Dendrobium is a kind of popular tropical orchid. In this experiment, the PLBs of Dendrobium nobile and Dendrobium candidum were used as the materials for the research of optimization of the conditions of rooting and shoot growth in the tissue culture in Dendrobium, in vitro flowering, gene cloning of LRR-RLK related to flower development, and gene expression of LRR-RLK at different stages of in vitro flowering in Dendrobium nobile. The main results were as follows:1. The regeneration system was optimized in Dendrobium.The PLBs of Dendrobium nobile were used as the materials for the studies on proliferation of protocorms, and the effects of medium, 6-BA and banana juice were studied. The results showed that the medium supplemented with banana juice could improve the proliferation coefficiency significantly. The best proliferation medium for protocorms was the KC medium supplemented with 1.0 mg/L 6-BA and 10g/L banana. The PLBs of Dendrobium nobile and Dendrobium candidum conserved for 5 years were used as the materials for the the orthogonal tests of optimization of the conditions of rooting and shoot growth in the tissue culture of Dendrobium nobile and Dendrobium candidum. The results showed that the best medium for rooting and shoot growth of Dendrobium nobile was 1/2MS medium with 6-BA0 mg/L, sugar 20 g/L, and banana 50 g/L, and the best medium for rooting and shoot growth of Dendrobium candidum tissue culture seedling was 1/2MS with 6-BA0 mg/L and sugar 10 g/L.2. Research of in vitro flowering of plantlets in Dendrobium.The effects of basal medium, 6-BA, modified MS medium, ZT, TDZ, potato juice on in vitro flowering were compared in Dendrobium nobile. The results showed that multi-factors combination was easier to induce flowering and obtain normal in vitro flowers than single factor in Dendrobium nobile. The modified MS (1/10 N,NH4+-N:NO3--N 4:1) supplemented with TDZ0.1 mg/L, sugar 20 g/L, CW100 mL/L, PP3331.0 mg/L and 14℃treated for 20 days was the best for in vitro flowering, and the inducing rate was 31.91%. The frequency of floral bud induction was significantly affected by NH4+-N:NO3--N, N, TDZ, PP333, sugar, CW, low temperature treatment and PP333; and the modified MS (1/10 N, NH4+-N:NO3--N 2:1, 5 P) supplemented with TDZ0.2 mg/L, sugar 40 g/L, CW100 mL/L, PP3331.0 mg/L and 14℃treated for 30 days was the best for inducing normal flower buds, and the influence degree of the above factors was sugar >PP333 >low temperature treatment>P >TDZ=CW>NH4+-N:NO3--N>N. The effects of 6-BA and TDZ on in vitro flowering were also compared in Dendrobium candidum. The results showed that TDZ was easier to induce flowering than 6-BA, and the best medium was 1/2MS supplemented with TDZ0.2 mg/L, CW100 mL/L and sugar20 mg/L, and the flowering rate was 94.79% (counted by the number of flower buds) or 62.50%( counted by the number of flowering plantlets) in Dendrobium candidum.3.Gene cloning of LRR-RLK in Dendrobium nobile PLBs.The PLBs of Dendrobium nobile were used as the materials for RNA extraction and further gene cloning of LRR-RLK in Dendrobium nobile. The results showed that the RNA strips extracted by general total RNA kit were bright and clear, but with DNA contamination; the strips extracted by modified Trizol were without DNA contamination, but unstable. The suitable method for RNA extraction should be selected based on the requirement of different experiments. A pair of specific primers of were designed according to LRR-RLK in other plants to amplify fragments, conserved sequence and 3'sequence were obtained, and the total sequence was 1034 bp. Sequence analysis revealed that the full length cDNA contained a 531 bp open reading frame and encoded 176 amino acids. The deduced amino acid sequence was highly homologous with LRR-RLK in other plants. The registered No. of the gene was GU357498.1 in GenBank.4. Expression analysis of LRR-RLK gene by real time RT-PCR during in vitro flowering in Dendrobium nobile.The expression analysis of LRR-RLK gene by real time RT-PCR during in vitro flowering was carried out in Dendrobium nobile. EF-1αfragment was first cloned as a candidate reference gene, and it was 716 bp, encoded 237 amino acids. Sequence analysis revealed that amino acid sequence was highly homologous with EF-1αin other plants, which was suitable to be tested as a candidate reference gene. The registered No. was GU357498.1 in GenBank. To select a more stable reference gene for expression analysis on LRR-RLK gene, 18S rRNA and EF-1αwere both compared, and the result showed that 18S rRNA was more suitable. The analysis on LRR-RLK gene during in vitro flowering by real time RT-PCR in Dendrobium nobile showed that the expression of LRR-RLK was at a relatively high level in PLBs, and at a higher level in general test-tube plantlets, but when the plantlets were transferred onto floral induction medium, the expression levels decreased, and the lowest in flower bud, however, it was at a higher level in flower, which suggested that LRR-RLK play an important role in shoot differentiation and flower bud development. |