Abstract
Following previously described Agrobacterium tumefaciens-mediated transformation procedures for Fragaria × ananassa Duch. ‘Chandler’, we undertook several experiments to establish the importance of some parameters affecting transformation. The most important factor that increased the percent recovery of transformants was the introduction of a pre-selection phase, in-between co-cultivation and selection, in which leaf disks were cultured on pre-selection regeneration medium containing validamycin A, timentin, and cefotaxime. The average percentage of leaf disks forming shoots on selection medium containing cefotaxime (250 mg l−1) + timentin (250 mg l−1) was 5.4% and about three shoots per regenerating leaf disk. Maximum transformation percentage, based on polymerase chain reaction, was 31.25%. Transgene integration and copy number were assessed by Southern hybridization confirming single copy as well as multiple copies of transgene integration in shoots as well as roots separately. This confirmed the non-chimeric nature of these transgenic plants. The system is very promising for the regeneration of genetically transformed cells and obtaining transgenic strawberry plants at high efficiency.
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References
Alsheikh MK, Suso HP, Robson M, Battey NH, Wetten A (2002) Appropriate choice of antibiotic and Agrobacterium strain improves transformation of antibiotic-sensitive Fragaria vesca and F.v. semperflorens. Plant Cell Rep 20:1173–1180
AOAC (1990) Official and tentative methods of analysis, 15th edn. Association of official Agricultural Chemists, Washington, DC, p 850
Arquelles JC (2000) Physiological roles of trehalose in bacteria and yeasts: a comparative analysis. Arch Microbiol 174:217–224
Asao H, Nishizawa Y, Arai S, Sato T, Hirai M, Yoshida K, Shinmyo A, Hibi T (1997) Enhanced resistance against a fungal pathogen Sphaerotheca humuli in transgenic strawberry expressing a rice chitinase gene. Plant Biotechnol 14:145–149
Bachelier C, Graham J, Machray G, Du Manoir J, Roucou JF, McNicol RJ, Davies H (1997) Integration of an invertase gene to control sucrose metabolism in strawberry cultivars. Acta Hortic 439:161–163
Barceló M, Mansouri EL, Mercado JA, Quesada MA, Alfaro FP (1998) Regeneration and transformation via Agrobacterium tumefaciens of the strawberry cultivar Chandler. Plant Cell Tissue Organ Cult 54:29–36
Bevan M (1984) Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res 12:8711–8721
Birch RG (1997) Plant transformation: problem and strategies for practical application. Annu Rev Plant Physiol Plant Mol Biol 48:297–326
Chalavi V, Tabaeizadeh Z, Thibodeau P (2003) Enhanced resistance to Verticillium dahliae in transgenic strawberry plants expressing a Lycopersicon chilense chitinase gene. J Am Soc Hortic Sci 128:747–753
Cochram WG, Cox GM (1957) Experimental designs. Wiley, New York, p 611
Cordero de Mesa M, Jiménez-Bermúdez S, Pliego-Alfaro F, Quesada MA, Mercado JA (2000) Agrobacterium cells as microprojectile coating: a novel approach to enhance stable transformation rates in strawberry. Aust J Plant Physiol 27:1093–1100
Crowe JH, Crowe LM, Chapman D (1984) Preservation of membranes in anhydrobiotic organisms: the role of trehalose. Science 223:701–703
Debnath SC (2005) Strawberry sepal: another explant for thidiazuron-induced adventitious shoot regeneration. In Vitro Cell Dev Biol Plant 41:671–676
Debnath SC, Teixeira da Silva JA (2007) Strawberry culture in vitro: applications in genetic transformation and biotechnology. Fruit Veg Cereal Sci Biotechnol 1:1–12
Dong JZ, Yang MZ, Jia SR, Chua NH (1991) Transformation of melon (cucumis melo l.) and expression from the cauliflower mosaic virus 35s promoter in transgenic melon plants. Bio/Technology 9:858–863
Drennan PM, Smith MT, Goldsworthy D, Van Staden J (1993) The occurrence of trehalose in the leaves of the desiccation-tolerant angiosperm Myrothamnus flabellifolius Welw. Plant Physiol 142:493–496
Du Plessis HJ, Brand RJ, Glyn-Woods C, Goedhart MA (1997) Efficient genetic transformation of strawberry (Fragaria × ananassa Duch.) cultivar Selekta. Acta Hortic 447:289–294
El Mansouri I, Mercado JA, Valpuesta V, Lopez-Aranda JM, Pliego-Alfaro F, Quesada MA (1996) Shoot regeneration and Agrobacterium-mediated transformation of Fragaria vesca L. Plant Cell Rep 15:642–646
Finstad K, Martin RR (1995) Transformation of strawberry for virus resistance. Acta Hortic 385:86–90
Folta KM, Dhingra A (2006) Transformation of strawberry: the basis for translational genomics in rosaceae. In Vitro Cell Dev Biol Plant 42:482–490
Folta KM, Dhingra A, Howard L, Stewart P, Chandler CK (2006) Characterization of LF9, an octoploid strawberry genotype selected for rapid regeneration and transformation. Planta 224:1058–1067
Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soyabean root cells. Exp Cell Res 50:151–158
Goddijn OJM, VanDun K (1999) Trehalose metabolism in plants. Trends Plant Sci 4:315–319
Goddijn OJM, Verwoerd TC, Voogd E, Krutwagen RWHH, de Graaf PTHM, Poels J, van Dun K, Ponstein AS, Damm B, Pen J (1997) Inhibition of trehalase activity enhances trehalose accumulation in transgenic plants. Plant Physiol 113:181–190
Godwin I, Todd G, Ford-Lloyd B, Newbury JH (1991) The effects of acetosyringone and pH on Agrobacterium-mediated transformation vary according to plant species. Plant Cell Rep 9:671–675
Graham J, McNicol RJ, Greig K (1995) Towards genetic based insect resistance in strawberry using the cowpea trypsin inhibitor gene. Ann Appl Biol 127:163–173
Gruchala A, Korbin M, Zurawicz E (2004a) Conditions of transformation and regeneration of ‘Induka’ and ‘Elista’ strawberry plants. Plant Cell Tissue Organ Cult 79:153–160
Gruchala A, Korbin M, Zurawicz E (2004b) Suitability of selected strawberry cultivars for genome modification by Agrobacterium tumefaciens. Acta Hortic 663:491–494
Guivarc’h A, Caissard JC, Brown S, Marie D, Dewitte W, Van Onckelen H, Chriqui D (1993) Localization of target cells and improvement of Agrobacterium-mediated transformation efficiency by direct acetosyringone pretreatment of carrot root discs. Protoplasma 174:10–18
Höfgen R, Willmitzer L (1990) Biochemical and genetic analysis of different patatin isoforms expressed in various organs of potato (Solanum tuberosum). Plant Sci 66:221–230
Horsch RB, Fry JE, Hoffman NL, Wallroth M, Eichholtz D, Rogers SG, Fraley RT (1985) A simple and general method for transferring genes into plants. Science 227:1229–1231
Hottiger T, Boller T, Wiemken A (1987) Rapid changes of heat and desiccation tolerance correlated with changes of trehalose content in Saccharomyces cerevisiae cells subjected to temperature shifts. FEBS Lett 220:113–115
Hounsa CG, Brandt EV, Thevelein J, Hohmann S, Prior BA (1998) Role of trehalose in survival of Saccharomyces cerevisiae under osmotic stress. Microbiology 144:671–680
Husaini AM, Abdin MZ (2007) Interactive effect of light, temperature and TDZ on the regeneration potential of leaf discs of Fragaria × ananassa Duch. In Vitro Cell Dev Biol Plant 43:567–584
Husaini AM, Abdin MZ (2008) Development of transgenic strawberry (Fragaria × ananassa Duch.) plants tolerant to salt stress. Plant Sci 174:446–455
Husaini AM, Srivastava DK (2006) Genetic transformation in strawberry—a review. Asian J Microbiol Biotech Env Sc 8:75–81
Husaini AM, Aquil S, Bhat M, Qadri T, Kamaluddin TA, Abdin MZ (2008) A high-efficiency direct somatic embryogenesis system for strawberry (Fragaria × ananassa Duch.) cultivar Chandler. J Crop Sci Biotech 11:107–110
Iordachescu M, Imai R (2008) Trehalose biosynthesis in response to abiotic stresses. J Integr Plant Biol 50:1223–1229
James DJ, Passey AJ, Barbara DJ (1990) Agrobacterium mediated transformation of apple and strawberry using disarmed Ti-binary vectors. Acta Hortic 280:495–502
James DJ, Uratsu S, Cheng J, Negri P, Viss P, Dandekar AM (1993) Acetosyringone and osmoprotectents like betaine or proline synergistically enhance Agrobacterium mediated transformation of apple. Plant Cell Rep 12:559–563
Jiménez-Bermúdez S, Redondo-Nevado J, Munoz-Blanco J, Caballero JL, Lopez-Aranda JM, Valpuesta V, Pliego-Alfaro F, Quesada MA, Mercado JA (2002) Manipulation of strawberry fruit softening by antisense expression of a Pectate lyase gene. Plant Physiol 128:751–759
Khan S, Qureshi MI, Kamaluddin TA, Abdin MZ (2007) Protocol for isolation of genomic DNA from dry and fresh roots of medicinal plants suitable for RAPD and restriction digestion. Afr J Biotechnol 6:175–178
Landi L, Mezzetti B (2006) TDZ, auxin and genotype effects on leaf organogenesis in Fragaria. Plant Cell Rep 25:281–288
Larkin PJ, Scowcroft WR (1981) Somaclonal variation—a novel source of variability from cell cultures for plant improvement. Theor Appl Gen 60:197–214
Li XG, Liu CN, Ritchie SW (1992) Factors influencing Agrobacterium-mediated transient expression of gusA in rice. Plant Mol Biol 20:1037–1048
Manickavasagam M, Ganapathi A, Anbazhagan VR, Sudhaka RB, Selvaraj N, Vasudevan A, Kasthurirengan A (2004) Agrobacterium-mediated genetic transformation and development of herbicide-resistant sugarcane (Saccharum species hybrids) using axillary buds. Plant Cell Rep 23:134–143
Martinelli A, Gaiani A, Cella R (1997) Agrobacterium-mediated transformation of strawberry cultivar Marmolada onebar. Acta Hortic 439:169–173
Mathews H, Bharathan N, Litz RE, Narayanan KR, Rao PS, Bhatia CR (1990) The promotion of Agrobacterium mediated transformation in Atropa belladonna L. by acetosyringone. J Plant Physiol 136:404–409
Mathews H, Wagoner W, Kellogg J, Bestwick R (1995) Genetic transformation of strawberry: stable integration of a gene to control biosynthesis of ethylene. In Vitro Cell Dev Biol Plant 31:36–43
Mathews H, Dewey V, Wagner W, Bestwick RK (1998) Molecular and cellular evidence of chimaeric tissues in primary transgenics and elimination of chimaerism through improved selection protocols. Transgenic Res 7:123–129
Mercado JA, Pliego-Alfaro F, Quesada MA (2007) Strawberry. In: Pua EC, Davey MR (eds) Transgenic crops V. Biotechnology in agriculture and forestry, vol 60. Springer, Berlin, pp 309–328
Mezzetti B, Costantini E, Chionchetti F, Landi L, Pandolfini T, Spena A (2004) Genetic transformation in strawberry and raspberry for improving plant productivity and fruit quality. Acta Hortic 649:107–110
Monticelli S, Gentile A, Damiano C (2002) Regeneration and Agrobacterium-mediated transformation in stipules of strawberry. Acta Hortic 567:105–107
Montoro P, Rattana W, Pujade-Renaud V, Michaux-Ferrieere N, Monkolsook Y, Kanthapura R, Adunsadthapong S (2003) Production of Hevea brasiliensis transgenic embryogenic callus lines by Agrobacterium tumefaciens: roles of calcium. Plant Cell Rep 21:1095–1102
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497
Murthy BNS, Murch SJ, Saxena PK (1998) Thidiazuron: a potent regulator of in vitro plant morphogenesis. In Vitro Cell Dev Biol Plant 34:267–275
Nehra NS, Chibber RN, Kartha KK, Datla RSS, Crosby WL, Stushnoff C (1990a) Genetic transformation of strawberry by Agrobacterium tumefaciens using a leaf disk regeneration system. Plant Cell Rep 9:293–298
Nehra NS, Chibber RN, Kartha KK, Datla RSS, Crosby WL, Stushnoff C (1990b) Agrobacterium transformation of strawberry calli and recovery of transgenic plants. Plant Cell Rep 9:10–13
Oosumi T, Gruszewski HA, Blischak LA, Baxter AJ, Wadl PA, Shuman JL, Veilleux RE, Shulaev V (2006) High-efficiency transformation of the diploid strawberry (Fragaria vesca) for functional genomics. Planta 223:1219–1230
Passey AJ, Barrett KJ, James DJ (2003) Adventitious shoot regeneration from seven commercial strawberry cultivars (Fragaria × ananassa Duch.) using a range of explant types. Plant Cell Rep 21:397–401
Pramanik MHR, Imai R (2005) Functional identification of a trehalose-6-phosphatise gene that is involved in transient induction of trehalose biosynthesis during chilling stress in rice. Plant Mol Biol 58:751–762
Puite K, Schaart J (1998) Agrobacterium-mediated transformation of the apple cultivars Gala, Golden delicious and Elstar, and the strawberry cultivars Gariguette, Polka and Elsanta. Acta Hortic 484:547–556
Qin YH, Zhang SL, Asghar S, Zhang LX, Qin QP, Xu CJ, Chen KS (2005) Regeneration mechanism of Toyonoka strawberry under different color plastic films. Plant Sci 168:1425–1431
Qin YH, Teixeira da Silva JAT, Zhang LX, Zhang SL (2008) Transgenic strawberry: state of the art for improved traits. Biotech Adv 26:219–232
Quesada MA, Martín-Pizarro C, García-Gago JA, Posé S, Santiago N, Sesmero R, Pliego-Alfaro F, Mercado JA (2007) Transgenic strawberry: current status and future perspectives. Transgenic Plant J 1:280–288
Rangana S (1986) Handbook of analysis and quality control for fruit and vegetable products, 2nd edn. Tata McGraw Hill Publishing Co. Ltd, New Delhi, p 1112
Ricardo VG, Coll Y, Castagnaro A, Diaz Ricci JC (2003) Transformation of a strawberry cultivar using a modified regeneration medium. HortScience 38:277–280
Schaart JG, Salentijn MJ, Krens FA (2002) Tissue specific expression of the β-glucuronidase reporter gene in transgenic strawberry (Fragaria × ananassa) plants. Plant Cell Rep 21:313–319
Shestibratov KA, Dolgov SV (2005) Transgenic strawberry plants expressing a thaumatin II gene demonstrate enhanced resistance to Botrytis cinerea. Sci Hortic 106:177–189
Shiekholeslam SN, Weeks DP (1987) Acetosyringone promotes high efficiency transformation of Arabidopsis thaliana explants by Agrobacterium tumefaciens. Plant Mol Biol 8:291–298
Sorvari S, Ulvinen S, Hietaranta T, Hiirsalmi H (1993) Preculture medium promotes direct shoot regeneration from micropropagated strawberry leaf disks. HortScience 28:55–57
Sriskandarajah S, Frello S, Jorgensen K, Serek M (2004) Agrobacterium tumefaciens-mediated transformation of Campanula carpatica: factors affecting transformation and regeneration of transgenic shoots. Plant Cell Rep 23:59–63
Styrvold OB, Strom AR (1991) Synthesis, accumulation, and excretion of trehalose in osmotically stressed Escherichia coli K-12 strains: influence of amber suppressors and function of the periplasmic trehalase. J Bacteriol 173:1187–1192
Tanprasert P, Reed BM (1998) Detection and identification of bacterial contaminants of strawberry runner explants. Plant Cell Tissue Organ Cult 52:53–55
Van Wordragen MF, Dons HJM (1992) Agrobacterium tumefaciens-mediated transformation of recalcitrant crops. Plant Mol Biol Rep 10:12–36
Vergauwe A, Van Geldre E, Inze D, Van Montagu M, Van den Eeckhout E (1998) Factors influencing Agrobacterium tumefaciens-mediated transformation of Artemisia annua L. Plant Cell Rep 18:105–110
Voth V, Bringhurst RS (1984) Strawberry plant ‘Chandler’. United States Patent PP05262. http://www.freepatentsonline.com/PP05262.html
Wang J, Ge H, Peng S, Zhang H, Chen P, Xu J (2004) Transformation of strawberry (Fragaria ananassa Duch.) with late embryogenesis abundant protein gene. J Hortic Sci Biotechnol 79:735–738
Wawrzyńczak D, Sowik I, Michalczuk L (2000) Agrobacterium-mediated transformation of five strawberry genotypes. J Fruit Ornam Plant Res 8:1–8
Weir B, Gu X, Wang MB, Upadhyaya N, Elliott AR, Brettell RIS (2001) Agrobacterium tumefaciens-mediated transformation of wheat using suspension cells as a model system and green fluorescent protein as a visual marker. Aust J Plant Physiol 28:807–818
Yonghua Q, Shanglong Z, Asghar S, Lingxiao Z, Qiaoping Q, Kunsong C, Changjie X (2005) Regeneration mechanism of Toyonoka strawberry under different color plastic films. Plant Sci 168:1409–1424
Zhang HM, Wang JL (2005) Establishment of genetic transformation system of “Allstar” strawberry leaf. Biotechnology 15:68–70
Zhao Y, Liu Q, Davis RE (2004) Transgene expression in strawberries driven by a heterologous phloem-specific promoter. Plant Cell Rep 23:224–230
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Amjad M. Husaini is thankful to Council of Scientific and Industrial Research, Government of India, for the financial support to this project. He is highly thankful to Dr. Ab. Matteen Rafiqi, University of Chicago, for his critical review and valuable help extended during the preparation of this research paper.
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Husaini, A.M. Pre- and post-agroinfection strategies for efficient leaf disk transformation and regeneration of transgenic strawberry plants. Plant Cell Rep 29, 97–110 (2010). https://doi.org/10.1007/s00299-009-0801-4
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DOI: https://doi.org/10.1007/s00299-009-0801-4