| Pomegranate belongs to the Punica L. genus, Punicaceae family. According to reports, the fruit has abundant antioxidant substance, which has therapeutic effects on cardiovascular diseases and cancer. Pomegranate is resistant to drought, barren and extensive management. The flower and fruit are ornamental. Therefore, pomegranate has value of economy, officinal and ornamental.Pomegranate has a lot of varieties. Tunisia soft-seed pomegranate’s testa is the sofest cultivar in China. It is early-ripening and high yield. The fruit has gorgeous appearance and excellent flavor. It is always popular between consumers. The application of the tunisia soft-seed pomegranate on production and cultivation was limited by its poor cold tolerance.The traditional method of breeding has several disadvantages, including poor breeding efficiency and long period. While application of transgenic technology provide the means for breeding new pomegranate cultivar with cold tolerance. In this study, we compared the difference on regeneration efficiency among three explants by using epicotyl, hypocotyl and cotyledon of tunisia soft-seed pomegranate as explants.Our objective was to establish an efficient and stable regeneration system and obtain an cold-resistance transgenic lines by transferring ICE1 gene. The main results are as follows:1 The sterilization treatment. The whole fruit was sterilized before the middle of August. The seeds were sterilized by 5% Na Cl O treatment for 30 min afterwards. The storaged fruits were sterilized by 1% Hg Cl2 for 8 min.2 The germination treatment. The germination efficiency was improved to 62.67% and the initial germination in 20 d after inoculation by 5% Na Cl O treatment for 15 min and cultured in 25±2°C for 7 d.3 The embryo culture. The length of epicotyl was increased to 0.5 to 1.0 cm by inoculating the medium that was MS containing 3.0mg/L GA3 after germination treatment. The germination efficiency was 86.67% after inoculate 20 d.4 The regeneration of adventitious shoots from epicotyl segments. The best growth quality of adventitious shoots were obtained when epicotyl segments placed in MS medium supplemented with 0.5 mg/L NAA and 1.0 mg/L 6-BA. The frequency of callus formation and adventitious shoots regeneration were up to 92.22% and 71.11%, respectively.5 The regeneration of adventitious shoots from hypocotyl segments. The best growth quality of adventitious shoots were obtained when epicotyl segments placed in MS medium supplemented with 0.5 mg/L NAA and 1.0 mg/L 6-BA. The frequency of callus formation and adventitious shoots regeneration were up to 95.56% and 81.11%, respectively.6 The regeneration of adventitious shoots from cotyledon. The cotyledons were cut in half. The most appropriate medium for the regeneration of adventitious shoots on cotyledon were obtained when cotyledon placed in MS medium supplemented with 0.1 mg/L NAA and 1.0 mg/L 6-BA. The frequency of callus formation and adventitious shoots regeneration were up to 78.89% and 37.78%, respectively.7 The induction of adventitious roots. The most appropriate medium for the regeneration of adventitious roots from adventitious shoots on three explants was 1/2MS containing 0.1 mg/L NAA and 0.5 g/L AC. The frequency of adventitious roots on cotyledon was better than the others.8 Comparison of regeneration capacity of three explants. Hypocotyls > epicotyl > cotyledon.9 The sensitivity of three explants to Agrobacterium-mediated transformation. The browning phenomenon was founded on epicotyl and hypocotyl after cocultivation in 3 d when ICE1 gene was introduced into three explants. The browning phenomenon was also found even to death in all of epicotyls after 10 to 15 d selection culture. The browning phenomenon was found in most of hypocotyls but not in cotyledon. The regeneration of adventitious shoots from cotyledon was obtained in 45 d. Therefore, the resistant strength of Agrobacterium-mediated transformation on three explants was considered that: cotyledon > hypocotyl > epicotyl. |
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