Chicory Protoplast Culture And Establishment Of Transient Expression System

Chloroplast is a type of very critical organelles in plant cell, it is the reaction site of photosynthesis and it has been attached importance to related research. Chloroplast inheritance is maternal hereditary process, compared to traditional nucleus transformation it has such advantages include exogenous gene expression in chloroplast can reach high level, biological safety and no possibility of gene flow, and there is no gene silencing has been observed whether at the transcriptional or translational level. Chloroplast transformation has become a new trend in genetic engineering research. This study obtained the following results:(1) Optimized chicory leaf in vitro regeneration system. We studied the influence of different hormones combination and different concentration to chicory leaf adventitious bud and root occurrence. The results showed that optimum medium for adventitious bud occurrence is MS medium supplemented with 1.5 mg/L BAP+0.5 ml/L IBA+3%Sucrose, and optimum medium for root occurrence is 1/2 strength MS medium supplemented with 0.2 mg/L NAA+3%Sucrose.(2) Induced chicory embryogenic callus, induced effects of cotyledon is preferable than other types of explant. The culture medium for embryogenic callus induction is MS medium supplemented with 0.2 mg/L BAP+2.0 mg/L 2,4-D+2%Sucrose+0.5% Casein hydrolysate.(3) Studied influence of different factors include enzyme type combination, mannitol concentration, different time of enzyme digestion to chicory mesophyll protoplast yield and activity. Optimized time of digestion is 10 h, optimized enzyme combination is 1%cellulase+0.5%himecellulase+1%pectinase, optimized mannitol concentration is 0.5mol/L, by tear off epidermal can harvest more chicory mesophyll protoplast.(4) Culture of chicory protoplasts from mesophyll cells. Chicory mesophyll protoplast cell division can be observed on DPD medium supplement with 0.5 mg/L BAP+2.0mg/L 2, 4-D+2% sucrose+0.45mol/L mannitol, yet regeneration system is not established. Other culture mediums include MS medium, B5 medium,1/2 strength MS medium are unsuitable for chicory mesophyll protoplast culture.(5) Transformed chicory chloroplast via PEG treatment and by biolistic bombardment and established chicory protoplast mesophyll protoplast transient expression system. Transformation of chicory protoplast via PEG treatment can be successfully proceed, efficiency is approximately 40%. GFP fluorescence signal driven by plasmid containing 35S promoter can be observed in chicory protoplast under laser confocal microscope sight.