Auxin type and timing of application determine the activation of the developmental program during in vitro organogenesis in apple Svetla D. Yancheva a , Sara Golubowicz b , Ervin Fisher c , Simcha Lev-Yadun d , Moshe A. Flaishman b, * a Plant Biotechnology Laboratory, Agricultural University, 12 Mendeleev Street, 4000 Plovdiv, Bulgaria b Department of Fruit Trees, Institute of Horticulture, ARO, The Volcani Center, P.O. Box 6, Bet Dagan, 50250, Israel c Institute of Soil Water and Environmental Science, ARO, The Volcani Center, P.O. Box 6, Bet Dagan, 50250, Israel d Department of Biology, Faculty of Science and Science Education, University of Haifa-Oranim, Tivon 36006, Israel Received 3 December 2002; received in revised form 7 February 2003; accepted 17 February 2003 This paper is dedicated to the memory of Ervin Fisher who passed away recently Abstract The effects of auxin and cytokinin on the regulation of de novo apple (cv. Topred) shoot development have been studied through shoot induction, differentiation and development. Leaves were cultured in vitro with Thidiazuron (TDZ) in combination with various auxins. Histological observations showed that within 3 days TDZ with 2,4-dichlorophenoxyacetic acid (2,4-D) enhanced more cell divisions than TDZ with indole-3-butyric acid (IBA). One day of exposure to either TDZ /IBA or TDZ /2,4-D, followed by culture on hormone free medium, initiated cell determination towards shoot regeneration but with only low rates of shoot formation (5  /6%). Extension of the culture with TDZ /IBA to 6 days, increased shoot regeneration to approximately 30% and treatment with TDZ /IBA for the whole experimental period (35 days) increased it to 72%. However, a 2-day culture with TDZ / 2,4-D, followed by TDZ /IBA for 33 days increased regeneration to approximately 80%. TDZ with IBA or with IAA determined cell fate to shoot development, whereas TDZ /2,4-D application for more than 6 days led to the decline of shoot development. The first 6 days of the process form the ‘window’ of determination of cell fate. We conclude that in apple, the type of auxin, the timing of its application and the length of explant exposure to the specific auxin are critical for the activation and progression of the developmental program. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Apple; Auxin; IBA; 2,4-D; TDZ; Differentiation; Organogenesis 1. Introduction Organogenesis de novo in tissue culture has provided a useful system for studying development of higher plants [1]. Early observations by Skoog and Miller [2] presented the evidence that shoot organogenesis is regulated by the balance of auxin and cytokinin in the culture medium. In recent years, shoot induction, differentiation and development were intensively studied using various annual plants by genetic, molecular, biochemical and cellular approaches [3  /6]. The process of organogenesis from plant tissue cultures has three main phases: (I) induction of explant competence, (II) determination of cell fate following the exogenous application of the growth regulators, and (III) morpho- genesis [7  /9]. Using Convolvulus arvensis , it has been established that shoot meristem initiation and develop- ment follows an ordered sequence of changes in tissue organization before they emerge from the explant. Later experiments determined the sequence of stages in the initiation and developmental pattern of root and shoot organogenesis in cultured leaves of Nicotiana tabacum * Corresponding author. Tel.:  /972-3-968-3394; fax:  /972-3-966- 9583. E-mail address: vhmoshea@agri.gov.il (M.A. Flaishman). Plant Science 165 (2003) 299  /309 www.elsevier.com/locate/plantsci 0168-9452/03/$ - see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0168-9452(03)00084-0