Vitrification Cryopreservation Of Embryonic Callus Of Agapanthus Praecox

In this paper, cryopreservation of embryogenic callus of Agapanthus praecox ssp. orientalis’Big Blue’was studied. The results are as follow:(1) Embryogenic callus of Agapanthus praecox were successfully cryopreserved by vitrification procedure. Small, fragile, loose embryogenic callus clumps of50±10mg in subcultured for20days were cultured for2days on a solid Murashige and Skoog (MS) medium containing0.5mol/L sucrose with no plant growth regulators at4℃, and250±10mg embryogenic callus were then placed in2mL cryovials, to each of which2.0mL of loading solution or vitrification solution (PVS2) was added. After either60min at room temperature or40min at0℃in these solutions, the callus were then plunged into liquid nitrogen. After1h, they were rapidly rewarmed by90s immersion in a40℃water bath and subsequently the PVS2was drained off. Embryogenic callus clumps were then washed in the cryovials with MS liquid medium supplemented with1.2mol/L sucrose,10mmol/L KNO3with three changes of liquid medium, each of10min, before being placed on proliferation medium. The highest survival (TTC) of embryogenic callus was56.9%, embryogenic callus were proliferated normally.(2) Genetic stability of cryopreserved embryogenic callus after55days were assessed by AFLP molecular marker technique with64combination formed8forward primer and8reverse primer. Nearly3086bands were scored in total, which6bands were differential in6%nondenaturing polyacrylamide gel. Less1%differences of polymorphic bands was observed between the band patterns of the control and that of the cryopreserved samples.(3) Physiological and biochemical changes in vitrification cryopreservation, including the water content, relative conductivity, malondialdehyde concentration, soluble sugar, starch and soluble protein levels were studied during process of preculture, osmoprotection, dehydration, rapid warming, recovery after24h. Further, the correlation between physiological indexes, physiological indexes and cell survival rate were analysed. The results indicated that the water content of embryogenic callus fell significantly before freezing. The water content decrease into41.5%before refrigeration in liquid nitrogen, while a drastic increase in recovery after24h. The variables of relative conductivity coincided with malondialdehyde, which the trend continued to rise from preculture to rapid thaw until declined slowly in recovery after24h. The soluble sugar content was bimodal curve changes. The changes of starch is consistent with soluble protein, which their changes reduced slowly, then increase significantly, finally descended markedly. Moreover, the multiple correlation analysis suggested that relative conductivity and malondialdehyde were positive correlation dramaticlly, and the cell survival rate and the relative conductivity, malondialdehyde were significantly negative correlation and negative correlation significantly, which correlation coefficient were0.999and0.896, respectively. Plasma membrane injury was a key factor, which influenced the survival rate in vitrification cryopreserved embryogenic callus significantly.(4) It is remarkable to add exogenous antioxygen for improving the survival rate of callus cell by contrast with procedure without exogenous antioxygen, melatonin, abscisic acid, glutathione optimized the system of vitrification cryopreservation of embryogenic callus by improving the cell survival rate74.7%,71.6%and59.2%, respectively. The result demonstrated that oxidative damage is the main factor in vitrification cryopreservation of Agapanthus praecox embryogenic callus.