Agrobacterium tumefaciens-Mediated Transformation of the Lichen Fungus, Umbilicaria muehlenbergii
Figure 3
Growth, transformation efficiency, and Southern analysis.
(A) Sensitivity of a wild-type U. muehlenbergii strain to hygromycin B was evaluated by culturing it on PDA amended with 0, 5, 10 and 20 µg/ml of hygromycin B. (B) Membranes overlaid with only fungal cells (1st on the left), and a mix of A. tumefaciens and fungal cells (2nd, 3rd and 4th) were placed on PDA containing 20 µg/ml hygromycin B after culturing on co-cultivation medium for 24, 36 and 48 hrs (2nd, 3rd and 4th). The pictures were taken after 28 days of culture on this selection medium. (C) Effect on the transformation efficiency by increasing the co-cultivation time. Data presented as the average of eight plates per treatments. Error bars indicates standard error. (D) Distribution of T-DNA copy number among transformants of different time interval of co-cultivation. Genomic DNAs were digested with HindIII, a restriction enzyme that does not cut the hph cassette (see Figure 2). The digested DNAs, fractionated using 0.7% agarose gel and transferred to a nylon membrane, were probed with a labeled hph cassette. (E) Representative result of Southern blot analysis from 15 randomly selected transformants and a wild-type strain. (F) Distribution of T-DNA copy numbers among 784 transformants.