Study On Oriental Lily Virus Detection And Shoot Tip Virus Elimination By Cryopreservation

In this experiment, Sorbonne and Siberia , two varieties of Oriental lily were used as materials for virus detection and shoot tip virus elimination by cryoperservation. The main contents of the experiment were as follows: establishment of the system of the regeneration in vitro in Oriental lily; virus detection of Oriental lily; establishment of the system of shoot tip virus elimiantion by cryopreservation; shoot tip virus elimination by cryopreservation and effect detection; detection of genetic stability on regeneration plants after virus elimination by cryopreservation. The main results were described as follows:1. Establishment of the system of the regeneration in vitro in Oriental lily. The Sorbonne and Siberia scales were used as materials to study the adventitious bud differentiation, proliferation, rooting, bulblet formation and transplantation in Oriental lily. The result showed that the Sorbonne adventitious bud differentiative effect was the best on the MS medium supplemented with 1.0 mg/L BA and 0.5 mg/L NAA, the differentiation coefficient was up to 7.32, and the proliferative effect was the best on the MS medium supplemented with 0.5 mg/L BA, 0.3 mg/L NAA, 0.5 mg/L IAA and 0.05 mg/L KT, the proliferative coefficient was up to 3.59; and the Siberia adventitious bud differentiative effect was the best on the MS medium supplemented with 1.0 mg/L BA and 0.3 mg/L NAA, the differentiation coefficient was up to 7.11, and the proliferative effect was the best on the MS medium supplemented with 0.5 mg/L BA, 0.1 mg/L NAA, 0.3 mg/L IAA and 0.2 mg/L KT, the proliferative coefficient was up to 3.74. The best rooting and bulblet formation medium of Oriental lily adventitious bud was the 1/2MS supplemented with 0.3 mg/L NAA and 90 g/L sucrose, the rooting rate was 100%, and the diameter of the scales were between 1.20-1.25cm. The survival rate was up to 93.33% after transferring into the mixture of perlite,sand and peat soil(2:1:1).2. Virus detection of Oriental lily. The leaves of tissue culture seedlings of Oriental lily were used as materials to detect main virus on lily, such as cucumber mosaic virus(CMV), lily mottle virus(LMOV) and lily symptomless virus(LSV). The result showed that both Sorbonne and Siberia had LMOV and LSV, but CMV was not picked up by multiplex-RT-PCR detection. The homolgy relationship identification showed that the LMOV and LSV had high homolgy among other lily virus home and abroad. The homolgy was up to 97%, and the LSV was up to 100%. So they were highly conserved and without district difference. 3. Establishment of the system of shoot tip virus elimiantion by cryopreservation of Oriental lily. The Sorbonne shoot tips were used as materials to study some main factors on shoot tip survival rate in the cryopreservation treatment. The result showed that the highest shoot tip survival rate combination was: preculture 1 day, 0.5mol/L sucrose, loading time 30 minutes and PVS2 treatment in 60 minutes, the survival rate was up to 54.25%. At the same time, we also compared other factors: culture time, shoot tip size, liquid nitrgen treated time and throwing methords. The result showed that highly shoot tip survival rate were: culture 20 days, with 2 to 3 leaf primordium, liquid nitrgen treated 24 hour and throwing in 40℃water. Moreover, we proved the virus elimination mechanism by cryopreservation through shoot tip paraffin section observation at different stages.4. Shoot tip virus elimination by cryopreservation and effect detection. The tissue culture seedlings of Sorbonne and Siberia with LSV and LM0V were used as materials to study the influence factors on survival rate and virus elimination rate , the length of shoot tips were <0.3mm, 0.3-0.5mm, 0.5-0.8mm, 0.8-1.0mm and 1.0-2.0mm respectively. The result showed that it was the best to choose 0.5-0.8mm shoot tip as virus elimination materials based on the high survival rate and virus elimination rate, the virus elimination rate was up to 43.33% in Sorbonne and 40% in Siberia, the survival rate were 50%. The length can not only guarantee survival rate, but also virus elimination rate, conformed to large scale production requirements.5. Detection of genetic stability on regeneration plants after virus elimination by cryopreservation. The experiment choosed 10 primers used for RAPD analysis of randomly selected 10 Oriental lily regeneration seedlings after virus elimination by cryopreservation. The result proved that somaclonal hereditary stability after virus elimination by cryopreservation was almost preserved, only appearing little variation. No band differences were found between regeneration seedlings and contrast seedlings in Sorbonne. But there were little differences in Siberia, the mutatioal rate was 1.6%. The reason of mutational may realated to varities or low temperature stress.As a whole, in this study the system of the regeneration in vitro was established in Oriental lily; lily virus were detected by multiplex-RT-PCR;Shoot tip virus elimination by cryopreservation was first used to Oriental lily, and virus elimination technical system was established.It proved that shoot tip virus elimination by cryopreservation was feasible through the virus elimination rate detection on the regeneration seedlings; it used RAPD technique to analysis the genetic stability on regeneration plants after virus elimination by cryopreservation, which would be of great importance for rapid propagation in vitro in Oriental lily, virus detection and virus elimiantion.