Studies On Tissue Culture, Cell Culture And Metabolism Regulation Of Flavonoids Production In Ginkgo Biloba L.

Ginkgo(Ginkgo biloba L.) is a sole representative of the genus Ginkgoales that appeared in the Permain era. It is a valuable resource that has been used in health care and fruit industry, and also was deemed as a beautiful plant in landscape art. Ginkgo leaves contain some pharmacological activity constituents such as flavonoids and terpene trilactones (ginkgolides and bilobalide), which are functional in treating the cerebral and peripheral arterial vascular disturbances of the blood supply, especially for people at senior age. To facilitate the industrialization process of this valuable secondary metabolism product by means of cell culture, we established the callus induction protocol to obtain the asepsis leaves resources by using mature embryos and young shoots from female or male trees as initial explants. Callus and suspension culture systems using these asepsis leaves as initial explants were also established. Effects of two elicitors, La(NO3)3 and Salicylic acid, on callus and suspension cells growth and the flavonoids synthesis were studied by adding different concentration of these chemicals. The main results from study are as follows: 1. In tissue cultivars of Ginkgo biloba L. ,three basic mediums B5, WPM and DCR for mature embryos were screened. There was no significant difference in their effects among the cultivars. The results in this study suggested that DCR was the most suitable cultural medium for mature embryos. 2. Adventitious roots were found from ten days old cotyledons cultured in MS+NAA0.2mg/L+BA0.5mg/L and MS+NAA0.2mg/L+KT0.5~1.0mg/L,whereas adventitious buds were found in cotyledons cultured in MS+NAA1.0 mg/L +KT2.0 mg/L. The induction frequency was about 5%. In addition, adventitious roots were also found in young leaves and immature pollens. 3.There existed position effect for young Ginkgo shoots. Tip of the Ginkgo shoot with terminal buds showed a height growth and leaf expansion; the middle section could induced axillary buds and adventitious buds; and the bottom section induced two or three cotyledon axillary buds and adventitious buds. Results showed that N6 and modified MS were the most suitable cultural medium for terminal buds, axillary buds and cotyledon axillary buds. The optimal concentration of NAA was 0.1~0.5 mg/L and that of BA was 0.5~1.0mg/L. The number and area of leaves from terminal buds cultured on media with hormone were more and larger than that no hormone if consequently subcultured in MS medium without hormone . The induction rate of axillary buds and area of leaves on media with La(NO3)3 was higher than that on media without it . This result will help supply resources of explants for producing more secondary metabolism in cell culture. Auxion content among three cut sections of one-month-old Ginkgo shoots showed that was highest in the terminal buds; cytokinin was highest in the middle sections , bottom sections and cotyledons. 4. Among the five cultivars, cultivarl No. 44 grown on 1/2MS+0.5mg/L IBA had the highest induction rate (33.3%) of adventitious roots. Not only the rates of adventitious roots were significantly increased, but also rooting time was 7 days earlier than control when adding 0.5~5mg/L of rare earth. The highest rate of rooting was up to 66.7%. However, high concentration (10~20mg/L) of rare earth caused a decrease in the rooting rate. 5. Results showed that the optimal medium for leaves induction callus was MS+NAA1.0+BA0.5; The optimal medium for subculture callus was MS+NAA1.0+KT0.5; The contents of flavonoids in callus induced from roots, leaves and cotyledons were higher than those of callus induced from other tissues. After adding La3+ to callus with different concentration , the content of soluble protein, nucleic acid, Polyamines and cell activity in callus increased in early days and decreased in late days in 20 days'cultural period. The peak of peroxidase (POD) activity appeared in the tenth day and was 5 days earlier than that of phenylalanine-amonialysae ( PAL) activity, which appeared in the fifteen days . 1 mg/L La3+ increased the activity of peroxidase isoenzymes in band Ⅲ, band Ⅳand band Ⅴ. Especially, isoenzymes band Ⅲwere observed in 10~15d, but this band was absent in the control. This phenomenon suggested that the tenth day of callus culturing might be the key time that primary metabolism changed to secondary metabolism. The flavoniods content was 1.25 % under 0.5 mg/L of La3+, which was 24% higher than control. However, high concentration La3+ (10~20mg/L) caused a decrease in flavonoids content. 6. Addition of salicylic acid increased soluble protein, nucleic acid content, the callus growth and cell activity in early days and then decreased . The peak of POD activity, CAT (Catalase ) activity ,PAL activity and Put(putrescine) content were observed at the sixth day or the ninth day during the culture. These peaks appeared earlier than those of the control. The results indicated that adding salicylic acid could shorten the time of primary metabolism changing into secondary metabolism, showing significant correlations among these indications at the 12th day. The content of flavonoids reached the highest level at a concentration of 0.5~5mg/L salicylic acid treatment. It was 1.87% at 0.5 mg/L and 1.90% at 5mg/L, respectively, which was 19% and 21% higher than that observed in the control. 7. Three flavonoids constituents (quercetin, kaempferol, isorhamnerin )were isolated from Ginkgo suspension cells. Total flavonoids content cultured under light was higher than in thedark. Not only the total flavonoids increased, and the proportion of three flavonoids constituents changed after the addition of rare earth and salicylic acid into the liquid mediums. 8. The results indicated green callus cells were in meristematic tissue period. It contained more protein and observed more bands than….. These cells were rich in mitochondrions, dictyosome and endoplasmic reticulum, while flavonoid content was observed lower than that in the yellow callus. The yellow callus cells were in period of primary metabolism changing into secondary metabolism. The content of protein was lower than that in green callus, especially 45.7KD ,38.4 KD ,33.4 KD were decreased. But it had higher content of flavonoids. Caryotheca was not in its integrity, and had big Starch grain. These were characteristics of callus in secondary metabolism period. The brown callus contained lowest protein and flavonoids. We observed that its protein bands decreased and five of the bands disappeared. It was also observed that the plasmolysis was appeared and many cellular organs were in disintegration, thus these cells were in senescence period.