Study on Key Technology and Its Physiological Basis of Virus Elimination and Rapid Propagation of Phalaenopsis
|School||Nanjing Agricultural College|
|Keywords||Phalaenopsis Virus elimination Rapid propagation RT-PCR Virus detection Heat treatment Chemical treatment Physiological basis|
Phalaenopsis, is also known as butterfly orchid, because of its unique flower-shaped, colorful, has high ornamental value, and occupies an important position in ornamental flowers. Generally, Phalaenopsis is cultured in protected condition, vulnerable to viruses in cultivation process. Then the viruses are quickly spread causing severe damage. After plants were infected with viruses, it can cause leaf spots, necrosis, and flower color, deformities and other symptoms, severely affecting their quality. Therefore, it has important application value that study on the effect of different virus eliminated technology on Phalaenopsis detoxification efficiency and the rapid propagation of virus-free plantlets.In this study, used Phalaenopsis strain’EG-727’plantlets as materials, on the basis of detection Cymbidium mosaic virus(CymMV) in Phalaenopsis, we applied secondary shoot-tip culture, shoot-tip culture combined with chemical treatment and shoot-tip culture combined with heat treatment to eliminate CymMV, to define their detoxification efficiency, and some key technologies such as virus-free plantlets obtained, buds germination and buds multiplication. We initially established a technical system for Phalaenopsis strain’EG-727’ efficient detoxification and rapid propagation of virus-free plantlets, to provide a scientific evidence and technical basis for rapid propagation of Phalaenopsis virus-free plantlets. The major results are as follows:1.49d heat treatment in 38/32℃(day/night), Phalaenopsis plantlets leaves appeared a large area yellow and wilt, even decay; soluble protein content first decreased and then increased; MDA content raised within 49d, significantly dropped after 49d; the change of relative conductivity was little difference before 35d, a significant increase after 35d; before 49d, SOD, POD and APX activity showed different degrees of increase, declined after 49d; while CAT activity reduced significantly within 49d, and raised rapidly afterwards. The results showed that:38/32℃heat stress caused significant oxidative damage, the time node may be 49dth. Therefore, Carrying out Phalaenopsis plantlets detoxification with 38/32℃(day/night) heat treatment, the treatment time should not exceed 49d.2. We applied RT-PCR to detect CymMV, and established a RT-PCR rapid detection system, which can be used in Phalaenopsis CymMV. To study the effects on CymMV detoxification efficiency of the second shoot-tip culture, shoot-tip culture combined with chemical treatment and shoot-tip culture combined with heat treatment, the virus tips were stripped from plantlets. The results suggested that:tips size significantly affected virus-free rate and plantlets rate, the smaller the tips, the lower survival rate, the better virus-free. Stripping 2.0mm size tips, one time or secondary shoot-tip culture was not able to effectively remove the virus. Although the shoot-tip culture combined with chemical treatment and heat treatment reduced the survival rate, but could significantly increase the virus-free rate. The virus-free rate of 6.0g·L-1Banlangen treatment was 77.33%; 50mg·L-1 Ribavirin treatment was 33.33%.38/32℃(day/night) alternating temperature heat treatment 35d, virus-free rate of 46.67%, virus-free rate and survival rate were significantly higher than 38℃constant temperature heat treatment.3. Selecting 2.0mm shoot-tip as explants, we studied the effects of different the basic medium, different 6-BA and NAA concentration ratio, organic additives and carbon concentration on the buds germination and proliferation. The results showed that:basic medium ND was the most suitable to buds germination of shoot-tip, MS medium was conducive to the proliferation and growth of buds. The buds were thick, forming a large number of clustered-like buds; the combination of 1.0mg·L-16-BA and 0.1mg·L-1NAA, was the best one of sprouting of buds, the combination of 8.0mg·L-16-BA and 0.5mg·L-1NAA was able to effectively stimulate the buds proliferation; adding coconut water (CW) 100ml·L-1 was conducive to the germination of buds, 150g·L-1banana could significantly promote the proliferation of buds. For Phalaenopsis strain’EG-727’plantlets buds germinated from shoot-tips, the suitable medium was ND+1.0mg·L-1 6-BA+0.1mg·L-1 NAA+100ml·L-1CW+1.0g·L-1AC+20g·L-1Sucrose+8.0g·L-1Agar, pH5.4; for virus-free buds multiplication and growth of plantlets, the suitable medium was MS+8.0mg·L-16-BA+0.5 mg·L-1NAA+150g·L-1Banana+30g·L-1 Sucrose+6.5 g·L-1 Agar, pH 5.7.