Pak. J. Bot., 50(3): 1203-1210, 2018. ESTABLISHMENT OF AN EFFICIENT PROTOCOL FOR PLANTLETS REGENERATION VIA DIRECT AND INDIRECT ORGANOGENESIS IN CITRUS RETICULATE BLANCO (KINNOW MANDARIN) MUBASHIR HUSSAIN 1 , NAVEED IQBAL RAJA 1 , HAMID RASHID 2 *, ZIA-UR-REHMAN MASHWANI, ASIM MEHMOOD 2 AND MUHAMMAD IQBAL 1 1 Department of Botany, PMAS Arid Agriculture University Rawalpindi, Pakistan 2 Department of Bio Sciences, COMSATS Institute of Information Technology, Sahiwal, Pakistan * Corresponding author’s: email: hamidrashid@ciitsahiwal.edu.pk Abstract Studies were initiated to explore the role of plant growth regulators and explant types on efficient plantlets regeneration via direct and indirect organogenesis in Kinnow mandarin [Citrus reticulata L. (Blanco)]. Explants were cultured on MS medium containing varying concentrations of phytohormones. The best callus induction response was obtained in MS medium containing 5 mg/l 2,4-D and 1 mg/l BAP where 90% from nucellus tissue, 58% from shoot apical meristem and 56% from nodal segments explants showed callogenic response after 2 weeks of inoculation.Best shoot induction medium via indirect organogenesis was found for nucellus tissue when MS medium was supplemented with 1.5 mg/l Kin and 500 mg/l malt extract which was 46% whereas for SAM and nodal segments, best response was obtained when MS medium was supplemented with BAP and NAA (3.0 + 0.5) mg/l which was 40% and 48% from SAM and nodal segments respectively after 11 weeks of inoculation.The shoot induction as well as multiplication were also studied by using different combinations and different concentrations of phytohormones in growth medium. The best medium for shoot induction percentage via direct organogenesis was [M3= MS + BAP (1.0 mg/l)] whereas 92%, 90% and 82% from nucellus tissue, SAM and nodal segments respectively showed organogenic response after 4 weeks of inoculation, whereas maximum number of shoots per explants was obtained in medium (M9)containing MS + Kin where 4, 7 and 6 average number of shoots were obtained from nucellus tissue,SAM and nodal segments respectively after 11 weeks of inoculation.Best rooting medium was found when MS medium was supplemented with 2 mg/l NAA where 88% from nucellus tissue and 80% from the nodal segments showed rooting response whereas for SAM best rooting response was obtained when supplemented with 1.5 mg/l NAA i.e 78%. The plantlets were successfully acclimatized in different potting mixtures and highest survival rate (100%) was achieved in potting mixture containing sand and peat moss (2:1). Key words: Tissue culture, MS medium, Callogenesis, Callus regeneration, Direct regeneration. Introduction Citrus is economically one of the most important fruit crop all over the world including Pakistan. The genus citrus belongs to family Rutaceae and is the most extensively cultivated fruit crop all over the world. The citrus production of the world was relatively invariable during last decade of 20 th century. Citrus origin is supposed to be southern slope of Himalayan region and northFinally Check Finally Check-eastern area of China and neighboring India (Gmitteret al., 1990). Pakistan is included in the ten major producers of citrus all over the world. Citrus is grown in Pakistan on area of 193,212 hectares and is increasing 5% annually. Pakistan contributes 95% of the total Kinnow mandarin and supplied to all over the world (Sharif & Waqar, 2005). Punjab province is considered as the main hub of citrus fruit crop and provides citrus of high excellence to all over the world. In Pakistan 86% of the citrus grown is Kinnow mandarin (Citrus reticulate Blanco) followed by Musambi, Feutral and Blood red in proportion of 10%, 4% and 1% respectively. Kinnow is a hybrid of two varieties, namely Citrus deliciosa and Citrus nobilis. The export of Kinnow mandarin can be increased by introducing seedless variety. There is a high demand of seedless varieties in the international market and Pakistan’s Kinnow mandarin has about 22-26 seeds per fruit. There is a report regarding seedless varieties in Pakistan (Khalil et al., 2011). Propagation of Kinnow mandarin is not very fast through traditional methods of propagation. To cope up with this problem, it is essential that Kinnow mandarin can be propagated through tissue culture methods such as direct and indirect organogenesis so that numerous numbers of plants can be produced in a limited period. Gene transfer, selection and regeneration of transformantsis now a day & employed by the plant tissue culture techniques (Shah et al., 2009). According to Helal (2011) tissue culture can be employed as substitute to conservative methods In vitro propagation with the purpose of increasing the developmental rate of preferred genotypes and commercial micropropagation. Tissue culture of citrus has attracted a considerable attention because of its commercial values and due to the important fruit crop of the world. In vitro regeneration of citrus cultivars has been discussed in various species of citrus mainly Citrus reticulate (Hassanein & Azooz, 2003); Citrus sinensis (Costa et al., 2004); Citrus aurantium L. (Bordon et al., 2000) and Citrus grandis (Huang et al., 2002). Micropropagation protocols have been described in various citrus species and explant sources (Usman et al., 2005; Khan et al., 2009; Laskar et al., 2009; Sharma et al., 2009; Pe´rez- Tornero et al., 2010, Hussain et al., 2016). The research work was carried out to explore the response of explants type and medium composition on callogenesis, frequency of plantlets regeneration and multiple shoot induction in Citrus reticulata L.