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. 2006 Nov 28;103(48):18054-9.
doi: 10.1073/pnas.0605389103. Epub 2006 Nov 16.

Engineering cottonseed for use in human nutrition by tissue-specific reduction of toxic gossypol

Ganesan Sunilkumar  1 , LeAnne M Campbell, Lorraine Puckhaber, Robert D Stipanovic, Keerti S Rathore
Affiliations

Engineering cottonseed for use in human nutrition by tissue-specific reduction of toxic gossypol

Ganesan Sunilkumar et al. Proc Natl Acad Sci U S A. .

Abstract

Global cottonseed production can potentially provide the protein requirements for half a billion people per year; however, it is woefully underutilized because of the presence of toxic gossypol within seed glands. Therefore, elimination of gossypol from cottonseed has been a long-standing goal of geneticists. Attempts were made to meet this objective by developing so-called "glandless cotton" in the 1950s by conventional breeding techniques; however, the glandless varieties were commercially unviable because of the increased susceptibility of the plant to insect pests due to the systemic absence of glands that contain gossypol and other protective terpenoids. Thus, the promise of cottonseed in contributing to the food requirements of the burgeoning world population remained unfulfilled. We have successfully used RNAi to disrupt gossypol biosynthesis in cottonseed tissue by interfering with the expression of the delta-cadinene synthase gene during seed development. We demonstrate that it is possible to significantly reduce cottonseed-gossypol levels in a stable and heritable manner. Results from enzyme activity and molecular analyses on developing transgenic embryos were consistent with the observed phenotype in the mature seeds. Most relevant, the levels of gossypol and related terpenoids in the foliage and floral parts were not diminished, and thus their potential function in plant defense against insects and diseases remained untouched. These results illustrate that a targeted genetic modification, applied to an underutilized agricultural byproduct, provides a mechanism to open up a new source of nutrition for hundreds of millions of people.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
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.
Fig. 2.
Fig. 2.
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.
Fig. 3.
Fig. 3.
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).
Fig. 4.
Fig. 4.
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.
Fig. 5.
Fig. 5.
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.
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