IN VITRO CULTURE OF FLORAL BUDS OF AQUILEGIA

TEPFER, S. S., R. I. GREYSON, W. R. CRAIG, and J. L. HINDMAN. (U. Oregon, Eugene.) In vitro culture of floral buds of Aquilegia. Amer. Jour. Bot. 50(10):1035-1045. Illus. 1963.-Floral buds at various stages of development, from early stages before sepal initiation to late stages with young carpel primordia present, were grown in culture on various agar media. A basic medium containing White's minerals, Nitsch's trace elements, coconut milk, sucrose, and assorted water-soluble vitamins was developed for growth of the buds. The addition of indoleacetic acid, gibberellic acid, and kinetin to the basic medium extended the developmental limits of buds at nearly all stages and decidedly improved the continued development of carpels. On this medium buds produce all of their organ primordia, growing from early stages to about the size of flowers at anthesis, but will not develop unless the sepals are removed. This inhibiting effect of sepals is not understood at this time. Stamen development is consistently poor beyond the point of differentiation of anther and filament, even with the addition of hormones. The development of buds in culture is illustrated and compared with development in intact plants. With further improvement of the medium, it is hoped that these buds may be used for experiments testing theories of floral morphogenesis. THIS PAPER is our first report on studies of the development of floral buds of Aquilegia grown in sterile culture. A long-range objective of this work is to devise and to test theories of floral morphogenesis by the use of materials and methods that permit complete control of the factors that influence growth and development of floral buds, while allowing observation of the stages in development. The study was stimulated by papers by Wardlaw (1957) and Heslop-Harrison (1959) which propose and develop a theory of floral morphogenesis in which the floral meristem is interpreted as a chemical reaction system that passes through several phases, corresponding with the formation, in sequence, of the various kinds of floral-organ primordia. According to this theory, specific genes or groups of genes become active in development, or cease activity, in response to the metabolism of the floral apex at any given moment. The immediate objectives of the study are less ambitious, yet their attainment is essential before general theories of morphogenesis may be considered. First, it is necessary to be able to grow excised floral buds in culture through all of the stages of initiation and development of floral organs; normal or near-normal growth in culture must be achieved to serve as a control in experiments. Second, as an immediate offshoot of the problem of development of techniques and media I Received for publication May 20, 1963. 2 Present address: State University of New York, University of Buffalo, Buffalo, N. Y. Supported by grants from the National Science Foundation (G5951; G14497) to the senior author. Mr. Craig worked as an Undergraduate Research Participant, N. S. F. We would like to thank Mr. Arnold J. Karpoff for his assistance in the latter part of the work. for supporting normal growth, certain preliminary results are available showing the effects of growth substances on floral development. The next phase of the work, already under way, is the detailed study of the effects of varying concentrations of growth substances on floral development, in general, on possible changes in the sequence of primordia, and on sex expression. Preliminary work on the surgical alteration of buds grown in culture has also been completed, but a report on it must await further experiments, using more sophisticated equipment that has just become available. Since 1933, when White reported his early experiments on the culture of isolated stem tips, a number of reports have appeared dealing with the growth of vegetative buds and of detached foliar primordia. Steeves and Sussex (1957) and Steeves, Gabriel, and Steeves (1957) have summarized this work to that time. More recently a number of papers have appeared describing experiments in which flowering has been induced in vegetative buds grown in culture. White (1933) described in general terms the development of flowers of Stellaria, which had either been preinduced to flowering, or had flowered spontaneously in culture without deliberate control. This pioneer effort preceded by many years a number of papers on the induction of flowering in culture reviewed by Raghavan and Jacobs (1961). A pertinent paper by Baldev (1962) has appeared since the review. Until recently, we were unable to find any reports of the growth and development of excised floral buds, in contrast to buds that became floral while in culture; Galun, Jung, and Lang (1962) however, have reported success with detached floral buds of Cucumis. This paper also states