Production and characterization of somatic hybrids between <Emphasis Type="Italic">Solanum tuberosum</Emphasis> L. and <Emphasis Type="Italic">S. pinnatisectum</Emphasis> Dun.

Interspecific somatic hybrids between the dihaploid Solanum tuberosum and the wild species S. pinnatisectum Dun. were produced via protoplast fusion. Protoplast isolation, electrofusion, culture of post-fusion products and regeneration of calli/shoots were undertaken following optimized protocols. Regenerants were characterized for hybridity, ploidy and resistance to Phytophthora infestans (Mont.) de Bery, causal fungal pathogen of late blight disease. From a total of 126 regenerated macrocalli, 12 somatic hybrids were confirmed by possessing species-specific diagnostic bands of their corresponding parents as revealed by RAPD, SSRs and cytoplasmic-DNA analyses. Tetraploid status of the 12 hybrids was determined using flow cytometry analysis. Intermediate phenotypes for leaf, flower, and tuber characteristics and high male fertility were observed in field-grown hybrid plants. Hybrids were highly resistant to foliage late blight based on field assessment for two seasons. In contrast, moderate level of resistance to foliage blight was observed in hybrids based on the detached leaf assay under laboratory conditions. Overall, somatic hybrids with moderate levels of resistance to foliage blight were identified, and these will be useful for in situ hybridization in potato breeding efforts.     

Proliferation of germ cells and somatic cells in first trimester human embryonic gonads as indicated by S and S+G2+M phase fractions

Objectives: The number of germ cells and somatic cells in human embryonic and foetal gonads has previously been estimated by stereological methods, which are time‐ and labour‐consuming with little information concerning cell proliferation. Here, we studied whether flow cytometry could be applied as an easier method, also enabling estimation of the fraction of cells in S or S+G₂+M (SG₂M) cell‐cycle phases as indicators of cell proliferation. Methods: Cell suspensions from 35 human embryonic gonads at days 37 to 68 post‐conception (pc) were immunomagnetically sorted into C‐KIT positive (germ) cells and negative (somatic) cells. They were stained for DNA content and analysed by flow cytometry. S and SG₂M fractions could be measured for 13 of the female and 20 of the male gonads. The number of cells was estimated using fluorescent reference beads. Results: During the period from 37 to 68 days pc, female germ and somatic cells had a stable S and SG₂M fractions indicating steady growth of both subpopulations, whereas they decreased in both male germ and somatic cells. The number of germ and somatic cells estimated by flow cytometry was significantly lower than in stereological estimates, suggesting loss of cells during preparation. Conclusions: Cell proliferation as indicated by S and SG₂M fractions could be estimated specifically for primordial germ and somatic cells. Estimation of total number of germ and somatic cells was not feasible.     

There is No Somatic Meiosis in Embryogenic Leaves of Cichorium

Several papers dealing with carrot cell cultures describe meiosis-likedivisions and haploid cells prior to somatic embryogenesis.We have studied the first division in embryogenic mesophyllcells of a diploidCichorium intybus L. and of a tetraploid hybridC.intybus L.xC. endivia L. which undergo direct somatic embryogenesisfrom single cells when leaf fragments are placed in a liquidagitated inductive medium (modified MS with 1x10^-7M NAA and2.5x10^-6M 2-iP), in darkness, at 35°C. MicrosporogenesisinC. intybus provided aspects of meiosis for comparison. Inleaves incubated in inductive conditions, DAPI staining of nucleishowed normal mitosis on days 3–6; about 0.6% cells inprophase had undergone spontaneous endoreduplication leadingto a tetraploid somatic embryo. Immunocytochemistry of tubulinrevealed the constant presence of a preprophase band, as ina normal mitosis. The first pluricellular somatic embryos becamevisible on day 5 of culture. Flow cytometric determination ofnuclear DNA on days 4, 5 and 6 did not show any peak correspondingto the 1C DNA level for the diploid plant or to the 2C DNA levelfor the tetraploid. Instead there was a weak but constant peakat the 4C and 8C levels. We conclude that inCichorium leaves,the first division of somatic embryogenesis is a normal mitosis,with a small shift to endoreduplication. In our opinion, somaticmeiosis is not a prerequisite during direct somatic embryogenesis. Cichorium ; chicory; somatic embryogenesis; cell division; flow cytometry; tubulin     

Experimentally Synthesized Plant Chimeras 2. A Comparison of In vitro and In vivo Techniques for the Production of Interspecific Nicotiana Chimeras

In vitro and in vivo techniques were compared for synthesizingchimeras between Nicotiana glauca Grahm and N tabacum L Interspecificchimeral callus, produced from mixed callus cultures in vitro,was placed on media which favoured only N tabacum shoot formationNone of the 474 regenerated N tabacum shoots incorporated Nglauca cells into their meristems When chimeral callus was regeneratedunder hormonal conditions favouring simultaneous organogenesis,of 397 shoots, only non-chimeral shoots of both species aroseIn vivo, reciprocal splice grafts between species were decapitatedjust above the graft union and treated with or without auxin—lanolinpastes Auxin increased callus formation but inhibited adventitiousshoot formation Three of 209 adventitious shoots arising fromthe graft union were interspecific mericlinal chimeras whichwere later stabilized as periclinal chimeras All three chimerasformed when N glauca was the understock Two of the chimerasarose on untreated shoots which produced no visible callus,indicating that excessive callus formation may be unnecessaryfor multiple cell origin of adventitious shoots to occur Chimeras, tobacco, Nicotiana glauca, Nicotiana tabacum, tissue culture, graft chimeras, callus cultures     

The Significance of Root Starch in Post-fire Shoot Recovery of the Resprouter Stirlingia latifolia R. Br. (Proteaceae)

Stirlingia latifolia, a common shrub of Banksia woodlands ofSW Australia, is a highly successful resprouter species recoveringfrom fire by multiple sprouting of new shoots from its upperroot stock. in comparison with the congeneric fire-sensitive(obligate seeder) species Stirlingia tenuifolia it exhibitsa low shoot:root dry weight ratio and high concentrations ofstored starch in the cortical tissue of its roots. The relationshipbetween root reserves of starch and development of newly sproutingshoot material following fire is examined in S. latifolia afterspring and summer burns. During the initial 2-5 month periodafter fire, levels of stored starch in the roots fall by 50-75%,followed by a slow increase as plants reproduce and the attainmentof pre-fire starch levels by 1·5-2 years after the fire.Starch reserves of roots can be further reduced by shading theregenerating shoots to limit their input of photosynthates andalmost totally eliminated by monthly removal of successive flushesof new shoots over a 10-12 month period. New shoots continueto sprout until all the starch is eliminated. The data are discussedin relation to the fire-induced reproduction of S. latifoliaand its ability to thrive in very frequently burnt habitats.Copyright1993, 1999 Academic Press Fire response, Proteaceae, resprouter, shoot:root ratio, starch storage, Stirlingia latifolia     

Somatic polyploidy examined by flow cytometry in Daphnia

Flow cytometry was used to examine the patterns of somatic polyploidyand to determine the haploid genome sizes in Daphnia. The averageproportions of polyploid nuclei among adult animals of D.pulex,D.longispina, D.pulex x D.longispina and D.magna equalled 27,24, 24 and 24%, respectively. Both interclonal and developmentallyregulated variation were observed in the level of somatic polyploidy.Adult animals expressed more extensive polyploidy than did juveniles.The estimates of the haploid genome sizes (C values) of D.pulex,D.longispina, D.pulex x D.longispina and D.magna equalled 0.32,0.27, 0.26 and 0.37 pg, respectively. These are the smallestgenomes reported in crustaceans. Apparently, somatic polyploidycompensates for the loss of genetic material and gives riseto genetic plasticity which may contribute to the considerablelevel of phenotypic plasticity observed in Daphina.     

Seed storage proteins in developing somatic embryos of alfalfa: defects in accumulation compared to zygotic embryos

Somatic embryos of alfalfa (Medicago sativa L.) synthesizedall of the major storage proteins of zygotic embryos; an 11Sglobulin (medicagin), a 7S globulin (alfin), and a 2S albumin(LMW). In zygotic embryos (cotyledons and/or axis) these storageproteins accounted for 30%, 10%, and 20%, respectively, of thetotal extractable protein. In somatic embryos the 7S proteinwas predominant while the 11S (particularly subfamily I) and2S proteins were present in lower amounts. Analysis of cultivarsand selfed seed of the embryogenic clone (RL34) demonstratedthat these differences were predominantly physiologically, ratherthan genetically, based. The accumulated 7S and 11S storageproteins of somatic embryos were processed normally, aggregatedas oligomers, and were deposited in protein bodies. This wasnot the case for the 2S storage protein. In somatic embryosthat protein was localized in the cytoplasm rather than in proteinbodies, the site of deposition in zygotic embryos. Key words: Medicago (alfalfa), zygotic/somatic embryos (seeds), storage proteins, immunolocalization     

Limited Genome Size Variation in Sesleria albicans

The extent and significance of intraspecific genome size variationin plants continues to be a matter of discussion: in some speciesconsiderable variation has been described, while no variationhas been detected in other taxa. In the present study, intraspecificgenome size variation was analysed in a perennial grass Sesleriaalbicans Kit. ex Schult. (Poaceae). Flow cytometry was usedfor the analysis of nuclear DNA content in ten geographicallyisolated populations ofS. albicans . Despite long-term isolationand lack of gene-flow between the populations, only negligibleinter-population differences were found. Although the differencesbetween the populations were statistically significant, themaximum inter-population difference reached only 1.6% of themean 2C value (9.78 ± 0.04 pg). The variation was notcorrelated with geographical location or with altitude of thepopulations analysed. The present study clearly demonstratesthat S. albicans belongs to the plant taxa with a highly stablegenome size. Copyright 2000 Annals of Botany Company Sesleria albicans, genome size, nuclear DNA content, intraspecific variation, flow cytometry, Europe     

Genome Size in Chinese Soybean Accessions--Stable or Variable?

Variation in genome size up to 1.12-fold has been recently reportedin 90 Chinese accessions ofGlycine max (soybean). Generallysuch results have to be viewed with reservation if rigorousinternal standardization and control tests for the repeatabilityof the results have not been done. Therefore, we reinvestigatedten accessions (five allegedly ranking high and five low) forgenome size using propidium iodide flow cytometry and Feulgendensitometry. Using flow cytometry, the maximum difference betweenaccessions was 1.018-fold (non-significant); the differencebetween the means of the high-ranking and low-ranking groupwas 1.002-fold (non-significant). With Feulgen densitometry,the maximum difference between accessions was 1.034-fold (non-significant).The present data suggest genome size constancy, in terms ofcytometric evidence, for the Chinese soybean accessions in question.Likewise, no reasonable evidence was obtained for a differencebetween Chinese and American soybeans. Copyright 1999 Annalsof Botany Company Glycine max, Chinese soybeans, U.S. soybeans, genome size variation, propidium iodide flow cytometry, Feulgen densitometry.     

Hormonal Control of Organogenesis and Somatic Embryogenesis inBeta vulgarisCallus

Tétu, T., Sangwan, R. S. and Sangwan-Norreel, B. S. 1987.Hormonal control of organogenesis and somatic embryogenesisin Beta vulgaris callus.—J. exp. Bot. 38: 506–517. Three main pathways of morphogenesis viz: root formation, shootformation and somatic embryogenesis, have been observed in thecallus derived from various explants of Beta vulgaris L. Growthhormones but not the basal media, determined the morphogeneticpotentiality of the callus. Auxin alone induced root formation.A combination of an auxin (naphthalene acetic acid) and a cytokinin(6-benzylaminopurine) gave only infrequent bud formation withvery low percentages (a maximum of 12%). Regular bud formationwith high percentages (52%) occurred when an anti-auxin (2,3,5-triiodobenzoicacid) with a cytokinin (BAP) was used. Shoots (2–3 cm)were transferred to a rooting medium. Roots were formed readilyin about 95% of the shoots. Histological studies showed thatcallus first formed meristematic zones and then shoot primordiadeveloped in these zones. Somatic embryos were formed only inthe calli derived from petiole explants. Multiple hormonal sequenceswere necessary for the induction and development of these somaticembryos. The embryos developed into normal plants when transferred,at the cotyledonary stage, to a hormone free basal medium. Key words: Beta vulgaris, organogenesis, somatic embryogenesis     

Direct Organogenesis and Plant Regeneration in Preconditioned Tissue Cultures of Lathyrus cicera L., L. ochrus (L.)DC. and L. sativus L.

This study demonstrates the importance of preconditioning ofsource tissue in regeneration of multiple shoot buds from severalspecies of Lathyrus. Preconditioned multiple shoots of Lathyruscicera L., L. ochrus (L.) DC. and L. sativus L. were obtainedby germinating seeds on Murashige and Skoog (MS) medium containing50 µM N⁵-benzylaminopurine (BAP) for 2 to 3 weeks. Multipleshoot bud formation occurred when epicotyl explants of preconditionedshoots were cultured on MS medium containing 5–50 µMBAP. No shoot regeneration was observed from epicotyl explantswhich were obtained from non-preconditioned shoots. Shoot budswere formed directly on explants without an intervening callusphase after 2 to 3 weeks of culture. Regenerated shoot budsformed healthy shoots which developed prolific and strong rootswhen transferred to MS medium supplemented with 2.5 µMnaphthaleneacetic acid (NAA). Lathyrus cicera L., L. ochrus (L.) DC., Ochrus Vetch, L. sativus L., Lathyrus pea, de novo differentiation, epicotyl, preconditioning with BAP     

Regeneration of Plants from Protoplasts of Arabidopsis thaliana L. cv. Columbia (C24), Via Direct Embryogenesis

This report describes a protocol for the regeneration of fertileplants from mesophyll protoplasts of Arabidopsis thaliana raceColumbia (C24). Regeneration was rapid and reproducible. Theprotocol is especially novel in that a large proportion of regeneratingprotoplasts regenerated via direct somatic embryogenesis. Protoplastsisolated from in vitro-grown plants entered sustained divisionafter 3–5 d in culture medium and over a period of severaldays 6–22% of protoplasts underwent at least one celldivision. Approximately 2–16% of these protoplasts continuedto divide and after 3 weeks in culture had formed macroscopiccolonies, of which 70–80% were regular embryo-like structures.Four weeksafter release from the alginate culture matrix andtransfer to solid medium in the light, 68–88% of thesestructures had produced well-developed shoots. Shoots couldbe maintained in culture or established in peat blocks. Theregenerated plants were fertile. Key words: Arabidopsis thaliana, protoplast, regeneration, embryogenesis, dicamba     

Organogenesis, Differentiation and Histolocalization of Alkaloids in Cultured Tissues and Organs of Duboisia myoporoides R. Br.

Duboisia myoporoides R. Br. shoots were regenerated from non-organogenicand organogenic calli induced with nine different cytokinin/auxincombinations. Alkaloid colour reagents localized tropane alkaloidsin the vascular regions which had large cells in the secondaryxylem of the basal stem sections of the non-rooted shoots. Tropanealkaloids were localized in shoots regenerated from calli inducedwith two different cytokinin/auxin combinations. No alkaloidswere localized in shoots regenerated from calli induced withother cytokinin/auxin combinations. However, only nicotine wasdetected by GC-MS in the non-rooted shoots regenerated fromcalli induced with three different cytokinin/auxin combinations.Tropane alkaloids were also localized in xylem cells of rootsregenerated from calli induced with two different cytokininand auxin combinations independently. The presence or absenceof nicotine, hyoscyamine and scopolamine in different culturedplant materials was confirmed by GC-MS, indicating that althoughthe root is the main site for alkaloid biosynthesis, with suitablecell differentiation, alkaloid biosynthesis may take place incultured shoots without root initiation. Copyright 2000 Annalsof Botany Company Duboisia myoporoides, Corkwood tree, Solanaceae, tropane alkaloid, alkaloid localization, shoot culture, root culture, iodoplatinate     

Endopolyploidy in Diploid and Tetraploid Maize (Zea mays L.)

Nuclei from different tissues such as stem, mesocotyl, nodalroot and root tip of diploid and tetraploid maize were isolated,stained with propidium iodide and passed through an EPICS-751flow-cytometer cell sorter. Variations in flow histograms wereobserved in different tissues. Stem tissues of both the diploidand tetraploid had two peaks representing G1 and G2 somaticnuclei. The remaining tissues in both the diploids and tetraploidsexhibited three peaks. The first peak observed in these tissuesrepresents G1 somatic nuclei of the lowest ploidy level. Thesecond peak represent G2 somatic nuclei of the lowest ploidylevel+G1 somatic nuclei of the next ploidy level. The thirdpeak represents G2 of the higher ploidy level+G1 somatic nucleiof the next higher ploidy level. Statistically significant differenceswere observed between the diploid and tetraploid maize tissueswith respect to nuclei distribution in the higher ploidy levelpeaks implying variation in the degree of endopolyploidy inthe diploid and tetraploid maize. The results of this studysuggest that the amount of endopolyploid observed in maize genotypeshas an effect on their overall agronomic performance under thefield conditions.Copyright 1993, 1999 Academic Press Zea mays L., maize, endopolyploidy, diploid, tetraploid, flow cytometry     

不同生态型东南景天对土壤中Cd的生长反应及吸收积累的差异性

 采用盆栽方法研究了两种生态型东南景天(Sedum alfredii)对土壤中不同含量Cd(即对照, 12.5, 25, 50, 100, 200, 300, 400 mg&;#8226;kg^－1)的生 长反应、吸收和积累Cd的差异性。结果表明,土壤添加重金属Cd后,矿山生态型东南景天生长正常,地上部和根系Cd含量随着土壤中Cd含量的 增加而增加,在400 mg&;#8226;kg^－1 Cd处理下含量分别高达2 900和500 mg&;#8226;kg^－1,其地上部显著大于根部;然而,土壤添加Cd后,非矿山生态型东 南景天的生长受到抑制,地上部和根部的生物量显著降低。当土壤Cd含量为50～100 mg&;#8226;kg^－1 时,非矿山生态型东南景天的地上部和根系Cd含 量随着土壤中Cd含量的增加而增加,而且根系Cd含量则大于地上部。当土壤Cd≤50 mg&;#8226;kg^－1时,矿山生态型东南景天根系Cd含量比非矿山生态 型高 ,但当土壤Cd≥100 mg&;#8226;kg^－1,两者之间无显著差异;然而,但在同一Cd处理水平下,矿山生态型东南景天地上部Cd含量总是高于非矿山 生态型。这些结果表明,矿山生态型东南景天有很强的忍耐和吸收土壤Cd的能力,再次证明其为一种Cd超积累植物。     

Flow cytometric analysis of somatic embryos, shoots, and calli of the cactus Copiapoa tenuissima Ritt. forma monstruosa

Flow cytometry was used for analyzing DNA contents of nuclei isolated from in vitro grown somatic embryos, shoots and calli, as well as mammillae of in vivo grown shoots of cactus, Copiapoa tenuissima Ritt. forma monstruosa. Endoreduplication was detected in both in vitro grown somatic embryos, shoots and calli and in mammillae derived from in vivo grown shoots. However, the lowest ploidy levels ranged from 2C to 4C in somatic embryos, and reaching up to 32C for in vitro grown shoots and calli from mammillae. Whereas, ploidy levels of in vivo-derived mammillae ranged from 2C to 16C. The presence of 2,4-dichlorophenoxyacetic acid (2,4-D) in the culture medium had no influence on levels of ploidy of regenerated tissues. The mean genome size of cactus was calculated as 2C = 2.87 ± 0.05 picogram (pg).     

In Vitro Culture and Plant Regeneration in Vicia faba subsp. Equina (var. Spring Blaze)

Explants of stem, leaves, roots, and cotyledons from etiolatedaxenically grown Vicia faba seedlings were cultured on a rangeof media. Shoot organogenesis was only obtained with nodal stemand cotyledonary node explants when cultured on MS medium with3% sucrose, 2.0 mg 1^–1 BAP and 02 mg 1^–1 NAA. Callusproliferation accompanied shoot organogenesis from nodal stemexplants. Successive subculture of nodal stem callus resultedin proliferation of regenerative callus which contained severalshoot bud initials. The capacity for shoot regeneration fromthis callus was maintained for 9 months. Histological studiesreveal de novo formation of meristematic centres in callus andtheir further development into bud primordia. High frequencyrooting of these adventitious shoots was obtained on half-strengthMS medium with 1.5% sucrose, 0.1 mg 1^–1 NAA and 0.5 mg1^–1 kinetin. Key words: Vicia faba, adventitious shoots, axillary shoots, de novomeristem formation, organogenesis, tissue culture     

Somatic hybrids between the cultivated potato Solanum tuberosum L. and the 1EBN wild species Solanum pinnatisectum Dun.: morphological and molecular characterization

Interspecific somatic hybrids between the 1EBN-wild species Solanum pinnatisectum (S. pnt) and four different diploid breeding lines of Solanum tuberosum (S. tbr) were produced by electrofusion. S. pnt exhibits resistance to Phytophthora infestans and Erwinia blackleg. Somatic hybrids were identified by RFLP analysis using the oligonucleotide (GATA)₄ as a probe. In three of four combinations all regenerates obtained were somatic hybrids. All 86 somatic hybrids between the breeding line H256/1 and S. pnt were analyzed in detail with respect to morphological and molecular characters; 50% of the somatic hybrids showed normal intermediate leaf morphology. Tubers of somatic hybrid plants grown in the greenhouse as well as in the field were evenly shaped and remarkably similar to those of the S. tbr breeding line. Analysis of relative DNA content by flow cytometry revealed that 75% of the somatic hybrids were tetraploid, some were hypotetraploid and others polyploid or mixoploid. Slotblot and RFLP analyses were carried out using repetitive and some single-copy DNA probes. The genome portion of the S. tbr breeding line was determined by slot-blot analysis using the species-specific repetitive probe pSA287. Obviously, most somatic hybrids contain the complete genomes of both fusion partners. In some of the somatic hybrids, a significantly lower intensity of the S. pnt-specific hybridization signal indicated a certain degree of asymmetry.Dedicated to Prof. Melchers on the occasion of his 90th birthday     

Relative Importance of Ion Exclusion, Secretion and Accumulation in Spartina alterniflora Loisel.

The extent to which Spartina alterniflora Loisel. excluded,secreted or accumulated the major seawater ions (Cl^-, SO^2-₄,Na⁺, K⁺, Mg²⁺, and Ca²⁺) was investigated under varying salinitytreatments. From a quantitative viewpoint, ion exclusion wasmost prominent and accounted for 91–97% of the theoreticalmaximum ion uptake as a result of transpiration and growth.Of those ions taken up, approximately half was secreted fromthe shoots. Relative to K⁺, a disproportionate amount of Na⁺was excluded at the roots and secreted by the shoots. The concentrationwithin the tissues of S. alterniflora did not change with salinitytreatment for the majority of the ions examined, but Na⁺ wasmore than twice as concentrated at 40 g dm^-3 than at lOgdm^-3.Calculations of the flux of ions from salt marsh sediments tothe flood water via shoot secretion or stem/leaf turnover indicatethat these processes may be important to the ecology of S. alternifloraas mechanisms that limit the accumulation of salt within theroot zone.     

Histological Analysis of Organogenesis and Somatic Embryogenesis Induced in Immature Tissues of Stylosanthes scabra

A well established protocol for in vitro germination of Stylosanthesscabra zygotic embryos was achieved. The response of S. scabraembryonic tissues cultured in vitro was highly dependent onthe kind of growth regulator used. Organogenesis was obtainedby using BAP, otherwise somatic embryogenesis was induced by2, 4-D. Histological aspects of both methods of regenerationwere evaluated. Endogenous neoformed buds seem to develop fromdeepseated vascular nodule structures into callus tissue. Besides,a direct somatic embryogenesis of a multicellular origin issuggested. Stylosanthes scabra, histology, organogenesis, somatic embryogenesis