Induction of Tumors in Anthurium andraeanum by Agrobactemium tumefaciens

A method was devised for infecting Anthurium andraeanum Linden ex André, an economically important ornamental monocot, with Agrobacterium tumefaciens. Tumors were obtained on plant stems 7 to 10 weeks after inoculation with oncogenic A. tumefaciens strains C58 and A281 cultured previously in an induction medium containing 200 μM acetosyringone at pH 5.5. A higher percentage of tumors were formed in vitro on etiolated internodes (32%) than on green leaf (2%) or petiole explants (3%) 4 weeks after inoculation with induced C58. All explants treated with nontumorigenic A. radiobacter or with induction medium alone failed to produce tumors. Chromatograms showed an accumulation of nopaline in internode explant tumors induced with C58. DNA amplification and hybridization studies showed that the DNA from these tumors, but not from noninoculated anthurium tissue, contained sequences homologous to the nopaline synthase gene of A. tumefaciens T-DNA. Chemical names used 3,5dimethoxy 4-hydroxyacetophenone (acetosyringone). Table 1. Response of Anthurium andraeanum selection UH965 explants to Agrobacterium tumefaciens strain C58 induced with acetosyrincone. Improvement of anthuriums, an important flower crop in Hawaii and Holland, through hybridization and selection is a long-term effort requiring 2 to 3 years for plants to mature and produce seed. A reliable gene transfer system for anthuriums could be convenient to breeders interested in improving flower color and bacterial disease resistance. The soil bacterium Agrobacterium tumefaciens can naturally transfer a segment of plasmid DNA, T-DNA, into the genome of a wounded plant (Chilton et al., 1977) and is well established as a vector for plant genetic transformation. Expression of some wild-type T-DNA genes can cause overproduction of auxins and cytokinins in plant cells, leading to the growth of tumors (Braun, 1958; Garfinkel et al., 1981; Ooms et al., 1981). Other T-DNA genes are responsible for the synthesis of opines, such as nopaline, in infected plant cells (Bomhoff et al., 1976). The vir gene region of plasmid DNA affects T-DNA transfer and integration; while it is required for plant infection, it is not integrated into the host genome (Horsch et al., 1986). Expression of vir genes can be induced by various phenolic compounds, including acetosyringone (Bolton et al., 1986; Stachel et al., 1985). Agrobacterium-mediated transformation has been most useful with dicotyledonous plants Received for publication 20 Nov. 1990. Journal Series no. 3549 of the Hawaii Institute of Tropical Agriculture and Human Resources. Research supported in part by the State of Hawaii Governor’s Agricultural Coordinating Committee and the Hawaiian Anthurium Industry Association. We thank A. Alvarez for her assistance. The coat of publishing this paper was defrayed in part by the payment of page charges. Under postal regulations, this paper therefore must be hereby marked advertisement solely to indicate this fact. lAssistant Horticulturist. 2Research Associate. HORTSCIENCE , VOL. 26(10), OCTOBER 1991 that, upon wounding, can induce expression of the vir genes. Recent studies have supported infection by Agrobacterium of monocots found in the Liliaceae, Iridaceae, Gramineae, and Dioscoreaceae (Bytebier et al., 1987; Graves and Goldman, 1986, 1987; Grimsley et al., 1987; Hemalsteens et al., 1984; Raineri et al., 1990; Schafer et al., 1987; Woolston et al., 1988). Of the 13 genera examined within the monocotyledonous family Araceae, only CalIa and Philodendron were reported to be susceptible to infection by A. tumefaciens strain B6 (de Cleene, 1985); however, anthurium was not susceptible when inoculated with uninduced A. tumefaciens strains C58, 920, A4, CG935, 937, K27, CG108, and 49 (Kuehnle, unpublished data). In this study, we examined the susceptibility of Anthurium andraeanum plants and explants to infection by induced oncogenic strains of Agrobacterium tumefaciens. Agrobacterium biovar 1 strains, from the collections of L. Moore (Oregon State Univ., zInduction medium containing 200 μM acetosyringone. Corvallis) and A. Alvarez (Univ. of Hawaii, Honolulu), were grown for 24 to 36 h at 50 rpm, 28C, in liquid YEP medium consisting of 1% Bactopeptone, 1% Bacto-yeast extract, and 0.5% NaCl (An et al., 1988) with 200 μM acetosyringone (Aldrich Chemical, Milwaukee) at pH 5.5 (YEP induction medium). Two common tumor-inducing strains with a wide host range were tested: C58 harboring the nopaline plasmid pTi-C58 and A281 combining the chromosomal background of C58 with the succinamopine plasmid pTi-Bo542. To ensure that the C58 and A281 cultures were virulent at the time of experimentation, inoculations were performed on young plants of Turkish tobacco (Nicotiana tabacum L.) as positive controls. Anthurium seedlings from four genetically diverse crosses, referred to by cross numbers 926,932,935, and 976 hereafter, were grown in flats in a glasshouse with 80% shade and 29C day/23C night with a natural photoperiod. Stems of 8to 12-month-old juvenile plants of all four crosses were punctured with a sterile dissecting needle dipped in heavy aqueous suspensions of strain C58. Other seedlings of crosses 926 and 976 were treated with strain A281. Stems wounded with a dissecting needle dipped in YEP induction medium served as controls. Wound sites were covered with petroleum jelly to maintain a humid environment favorable for bacterial proliferation and infection. The tumorigenic response was evaluated after 7 to 10 weeks by use of a 0 to 5 scale, where 0 = a smooth swelling of <1 mm in diameter at the wound site and ratings of 1 to 5 = rough, raised growths 1 to 5 mm in diameter, respectively. Sample sizes were n = 13 controls and 14 inoculations for 935 and 932; n = 9 controls and 10 inoculations each for C58and A281-exposed 926; and n = 27 controls Fig. 1. Mean response rating of seedlings from four anthurium crosses 7 to 10 weeks after inoculation with Agrobacterium tumefaciens strain C58 induced with acetosyringone. Controls were treated with YEP induction medium lacking bacteria. Ratings reflect tumor size and appearance, with large raised tumors receiving higher ratings than small, smooth tumors. Means were compared using the Mann-Whitney test; (NS) nonsignificant, (**) significant at P < 0.001.