Different Virus Elimination Methods In Species Variation Of Citrus Siensis Osbeck Cv. 'Hongjiang' And The Effect Of The Virus Of Liberobacter Asiaticum

The raging epidemic of Coulddidatus liberobacter asiaticum causes Hongjang Orange endangered and the chimerism of Hongjang Orange brings the instability properties of fruit. These two affected the development of Hongjiang Orange seriously. Therefore, it was urgently needed to establish a set of virus-free citrus nursery propagation system and to distinguish whether Hongjang Orange separated in tissue culture or not.This research we used tissue culture to cultivate Hongjang Orange abortive embryos for virus-free nursery; we taked shoot tips from adult stem with virus and healthy Hongjang Orange shoot tips as materials to micro-grafting for seedlings without virus; we exploitated Hongjang Orange explanted stem for carrying on rapid propagation and germplasm preservation; we detected infected materials and grafted survival by polymerase chain reaction (PCR); we used SSR molecular markers method to identify whether Hongjang Orange stem separated in tissue culture or not. The results shown below:1. In this work, according as abortive embryos remain the characteristics of the female parent. We tried to cultivate virus-free seedlings in embryonic callus culture, But the results showed that the gathered seedlings from abortive embryos was orange by polymerase chain reaction (PCR) detection.2.In Hongjiang Orange explanted stem sterilized experiment, the treatment was best and the rate of survival was 90% or more, which used 1/200 Xiaojiajing solution to soak for 10min,washed times with sterile water, treated with 70% ethanol 1min and then soaked in 0.1% HgCl2 for 15min.3. In this experiment with improved method we raised shoot-tip grafting survival rate. Inverted "T"-type method had the lowest grafting survival rate of only 10%; Followed by cross-pressure method, 10.4%; the highest survival rate for the triangular method, the survival rate reached 28.6%; Point interpolation method had survival rate of 20%.4. The best hormone combination for induced sprouting from explanted stem of Hongjiang Orange was MT+6-BA1.0 mg.L-1+IBA0.5 mg.L-1+GA30.5 mg.L-1;The best hormone combination for virus-free seedlings from abortive embryos in embryonic callus culture was MT+IAA0.2mg.l-1+6-BA0.5mg.L-1 +ZT0.5mg.l-1.5. In the experiment, we found Hongjiang Orange continue to separating in tissue culture by PCR detection. Relying on tissue culture for rapid propagation and germplasm reserves was not feasible.6. We designed PCR primers by Huanglongbing pathogen's nucleotide sequences. We detected infected Hongjiang Orange by PCR before virus elimination. Results amplified specific electrophoresis zone, which is consistent with the design of the amplified fragment. It proved to be the plant infected with Huanglongbing pathogen.7. We did not detect the Huanglongbing pathogen from the survival of grafted seedlings. That indicated for removal of Huanglongbing pathogen tip grafting was effective and could provide healthy scion materials for the propagation of virus-free seedlings.In this study, the conclusion followed: First, with abortive embryos cultivating virus-free seedlings was not feasible; second, exploiting Hongjiang Orange explanted stem to preserve germplasm or to rapidly propagate was not feasible also; third, the point interpolation method and the triangle method were worth promoting in the micro-grafting; fourth, micro-grafting technique could eliminate Hongjiang Orange Huanglongbing virus; fifth, employing the right explant disinfection could achieve the lowest contamination rate...