| Sugarcane is a vegetatively propagated crop, vulnerable to disease infection for years of continuous cropping. Utilizing tissue culture method to produce pathogen-free and healthy seedcane is one of the most effective ways to control diseases in sugarcane. Extensive application of tissue culture techniques provides an effective means of rapid propagation and extension of improved sugarcane varieties, not only greatly increases the propagation coefficient, but also saves seeds and improves cane and sugar production because of removing seedcane pathogens and improving the resistance of sugarcane. However, browning of explants is the main cause of failure of tissue culture in healthy sugarcane seedcane production.In the present study, we used the sugarcane variety ROC22 as the explants, the browning rate and the content of polyphenols, and the activity of polyphenol oxidase (PPO) and peroxidase (POD) in the explants from buds at different positions and different time of budding, and the contents of gallic acid, catechin, chlorogenic acid, and epicatechin gallate in the browning and normal tissues were determined with HPLC, and the ultrastructure and microstructure of browning and normal materials were also observed. The relationship between browning and the physiological and biochemical parameters were analyzed. The main results are as follows.1. The content of polyphenols and the activities of PPO and POD in the explants varied for buds at different position of stalk and different time of budding. The content of polyphenols showed changes from high to low and then high again. The activity of PPO also had a falling domw and rising up process. The activity of POD changed more complicatedly, but it showed consistent for the buds on the nodes 2,3,5 and 6 (namely, buds 2,3,5 and 6). The content of polyphenols was alwasys higher in bud 1 than in other buds, but the activity of POD was just the oPPOsite, always higher in bud 1 at any time of budding. The analysis of significance showed that the browning rate had a significant and positive correlation with the activity of PPO and POD, but not with the content of polyphenols. The correlation between the content of polyphenols and the activity of POD was significant and negative.2. The ultrastructure and microstructure observations for the explants at different growth status and different culture stages showed that the normally growing cells maintained basic normal morphology for their internal organelle. Microstructure observation show that, whether normally growing or browning tissues, the tips kept developing, but the browning tips grew more slowly than the normal tips. Ultrastructure observation showed that the functions of various organelle are well inside the cells in the normal materials, and the cells and tissues showed normal growth and differentiation. For the organelles inside the cells in the browning materials, however, the organelle showed changes in varying degrees, for example, the tonoplast’s boundaries began blurring, the structure of mitochondria was deforming and breaking from the outside to inside, the nucleus and nucleolus are deforming or degrading, the cell wall is expanding and fracturing, and the cells died eventually.3. The composition and contents of polyphenols, were quite different between the normal and browning sugarcane stem tips at different culture periods. The contents of the tested four polyphenols, that is, gallic acid, catechin, chlorogenic acid and epicatechin in sugarcane stem tip were found the highest in 0 day. The contents of gallic acid and chlorogenic acid were higher in the normally growing materials than in the browning materials for those inoculated at the same time. The contents of catechin and epicatechin were also higher in the normally growing materials than the browning materials except at day 9. At different days of culturing, the content of epicatechin was the highest among the four polyphenols in sugarcane stem tip. |
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