Loss of Sprouty2 partially rescues renal hypoplasia and stomach hypoganglionosis but not intestinal aganglionosis in Ret Y1062F mutant mice

The glial cell line-derived neurotrophic factor (GDNF)/RET tyrosine kinase signaling pathway plays crucial roles in the development of the enteric nervous system (ENS) and the kidney. Tyrosine 1062 (Y1062) in RET is an autophosphorylation residue that is responsible for the activation of the PI3K/AKT and RAS/MAPK signaling pathways. Mice lacking signaling via Ret Y1062 show renal hypoplasia and hypoganglionosis of the ENS although the phenotype is milder than the Gdnf- or Ret-deficient mice. Sprouty2 (Spry2) was found to be an antagonist for fibroblast growth factor receptor (FGFR) and acts as an inhibitory regulator of ERK activation. Spry2-deficient mice exhibit hearing loss and enteric nerve hyperplasia. In the present study, we generated Spry2-deficient and Ret Y1062F knock-in (tyrosine 1062 is replaced with phenylalanine) double mutant mice to see if abnormalities of the ENS and kidney, caused by loss of signaling via Ret Y1062, are rescued by a deficiency of Spry2. Double mutant mice showed significant recovery of ureteric bud branching and ENS development in the stomach. These results indicate that Spry2 regulates downstream signaling mediated by GDNF/RET signaling complex in vivo.     

Developmental processes of achlorophyllous orchid, <Emphasis Type="Italic">Epipogium roseum</Emphasis>: from seed germination to flowering under symbiotic cultivation with mycorrhizal fungus

We have achieved the symbiotic cultivation of an apparently achlorophyllous orchid, Epipogium roseum Lindl., with a mycorrhizal fungus isolated from an underground organ of this orchid. Although the seed germination rate was extremely low, subsequent growth from protocorm to flowering was induced in a medium containing volcanic soils and sawdust. Stolons elongated from each protocorm, and rhizomes were formed at certain intervals on the stolons. Some of the rhizomes developed into a coralloid form, and tubers were formed from the coralloid rhizomes. The coralloid rhizomes degenerated concurrently with maturation of the tubers. Six months after seed sowing, around 80 tubers were produced from a single protocorm. An inflorescence appeared from each of the large tubers, and the process to flowering was observed in one of these. Consequently, the developmental processes from seed to flowering in E. roseum was clearly revealed in this study.     

A simple and efficient plant regeneration system for leaf protoplasts ofNierembergia repens by inducing single shoots on the microcolonies

A simple and efficient protocol for plant regeneration from mesophyll protoplasts ofNierembergia repens is described. The protoplasts divided in modified half-strength MS (/12 MS) medium containing benzylaminopurine (BA) and a-naphthaleneacetic acid (NAA) and formed visible colonies after 2 weeks which produced single adventitious shoots 4 weeks later. Plating efficiency (11.2%), percent colony formation (0.84%), and the number of shoot-forming colonies (368/dish) were highest in /12 MS containing 0.1 mg/l BA and 0.05 mg/l NAA. However, the percentage of colonies with shoot formation was highest (31.8%) in /12 containing 0.05 mg/l BA and 0.01 mg/l NAA. Almost all of the remaining colonies (97.5%) also regenerated shoots upon transfer onto MS medium containing 0.05 mg/l BA. The shoots with 2–3 leaves readily rooted 3–5 days after insertion in /12MS lacking plant growth regulators. Regenerated plantlets were easily established in soil 50 days after protoplast isolation. All the regenerants were normal and possessed diploid chromosome numbers.Abbreviations BA Benzylaminopurine - FDA fluorescein diacetate - MES 2-(Morpholino)ethanesulfonic acid - MS Murashige and Skoog (1962) medium - /12MS half strength MS - NAA -naphthaleneacetic acid - PGR plant growth regulator     

Shoot regeneration from spinach hypocotyl segments by short term treatment with 5,6-dichloro-indole-3-acetic acid

Factors affecting shoot regeneration from hypocotyl segments of spinach (Spinacia oleracea L.) were investigated. When expiants were cultured on medium containing 10 mg/l IAA for 7 weeks, 3 out of 9 cultivars showed relatively high shoot regeneration response (15 – 35%). The other PGRs tested had no effect on shoot regeneration. However, the transfer of explants from auxin-containing medium to auxin-free medium 20 d after culture induced shoot formation from expiants cultured on media containing each of the auxin sources tested individually. By applying this short term auxin treatment, more than 80% shoot regeneration was obtained on medium containing 5–20 mg/l 5,6-Cl₂-IAA, compared to less than 30% with 10–20 mg/l IAA treatment.Abbreviations BAP 6-benzylaminopurine - NAA 1naphthaleneacetic acid - IAA indole-3-acetic acid - 2,4D 2,4-dichlorophenoxyacetic acid - 5,6-Cl₂-IAA 5,6dichloro-indole-3-acetic acid - PGR plant growth regulator - A-PGR auxin-like plant growth regulator     

Plant regeneration from hairy roots induced by infection with Agrobacterium rhizogenes in Crotalaria juncea L.

 Hairy roots were induced from leaf segments of Crotalaria juncea, which is used as a green manure crop antagonistic to nematodes, by infection with a mikimopine type wild strain of Agrobacterium rhizogenes A13 (MAFF02-10266). These roots exhibited vigorous growth and abundant lateral branching on half-strength Murashige and Skoog (1/2MS) medium without phytohormones. The adventitious shoots were induced from 30% of root segments 3 months after transfer onto medium containing 3 mg/l benzyl adenine. These shoots produced roots 1 month after transfer onto 1.2% agar-solidified 1/2MS medium without phytohormones. Regenerated plants were successfully grown under greenhouse conditions. The transgenic nature of the regenerated plants was confirmed by Southern-blot analysis. Received: 13 February 1999 / Revision received: 12 August 1999 / Accepted: 17 August 1999     

Construction of synteny groups of Brassica alboglabra by RAPD markers and detection of chromosome aberrations and distorted transmission under the genetic background of B. campestris

Interspecific hybrids were produced by crosses between the inbred lines of B. campestris and B. alboglabra, and were backcrossed twice to B. campestris. Genetical constitutions of the BC₂ plants were analyzed by RAPD (random amplified polymorphic DNA), flow cytometry and cytological observations. By using 140 arbitrary primers, a total of 137 polymorphic bands were obtained and 125 were found to be specific to B. alboglabra. Based on the presence and absence of the specific RAPD markers of B. alboglabra, 13 synteny groups were constructed. The number of markers in each synteny group was found to be different and varied from 2 to 28. This reflects the difference in the degree of genetic variability among the B. alboglabra chromosomes from those of B. campestris. Losses or gains of RAPD markers were observed frequently in most of the synteny groups, which indicated the occurrence of chromosome translocations and/or deletions in the chromosomes of B. alboglabra. In a population of 40 BC₂ plants, chromosome transmission rates were analyzed by using the RAPD markers in each synteny group. Most of the chromosomes of the synteny groups were transmitted with rates of 0.37–0.68. An extremely high transmission rate, 0.98, was however observed in one of the synteny groups. Inheritance data of the synteny groups revealed relationships among themselves. The plants lacking the RAPD markers of two synteny groups tended to lose others belonging to the rest of the synteny groups, indicating the effects of these groups on the transmission of B. alboglabra chromosomes to the B. campestris background. Received: 26 February 1999 / Accepted: 30 December 1999     

The Holocene Pulleniatina Minimum Event revisited: Geochemical and faunal evidence from the Okinawa Trough and upper reaches of the Kuroshio current

The Holocene Pulleniatina Minimum Event (PME) is characterized by a very low abundance of the planktonic foraminifer Pulleniatina obliquiloculata between  4.5 and 3 ka. The PME occurs widely in the Okinawa Trough and the South China Sea, and can be correlated throughout this area; it has been related to variability in the Kuroshio current. To further explore the nature of the PME, we studied cores obtained from the southern Okinawa Trough and the upper reaches of the Kuroshio current. Faunal census data indicate that all cores record the PME between  4.5 and  3 ka. The relative abundance of Neogloboquadrina dutertrei is negatively correlated to that of P. obliquiloculata in the southern Okinawa Trough, but not in the sites at the upper reaches. Mg/Ca and δ¹⁸O measurements on Globigerinoides ruber shells from the southern Okinawa Trough indicate that there was no change in sea surface temperature or sea surface salinity during the PME. The vertical structure of the water column as reconstructed by multispecies δ¹⁸O and δ¹³C profiles shows no consistent anomalies in the southern Okinawa Trough and western Philippine Sea during the PME. These observations suggest that: (1) the PME was not restricted to marginal seas, but widespread in the western North Pacific. (2) The high abundance of N. dutertrei during the PME in the Okinawa Trough may be a result of higher food-availability in the absence of P. obliquiloculata. (3) No distinctive, consistent anomalies in the paleoceanographic proxies are associated with the PME, implying there were no changes in hydrography and productivity. The absence of a linkage between faunal variation and paleoceanographic proxies indicates that we do not yet understand what causes changes in planktonic foraminiferal assemblages. This lack of understanding implies that we cannot always trust fauna-based paleothermometry at millennial timescales.     

Production of marker-free transgenic Nierembergia caerulea using MAT vector system

Agrobacterium tumefaciens strain EHA105 harboring an ipt-type MAT vector, pNPI132, was used to produce morphologically normal transgenic Nierembergia caerulea cv. Mont Blanc employing ipt gene as the selectable marker gene. β-glucuronidase (GUS) gene was used as model gene of interest. The MAT vector system is a positive selection system that gives the advantage of regeneration to the transgenic cells without killing the non-transgenic cells. Infected explants were cultured on hormone- and antibiotic-free MS medium, and 65% of the regenerated shoots developed ipt shooty phenotype-morphologically abnormal shoots, within approximately 3 months after co-cultivation. Twenty morphologically normal shoots were produced from 12 transgenic ipt shoots 7 months after co-cultivation. The normal shoots rooted well on hormone-free MS medium. Ninety percent of the normal shoots were ipt ⁻, GUS⁺ and excision⁺ as determined by PCR and Southern blot analyses. These results indicate that ipt-type MAT vector system can be used successfully in Nierembergia to produce marker-free transgenic plants without using phytohormones and selective chemical agents.     

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.