The regulation of solasodine production by Agrobacterium rhizogenes-transformed roots of Solanum aviculare.

Transgenic roots of Solanum spp. containing extra copies of an hmgr gene derived from Artemisia annua have been obtained via transformation with Agrobacterium rhizogenes. Hairy root clones of Solanum aviculare which were transgenic for hmgr typically grew faster than those which did not contain extra copies of this gene and also accumulated up to 4.2 times more solasodine when grown under dark, but not light, conditions. The implications of these findings with respect to the regulation of solasodine production in Solanum spp. are considered.

Novel biotechnological approaches in environmental remediation research.

Two novel approaches, the use of Agrobacterium-transformed plant roots and mycelia cultures of fungi, are considered as research tools in the study of the remediation of soil, groundwater, and biowastes. Transformed roots are excellent model systems for screening higher plants that are tolerant of various inorganic and organic pollutants, and for determining the role of the root matrix in the uptake and further metabolism of contaminants. Edible and/or medicinal fungi may also be natural environmental remediators. Liquid cultures of fungal mycelia are appropriate model systems with which to commence screening and biochemical studies in this under-researched area of biotransformation.

CaMV 35S promoter directs ß-glucuronidase expression in the laticiferous system of transgenic Hevea brasiliensis (rubber tree).

Hevea brasiliensis anther calli were genetically transformed using Agrobacterium GV2260 (p35SGUSINT) that harboured the ß-glucuronidase (gus) and neomycin phosphotransferase (nptII) genes. ß-Glucuronidase protein (GUS) was expressed in the leaves of kanamycin-resistant plants that were regnerated, and the presence of the gene was confirmed by Southern analysis. GUS was also observed to be expressed in the latex and more importantly in the serum fraction. Transverse sections of the leaf petiole from a transformed plant revealed GUS expression to be especially enhanced in the phloem and laticifers. GUS expression was subsequently detected in every one of 194 plants representing three successive vegetative cycles propagated from the original transformant. Transgenic Hevea could thus facilitate the continual production of foreign proteins expressed in the latex.

Cloning and analysis of a cDNA encoding farnesyl diphosphate synthase from Artemisia annua.

A cDNA encoding farnesyl diphosphate (FPP) synthase (FPPS) has been cloned from a cDNA library of Artemisia annua. The sequence analysis showed that the cDNA encoded a protein of 343 amino acid (aa) residues with a calculated molecular weight of 39420 kDa. The deduced aa sequence of the cDNA was highly similar to FPPS from other plants, yeast and mammals, and contained the two conserved domains found in polyprenyl synthases including FPPS, geranylgeranyl diphosphate synthases and hexaprenyl diphosphate synthases. The expression of the cDNA in Escherichia coli showed enzyme activity for FPPS in vitro.

Expression of bacterial isochorismate synthase (EC 5.4.99.6) in transgenic root cultures ofRubia peregrina.

The gene coding for isochorismate synthase (EC 5.4.99.6) was amplified by the polymerase chain reaction fromEscherichia coli, cloned into a binary vector. and mobilised intoAgrobacterium rhizogenes LBA9402 which was used to transformRubia peregrina. Transgenic roots containing bacterial isochorismate synthase cDNA expressed twice as much isochorismate synthase activity (4.88 pkat/mg protein) as the control roots (2.45 pkat/mg protein) after 10 days in culture, and accumulatedca. 20% higher levels of total anthraquinones after 30 days in culture. Whilst the amount of total alizarin (free and bound) in the transgenic roots was 30% higher than in control roots, the level of free alizarin wasca. 85% higher.

Gene insertion into Hevea brasiliensis.

A transformation system has been developed for Hevea brasiliensis using the particle gun method. Anther derived calluses were transformed with vectors harbouring the ß-glucuronidase (gus) gene, the neomycin phosphotransferase (nptII) gene, and the chloramphenicol acetyl transferase (cat) gene. Gene transfer was determined by histochemical staining and fluorometric assay for ß-glucuronidase activity, enzyme linked immunosorbent assay for detecting neomycin phosphotransferase II gene and direct enzyme assay for detection of expression of the cat gene. These independent assays all showed a several-fold increase, compared to control values, in gene product level and enzyme activity in extracts from transformed callus and embryoids of Hevea. These results were confirmed using polymerase chain reaction with primers designed to amplify an internal gus fragment. Together, the results show the feasibility of the particle gun method for the introduction of foreign genes into Hevea.

Organogenesis in callus culture.

Plant cells can be totipotent, i.e., each cell may be capable of developing into an entire plant when provided with the correct environmental stimuli. Research during the last 30 yr has demonstrated that successful organogenesis in callus cultures can be achieved by the correct choice of medium components, selection of a suitable inoculum, and control of the physical environment (1). The manipulation of plant growth regulator concentration is probably the most widely used technique for the induction of organogenesis, and this methodology has formed the basis of the propagation of commercially important plants via tissue culture in recent years (2).

Investigation of the enzymatic digestion of plant cell walls using reflectance Fourier Transform Infrared spectroscopy.

A method has been developed to determine the reflectance Fourier Transform Infrared spectra of plant cells grown in vitro and of the protoplasts released from such cells by enzymatic digestion. It is demonstrated that there is a smooth and reproducible transition in spectral detail as enzymatic digestion procedes. Reflectance Fourier Transform Infrared spectroscopy has been used to monitor the progress of protoplast release during enzymatic digestion of cell wall material.