Life Science Toyo University Italura-machi, Ora-gun, Gumma, Japan
Body of Abstract: In general, de novo organogenesis from plant tissues requires the addition of phytohormones such as auxin and cytokinin (CK) into the culture media. However, ipecac (Carapichea ipecacuanha (Brot.) L. Andersson), one of the medicinal plants growing in tropical rainforests, can be induced de novo shoots (adventitious shoots) easily on the internodal segment without phytohormone treatment. Understanding the molecular mechanism of adventitious shoot formation in ipecac contributes to improving the efficiency of adventitious shoot formation in other plant species. Increasing endogenous CK levels is one of the triggers to induce adventitious shoots in ipecac. In this study, we investigated in which tissues expression of CK biosynthesis genes were upregulated in the internodal segments of ipecac. First, the internodal segments were cut into two sections; tissue containing vascular bundles and non-vascular tissue. Then, they were cultured on phytohormone-free or CK-containing culture medium. In tissue containing vascular bundles, adventitious shoots were formed on epidermal cells with or without phytohormone treatment. On the other hand, no adventitious shoots were observed on non-vascular tissue cultured on phytohormone-free culture media. However, adventitious shoots were formed on the non-vascular tissue cultured on CK-containing culture media. Expression of CK biosynthesis genes, ISOPENTENYLTRANSFERASE3 (IPT3) and LONELY GUY7 (LOG7), in vascular tissue was higher than that in non-vascular tissue. We also prepared frozen sections of internodal segments and cut them into three regions, pith, cortex, and epidermis using Laser Microdissection. Then we analyzed the expression of IPT3 and LOG7 by qRT-PCR. IPT3 and LOG7 were respectively expressed in the pith and the epidermis. LOG7 expression in the epidermis was confirmed by in situ hybridization. These data indicate that CK precursors are produced in the pith, transferred to the epidermis, and then converted to active CKs in the epidermis.