|Ozel, Cigdem -|
Submitted to: Acta Horticulturae
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 20, 2014
Publication Date: June 2, 2014
Citation: Ozel, C.A., Kamo, K.K. 2014. Agrobacterium-mediated transformation of Easter lily (Lilium longiflorum cv. Nellie White). Acta Horticulturae. 1002:231-236. Interpretive Summary: Two methods generally used for genetic engineering of plants utilize the gene gun or Agrobacterium. The benefit of using Agrobacterium is that it typically results in a lower copy number of the transgene to be inserted, and this often results in higher levels of transgene expression rather than the multicopy insertion characteristic of the gene gun. Also, many genes are already present in the binary vectors used for Agrobacterium-mediated transformation and do not have to be subcloned into smaller vectors needed for gene gun-mediated transformation. This study was done to determine the conditions needed to obtain transient expression of a reporter gene (GUS) in Easter lilies. It was found that growing the plant in the dark was critical to achieve high levels of transient GUS expression. Both bulb scales and basal meristems were used for inoculation by Agrobacterium, and these two tissues had to be incubated in the dark on Murashige and Skoog’s medium containing dicamba. The levels of transient GUS expression were high enough to make it feasible to attempt the next step - stable transformation. Once stable transformation is achieved, it will be possible to genetically engineer lilies for disease resistance, and this will help improve the environment and lower costs for the growers.
Technical Abstract: Conditions were optimized for transient transformation of Lilium longiflorum cv. Nellie White using Agrobacterium tumefaciens. Bulb scale and basal meristem explants were inoculated with A. tumefaciens strain AGL1 containing the binary vector pCAMBIA 2301 which has the uidA gene that codes for ß-glucuronidase, GUS expression. Transformed bulb scales showed GUS expression when they had been precultured 11 days on Murashige and Skoog’s (MS) medium supplemented with 2 mg/liter dicamba. The outer, larger-sized bulb scales were not infected nearly as well as the inner, smaller bulb scales. Maximum GUS expression occurred when bulb scales had been obtained from plants that had been grown in the dark for at least 2 months rather than in the light. There was minimal GUS expression when bulb scales were used from plants grown one month in the dark indicating the importance of growing plants in the dark for Agrobacterium-mediated transient transformation of bulb scales. Basal meristems taken from plants grown in the dark for 4 months showed 3 times as much GUS expression as basal meristems from plants grown in a 12 h light photoperiod. The frequency of transient GUS expression achieved in this study indicated that it should be possible to achieve stable transformation of ‘Nellie White’ which is the cultivar that dominates the U.S Easter lily market. Experiments for stable transformation are in progress.