Establishment of in Direct Propagation of Mandarin (Citrus reticulata L.) Using Tissue Culture

Abstract

THE PRESENT investigation deals with development of an efficient micropropagation protocol for mandarin (Citrus reticulata L.) via in direct propagation using cotyledons and juice vesicles as explant. The establishment of in vitro callus induction from leaf, stem segments and cotyledon system were excised from seedlings has been done. Best results for induction of callus were observed from cotyledons and stem segments 100% on MS medium supplemented 1 or 2mg/l 2,4-D. MS medium supplemented with 300mg/l glutamine or 1.5mg/l casein hydrolysate proved to be the most efficient additive in promoting callus formation from juice vesical with 4.22, 4.23g/callus fresh weight, 100% response and was the most favorable medium. Maximum callus fresh weight (7.96 g) and 100% response were obtained from juice vesicles when cultured on MS medium supplemented with 2mg/l 2,4-D and 10% coconut water. The highest callus regeneration and weight of callus observed from juice vesicles by adding 2mg/l 2,4-D, 300 or 400 mg/l malt extract. Callus raised from juice vesicles showed maximum shoots (96.67%) with 0.5mg/l Kin, 400mg/l malt extract and 2mg/l NAA. Regenerated shoots raised from juice vesicles showed better shoot multiplication and highest length of shoots on MS medium with 2mg/l BA and 0.4mg/l NAA. Regenerated shoots were rooted on MS medium supplemented with different concentrations of NAA and best response (98%) was observed with NAA (2 mg/l) and gave maximum, roots number, roots length and leaves number. Rooted plantlets were successfully acclimatized with survival rate reaching almost 87%. These plants grew normally without showing any morphological variation. Developed protocol can be useful for application of somatic embryogenesis from juice vesicles to improve mandarin.