Research On Mature Endosperms Of Sapium Sebiferum In Plant Regeneration And Genetic Transformation

Sapium sebiferum is an important oil tree as well as ornamental plants. It has a very long history of cultivation and utilization.In recent years, has been considered as an idle and potential bio energy plant, not only because it yields a lot of seeds containing more than45%oil and the oil can be used for biodiesel production, but also because it produces huge amount of biomass annually that can be sued for direct burning or as feedstock for production of ethanol, methanol and carbon. So far, all Sapium sebiferum plants are diploid and there have been no reports on its triploid plants.In this study, an efficient protocol for producing trip lo id Sapium sebiferum plants from mature endosperms were established, and its Agrobacterium-mediated transformation was further investigated using endosperm-derived calli as explants. The results showed that:(1)70%of the mature endosperms produced green and compact calli when cultured on MS medium supplemented with1.0-5.0mg/L BA and0.2mg/L NAA for30days.(2) Cutting the calli into pieces and culturing them on MS medium containing1.0-2.0mg/L BA and0or0.1mg/L NAA for45days led to approximate60%of callus explants to initiate adventitious buds, each having2-5buds.(3)42%of the buds elongated into shoots after they were transferred to MS medium supplemented with0.1mg/L BA and1g/L activated charcoal.(4) All the shoots rooted well and formed intact plantlets within30days of culture on MS medium supplemented with2.0mg/L IBA and1g/L activated charcoal.(5) the ploidy level of some endosperm-derived plants were determined by flow cytometry using the diploid plants germinated from seeds as controls and all of the plants were identified as triploid.(6) The endosperm-derived callus pieces about2mm in thickness were sensitive to kanamycin and the crucial concentration was50mg/L.(7)9kanamyc in-resistant calli were obtained by infecting the endosperm-derived callus pieces in Agrobacterium cells for30min and culturing them on MS medium containing2.0mg/L BA,0.2mg/L NAA and100pM acetosyringone for2days and then on MS medium containing2.0mg/L BA,0.2mg/L NAA,50mg/Lkanamycin and100mg/L Timentin for3months; these kanamycin-resistant calli were transgenic first by GUS staining and the PCR.