Overexpression of farnesyl pyrophosphate synthase (<Emphasis Type="Italic">FPS</Emphasis>) gene affected artemisinin content and growth of <Emphasis Type="Italic">Artemisia annua</Emphasis> L.

Transgenic plants of Artemisia annua L., a medicinal plant that produces the compound artemisinin which has an anti-malarial activity, were developed following Agrobacterium tumefaciens-mediated transformation of leaf explants. A. tumefaciens strain EHA105 carrying either pCAMBIA1301 or pCAMBIAFPS was used. Both plasmids harbored the hygromycin phosphotransferase II (hptII) gene as a selectable gene, but the latter plasmid also harbored the gene encoding for farnesyl pyrophosphate synthase (FPS), a key enzyme for artemisinin biosynthesis. Shoot regeneration was observed either directly from leaf sections or via intervening callus when explants were incubated on solidified Murashige and Skoog (MS) (1962) medium containing 0.1 mg l⁻¹ α-naphthaleneacetic acid (NAA), 1 mg l⁻¹ N⁶-benzyladenine (BA), 30 mg l⁻¹ meropenem and 10 mg l⁻¹ hygromycin. Applying vacuum infiltration dramatically increased transformation efficiency up to 7.3 and 19.7% when plasmids with and without FPS gene were used, respectively. All putative transgenic regenerants showed positive bands of hptII gene following Southern blot analysis. Expression of FPS was observed in all transgenic lines, and FPS over-expressed lines exhibited higher artemisinin content and yield, of 2.5- and 3.6-fold, respectively, than that detected in wild-type plants. A relatively high correlation (R ² = 0.78) was observed between level of expression of FPS and artemisinin content. However, gene silencing was detected in some transgenic lines, especially for those lines containing two copies of the FPS transgene, and with some lines exhibiting reduced growth.     

Nuclear DNA content in the genus Hepatica (Ranunculaceae)

Using flow cytometry, we measured the nuclear DNA contents of all known taxa in Hepatica. Nuclear DNA content of Hepatica falconeri (diploid, crenate leaf lobes) was significantly lower than that of diploid entire species. Among the tetraploid species, crenate species had lower DNA contents than the entire taxon H. nobilis var. pubescens. The DNA content of the tetraploid species was more than double that of the diploid species among the same leaf-type groups.     

Unilateral compatibility and genotypic difference in crossability in interspecific hybridization between Dianthus caryophyllus L. and Dianthus japonicus Thunb

Reciprocal interspecific crosses were carried out between six lines of Dianthus caryophyllus L. and one line of Dianthus japonicus Thunb. Although no seed was set when D. japonicus was used as the seed parent, six seedlings were successfully obtained from 2,380 immature ovules by applying the embryo-rescue technique. However, they showed seed parent-like morphology and no evidence for the hybridity by flow cytometry and RAPD analyses. When six lines of D. caryophyllus were used as seed parents, a total of 192 seedlings were successfully obtained without using the embryo-rescue technique. Among these seedlings, 12 out of 25 progenies obtained from the carnation line '98sp1651' were confirmed to be the hybrids. The remaining 13 progenies of this line, and the total 167 progenies obtained from the other carnation lines, had carnation-like morphology without any evidence of hybridity by flow cytometry and RAPD analyses. The progenies confirmed as hybrids had intermediate characters of the parents with respect to leaf width and flower size, but they had a uniform flower color, reddish purple, which was different from that of either parent. Since the hybrids obtained in the present study have some profitable characters such as vigorous growth in summer time, upright robust stem, broad leaves and early flowering, they are expected to be used for the breeding of carnation which is suitable for growing under the Japanese climate.     

The Wilms' tumor gene WT1 is a common marker of progenitor cells in fetal liver

It is well known that the Wilms' tumor gene WT1 plays an important role in cell proliferation and differentiation, and in organ development. In this study, to examine the role of the WT1 gene in lineage determination, fetal liver cells from LacZ-transgenic mice, in which WT1 expression was marked by the expression of the LacZ gene driven by WT1 promoter, were FACS-sorted according to LacZ expression of high (LacZ(++)) or undetectable (LacZ(-)) levels, which paralleled endogenous WT1 expression levels. LacZ(++) fetal liver cells were enriched by hepatocyte and endothelial progenitor cells. These results indicated that WT1 expression is a common marker of both hepatocyte and endothelial progenitors. These results also implied a role of the WT1 gene in lineage determination.     

Nestin-Expressing Stem Cells Promote Nerve Growth in Long-Term 3-Dimensional Gelfoam?-Supported Histoculture

We have previously reported that hair follicles contain multipotent stem cells which express nestin. The nestin-expressing cells form the hair follicle sensory nerve. In vitro, the nestin-expressing hair follicle cells can differentiate into neurons, Schwann cells, and other cell types. In the present study, the sciatic nerve was excised from transgenic mice in which the nestin promoter drives green fluorescent protein (ND-GFP mice). The ND-GFP cells of the sciatic nerve were also found to be multipotent as the ND-GFP cells in the hair follicle. When the ND-GFP cells in the mouse sciatic nerve cultured on Gelfoam® and were imaged by confocal microscopy, they were observed forming fibers extending the nerve. The fibers consisted of ND-GFP-expressing spindle cells, which co-expressed the neuron marker β-III tubulin, the immature Schwann-cell marker p75^NTR and TrkB which is associated with neurons. The fibers also contain nestin-negative spherical cells expressing GFAP, a Schwann-cell marker. The β-III tubulin-positive fibers had growth cones on their tips expressing F-actin, indicating they are growing axons. When the sciatic nerve from mice ubiquitously expressing red fluorescent protein (RFP) was co-cultured on Gelfoam® with the sciatic nerve from ND-GFP transgenic mice, the interaction of nerves was observed. Proliferating nestin-expressing cells in the injured sciatic nerve were also observed in vivo. Nestin-expressing cells were also observed in posterior nerves but not in the spinal cord itself, when placed in 3-D Gelfoam® culture. The results of the present report suggest a critical function of nestin-expressing cells in peripheral nerve growth and regeneration.     

Enhancement of artemisinin content and biomass in <Emphasis Type="Italic">Artemisia annua</Emphasis> by exogenous GA<Subscript>3</Subscript> treatment

Artemisinin is a promising and potent antimalarial drug naturally produced by the plant Artemisia annua L. but in very low yield. Its artemisinin content is known to be greatly affected by both genotype and environmental factors. In this study, the production of artemisinin and leaf biomass in Artemisia annua L. was significantly increased by exogenous GA₃ treatment. The effect of GA₃ application on expression of proposed key enzymes involved in artemisinin yield was examined in both wild type (007) and FPS-overexpression (253-2) lines of A. annua. In the wild type (007) at 6 h post GA₃ application there was an abrupt rise in FPS, ADS and CYP71AV1 expression and at 24 h a temporary and significant peak in artemisinin (1.45-fold higher than the control). After GA₃ application in line 253-2, there was a dramatic rise in expression of FPS at 3 h, CYP71AV1 at 9 h and ADS at 72 h and accumulation of artemisinin after 7 days, which was a delay when compared with the wild type plant. Thus, increased artemisinin content from exogenous GA₃ treatment was associated with increased expression of key enzymes in the artemisinin biosynthesis pathway. Interestingly, exogenous GA₃ continuously enhanced artemisinin content from the vegetative stage to flower initiation in both plant lines and gave significantly higher leaf biomass than in control plants. Consequently, the artemisinin yield in GA₃-treated plants was much higher than in control plants. Although the maximum artemisinin content was found at the full blooming stage [2.1% dry weight (DW) in 007 and 2.4% DW in 253-2], the highest artemisinin yield in GA₃-treated plants was obtained during the flower initiation stage (2.4 mg/plant in 007 and 2.3 mg/plant in 235-2). This was 26.3 and 27.8% higher, respectively, than in non-treated plants 007 and 253-2. This study showed that exogenous GA₃ treatment enhanced artemisinin production in pot experiments and should be suitable for field application.     

Cryopreservation of Doritaenopsis suspension culture by vitrification

 Cells of a suspension culture of Doritaenopsis cv. New Toyohashi were placed in a mixture of 2 M glycerol and 0.4 M sucrose for 15 min at room temperature and then dehydrated with a vitrification solution (PVS2) for 1–3 h on ice and plunged into liquid nitrogen. The highest viability (64% by 2,3,5-triphenyltetrazolium chloride stainability) was obtained when the cells were precultured in liquid New Dogashima medium with 0.1 M sucrose and 1.0 mg/l abscisic acid for 1 week at 25  °C in the light. Dehydration by PVS2 was important for the cryopreservation of Doritaenopsis cells. Protocorm-like bodies were induced from cryopreserved cells without morphological variations. Received: 18 January 2000 / Revision received: 16 June 2000 / Accepted: 22 June 2000