In vitro mass multiplication with genetic clonality in elephant garlic (Allium ampeloprasum L.) S GANTAIT 1 , N. MANDAL 1 , S. BHATTACHARYYA 2 AND P. K. DAS 2 1 Department of Biotechnology, Instrumentation and Environmental Science, 2 Department of Genetics, Bidhan Chandra Krishi Viswavidyalaya., Mohanpu-741235r, West Bengal, India ABSTRACT A novel protocol was developed for Allium ampeloprasum L. to enhance in vitro cloning through multiple shoot induction. First bud induction was recorded from shoot tip within 6 days in MS with 0.25 mgl -1 NAA and 2 mgl -1 Kn. A maximum of 3 buds from a single explant appeared within the 15 days after the first bud induction. MS with 2.5 mgl -1 Kn plus 60 mgl -1 of adenine sulphate proved best for multiple shoot proliferation resulting in 6 shoots per inoculated shoot bud within next 30 days. Maximum 5 roots per shoot were recorded when cultured on MS with 0.5 mgl -1 IAA for 20 days. The combination of soil, sand and vermicompost with intermittent water spraying proved to be the best for hardening of micropropagated plantlets ensuring 90% success in next 25 days. Selected ISSR primers were used to ensure genetic clonality for the in vitro generated propagules. Key words: Acclimatization, Allium, in vitro cloning, ISSR, multiple shoot Allium ampeloprasum L, commonly known as elephant garlic, is an important family member of Alliaceae. It is more closely related to the leek than to ordinary garlic. A single clove of elephant garlic can be as large as a whole bulb of ordinary garlic. It is more perishable than ordinary garlic so it is difficult to keep as long. Elephant garlic is considered to be nature's very own antibiotic. It has also been used for lowering cholesterol, reducing high blood pressure, and treating respiratory problems such as bronchitis and asthma. Conventional propagation of elephant garlic by planting of cloves, used for both culinary purposes and propagation is slow, labour-intensive, time-consuming and non-economical like anyother Allium sp. (Robledo-Paz et al., 2000). To overcome this drawback in vitro mass multiplication was fruitfully attempted in several Allium spp. using root tip (Haque et al., 1997), shoot tip (Myers and Simon, 1998), seed (Wawrosch et al., 2001), mature clove (Roksana et al., 2002) and stem dome (Kamstalityte and Stanys, 2004) explants. The present study aims at to develop a novel protocol of in vitro cloning and establishment of an effective ex vitro acclimatization process; to enhance the multiplication rate through induction of multiple shoots; to raise and maintain a sustainable pool of propagules for further multiplication and constant supply and to ensure the genetic clonality of the regenerated propagules through DNA fingerprinting. MATERIALS AND METHODS Thirty days-old shoot tips of elephant garlic were collected from healthy disease free plants. Collected explants were surface sterilized with10% (v/v) NaOCl for 5 min, washed with sterile water and treated again with 0.1% (w/v) HgCl 2 for 3 min. The explants were then thoroughly washed 4-5 times in sterile double distilled sterile water, and finally trimmed to 2 cm. The whole process was performed under strict aseptic conditions. To induce bud break from shoot tips the explants were inoculated in MS basal medium (Murashige and Skoog, 1962) plus 3% (w/v) sucrose supplemented with different levels of α- naphthalene acetic acid (NAA) and Kinetin (Kn). The MS basal media (consisted of salts, vitamins and 3% sucrose) was solidified with 0.7% (w/v) agar. The growth regulators NAA, Kn, IAA and IBA were added in the medium before the pH adjustment to 5.7 (with 0.5 N NaOH) and autoclaving at 1.06 kg. cm -2 ; 121°C for 15 min. MS basal salts, sucrose, agar, vitamins and growth regulators were obtained from SRL, India. Incubations of in vitro cultures were done at 25± 2°C temperature with 60% RH under a 16 hr photoperiod (using white fluorescent tubes) and 3000 lux light intensity. Excised explants were inserted in the media dipping approximately up to 0.5 cm for good contact and anchor. The induced buds were separated and cultured on MS with different concentrations of Kn plus adenine sulphate for multiple shoot proliferation where MS devoid of growth regulator act as a control. During shoot multiplication and proliferation the cultures were incubated for 30 days. The best resulting media formulation was identified in respect of response to multiple shoot formation, number and length of shoots. For root induction and elongation MS basal medium was supplemented with different concentrations of indole acetic acid (IAA) or indole butyric acid (IBA). Multiple shoots were separated and then transferred individually to the rooting media. The efficiency of IAA and IBA were tested in respect of response of shoots to root induction, days to root induction, number and length of roots which ultimately determined the most successful media E.mail : saikatgantait@yahoo.com Journal of Crop and Weed, 5(1):92-96 (2009)