Plant Cell Reports (1988) 7:100-103 Plant Cell Reports © Springer-Verlag1988 Celery transformation by A grobacterium tumefaciens: cytological and genetic analysis of transgenic plants D. Catlin 1, O. Ochoa 1, S. McCormick 2, and C.F. Quiros 1 1 Department of Vegetable Crops, University of California, Davis, CA 95616, USA 2 Plant Gene Expression Center, USDA-ARS, Albany, CA 94710, USA Received October 19, 1987 / Revised version received December 22, 1987 - Communicated by L. K. Grill ABBOT Transgenic celery plants were obtained followlng co-cultivation of petiole explants with ~grobacterlum tumefaciens containing pMON200, a cointegrate vector carrying genes for kanamycln resistance and nopaline synthase. Transformants were selected by ability of callus to grow in the presence of 50mg/l kanamycin. Transformation was confirmed either by the presence of nopaline or by Southern blots. Cytological analysis of 14 transformed plants revealed chromosomal aberrations, both In structure and number. Only 20% of the regenerated plants had the normal karyotype. Kanamycln resistance behaved as a monogenlc, dominant trait, segregating in a 3:1 ratio in three families derived from plants with normal karyotypes. /~IBREVIATIONS KB: Kilobases, 2-4D: 2,4-dlphenoxyacetic acid I NTRODL~TION Genetlc transformation by Agrobacterlum is becoming a routine technique in plants (Fraley et al, 1986). The development of a crop specific protocol requires the determination of the best explant source, optimal conditions for co-cultivatlon and regeneration and a usable selectable marker. Celery, Aplum 9raveolens L., Is a good candidate for transformetlon because of its abillty to regenerate In culture from tissue explants. In thls paper, we report successful transformation by co-culIivation of celery petiole sections wlth Agrobacterium tumefaclens carrying the Monsanto pMON200 vector. The resulting plants were analyzed cytologically and genetically In order to determine the inheritance of kanamycln resistance. NATERIALS AND NETHODS PLANT NATERI AL Celery seedlings of an annual strain from Thailand (P1257228) were grown in the greenhouse under optimal conditions. This strain does not require vernalization for flowering and therefore has a shorter llfe cycle than commercial celery varieties, which are biennial. Thls annual strain can be crossed with the commercial varieties and its use accelerates genetics analysis. Explant Source and Kanamycin Sensitivity We tested two explant sources, leaf and petiole. Leaf discs from the second or third true leaves (5 mm In diameter) or young petiole explants (cross sections 2-3mm thick) were surface sterilized (70% ethanol for I minute, 10% Chlorox for 5 minutes), and rinsed three times In sterile water. For the kanamyein sensitivity tests, explants were cultured on medium (medium K) composed of MS salts (Murashlge and Skoog, 1962), B5 vitamins (Gamborg et al, 1968), 3% sucrose, 100 mg/l serlne, 0.8% agar, 0.5 mg/l 2,4-D and 0.6 mg/l klnetin (Williams and Collin, 1976). Kanamycin sensitivity of these explants was determined by supplementing the medium with kanamycTn at concentrations of 0,25,50,75,100,200,300,400 and 500 mg/l. The cultures were maintained at 27°C in the dark. The sensitivity of celery seed germination to kanamycin was determined in a replicated trial on the same medium wlth the same range of kanamycln concentrations. Transformation procedure Petiole explants were co-cultivated with Agrobacterlum tumefaclens carrying the kanamycln resistance vector pMON200, following the techniques of Horsch et al (1985) and McCormick et al (1986). Co-cultivation with the pMON120 vector (lacking the kanamycin resistance marker), and unlnoculated celery explants were used as controls. Explants were dipped In a diluted (1:30 In sterile water) overnight culture of Aarobacterium for two minutes, blotted dry and transferred to callusing medium (medium C, containing either 50 or 100 mg/l kanamycin and 500 mg/i oarbeniclllin). Because Horsch et al (1985) and McCormick et al (1986) stated that feeder cell layers are beneficial for transformation, we tested four feeder protocols (Table I) during the two day co-cultivation period. Explants were transferred to new selection media every four weeks. The plates were cultured in the dark at 27°C. As calll developed, I-2 mm portions were transferred to shoot/root regeneration medium (R) and cultured under 16 hr light at 27°C. Medium R Is the same as medium C, but with 0.04 mg/l kinetin. Regenerated plants were transferred to GA7 containers (Magenta Corp., Chicago, IL, USA) containing R medium for further growth, transferred to vermiculite and then to the greenhouse. Transformation was Indicated by continued growth in the presence of kanamycln and by the presence of nopallne In callI and in the leaves of regenerated plants. Offprint requests to: C. F. Quiros