247 N Save Nature to Survive 7(2) : 247-250, 2012 www.thebioscan.in ZEATIN INDUCED DIRECT IN VITRO SHOOT REGENERATION IN TOMATO (SOLANUM LYCOPERSICUM L.) B. D. PAWAR*, A. S. JADHAV, A. A. KALE, V. P. CHIMOTE AND S. V. PAWAR State Level Biotechnology Centre, Mahatma Phule Krishi Vidyapeeth, Rahuri - 413 722, Maharashtra, INDIA E-mail: bhau.raje@gmail.com INTRODUCTION Tomato ( Solanum lycopersicum L. formerly known as Lycopersicon esculentum Mill) (2n =2x =24) is commercially important vegetable crop throughout the world. It is grown under a wide range of climates (temperate or tropical) in the open field or under protected cultivation. Tomatoes are very valuable for human health since they are low in fat and calories, free of cholesterol and rich in vitamins A and C, b-carotene, lycopene and potassium, as well as octadecadienoic acid (Kim et al., 2011). There is a great potential for genetic manipulation in tomato to enhance productivity through increasing pest and disease resistance, environmental stress tolerance and to study gene function and regulation. Establishment of an efficient in vitro regeneration protocol is an essential prerequisite for harnessing the advantage of cell and tissue culture for genetic improvement. Till date several methods for in vitro regeneration of tomato have been described and cotyledons are the choice explant as they are quickly established and possess a high morphogenetic potential (Kaur and Bansal, 2010). However limitations with tomato regeneration system includes, morphogenetic response is highly growth regulator-dependent (Bhatia et al., 2004), genotype specific response (Park et al., 2003) and in most cases regeneration of shoots has been obtained through callus (Singh et al., 2010; Kaur and Bansal, 2010). Since tomato regeneration is genotype and procedure dependent, standardization of regeneration protocol for desired genotype is very important for genetic transformation. Therefore the aim of the present study was to simplify tomato regeneration procedure by simplifying medium conditions, avoiding callusing phase, eliminating frequent medium changes and systematically applying these conditions to achieve high regeneration efficiency. MATERIALS AND METHODS The seeds of Tomato ( Solanum lycopersicum L.) variety “Dhanshree” were obtained from All India Coordinated To- mato Improvement Project, Mahatma Phule Krishi Vidyapeeth, Rahuri. These seeds were surface sterilized with 70% (v/v) ethanol for 1 min followed by 4% (w/v) sodium hypochlorite (NaClO) or 0.1% (w/v) mercuric chloride (HgCl 2 ) solution for 5/10 min. They were further rinsed five times with sterile dis- tilled water and then inoculated on a Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) supplemented with 3% (w/v) sucrose and 0.8% (w/v) agar; and incubated at 25°C temperature with 16h light period and 8h dark period. Cotyledons and hypocotyls explants were obtained from 7- 14 days old seedlings and leaves from 4-week old seedlings. Cotyledons, hypocotyls and leaves were aseptically excised from the in vitro grown seedling cultured on MS medium fortified with different levels of BAP, zeatin, IAA and naphtha- lene acetic acid (NAA). Those shoots greater than 2cm in length were excised and cultured on MS medium without any growth regulator or with either of 1mg/L of IAA, NAA and indole bu- tyric acid (IBA). Plantlets with well developed roots were trans- ferred to plastic pots containing coco peat: perlite (2:1). Those were initially covered with plastic bags for 5–7 days, and kept ABSTRACT The present investigation was undertaken to develop a highly efficient direct in vitro regeneration system, as an essential pre-requisite for the genetic transformation in tomato. Comparative studies on in vitro regeneration from cotyledon, hypocotyls and leaf explants of tomato revealed considerable variability in response to plant growth regulators. Direct shoot initiation was observed in medium supplemented with zeatin, while indirect shoot initiation with intermediate callusing phase, was observed in medium supplemented with benzylaminopurine (BAP) as cytokinin source. The MS medium supplemented with 2.0 mg/L zeatin + 0.2 mg/L indole acetic acid (IAA) was found to be the best regeneration medium with regards to regeneration efficiency, days to shoot initiation and number of shoots per explant. Among explants used cotyledons showed highest shoot regeneration efficiency (91.11%) followed by hypocotyls (87.77%) and leaf explant (85.55%). Earliest shoot initiation was observed in cotyledon explants (8.2 days), while highest numbers of shoots per explant (7.2) were obtained with hypocotyl explants. In vitro rooting initiation was observed within 14 days on MS medium devoid of any growth regulator. The in vitro rooted plantlets were successfully established in polycarbonated polyhouse with 86% survival rate. KEY WORDS Solanum lycopersicum L. Direct shoot induction Zeatin Received on : 15. 03.12 Accepted on : 07. 05.12 *Corresponding author