Tobacco (Nicotiana tabacum) cv. ‘Bright Yellow 2’ (BY2) cell suspension is a useful system to study the structure and function of plant cell. However, low efficiency of Agrobacterium-mediated transformation, and transgene silencing during subculture limit its application. Here we present optimization of the traditional protocols of Agrobacterium-mediated transformation and genomic DNA extraction. The transforming efficiency and recovery ratio of genomic DNA extraction were substantially increased by these improvements. Southern assay demonstrated that copy number of transgene could be determined unambiguously. Meanwhile by monitoring the GFP fluorescence we found that the GFP expression can keep stable in suspension culture cells for at least 20 days in liquid medium. Finally, applicability of constitutive promoters of Arabidopsis thaliana UBIQUITIN10 (AtUBQ10) and ARABIDOPSISSKP1 HOMOLOGUE1 (AtASK1) also can drive stable GFP expression in vivo of BY2 cells like CaMV 35S promoter in this plant system./span>
A rapid and inexpensive one-tube genomic DNA extraction method from Agrobacterium tumefaciens
