A rapid, novel and high-throughput identification of putative bell pepper transformants generated through in planta transformation approach

The recent progress on bell pepper transformation through in planta approach is incredibly simple with high transformation efficiencies in indigenous varieties. This method produces chimeric primary transformants (T0) hence, a large number of T0 plants and an efficient high throughput screening strategy is necessary to be developed. In the present investigation, we have standardized a rapid and highly efficient method of screening putative bell pepper transformants. Bell pepper transformants harbouring gfp genes was developed using in planta approach and 1050 etiolated T1 seedlings were screened using fluorescent microscope to select GFP positive plants. The molecular analysis such as, gene specific PCRs, semiquantitative RT-PCR and Southern blots analysis corroborated the transgene integration in selected T1 transformants. Based on progressive selection strategy, the transformation efficiency was 27.4% in gfp screened plants. The fluorescence based screening is viable, rapid, requires minimal labour, expense, and expertise with less percent of non-transformant escapes; further, this method can be extended from bell pepper to other crops to identify putative transformants developed through in planta approach.

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► T0 plants being chimeric this method needs large number of primary transformants.
► So far, no viable method of screening putative pepper transformants are available.
► 1050 etiolated seedlings screened based on gfp fluorescence using microscope.
► PCR, RT-PCR, Southern analysis confirmed the transgene integration in T1gfp plants.
► Overall transformation efficiency was 27.4% based on progressive selection strategy.