Expression Of Stress Inducible Transcription Factor Gene AtDREB1A Gene In Ground-cover Chrysanthemum Enhances Drought Salt And Cold Stress Tolerance

Young leaf explants of ground cover chrysanthemum (Dendranthema grandiflorum (Ramat.) Kitamura) 'Fall Color' were used to develop the regeneration system with an efficient, high-frequency by somatic embryogenesis. Leaf discs were excised and inoculated on induction medium (IM), which was based on MS medium and containing 1.5mg/L IBA and 0.5 mg/L BA and 0.75 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) for 15d induction of pre-embryogenic callus. The induced pieces of pre-embryo callus tissue were then inoculated onto regeneration mediun (RM), which was MS medium with addition of 1.5mg/L IBA and 0.5mg/L BA without 2,4-D, after 8 weeks, somatic embryo emerged and primordial shoots developed. Somatic embryos were produced from the explants, and 93% of shoot regeneration rate was obtained through further regeneration culture.AtDREBIA, a stress-inducible transcription factor, driven by 35S cauliflower mosaic virus (CaMV) promoter or stress inducible rd29A promoter, was transferred into chrysanthemum plants through Agrobacterium-mediated transformation. A predicted 453 bp internal fragment and a positive signal for the DREB1A gene was amplified by PCR and integration of target gene of DREB1'A was confirmed by PCR-Southern blotting. Among 24 antibiotic-resistant S₀ plants, 19 plants harboring 35S:DREB1A were obtained. To identify their genetic stability, 6 plants were randomly chosen from 19 S₁ chrysanthemums harboring the 35S:DREB1A, and 5 plants were further confirmed by PCR and PCR-Southern. The same procedures were performed in S₀ and S₁ antibiotic resistant plants harboring rd29A:DREBlA, 15 plants from 18 plants with antibiotic resistance were identified, three of S₁ plants were further confirmed. RT-PCR analysis revealed that DREB1A gene was over-expressed constitutively in 35S.DREB1A plants, but was inductively in rd29A:DREBlA plants. When exposed to cold, drought and salt stress conditions, DREB1A gene expression was enhanced in young plant roots of transgenic 35S.DREB1A lines, and induced obviously in that of transgenic rd29A:DREBlA lines.A severe retardation phenomenon was observed in 35S.DREB1A plants, but not in rd29A:DREBlA plants compared with WT plants. The transgenic plants exhibited tolerance to drought, salt and cold stress, and the tolerance was significantly stronger in rd29A:DREBlA plants than in 35S.DREB1A plants. Electrolyte leakage in leaves and membrane damage rate were obviously lower in transgenic plants than in WT plants under the stress conditions.Proline content and SOD activity were much increased inductively in rd29A:DREBlA plants than in 35S.DREB1A plants and WT under drought, salt and cold stress conditions. These results indicated that hererologous AtDREBIA could confer drought , salt and cold tolerance in transgenic chrysanthemums, and improvement of the stress tolerance might be related to enhancement of proline content and SOD activity.