Adventitious shoot formation from leaf explants of carnation (Dianthus caryophyllus L.) was investigated. The two leaves from one node of in vitro-grown plants showed different shoot-forming potential, depending on the order in which the leaves were removed from the stem. The leaf removed second formed more shoots and also had a large amount of adhering stem tissue. Explants with equal amounts of adhering stem tissue were obtained by making two incisions through the fused leaf bases, prior to their removal, resulting in an improved shoot formation. The procedure developed for leaf explants from in vitro-grown plants was also applied to leaf explants from greenhousegrown plants. Shoot formation from leaf explants taken from greenhouse-grown plants was further improved by cutting the leaf explant longitudinally into two parts.Abbreviations: BA -benzyladenine, NAA -a-naphthaleneacetic acid Adventitious shoot formation for carnation (Dianthus caryophyllus L.) has been obtained from petals (Kakehi 1979), axillary buds (Miller et al. 1991), stems (Nugent et at. 1991 and leaves from in vitro-grown plants (van Altvorst et at. 1992). The procedure described by van Altvorst et at. (1992) allowed production of more than 10 adventitious shoots per explant with up to 65 % of the explants showing adventitious shoot formation. This procedure was also successfully applied to a wide range of cultivars belonging to all major carnation types. Since shoot formation was restricted to the base of the leaf, the best way to isolate the leaves was to carefully tear them from the stem using forceps while keeping the leaf base intact. The leaf that was removed second gave a much higher shoot regeneration response, possibly because of the larger amount of basal leaf tissue that remained attached to the leaf explant. In this paper, we further analyze the relation between shoot regeneration response and the adhering stem tissue. The shoot regeneration responses of leaf explants from in vitro-and greenhouse-grown plants were compared. The efficiency of adventitious shoot formation was further improved by modifying the preparation of the explants.The carnation plants (Diantini type, CPRO clone 89086) were obtained as rooted cuttings from a commercial source and were grown in a greenhouse at 16/14 °C (day/night). Nodal cuttings from greenhousegrown plants were surface-disinfested and cultured in vitro as described by van Altvorst et al. (1992). Explants from greenhouse-grown cuttings and from in vitro-grown plants were taken from the three leaf pairs just below the apex. The two leaves of one pair were carefully removed from the stem using forceps in such a way that the axillary bud remained attached to the stem (Fig. la). The order in which the leaves of each pair (first and second) were removed from the stem was recorded and the explants were cultured on shoot regeneration medium. This shoot regeneration medium contained MS salts and vitamins (Murashige & Skoog 1962) supplemented with 3% (w/v) sucrose (MS-30), 4 ~tM BA, 1.6 ~tM NAA and 7 g 1-...

For the development of an Agrobacterium-mediated transformation procedure of carnation (Dianthus caryophyllus L.), an intron-containing ~-glucuronidase (gus) gene was used to monitor the frequency of transformation events soon after infection of leaf explants. The efficiency of gene transfer was dependent on the carnation genotype, explant age and cocultivation time. Leaf explants from the youngest leaves showed the highest number of GUS-positive spots. After selection on a kanamycin-containing medium, transgenie shoots were generated among a relatively high number of untransformed shoots. The selection procedure was modified in such a way that the contact between explant and medium was more intense. This improved the selection and decreased the number of escapes. Kanamycin-resistant and GUS-positive plants were obtained from five cultivars after infection of leaf explants with the supervirulent Agrobacterium strain AGLO. A higher transformation frequency was observed with the binary vector pCGN7001 than with the p35SGUSint vector. Integration of the genes into the carnation genome was demonstrated by Southern blot hybridization. The number of incorporated T-DNA insertions varied between independent transformants from one to eight. Transformants were morphologically identical to untransformed plants. Segregation of the genes occurred in a Mendelian way.

Transgenic carnation plants were obtained after infection of petal explants with the supervirulent Agrobacterium tumefaciens strain AGLO. Southern blot techniques confirmed the transgenic nature of four transformed plants. The expression of the gus gene was verified in these plants by histochemical assays on selected shoots. It was very difficult to transfer the transgenic plants to the greenhouse due to vitrification and premature flowering.

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