Transgenic tetraploid Isatis indigotica expressing Bt Cry1Ac and Pinellia ternata agglutinin showed enhanced resistance to moths and aphids.

Co-expression of multiple genes encoding different kinds of insect resistant proteins has been developed to confer a broader spectrum of pest control. Tetraploid Isatis indigotica Fort was transformed with a plasmid, p3300BP, containing Bacillus thuringiensis Cry1Ac gene (Bt) and Pinellia ternata agglutinin gene (Pta) and the selectable marker herbicide resistance gene (Bar) driven by the CaMV35S promoter via Agrobacterium tumefaciens-mediated transformation. The integration and expression of introduced genes in regenerated transgenic plants were confirmed by PCR and Western blot assays.

Tropane alkaloids production in transgenic Hyoscyamus niger hairy root cultures over-expressing putrescine N-methyltransferase is methyl jasmonate-dependent.

The cDNA from Nicotiana tabacum encoding Putrescine N-methyltransferase (PMT), which catalyzes the first committed step in the biosynthesis of tropane alkaloids, has been introduced into the genome of a scopolamine-producing Hyoscyamus niger mediated by the disarmed Agrobacterium tumefaciens strain C58C1, which also carries Agrobacterium rhizogenes Ri plasmid pRiA4, and expressed under the control of the CaMV 35S promoter. Hairy root lines transformed with pmt presented fivefold higher PMT activity than the control, and the methylputrescine (MPUT) levels of the resulting engineered hairy roots increased four to fivefold compared to the control and wild-type roots, but there was no significant increase in tropane alkaloids. However, after methyl jasmonate (MeJA) treatment, a considerable increase of PMTase and endogenous H6Hase as well as an increase in scopolamine content was found either in the transgenic hairy roots or the control.

Molecular cloning and characterization of a novel calcium-dependent protein kinase gene IiCPK2 Responsive to polyploidy from tetraploid Isatis indigotica.

A novel calcium-dependent protein kinase gene (designated as IiCPK2) was cloned from tetraploid Isatis indigotica. The full-length cDNA of IiCPK2 was 2585 bp long with an open reading frame (ORF) of 1878 bp encoding a polypeptide of 625 amino acid residues. The predicted IiCPK2 polypeptide included three domains: a kinase domain, a junction domain (or autoinhibitory region), and a C-terminal calmodulin-like domain (or calcium-binding domain), which presented a typical structure of plant CDPKs.

Engineering tropane biosynthetic pathway in Hyoscyamus niger hairy root cultures.

Scopolamine is a pharmaceutically important tropane alkaloid extensively used as an anticholinergic agent. Here, we report the simultaneous introduction and overexpression of genes encoding the rate-limiting upstream enzyme putrescine N-methyltransferase (PMT) and the downstream enzyme hyoscyamine 6 beta-hydroxylase (H6H) of scopolamine biosynthesis in transgenic henbane (Hyoscyamus niger) hairy root cultures. Transgenic hairy root lines expressing both pmt and h6h produced significantly higher (P < 0.