High efficiency transient and stable transformation by optimized DNA microinjection intoNicotiana tabacumprotoplasts

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Abstract
An efficient system has been established that allows well controlled DNA microinjection into tobacco (Nicotiana tabacum) mesophyll protoplasts with partially regenerated cell walls and subsequent analysis of transient as well as stable expression of injected reporter genes in particular targeted cells or derived clones. The system represents an effective tool to study parameters important for the successful transformation of plant cells by microinjection and other techniques. Protoplasts were immobilized in a very thin layer of medium solidified with agarose or alginate. DNA microinjection was routinely monitored by coinjecting FITC-dextran and aimed at the cytoplasm of target cells. The injection procedure was optimized for efficient delivery of injection solution into this compartment. Cells were found to be at the optimal stage for microinjection about 24 h after immobilization in solid medium. Embedded cells could be kept at this stage for up to 4 d by incubating them at 4 °C in the dark. Within 1 h successful delivery of injection, solution was routinely possible into 20–40 cells. Following cytoplasmic coinjection of FITC-dextran and pSHI913, a plasmid containing the neo (neomycin phosphotransferase II) gene, stably transformed, paromomycin-resistant clones could be recovered through selection. Transgenic tobacco lines have been established from such clones. Injection solutions containing pSHI913 at a concentration of either 50 μg ml−1 or 1 mg ml−1 have been tested. With 1 mg ml−1 plasmid DNA the percentage of resistant clones per successfully injected cell was determined to be about 3.5 times higher. Incubation of embedded protoplasts at 4°C before microinjection was found to reduce the percentage of resistant clones obtained per injected cell Protoplasts were immobilized above a grid pattern and the location of injected cells was recorded by Polaroid photography. The fate of particular targeted cells could be observed. Isolation and individual culture of clones derived from injected cells was possible. Following cytoplasmic coinjection of FITC-dextran and 1 mg ml−1 plasmid DNA on average about 20% of the targeted cells developed into microcalli and roughly 50% of these calli were stably transformed. Transient expression of the firefly luciferase gene (Luc) was nondestructively analysed 24 h after injection of pAMLuc. Approximately 50% of the injected cells that were alive at this time point expressed the Luc gene transiently. Apparently, stable integration of the injected genes occurred in essentially all transiently expressing cells that developed into clones.

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