Plant Growth Regulation 32: 59–63, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands. 59 Ethylene inhibitors enhance in vitro root formation on faba bean shoots regenerated on medium containing thidiazuron Mutasim Mohamed Khalafalla* & Kazumi Hattori Laboratory of Plant Genetics and Breeding, Graduate School of Bioagricultural Science, Nagoya University, Chikusa, Nagoya 464-8601 Japan (*author for correspondence, e-mail: i982004d@mbox.media.nagoya-u.ac.jp) Received 28 December 1999; accepted 6 January 2000 Key words: ethylene, faba bean, inhibitors, precursor, thidiazuron Abstract The possible involvement of ethylene in in vitro rooting of faba bean (Vicia faba L.) shoots regenerated on medium containing thidiazuron was investigated. The effects of the ethylene precursor 1-aminocyclopropane-1- carboxylic acid (ACC) and three ethylene inhibitors, silver nitrate (AgNO 3 ), acetyl salicylic acid (ASA) and cobalt chloride (CoCl 2 ) on root formation were tested in vitro using TDZ-induced shoots of faba bean accession 760. ACC inhibited root formation. In contrast, ethylene inhibitors promoted root formation, AgNO 3 at the appropriate concentrations enhanced root emergence and increased root number per shoot, root growth rate, and root length. Both CoCl 2 and ASA at the appropriate concentrations increased rooting efficiency. These promotive effects may result from a reduction in ethylene concentration or inhibition of ethylene action. The results offer a new approach to improve the rooting efficiency of TDZ-induced shoots of faba bean and possibly of other plant species. Abbreviations: ACC – 1-Aminocyclopropane-1-carboxylicacid; MS – Murashige and Skoog; BA – benzyladenine; TDZ – thidiazuron; ASA – Acetyl salicylic acid 1. Introduction Thidiazuron [TDZ: N-phenyl-N ′ -1, 2, 3-thidiazol- 5-ylurea] is a substituted phenylurea which was developed for mechanized harvesting of cotton bolls and has now emerged as a highly efficacious bioreg- ulant of morphogenesis in the tissue cultures of many plant species [13]. In tissue culture faba beans (Vicia faba L.) appear to be a recalcitrant towards in vitro regeneration. How- ever, in recent years the inclusion of TDZ in cul- ture media has been reported to be very effective in inducing plant regeneration from protoplasts [19] and seedling explants via organogenesis [8, 11]. Unfor- tunately, an important problem encountered in these regeneration methods is the poor rooting ability of the regenerated shoots. Efficient plant regeneration procedures, employing organogenesis leading to shoot regeneration with sub- sequent adventitious rooting are prerequisites for the application of modern gene transfer technologies in plant improvement [1]. Therefore, as part of our effort to develop an efficient transformation system for faba bean, we undertook the present study to improve in vitro rooting efficiency in a protocol previously developed in our laboratory for inducing multiple shoots from faba bean on a medium supplemented with TDZ. Ethylene has repeatedly been assigned a major role in the rooting of cuttings, but the experimental results have been contradictory [6]. Mensuali-Sodi et al. [10] did not find any effect of ethylene on rooting of tomato cotyledon and lavandin cuttings, whereas Nordstorm and Eliasson [15], Coleman et al. [5] and Ma et al. [9] reported inhibition and Bollmark and Eliasson [3] and Gonzalez et al. [6] stimulation. The accumulation of ethylene, a gaseous plant growth regulator, in excessive amounts in tightly