| Tree peonies are Chinese traditional famous flowers and medicine.Paeonia ostii var.lishizhenii is strong resistance to abiotic stress,widely cultivated throughout the country and mainly used as medicinal materials and horticultural rootstocks.The peony seed has developed into a new woody oil crop at the beginning of this century.It's multipurpose for medicine,ornamental and edible oil has become a high-quality tree species with stress resistance for accelerating rural rejuvenation in landscaping and seed oil production.This thesis based on the researches of P.ostii var.lishizhenii since 1996,established in vitro cell culture system,and explored shoot regeneration for genetic engineering and paeonol production for a secondary metabolite cellular engineering.The main results are follows.1.Establishment of aseptic in vitro cell culture system P.ostii var.lishizheniiThe mature and plump superior seeds of P.ostii var.lishizhenii were selected,peeled,soaked in 70%ethanol for 60 seconds and 1.5%sodium dichloroisocyanurate solution for 30 minutes,and inoculated on MS medium soiled with agar 6 g/L,the seed decontamination rate was as high as 98.89%.Sterilized embryos and tissue culture shoot leaves were placed on MS medium added 6-BA 1.5 mg/L and 2,4-D 2.0 mg/L,callus induced with rate of 90.00%and 82.22%,respectively.While the shoot stems placed on MS medium added 6-BA 1.0 mg/L and 2,4-D 2.0 mg/L,calli were induced with rate of 85.55%.2.In vitro cell culture system for shoot regenerationCalli of P.ostii var.lishizhenii were inoculated on the factorial experimental MS media cooperated with cytokine 6-BA(1.0,2.0,3.0 mg/L)and auxin NAA(0.1,0.2,0.3 mg/L),respectively.The plant growth regulators did not effected significant difference on shoot regeneration(p>0.05),though the highest rate of shoot regeneration achieved 13.33%on MS medium with 6-BA 2.0 mg/L,NAA 0.1 mg/L or 6-BA 3.0 mg/L,and NAA 0.3 mg/L.3.In vitro cell culture system for a secondary metabolite paeonol accumulationP.ostii var.lishizhenii callus was cultured on solid medium.Some factors,such as medium types,sucrose,hydrolyzed casein,agars,pH and sub-culture time effected significantly on cell proliferation(p<0.05);while media types,6-BA,sucrose and agar affected significantly on callus browning(p<0.05).Optimized medium for cell culture was this,the modified WPM(Wang and Staden,2001)cooperated with 6-BA 1.8 mg/L,NAA 0.6 mg/L,sucrose 30 g/L,hydrolyzed casein 0.4 g/L,agar 5 g/L and pH 7.3.The callus cultured in dark were milky white,grew with a S-shaped curve,logarithmically period in 10 to 30 days,a steady-state on 30 to 45 days.In the conditions cell proliferation ratio was 3.67 and browning rate was 11.78%at 35 days.While the secondary metabolite paeonol determined each 5 days was high at 25 d with 1.75±0.13 mg/g.P.ostii var.lishizhenii cell suspension culture was influenced significantly by initiation cell density,concentrations of 6-BA,sucrose,hydrolyzed casein and inositol in the cell fresh weight,dry weight and proliferation ratio of cell proliferation,while medium types had a significant effect on cell dry weight(p<0.05).Optimized culture system was that calli 12 g/L(2 mm x 2 mm x 2 mm)cooperated in the solution with 6-BA 0.5 mg/L,NAA 1.0 mg/L,sucrose 25 g/L,inositol 0.1 g/L,pH 5.8 in WPM medium,shaked 100 r/min,dark culture;suspension cells grow in S-shaped curve,0-10 d was in lag phase,10-30 d in logarithmic growth stage,and 30-40 d roughly in steady-state stage,respectively.When cultured for 30 days,the suspension cell proliferation ratio was 4.76,and the content of paeonol,a secondary metabolite,was the highest,achieved 2.65 ± 0.25 mg/g. |
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