The levels of gossypol and related terpenoids in the leaves of transgenic progeny from RNAi lines are not reduced. The levels of gossypol (G), hemigossypolone (HGQ), and total heliocides (H) in leaf tissues from 10 individual wild-type control plants and the T₁ progeny of the two RNAi transgenic lines. The results from PCR analysis on DNA from the same individual progeny plants from the RNAi lines are depicted under the respective graphs. Mean (±SEM) values for terpenoid levels in the leaf tissue of control plants (n = 10) and the transgene-bearing T₁ plants (n = 9) from each of the transgenic lines are shown with the respective graphs. The key to bar colors is consistent with Fig. 1A.

δ-Cadinene synthase transcripts and enzyme activity are significantly reduced in developing embryos from the RNAi lines. Separate sets of embryos (35 dpa) isolated from wild-type plants, null segregant plants, and homozygous T₁ plants from lines LCT66-2 and -32 were used for each type of analysis. (Top) The hybridization band (dCS) on a Northern blot; (Middle) ethidium bromide-stained RNA gel before blotting; (Bottom) δ-cadinene synthase activities. The enzyme activity is presented as total ion peak area of δ-cadinene generated min⁻¹· mg⁻¹ embryo. Enzyme activity results are mean (±SEM) of values obtained from three separate sets of embryo samples from each type of plant. ∗, The value for the transgenic line is significantly different from the control (wild-type and null segregant) value at P < 0.004.

Reductions in gossypol levels and target transcripts in the transgenic cottonseeds and developing embryos, respectively, from two RNAi lines. (A) Gossypol levels in 10 individual mature seeds each from wild-type control plants (red) and two independent RNAi transgenic lines, LCT66-2 (light green) and LCT66-32 (dark green). The results from PCR analysis on DNA from the same individual seeds from RNAi lines are depicted under the respective graphs. Note that the gossypol levels in the null segregant seeds (pink) are similar to control values. Mean (±SEM) gossypol values for control (n = 10) and the transgene-bearing seeds (n = 8) from each of the transgenic lines are shown with the respective graphs. ∗, The value for the transgenic line is significantly different from wild-type control value at P < 0.001. (B) Photomicrographs of sections of four mature T₁ seeds obtained from the transgenic line LCT66-32 (Left). The seed at the top was a null segregant, whereas the others were transgenic seeds. HPLC chromatograms (Right) show the gossypol levels in the extracts from the same four seeds. y axis, absorbance at 272 nm; x axis, elution time (min). Note the correlation between visible phenotype and gossypol level in the seed. (C) RT-PCR analysis of δ-cadinene synthase (dCS) expression in a separate set of 10 individual, developing embryos (35 dpa) each from a wild-type control plant and the two RNAi transgenic lines. Transcripts from histone 3 gene of cotton were amplified as internal controls in the duplex RT-PCR analyses. The results from PCR analysis on DNA from the same individual embryos from the RNAi lines are also shown to illustrate a correlation between reduced dCS transcripts and presence of the transgene.

The levels of gossypol and related terpenoids in terminal buds, bracts, floral organs, bolls, and roots of transgenic progeny from RNAi lines are not reduced. The levels of terpenoids in various organs of wild-type control plants (red), T₁ transgenic progeny from RNAi line LCT66-2 (light green), and T₁ transgenic progeny from RNAi line LCT66-32 (dark green). The results shown are mean (±SEM) terpenoid values in tissue samples taken from three individual plants in each category. Note that in petals, gossypol was the only terpenoid detected and in the root tissue, the terpenoids detected were: gossypol (G), gossypol-6-methyl ether (MG), gossypol-6,6′-dimethyl ether (DMG), hemigossypol (HG), desoxyhemigossypol (dHG), hemigossypol-6-methyl ether (MHG), and desoxyhemigossypol-6,6′-methyl ether (dMHG).

The low-seed-gossypol trait is successfully transmitted to T₂-generation seeds in the transgenic RNAi lines. Gossypol levels in 10 individual seeds each from wild-type control plant and a null segregant plant and 50 individual T₂ seeds each from homozygous T₁ plants that were derived from their respective parental transgenic lines, LCT66-2 and -32. Mean (±SEM) gossypol values for control (n = 10) and transgenic seeds (n = 50) are shown with the respective graphs. ∗, The value for the transgenic line is significantly different from the control (wild-type and null segregant) value at P < 0.001.