Studies On Protoplast Islation And Cuture Of Lilium Spp.

Lily is one of perennial bulbous flowers, which belong to the Liliaceae of Lilium and is one of the great demanded flower in the world. There are some orthodox breeding methods, such as rule of divide bulb, divide bulblets, scales cutting, scales embedment. But adopted these methods which propagation coefficient was even little, especially which usually resulted variety degeneration and virus buildup, and influenced yield and quality of lily. We can removed virus, refreshment varieties, accelerated speed of intermediate propagation of lily and decurtated breeding cycle of lily which adopted tissue culture technique.Research of protoplast succeed in protoplast culture with Takebe in 1971 when protoplast of tobacco had gotten regeneration plants. The protoplast is homoplasmic in heredity from the same plant, so it can provided favourable experiment system for cell biology, developmental biology, cell physiology, cell genetics and some other biology subjects; protoplast could overcame sexual cell incompatibility and progressed distance Cell Hybridization; protoplast was ideal recipient with studied on genetic transformation which could intake exogenous DNA, cell organ, virus and plasmid. So plant protoplast had wide usage in alteration heritage of plants, exploratory development of improved crop species and foundation theory research of biology.The experiment used three species Bernini, Elite and Lilium lancifolium Thunb. as explants, and set up direct differentiation regeneration system and callus regeneration system, base on this, we set up the system of protoplast isolation and protoplast regeneration. The result are as follows:1. Established direct differentiation and callus differentiation regeneration system: Taked bulbs as explants, direct differentiated adventitious bud on the culture medium of MS+6-BA0.5mg/L+KT 0.1mg/L+NAA1.0mg/L, induced roots on the culture medium of MS+IBA0.5mg/L. Taked young filements as explants, induced callus on the culture medium of MS+2,4-D2.0mg/L+LH300mg/L, differentiated on the culture medium of MS+6-BA 0.5 mg/L+KT 0.1mg/L+NAA 1.0mg/L and induced roots on the culture medium of MS, embryonic callus can shaped embryoid in continue culture.2. The experiment used mannitol as osmotic pressure stabilize, we adopted plasmolytic method to determine the osmotic pressure of lily leaves, was 0.6mol/L.3. The experiment indicated that digestion time was 2 hours, and the best enzyme solution was Cellulase"Onzuka"RS 2%(Yakult honsha, Tokyo)+Pectinase Y-23 0.1%(Yakult honsha, Tokyo)+Macerozyme R-10 0.5%(Yakult honsha, Tokyo)+CaCl2·2H2O 10mM+MES 5mM+mannitol 0.6mol/L(pH5.6). The digestion condition was incubated for 2 hours on a rotary shaker at 40 rpm. in dark conditions at 25℃.