HORMONAL REQUIREMENTS OF EXCISED DIANTHUS CARYOPHYLLUS L. SHOOT APICAL MERISTEM IN VITRO

Whereas a medium containing kinetin alone enabled a few Dianthus caryophyllus L. apical meristem dome explants to develop into rooted plants, the highest frequency of plants was obtained in one containing supplements of both IAA and kinetin. In an unsupplemented medium, continued development required that explants have 2 pairs of primordial and a pair of expanding leaves. Kinetin alone caused production of many new leaves, but the development was significantly less than when it was furnished in combination with IAA. IAA given alone caused meristem explants to develop primarily callus, roots, and a few leaves. Gibberellin and abscisic acid were without promotive effects on leaf and shoot formation. A balance of hormonal substances, synthesized in young leaf structures and relocated to the meristem, is proposed as the fundamental mechanism that regulates new leaf initiation in the shoot apex. INVESTIGATIONS by Ball (1946, 1960) suggested that perpetuation of growth and organogenesis in excised angiosperm shoot apical meristems required subjacent stem and leaf primordia. More recently, Smith and Murashige (1970) were able to show that the subjacent structures were unnecessary. Starting with excised meristem domes less than 0.1 mm tall they were able to obtain complete plants of several herbaceous angiosperms, by using a simple nutrient medium that contained IAA (indole-3-acetic acid) as the only hormonal addendum. Detailed study (Smith, 1970) with Coleus blumei confirmed Ball's observations that no exogenous growth substances were required, if the explant consisted of the apical meristem together with 2 or more pairs of primordial leaves. However, provision of IAA was critical when leaf primordia were absent from the meristem explant. The significance of other hormonal substances remained unestablished. Smith (1970) observed that low concentrations of kinetin increased the frequency of surviving explants, but did not enable development of complete plants. Exogenous gibberellin (GA3) was observed to repress new leaf and root initiation and adenine appeared to reverse the repression slightly. In this investigation the roles of auxin, cytoI Received for publication 14 July 1976; revision accepted 26 October 1976. 2This paper represents a portion of a dissertation submitted in partial fulfillment of the requirements for a Ph.D. degree in Botany at the University of California, Riverside. The research was supported by the Elvenia J. Slosson Fellowship in Ornamental Horticulture awarded to T. M. The authors wish to thank Yanai Floral, Gardena, California, for the carnation cuttings, H. Quick for the photographs, and S. Ham-man and S. Kearns Sharp for typing the manuscript. kinin and gibberellin, as well as of abscisic acid, in morphogenesis of the shoot apex of the angiosperm, Dianthus caryophyllus L., has been examined more thoroughly. Furthermore, the findings have been applied towards elaboration of certain concepts associated with leaf initiation in the shoot apex. The carnation was employed because of its large and relatively easily isolatable meristem dome. MATERIALS AND METHODS-Terminal cuttings, 15-20 cm long, were obtained from greenhousegrown plants. All large leaves and lateral shoots were removed until there remained only the terminal meristem subtended by leaf primordia and 1-2 pairs of small leaves. The stem was shortened to 2 cm. The prepared cuttings in groups of 10 were wrapped in 10-cm squares of cheesecloth and transferred to 25 x 150 mm test tubes. A solution of 0.5 % sodium hypochlorite (commercial bleach diluted 10-fold and containing 2 drops Tween 20 emulsifer/100 ml) was added to the tubes to cover their contents. After 5 min the disinfestant solution was decanted and the contents of the tube were rinsed 3 times with autoclaved distilled water. Excision of the shoot apex tissues was performed in a horizontal laminar air flow hood. A Wild M5 dissecting microscope, with magnifications of 12.5-25 x, was used during the surgical manipulations. The excision was performed in a glass petri dish, lined with sterile, moist filter paper to retard dessication of the small explant. Severing of the meristem dome was accomplished with microscalpels made from razor blade slivers mounted on a Beaver chuck handle. The explant was lifted on the tip of the knife and transferred to a nutrient tube. The carnation apical meristem dome measured about 0.1 mm in height and 0.2 mm wide at the base.