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Leaf blades of Crepis capillaris were cultured for one month in the dark on Linsmaier and Skoog's basic agar medium containing indoleacetic acid (IAA) and kinetin, singly or in combination, to examine a relation of plant hormones to organ differentiation. On a basal medium, leaf blades showed no morphological changes. IAA (0. 1-10 ppm) stimulated stronger rooting with an increase of concentration. Single addition of kinetin initiated adventitious bud on leaf blades isolated from the seedlings grown on basal medium. However, coexistence of IAA and kinetin was required for bud initiation on the leaf blades severed from leaves developing on kinetin medium. The optimum concentration of kinetin was 0.5 ppm. Kinetin suppressed the rooting at a higher concentration. Organ formation has been found to occurr in cultured tissues in many plants'-" Skoog and Miller4' revealed the effects of plant hormones on the differentiation of tobacco pith callus. They showed that the ratio of kinetin to indoleacetic acid in the medium was an important factor controlling three types of differentiation, i. e., bud, callus and root formation. Regeneration of begonia leaf cuttings was found to be regulated by a hormonal level affected by physiological conditions, such as temperature, photoperiod, and so forth5-7'. Such a hormonal regulation of organn differentation has been known in many other plant species3'. Reinert and Kuster8 reported development of root, bud and green callus tissues from isolated leaves of Cre pis ca pillaris. Yazawa9' observed in the same plant species that callus tissues which originated from root and stem segments produced roots after 4 to 8 months' culture. In the present paper, organ differentiation inn isolated leaf blades of Crepis capillaris and its relation to hormonal requirement will be described. Material and Methods A strain of Crepis capillaris stocked in the National Institute of Genetics was used. The seeds were soaked in 70% ethanol for 2 min, sterilized in 1% sodium hypochlorite for 5 min and washed three repeated times with water, then sown on an agar medium in test tubes. For germination, the basal medium of Linsmaier and Skoog10 containing organic constituents was used without hormones. For differentiation experiments, IAA and kinetin were added, singly or in combination, to the basal medium (hereafter called IAA, KT or IAA-KT medium, respectively). Agar was used at 0.7% concentration, and sucrose at 3%. The medium was adjusted to pH 5.8 with NaOH ar HCl before autoclaving. All media were autoclaved twice, each for * Contribution No. 729 from the National Institute of Genetics . ** Biological Institute , Department of Liberal Arts, Shizuoka University, Ooya 836, Shizuoka 420, Japan. May, 1969 Organ Formation in Cultured Leaf Blades of Crepis capillaris 205 15 min at 2 kgw/cm2 pressure. Seedlings were grown under continuous light after germination. The leaf blades were inoculated each into a test tube (16x 110 or 16x 160 mm) and incubated for 1 month at 27° in the dark and examined for differentiation. Thereupon, the containers were transferred, and kept under continuous light in order to stimulate leaf development. Observations and Results 1. Culture of seedling organs. Various organs of young seedlings grown on basal agar medium for two weeks were cut out, inoculated on IAA (10 ppm) or KT (0.5 ppm) medium, and cultured for one month in the dark. The cultured organs were leaves, cotyledons (both about 3 mm in midrib-length), hypocotyls (2-5 mm) and roots (5-15 mm in length). On KT medium, callus tissues were induced from leaf blades, from which new buds and leaves developed after about one month (Fig. lc). However, no morphological changes were observed in roots and hypocotyls. Cotyledons were swollen, while further differentiation did not occur. No root formation was observed in all organs examined on KT medium. On IAA-KT medium, both cotyledons and leaf blades were swollen and became callus tissues, from which new buds and roots developed (Fig. lb). New bud and leaf development was also found in callus tissues derived from hypocotyls after 2 months' culture. On IAA medium, a excellent root formation was observed in both cotyledons and leaf blades (Fig. la), but they scarcely produced new buds. Bud initiation was not found in root-derived tissues cultured on any kinds of media. Briefly saying, leaf blades easily produced new buds and leaves, especially in KT medium or IAA-KT medium. 2. Organ formation from seedling leaf blades. For further study of an effect of IAA and kinetin upon organ formation, only leaf blades of seedlings 5 weeks old were cultured on a medium containing either IAA (1 or 10 ppm) or kinetin (0.5 or 5 ppm). They were also incubated on a medium with 10 ppm IAA plus 0.5 ppm kinetin for one month in the dark. The inoculum of leaf blades were 2.5-3.0 mm in midrib-length and obovate in shape, representing a juvenile stage. The leaf blades were placed pointing the basal part down on the slant agar medium. The results are shown in Table 1. A high concentration such as 5 ppm of kinetin strongly inhibited callus formation, thus bringing about no differentiation of organs. 1 or 10 ppm IAA intensely stimulated root formation, but no bud initiation occurred. When IAA concentFig. 1. Organ formation from seedling leaf blades of Crepis capillaris. Continuous light was applied for two weeks after one month's culture in the dark. a,) 10 ppm IAA; b) 10 ppm IAA plus 0 .5 ppm kinetin ; c) 0.5 ppm kinetin, x l. 206 YONEDA, Y. Vol. 82 ration was appreciably high (10 ppm), the leaf blades became transparent as a whole within 10 to 20 days after inoculation. Over the veinlets of these leaf blades occurred many protrusions consisting of small cells seemingly meristematic. Many roots developed later seemed to have originated from some of them. The detail will be reported later. In contrast, 0.5 ppm kinetin induced the formation of callus and new leaf, but did not stimulate rooting. In this case, the callus appeared primarily on a basal cut surface of the midrib, and an initiation of adventitious bud occurred from this callus later (Fig. 2). On IAA-KT medium, the callus growth was intense, and the rooting and bud initiation occurred at a later stage. Namely, in young obovate leaf blades, callus and buds were induced by kinetin (0.5 ppm) and roots were induced by IAA (1, 10 ppm)• 3. Organ formation from secondarily developed leaf blades. As mentioned above, new buds were initiated from leaf blades of a seedling and grew quickly and abundantly on 0.5 ppm KT medium. Differentiation ability of these secondarily developed leaf blades was tested. As the leaves elongated to several centimeters, the terminal parts of the leaves, 1 cm long, were used for inoculation. Most of the leaves were serrate in the margin, which may indicate a grade of maturing. One month culture on the media containing varied concentrantions of IAA and kinetin showed the following results (Table 2). Almost no morphological change was observed in leaf blades on the basal medium and KT medium. Especially, the result that 0.5 ppm KT medium showed Table 1.