Somatic embryogenesis and plant regeneration in centipedegrass (Eremochloa ophiuroides [Munro] Hack.)

Centipedegrass (Eremochloa ophiuroides [Munro] Hack.) is an important warm-season turfgrass and pasture grass. To explore the potential use of biotechnical tools in breeding of centipedegrass, we established an efficient plant regeneration system for this species. Four basal media and 24 combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BAP) were examined for their effects on callus induction from mature seed explants. Twenty combinations of naphthaleneacetic acid (NAA) and BAP were tested for their effect on plant regeneration. Results indicated that Murashige and Skoog basal medium supplemented with 4.5 mg l⁻¹ 2,4-D and 1 mg l⁻¹ BAP was the best medium for callus induction, while the combination of 2 mg l⁻¹ BAP and 1 mg l⁻¹ NAA induced the highest rate of regeneration and development of shoots and roots. This work provides a basis for the breeding of centipedegrass through somaclonal variation and genetic transformation.     

Somatic embryogenesis and plantlet production using rejuvenated tissues from serial grafting of a mature <Emphasis Type="Italic">Kalopanax septemlobus</Emphasis> tree

An effective micropropagation technique via somatic embryogenesis has been developed using tissue from serially grafted shoots generated from a mature Kalopanax septemlobus tree (~40 y old). Callus was induced from leaf segments obtained from the grafts by culturing the explants in Murashige and Skoog (MS) medium supplemented with 2,4-D and 3% w/v sucrose under darkness. The effects of sucrose, coconut water, and polyethylene glycol (PEG-3350) were evaluated as factors to promote development of somatic embryos (SEs) from embryogenic callus. More than 90% of explants formed callus; however, only 2.5%, or 20 leaf segments out of 800 explants, formed embryogenic callus after 8 wk of culture. High sucrose concentrations (3% and 5% w/v) were effective in inducing SEs. Treatment with 2–10% v/v coconut water also had a positive effect on embryo induction. A synergistic effect on SE induction was obtained using sucrose and PEG, with presence of the latter compound resulting in smaller, more uniform SEs. Embryo germination and conversion to plantlets were significantly influenced by the gelling agents. In general, gelrite-gelled medium was superior to agar-gelled medium. In gelrite-gelled medium, gibberillic acid (GA₃) enhanced embryo germination. Converted plantlets in an artificial soil mixture showed a 91% survival rate and displayed no distinct morphological variations. Our results indicate that reliable somatic embryogenesis and plant production can be achieved with rejuvenated tissues after repeated grafting of shoots derived from a mature Kalopanax septemlobus tree.     

Biosynthesis of Dibenzyl Trisulfide (DTS) from somatic embryos and rhizogenous/embryogenic callus derived from Guinea hen weed (<Emphasis Type="Italic">Petiveria alliacea</Emphasis> L<Emphasis Type="Italic">.</Emphasis>) leaf explants

A procedure for producing somatic embryos enriched with dibenzyl trisulfide (DTS) using a hormone-dependent culture system is reported for Petiveria alliacea L. (Guinea hen weed). Leaf explants were cultured on a Murashige and Skoog medium supplemented with a range of naphthaleneacetic acid (NAA) concentrations and a fixed concentration of benzyladenine (BAP) at 11.0 μM and sucrose or glucose at 30 g l⁻¹. Leaf explants cultured on all media types started to form callus at the cut surfaces of the discs 10–14 d after initiation. The type of sugar used influenced average fresh weight, the propensity to form roots, as well as the embryogenic response. The highest mean fresh weight (337.7 ± 26.18 mg) and mean root number (23.7 ± 1.69) was produced on media enriched with sucrose and supplemented with 53.7 μM NAA and 11.0 μM BAP. An ethanol extract of rhizogenic/embryogenic callus or somatic embryos was subjected to high-performance liquid chromatography analysis, which revealed the presence of DTS in both extracts. UV spectral analysis and the use of standard quantitation procedures showed that the quantity of DTS in the somatic embryo extract, at 0.16% (w/v), was approximately 30-fold higher than in rhizogenic/embryogenic callus (0.0055% w/v) of similar fresh weight. These results indicate that it is possible to biosynthesize approximately 6 mg of natural DTS from 3,808 mg of fresh somatic embryos within 10 wk from less than three leaf explants.     

我国部分禽流感病毒H5N1之HA序列变异演化分析

从GenBank上获得我国人(Homo sapiens)、家禽和野鸟42株H5N1亚型禽流感病毒的HA基因核酸序列,利用DNAStar分析HA蛋白关键位点氨基酸残基的变化,比较HA基因核苷酸序列同源性,构建遗传进化树.探讨我国部分人、家禽和野鸟H5N1病毒基因的遗传进化关系.序列分析结果表明:禽流感病毒H5N1亚型的HA基因持续地发生着变异,但并非以均一速度进行,时间间隔愈长,核苷酸同源性愈低;我国同一地区或临近地区,当年或前后两年发生的人及家禽感染的禽流感病毒高度同源.推测我国部分人发生的禽流感可能是通过家禽感染的;候鸟的迁徙在传播病毒过程中所起的作用有待深入探讨.     

Multiple near-identical genotypes of Schistosoma japonicum can occur in snails and have implications for population-genetic analyses

We genotyped (using 16 or 17 microsatellite loci) numerous adult Schistosoma japonicum raised in rabbits exposed to pooled cercariae from small numbers of naturally infected snails from several localities in China. As expected, duplicate multi-locus genotypes (MLGs) were found among these worms. Additionally, many more MLGs, often near-identical, were found than snails used as sources of cercariae. Explanations for these results include (i) genotyping errors, (ii) development within each infected snail of multiple sibling miracidia and (iii) somatic mutation producing genetically varied cercariae from a single miracidium. To control for genotyping errors we re-analysed samples from many individual worms, including repeating the initial PCR. Explanations invoking the development of multiple sibling miracidia within a single snail are not likely to be correct because almost all duplicate MLGs fell within same-sex clusters in a principal coordinates analysis. We would expect both sexes to be represented in a multi-miracidium infection. In addition, we exposed several snails to infection by a single miracidium. One such snail, via an experimentally infected mouse, yielded 48 adult worms. The presence of at least nine near-identical MLGs among these worms was confirmed by re-genotyping. We regard somatic mutation as the most likely explanation for our results. The implications of multiple MLGs for population-genetic studies in S. japonicum are discussed.     

Plasticity in fitness and fitness-related traits at ramet and genet levels in a tillering grass Panicum miliaceum under patchy soil nutrients

Seedling and root sprout recruitment after a volcanic deforestation was examined in a Prunus ssiori population. According to genotypes of five microsatellite (SSR) loci, 44 genets were identified among 188 ramets ≥ 5 cm DBH in a 2.3-ha plot in 2000. The genets formed mutually exclusive patches. Because only one repeat change in an SSR locus distinguished five genets from adjacent genets, there is a possibility that the five genets derived from somatic mutations as well as sexual reproduction. Thus, at least 39 genets originated from seedlings, and at least 144 ramets derived from root sprouts. The seedlings were recruited after the volcanic eruptions in 1739 and before 1972 because trees of 5 cm DBH were 28 years of age, estimated from annual rings. As the largest ramet recruited from a root sprout was estimated to be 94 years old, root sprout recruitment began between 1739 and 1906. Thus, the estimated minimum rates of recruitment from seedlings and root sprouts were 0.167 y^–1 and 0.618-2.182 y^–1, respectively. The clonal diversity of the P. ssiori population (Simpson's D = 0.92) was higher than that of other clonal plant populations. The inbreeding coefficient was significantly negative (Wright's F _IS = −0.069). These results suggest that the frequent seedling recruitment in an outbreeding system maintains the clonal diversity in the population. This revised version was published online in June 2006 with corrections to the Cover Date.     

Role of permanent dicentric systems in carrot somatic embryogenesis

Summary A permanent dicentric chromosome system was studied on carrot cultures and regenerated somatic embryos at different stages of development. The large chromosomal variability of the cultures and the presence of the breakage-fusion bridge cycle did not interfere with the initial developmental process up to the seedling stage but subsequent growth proceeded only if healing of the broken ends or dicentric loss had occurred. The behaviour of the dicentric chromosome in culture and during somatic embryogenesis is discussed in relation to chromosomal variability, abnormal development and the somaclonal variation that such mechanisms may generate in regenerated plants.     

Sequence Variation in the Gpl20 region of SHIV-CN97001 during in vivo Passage

SHIV-CN97001 played an important role in assessing the immune effect and strategy of the AIDS vaccine which included genes of the predominant prevalent HIV-1 strain in China. In this study, SHIV-CN97001 was in vivo passaged serially to construct pathogenic SHIV-CN97001/rhesus macaques model. To identify variation in the gp120 region of SHIV-CN97001 during passage, the fragments of gp120 gene were amplified by RT-PCR from the plasma of SHIV-CN97001 infected animals at the peak viral load time point and the gene distances （divergence, diversity） were calculated using DISTANCE. The analysis revealed that the genetic distances of SHIV-CN97001 in the third passage animals were the highest during in vivo passage. It had a relationship between viral divergence from the founder strain and viral replication ability. The nucleic acid sequence of the V3 region was highly conservative. All of the SHIV-CN97001 strains had V3 loop central motif （GPGQ） and were predicted to be using CCR5 co-receptor on the basis of the critical amino acids within V3 loop. These results show that there was no significant increase in the genetic distance during serial passage, and SHIV-CN97001 gp120 gene evolved toward ancestral states upon transmission to a new host. This could partly explain why there was no pathogenic viral strain obtained during in vivo passage.     

Fertile fruit trees obtained by somatic hybridization: navel orange (Citrus sinensis) and Troyer citrange (C. sinensis x Poncirus trifoliata)

Summary Nucellar cell suspension protoplasts of navel orange (Citrus sinsensis Osb.) were chemically fused with mesophyll protoplasts of Troyer citrange (C. sinensis x Poncirus trifoliata) and cultured in hormone-free Murashige and Tucker medium containing 0.6 M sucrose. Two types of plant were regenerated through embryogenesis. One type showed intermediate mono-and difoliate leaves and the other types was identical to Troyer citrange. The regenerated plants with intermediate morphology were demonstrated by chromosome counts and rDNA analysis to be amphidiploid somatic hybrids. Five clones of these somatic hybrids were grafted in the field. After 4 years, they set flowers having a morphology intermediate between those of the two parents. The pollen grains showed high stainability and sufficient germinability, and were larger than those of Troyer citrange. The fruits of the somatic hybrids were large and spherical with thick rinds. Most of them contained seeds with normal germinability. These results indicate that somatic hybridization is a useful tool for Citrus breeding.     

Culture studies onCaulerpa (caulerpales,chlorophyceae) III. Reproduction,development and morphological variation of laboratory-culturedC. racemosa var.peltata

Reproduction, development and morphological variation of the marine green algaCaulerpa racemosa var.peltata from the southern part of Japan were studied in culture in the laboratory. Anisogamous biflagellate male and female gametes were produced monoeciously and copulated with each other. Settled zygotes became spherical and increased in volume. After five weeks, they formed two germ tubes which extended in opposite directions. Both germ tubes became elongated and branched, resulting in the formation of creeping, filamentous, protonema-like plants. These plants formed primary shoots which differentiated into creeping rhizomes and upright axes. Each upright axis successively formed ramuli and developed into an assimilator. The morphology of assimilators, i.e., shape and arrangement of ramuli, varied with culture coditions. The effects of temperature and light intensity on the formation of assimilators were investigated with 25 combinations of 5 temperatures (20.0–30.0C) and 5 light intensities (0.5–8.0 klux). The morphological plasticity of this alga is identical to that ofCaulerpa racemosa var.laetevirens, which was previously described by the present authors. Thus, apparently, the plasticity of this taxon is correlated with environmental factors. It appears, moreover, thatC. racemosa var.peltata andC. racemosa var.laetevirens are ecophenes (ecads) of a single species.