Study On The Establishment Of Aseptic System Of Grapefruit Adult Explants And Shoot Tip Micro Bud Grafting

In this study,we carried out induced experiments of mature Citrus paradisi stems in vitro using the stem segments of Flame,Hudson Foster,Ray Ruby,Rio Red and Star Ruby as experimental materials.Combining induced techniques in vitro and advanced grafting methods,our aim is to get adventitious regenerated buds and improve the survival rate of shoot tip grafting.Finally,the seedlings were detected by polymerase chain reaction?PCR?to obtain the grafted plants.The main findings are as follows:1.The disinfection methods of culture in vitro for mature grapefruit stem-segments were improved by comparing different effect factors.Results showed that the explant with2 cm in size is the best selection for its culture in vitro.Different breeds of grapefruits that were disinfected had a significant difference in both contamination and germination rates,with the highest contamination rate of 68.8%for Rio Red and the highest germination rate of 21.3%for Star Ruby.With the disinfection of 75%alcohol and 5%NaCIO for 20 s and20 min,respectively and then incubation for 48 h,re-disinfection methods could effectively decrease the pollution rates of stem culture?21.3%?and meanwhile increase the germination rate?52.5%?.Further,the stem segments of re-disinfection were inoculated into the media with WPM+100 mg/L cysteines.The stem segment contamination rate was15.6%and the germination rate was 83.1%.The results indicated that the medium could effectively relieve the browning after repeated sterilization and increase the germination rate.2.The results showed that components of the best medium for axillary bud induction were WPM+1.0 mg/L 6-BA+0.2 mg/L NAA+0.2 mg/L GA₃+sucrose 40 g/L.On such media,the axillary buds had thick and fast growth with the induction rate of 78.89%.3.In the experiment of rootstock germination rate,the MS medium is optimal for the seed germination of Mansailong pomelo,showing 81.7%germination rate.In addition,NAA,6-BA and GA3 were capable of reducing the time of seed germination.For rootstock seedlings,they had the best effects for seed germination rate and seedling emergence rate.4.Comparing four kinds of grafting methods,results showed that the puncture method was more suitable for the grapefruit micro-grafting,with the highest grafting survival rate for of 75%.In contrast,split grafting connection method had the lowestsurvival rate of only 18.67%.Different varieties of grapefruit bud grafting survival differences,five scion varieties of test bud grafting survival rate:Ray Ruby>Star RubyHudson Foster>Flame>Rio Red.The micro-grafting experiments of two stem tips from the field and induced treatments were carried out.The results showed that the grafting survival rate of stem segment of adventitious bud as a scion bud was 76.67%,and another treatment was 38.33%.This result exhibited significant difference between stem tips of field and treatments.5.According to the sequences of the pathogen of citrus huanglongbing disease,we designed gene specific primers using for PCR amplification.Using PCR to detect suspected infection citrus huanglong disease of plants,the results showed that only the plants with disease infection exhibited specific bands.Next,the stem segments of infected plants were grafted,and the survivals were further detected.The results showed that no specific bands were observed.Thus,it suggested that the STG could remove the pathogen of citrus huanglongbing virus from virus-infected apexes and will be considered as an effective method for propagating vitrus-free citrus cultivars in the future.