GUS AND GFP ANALYSIS IN HAIRY ROOTS OF UNIVERSITEIT GENT GUS AND GFP ANALYSIS IN HAIRY ROOTS OF SEVERAL PLANT SPECIES Nur KOYUNCU1, Mansour KARIMI2 and Godelieve GHEYSEN3 1Departmant of Field Crops, Faculty of Agriculture, University of Ankara 06110, Ankara TURKEY 2 Department of Plant System Biology (PSB), Vlaams Interuniversitair Instituut voor Biotechnologie, Universiteit Gent, K.L. Ledeganckstraat 35, B 9000, Gent, BELGIUM 3 Vakgroep Moleculaire Biotechnologie, Faculteit Landbouwkundige en Toegepaste Biologische Wetenschappen, Universiteit Gent, Coupure Links 65, B9000,Gent,BELGIUM UNIVERSITEIT GENT Results Abstract The Agrobacterium system is often used for plant transformation. Infection with A. tumefaciens results in a tumour, while A. rhizogenes induces hairy roots at the infection site. In comparison with the production of transgenic plants, the generation of hairy roots has the advantage that more independent transgenic lines can be produced in a shorter period of time. Therefore, we used this approach for expression analysis of five different promoters fused to gus or gfp in Arabidopsis thaliana and several plant species. All the binary vectors containing the promoter-reporter fusions were co-transformed by A. rhizogenes 15834 RifR into the plant species. Integration of the T-DNAs from the binary vector and the Ri plasmid results in the generation of kanamycin resistant hairy roots. To analyse the expression patterns of the promoters in the transformed hairy root lines they were subjected to a GUS histochemical assay or GFP analysis. Here we showed that many independent A. rhizogenes-transformed hairy roots can be directly used for the analysis of gene expression without the need to go over the time-consuming regeneration procedure. We have furthermore shown that the promoters that are nematode-responsive in A. thaliana can also be used in crop species. Analysis of transformed hairy root lines For the analysis of promoter activity in the transformed hairy root lines they were incubated on callus inducing medium ( 0.5 mg/l 2,4-D and 0.05mg/l kinetin) for 5 days and then they were subjected to a GUS histochemical assay according to the method of Jefferson et al. (1987). After the samples were incubated at 37 °C overnight, they were examined under the light microscope. For visualisation of GFP in the transformed hairy roots containing GFP construct a fluorescence microscope was used. The filter set for fluorescence microscopy consisted of excitation, cut-off and emission filters with 450-490, 510, 515-565 nm wavelength respectively. DNA was extracted from hairy roots and analyzed as described by Dellaporta et al. (1983). In order to check the presence of the T-DNA in the transgenic hairy roots, polymerase chain reaction (PCR) amplification was conducted with primers corresponding to the nptII. The PCR amplification was carried out in 50 μl reaction (94 C, 2 min; 62 C, 45 sec; 72 C, 45 sec, 30 cycles). HindIII SphI PstI SalI XbaI BamHI SmaI KpnI SacI EcoRI lacZ pW2S90EGFPERBinPlus LB RB SpeI XhoI HincII NPTII Tnos Pnos pW2 35S-3’ HindIII SphI PstI SalI XbaI BamHI SmaI KpnI SacI EcoRI lacZ p35S800EGFPERBinPlus LB RB SpeI XhoI HincII 35S (800) NPTII Tnos Pnos 35S-3’ EGFPER 35S (90) EGFPER pW2gusBin19 construct was used for transformation of following plant species; tomato, potato (Solanum tuberosum), radish (Raphanus sativus), tobacco (Nicotiana tubacum), and alfalfa (Medicago sativa). pW2GUSBin19 RB LB EcoRI HindIII BamHI SpeI XbaI NPTII Tnos Pnos pW2 NOS- ter gus Tnos pC2a EGFPER room at 22 °C (16 h light/8 h dark photoperiod) (Fig. a). Arabidopsis seedlings were incubated for 3 weeks and other species for about one week. Leaves from potato plantlets were used from an in vitro potato culture. The Arabidopsis and potato leaves and cotyledons of other species were transversely cut in two halves with a sterile scalpel. To avoid desiccation of tissue during cutting, leaves were floated on sterile water in Petri dishes. The leaf halves were placed upside-down on callus-inducing medium and were incubated for 4 days in the growth room (Fig. b) . A. rhizogenes was cultured in liquid LB medium and appropriate selective agents were added. The infection solution was made by diluting the bacterial culture in liquid standard medium (SM) to a final OD=0.1. Leaf pieces were incubated with infection solution and then transferred to solidified SM medium. The explants were incubated for 3 days in the growth room. Leaf pieces were washed 3 times with liquid SM medium a b c Transformation Sterilized seed of plant species were plated on germination medium and the Petri dishes were incubated in the growth Keywords: A. rhizogenes, Arabidopsis thaliana, gfp, gus, hairy root Fig. 7 PCR products from DNA of A. thaliana using different constructs; A and B) pcdc2aEGFPERBinplus, C and D) p35S800EGFPERBinplus, E) control reaction using DNA from pBinplus binary vector. Introduction Both Agrobacterium tumefaciens and Agrobacterium rhizogenes, causal agents of the crown gall and the hairy root disease respectively, have frequently been used for plant transformation. The virulent strains of A. tumefaciens or A. rhizogenes harbor a large tumor-inducing (Ti) or root-inducing (Ri) plasmid, respectively, responsible for the disease in host plants. We have used the hairy root production approach to analyze several nematode - inducible promoters in several plant species (Karimi et al., 1998; Karimi et al., 1999). Many independent A. rhizogenes-transformed hairy roots can be directly used for analyzes of gene expression in the roots without the need to go over the time-consuming regeneration procedure. Here, we present a method to obtain, propagate and analyze hairy roots from several plant species. CONCLUSION In conclusion We found that in comparison with the production of transgenic plants, the generation of hairy roots has the advantage that more independent transgenic line can be produced in a shorter period of time. This method is suited for promoter analysis in different plant species. Materials and Methods Baterial strains and plasmids The A. rhizogenes Ri15834 Rif R (Lippincott and Lippincott, 1969) was used for transformation of all plant species. All binary vectors were introduced into pRi15834 by three-parental mating. Fig.3 pW2GUSBin19 - tobacco Fig.1 pW2GUSBin19 - tomato Fig.2 p35S800EGFPERBinplus – arabidopsis Fig.4 p35SGiBin19 - tomato Fig.5 pcdc2aEGFPERBinplus– arabidopsis Fig.6 pW290EGFPERBinplus – arabidopsis Fig.7 pW2GUSBin19 - radish Tomato (Lycopersiconesculentum) plants were transformed by p35SGiBin19 (Karimi et al., 2000), which contains CaMV 35S promoter-gusintron fusion. The pcdc2aEGFPERBinplus, cdc2a promoter-gfp fusion , pW290EGFPERBinplus, the downstream region of the WRKYa promoter linked to the minimal 35S promoter fused to gfp and p35S800EGFPERBinplus, the CaMV 35S promoter-gfp fusion constructs were used for transformation of A. thaliana. References Dellaporta, S.L., J. Wood, J.B. Hicks. 1983. A plant DNA minipreparation: version II. Plant Mol. Biol. Rep. 1: 19-21. Jefferson, R.A., T.A. Kavanagh, M.V. Bevan. 1987. GUS fusions: -glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6: 3901-3907. Karimi, M., Van Poucke, K., Barthels, N., Van Montagu, M. and Gheysen, G.(1998) Introduction of nematode-inducible plant promoter-gus fusions in hairy roots of different plants. Med. Fac. Landbouww. Univ. Gent 63/3a, 609‑617. Karimi, M., Van Montagu, M. and Gheysen, G. (1999) Hairy root production in Arabidopsis thaliana: cotransformation with a promoter‑trap vector results in complex T‑DNA integration patterns. Plant Cell Rep., 19: 133-142. Karimi, M., M. Van Montagu and G. Gheysen. 2000. Nematodes as vectors to introduce Agrobacterium into plant roots. Mol. Plant Pathol. 1: 383‑387. Lippincott, B.B. and J.A. Lippincott. 1969. Bacterial attachment to a specific wound site as an essential stage in tumor initiation by Agrobacterium tumefaciens. J. Bacteriol. 97: 620-628. transformed hairy roots started to emerge from small calli on the cut sites of the leaves (Fig. c) and 2 weeks later they were big enough for subculturing (Fig. d). One-cm-long hairy roots were cut and were placed on hairy root elongation medium (H-EM) containing 300 mg/l carbenicillin. Transformants were subcultured every 2-3 weeks on H-EM medium without antibiotics. d containing 300 mg/l carbenicillin. After transfer to sterile filter paper, the explants were placed on solidified SM medium containing 300 mg/l carbenicillin and 50 mg/l kanamycin. After 2 weeks incubation on SM medium, HindIII SphI PstI SalI XbaI BamHI SmaI KpnI SacI EcoRI lacZ LB RB SpeI XhoI HincII NPTII Tnos Pnos pCDC2a EGFPER 35S-3’ pcdc2aEGFPERBinPlus2 p35SGiBin19 RB LB EcoRI HindIII 35S gusintron NOS-ter NPTII Tnos Pnos