GENETIC TRANSFORMATION OF OLIVE (Olea europaea L.) USING AGROBACTERIUM TUMEFACIENS M. Mencuccini, M. Micheli, A. Angiolillo and L. Baldoni ‘ Istituto di Ricerche sulla Olivicoltura-CNR, via Madonna Alta 128, 06128 Perugia, Italy Keywords: Callus, NPTII, rol A,B,C, cv. Dolce Agogia Abstract ‘The genetic improvement of olive varieties is significantly hampered by the extended juvenile phase, the size of the mature plants and the diffused self incompatibility. For these reasons the in vitro genetic transformation represents a very important alternative, considering the advantage to directly add specific characters in extensively tested genotypes. The Agrobacterium-mediated transformation technique has already been used successfully in some woody species like apple, walnut, grapevine and actinidia. Genetic transformation of olive somatic tissues and zygotic embryos of the cv Dolce Agogia were obtained using Agrobacterium tumefaciens cartying the plasmid pBIN19 containing rol A, B, C from Agrobacterium rhizogenes T-DNA and kanamycin resistance gene neomycin phosphotransferase II (NPT Il). The presence of the rol inserts in the genome somatic tissues was shown by their resistance to kanamycin and by the positivity to the NPT I enzyme activity assay. The Southern hybridization confirmed these results and showed the possibility to use exogenous genes to improve the olive cultivars performance. 1. Introduction To achieve in vitro olive genetic improvement is considered a valuable alternative to the conventional breeding approaches. Recently, very interesting results have been obtained by organogenesis (Mencuccini and Rugini, 1993) and somatic embryogenesis (Rugini and Caricato, 1995) from somatic tissues. The gene transfer technique by means of Agrobacteria vectors and the shoot regeneration from cultivar tissues can achieve the genetic improvement of agronomically selected genotypes which are capable of inserting genes of agronomic interest and of achieving targeted genetic improvement. The rol A.B.C genes are known to cause morphological alterations (White ef al., 1985) and particularly the rol B stimulates root morphogenesis in recalcitrant woody plants (Capone etal., 1991; Rugini et al., 1991) and this aspect represents a very important character for some of the most important olive cultivars with low rooting ability of their cuttings. The genetic transformation can be used to solve the problems related to the cultivar Propagation. The objective of the present study was to optimize the condition for the genetic transformation of olive cultivars as first step to obtain transgenic plants. 2. Material and methods 2.1. Plant material and culture media Leaf petioles of cv Dolce Agogia were collected from shoots growing in vitro on OM medium (Rugini 1984). For organogenesis and callus induction a solid regeneration medium was used, consisting of MS basal medium (Murashige and Skoog, 1962), supplemented with TDZ 20M, NAA 5 uM, 2% sucrose and 0.7% agar, pH adjusted to 5.5 before autoclaving at 121°C for 15 min. The callus proliferation medium consisted of MS basal medium supplemented with zeatin 0.5uM, NAA 27uM, 3% sucrose, 0.8% agar, pH adjusted to 5.5 after autoclaving. Regenerated shoots were grown on the OM medium: roe. 3* int. ISHS Symp. on Ove Growing 515 Eds LT. Metadakis nd 0. Voyiatzs ‘Acta Hor. 478, ISHS 1958,The concentration of kanamycin effective for selection of olive calluses and shoots was established in previous experiments (data not shown) and determined at 50 mg/ml in both cases. 2.2. Bacterial strains Rol A,B,C, (ORFs 10,11,12, fragment E15) containing T-DNA restriction fragrnents extracted from suitable pRi 1855 pBR322 clones (Pomponi ¢t al. 1983) and the NPT-IL gene for kanamycin resistance, cloned in binary vector pBIN19 (Bevan 1984) and transferred into Agrobacterium tumefaciens LBA 4404 were used to transform the olive explants. The bacteria were grown overnight at 28°C in 10 ml of YMB liquid medium containing 100 mg/ml kanamycin. The bacterial suspension was centrifuged and resuspended in 25 ml of MS basal medium before inoculation. 2.3. Transformation procedure and antibiotic selection Leaf petioles, approximately 3-4 mm long, were co-cultured for about 15 min with Agrobacterium suspension, blotted on a dry sterile filter paper and placed onto solid regeneration medium without antibiotics for three days. The experiment consisted of 400 petioles treated with bacteria (40 petri deshes x 10 explants each) and 120 petioles as untreated control (12 petri deshes x 10 explants each). To rule out the possibility of any bacterial contamination both treated explants and control were then subcultured on the same medium supplemented with 500 mg/ml carbenicillin. The obtained calluses were cultured in the callus proliferation medium supplemented with 100 mg/ml kanamycin and subcultured on the same medium. Regenerated shoots from basal petioles were cultured on the OM medium containing 50 mg/ml kanamycin. Some untreated calluses were cultured on the same medium without kanamycin and used as control. 2.4. Extraction of DNA from callus Genomic DNA was extracted from kanamycin resistant and untreated calluses using the modified method of Saghai-Maroof et al. (1984). Five-six grams of calluses were incubated with 2X CTAB buffer for Ih at 65°C. A chloroform/isoamyl extraction was repeated twice, and RNA was removed from the aqueous solution using RNase 25 mg/ml for Ih. After isopropanol/ethanol precipitations DNA was resuspended in 500 ml TE buffer. With this method about 8 mg of DNA per 1g of fresh tissue were obtained 2.5. Southern blot analysis 10 mg of DNA from both control and kanamycin resistant calluses were digested with the enzymes Eco RI, Hind Ill, Xba I and run on a 1% agarose gel. Southern hybridization was carried out according to Sambrook ef al. (1989). The E15 fragment, prepared by random priming from electroeluted insert, was used as a probe. Three separate experiments were performed. 2.6. Assay for NPT-IT The presence of NPT-II gene in the kanamycin resistant calluses was confirmed by enzyme expression assay. The Enzyme-Linked ImmunoSorbant Assay (ELISA) on both control and selected calluses was performed according to the procedure described by manufacturer of the NPT-II Elisa kit (593° Prime Inc., CO, USA). 5163, Results 3.1. Transformation with rol genes Calluses (16%) and shoot-like structures (7%) were obtained after 2-3 weeks from leaf petiole explants treated with bacteria. Most of the calluses transferred on selective medium with kanamycin turned brown and died, only few callus survived the selection treatment and formed compact white callus growing actively also on 100 mg/ml kanamycin (figure 1). Shoot-like structures did not survive in the kanamycin selection but bleached completely within 6 weeks. From the putatively transformed callus, adventitious root initials were obtained on the callus medium while the root formation was not observed in control callus. 3.2. Molecular characterization The presence of transferred T-DNA into the olive genome was demonstrated by Southern analysis (figure 2). The genomic DNA of both treated and untreated callus was digested with Eco RI which cuts outside the E15 fragment (3500 bp). Hind III and Xba I were used because the former has two cutting sites inside the E15 releasing a fragment of 1773 bp and the second linearizes the pBin19 plasmid permitting to evidence the eventual plasmidic contamination of callus DNA. The pBIN19 plasmid containing E15 fragment linearized with Xba I and the same fragment, excised from the pBINI9 after Eco RI digestion were used as homologous control. The E15 fragment was also used as a probe. Two bands were visible in the Eco RI digestion lane and one of about 1800 bp in the Hind II lane. The absence, in the Xba I digested DNA, of a band corresponding to the length of the pBIN19 + E15 may exclude the hypothesis of plasmid contamination of the callus. Multiple bands of different length were present instead. These results can be explained by the occurrance of multiple insertion of the E15 fragment into the genomic DNA. No signal was detected in DNA from untreated calli 3.3. NPT-II Assay The NPT-II enzyme expression assay, measured by ELISA kit, confirmed the presence of NPT-II enzyme in the transformed callus. Control calluses didn’t show any signal. The gene rol B cloned from Ri T-DNA of Agrobacterium rhizogenes strain 1855 is known as a root-inducing gene since it stimulates the natural induction and development of roots because possesses an auxin-like activity and it has been reported as the gene specifically involved in adventitious root formation typical of the “hairy root” pathogenesis (Cardarelli et al., 1987a). Organogenesis and somatic embryogenesis from petioles and olive calli increase the possibility to transform olive using Agrobacteria, This work represents the first result on olive transformation by means of Agrobacterium infection obtained on a somatic tissue. The antibiotics resistance, NPT-II assay, and the Southern analysis represent the first evidences of a DNA transfer into the olive genome. The possibility to produce olive transgenic plants allows to genetically improve the cultivars by introducing very important exogenous genes in their genome in a shorter time compared to traditional methods of crossbreeding. The possibility to obtain regeneration from callus will represent the real possibility to overcome every barrier to the olive genetic manipulation. S17References Bevan, M. 1984. Binary Agrobacterium vectors for plant transformation. Nucl. Ac. Res. 12: 8711-8721. Capone, L., Cardarelli, M., Mariotti, D., Pomponi, M., De Paolis, A. and Costantino, P. 1991. Different promoter regions control level and tissue specificity of expression of Agrobacterium rhizogenes rol B gene in plants. Plant Mol. Biol. 16: 427-436. Cardarelli, M., Spand, L., Mariotti, D., Mauro, M. L. and Costantino, P. 1987a. The role of auxin in hairy root induction. Mol. Gen. Genet. 208: 457-463 Mencuccini, M., Rugini, E. 1993. In vitro shoot regeneration from olive cultivars tissues. Plant Cell, Tissue and Organ Culture 32: 283-288. Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473-497. Pomponi, M., Spand, L., Sabbadini, M. G., Costantino, P. 1983, Restriction endonuclease mapping of root-inducing plasmid of Agrobacterium rhizogenes 1855. Plasmid 10: 119-129. Rugini, E. 1984. In vitro propagation of some olive (Olea europaea sativa L.) cultivars with different root-ability, and medium development using analytical data from developing shoots and embryos. Sci. Hort. 24: 123-134. Rugini, E., Caricato, G. 1995. Somatic embryogenesis and plant recovery from mature tissues of cultivars (Olea europaea L.) “Canino” and “Moraiolo”. Plant Cell Rep. 14: 257-260. Rugini, E., Pellegrineschi, A., Mencuccini, M. and Mariotti, D. 1991. Increase of rooting ability in the woody species kiwi (Actinidia deliciosa A. Chev.) by transformation with Agrobacterium rhizogenes rol genes. Plant Cell Rep. 10: 291-295. Saghai-Maroof, M. A., Soliman, K. M., Jorgensen, R. A., Allard, R. W. 1984, Ribosomal DNA spacer-length polymorphism in population dynamics. Proc. Natl. Acad. Sci. USA, 81: 8014-8018. Sambrook, J., Fritsch, E.F., Maniatis, T. 1989, Molecular cloning. A laboratory manual. Second Ed. Cold Spring Harbor Lab. Press, New York. White, F.F., Taylor, B.H., Huffman, G.A., Gordon, M.P., Nester, E.W. 1985. Molecular and genetic analysis of the transferred DNA regions of the root-inducing plasmid of Agrobacterium rhizogenes. J Bacteriol. 164: 33-44, S18Figure 1. Calluses on selective medium (100 mg/l kanamycin Abp 123456789 (23100 » pBin (rol A,B,C) 9400 6600 rol A,B,C 2645 bp 2300 2000 Tine 1(8) line 2(c) [EcoRI]; line 7 (pBint9 [Xba I]) ; Tine 3(0) line 4(c) [Hind TIT] ; fine 8 (pBin19 [EcoRI }) ; line 5(¢) line 6(c) [Xba]; line 9 (marker) ; t= transformed callus ¢= control callus Figure 2. Southern analysis of transformed and controll callus 519