Establishment Of Transient Expression System Of Camellia Oleifera Protoplast And Genetic Transformation Of Callus

Oil tea(Camellia oleifera)is an important woody oil tree species in our country,but its basic research and molecular breeding are restricted greatly because of the lack of genetic transformation platform.Plant protoplasts and callus are easy to absorb foreign genes and are ideal receptors for genetic transformation.Our research group has established an efficient protoplast separation technique for Camellia oleifera,but has not studied its genetic transformation method.Therefore,this study intends to take ’Hua Shuo’,a superior seed of Camellia oleifera approved by the State,as the material to explore the conditions of the instantaneous transformation of mesophyll protoplast/callus protoplast,so as to establish-an efficient instantaneous transformation technology-system of Camellia oleifera.At the same time,the method of agrobacterium tumefaciens mediated callus transformation was explored by using the ’Hua Shuo’ callus as the material,and the genetic transformation system of Camellia oleifera callus was preliminarily established,so as to carry a technology platform for the genetic transformation of Camellia oleifera.The main research results are as follows:(1)The efficient transient transformation technology system of Camellia oleifera mesophyll protoplasts was established.Mesophyll protoplasts were isolated from the leaves of tissue culture seedlings of Camellia oleifera ’Hua Shuo’,and the effects of PEG type,PEG treatment concentration,PEG transformation time,mass of transformed plasmid,and co-culture time after transformation on the instantaneous transformation of mesophyll protoplasts were studied by PEG mediated method.The results showed that:With 40%PEG 4000 treatment,when 15 μg plasmid was added,the transformation time was 20 min,the co-culture time was 14 h,and the co-culture solution was W5 solution with 0.4 M mannitol,the instantaneous transformation efficiency of the mesophyll protoplasts was the highest,which could reach 75%.(2)The efficient transient transformation technology system of protoplast in suspension line of Camellia oleifera was established.Using the suspension cells of Camellia oleifera ’Hua Shuo’ as materials,protoplasts were isolated.PEG mediated method was used to study the effects of PEG type,PEG treatment concentration,PEG transformation time,mass of transformed plasmid,co-culture time after transformation,type of co-culture solution after transformation and other factors on the instantaneous transformation of suspension protoplasts of Camellia oleifera.The results showed that:Using 40%PEG 6000 treatment,when adding 5 μg plasmid,the conversion time was 15 min,the co-culture time was 14 h,and the co-culture solution was W5 solution with 0.4 M mannitol,the instantaneous transformation efficiency of the protoplasts of Camellia oleifera suspension system was the highest,which could reach 80%.(3)The factors influencing the genetic transformation system of Camellia oleifera callus mediated by Agrobacterium tumefaciens were investigated.The effects of callus type,pre-culture time,bacterial concentration,infection time and co-culture conditions on the transformation of Camellia oleifera callus were studied.The results showed that the callus induced by seed embryo was used for transformation,and the embryogenic ability was good.After 10 d of pre-culture,the concentration of bacterial solution was OD600=0.3,the infection time was 15 min,the co-culture medium type was solid medium,and the coculture time was 3 d,the transformation was more favorable.