Analysis of ultrastructure and reactive oxygen species of hyperhydric garlic (Allium sativum L.) shoots

Hyperhydricity is a physiological disorder frequently affecting shoots propagated in vitro. Since it negatively affects shoot multiplication vigor, and impedes the successful transfer of micropropagated plants to in vivo conditions, hyperhydricity is a major problem in plant tissue culture. In commercial plant micropropagation, there are reports of up to 60% of cultured shoots or plantlets which demonstrate hyperhydricity, which reflects the pervasiveness of this problem. The phenomenon has been correlated to water availability, microelements, and/or hormonal imbalance in the tissue culture. In this study, the ultrastructure and the characteristics of reactive oxygen species between hyperhydric and normal shoots of garlic were studied. We observed that in some cells of hyperhydric tissues, the intranuclear inclusion was separated, the mitochondrion was swollen and its intracristae had splits, the organelles were compressed against the cell wall, and the chloroplasts and intergranal thylakoids were also compressed. Additionally, the content of chlorophyll and soluble protein in hyperhydric shoots decreased significantly. For instance, chlorophyll a decreased 43.61%, chlorophyll b decreased 49.29%, chlorophyll a+b decreased 48.10%, and soluble protein dropped 47.36%. In contrast, the O₂ generation rate and H₂O₂ level increased 45.36% and 63.98%, respectively, obviously higher than the normal shoots. Lipoxygenase activity and malondialdehyde content in the hyperhydric shoots increased significantly, while the electrolyte leakage rose, indicating a serious membrane lipid peroxidatic reaction. Superoxide dismutase, peroxidase, catalase, glutathione peroxidase, and ascorbate peroxidase activities in hyperhydric tissue were all significantly higher than in normal leaf tissue. The antioxidant metabolism demostrated a close connection between hyperhydricity and reactivated oxygen species.