Research Of Embryogenic Cells Suspension Culture And Protoplast Fusion On Dendranthema Indicum Var. Aromaticum

Dendranthema indicum var. aromaticum is one of the Chrysanthemum family and it is a new plant of wild resources. Its flowers, leaves and roots have the abundant of fragrance Folks often make sachets by the shade desiccated flowers、leaves.Now Dendranthema indicum var. aromaticum is one new of medicinal plant and it be worth of researching, developing and using,because its flowers and leaves have the effect of clearing heat and detoxicating. At present if we can transform the optimizing gene into the cultivation chrysanthemum, acquire the varieties of aroma,and achieve the new cultivating kinds. This thesis mainly discusses the established of the embryogenic cells suspension culture and protoplast culture system of Dendranthema indicum var. aromaticum, and preliminary discussion the protoplast fusion of Dendranthema indicum var. aromaticum and the chrysanthemum’xuegongzhu’culture in the open, this provide certain theoretical basis of the new varieties which have the good characters from not only the Dendranthema indicum var. aromaticum but also the chrysanthemum culture in the open. The main research results are as follows:(1) Established the cell suspension culture system by used the subculture callus of Dendranthema indicum var. aromaticum for material, the results show that:the suitable cell suspension culture of the initial quantity and liquid medium is2.0g/40mL liquid medium/100mL triangle bottles, the fresh weight of cells growth rate can amount to88.5%. The optimal cell suspension culture medium for Dendranthema indicum var. aromaticum is MS+0.5mg/L2,4-D+0.2mg/L6-BA,the subculture cycle is15～18d, and the cells vitality appeared first rise andthen decline with the extension of training time, the largest in the3d.(2) Using the tender leaves as materials, discusses the various factors what influence Dendranthema indicum var. aromaticum protoplast isolation, such as osmotic pressure regulator,enzyme varieties and concentration and so on. The experimental results show that: the advisable concentration of mannitol for Dendranthema indicum var. aromaticum protoplast isolation is0.6mol/L, the protoplast yield can reached4.39x106/g. The best combination of enzymes is:cellulose1.5%+pectinase0.8%+0.6mol/L mannitol+5mol/L MES+CPW solution, temperature (25±2)℃. The protoplast of Dendranthema indicum var. aromaticum appeared rising with the increasing of the enzyme concentration, and it vitality appeared descending trend with increased of enzyme concentration.(3) Research the enzymolysis time and method of Dendranthema indicum var. aromaticum protoplast,used the asepsis leaves as material. The results show that:the protoplast yield of Dendranthema indicum var. aromaticum leaves appeared first rising andthen decline with the extension of enzymolysis time,it peak arise at10h, reached4.36x106/g, and it vitality decreased with the extension of enzymolysis time. The protoplast yield of suspension culture cells appeared the same that is first rising and decline, its peak appeared in12h, the highest yield can reach4.69x106/g, it vitality also gradually down with the extension of time.(4) The best medium for Dendranthema indicum var. aromaticum protoplast cultivation is KM8P+1.0mg/L2,4-D+0.5mg/L6-BA+0.5mg/L NAA, PH5.8.It protoplast split rate can reach to7.26%. Protoplast training have used the methods of solid and liquid combined, the first division appear at the3d, after15d the split rate was6.23%, there has0.48%can form mass of cells.(5) Fusion the two protoplasts from Dendranthema indicum var. aromaticum and the asepsis leaves of chrysanthemum ’xuegongzhu’ culture in the open as material,used PEG combined Ca2+and PH method. The results show that:the best fusion processing time is30min for Dendranthema indicum var. aromaticum and ’xuegongzhu’, the rate of fusion is5.20%.