| It is a new method to produce some secondary metabolities by using cell suspension culture to enhance the contents of plant secondary metabolities. In this text, choose the table grape Kyoho, wine grape Cabernet Suavignon and Pinot Noir as materials, B5 medium as basic medium, by modulate the hormone concentration and ratio to establishe the suspension culture, and optimized the condition of suspension culture. Through added different elicitors such as sucrose, phenylalanine, methyl jasmonate and UV-C treatment to the cell suspension culture to study the influence on the cell growth and the total phenol, resveratrol and anthocyanins contents of grape Kyoho. It supplied the industrialization theory for using cell suspension culture to produce secondary metabolities.The results showed as below:1. Established the stable grape cell suspension culture: Choosed the hormone concentration and ratio, pH, sucrose concentration, and etc that suit the grape calluses growth. Different grape varieties need different hormone ratio and concentration. The suitable medium for inducing calluses is B5+250㎎·L-1 hydrolyze casein+0.1㎎·L-1 NAA +0.2㎎·L-1 KT +30g·L-1sucrose +6g·L-1agar, while suitable liquid medium for suspension culture for Kyoho is B5+0.5㎎·L-1 6-BA+1㎎·L-1 NAA.+30 g·L-1sucrose, for Cabernet Sauvignon is B5+250㎎·L-1 hydrolyze casein +2㎎·L-1 2,4-D+0.2㎎·L-1 KT+1% PVP+30g·L-1sugar, for Pinot Noir is B5+1㎎·L-1 NAA+0.5㎎·L-1 6-BA+0.2% PVP+30g·L-1sugar.The vigor of both calluses and suspension cells are Kyoho > Cabernet Suavignon > Pinot Noir. The suspension cell of Kyoho is thick, equal, stable with yellow-white color, existed in the form of single cell and little cell congeries that suit to have induce treatment, While CS and Pinot Noir have some tissue congeries, dark color, and easy to browning.2. Selected the suitable inoculation method for suspension culture: Selected the fresh, soft calluses 1520g that have continue inoculated at least 3 times.and added them into triangle bottle to have surge culture in the rocking incubator at the condition of darkness, 25℃, and 120rpm, .Keep them quiet after 24h, and transfered the upper cell into empty triangle bottle continue cultivate and abandon the large cell congeries. Repeat the action after 3 to 4 days if necessary. After one week some fresh liquid culture medium were added till get to a certain quantity equal and stable suspension culture. In the future when inoculate, 5～10g fresh cell per 40mL medium liquid culture is enough.3. The method of preventing or alleviating the browning: Studyed the methods of preventing &alleviating the cell browning through the calluses selection, inoculation control, bottle changing, PVP and adsorptive resin utilizing, saving, and etc.4. The influence of inducing treatment on cell growth:All kinds of inducing treatment have no advance for suspension celles growth, on the other way around they can aggravate browning.5. The influence of sucrose on polyphenol synthesis of Kyoho suspension cell: The total phenol contents increase along with the sucrose increase yet it make browning heavy. there are little influence on anthocyanin, 30g·L-1 sucrose can light improve while 7.5 g·L-1 and 15g·L-1 can light inhibit the anthocyanin contents. For stilbenes, the sucrose concentration if proper can enhance the stilbenes contents, if too high,it will inhibit stilbenes contents. 15g·L-1sucrose can notablely enhance the resveratrol contents.6. The influence of phenylalanine on polyphenol synthesis of Kyoho suspension cell: Adding phenylalanine can notablely enhance the contents of total phenol, anthocyanin and resveratrol.7. The influence of methyl jasmonate on polyphenol synthesis of Kyoho suspension cell: Adding methyl jasmonate can enhance the contents of total phenol and resveratrol, but have slight inhibition on anthocyanin.8. The influence of UV-C treatment on polyphenol synthesis of Kyoho suspension cell: The polyphenol contents can be enhanced by certain intensity UV-C treatment. total phenol, anthocyanin and resveratrol can be notablely enhanced at the condition of 41.5μW·cm-2, radiate for 2 hours, at the distance of 35cm. The UV light is: D=2.5cm, L=88 cm , P=30W.
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