AN EFFICIENT PROTOCOL FOR PLANT REGENERATION OF CAM SANH (CITRUS NOBILIS LOUREIRO) VIA MULTI-SHOOT INDUCTION FROM EPICOTYL SEGMENTS

Abstract

Citrus transfonnation has now been achieved in a number of laboratories by various methods, including the direct uptake of naked DNA by protoplasts, the Agrobacterium-mediated transformation of embryogenic suspension-cultured cells and particle bombardment of similar tissues. At present the use of seedling or juvenile plant explant tissues and an Agrobacterium-mediated transformation is the most efficient method for producing transgenic Citrus plants or plants of the more regenerable Citrus relative. Therefore, the purpose of this study is to develop a culture system for Agrobacteriurn tumefaciens mediated gene transformation in Cam Sanh (Citrus nobilis Loureiro) through multi-shoot induction method. The multi-shoot induction protocol includes the following major steps: 10 mm epicotyl stem segments were cut from 20 - 25 days old seeding and cut longitudinally: explants were placed on germinating medium containing Murashige and Skoog's (MS) salts, 2 mg/I BAP. 5% sucrose and 8g/l agar. Shoot differentiation was observed in 3 - 4 weeks after transfer onto shoot elongation medium containing MS supplemented with 0.5 mg/I NAA, 0.1 mg/I GA3 and 2 g/I activated charcoal; for rooting, 3 - 4 cm long shoots were cultivated on root induction medium containing O.5"mg/1 NAA; rooted plants were transferred onto soil in green house. The frequency of plant regeneration was 98.5%, the average efficiency for regeneration was 8,4 regenerated shoots per epicotyJ stem segment. the frequency of plant rooting was 71.6% and then plantlets were successfully transferred into soil in greenhouse condition