Abstract
The effect of chemical additives (acetosyringone, AS; L-cysteine, CYS; dithiothreitol, DTT; glutathione, GSH; cellulase, CEL; pectinase, PEC) and light regimes (16/8 light/dark photoperiod, 16L/8D; continuous light, 24L; continuous dark, 24D) applied during cocultivation procedure of pea explants with Agrobacterium tumefaciens on transformation efficiency was studied. A hypervirulent strain of A. tumefaciens EHA 105 with two plasmids, namely pGT89 and pBIN19, both carrying reporter gus-int gene, and bar or nptII selectable marker gene, respectively, was used for genetic transformation of cotyledonary node explants of three dry seed pea cultivars Adept, Komet and Menhir. The focus was laid on cocultivation step (48 h) of transformation protocol. After chemical or physical treatments, transient GUS expression was recorded 20 days after cocultivation as a measure of successful transformation, using a four category scale (0 – without GUS expression, 1 – weak, 2 – medium and 3 – strong GUS expression) for calculation of IGE (Intensity of GUS Expression). Of the tested chemical cocultivation additives, 100 μM AS and 50 mg CYS significantly improved GUS expression (IGE value), while DTT, GSH and both macerating enzymes (CEL, PEC used either separately or in combination) either had no positive effect or were even negative. There were no statistically significant differences between the light regimes tested. Nevertheless, cocultivation in 24L, without chemical additives, reproducibly resulted in the highest frequency of explants scored in category 3 of GUS expression (followed by 24D and 16L/8D treatment). However, application of 100 μM AS reverted this trend. Cv. Adept yielded higher transformation frequencies than cvs. Menhir and Komet. Plasmid pGT89 produced a higher IGE value than pBIN19. Based on our results, the improved cocultivation step for pea consists of 48 h cocultivation at 20 ± 2°C, with 50 mg l−1 CYS and 100 μM AS, 16L/8D photoperiod (or without AS in continuous light).
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Abbreviations
- AS:
-
Acetosyringone
- CCM:
-
Cocultivation medium
- CEL:
-
Cellulose
- CYS:
-
L-Cysteine
- d/n:
-
Day/night
- DTT:
-
Dithiothreitol
- GSH:
-
Glutathione
- IGE:
-
Intensity of GUS Expression
- PEC:
-
Pectinase
- 24L:
-
Continuous light regime
- 24D:
-
Continuous dark regime
- 16L/8D:
-
16 h light/8 h dark photoperiod
References
Bean SJ, Gooding PS, Mullineaux PM, Davies DR (1997) A simple system for pea transformation. Plant Cell Rep 16:513–519
Bevan M (1984) Binary Agrobacterium vectors for plant transformation. Nucl Acids Res 12:8711–8721
Dandekar AM, Fisk HJ (2005) Plant transformation – Agrobacterium-mediated gene transfer. In: Peña L (ed) Transgenic plants. Methods and protocols. Methods in molecular biology, vol 286. Humana Press, Totowa, New Jersey, pp 35–46
Davies DR, Hamilton J, Mullineaux P (1993) Transformation of peas. Plant Cell Rep 12:180–183
De Clercq J, Zambre M, Van Montagu M, Dillen W, Angenon G (2002) An optimized Agrobacterium-mediated transformation procedure for Phaseolus acutifolius A. Gray. Plant Cell Rep 21:333–340
DEFRA project AR1004 final report: an investigation into strategies to improve pea transformation. John Innes Centre, Norwich, UK (2003) http://www.defra.gov.uk/science/project_data/DocumentLibrary/AR1004/AR1004_756_FRP.doc
De Kathen A, Jacobsen H-J (1990) Agrobacterium tumefaciens-mediated transformation of Pisum sativum L. using binary and cointegrate vectors. Plant Cell Rep 9:276–279
De Kathen A, Jacobsen H-J (1995) Cell competence for Agrobacterium-mediated DNA transfer in Pisum sativum L. Transgenic Res 4:184–195
Drake PMW, John A, Power JB, Davey MR (1997) Expression of the gus A gene in embryogenic cell lines of Sitka spruce following Agrobacterium-mediated transformation. J Exp Bot 48:151–155
Flores Solís JI, Mlejnek P, Studená K, Procházka S (2003) Application of sonication-assisted Agrobacterium-mediated transformation in Chenopodium rubrum L. Plant Soil Environ 49:255–260
Frame BR, Shou H, Chikwamba RK, Zhang Z, Ch Xiang, Fonger TM, Pegg SEK, Li B, Nettleton DS, Pei D, Wang K (2002) Agrobacterium tumefaciens-mediated transformation of maize embryos using a standard binary vector system. Plant Physiol 129:13–22
Fütterer J, Gisel A, Iglesias V, Klöti A, Kost B, Mittelsten Scheid O, Neuhaus G, Neuhaus-Url G, Schrott M, Shillito R, Spangenberg G, Wang ZY (1995) Standard molecular techniques for the analysis of transgenic plants. In: Potrykus I, Spangenberg G (eds) Gene transfer to plants, Springer lab manual. Springer-Verlag, Berlin, pp 215–263
Grant JE, Cooper PA, McAra AE, Frew TJ (1995) Transformation of peas (Pisum sativum L.) using immature cotyledons. Plant Cell Rep 15:254–258
Grant JE, Thomson LMJ, Pither-Joyce MD, Dale TM, Cooper PA (2003) Influence of Agrobacterium tumefaciens strain on the production of transgenic peas (Pisum sativum L.). Plant Cell Rep 21:1207–1210
Hansen G, Wright MS (1999) Recent advances in the transformation of plants. Trends Plant Sci 6:226–231
Heeres P, Schippers-Rozenboom M, Jacobsen E, Visser RGF (2002) Transformation of a large number of potato varieties: genotype-dependent variation in efficiency and somaclonal variability. Euphytica 124:13–22
Hood EE, Gelvin SB, Melchers LS, Hoekema A (1993) New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Res 2:208–218
Jones AL, Johansen IE, Bean SJ, Bach I, Maule AJ (1998) Specificity of resistance to pea seed-borne mosaic potyvirus in transgenic peas expressing the viral replicase (NIb) gene. J Gen Virol 79:3129–3137
Ko TS, Korban SS (2004) Enhacing the frequency of somatic embryogenesis following Agrobacterium-mediated transformation of immature cotyledons of soybean [Glycine max (L.) Merrill]. In vitro Cell Dev Biol Plant 40:552–558
Kosugi S, Ohashi Y, Nakajima K, Arai Y (1990) An improved assay for β-glucuronidase in transformed cells: methanol almost completely suppresses a putative endogenous β-glucuronidase acitivity. Plant Sci 70:133–140
Krejčí P, Matušková P, Hanáček P, Reinöhl V, Procházka S (2007) The transformation of pea (Pisum sativum L.): applicable methods of Agrobacterium tumefaciens-mediated gene transfer. Acta Physiol Plant 29:157–163
Kumar V, Sharma A, Prasad BCN, Gururaj HB, Ravishankar GA (2006) Agrobacterium rhizogenes mediated genetic transformation resulting in hairy root formation is enhanced by ultrasonication and acetosyringone. Electr J Biotechnol 9:349–357
Langley RA, Kado CI (1972) Studies on Agrobacterium tumefaciens: conditions for mutagenesis by N-methyl-N-nitrosoquanidine and relationship of A. tumefaciens mutants to crown gall induction. Mutat Res 14:277–286
Li ZT, Dhekney S, Dutt M, van Aman M, Tattersall J, Kelley KT, Gray DJ (2006) Optimizing Agrobacterium-mediated transformation of grapevine. In Vitro Cell Dev Biol-Plant 42:220–227
Lulsdorf MM, Rempel H, Jackson JA, Baliski DS, Hobbs SLA (1991) Optimizing the production of transformed pea (Pisum sativum L.) callus using disarmed Agrobacterium tumefaciens strains. Plant Cell Rep 9:479–483
Mondal TK, Bhattacharya A, Ahuja PS, Chand PK (2001) Transgenic tea [Camelia sinensis (L.) O. Kuntze cv. Kangra Jat] plants obtained by Agrobacterium-mediated transformation of somatic embryos. Plant Cell Rep 20:712–720
Nadolska-Orczyk A, Orczyk W (2000) Study of the factors influencing Agrobacterium-mediated transformation of pea (Pisum sativum L.). Mol Breed 6:185–194
Olhoft PM, Somers DA (2001) L-Cysteine increases Agrobacterium-mediated T-DNA delivery into soybean cotyledonary-node cells. Plant Cell Rep 20:706–711
Olhoft PM, Lin K, Galbraith J, Nielsen NC, Somers DA (2001) The role of thiol compounds in increasing Agrobacterium-mediated transformation of soybean cotyledonary-node cells. Plant Cell Rep 20:731–737
Olhoft PM, Flagel LE, Donovan CM, Somers DA (2003) Efficient soybean transformation using hygromycin B selection in the cotyledonary-node method. Planta 216:723–735
Opabode JT (2006) Agrobacterium-mediated transformation of plants: emerging factors that influence efficiency. Biotechnol Mol Biol Rev 1:12–20
Paz MM, Shou H, Guo Z, Zhang Z, Banerjee AK, Wang K (2004) Assessment of conditions affecting Agrobacterium-mediated soybean transformation using the cotyledonary node explant. Euphytica 136:167–179
Perl A, Lotan O, Abu-Abied M, Holland D (1996) Establishment of an Agrobacterium-mediated transformation system for grape (Vitis vinifera L.): the role of antioxidants during grape-Agrobacterium interactions. Nat Biotechnol 14:624–628
Polowick PL, Quandt J, Mahon JD (2000) The ability of pea transformation technology to transfer genes into peas adapted to western Canadian growing conditions. Plant Sci 153:161–170
Puonti-Kaerlas J, Eriksson T, Engström P (1990) Production of transgenic pea (Pisum sativum L.) plants by Agrobacterium-mediated gene transfer. Theor Appl Genet 80:246–252
Qiusheng Z, Bao J, Likun L, Xianhua X (2005) Effects of antioxidants on the plant regeneration and GUS expressive frequency of peanut (Arachis hypogaea) explants by Agrobacterium tumefaciens. Plant Cell Tiss Organ Cult 81:83–90
Rao MVR, Rao GJN (2007) Agrobacterium-mediated transformation of indica rice under acetosyringone-free conditions. Plant Biotechnol 24:507–511
Reyes SM, Rencoret HJ (2008) Bactericidal, bacteriostatic and fungicidal composition comprising two or more live species of trichoderma. European patent EP 1384405
Santarém ER, Trick HN, Essig JS, Finer JJ (1998) Sonication assisted Agrobacterium-mediated transformation of soybean immature cotyledons: optimization of transient expression. Plant Cell Rep 17:752–759
Schroeder HE, Scholtz AH, Wardley-Richardson T, Spencer D, Higgins TJV (1993) Transformation and regeneration of two cultivars of pea (Pisum sativum L.). Plant Physiol 101:751–757
Sonntag K, Döscher B, Sellner M (2001) Genotype effects on Agrobacterium mediated genetic transformation of Brassica napus. Acta Hort (ISHS) 560:215–218
Švábová L, Lebeda A, Griga M (1998) Comparison of in vitro testing methods for screening of resistant peas to Fusarium spp. Acta Phytopath Entomol Hung 33:275–284
Švábová L, Smýkal P, Griga M, Ondřej V (2005) Agrobacterium-mediated transformation of Pisum sativum in vitro and in vivo. Biol Plant 49:361–370
Švábová L, Smýkal P, Griga M (2008) Agrobacterium-mediated transformation of pea (Pisum sativum L.): Transformant production in vitro and by non-tissue culture approach. In: M.C. Kharkwal (ed.), Food Legumes for Nutritional Security and Sustainable Agriculture, ISGPB, New Delhi, India (in press)
Torregrosa P, Iocco P, Thomas MR (2002) Influence of Agrobacterium strain, culture medium, and cultivar on transformation efficiency of Vitis vinifera L. Am J Enol Vitic 53:183–190
Townsend JA, Thomas LA (1996) Method of Agrobacterium-mediated transformation of cultured soybean cells. U.S. patent No. 5,563,055
Trinh TH, Ratet P, Kondorosi E, Durand P, Kamaté K, Bauer P, Kondorosi A (1998) Rapid and efficient transformation of diploid Medicago truncatula and Medicago sativa ssp. falcata lines improved in somatic embryogenesis. Plant Cell Rep 17:345–355
Vancanneyt G, Schmidt R, O′Connor-Sanchez A, Wilmitzer L, Rocha-Sosa M (1990) Construction of an intron-containing marker gene: splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium-mediated plant transformation. Mol Gen Genet 220:245–250
Weber S, Friedt W, Landes N, Molinier J, Himber C, Rousselin P, Hahne G, Horn R (2003) Improved Agrobacterium-mediated transformation of sunflower (Helianthus annuus L.): assessement of macerating enzymes and sonication. Plant Cell Rep 21:475–482
Xinping YI, Deyue YU (2006) Transformation of multiple soybean cultivars by infecting cotyledonary-node with Agrobacterium tumefaciens. Afr J Biotech 5:1989–1993
Yamada T, Teraishi M, Hattori K, Ishimoto M (2001) Transformation of azuki bean by Agrobacterium tumefaciens. Plant Cell Tiss Org Cult 64:47–54
Yeung EC, Belmonte MF, Tu LTT, Stasolia C (2005) Glutathione modulation of in vitro development. In Vitro Cell Dev Biol-Plant 41:584–590
Zambre M, Terryn N, De Clerq J, De Buck S, Dillen W, Van Montagu M, Van Der Straeten D, Angenon G (2003) Light strongly promotes gene transfer from Agrobacterium tumefaciens to plant cells. Planta 216:580–586
Zeng P, Vadnais DA, Zhang Z, Polacco JC (2004) Refined glufosinate selection in Agrobacterium-mediated transformation of soybean [Glycine max (L.) Merill]. Plant Cell Rep 22:478–482
Acknowledgements
The authors wish to thank Ms. J. Vychodilová for excellent technical assistance and Dr. Sergio Ochatt for critical reading of manuscript and helpfull comments. This research was financially supported by the National Agency for Agricultural Research (grant QF 3072) and Ministry of Education of Czech Republic (grants MSM 2678424601 and 1M06030).
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Švábová, L., Griga, M. The effect of cocultivation treatments on transformation efficiency in pea (Pisum sativum L.). Plant Cell Tiss Organ Cult 95, 293–304 (2008). https://doi.org/10.1007/s11240-008-9443-4
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DOI: https://doi.org/10.1007/s11240-008-9443-4