Clone Cytoplasmic Male Sterile Nad Gene And Studies On Regeneration Of Kenaf (Hibiscus Cannabinus L.)

As one of the most important fibre cash crops in our country, Kenaf (Hibiscus cannabinus L.) has excellent fiber quality and it was widely used.Therefore,kenaf was regarded as the most potential multi-use crops in the 21st century. As other plant, the cultivation of new kenaf variety to solve the question is an inevitable way.Using cytoplasmic male sterility of crops is the main way of heterosis, however its mechanism is very complex.Therefore,research related to CMS becomes the hot and difficult issues in national and international scholars.A large number of research indicate that cytoplasmic male sterility has the close relation with the mitochondrial DNA variation. Our group confirmed the difference of nad gene fragment in cytoplasmic male sterile line and their maintainer lines by SRAP markers techology. In order to research cytoplasmic male sterility with mitochondrial DNA between relations further, we have experimented by using cytoplasmic malesterile line P3A and their maintainer lines P3B as materials, then we established the method of isolation of high purity mitochondrial DNA and clone the cytoplasmic male sterile related gene nadl-nad3. We also toke semi-quantitative RT-PCR to analyse nadl-nad9 gene and study preliminarily the regeneration of kenaf.The main results are shown as follows:1.An efficient method for isolation of mitochondrial DNA (mtDNA) from etiolated tissues of kenaf for sequencing was developed. The result showed that sucrose gradient centrifugation was better to Percoll as for mitochondria isolation of kenaf. The mtDNA was detected by gelose electrophoresis and ultraviolet spectrophotometer, furthermore a chloroplast and a nuclear-specific primers were designed to detect mtDNA purity. The result showed that the mtDNA extracting by this way have high purity and no contaminant. 2.We designed degenerate primers according to the conserved sequence of the nad gene published in NCBI database, and isolated the 817bp,1052bp,235bp produets (conserved sequence) of the P3A and P3B using PCR amplification. Comparied with nad gene of other plants,the nucleotide sequence of these fragments shares,respectively,98%,97% and 97% identity with that of Beta vulgaris andPhyllostachys, Nicotiana and Citrullus lanatus So these fragments were the correct sequence. Compared the same nad gene between P3A and P3B show that there was only litter nucleotide different. The expression of the nad gene between P3A and P3B were further analysed via semi-quantitative RT-PCR.3.In the process of callus induction, callus of kenaf was inducted by embryo. The results showed that number A6 meedium(MS+0.5mg/L6-BA+1.0mg/L2,4-D) was suitable for callus induction.The callus induction rate was up to 100%. The callus some advantages such as major, yellow-green colour and close texture. The callus were cut into knob and cultivated in the medium MS adding 0.5-2.5mg/mL 6-BA and 0.1-0.5mg/mLNAA. With increasing concentration of 6-BA, the green callus was more in higher concentration of NAA. The result show that the combination of 6-BA and NAA can induce differentiated cell, and the best media is number B20 medium (MS+2mg/L6-BA+0.5mg/LNAA). But the ratio of 6-BA and NAA will study further in order to induce adventitious bud.