Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method
Collective efforts of several laboratories in the past two decades have resulted in the development of various methods for Agrobacterium tumefaciens–mediated transformation of Arabidopsis thaliana. Among these, the floral dip method is the most facile protocol and widely used for producing transgenic Arabidopsis plants. In this method, transformation of female gametes is accomplished by simply dipping developing Arabidopsis inflorescences for a few seconds into a 5% sucrose solution containing 0.01–0.05% (vol/vol) Silwet L-77 and resuspended Agrobacterium cells carrying the genes to be transferred. Treated plants are allowed to set seed which are then plated on a selective medium to screen for transformants. A transformation frequency of at least 1% can be routinely obtained and a minimum of several hundred independent transgenic lines generated from just two pots of infiltrated plants (20–30 plants per pot) within 2–3 months. Here, we describe the protocol routinely used in our laboratory for the floral dip method for Arabidopsis transformation. Transgenic Arabidopsis plants can be obtained in approximately 3 months.
Comments
Dear Professor Chua and friends working on Arabidopsis,
I worked on Arabidopsis tranformation before with the similar floral dip method (FDM) described in this paper. It worked well for my experiment. I also tested the floral spray method (FSM),and it worked much better.
In FSM, all are same as FDM, except delivering Agrobacterium to Arabidopsis inflorescences by a sprayer. FDM usually needs more than 500ml of Agro suspension, while FSM is very flexible, can be as low as 10ml. You can also repeat spay after a few days in case some new inflorescences grow up. It is hard to dip the small inflorescences into Agro suspension, and even harder to repeat dip with FDM when plants are longer than 15cm.
It is notable that I can harvest seeds from three to four weeks after spray if growing plants under 27 degree Celsius and 3000Lx of light. My transformation efficientcy can reach 11% (not all the time). I believe it is worth to try for related researchers.
Thanks.
Posted by: Chang-Ming Ye | October 20, 2008 08:27 PM