Non-Plant Tissue Culture Genetic Transformation Systems For Mulberry

In recent years,plant genetic transformation methods that do not depend on plant tissue culture have been developed.Their advantages are their simplicity,speed,and lack of a requirement for tissue culture.Mulberry,as a native tree species in China,is an important eco-economic forest tree.At present,there is no mature and stable regeneration system for mulberry.Therefore,the establishment of a genetic transformation method that independent on a tissue culture is of great significance for studies on mulberry gene functions.In this study,the new genetic transformations of mulberry,nano-material mediated plant transformation and virus-induced gene silencing?VIGS?technology,were used to genetically transform mulberry.The main results obtained are as follows:1.Pollen magnetofection can be used for genetic transformation of mulberry.The structure of mulberry pollen was detected under a cryo-scanning electron microscope.The results showed that the pollen of mulberry has one germination hole,indicating that pollen magnetofection may be applied to mulberry transformation.To increase the efficiency of pollination,we optimized the culture medium of mulberry pollen in vitro by a single-factor experiment.A plant expression vector Gh PAP1 for the overexpression of the cotton anthocyanin synthesis transcription factors PAP1 and TT8was constructed and transfected by pollen magnetofection.In total,554 transformed mulberry seeds were collected,germinated,and then grew into mulberry seedlings.The seedlings were screened by GUS staining.The GUS positive stainings were observed,however no anthocyanin accumulation phenotypes were detected.The plant gene expression vector cas9-MaPDS was constructed for editing hydrogen lycopene dehydrogenase?PDS?gene of mulberry and transfected by pollen magnetofection.We obtained 1,031 mulberry seeds.After being germinated and grew into green mulberry seedlings,no albino phenotypes were detected.Among them,only two mCherry positive seedlings were identified using the genomic PCR method.2.Mulberry mosaic dwarf associated virus?MMDaV?is able to infect mulberry.The infectious clone of MMDaV was infected cowpea cotyledons,a susceptible plant of MMDaV virus.The infected cowpea leaves showed obvious symptoms after one week.The results of genomic PCR analyses showed that MMDaV infectious clone could infect cowpea and move between plant cells.Then the white mulberry?Morus alba,M.alba?was infected by cotyledons with different Agrobacterium concentrations(OD₆₀₀ values 1.0,1.5,and 2.0).The results showed that MMDa V had the highest infection rate and no toxic effect on mulberry when OD₆₀₀=1.0.Cotyledons of 12mulberry varieties were inoculated with MMDaV,but none showed obvious disease symptoms.The results of PCR analyses showed that all mulberry varieties could be infected by MMDaV,with infection rates ranging from 31%to 83%.In repeated experiments on seven mulberry varieties,all varieties could be infected by MMDaV,and there was no difference in resistance levels among the mulberry varieties.3.MMDaV and 2mDNA1 silencing vectors in the studies of mulberry gene functions.When cowpea leaves infected with MMDaV plus 2mDNA1,the plants showed dark necrotic lesions,while those infected with 2mDNA1 alone showed wild-type phenotype.Seven mulberry resources were used to investigate the infectious capacity of MMDaV and 2mDNA1.After two weeks,the infected mulberry seedlings exhibited no typical MMDaV infectious phenotype,and the infection rates ranged from 67%to 87%among the seven resources,which is higher than that of MMDaV alone.In order to investigate the function of MMDaV infectious clone as a VIGS vector,we constructed a MaPDS gene silencing vector?2mDNA1-PDS?and infected recombinant2mDNA1-PDS with MMDaV in cotyledons of five mulberry resources.The infection rate ranged from 33%to 70%among the five mulberry resources.Furthermore,the phenotype of silenced MaPDS was observed in the first and second leaves of the infected mulberry seedlings.The results of RT-PCR and qRT-PCR confirmed that the expression levels of MaPDS were significant down-regulation.A conclusion can be drawn that the present study provided the primary data for mulberry non-plant tissue culture genetic transformation systems.