Abstract:  【Objective】 To establish a high-efficiency plant regeneration and transformation procedure of switchgrass.【Method】mature seeds of three different switchgrass (Panicumvirgatum L.) varieties were used as explants for callus induction. After six weeks of callus induction, the phenotype of the callus was observed under a dissecting microscope, we discarded the out-layer sponge-like, watery callus and selected the core-like, highly compact callus that lies in the middle of callus clump for further subculture, after the sub-culture was in the dark for three weeks, calluses grew fast, compact and grainy like were selected for further culture, calluses generated from one seed as a cell line and were kept together. This type of callus was highly regenerable, and was characterized as Type Ⅱ callus of monocot plant. After two rounds of selection, enough type Ⅱ callus could be obtained for plant regeneration and transformation. To optimize the procedure of Agrobacterium-mediated transformation, transformation efficiencies under three different Agrobacterium-infection procedures were evaluated. They are vacuum-drying treatment (VD): That was in a 50 mL centrifuge tube, the calluses that merged in Agrobacterium suspension were given a vacuum treatment (-0.8 Mpa) for 10 min, then slight agitated in a incubator-shaker at 80 r/mim, 28℃ for 20 min. Then drainage the bacteria and dry on a sterilized filter paper tower, the calluses were transfer onto two layers of filter paper soaked with 500 mL sterile water in a petri dish for co-cultivation. Cold combined with vacuum-drying treatment (CVD): calluses were given a cold treatment on ice in a solution of 3% maltose and 300 μmol·L^-1 of glutamine (Gln) for 20 min before conducted as VD process. A slightly different procedure was used instead of sterile water MP liquid medium was used for cocultication. Osmotic treatment combined with the cold and vacuum-drying treatment (PVD): 6% maltose was used in CVD process. The transgenic transformation efficiencies were evaluated by GUS staining assay and PCR tests.【Result】A regeneration efficiency of over 95% was reached for the type Ⅱ callus selected from the lowland switchgrass cultivar “Alamo” and “Performer”; 50% was reached for upland cultivar “Blackwell”. The transformation rate of switchgrass cultivar “Alamo” reached to 72% under CVD procedure, significantly higher than that was obtained under the procedure VD (53%) and PVD (44%). Under CVD procedure, transformation efficiency of lowland cultivars “Performer” reached to 96.7%; for the upland cultivar “Blackwell”, the transformation efficiency reached to 5.6%. This was the first time of obtaining the transgenic plant of the upland cultivar “Blackwell”.【Conclusion】Highly regenerative type Ⅱ calluses can be obtained from different ecotypes of switchgrass with a stereo-microscope. Agrobacterium- mediated transformation efficiency could be significantly improved under a CVD procedure for infection. This was the first report on how to select the type Ⅱ callus derived from mature seeds of switchgrass. A high-efficiency plant regeneration and transformation procedure that was suitable to lowland and upland switchgrass cultivars was developed, which made a foundation for switchgrass genetic improvement and functional gene research. The method presented here is also helpful in establishing a plant regeneration and transformation system for other recalcitrant monocot.

Key words: switchgrass, upland cultivars, typeⅡ callus, Agrobacterium-mediated transformation, Agrobacterium-mediated