INTERNATIONAL JOURNAL OF AGRICULTURE & BIOLOGY 1560–8530/2004/06–4–704–707 http://www.ijab.org In Vitro Regeneration and Multiple Shoot Induction in Upland Cotton (Gossypium hirsutum L.) SAEED RAUF , MUHAMMAD USMAN† 1 , B. FATIMA†, TARIQ MANZOOR KHAN AND IQRAR A. KHAN‡ Department of Plant Breeding & Genetics, University of Agriculture, Faisalabad-38040, Pakistan †Plant Tissue Culture Cell, Institute of Horticultural Sciences; University of Agriculture, Faisalabad-38040, Pakistan ‡Department of Crop Science, College of Agriculture, Sultan Qaboos University, Muscat-Oman 1 Corresponding author’s e-mail: muhammadusm@hotmail.com ABSTRACT Multiple shoot induction was studied in upland cotton. Cotyledonary nodes obtained from aseptically raised seedling were cultured on modified Murashige and Skoog media supplemented with different doses of Kinetin. Cotyledonary nodes produced maximum number of shoots (3.43 shoots/explant) when cultured on MS medium supplemented with 0.25 mgL -1 Kinetin. Highest percentage (93.3 %) of root development and root length (5.85 cm) was obtained when shoots were cultured on MS media supplemented with 0.5 mgL -1 NAA and 0.1 mgL -1 Kinetin. Key Words: Cotton; Cotyledonary nodes; Caulinar apex; Multiple shoots INTRODUCTION Cotton is an excellent source of textile fiber and is cultivated in many countries. Because of its high economic value considerable attention has been paid to improve cotton plant by conventional plant breeding methods. However genetic improvement of cotton through conventional means is limited due to many factors like absence of necessary variation, especially against the pests and diseases causing major threats to cotton plant. Plant Tissue Culture provides an alternative mean of improvement. In vitro culture can also be utilized for cotton genetic and physiological development and improvement but it requires the availability of effective regeneration system through somatic embryogenesis which is quite difficult in cotton. Cotton regeneration was first observed in Gossypium hirsutum cv. Coker 310 (Davidonis & Hamilton, 1983) since then major work has been carried out for the development of protocol for an efficient regeneration system in cotton. Several scientists have successfully produced somatic embryoids and multiple shoots using various methods and media from somatic tissues of cotton plants (Shoemaker et al. 1986; Chen et al., 1987; Trolinder & Goudin, 1987; Zhang & Wang, 1989; Voo et al., 1991; Kolganova et al., 1992). Although efficiency of cotton regeneration have been significantly improved but some difficulties still remains. Regeneration in cotton is limited to few cotton cultivars (Trolinder & Xhixian, 1989). In order to use different techniques of biotechnology, broad range of genotypes must be responsive to the regeneration. The purpose of this research was to study the effect of various concentrations of kinetin on multiple shoot induction. MATERIALS AND METHODS The present research was conducted in Plant Tissue Culture Cell, Institute of Horticultural Sciences, University of Agriculture, Faisalabad-Pakistan. Seed material. Seeds of Cotton Cultivar NIAB-999 were obtained from Nuclear Institute of Agriculture and Biology (NIAB), Faisalabad. Preparation of plant material. Seeds were delinted using H 2 SO 4 @ 15 ml per 100 g of seeds. Seeds were disinfected with 0.1% HgCl 2 for 20 minutes followed by 70% ethanol for 10 minutes. Sterilized seeds were rinsed with double distilled water for 2-3 times and cultured on MS media to raise seedlings in aseptic condition at 25 ± 2 °C for 72 h. Explants comprised. Shoot apex along with cotyledonary nodes; along with 0.5 cm hypocotyl with out cotyledons, with both cotyledons and with single cotyledon attached. Media composition. Seedlings were grown on MS basal salts (Murashige & Skoog, 1962). Explants were cultured on MS macro and micro salts, vitamins of B5 medium (Gamborg et al., 1968), glucose 30%, solidified on Phytagel 1.4 gmL -1 . The medium pH was adjusted to 5.8 before autoclaving. Effects of various growth regulators on shoot induction were observed. Explants were embedded in 250 ml glass bottles containing 50 ml MS medium supplemented with following doses of Kinetin: K1= MS media (Control); K2 = MS+0.10 mg L -l kinetin; K3 = MS+0.25 mg L -l kinetin; K4 = MS+0.50 mg L -l kinetin; K5. MS+1.00 mg L -l kinetin Rooting of shoots. Elongated shoots (4-5 cm) were excised and cultured on MS basal media with following concentrations of growth hormones.