Abstract
Genetic manipulation of gymnosperms using Agrobacterium—mediated system, a powerful field in forest biotechnology, has a potential to accelerate forest tree improvements with desired attributes. However, most conifers such as pine have been considered recalcitrant to genetic transformation. The present study reports the successful production of transgenic Pinus massoniana from zygotic embryos using Agrobacterium tumefaciens harboring the pBI121:CslA2 binary vector. Various independent parameters were tested for their effects on transformation efficiency in P. massoniana. According to our results, combination of Agrobacterium density at OD600 of 0.5, cold treatment of Agrobacterium suspension at 4 °C for approximately 5 h, inoculation period of 5 h, and the addition of 100 µM acetosyringone in co-cultivation medium significantly enhanced the transformation efficiency. The stable integration of CslA2 gene into the genome of putative transgenic plants was confirmed by polymerase chain reaction and Southern blot hybridization, and the expression levels were determined using quantitative reverse transcription-PCR. Our optimized transformation procedure could provide an opportunity for transferring economically important genes into P. massoniana and other conifer species as well.
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Abbreviations
- 2,4-D:
-
2,4-Dichlorophenoxy acetic acid
- 6-BA:
-
Benzylaminopurine
- NAA:
-
Naphthalene acetic acid
- AS:
-
Acetosyringone
- OD:
-
Optical density at 600nm
- NPTII :
-
Neomycin phosphotransferase
References
Alvarez JM, Ordás RJ (2013) Stable Agrobacterium-mediated transformation of maritime pine based on kanamycin selection. Sci World J. https://doi.org/10.1155/2013/681792
Cerda F, Aquea F, Gebauer M, Medina C, Arce-Johnson P (2002) Stable transformation of Pinus radiata embryogenic tissue by Agrobacterium tumefaciens. Plant Cell Tiss Org 70:251–257. https://doi.org/10.1023/A:1016508031151
Choudhury H, Kumaria S, Tandon P (2014) Pinus biotechnology: progress and prospects. In: Ramawat KG, Merillon J-M, Ahuja MR (eds) Tree biotechnology, 1st edn. CRC Press, Taylor & Francis Group, Boca Raton, London, pp 223–247
Dutt M, Grosser JW (2009) Evaluation of parameters affecting Agrobacterium-mediated transformation of citrus. Plant Cell Tiss Org 98:331–340. https://doi.org/10.1007/s11240-009-9567-1
Dutta I, Kottackal M, Tumimbang E, Tajima H, Zaid A, Blumwald E (2013) Sonication-assisted efficient Agrobacterium-mediated genetic transformation of the multipurpose woody desert shrub Leptadenia pyrotechnica. Plant Cell Tiss Org 112:289–301. https://doi.org/10.1007/s11240-012-0236-4
Fortin C, Nester EW, Dion P (1992) Growth inhibition and loss of virulence in cultures of Agrobacterium tumefaciens treated with acetosyringone. J Bacteriol 174:5676–5685. https://doi.org/10.1128/jb.174.17.5676-5685.1992
Grace LJ, Charity JA, Gresham B, Kay N, Walter C (2005) Insect-resistant transgenic Pinus radiata. Plant Cell Rep 24:103–111
Grant JE, Cooper PA, Dale TM (2015) Genetic transformation of micropropagated shoots of Pinus radiata D. Don. bioRxiv. https://doi.org/10.1101/030080
Holsters M, Waele DD, Depicker A, Messens E, Montagu MV, Schell J (1978) Transfection and transformation of Agrobacterium tumefaciens. Mol Gen Genet 163:181–187. https://doi.org/10.1007/BF00267408
Hood EE, Gelvin SB, Melchers LS, Hoekema A (1993) New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Res 2:208–218. https://doi.org/10.1007/BF01977351
Hou W, Shakya P, Franklin G (2016) A perspective on hypericum perforatum genetic transformation. Front Plant Sci 7:879. https://doi.org/10.3389/fpls.2016.00879
Isah T (2016) Induction of somatic embryogenesis in woody plants. Acta Physiol Plant 38:118–139. https://doi.org/10.1007/s11738-016-2134-6
Kuta DD, Tripathi L (2005) Agrobacterium induced hypersensitive necrotic reaction in plant cells: a resistance response against Agrobacterium-mediated DNA transfer. Afr J Biotechnol 4:752–757
Levee V, Garin E, Klimaszewska K, Seguin A (1999) Stable genetic transformation of white pine (Pinus strobus L.) after cocultivation of embryogenic tissues with Agrobacterium tumefaciens. Mol Breed 5:429–440. https://doi.org/10.1023/A:1009683605841
Li F, Li M, Zhan C, Wang S (2015) A reliable and high-efficiency Agrobacterium tumefaciens-mediated transformation system of Pogonatherum paniceum embryogenic callus using GFP as a reporter gene. Plant Cell Tissue Organ Cult 120:155–165. https://doi.org/10.1007/s11240-014-0589-y
Li S, Cong Y, Liu Y, Wang T, Shuai Q, Chen N, Ga J, Li Y (2017) Optimization of Agrobacterium-mediated transformation in Soybean. Front Plant Sci 8:246. https://doi.org/10.3389/fpls.2017.00246
Maheshwari P, Kovalchuk I (2016) Agrobacterium-mediated stable genetic transformation of Populus angustifolia and Populus balsamifera. Front Plant Sci 7:296. https://doi.org/10.3389/fpls.2016.00296
Malabadi RB, Nataraja K (2007) Genetic transformation of conifers: applications in and impacts on commercially forestry. Transgenic Plant J 1:289–313
Malabadi RB, da Silva JAT, Nataraja K (2008) Agrobacterium tumefaciens-mediated genetic transformation of Pinus kesiya Royle ex Gord (Khasi Pine). Asian Aust J Plant Sci Biotech 2:7–14
Nyaboga EN, Njiru JM, Tripathi L (2015) Factors influencing somatic embryogenesis, regeneration, and Agrobacterium-mediated transformation of cassava (Manihot esculenta Crantz) cultivar TME14. Front Plant Sci 6:411. https://doi.org/10.3389/fpls.2015.00411
Ombori O, Muoma JMO, Machuka J (2013) Agrobacterium-mediated genetic transformation of selected tropical inbred and hybrid maize (Zea mays L.) lines. Plant Cell Tissue Organ Cult 113:11–23
Porebski S, Bailey LG, Baum BR (1997) Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components. Plant Mol Biol Rep 15:8–15. https://doi.org/10.1007/BF02772108
Rashid H, Afzal A, Khan MH, Chaudhry Z, Malik SA (2010) Effect of bacterial culture density and acetosyringone concentration on Agrobacterium mediated transformation in wheat. Pak J Bot 42:4183–4189
Sainger M, Chaudhary D, Dahiya S, Jaiwal R, Jaiwal PK (2015) Development of an efficient in vitro plant regeneration system amenable to Agrobacterium- mediated transformation of a recalcitrant grain legume blackgram (Vigna mungo L. Hepper). Physiol Mol Biol Plants 21:505–517. https://doi.org/10.1007/s12298-015-0315-1
Sarmast MK (2016) Genetic transformation and somaclonal variation in conifers. Plant Biotechnol Rep 10:309–325. https://doi.org/10.1007/s11816-01
Singh B (2014) Effect of desiccation and chilling treatment on somatic embryo development and germination in Rough Lemon (Citrus jambhiri Lush.). Br Biotechnol J 4:136–148
Song ZY, Tian JL, Fu WZ, Li L, Lu LH, Zhou L, Shan ZH, Tang GX, Shou HX (2013) Screening Chinese soybean genotypes for Agrobacterium-mediated genetic transformation suitability. J Zhejiang Univ Sci B 14:289–298. https://doi.org/10.1631/jzus.B1200278
Stachel SE, Messens E, Van Montagu M, Zambryski P (1985) Identification of the signal molecules produced by wounded plant cells that activate T-DNA transfer in Agrobacterium tumefaciens. Nature 318:624–629. https://doi.org/10.1038/318624a0
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
Tang W, Sederoff R, Whetten R (2001) Regeneration of transgenic loblolly pine (Pinus taeda L.) from zygotic embryos transformed with Agrobacterium tumefaciens. Planta 213:981–989. https://doi.org/10.1007/s004250100566
Tang W, Luo H, Newton RJ (2004) Effects of antibiotics on the elimination of Agrobacterium tumefaciens from loblolly pine (Pinus taeda) zygotic embryo explants and on transgenic plant regeneration. Plant Cell Tiss Org 79:71–81. https://doi.org/10.1007/s11240-004-4657-6
Tang W, Newton RJ, Weidner DA (2007) Genetic transformation and gene silencing mediated by multiple copies of a transgene in eastern white pine. J Exp Bot 58:545–554
Tang W, Xiao B, Fei Y (2014) Slash pine genetic transformation through embryo cocultivation with A. tumefaciens and transgenic plant regeneration. In Vitro Cell Dev Biol Plant 50:199–209. https://doi.org/10.1007/s11627-013-9551-7
Tereso S, Miguel C, Zoglauer K, Valle-Piquera C, Oliveira MM (2006) Stable Agrobacterium-mediated transformation of embryogenic tissues from Pinus pinaster Portuguese genotypes. Plant Growth Regul 50:57–68. https://doi.org/10.1007/s10725-006-9126-2
Wenck RA, Quinn M, Whetten RW, Pullman G, Sederoff R (1999) High-efficiency Agrobacterium-mediated transformation of Norway spruce (Picea abies) and loblolly pine (Pinus taeda). Plant Mol Biol 39:407–416. https://doi.org/10.1023/A:1006126609534
Zhang SQ, Fei BH, Yu Y, Wang HK (2012) Fundamental properties of Masson pine (Pinus massoniana Lamb.) wood from plantation. In: Proceedings of the 55th international convention of society of wood science and technology, August 27–31, Beijing, China
Zhang WJ, Dewey RE, Boss W, Phillippy BQ, Qu R (2013) Enhanced Agrobacterium-mediated transformation efficiencies in monocot cells is associated with attenuated defense responses. Plant Mol Biol 81:273–286. https://doi.org/10.1007/s11103-012-9997-8
Zhu LH, Wu XQ, Qu HY, Ji J, Ye JR (2010) Micropropagation of Pinus massoniana and mycorrhiza formation in vitro. Plant Cell Tiss Org 102:121–128. https://doi.org/10.1007/s11240-010-9711-y
Acknowledgements
This study was supported by “The Thirteenth 5-year Plan” National Key Research Project “Pinus massoniana sustainable management technology research on Pinus massoniana”,and A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
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SM and KJ designed the experiments. SM conducted the experiments and collated the results. KM performed the statistical analysis and prepared the graphs. Drafting of manuscript and reading and final approval of the version to be published; SM, KM and KJ.
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Communicated by Sergio J. Ochatt.
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Maleki, S.S., Mohammadi, K. & Ji, K.S. Study on factors influencing transformation efficiency in Pinus massoniana using Agrobacterium tumefaciens. Plant Cell Tiss Organ Cult 133, 437–445 (2018). https://doi.org/10.1007/s11240-018-1388-7
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DOI: https://doi.org/10.1007/s11240-018-1388-7