Research About The Isolation,Purification And Regeneration Of Carnation'master' Protoplast

Carnation(Dianthus caryophyllus)is one of the world's largest selling flowers which can also refine the essence.In the breeding work,no artificial processing conditions of carnation usually can not produce seeds,which is the breeding limit.Protoplast fusion breeding can overcome the limitations of carnation breeding,carnation high temperature resistance,high ornamental breeding traits provide beneficial way.The establishment of purification and regeneration system of carnation protoplast isolation,high efficiency,will provide the basis for breeding.In this experiment,carnation cultivated varieties 'Master' aseptic line leaves were used as material,we analysed the related conditions about protoplast isolation and regeneration system.Establish the best method of enzymatic hydrolysis,enzyme solution composition,concentration,enzymatic hydrolysis time,the results are as follows:1.Separation of carnation protoplasts:1)Osmotic stabilizers:the carnation leaf osmotic pressure is 0.5 mol/L,so we use 05.M mannitol solution as osmotic stabilizer.2)Size of protoplast:By means of microscope scale measurement,carnation protoplast particle size is between 40-70 ?m,which was purified by 200 mesh nylon net;3)We use two different methods to isolate the protoplast:Static interface enzyme purification method,the optimum condition is 0.3%cellulase and 0.5%macerozyme,enzymolysis time is 12h.Maximum yield is 4.33×105Cell/g FW,the vitality is 83.87%;Low speed oscillation settlement purification method:the optimum conditions is 1.5%cellulase and 0.2%pectinase,enzymatic hydrolysis time was 3.5h,the highest yield was 1.83 ×107Cell/g FW,the vitality of 96.3%.This method greatly shortened the effect of enzyme liquid,in operation and protoplast yield,viability are better than the static interface purification method.2.Culture of 'Master,Protoplast:1)On the basis of MPSCul and KM8P culture media,hormone species,concentration,culture methods,achieve a continuous division of carnation 'masters' protoplast.As complex as protoplasts in the culture process,there are different products and characteristics in different stages which need different medium:cell clusters and small callus formation period of LMP medium(KM8P+5mg/L NAA +lmg/L ZT),the proliferation of callus culture medium DMP4-1(8%sucrose + 1.0mg/l 2,4-D+0.5mg/L ZT),DMP4-2(MS+6%sucrose + 1.0mg/l 2,4-D+0.5mg/L ZT).Different culture period,according to the characteristics of different,there are differences in the way of training.2)Establishment of protoplast regeneration system:The callus,which was formed by protoplast proliferation,was transferred into the plant regeneration medium,and gradually proliferated,and the color changed to be green.The callus in medium contain TDZ are the best.The plant regeneration was still in the process because of the longer cycle of plant regeneration.