재래감귤 미성숙 배주 이용 Embryogenic Callus 유도 및 감귤 형질전환

Abstract

PART Ⅰ. Embryogenic callus induction from undeveloped ovules of Jeju native citrus species There are 12 kinds of native citrus species in Jeju island. They have been well known for their physiological active substances. With increasing of the cultivation area for commercial citrus cultivars, the numbers of the Jeju native citrus species have lowered to an extremely dangerous level. Citrus callus can be applied widely to somatic hybridization, genetic transformation, and in vitro germplasm conservation. To obtain calluses, the undeveloped ovules of 12 different citrus species were cultured on 5 different media, respectively; MSI (MS, modified, with the addition of malt extract 500 mg/L and sucrose 50 g/L), MSII (MS, modified, with the addition of malt extract 500 mg/L, kinetin 1 mg/L and sucrose 50 g/L), MSIII (MS, modified, with the addition of malt extract 500 mg/L, 6-benzyladenine, 3 mg/L and sucrose 50 g/L), EME (MT, modified, with the addition of malt extract 500 mg/L and sucrose 50 g/L), 1/2 EME (half concentration of MT marcronutrients, half concentratrion of BH3 marcronutrients, malt extract 500 mg/L, glutamine 1.55 g/L and sucrose 50 g/L). The embryogenic calluses of Citrus grandis Osbeck and Citrus platymamma Hort . et Tanaka were obtained from media MSIII and MSII for two month culture, respectively. Embryogenic calluses were observed from surface of undeveloped ovules.

PART Ⅱ. citrus unshiu Marc. transformation Carotenoids are a large family of isoprenoid compounds which provide attr- active coloration to flowers and fruits, ranging from yellow, orange to deep red. High antioxidant activities also have effects on the prevention of heart disease and various cancers, offering useful functions for health. Ggps (geranylgeranyl diphosphate synthase) is known as a key enzyme for carotenoid biosynthesis pathway and ZDS (ζ-carotene desaturase) is responsible for lycopene biosynthesis. To obtain trangenic citrus trees expressing Ggps and ZDS respectively, agrobacterium-mediated transformation was performed. After transformation by cocultivation Citrus unshiu callus and agrobacterium, transgenic plant candidates were selected with hygromycin selection medium，elongation medium and germination medium. Four Ggps gene transgenic target plants all confirmed and ten ZDS gene transgenic target plants eight confirmed by PCR (polymerase chain reaction).