Study On Protoplast Fusion Of Panax Quinquefolium And Panax Notoginseng

Panax quinquefolium L.and Panax notoginseng.are perennial upright herbs of Araliaceae.Somatic hybridization can overcome the incompatibility and abortion of distant hybridization,and lay a theoretical foundation and provide technical support for breeding new varieties and germplasm innovation.In this experiment,the leaves of Panax quinquefolium L.and Panax notoginseng.were used as materials to induce callus through tissue culture technology.The main conditions and factors of callus induction were further analyzed.The obtained callus and embryogenic callus of Panax quinquefolium L.were successfully separated by enzymatic hydrolysis,and then the protoplasts of Panax quinquefolium L.and Panax notoginseng were fused by PEG,high Ca²⁺high p H PEG and electrofusion,Finally,the regeneration system of Panax quinquefolium L.protoplast,Panax notoginseng protoplast and Panax quinquefolium L.Panax notoginseng fusion was established.The main results are as follows:1.The results showed that different hormone types and hormone concentrations affected the induction of Panax quinquefolium L.and Panax notoginseng leaves.Panax quinquefolium L.leaves were induced in MS+0.5mg/l 6-BA+1mg/L 2,4-D medium,and the callus induction rate was the highest,75.5%.Panax notoginseng leaves were induced in MS+0.5 mg/L 2,4-D+0.5 mg/L 6-BA medium,and the callus induction rate was as high as 87.72%.The best condition for callus differentiation of Panax notoginseng was MS+30g/L sucrose+1mg/L6-BA,and the differentiation rate was as high as 77.5%.2.The factors affecting the separation,purification and regeneration of protoplasts of Panax quinquefolium L.and Panax notoginseng were analyzed and discussed by enzymatic hydrolysis.The results showed that the ratio of enzyme solution,enzymatic hydrolysis time,osmotic pressure of enzyme solution,purification method and callus culture days all affected the protoplast separation.Panax quinquefolium L.callus was cultured for 15 days,2%cellulase,0.5%pectinase and 13%mannitol were used to adjust the osmotic pressure.After enzymatic hydrolysis for 8 hours,25%sucrose was used,the volume ratio of sucrose to suspension was 1:2800 r/min and centrifugation for 5 minutes,the protoplast preparation effect of Panax quinquefolium L.was the best,and Panax quinquefolium L.protoplasts with high yield and high activity could be isolated;The embryogenic callus of Panax notoginseng was cultured for 30days.The osmotic pressure was adjusted by using 2%cellulase,0.1%segregation enzyme,0.75%pectinase and 13%mannitol.After enzymatic hydrolysis for 7 hours,centrifugation for 5minutes.The protoplast preparation effect of Panax notoginseng was the best,and the protoplast of Panax notoginseng with high yield and high activity was obtained.3.In this experiment,three fusion methods of PEG,high Ca²⁺high p H-PEG and electrofusion were used to explore the protoplast fusion of Panax quinquefolium L.and Panax notoginseng.After the protoplasts of Panax quinquefolium L.and Panax notoginseng obtained by enzymatic hydrolysis were mixed 1:1,when peg fusion was used,the protoplast density was4×10⁵/ml,treated with 40%PEG for 30 min,the protoplast fusion rate reached the maximum of 1.7%.In the fusion test of high Ca²⁺and high PEG-PH,the protoplast density was adjusted to5×10⁵/ml,0.12mol/l Ca Cl₂·2H₂O was added to 40%PEG,and the fusion rate reached the maximum value of 5.03%when treated for 20min.Protoplast Electrofusion test showed that the AC electric field intensity was 50 V/cm,the DC pulse voltage was 1250v/cm,the number of DC pulses was 5,and the width of DC pulse was 40μs,the fusion rate reached 1.07%.4.This experiment adopts liquid shallow culture.Through repeated experiments,it is concluded that the suitable plate planting rate density of Panax quinquefolium L.and Panax notoginseng fusion is 1×10⁶/ml,cultured in liquid medium containing 210g/L sucrose+0.2mg/L KT+0.2mg/L 2,4-D,it is easier to divide.The suitable density of planting plate for protoplast culture of Panax quinquefolium L.was 5×10⁵/ml,which is more suitable for American ginseng protoplast culture in the liquid medium containing 210g/L sucrose+0.2 mg/L KT+0.2mg/L 2,4-D.The most suitable density of protoplast of Panax notoginseng was 1×10⁶/ml,which is more suitable for protoplast culture of Panax notoginseng in liquid medium containing 210g/L sucrose,0.1 mg/L KT+0.2mg/L 2,4-D.5.Flow cytometry was used to identify the ploidy of the fusion.American ginseng was tetraploid,with a peak value of 2000000 in G1 phase,Panax notoginseng was diploid,with a peak value of 800000 in G1 phase,and American ginseng was Panax notoginseng fusion callus,with a peak value of 2800000 in G1 phase,which was Hexaploid.