Barry V. Charlwood

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.

The control of callus formation and differentiation in scented pelargoniums

Summary Long-term cultures have been established from stem tissue of 27 variants of scented Pelargoniums. The effects of a wide range of concentrations of growth regulators on growth and differentiation of P. australe, P. citriodorum, P. crispum, P. graveolens, P. filifolium, P. quercifolium, P. tomentosum and the cultivars «Duke of York», «Miss Australia» and «Prince of Orange» are reported and discussed. Shoot regeneration could be induced in calli of all variants (with the exception of P. echinatum) by transferrence to a medium containing BAP (0.50 mg · 1 - 1 ) and NAA (0.05 mg · 1 - 1 ). Callus lines of P. australe and P. tomentosum , which had been maintained on a medium free of 2,4-D, were still highly morphogenic after 1.5 years in culture. Shoot morphogenesis has been efficiently induced in submerged cultures of P. australe, P. jragrans, P. tomentosum and the cultivar «Lillian Pottingen». The accumulation of monoterpenes in callus cultures grown under maintenance conditions was negligible, but morphogenic cultures, particularly those in liquid media, accumulated significant amounts of secondary compounds.

Accumulation of essential oils by Agrobacterium tumefaciens-transformed shoot cultures of Pimpinella anisum

Axenic transformed shoot cultures of Pimpinella anisum (anise) were established following inoculation of plant stems with the nopaline strain T37 of Agrobacterium tumefaciens. The stable incorporation of T-DNA in the transformed tissues was demonstrated by polymerase chain reaction. Total essential oil accumulated by transformed shoot cultures grown under continuous light was found to be 18% lower (per unit fresh weight of tissue) than that produced by untransformed shoot cultures incubated under similar conditions, but more than 89% lower than the yield of oil from the intact plant. The relative amounts of the principal components of the essential oil of the transformed shoot cultures, namely geraniol, β-bisabolene, trans-pseudoisoeugenol-2-methylbutyrate and transanethole, were similar to those present in the parent plant, but significantly different from those of the untransformed shoot cultures.

Agrobacterium rhizogenes-mediated transformation of Rubia peregrina L.: in vitro accumulation of anthraquinones

An Agrobacterium rhizogenes-mediated transformation system for Rubia peregrina L. has been established by co-cultivation of callus cultures or by direct infection of explants with A. rhizogenes LBA 9402 harbouring the binary vector pMON 9703 containing gus and npt-II genes as markers. The putative transformed roots were selected on medium containing kanamycin (25 mg l-1). Antibiotic resistant root clones were subjected to histochemical analysis for the localisation of β-glucuronidase activity. Polymerase chain reaction was used to confirm the presence of gus, npt-II and TL border sequences in the transformed root clones. Spontaneous regeneration of shoots was observed from 30 day-old transgenic roots. Total anthraquinone and alizarin contents of transgenic root cultures were measured by spectrophotometry and by high performance liquid chromatography. The accumulation of total anthraquinones in transformed roots was found to be approximately 2-fold higher than that found in one year-old field grown roots (2.12±0.12 and 1.23±0.12 mg g-1 dry weight, respectively). Alizarin was found to be the major anthraquinone in transformed root cultures and was found to be approximately 3-fold higher than in field grown roots.

The accumulation of phenylpropanoid glycosides in tissue cultures of Tecoma sambucifolium

Abstract Callus cell lines of Tecoma sambucifolium obtained from stem explants accumulate up to 8.0% of their dry weight as phenylpropanoid glycosides: the main components were identified as verbascoside, orobanchoside, isoverbascoside and rhodioloside. In fast growing cell lines, selected on medium containing 2,4-dichlorophenoxyacetic acid and kinetin in comparable concentrations, the ratio of the sugar esters was 60:20:3:1, respectively, a product profile similar to that found in the leaves of the intact plant. When cells were selected on medium containing much lower levels of cytokinin compared with auxin, the growth rate was considerably reduced and the product ratio was found to be 25:60:6:4, respectively. None of the cell lines accumulated iridoid glycosides characteristic of the parent plant.

The regulation of accumulation of lower isoprenoids in plant cell cultures

In comparison with other classes of natural products, few studies have been carried out concerning the accumulation of monoterpenes in plant cells in culture. The subject has been reviewed (Charlwood et al. 1986) and a compilation of data is available (Koch-Heitzmann and Schultze 1988).

Accumulation of diterpenoids in cell and root-organ cultures of Jatropha species

In a number of species of the genus Jatropha, used in folk medicine in tropical areas, the active principle is the macrocyclic diterpenoid jatrophone, which co-occurs with the inactive diterpenoids jatropholone A and B. Callus and suspension cultures of Jatropha cathartica, J. cinerea, J. curcas, J. dioica, J. elliptica, J. multifida, J. podagrica and J. mollissima were established, but most of the cell lines tested accumulated no, or only small quantities of jatrophone. The maximum jatrophone accumulation (3 mu g . g(-1) dry weight) was obtained with callus and suspension cultures derived from J. curcas and J. elliptica. Root-organ cultures of J. elliptica formed following incubation of suspension cells in the presence of 3-indoleburyric acid (5 mg . L(-1)). The maximum accumulation of jatrophone and the jatropholones in newly initiated roots grown in vitro was 329 and 458 mu g . g(-1) dry weight, respectively. The effects on diterpenoid accumulation root-organs of alteration of environmental parameters, and of the addition of chitosan, Benomyl(R) and Al3+ to the growth medium were investigated. The yield of jatrophone was significantly increased in the presence of 0.07-0.7 mmol/L Al3+, and the product profile was altered concomitantly.

The l ocalisation and a ccumulation of v alepotriates in h airy r oots of Valerianella discoidea (L.) Loisel

A cytochemical and biochemical study of Agrobacterium rhizogenes-transformed (hairy) roots of Valerianella discoidea has demonstrated: (a) the presence of a soluble valepotriate fraction in root sections in addition to the stored valepotriates in lipid droplets; (b) that roots need to be at least 3.5 cm long before they will synthesise/store valepotriates; (c) that there is a loss of valepotriates from the pericycle cells about to form lateral roots; and (d) an accumulation of seven times more monoene and diene valepotriates compared with non-transformed, control roots. Copyright