Transgenic PNA plants expressed an antifungal peptide from the Japanese morning glory Ipomoea nil. Transgenic ICE plants Bortezomib IC50 expressed an anti microbial peptide from the common ice plant Mesembryanthemum crystallinum and transgenic FAB plants expressed an antimicrobial peptide Inhibitors,Modulators,Libraries from the broad bean Vicia faba. The sequences of the PNA and FAB con structs were manually adapted to the codon usage table of N. tabacum. Plant transformation and line screening N. attenuata Torr. ex S. Watson seeds were originally col lected in 1988 from a natural population at the DI Ranch in Southwestern Utah. Wild type seeds from the 30th inbreed generation were used for the construction of transgenic plants and as WT controls in all experiments. Plant trans formation was performed by Agrobacterium tumefaciens mediated gene transfer as previously described.
Ex plant cultures were regenerated from Inhibitors,Modulators,Libraries elongated hypocotyl tissue and the selection for correct T DNA integrations was performed on phytagel based media supplemented with 20 mg/L hygromycin B. For germination seeds were sterilized for 5 min with a 2% aqueous solution of sodium dichloroisocyanuric acid and treated for 1 h with 0. 1 M gibberelic acid in 50 diluted liquid smoke solution. Inhibitors,Modulators,Libraries At least 60 seedlings per plant were germinated on Gamborgs B5 Medium supplemented with 35 mg/L hygromycin B and incubated in a growth chamber. After 10 days the segrega tion rate was determined and re sistant seedlings transferred to the glasshouse under constant temperature and light conditions. Since N.
attenuata is self compatible, the collected seeds result generally from self pollination, except Inhibitors,Modulators,Libraries if crossings with different lines are indicated. For crossings, the flowers were antherectomized before opening and hand pollinated using pollen from either homozygous transgenic or wild type plants. Independent overexpression plant Inhibitors,Modulators,Libraries lines used in this study. The plant generations were indicated within the line number as follows T1 seeds or plants have only the line number, T2 seeds were indicated by an extra number to identify the plant from which seeds were collected from, T3 seeds were additionally numbered. Two lines harboring an inverted repeat construct for silen cing the expression of N. attenuata acetyl CoA transferase Genomic DNA isolation Genomic DNA was isolated with a modified hexa decyltrimethylammonium bromide method de scribed in.
For Southern blotting 15 day old seedlings were ground in liquid nitrogen to a fine powder and exactly 300 mg used for DNA isolation. The quality and concen tration was estimated by agarose gel electrophoresis. For bisulfite sequencing gDNA was isolated from cotyledons and first true leaves of seedlings 15 days post germination, leaves of rosette stage plants, cauline leaves of elongating plants and cauline leaves of flowering plants. The last three time points were successively sampled from the same plants.