The Establishment Of Plant Virus-Elimination And Micropropagation In Taro ’Hongxiang’

Taro (Colocasia esculenta(L.) Schott), also known as yutou, yunai and maoyu, is a typical vegetative propagation plant with corm as the organ of product and propagation. The corm of taro is rich in nutrients which could not only be used as vegetable or food, but also has a high medicinal value. The long-term vegetative propagation mode make taro easily infected by virus, and once be infected, the virus spread quickly which would make a serious loss. Among those virus infected taro, DMV (Dasheen Mosaic Virus, DMV) is the one whose infection is the widest and the damage is the most serious. After being infected, the plant shows plumatus, mosaic or wrinkled, the leaf vein and stem represent necrosis or other symptoms, which affect the production and quality seriously. Therefore, the establishment of virus-elimination and rapid propagation of taro is very significant to the purification and rejuvenation of the excellent taro germplasm resources.In this study, Jianchang taro ’Hongxiang’, the good local resource of Jintan in Jiangsu province, was adopted as the material, and a RT-PCR rapid detection system of taro ’Hongxiang’DMV was established, then we screened out the most appropriate method to detect DMV from taro’Hongxiang’plantlet in vitro from electron microscope, DAS-ELISA and RT-PCR. Subsequently, the effectiveness of removing DMV from taro plantlet in vitro was compared between four virus-elimination methods, specifically two time shoot-tip dissection culture, modified heat therapy with micro-tip culture, ribavirin treatment with micro-tip culture and modified heat therapy, ribavirin treatment with micro-tip culture. After that, virus-free plantlet in vitro was applied to get corm in vitro, and the corm in vitro was used to induce somatic embiyogenesis and plant regeneration. At the end, the effect of 5-aminolevulinic acid (ALA) on the cold resistance of taro plantlet in vitro was studied. Finally, we established an efficient detoxification and rapid propagation technical system of taro ’Hongxiang’ which provide a scientific evidence and technical basis for rapid propagation of taro virus-free plantlet in vitro. The main results are as follows:1. We established a RT-PCR rapid detection system which could be used in taro ’Hongxiang’ DMV detection. The result of three detection method showed that electron microscope, DAS-ELISA and RT-PCR could all be used in taro ’Hongxiang’ DMV detection, but RT-PCR was more accurate, and the repeatability of electron microscope and DAS-ELISA was lower which might lead false-negative result. Therefore, RT-PCR was the most appropriate method applied in taro ’Hongxiang’ plantlet DMV detection in vitro.2. The two time shoot-tip dissection culture of 0.5 mm and 1.0 mm size,21d～42d modified heat therapy with 2.0 mm size micro-tip culture,25 mg·L-1 and 50 mg·L-1 ribavirin treatment with 2.0 mm size micro-tip culture,28 d modified heat therapy,25 mg·L-1 ribavirin treatment with 2.0 mm size micro-tip culture could all make the better detoxification effect. Among these method the synthesize treatment was the best, the ratios of plantlet and free-virus were achieved 88.00% and 66.67%, the ratio of free-virus plantlet was up to 58.67%. Synthesize the ratios of plantlet formation and virus-free, the 28 d modified heat therapy,25 mg·L-1 ribavirin treatment with 2.0 mm size micro-tip culture was the most suitable method of virus-elimination and rapid propagation of taro ’Hongxiang’.3. Adopted 1-2 mm size corm slices as the explant and 1/2MS as the basic culture medium, firstly the taro corm slices were cultured in the medium with 1.0 mg·L-12,4-D for 20 days, and then were transferred to the medium with 0.5 mg·L-1 TDZ. After 110 days, 48.81% corm slices were induced to be embryogenic callus which was significantly higher than other concentration combination. Under the best concentration combination of 2,4-D and TDZ,20 d of the 2,4-D treatment was better to induce embryogenic callus than 10 d and 30 d. The formed embryogenic callus was transferred to different development medium of somatic embryo and on the free-plant growth regulator 1/2MS medium the ratio of incidence of somatic embryos was achieved to 54.44% and the regeneration coefficient was 8.17 which were significantly higher than other medium.4. Taro ’Hongxiang’ was adopted as the material. The changes of physiological indexes of taro plantlet in vitro were studied by using different concentrations of ALA under the low temperature stress simulated artificially. The results showed that:under short-term (16 h) low temperature stress, the 2～20 mg·L-1 exogenous ALA treatment could reduce the chilling injury degree of taro plantlet in vitro, promote the activities of SOD, POD, CAT and APX, decrease the production speed of ·02- and H2O2 content, suppress the increment of MDA and membrane permeability while enhancing the ability of cell osmoregulation. The above results indicated that the ALA with suitable concentration could improve the cold resistance of taro plantlet in vitro to relieve the chilling injury under low temperature stress and ALA at 10 mg·L-1 could make the best effect.