. Dutt M, Vasconcellos M, Grosser JW (2011) Effects of antioxidants on Agrobacterium-mediated transformation and accelerated production of transgenic plants of Mexican lime (Citrus aurantifolia Swingle). Plant Cell, Tissue Organ Cult 107:79–89CrossRef
22. Emani C, García JM, Lopata-Finch E, Pozo MJ, Uribe P, Kim D-J, Sunilkumar G, Cook DR, Kenerly CM, Rathore KS (2003) Enhanced fungal resistance in transgenic cotton expressing an endochitinase gene from Trichoderma virens. Plant Biotechnol J 1:321–326CrossRefPubMed
23. García-Casado G, Collada C, Allona I, Soto A, Casado R, Rodríguez-Cerezo E, Gómez L, Aragoncillo C (2000) Characterization of an apoplastic basic thaumatin-like protein from recalcitrant chestnut seeds. Physiol Plant 110:172–180CrossRef
24. Girhepuje PV, Shinde GB (2011) Transgenic tomato plants expressing a wheat endochitinase gene demonstrate enhanced resistance to Fusarium oxysporum f. sp. lycopersici. Plant Cell, Tissue Organ Cult 105:243–251CrossRef
25. Han JS, Kim CK, Park SH, Hirschi KD, Mok IG (2005) Agrobacterium-mediated transformation of bottle gourd (Lagenaria siceraria Standl). Plant Cell Rep 23:692–698CrossRefPubMed
26. Han KM, Dharmawardhana P, Arias RS, Ma C, Busov V, Strauss SH (2011) Gibberellin-associated cisgenes modify growth, stature and wood properties in Populus. Plant Biotechnol J 9:162–178CrossRefPubMed
27. Hebard FV (2006) The backcross breeding program of the American Chestnut Foundation. In: Steiner KC, Carlson JE (eds) Restoration of American chestnut to forest lands. Proceedings of a conference and workshop, May 4–6, 2004, The North Carolina Arboretum, Asheville. Natural Resources Report NPS/NCR/CUE/NRR – 2006/01. Washington, DC: National Park Service, pp 61–77
28. Heiniger U, Rigling D (1994) Biological control of chestnut blight in Europe. Ann Rev Phytopathol 32:581–599CrossRef
29. Holme IB, Dionisio G, Brinch-Pedersen H, Wendt T, Madsen CK, Vincze E, Holm PB (2012) Cisgenic barley with improved phytase activity. Plant Biotechnol J 10:237–247CrossRefPubMed
30. Hood EE, Gelvin SB, Melchers LS, Hoekema A (1993) New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Res 2:208–218CrossRef
31. Hou H, Atlihan N, Lu Z-X (2014) New biotechnology enhances the application of cisgenesis in plant breeding. Front Plant Sci 5:389PubMedPubMedCentral
32. Jacobs DF (2007) Toward development of silvical strategies for forest restoration of American chestnut (Castanea dentata) using blight-resistant hybrids. Biol Conserv 137:497–506CrossRef
33. Jacobs DF, Dalgleish HJ, Nelson CD (2013) A conceptual framework for restoration of threatened plants: the effective model of American chestnut (Castanea dentata) reintroduction. New Phytol 197:378–393CrossRefPubMed
34. Jacobsen E, Schouten HJ (2008) Cisgenesis, a new tool for traditional plant breeding, should be exempted from the regulation of genetically modified organisms in a step by step approach. Potato Res 51:75–88CrossRef
35. Jia Z, Sun Y, Yuan L, Tian Q, Luo K (2010) The chitinase gene (Bbchit1) from Beauveria bassiana enhances resistance to Cytospora chrysosperma in Populus tomentosa Carr. Biotechnol Lett 32:1325–1332CrossRefPubMed
36. Joshi SG, Schaart JG, Groenwold R, Jacobsen E, Schouten HJ, Krens FA (2011) Functional analysis and expression profiling of HcrVf1 and HcrVf2 for development of scab resistant cisgenic and intragenic apples. Plant Mol Biol 75:579–591CrossRefPubMedPubMedCentral
37. Khan RS, Kameya N, Mii M, Nakamura I (2012) Transgenic Petunia hybrida expressing a synthetic fungal chitinase gene confers disease tolerance to Botrytis cinerea. Plant Biotechnol 29:285–291CrossRef
38. Kong LK, Holtz CT, Nairn CJ, Houke H, Powell WA, Baier K, Merkle SA (2014) Application of airlift bioreactors to accelerate genetic transformation in American chestnut. Plan Cell Tissue Organ Cult 117:39–50CrossRef
39. Kovács G, Sági L, Jacon G, Arinaitwe G, Busogoro J-P, Thiry E, Strosse H, Swennen R, Remy S (2013) Expression of a rice chitinase gene in transgenic banana (‘Gros Michel’, AAA genome group) confers resistance to black leaf streak disease. Transgenic Res 22:117–130CrossRefPubMedPubMedCentral
40. Kubisiak TL, Hebard FV, Nelson CD, Zhang J, Bernatzky R, Huang H, Anagnostakis SL, Doudrick RL (1997) Molecular mapping of resistance to blight in an interspecific cross in the genus Castanea. Phytopathology 87:751–759CrossRefPubMed
41. Leclercq J, Lardet L, Martin F, Chapuset T, Oliver G, Montoro P (2010) The green fluorescent protein as an efficient selection marker for Agrobacterium tumefaciens-mediated transformation in Hevea brasiliensis (Müll. Arg). Plant Cell Rep 29:513–522CrossRefPubMed
42. Liu X, Pijut PM (2010) Agrobacterium-mediated transformation of mature Prunus serotina (black cherry) and regeneration of transgenic shoots. Plant Cell, Tissue Organ Cult 101:49–57CrossRef
43. Mallón R, Valladares S, Corredoira E, Vieitez AM, Vidal N (2014) Overexpression of the chestnut CsTL1 gene coding for a thaumatin-like protein in somatic embryos of Quercus robur. Plant Cell, Tissue Organ Cult 116:141–151CrossRef
44. Maynard CA, Powell WA, Polin-McGuigan LD, Vieitez AM, Ballester A, Corredoira E, Merkle SA, Andrade A (2008) Chestnut. In: Kole C, Hall TC (eds) Compendium of transgenic crop plants: transgenic forest tree species. Blackwell, Chichester, pp 169–192CrossRef
45. Milgroom MG, Cortesi P (2004) Biological control of chestnut blight with hypovirulence: a critical analysis. Ann Rev Phytopathol 42:311–338CrossRef
46. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15:473–497CrossRef
47. Newhouse AE, Polin-McGuigan LD, Baier KA, Valletta KER, Rottmann WH, Tschaplinski TJ, Maynard CA, Powell WA (2014) Transgenic American chestnut show enhanced blight resistance and transmit the trait to T1 progeny. Plant Sci 228:88–97CrossRefPubMed
48. Nonaka S, Ezura H (2014) Plant-Agrobacterium interaction mediated by ethylene and super-Agrobacteriumconferring efficient gene transfer. Front Plant Sci 5:681CrossRefPubMedPubMedCentral
49. Nonaka S, Yuhashi K, Takada K, Sugaware M, Minamisawa K, Ezura H (2008) Ethylene production in plants during transformation suppresses vir gene expression in Agrobacterium tumefaciens. New Phytol 178:647–656CrossRefPubMed
50. Pasonen H-L, Seppänen S-K, Degefu Y, Rytkönen A, von Weissenberg K, Pappinen A (2004) Field performance of chitinase transgenic silver birches (Betula pendula): resistance to fungal diseases. Theor Appl Genet 109:562–570CrossRefPubMed
51. Polin LD, Liang H, Rothrock RE, Nishii M, Diehl DL, Newhouse AE, Mairn CJ, Powell WA, Maynard CA (2006) Agrobacterium-mediated transformation of American chestnut (Castanea dentata (Marsh) Borkh) somatic embryos. Plant Cell, Tissue Organ Cult 84:69–79CrossRef
52. Pourhosseini L, Habashi AA, Kermani MJ, Khalighi A, Tahmasbi Z (2012) Agrobacterium-mediated transformation of chitinase gene in Rosa damascena cv. Ghamsar. Ann Biol Res 3:2843–2850
53. Roberts WK, Selitrennikoff CP (1990) Zeamatin, an antifungal protein from maize with membrane-permeabilizing activity. J Gen Microbiol 136:1771–1778CrossRef
54. Rohini VK, Rao KS (2001) Transformation of peanut (Arachis hypogea L.) with tobacco chitinase gene: variable response of transformants to leaf spot disease. Plant Sci 160:889–898CrossRefPubMed
55. Rothrock RE, Polin-McGuigan LD, Newhouse AE, Powell WA, Maynard CA (2007) Plate flooding as an alternative Agrobacterium-mediated transformation method for American chestnut somatic embryos. Plant Cell, Tissue Organ Cult 88:93–99CrossRef
56. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
57. Schouten H, Krens FA, Jacobsen E (2006a) Cisgenic plants are similar to traditionally bred plants. EMBO Rep 7:750–753CrossRefPubMedPubMedCentral
58. Schouten HJ, Krens FA, Jacobsen E (2006b) Do cisgenic plants warrant less stringent oversight? Nat Biotechnol 24:753CrossRefPubMed
59. Sharma M, Kothari-Chajer A, Jagga-Chugh S, Kothari SL (2011) Factors influencing Agrobacterium tumefaciens-mediated genetic transformation of Eleusine coracana (L.) Gaertn. Plant Cell, Tissue Organ Cult 105:93–104CrossRef
60. Sokal RR, Rohlf FJ (1981) Biometry: the principles and practice of statistics and biological research. W. H. Freeman & Company, New York 776 pp
61. Tang G-X, Knecht K, Yang X-F, Qin YB, Zhou W-J, Cai D (2011) A two-step protocol for shoot regeneration from hypocotyl explants of oilseed rape and its application for Agrobacterium-mediated transformation. Biol Plant 55:21–26CrossRef
62. Toldi O, Tóth S, Pónyi T, Scott P (2002) An effective and reproducible transformation protocol for the model resurrection plant Craterostigma plantagineum Hochst. Plant Cell Rep 211:63–69
63. Van Alfen NK, Jayners RA, Anagnostakis SL, Day PR (1975) Chestnut blight: biological control by transmissible hypovirulence in Endothia parasitica. Science 189:890–891CrossRefPubMed
64. Vanblaere T, Szankowski I, Schaart J, Schouten H, Flachowsky H, Broggini GAL, Gessler C (2011) The development of a cisgenic apple plant. J Biotechnol 154:304–311CrossRefPubMed
65. Vannini A, Caruso C, Leonardi L, Ruggini E, Chiarot E, Caporale C, Buonocore V (1999) Antifungal properties of chitinases from Castanea sativa against hypovirulent and virulent strains of the chestnut blight fungus Cryphonectria parasitica. Physiol Mol Plant Pathol 55:29–35CrossRef
66. Veluthakkal R, Dasgupta MG (2010) Pathogenesis-related genes and proteins in forest tree species. Trees 24:993–1006CrossRef
67. Veluthakkal R, Karpaga Raja Sundari B, Ghosh Dasgupta M (2012) Tree chitinases—stress- and developmental-driven gene regulation. For Path 42:271–278CrossRef
68. Vieitez AM, San José MC, Corredoira E (2011) Cryopreservation of zygotic embryo axes and somatic embryos of European chestnut. In: Thorpe TA, Yeung EC (eds) Plant embryo culture: methods and protocols, methods in molecular biology, vol 710. Springer, New York, pp 201–213CrossRef
69. Wally O, Punja ZK (2010) Genetic engineering for increasing fungal and bacterial disease resistance in crop plants. GM Crops 1:199–206CrossRefPubMed
70. Wheeler N, Sederoff R (2009) Role of genomics in the potential restoration of the American chestnut. Tree Gen Genom 5:181–187CrossRef
71. Xu R, Li QQ (2008) Protocol: Streamline cloning of genes into binary vectors in Agrobacterium via the Gateway®TOPO vector system. Plant Methods 4:4CrossRefPubMedPubMedCentral
72. Zhan B, Oakes AD, Newhouse AE, Baier KM, Maynard CA, Powell WA (2013) A threshold level of oxalate oxidase transgene expression reduces Cryphonectria parasitica-induced necrosis in a transgenic American chestnut (Castanea dentata) leaf bioassay. Transgenic Res 22:973–982CrossRef
73. Zhiying W, Fuli Z, Zhanbin W (2010) Transformation of chitinase gene into Populus simonii × P. nigra and chitinase activity of transgenic plants. Sci Silvae Sin 46:147–151

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