Improvement Of Mantle Cell Culture And Technique For Nucleated And Large Pearl Producing In Hyriopsis Cumingii

Hyriopsis cumingii Lea is one of the most important species of freshwater pearl oyster in China. It becames the first choice because of the shining, glossy, smooth pearl it produces with the highest quality. At present, China produces the most amount of world fresh water pearls, but sales only up to 18%, which is seriously unbalanced between the supply and demand. The small size and low quality are the main reasons leading to the contradiction.In order to obtain large and high quality pearls, the methods of the pearl culture in visceral mass combined with inserting large pearl-nucleus co-cultured by dissociating mantle cell were studied. The main contents and results are as followed:1. The impacts of Ca²⁺ and bFGF on the mantle cell culturea) The mantle cell viability and ALP activity were detected by adding different concentrations of Ca²⁺ (0 mmol / L, 0.5 mmol / L, 1.25 mmol / L, 3 mmol / L.) into the cell culture medium. The results showed that the variation tendency was similar:with the increasing of Ca²⁺ concentration , the two activities gradually increased and got up to the highest at the Ca²⁺ concentration of 1.25 mmol / L. while began to get lower at the concentration of 3 mmol/L. The ALP activity and cell viability showed significant difference when the Ca²⁺ concentration of 1.25 mmol/ L(P<0.05), and meanwhile the cell state and adherence were better then the other three groups under the microscope. So, we got a conclusion that the Ca²⁺ concentration of 1.25 mmol/ L is suitable for the mantle cell culture of Hyriopsis cumingii.b) The mantle cell viability and ALP activity were detected by adding different concentrations of bFGF (0μg / L, 0.3μg / L, 0.6μg / L, 0.9μg / L) into the cell culture medium. The results showed that the variation tendency was different: With the increasing of bFGF concentration, the cell viability rose gradually and peaked at the concentration of 0.6μg/ L, but obviously decreased at 0.9μg/ L; The ALP activity rose with the increasing of bFGF concentration, but it peaked at 0.9μg/ L. In all experimental groups, the capacity of cell proliferation had a rise especially in 0.6μg/ L, and that showed 0.6μg/ L was the most suitable concentration of bFGF. High concentration would have an inhibitory action in cell proliferation. While the ALP activity rose with the increasing of bFGF concentration and peaked in 0.9μg/ L2. nucleated and large pearl producing after improved cell cultureIn order to obtain large and high quality pearls, the methods of the pearl culture in visceral mass combined with inserting large pearl-nucleus co-cultured by dissociating mantle cell were studied. The mantle cell was cultured by two different culture mediums that were named for medium 1 and medium 2. The cell viability was detected at the different time points (2, 4, 6 and 12 h) during the process of culture. Then large pearl-nucleus was co-cultured by the two different dissociating cell groups. 500 Hyriopsis cumingiis, divided into two groups, were separately inserted with the two kinds of co-cultured pearl-nucleus in their visceral mass.The results showed that the cell viability significantly increased with the cultured time in both of culture mediums . The cell viabilities at 6h and 12h were both obviously higher than those at 4h and 2h (P<0.05), the difference of the cell viability was insignificant between the group of 6h and 12h (P>0.05). As for the two different culture mediums, the difference of the cell viability at 2h was insignificant (P>0.05). While, at 4h,6h and 12h, the cell viability of medium 2 was apparently higher than that of medium 1 (P<0.05).The pearl-nucleus co-cultured by the medium 2 was attached with more cells than that by medium 1. After 5-month cultivation , large and glossy pearl with 0.8 mm nacrum was obtained in the group of medium 2. While, the rate of pearl-nucleus without nacrum in the group of medium 1 was much higher than that in the group of medium 2. The research indicated that the improved culture medium was more beneficial to the mantle cell culture and the nucleated pearl culture in visceral mass of Hyriopsis cumingii, which provides practical basis for the mantle cell culture and theoretical foundation for the large nucleated pearl culture in freshwater mussels.