| Hyperhydricity is a morphological distortion and physiological disorder occurred generally in plant tissue-culture. It would badly result in abnormal growth and yield decline of the plantlets in vitro when it took place. Therefore, analysis the mechanism on physiological, biochemical and molecular levels of hyperhydricity shoots is very important for its control and recovery. Hyperhydricity is very easy to find in tissue culture of blueberry, and it would badly affect the factory production of blueberry. This research is about the changes on morphological structures, physiological properties and DNA methylation levels of hypethydric shoots of blueberry. Meanwhile, the effects on recovery for hypethydric shoots of dehydration, sugar and agar concentration, adding AgNO3and CoCl2in medium are also involved in this study. The main results are as follows:1. There had significant differences between normal shoots and hyperhydric shoots in microstructure and ultrastructure. Compared with normal leaves, hyperhydric leaves appeared thickened. The development of the epidermis was often discontinuous and the intercellular space was large. Stomata were abnormal. The leaf was characterized by the absence of palisade mesophyll tissue and the malformation of spongy mesophyll tissue. Vascular tissue of stem developed poorly. On the aspect of ultrastructure, the size of mesophyll cell and the thickness of cell wall increased, the nucleus and mitochondria were missed in some mesophyll cells in the hyperhydric leaves. Hyperhydric leaves had a significantly lower chloroplast number per cell and chloroplasts showed collapsed thylakoid stacking compared with normal leaves.2. Hyperhydricity resulted in a dramatic change about physiological and biochemical characteristics of blueberry shoots, such as a general increase of water content and decreases in the contents of chlorophyll, soluble sugar and protein. The production efficiency of O2ยท-, the level of H2O2, MDA and the relative conductivity in hyperhydric leaves were increased efficiently. The activities of antioxidant enzymes, such as catalase (CAT) and peroxidase (POD) were lower while superoxide dismutase (SOD) were higher in hyperhydric shoots than in normal shoots. The activities of phenylalanine ammonialyase (PAL) in hyperhydric leaves reduced. These results reflected a disorder in metabolic activity and a reduction in the ability of reactive oxygen removing in hyperhydric shoots of blueberry. 3. Dehydration, increasing agar contents (7.4g/L) and adding5mg/L AgNO3had the same efficacy in recovery reaserch. Some physiological indexes of recovered shoots were even higher than normal shoots. In addition, among all results the recovery rate was the highest when the materials were put in an empty flask for two days for reducing its water content4. Analyzing the differences in methylation patterns and levels of CCGG sequences between normal shoots and hyperhydric shoots with MSAP technology. The number and types of bands showed that the methylation ratio of the genome DNA in hypethydric shoots was66.22%, higher than it in normal shoots which was51.82%. Moveover, the ratio of the bands which were methylation-degree increased types (40.12%) was apparently higher than the decreased types (30.54%). Consequently, it seems that the change of methylation patterns and levels of genome DNA was closely related to the hyperhydricity phenomenon.This research laid the foundation for further elucidating the mechanism of hyperhydricity in blueberry shoots. At the same time, it contributed to studying the control and recovery methods of hyperhydricity phenomenon in blueberry tissue-culture, as well as other plantlets in vitro. |
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