IOSR Journal of Pharmacy and Biological Sciences (IOSR-JPBS) e-ISSN:2278-3008, p-ISSN:2319-7676. Volume 11, Issue 4 Ver. III (Jul. - Aug.2016), PP 72-78 www.iosrjournals.org DOI: 10.9790/3008-1104037278 www.iosrjournals.org 72 | Page Efficient Callus Initiation and Plantlet Regeneration of Citrus japonica Margarita Mohammed Raqibul Hasan 1 , Anup Gupta 1 , Md. Nazmul Hasan 1 , Shahariar Mohammed Fahim 2 , Hanif Mohammad Rejwan 1 , Md. Abdullah Shamim 2 , Md. Abu Talha Siddique 2 , Shamsul H. Prodhan 1 1 Department of Genetic Engineering & Biotechnology, School of Life Sciences, Shahjalal University of Science & Technology, Sylhet-3114, Bangladesh. 2 Pharmacy Discipline, Khulna University, Khulna-9208, Bangladesh. Abstract :In vitro approaches has become necessary for overcoming the hurdles of cultivation of Citrus, a commercially important fruit. The present study deals with establishment of protocol for micro propagation of Citrus Japonica Margarita through callus induction and regeneration. Mature seeds of this variety of citrus are used as explant. The Explants were cultured on Murashige and Skoog medium (MS) containing 30 g/L sucrose and 7 g/L agar supplemented with different concentrations and combinations of different phyto-hormones; 6- benzylaminopurine (BA), naphthalene acetic acid (NAA), 2,4-dichloro phenoxy acetic acid (2,4 D). The maximum callus induction (88%) was observed from the mature seeds of Citrus japonica observed on MS medium supplemented with 2,4 D 16 μM. Maximum shoot regeneration response (70%) was observed on MS medium supplemented with BA 13.0 μM. Maximum root regeneration response (80%) was observed on MS medium supplemented with IBA 10 μM and when it is supplemented with NAA 5 μM it is 70%. The regenerated plantlets were successfully acclimatized in pots containing sterile soil mixture to study their response in in vivo conditions. Keywords: Citrus japonica Margarita,In vitro, Callus induction, Shoot formation, Explant, Rooting. I. Introduction Citrus is considered as the number one fruit of the world due to its high nutritional value, great production potential and preparation of large number of fruit products from them. Citrus species are cultivated in most tropical and subtropical regions of the world. The genus citrus includes more than 162 species belonging to the Order Geraniales family Rutaceae and sub family Aurantoideae. Kumquat ( Citrus japonica), Sweet orange (C. sinensis L. Osbeck), Grapefruit (C. paradisiMacf.), Pummelo (C. grandisOsbeck), Acid Lime (C. aurantifoliaChristm) and Lemon (C. limon (L) Burn F.) etc. are commercially grown in the world. Citrus fruit has been cultivated in an ever-widening area since ancient times; the best-known examples are the oranges, lemons, grapefruit, and limes. Citrus japonica Margarita, or Fortunella japonica, is a species of kumquat, also known as Nagami Kumquat have been called "the little gems of the citrus family"(Morton, J. 1987. Kumquat. p. 182–185. In: Fruits of warm climates). Kumquat (cumquat) in Cantonese Chinese, means golden (Kum) and “Quat” meaning good fortune. It is regarded as the golden orange in China. This species lives only in climates with hot summers, with temperatures ranging from 25 to 30 °C (77° to 86 °F). However, it is remarkably tolerant of frost, sometimes withstanding temperatures as low as −8 °C (18 °F). Propagation of orange trees is deceptively difficult, because hardy edible oranges are not generally grown from seed. Cultivars that produce good quality fruit are highly susceptible to root diseases. Grafted trees also begin bearing fruit many years earlier than trees reproduced by seed. Other benefits of grafting include more accurate reproduction of good fruit traits than plants derived from seed, and the opportunity to alter tree size, productivity, and other traits through rootstock influence, while maintaining identical fruit characteristics. Almost all orange trees are propagated in two stages. First, rootstock is grown from seed. When the seedling is well-established, the leafy top is cut off, and bud wood from an existing tree is grafted onto the rootstock. It is the bud wood that determines the variety of orange that is grown. Plant tissue culture is a collection of techniques used to maintain or grow plant cells, tissues or organs under sterile conditions on a nutrient culture medium of known composition. Plant tissue culture is widely used to produce clones of a plant in a method known as micropropagation. Different techniques in plant tissue culture may offer certain advantages over traditional methods of propagation, including: The production of exact copies of plants that produce particularly good flowers, fruits, or have other desirable traits. To quickly produce mature plants. The production of multiples of plants in the absence of seeds or necessary pollinators to produce seeds. The regeneration of whole plants from plant cells that have been genetically modified. The production of plants in sterile containers that allows them to be moved with greatly reduced chances of transmitting diseases, pests, and pathogens. Citrus