| In order to establish a molecular genetic basis for modulating lignin biosynthesis in sweetgum (Liquidambar styraciflua), three integral components were investigated: plant tissue culture, genetic transformation, and characterization of a sweetgum laccase gene.; To establish an efficient plant regeneration system, thidiazuron (TDZ) was tested and was found to be most active in adventitious bud and shoot formation from hypocotyl segments in combination with 2,4-dichlorophenoxyacetic acid (2,4-D). For shoot elongation, lower concentrations of TDZ or no TDZ in combination with NAA and BA were required. Liquid culture significantly enhanced shoot production. Ex vitro rooting resulted in a dramatic improvement in growth rates and root development.; For genetic transformation, sweetgum nodule cultures were developed in liquid medium containing 0.1 mg/l TDZ and 0.01 mg/l 2,4-D or 0.1 mg/l 2,4-D. Sweetgum nodules were stably transformed by microprojectile bombardment using a 7.4 kb plasmid, pTRA 140, harboring CaMV 35S-HPH and CaMV 35S-GUS. Shoots differentiated in liquid medium containing BA (1 mg/l), BA (0.5 mg/l) and NAA (0.01 mg/l), or BA (0.5 mg/l), NAA (0.01 mg/l), and TDZ (0.05 mg/l) in the light and in the presence of hygromycin B. Differentiating shoots required optimally four weeks of dark treatment for further development on solid medium containing 1 mg/l BA. Elongated shoots were excised and planted in potting mix for ex vitro rooting. Roots differentiated from both transgenic and non-transgenic shoots in two weeks. PCR and Southern analyses confirmed the presence of the GUS gene in nodules and shoots.; A 360 bp cDNA fragment of a sweetgum laccase gene was obtained via reverse transcription-polymerase chain reaction (RT-PCR). Forty-one to 59% of the amino acid sequence translated from the initial 360 bp of the cDNA was identical to the same region of laccase genes from three other plant species. An approximately 2 kb laccase transcript was detected in xylem of sweetgum via northern blot analysis. Southern blot analysis of genomic DNA showed that the sweetgum laccase cDNA was derived from a single gene. Using sequence from the cDNA fragment, the putative promoter region was obtained, which contained TATA and CAAT boxes, as well as motifs corresponding to regulatory elements involved in the phenylpropanoid pathways of other species. In addition, an AG repeat sequence, which has been seen in the laccase genes from yellow-poplar, was found. |
