ADVENTIVE ORGANOGENESIS FROM SOMATIC TISSUE CULTURES OF SOLANUM CAROLINENSE: ORIGIN AND DEVELOPMENT OF REGENERATED PLANTS

The development of adventitious shoot formation from cultured somatic tissues of Solanum carolinense was studied using light microscopy. For purposes of comparison, callus initiation and proliferation were also followed. When stem segments of this plant were cultured on medium supplemented with 6 mg/l benzyladenine (BA), cell division was first observed after 48 hr in the external phloem and inner cortex of the segments. This division activity gave rise to meristematic zones which subsequently formed shoot primordia within 6 days of culture. While organogenic potential appears to be limited to specific regions, all tissues of the explant were capable of callus formation when cultured on medium containing 3 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). In conjunction with a previous study, it appears that unlike somatic embryogenesis in this species, organogenesis does not require an intermediate callus phase for differentiation to occur. SOMATIC TISSUES of Solanum carolinense L. have been shown to display a wide range of morphogenetic potentials in tissue culture including regeneration from callus via embryogenesis and organogenesis (Reynolds, 1986). Additionally, plants of this species can be regenerated directly from cultured stem segments through adventive organogenesis (Reynolds, unpublished results). This latter protocol may be especially suitable for developmental studies of adventive organogenesis since morphogenesis is rapid and does not involve the production of a bulky intermediate callus phase. This report describes the morphogenetic events which take place when stem segments of S. carolinense are subjectd to a regeneration procedure which induces adventive organogenesis. For comparative purposes, the induction of callus formation from stem segments was also followed. Knowledge of the developmental events occurring during organogenesis should help our understanding of the regeneration process and provide a baseline for future studies concerning the biochemistry of this phenomenon. MATERIALS AND METHODS-Plants of the perennial Solanum carolinense were raised from seed in a growth chamber under a photoperiod of 18 hr (120 ,uE m-2 sec-1) provided by fluorescent and incandescent lights. For tissue I Received for publication 14 June 1988; revision accepted 17 October 1988. This study was supported in part by the North Carolina Biotechnology Center, the Foundation of the University of North Carolina at Charlotte, and the State of North Carolina. culture, 1 cm stem segments from the terminal 3 internodes of 8 week old plants were surface sterilized in 20% (v/v) commercial bleach for 20 min and washed several times in sterile distilled water. Whole explants were cultured on 10 cm diam petri dishes containing agar solidified MS medium (Murashige and Skoog, 1962) supplemented with 6 mg/l benzyladenine (BA) or 3 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). Preliminary studies had shown these concentrations of BA and 2,4-D to be optimal for organogenesis or callus initiation, respectively. Cultures were maintained in a growth chamber under the same conditions as described above. For anatomical studies, cultured explants were fixed in formalin-acetic-alcohol (FAA; 50% ethanol, 5% acetic acid, 10% formaldehyde) for 24 hr, dehydrated in a graded ethanol series, and embedded in glycol methacrylate (Sorvall plastic embedding medium). Sections (2-3 ,um) were cut on a JB-4 microtome (Sorvall) using glass knives. The slides were subsequently stained with 0.05% toluidine blue and viewed using bright field microscopy. OBSERVATIONS AND DISCUSSIONOrigin of callus-At the time of culture, stem segments were 5-6 mm in diameter and well differentiated. The vascular organization was bicollateral and cell division was restricted primarily to the vascular cambium (Fig. 1). The cortex was largely parenchymatic with 3-4 layers of collenchyma making up the hypodermis. The pith displayed a weak perimedullary zone, lacked any specialized cell types, and contained little starch. Stem segments cultured on un-