Studies On Cryoperservation Of Petunia (Petunia×Hybrida Vilm.) Germplasm By Droplet-vitrification

Petunia (Petunia×hybrida Vilm.) is very famous flower beloved by the people all over the world because of its beautiful flower-shaped, color and diversity. Petunia is not only the important plant germplasm as for ornamental horticulture, but also the vital model materials for genetic engineering purpose, especially for the flowering genes. Petunia can be stored by seeds, however, for some hybrids, it is better that some unique characteristics of germplasm were kept through the vegetative propagation. The germplasm of petunia can be kept in the plant gardens and in vitro genebanks, however, it consumes lots of time, labour and material resources. With the rapid development of the cryopreservation techniques for the plant materials, the cryopreservation has become the crucial methods for safe and long-term conservation for plant germplasm. Until now, the cryopreservation has been used for more than200species. The shoot tips are the optimal materials for successful cryopreservation. Thus, the cryopreservation procedures needs to be developed for petunia. And furthermore, the petunia is an appropriate material for study on the mechanism of the cryopreservation in terms of its relatively clear genetic background knowledge and short-life cycle.In this study, shoot tips from in vitro petunia variety 'Niu2' were successfully cryopreserved by droplet vitrification method. Orthogonal design was used to analyze effects of dominant parameters on survival and regenerated rates of shoot tips, and to obtain the primary cryopreservation procedures. And then, the cryopreservation procedures were optimized by one-factor experiments and then used for the other3petunia cultivars. The ultrastructures of meristem cells during cryopreservation were observed and studied for further survey of mechanisms of cryopreservation. Finally, the genetic stability was studied by microsatellites markers in our experiment. The main results are as follows:1. Shoot tips of 'Niu2' were successfully cryopreserved with the droplet virtrification techniques. Procedure was established as follows:Shoot tips consisting2-3leaf primordia were excised from in vitro plants which had been subcultured in MS medium for about15-20days. Excised shoot tips were precultured in liquid MS medium supplemented with0.2M sucrose for2days, osmoprotected in loading solution for30min at25℃, dehydrated with PVS2solution for 30min at0℃, frozen in the micro-droplets of vitrification solution placed on aluminium foils, which were immersed rapidly in liquid nitrogen. Rapid rewarming was conducted in the MS liquid medium contained1.2M sucrose. Post-thawed shoot tips were transferred to regeneration medium and stored in the dark at25℃for lweek, then cultured under white fluorescent light at an intensity of700lux, with a16h photoperiod at25℃. The highest post-thaw survival rate and regeneration rate of cultivar'Niu2'was93%and81%, respectively.2The optimized droplet vitrification cryopreservation procedures were used for the other3petunia cultivars,'Niu1','Niu3'and'Niu4,'and the mean survival percentage is66%,78%and74%, respectively, and the mean regeneration percentage is55%,59%and53%, respectively.3Ultrastructure changes of apical meristem cells in the process of cryopreservation were observed using the transmission electron microscopy (TEM). The results showed that the main changes occured in preculture and dehydration before cryopreservation. The plasmolysis, the rapid development of mitochondrion, the swelling plasmid, the enriched nucleoplasm and the increased heterochromatin were observed and the tolerance for the cold and dehydration of the apical meristem cells might be promoted with the preculture and PVS2treatment. After cryopreservation, some cells were able to repair damage and regain their normal ultrastructure within2days in culture and could survive and regenerate plantlets, and some cells were extensively damaged and lethally injured.4The genetic stability of the regenerated plantlets for petunia after our droplet vitrification cryopreservation was detected by15pairs of microsatellite primers. PAGE gels showed that there are no obvious differences in the process of the tissue culutre for once and no obvious differences between the regenerated samples and controls.