EXPERIMENTAL INDUCTION OF VASCULAR TISSUES IN CALLUS OF ANGIOSPERMS

WETMORE, RALPH H. (Harvard U., Cambridge, Mass.), and JOHN P. RIER. Experimental induction of vascular tissues in callus of angiosperms. Amer. Jour. Bot. 50(5): 418-430. Illus. 1963.-Callus tissues in established maintenance culture lack morphological and physiological organization. Such callus consists of homogeneous parenchyma. Movement of auxin and sugar, therefore, must be along diffusion gradients. The only vascular tissues occurring in callus are induced. Experimental induction of vascular tissues has been successful in callus of 3 sp. of the Oleaceae: a tree, Fraxinus americana, and 2 shrubs, Syringa vulgaris and Ligustrum vulgare; another tree, Salix purpurea, var. lambertiana; a vine, Parthenocissus tricuspidata; and an herb, Helianthus tuberosus. In each of these species, an auxin (IAA or NAA in these studies) and a sugar (sucrose or glucose in these studies) prove necessary for the induction and complete differentiation of xylem and phloem in callus tissues. Varying concentrations of sugar alter the proportions of xylem to phloem: low concentrations, 1.5%-2.5%, favor xylem formation; high, 3%-4%, favor phloem. Middle concentrations, 2.5%0-3.5%, favor the presence of xylem and phloem, usually with a cambium between. The almost universal association of xylem and phloem may have its explanation in this middle concentration of sugar. Grafting of apices into callus or direct application of appropriate concentrations of an auxin and a sugar in agar to the surface of callus causes nodules of vascular tissue to be formed, mostly in a circular pattern when seen in section transverse to the axis of orientation of the callus in the medium. The diameter of this circle varies directly with the auxin conieentration at the place of application, 0.05 mg/liter giving a narrow, and 1 mg/liter, a wide pith. In individual nodules, xylem is characteristically oriented towards the center of the callus and the phloem towards the outside. Variable cross-sectional views of nodule distribution in calli under different treatments suggest experimental approaches to understanding stelar patterns. The induction and differentiation in callus of xylem and phloem tissues has no relation to conduction. Any use of vascular tissues can occur only after their induction. Anyone who has worked with vascular tissues is aware of their varied patterns in the different groups of plants, of the involved and diversified cytological details of their component cells, and also of the universality of their presence. This last characteristic serves as the basic dichotomy in the classification of plants as non-vascular or vascular. The probable and simple explanation for the complicated fabric of vascular tissues may lie in a long-evident phenomenon, namely that the component elements, xylem and phloem, are, with rare exception, closely associated, both in time of development and in position within any organ or part of the plant. However varied may be the types of elements comprising xylem and phloem, the fact of their association in time and place makes it clear that their joint induction and differentiation surely cannot depend upon satisfaction of too many variables. 1 Received for publication July 6, 1962. This project has been supported in part by the National Science Foundation (Grant G-10935 to the senior author). The senior author wishes to acknowledge the active participation in aspects of the investigations reported in this paper by a former student: Dr. William S. Kaden. He also is grateful for helpful discussion of problems raised in the investigations with Professor R. J. Gautheret of La Sorbonne, Paris, France, and Professor John G. Torrey, of Harvard University. Initial studies by Wetmore and Sorokin (1955), Torrey (1955, 1958, 1963), and Jacobs and Morrow (1957, 1958) have supported this interpretation. Jacobs and Morrow clearly established a quantitative correlation in the incidence and continued differentiation of xylem in young leaves and adjacent internodes of the stem with the concentration of auxin produced in the leaves. Sugar was definitely involved in these events. Torrey has pointed to corresponding correlations in root development though the quantitative relations were not as obvious. Wetmore and Sorokin, using callus of Syringa initially, have demonstrated that buds grafted into callus were capable of inducing xylem in the callus. Further, they showed that the buds could be replaced by auxin and sugar in agar and corresponding results could be obtained. The present paper reports on continuing studies of the differentiation of vascular tissues in callus. Investigations have been extended to include induction of phloem as well as that of xylem. Interpretation of the characteristic association of xylem and phloem in development has also become a part of the investigations. Incidental but important in itself is the possible opening to understanding of the patterns of vascular tissues invoked, patterns which are suggestive of time-honored stelar patterns. That these stelar patterns which have been