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3 . 2 . 3 5 . 3 .
Transformation of gene cassettes harboring cah marker gene and l i p
gene in tobacco
3 . 2 . 3 . 5 . 3 . 1
Development of tobacco plantlets
Tobacco seeds were surface sterilized with 0.1% HgCI2 for 5 min.. then rinsed
thrice for 5 min. in sterile distilled water. Seeds were then germinated under sterile
conditions in tissue culture bottles containing Murashige and Skoog (MS) basal
medium (Murasbige and Skoog, 1962) under light continuously at 25
+
2 "C in the
growth chamber (Sanyo MLR 351H Plant Growth Chamber, USA). Aseptically
grown 8 weeks old plants were used as a source of explants far leaf disc
transformation in tobacco.
3 . 2 . 3 . 5 . 3 . 2 .
Agrobaclerium-mediated transformation of gene cassette in tobacco
3 . 2 . 3 . 5 . 3 . 2 . 1 .
Freeze-thaw method
The gene cassettes, pCAMBI.4-cah-Ltp 3FI and pCAMBIA-cah-Bar2chi
were transformed into Agrobacteriurn strain LBA 4404 using the freeze-thaw method
as described (Chen et al., 1994). Briefly. Agrobacleriurn strain LBA 4404 helper Ti
plasmid were grown in
5
ml of yeast extract broth (YEB) medium overnight at 28 'C.
The overnight culture (2 ml) was added to 50 ml YEB medium in a 250 ml fliuk and
was shaked vigorously (250 rpm) at 28 "C until the OD of 0.5 to 1.0 at 600 nm. The
culture was chilled on ice and centrifuged at 3,000 g for 5 min. at 4 "C. The cells
were resuspended in 1 ml of 20 mM ice cold CaC12 solution. 0.1 ml aliquots were
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