In Vitro Culture Of Mature Citrus Stems And Studying On Virus-free Methods

The preventions and treatments of HLB still rely on traditional methods in china at present, such as cutting down diseased trees, prevention of Psyllids, cultivation of virus-free seedlings. The aim of this study is to make use of drugs to do a series of researches with the use of traditional methods, and by combination of in vitro culture of mature adult phase as well as shoot micro-grafting to achieve the toxin-free plants. More details are as follows:1、Multiple shoots derived from the in vitro culture of mature citrus nodal stem segments. The study provides insights into the contamination rates and survival rates of mature citrus nodal stems of Seike Navel Orange with different sterilization methods, the emergence of shoots under different concentration ratioes of hormones. The data revealed that the best sterilization method is 10% Na Cl O(V/V)for 30 min, 2 h interval, 10% Na Cl O(V/V)for another 20 min, when concentration of hormones is 2.0 mg/L for 6-BA, 0.2 mg/L for NAA, with the most shoots emergence for 3.97, the lower utilization, and when 1.5 mg/L for 6-BA, 0.3 mg/L for NAA, with the shoots emergence for 2.57, the best utilization.2、Eradication of citrus HLB with antibiotics. The broad-spectrum antibiotics, such as Metronidazole and Fenbufen Capsules 200 mg/L, Terbinafine Hydrochloride Tablets 100 mg/L, Ornidazole 200 mg/L can suppress HLB effectively. The antibiotics targeted for Gram bacilli, such as cephalosporins, Fluoroquinolones, Penicillin 200 mg/L can reduce the percent incidence of the disease range from 100 % to 0 %, 0 %, 25% respectively, and combined processing of the three specific antibiotics can reduce it to 0%. It suggests that the professional antibiotics have great prospects to prevent the spread of HLB.3、The experiment adopting for shoot-tip micro-grafting technique obtained 52 virus-free seedings, which include 12 varieties, with the three better varieties C. unshiu Marc. ?Dapu?, C. sinensis Osbeck.?Newhall?, C. unshiu Marc. ?Cuipimiju?, the survival rates is 6.36%, 5.53%, 5.44% respectively. After 8-12 monthes cultivation, one or two blades are removed for q-PCR test, the result shows that the elimination rates of HLB is 89.29%.4、The clone of 16 S r DNA fragment in HLB agent from Qiongzhong, Hainan. The specific primer pair of OI1/OI2 c was used to amplify and clone the 16 S r DNA fragment. Homology analysis and cluster analysis indicated that HLB from Hainan was more closed to Okinawa lines from Japan and the homology with it is 99 %.5、Quantities of citrus samples from Hainan were detected for two years running, the datas show that the comprehensive average detection ratio of HLB is 41.2 %, CTV is 92.5 %. All above tells that HLB and CTV gradually cause serious losses to the orchard in Qiongzhong, Hainan,and the reason for this is because that when farmers find branches with symptoms, they don?t remove it timely,which may increase the spread of Las through the canopy to other tree pushes, then enhance the infection process, coming with the destroy of the whole orchard.