Abstract
Transient transformation is simpler, more efficient and economical in analyzing protein subcellular localization than stable transformation. Fluorescent fusion proteins were often used in transient transformation to follow the in vivo behavior of proteins. Onion epidermis, which has large, living and transparent cells in a monolayer, is suitable to visualize fluorescent fusion proteins. The often used transient transformation methods included particle bombardment, protoplast transfection and Agrobacterium-mediated transformation. Particle bombardment in onion epidermis was successfully established, however, it was expensive, biolistic equipment dependent and with low transformation efficiency. We developed a highly efficient in planta transient transformation method in onion epidermis by using a special agroinfiltration method, which could be fulfilled within 5 days from the pretreatment of onion bulb to the best time-point for analyzing gene expression. The transformation conditions were optimized to achieve 43.87% transformation efficiency in living onion epidermis. The developed method has advantages in cost, time-consuming, equipment dependency and transformation efficiency in contrast with those methods of particle bombardment in onion epidermal cells, protoplast transfection and Agrobacterium-mediated transient transformation in leaf epidermal cells of other plants. It will facilitate the analysis of protein subcellular localization on a large scale.
Highlights
Onion (Allium cepa L.), one kind of biennial herb Liliaceae plant, has been used as classical experimental materials in analyzing structure of plant cells, distribution location of DNA and RNA, reducing sugar of plant tissues [1], plasmolysis and recovery of plant cells [2,3], karyotype [4], protein subcellular localization and interaction [5,6,7].Imaging subcellular localization of proteins in living cells has become an important tool for defining protein function
Most transient transformation methods have certain disadvantages, such as the lower transformation efficiency, equipment dependency and auxiliary material needed for particle bombardment, complex preparation procedures required for protoplasts transfection
We developed an in planta transient transformation method in living onion epidermal cells by using Agrobacterium-mediated infiltration
Summary
Onion (Allium cepa L.), one kind of biennial herb Liliaceae plant, has been used as classical experimental materials in analyzing structure of plant cells, distribution location of DNA and RNA, reducing sugar of plant tissues [1], plasmolysis and recovery of plant cells [2,3], karyotype [4], protein subcellular localization and interaction [5,6,7].Imaging subcellular localization of proteins in living cells has become an important tool for defining protein function. Most transient transformation methods have certain disadvantages, such as the lower transformation efficiency, equipment dependency and auxiliary material needed for particle bombardment, complex preparation procedures required for protoplasts transfection. For transient transformation in plants having complex outline of epidermal cells and the bushy epidermal hairs, laser scanning confocal microscope was usually needed to get ideal micro-images, which increased the reliability on expensive equipments. To avoid these disadvantages, we developed an in planta transient transformation method in living onion epidermal cells by using Agrobacterium-mediated infiltration. Infiltration liquid of Agrobacterium carrying constructed vectors were injected into the interface between adaxial epidermis and mesophyll of onion bulb scales, which played an important role in yielding high transformation efficiency, and kept in the living onion bulb for about three days
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