The Preliminary Study On Establishment Of Genetic Transformatio System Of Callus Of Paeonia

The study used agrobacterium strain GV3101 and LBA4404,plasmid pCAMBIA1301,callus induced by the plantlets's petiole of Tree peony cultivars Paeonia ostii 'Fengdan' as test material,accordingly,the study preliminary researched on the establishment of genetic transformation systerm of Callus Paeonia and construction agrobacterium expression vector.Then received transformation agrobacterium GV3101 and LBA4404.Further,putting the GUS gene into peony callus,and detecting successfully transient expression activity of GUS after co-cultue.Meantime,the experiment get transformed co-cultured callus for GUS's histochemical detection and completely put the GUS gene's activity detection.The experiment put the next step some foundation and technical basis on transferring to other functional genes.The main results and conclusions as follows:1.Establishment of transformation agrobacterium expression vector and screening strainsThrough a series of process such as extracting plasmid pCAMBIA1301's DNA,Preparating of agrobacterium GV3101 and LBA4404's competent,and plasmid DNA's transformation for agrobacterium,the study has put the plasmid pCAMBIA 1301 into agrobacterium and received agrobacterium strain GV3101 and LBA4404 expression vector containing.Through a series of experiment like that two strain infect the poney callus,the result shows that:the strain GV3101 has strong toxicity and different to restrain strain.After infecting,it can hurt poeny callus's viability.The callus start to turn red after GUS's staining,and the time of staining is too long.LBA4404 has little toxicity.it has not effect on poeny callus's viability definitely and has higher rate of transformation.Therefore,the study considered that strain LBA4404 is fit for Peony callus's genetic transformation.2.Determination of selective antibioticThe experiment filtered the common selective antibiotic like Kan and Hyg,and tested its sensitivity of peony callus.The results show that,the peony callus is not sensitivity for Kan.Kan is not fit for peony callus's selective antibiotic.But the peony callus is deeply sensitivity for Hyg that the concentration of 3mg/l can drop the good rate of peony callus significantly.So,Hyg,concentration of 3mg/l is the selective antibiotic which are fit for peony callus.3.Determination of bacteriostatic antibioticsThe experiment selected bacteriostatic antibiotics such as Cef and Cb,tested its sensitivity of peony callus,and tested two kinds of bacteriostatic antibiotics's inhibitory effect.The result shows that,the peony callus is more sensitivity for Cef than Cb.The result of inhibitory effect shows that,Cef's inhibitory effect is better than Cb.Therefore,Cef is fit for peony callus's bacteriostatic antibiotics,and the concentration Preferably not exceed 200mg/l.4.The preliminary construction on establishment of Genetic transformation systerm of Callus PaeoniaThe experiment preliminary researched Genetic transformation systerm of Callus Paeonia through choosing strain LBA4404 by agrobacterium-mediated.It Initially established Genetic transformation systerm of Callus Paeonia by bacteria plant tissue culture suspension infect Act;It choosed single colony LBA4404 and added to YEB Liquid medium.At meantime,put it to the Shaker whole night at 28?,220rpm and put the Bacilli Centrifugal at 4000rpm,10min,then abandon waste.And suspended thallus to the Bacilli OD600=0.6 by Plants broth WO.Without pre-incubation,we cut the peony callus's size to 0.2*0.2cm,and use the Bacilli OD600=0.6 to infect directly in 20 min,put the infected callus to filter paper and seeded in Co-culture medium,co-cultured in dark 3 days at 22?.Then rinsed and soaked the callus in 30 min by 200mg/lCef so as to wash residual bacteria.After that,put it to liquid proliferation medium 100mg/lCef.meantime shaked 24h.Then removed the callus,washed it and put it to the filter paper.After that we put it to the solid selective medium such as 100mg/lCef and 3mg/lHyg once in 20 days.5.Optimization on GUS's tissue staining methodThe experiment opetimizated GUS's tissue staining method.According to Jefferson's method,the experiment shows that the stained peony callus are all dead at 37 ? and no transient expression.Therefore,when opetimizating the temperature to 24?,we are all successfully detected GUS's transient expression activity.