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The cDNAs from Hyoscyamus niger, encoding two enzymes of tropane-alkaloid biosynthesis, tropinone reductase I (TR-I) and hyoscyamine-6beta-hydroxylase (H6H), have been simultaneously introduced into Nicotiana tabacum using particle bombardment and expressed under the control of the CaMV 35S promoter. Southern and Northern analyses confirmed integration and transcript accumulation for both trl and h6h. Fertile tobacco plants expressing both transgenes were regenerated and detached leaves of these plants were fed with tropinone and hyoscyamine, the substrates, respectively, of TR-I and H6H. Besides the expected TR-I and H6H reaction products, acetylated forms of tropine were also generated in these experiments, indicating that the expression of alkaloid-pathway enzymes in a transgenic background can produce unexpected substances. In addition, leaves of the transgenic plants showed in most cases higher nicotine content than leaves of control plants. Nicotine levels were approximately 3- to 13-fold higher in both the parental transgenic lines and in T1 progeny expressing functional TR-I and H6H, compared to levels in wild-type plants and in transgenic plants carrying the nptII transgene alone. In addition, nicotine related compounds such as anatabine, nornicotine, bipyridine, anabasine, and myosmine were identified in transgenic tobacco lines and below detection limit in wild-type plants, suggesting changes in the activity of the enzymes in the nicotine biosynthetic pathway in the transgenic background.

This report describes the stable expression of a medically important antibody in the staple cereal crops rice and wheat. We successfully expressed a single-chain Fv antibody (ScFvT84.66) against carcinoembryonic antigen (CEA), a well characterized tumor-associated marker antigen. scFv constructs were engineered for recombinant antibody targeting to the plant cell apoplast and ER. Up to 30 mg/g of functional recombinant antibody was detected in the leaves and seeds of wheat and rice. We confirmed that transgenic dry seeds could be stored for at least five months at room temperature, without significant loss of the amount or activity of scFvT84.66. Our results represent the first transition from model plant expression systems, such as tobacco and Arabidopsis, to widely cultivated cereal crops, such as rice and wheat, for expression of an antibody molecule that has already shown efficacy in clinical applications. Thus, we have established that molecular pharming in cereals can be a viable production system for such high-value pharmaceutical macromolecules. Our findings provide a strong foundation for exploiting alternative uses of cereal crops both in industrialized and developing countries.

Transgenic rice indica varieties Basmati 370 and M 7 expressing the novel cry2A (Bt) insecticidal gene were generated by particle bombardment. Molecular and biochemical analyses in R0 and R1 populations confirmed stable integration and expression of this novel Bt transgene. We estimated that the gene product was expressed up to 5% of total leaf protein. Insect feeding bioassays demonstrated that the Cry2A protein was effective against the yellow stem borer and the rice leaf folder, two major rice pests in the Indian Subcontinent. This is the first report of the control of major rice pests using this specific Bt gene. The cry2A gene can now be used in combination with other insecticidal genes for pyramiding resistance against insect pests. This will delay, or perhaps in combination with integrated pest management practices, prevent evolution of insect populations resistant to single insecticidal genes.