Abstract
Repeated attempts to genetically transform Pinus radiata embryonal masses through cocultivation with Agrobacterium tumefaciens on MSG medium were unproductive due to Agrobacterium overgrowth. Timentin at either 200 or 400 mg l−1 was ineffective in inhibiting bacterial growth after cocultivation. In this study, the causes of the abundant bacterial growth were investigated by comparing MSG medium with two other media (mLV and DCR) commonly used in conifer somatic embryogenesis. Statistical analysis of the growth data (optical density and number of cell-forming units) showed that bacterium grew significantly more on MSG than on mLV or DCR during the 48-h cocultivation. This enhanced growth was attributed to the higher concentration of L-glutamine in MSG. Lowering the concentration of L-glutamine in MSG to 0.5 g l−1 resulted in similar growth of Agrobacterium compared with the other two media. MSG was also superior for the growth of radiata pine cells, with a statistically significant difference after 14 d of culture. Hence, to avoid bacterial overgrowth during and after cocultivation, a two-medium protocol was developed in which cocultivation was carried out on mLV, followed by 5 d on mLV with 400 mg l−1 Timentin. Selection for transformed cells and further control of bacterial growth was then performed using MSG with Timentin and Geneticin. By sequential application of these two media, 2,096 cell colonies were selected; of these, 94 were analyzed and 49 were transgenic. These results highlight yet another factor that might be critical for the success of transformation experiments but has not been sufficiently studied until now: the growth dynamics and ability to eliminate A. tumefaciens on various plant tissue culture media.
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References
Becwar M, Nagmani R, Wann S (1990) Initiation of embryogenic cultures and somatic embryo development in loblolly pine (Pinus taeda). Can J Forest Res 20:810–817
Chambers JM, Hastie TJ (eds) (1992) Statistical models in S. Wadsworth & Brooks/Cole, Pacific Grove, California, 608 p
Charity JL, Holland L, Grace LJ, Walter C (2005) Consistent and stable expression of the nptII, uidA and bar genes in transgenic Pinus radiata after Agrobacterium tumefaciens-mediated transformation using nurse culture. Plant Cell Rep 23:606–616
Charity JL, Klimaszewska K (2005) Persistence of Agrobacterium tumefaciens in transformed conifers. Environ Biosafety Res 4:167–177
Cheng ZM, Schnurr JA, Kapaun JA (1998) Timentin as an alternative antibiotic for suppression of Agrobacterium tumefaciens in genetic transformation. Plant Cell Rep 17:646–649
Gleeson D, Lelu-Walter MA, Parkinson M (2005) Overproduction of proline in transgenic hybrid larch (Larix x leptoeuropaea (Dengler)) cultures renders them tolerant to cold, salt and frost. Mol Breed 15:21–29
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 (2004) Transgenic Pinus radiata from Agrobacterium tumefaciens-mediated transformation of cotyledons. Plant Cell Rep 22:894–902
Gupta P, Durzan D (1985) Shoot multiplication for mature trees of Douglas fir (Pseudotsuga menziesii) and sugar pine (Pinus lambertiana). Plant Cell Rep 4:177–179
Hargreaves CL, Reeves CB, Find JI, Gough K, Josekutty P, Skudder DB, van der Maas SA, Sigley MR, Menzies MI, Low CB, Mullin TJ (2009) Improving initiation, genotype capture, and family representation in somatic embryogenesis of Pinus radiata by a combination of zygotic embryo maturity, media, and explant preparation. Can J Forest Res 39:1566–1574
Klimaszewska K, Lachance D, Pelletier G, Lelu MA, Séguin A (2001a) Regeneration of transgenic Picea glauca, P. mariana, and P. abies after cocultivation of embryogenic tissue with Agrobacterium tumefaciens. Vitro Cell Dev Biol-Plant 37:748–755
Klimaszewska K, Park YS, Overton C, Maceacheron I, Bonga J (2001b) Optimized somatic embryogenesis in Pinus strobus L. Vitro Cell Dev Biol-Plant 37:392–399
Klimaszewska K, Sutton B, Polonenko D, Cyr D, Stodola T (2001c) Maturation of somatic embryos. US Patent 6,200,809 B1
Koncz C, Schell J (1986) The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimeric genes carried by a novel type of Agrobacterium binary vector. Mol Gen Genet 204:383–396
Levee V, Lelu MA, Jouanin L, Cornu D, Pilate G (1997) Agrobacterium tumefaciens mediated transformation of hybrid larch (Larix kaempferii x L. decidua) and transgenic plant regeneration. Plant Cell Rep 16:680–685
Lin YJ, Zhang Q (2005) Optimizing the tissue culture conditions for high efficiency transformation of indica rice. Plant Cell Rep 23:540–547
Littell RC, Milliken GA, Stroup WW, Wolfinger RD, Schabenberger O (2006) SAS for mixed models, 2nd edn. SAS Institute Inc., Cary
Litvay J, Verma D, Johnson M (1985) Influence of a loblolly pine (Pinus taeda L.) culture medium and its components on growth and somatic embryogenesis of the wild carrot (Daucus carota L.). Plant Cell Rep 4:325–328
Mohammed GH, Dunstan DI (1986) Influence of nutrient medium upon shoot initiation on vegetative explants excised from 15- to 18-year-old Picea glauca. New Zeal J Forest Sci 16:297–305
Montalbán IA, De Diego N, Moncaleán P (2010) Bottlenecks in Pinus radiata somatic embryogenesis: improving maturation and germination. Trees 24:1061–1071
Pinheiro JC, Bates DM (2000) Mixed-effects models in S and S-PLUS. Springer, New York
Quoirin M, Lepoivre P (1977) Improved media for in vitro culture of Prunus sp. Acta Hortic 78:437–442
Smith D (1996) Growth medium. US Patent 5:565,355
Starr MP (1946) The nutrition of phytopathogenic bacteria: the genus Agrobacterium. J Bacteriol 52:187–194
Tereso S, Miguel C, Zoglauer K, Valle-Piquera C, Oliveira MM (2006) Stable Agrobacterium-mediated transformation of embryogenic tissue from Pinus pinaster Portuguese genotypes. Plant Growth Regul 50:57–68
Trontin JF, Walter C, Klimaszewska K, Park YS, Lelu-Walter MA (2007) Recent progress in genetic transformation of four Pinus spp. Transgenic Plant J 1:314–329
Zimmerman TW (1995) Effect of Timentin for controlling Agrobacterium tumefaciens following cocultivation on select plant species. Vitro Cell Dev Biol 31:70A
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Financial support from Genómica Forestal S.A. and INNOVA-CHILE (grant number 05CTE04-04) is gratefully acknowledged. We thank the reviewers, editor, and copy-editor for their helpful suggestions. KK and MBC were supported by Natural Resources Canada, Canadian Forest Service, Canada.
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Editor: Ming Chen
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Le-Feuvre, R., Triviño, C., Sabja, A.M. et al. Organic nitrogen composition of the tissue culture medium influences Agrobacterium tumefaciens growth and the recovery of transformed Pinus radiata embryonal masses after cocultivation. In Vitro Cell.Dev.Biol.-Plant 49, 30–40 (2013). https://doi.org/10.1007/s11627-013-9492-1
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DOI: https://doi.org/10.1007/s11627-013-9492-1