Studies On The Mechanism Of Leaf Explant Browning In Phalaenopsis In Vitro

Tissue browning and blackening are major impediments of in vitro culture of plant. When cells are damaged, the contents of cytoplasm and vacuoles are mixed. Oxidized phenolic compounds may inhibit enzyme activity and result in darkening of the culture medium and subsequent lethal browning of explants. The objective of our study was to elucidate the harmful browning of tissue culture of Phalaenopsis. The main results were as following:Medium has enflencen on explant browning. The browning ratio of Phalaenopsis leaf explant in vitro growning on MS liquid paper bridge and half-strengh MS medium (1/2MS) is less than that growning on MS, B5 and N6. Explant on the medium turned yellow without 6-BA. With the change of the components of MS which can be achieved by doubling the content of FeS04 or exorcize the miroelement, the degree of explant browning became more obvious and explant excreted lots of brown substances in medium. While the microelement in MS medium doubled, the browning of explant on medium alleviated. Compare to control, the content of agar was reduced to half, explant browning became serious. The higher the intensity of light, the more serious explant browning. Treated with AA or PVP had proved to be no influence on explant browning. When the medium was added 2000mg/L or 1000mg/L AC, the explant cultured there turned yellow, while explantcultured on medium supplemented with citric acid showed a considerable reduction in browning.In the early stage of explant browning, the activity of PPO & POD became high, while in the later stage, their activitives declined. Study PPO & POD isoenzyme, the results showed that after 2 days' culture, four bands of POD in browning cell appeared and they exhibited no obvious change as the culture period extended. For PPO, the number of its bands varied during the culture period.As explant turned black, the content of total phenol compounds was increasing and the activity of PAL was rising. The content of chlorophyll a and b decreased as explant turned black, in accordance with the colour change of explant. This phenonmenon demonstrated that the content of total phenol and the activity of PAL had a very important relationship with explant browning.The activity of CAT and SOD decreased after inoculting and increased as browning initiated. The content of MDA in explant was two times as that of control after 2 days' culture, and maintained a high level during explant browning. This demonstrated that the membrane has been peroxidation and the intact of membrane has been destroyed.Stereoscan photograph showed that the vascular bundle of brown cell filled with tannin, and the epicuticula shriveled, but the lower epidermis did not exhibit any changes. There were something deposited in parenchyma cell. Under the light microscope, section of browning explant showed that the number of chloroplast was less than that of control. The vascular handletook on deep colour when stain with FeCl3.The colorimetric assay demonstrated that the substance extracted from medium was condensed tannins, and there were four elements in acetone extract through mass spectrography method.