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When the embryo of Sesamum indicum, peeled out of seed coat and deprived of one of the cotyledons, is treated with Amo-1618 at the concentration of 200 ppm for 48 hours, the first leaf primordia develop to a double leaf on the decotylated side of the shoot apex. The formation of double leaf is restricted to the case in which one cotyledon is removed and the treatment with Amo-1618 is made within 6 hours after sowing. 2, 4-D induces gamophylls, which partly resemble the double leaf, when it is applied to the embryo deprived of one cotyledon. It also induces gamophylls in the presence of both cotyledons and even when applied to the embryos grown for 24 hours after sowing. Histological observations on the shoot apices of the treated embryos suggest that Amo-1618 induces double leaf by causing structural changes in the apical meristem and leaf primordia, whereas 2, 4-D induces gamophyll by stimulating the interprimordial region in the peripheral zone of the shoot apex. All other chemicals examined, CCC, IAA, NAA, GA, and MH, have no effect of producing double leaf or gamophyll when used in the same manner as Amo-1618 or 2,4-D was. Since Mitchell et al.'' investigated the plant growth reducing properties of some nicotinium compounds, various chemicals having such properties have been listed up as growth retardants, and a vast amount of investigations on the physiology of those substances have been accumulated (cf. review by Cathey, 19642'). In general, growth retardants reduce stem elongation but never cause malformation or other formative changes. A growth retardant Amo-1618 was reported to inhibit particularly cell expansion and division of the subapical meristem without inhibiting leaf initiation in Chrysanthemum morif olium3-5'. It was also shown that Amo-1618 was highly effective to sesame plant in reducing stem growth. When it was applied to sesame seedlings through soil treatment, it induced in them dwarf characters such as short internode, thick stem and dark-green leaves6'. Recently, Courduroux7' reported that apical as well as subapical cells of the buds of Helianthus tuberosus cultured for long period on a medium containing Amo-1618 were affected and the buds lost the ability to sprout and continued to form tubers. * Partly supported by the Grant in Aid for Scientific Researches from the Ministry of Education, No. 4007. ** Department of Biology, Faculty of Science, Tokyo Metropolitan University, Setagaya-ku, Tokyo. 546 Bot. Mag. Tokyo Vol. 81 Some authors have shown that 2, 4-D induced gamophyllous cohesion of leaves when it was applied to the bud primordium8'9' or shoot tip10' in several plants. It is of interest above all that Haccius11' and Haccius and Trompeter12' induced syncotyly, i. e. cohesion of cotyledons by one margin only, in Eranthis hiemalis by treating immature seeds with 2, 4-D. The induced syncotyly strikingly resembles in various respects the state of double leaf of sesame seedling, which is formed by cohesion of the first leaf primordia when the shoot apex of embryo is incised along the intercotyledonary planel3'. In the present investigation, it is attempted to get the double leaf by treating the embryo with Amo-1618 and 2, 4-D. In addition, some other chemicals including growth retardant and auxins are likewise tested. The results of application of those substances to the sesame embryos in various conditions will be presented. Material and Methods Seeds of Sesamum indicum were stripped of seed coat with a dissecting needle after being immersed into water for about 30 minutes. Embryos thus peeled out were treated with chemicals. Treatment with Amo-161.8*. The treatment with Amo-1618 was carried out immediately after the embryo was peeled out, or after varying periods from the time of sowing. In the former case, embryos were immediately deprived of one of the cotyledons (Fig. 2A), and then placed on the filter paper soaked with 5 ml of 200 ppm solution of Amo-1618 in a Petri dish. The treatment was continued for 48 hours. Embryos deprived of both cotyledons and those bearing both cotyledons were also treated in the same way. When the treatment was started at varying times after sowing, embryos were kept for varying periods after stripping of seed coat on a wet filter paper with water in a Petri dish, then they were deprived of one of the cotyledons and subjected to the treatment. Eight series of treatment were started at 3, 6, 9, 12, 15, 18, 21, and 24 hours after sowing, and continued for the subsequent 48-hour period respectively. Treatment with 2, 4-D*. 2, 4-D was applied at the concentrations of 2 and 0.5 ppm for 24 hours to the embryos at 0and 24-hour stages in the same way as Amo-1618 was done. Following the treatment, embryos were transferred on to the wet filter paper with water in Petri dishes, allowed to grow for several days, and then transplanted on vermiculite in glass pots poured with 0.1 solution of " Hyponex "**. Whole course of culture of the embryos and seedlings, including the treatment period, was carried out at 28-30' under continuous illumination at 4000 lux with day-light fluorescent tubes. Developmental changes occurring in the shoot apices of the growing embryos and seedlings were observed every day during 10 days with a binocular microscope, and some of the apices were sampled and fixed with FAA (5 ml of formalin, 5 ml of glacial acetic acid, and 90 ml of 70°o alcohol) and prepared for histological examination following the usual paraffin section procedure. * Amo-1618: 4-hydroxyl-5-isopropyl-2-methylphenyl trimethyl ammonium chloride , 1piperidine carboxylate. 2, 4-D: 2, 4-dichlorophenoxyacetic acid. ** Manufactured by Hydroponic Chemical Co ., Inc., Copley Ohio, U. S. A. November, 1968 HANAWA, J. 547 Other chemicals, CCC, NAA, IAA, GA, and MH*, were also tested as to the effect to produce double leaf. These were applied to the embryos deprived of one of the cotyledons in the same manner as that of Amo-application. Concentrations and duration of application are described in respective paragraphs later on.

Formation of Double Leaf by Amo-1618 and 2, 4-D in Sesamum indicum