Auxin-Cytokinin Control of Secondary Vascular Tissue Formation in Isolated Roots of Raphanus

A comparative study was made of the effectiveness of various horinoine anid metabolite mixtures in inducing vascular cambium initiation and secondary vascular tissue formation in isolated first-transfer oots of the radish, Raphanus sativus L. 'White Icicle,' when provided to the cut basal end of the root grown in sterile culture. An auxin, such as indoleacetic acid (IAA) at 10-5 M, a cytokinin, such as 6-benzylamino purine at 5 X 10M, a cyclitol, such as myo-inositol at 5 X 10-1 M and sucrose at 8% were all required for maximum response. Requirements for auxin and cytokinin were absolute; in their absence no cambium was formed. The addition of cyclitol, while not an absolute requirement for cambium initiation, increased the magnitude of the response markedly. Alternative auxins such as a-naphthaleneacetic acid and 2,4-dichlorophenoxyacetic acid were equally as effective as IAA. Alternative effective cytokinins included 6-furfurylaminlopurine, 6-phenylaminopurine and 6-(y,--dimethylallylaminio)purine. Alternative cyclitols equivalent to myo-inositol were seyllitol and pinitol. Other related cyclitols tested were much less effective or totally inactive. ISOLATED EXCISED ROOTS growIn in sterile nutrient culture usually do Inot form a vascular cambium or secondary vascular tissues. Only under unusual culture conditionis (Dormer and Street, 1948; Seeliger, 1956; Torrev, 1951) are secondary vascular tissues observed. Using a technique devised by Raggio and Raggio (1956) for culturing excised roots by providing organic nutrients via medium iintroduced into the cut basal enid of the root, Torrey (1963) was able to induce vascular cambium and limited secondary vascular tissue formation in excised roots of Pisum sativum L. Loomis and Torrey (1964) demonstrated with the same techniques that subcultured isolated I Received for publication 21 November 1966. This investigation was supported in part by research grant (GM-08145) and a special fellowship (no. 1F3GM2024-01) to RSL from the National Institutes of Health, U. S. Public Health Service. The technical assistance of Sharon Hosley and Pauline Borsari is gratefully acknowledged. roots of the radish, Raphanus sativus L., could be induced to form cambium and secondary vascular tissues bv introducinig appropriate metabolites and hormone mixtures through the cut basal end. They showed that, in additon to sugar in the form of sucrose, the excised radish roots must be provided an auxiin such as indoleacetic acid and a cytokinin, such as 6-beiizylaminopurine, both at appropriate low coincenitrations, in order to induce vascular camabium iniitiation. The addition of myo-iiiositol increased the response markedly. A further study has been made to determine the optimum coniditions in vitro for cambial initiation and for extenided vascular cambium activity in the excised radish root. A broad range of concentrations of the essenitial components, alone and in combinationi, was tested on first-transfer radish roots in. culture, and the ailatomical responses were determined in fixed and sectioned material. In the studies presenited below a comparisoin is made of ainatomiical responses observed October, 1967] TORREYl AND LOOM1-VASCULAR TISSUE FORMATION 1099 at the end of a period of approximately 4 weeks of treatment of roots subjected to a wide range of experimental conditions. Conclusions concerning the optimum concentrations of the chemical constituents required for cambial initiation and maintained activity are presented. Detailed considerations of the ontogenetic sequence in cambial initiation and secondary vascular tissue formation are presented elsewhere (Torrey and Loomis, in press). MATERIALS AND METHODS Ro0ots of radish, Raphanus sativus L. 'White Icicle,' were used in the anatomical studies described here. Mlethods of culturing and treating the excised roots have already been described in detail (Loomis and Torrey, 1964) and may be summarized briefly. Ten-mm root tips from 3-day-old seedlings germinated aseptically in sterile distilled water in the dark at 23 C were excised and cultured in a modified Bonner nutrient agar medium. After 4 days, when the isolated root tips were about 50 mm in length, 15-mm first-transfer tips were excised with a sharp scalpel and transferred to fresh nutrient medium in 11-cm petri dishes. The basal 5-mm portion of each root wtas inserted into a separate agar medium contained in a 12 X 35 mm glass vial. Ussually two roots were cultured in each 11-cm dish. The roots N-ere grown in the dark at 23 C for additional periods up to about 5 weeks. After various periods of culture, whole roots were removed, fixed in formalin-acetic acid-alcohol, dehydrated through an ethyl-butyl alcohol series, embedded in Tissuemat, and sectioned at 10 p. Sections were stained for anatomical study using Heidenhaiin's iron haematoxylin and safranin. From the studies reported earlier (Loomis and Torrey, 1964), it was know-n that aniatomical differences, especially with respect to the initiation of the vascular cambium and the development of secondary vascular tissuies, were dependent upon the preseince of hormones and hormone-like materials in the vial medium. Auxins, cytokinins, and cyclitols, as well as the concentration of sugar provided, appeared especially important. A large number of different combinations of nutrients both in the vial and in the plate medium had been tested for physiological response. Macroscopic observation of roots in culture usually showed whether secondary thickening of the roots had occurred, but it was necessary to make careful anatomical investigations in order to assess the extent and nature of the response to the various treatments. In the present study anatomical analyses were made of roots taken from approximately 64 different treatments. In the results described below, emphasis has been placed on the following aspects: the effects of auxin concentration including omission, auxin type; cytokinin concentration including omission, cytokinin type; cyclitol omission and type of cyclitol. A comparative study was made of structures formed in response to optimum concentration of these factors and to their absence from the vial medium. In making such an anatomical study large numbers of roots must be sampled and some variation in response is inevitable. Some arbitrary selection must be made of roots to be studied in this way. Usually each treatment involved 8-16 roots, but fewer than half of the roots wiere fixed and even fewer were selected for sectioning. Among those sectioned were the roots showing the maximum response as measured by root diameter. Despite this selection, the number of different reatments was sufficiently great so that a wide range of responses could be studied. The anatomical responses to similar treatments were remarkably consistent. Photographs of sections are selected as typical of the particular treatment and as showing good physiological response. In most cases any of a number of roots sectioned could have been used as illustrations. In the presence of optimum concentratioins of substances stimulatory to cambial activitv, the anatomical response was dramatic and a large root diameter resulted; in the absence of any one critical component, the response was markedly reduced or absent. IPhotographic comparison among roots from various treatments was made difficult, since useful magnifications varied markedly. In the accompanying photographs some compromise has been made in choice of magnification so that, where possible, cell detail can be seen. Large-diameter oots are reproduced to show overall response rather than cell detail. Some attention must be paid by the reader to the magnification of the photographs in comparing responses. In most instanices, the thickeening was macroscopically complete wN-ithin 14-21 days, but to be sure that all effects were observed the experiments were sometimes continued for longer periods. Some of the cambial derivatives continued to differentiate after cambial activity ceased, but the anatomical patterns in young (14-21 days) and old (28-35 days) roots were generally quite similar. The principal difference was that a cambial region of small, rapidly dividing cells was not as clearly evident in the old roots. The reasons for cessation of cambial activity constitute an important problem (Loomis and Torrey, 1964) w-hich is being investigated further. RESULTS As has been described briefly by Loomis and Torrey (1964), isolated 'White Icicle' radish roots in culture show maximum secondary vascular-tissue formation under the following cultural conditions. Plate medium: M\odified Bonner medium containing 2 % sucrose and vitamins (Bonner and Devirian, 1939). 1100 AMERICAN JOURNAL OF BOTANY [Vol. 54