TISSUE CULTURE OF CALLUS FROM SEEDLING AND ADULT STAGES OF HEDERA HELIX

Callus from stem pieces of the juvenile (seedling) and adult stages of Hedera helix L. were grown for up to 17 passages on a medium containing mineral salts, vitamins, sucrose, and coconut water and for fewer passages on media which lacked coconut water. Seedling callus differed in its growth in culture from adult. It was less stable than the adult; i.e., it developed vigorous variants earlier and more freely than the adult callus. The tendency of seedling callus toadapt to unfavorable media was greater. On low salt media root formation by callus was infrequent. Adult calluses formed roots more freely on high salt media than seedling calluses did. IN THEIR normal ontogeny from seed to flower many kinds of plants, both herbaceous and woody, evidence more or less well defined juvenile and adult stages which are distinguished by differences in morphology and physiology. The morphology and physiology which characterize the juvenile and adult stages originate in the apical meristem. Its activity changes or is changed with the age of the plant. What causes the change in the activity of the apical meristem? The explanations which have been offered fall into two more or less distinct categories. O(ne assumes that the kind of structures produced by the meristem is determined by the materials which come to it from the balance of the plant or by the eff ect of changes in the external environment; the meristem itself does not change during the life of the plant; it does not age. Another hypothesis assumes that the meristem ages; it changes in character during the life of the plant. There are young meristems and adult or old meristems. Although the activity of each is affected by the internal and external environment, the response differs because the juvenile meristem differs in character from the adult meristem (Brink, 1962; Robbins, 1964). Stoutemyer and Britt (1963, 1965) reported that calluses from pieces of stem (explants) of seedling Hedera helix grew in tissue culture more rapidly than those from stems of adult Hedera. Calluses from reversion stages were intermediate in vigor. These reports suggested that meristems of adult and juvenile stages of H. helix are different. We realize that the juvenile and adult stages arise from the apical meristem, not from the callus, although the tissues from which the callus is derived originate in the last I Received for publication 15 August 1969. This investigation was supported in part by a grant from The Conservation and Research Foundation and grant GB-3007 from The National Science Foundation. analysis from the apical meristem. We recognize also that a meristem in culture may not act as it does in situ. Nevertheless, differences in the development in tissue cultures of callus from adult stages would support the second of the hypotheses summarized above. MATERIALS AND METHODS-We have compared the growth of callus of stem explants from four shrubby plants of H. helix grown in the greenhouses of The Rockefeller University, and of callus from explants of seedlings derived from each of the four plants. The plants were labeled for convenience A, B, C, and D. Seeds from the four adult plants were planted 6 January 1965. Explants were made from adult plant C and its seedlings 26 August 1966. Callus from the explants was removed when explants were 35 days old. Explants of seedlings and of adult plants, A, B, and D were made 20 October 1966. Callus was removed when the explants were 36 days old. Explants were taken from more than one branch of an adult plant and from more than one seedling of each plant. Each of the adult plants had produced reversion shoots and some branches had characters intermediate between adult and juvenile, for example, adult foliage and phyllotaxy but aerial roots. Care was taken to select branches which showed no juvenile characters for explants from the shrubby plants. Stem pieces were sterilized by dipping in 95 % ethanol, washing in sterile water, treating with 1 % Clorox for 5 or 10 min, washing in sterile water, and flaming after dipping in 95 % ethanol. Explants were about 5 mm in length. Pieces with nodes were usually discarded. The explants were placed on the (B-) medium (see below) to which 100 mg/liter of casein hydrolysate (Edamin, Sheffield) was added. This medium (eB -) permitted the detection of contaminants more