Somatic Embryogenesis In Dendrobium Candidum Wall Ex Lindl

Effects of plant growth regulators on callus induction and plant regeneration, morphogenetic process of somatic embryogenesis, patterns of isozymatic and gene expression during somatic embryogenesis in Dendrobium candidum Wall ex Lindl. were studied in this paper. The main results were as follows:1. An efficient system was established with a high frequency of protocorm-like bodies (PLBs) regeneration from the callus of D. candidum. The calli were induced with the highest frequency from longitudinally bisected segments of protocorms on 1/2MS medium, which was the MS medium with macronutrients at half strength and 2% sucrose, supplemented with 2.0 mg l^-1 6-BA, and then subcultured 2 times every 40 days on the same medium. PLBs formation was achieved when calli were transferred to 1/2MS medium without any plant growth regulators. PLBs developed into intact plantlets with four to five leaves and three to four roots on 1/2MS medium with 0.2 mg l^-1 NAA. Plantlets were transplanted into vermiculite with a 95% survival rate in a greenhouse.2. From histological observations, PLBs developed from the somatic embryos derived from the inner or outer region of the callus cultured on 1/2MS medium without any plant growth regulators. The somatic embryos derived from the single embryogenic cell of the inner region of the callus showed the similar developmental pathways with the zygote in vivo, and the developmental process after globular embryo is consistent with that of germination of the seeds. As the differentiation of the callus progressed, these somatic embryos passed through the single-celled embryo, two-celled embryo, four-celled embryo, five-celled embryo, globular embryo, heart-shaped protocorm-like body (PLB) and PLB with a cotyledon until an intact plant.3. The soluable proteins and isoenzymes were studied during somatic embryogenesis. Patterns in Peroxidase (POD), Catalase (CAT), Cytochrome Oxidase (COD), Superoxide Dismutase (SOD), Esterase (EST), Malate Dehydrogerase (MDH), Amylase (AMY), Alcohol Dehydrogenase (ADH) varied to different degrees during somatic embryogenesis. New bands of EST, COD and ADH appeared specifically at heart-shaped protocorm-like body stage, and a MDH band disappeared at the same stage. The activity of antioxidant enzyme of POD, CAT and SOD showed the same changing tendency during somatic embryogenesis, the activity of them reached the highest at the single-celled embryo satge. These results shows that the single-celled embryo and heart-shaped PLB satges are the key stages of somatic embryogenesis in D. candidum, and it is possible to use these isoenzymes as molecular marker of somatic embryogenesis.4. Two genes, TIM and CUC, related to embryo development were detected by RT-PCR technique. The mRNA of TIM and CUC obtained through 3'-RACE and 5'-RACE were cloned, sequenced and identified primarily through sequence analysis.In conclusion, an indirect somatic embryogenesis system was established in D. candidum.