Studies On Virus Eradication By Shoot-tip Micrografting And PCR Detection Of Citrus Huanglongbing

This study was done in the National Indoor Conservation Center of Virus-free Germplasm of Fruit Crops of Huazhong Agriculture University from 2002.10 to 2004.3, the healthy and virulent different citrus breeds' apex were used as explants to proceed shoot-tip grafting. Furthermore, we identified the generated plantlets by specific PCR and observation of refluorescence to detect whether there was citrus Huanglongbing virus in them. The system of production on citrus Huanglongbing virus-free using the technique STG were established. The main results are as follows:1. Improved the shoot-tip grafting survival rate by ameliorative method, the highest survival rate of the inverted " " shaped method was 37.5%, while the highest survival rate was 75% by ameliorative method.2. To treat the apexes with 6-BA with different concentration for different time, we found that the effect was distinct with different varieties. It was propitious to improve the survival rate of 5 varieties by immersing the apexes with 1mg/L 6-BA for ten minutes.3. To treat the apexes with GA3 with different concentration for different time, we found that the effect was distinct with different varieties. It was propitious to improve the survival rate of 3 varieties by immersing the apexes with 2mg/L GA3 for twenties minutes.4. The primers of the PCR were designed based on the sequence information of In-2.6 (Gene bank accession number M9491). Before elimination the virus, polymerase chain reaction(PCR) was performed for detection of the bacteria-like organism(BLO) that caused citrus Huanglongbing disease. By using the PCR procedure, the presence of citrus Huanglongbing BLO in some citrus' seedlings could be detected in samples of infected citrus. Such as hongjiangcheng, gongchuan, Nawhall.5. Another method to detect citrus Huanglongbing was named "direct fluorescence detection method". The exceptional fluorescence in sieve cells of plants that infected citrus Huanglongbing was caused by a group of putrescence cells, when observed under a flourescence microscope (Type: Olympus),a yellow fluorescence was distinctly observed in the viral phloem tissues and not in the phloem tissues of healthy plants.6. We couldn't detect the citrus Huanglongbing pathogen in the STG plants through PCR method and direct fluorescence detection method. It was proved that the STG could eliminate the pathogen of citrus Huanglongbing from virus-infected apexes.Thus, the survival of virus-free plants would be used as materials for propagating virus-free citrus cultivars in the future and improving the progress of citrus breeding.