Tracking individual wheat microspores in vitro: identification of embryogenic microspores and body axis formation in the embryo

 The development of isolated, defined wheat microspores undergoing in vitro embryogenesis has been followed by cell tracking. Isolated wheat (Triticum aestivum L.). microspores were immobilized in Sea Plaque agarose supported by a polypropylene mesh at a low cell density and cultured in a hormone-free, maltose-containing medium in the presence of ovaries serving as a conditioning factor. Embryogenesis was followed in microspores isolated from immature anthers of freshly cut tillers or from heat- and starvation-treated, excised anthers. Three types of microspore were identified on the basis of their cytological features at the start of culture. Type-1 microspores had a big central vacuole and a nucleus close to the microspore wall, usually opposite to the germ pore. This type was identical to the late microspore stage in anthers developing in vivo. Microspores with a fragmented vacuole and a peripheral cytoplasmic pocket containing the nucleus were defined as type 2. In type-3 microspores the nucleus was positioned in a cytoplasmic pocket in the centre of the microspore. Tracking revealed that, irrespective of origin, type-1 microspores first developed into type 2 and then into type-3 microspores. After a few more days, type-3 microspores absorbed their vacuoles and differentiated into cytoplasm-rich and starch-accumulating cells, which then divided to form multicellular structures. Apparently the three types of microspore represent stages in a continuous process and not, as previously assumed, distinct classes of responding and non-responding microspores. The first cell division of the embryogenic microspores was always symmetric. Cell tracking also revealed that the original microspore wall opened opposite to a region in the multicellular microspore which consisted of cells containing starch grains while the remaining cells were starch grain-free. The starch-containing cells were located close to the germ pore of the microspore. In more advanced embryos the broken microspore wall was detected at the root pole of the embryo. Received: 27 December 1999 / Accepted: 11 May 2000     

视黄酸缺乏对斑马鱼心脏房室分化的影响

目的 通过化学遗传学方法建立视黄酸缺乏的斑马鱼模型,探讨视黄酸缺乏对斑马鱼胚胎心脏前后轴发育即房室分化的影响.方法 在斑马鱼胚胎孵育的5 hpf,用不同浓度梯度的视黄醛脱氢酶2抑制剂DEAB(1×10-6、5×10-6、10×10-6、25×10-6 mol/L)处理斑马鱼胚胎,实时观察斑马鱼胚胎发育的全过程.通过给予斑马鱼胚胎外源性视黄酸,观察其对DEAB的拮抗作用.应用胚胎整体原位杂交观察视黄酸缺乏对心脏特异基因vmhc和amhc表达的影响.结果 斑马鱼胚胎的生存率随着DEAB处理浓度的增加而降低,当DEAB浓度≥5×10-6 mol/L时,斑马鱼的畸胎率达100%.5×10-6 mol/L DEAB的致畸作用能够被1×10-9mol/L外源性视黄酸所拮抗.整体原位杂交结果显示视黄酸缺乏会导致斑马鱼胚胎心脏房室分化异常,表现为vmhc表达细胞的范围增大,amhc表达细胞的范围缩小.结论 通过外源性DEAB处理能有效地建立视黄酸缺乏的斑马鱼模型,DEAB影响胚胎发育存在剂量依赖性.视黄酸在斑马鱼心脏前后轴发育过程中起重要调控作用,心脏发育早期视黄酸缺乏会抑制心房的发育而支持心室的发育,出现房室分化异常.     

应用原位杂交及RAPD技术标记抗黄矮病小麦一中间偃麦草染色体异附加系

以生物素（Ｂｉｏｔｉｎ－１６－ｄＵＴＰ）标记中间偃麦草基因组 ＤＮＡ为探针,与抗黄矮病小麦－中间偃麦草染色体异附加系Ｚ６进行原位杂交,鉴定出附加的１对中间偃麦草染色体。对异附加系 Ｚ６和 Ｌ１及它们的小麦亲本进行了 ＲＡＰＤ分析,从 １２０个随机引物中,筛选出 ２个引物可以扩增出附加染色体的特异ＤＮＡ片段,可作为鉴定寻人小麦的中间偃麦草染色质的分子标记。     

Identification of differentially expressed cDNA sequences and histological characteristics of Hevea brasiliensis calli in relation to their embryogenic and regenerative capacities

Different friable Hevea callus lines from the same genotype can display different embryogenic and regenerative potentials. These lines can not be distinguished on the basis of macroscopic criteria. A histological and molecular study was undertaken to characterize the differences existing between five callus lines with different potentials. The genes differentially expressed during induction were analyzed using the differential display (DD-RT) technique. Twenty-eight cDNAs were found to be differentially expressed during induction in the embryogenic regenerating line (ER). Embryogenic nodules were formed earlier in the ER lines than in the embryogenic non-regenerating lines and were completely absent in the non-embryogenic line. Of these 28 cDNAs, five could be used to distinguish between calli prior to induction, thereby enabling an early diagnosis of friable Hevea callus embryogenic potential.Abbreviations DD-RT: Differential display technique - ENR: Embryogenic non-regenerating line - ER: Embryogenic regenerating line - NE: Non-regenerating line Communicated by P. DeberghThis work was funded by a grant from the French Ministry of National Education, Research and Technology and also by the Centre de Coopération International en Recherche Agronomique pour le Développement, Département des Cultures Pérennes (CIRAD-CP) at Montpellier-France.     

Plant regeneration in vitro from embryogenic cultures of spring- and winter-type barley (Hordeum vulgare L.) varieties

Summary Immature embryos of 41 lines of barley were screened in vitro for callus induction and somatic embryogenesis on different media to establish totipotent cultures. The use of modified MS and CC media, both supplemented with 1 g/l casein hydrolysate, and the substitution of agarose for agar resulted in the highest frequencies of somatic embryo induction. Embryogenic callus was induced and plants regenerated from 23 of the lines tested. The auxins 2,4-D, dicamba, picloram and 2,4,5-T were suitable for embryogenic callus induction. High frequencies of somatic embryo germination occurred on CC medium supplemented with 1 mg/l IAA and 0.05 mg/l zeatin. A strong genotypic effect on the capacity and frequency of embryogenic callus formation was found. Cultivar Golden Promise always gave the best results. Experiments with field grown material in 3 consecutive years showed that environmental factors also strongly influenced the induction of somatic embryogenesis and plant regeneration.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - dicamba 3,6-dichloro-o-anisic acid - picloram 4-amino-3,6,6-trichloropicolinic acid - NAA naphtaleneacetic acid - IAA indole-3-acetic acid - ABA abscisic acid - BAP 6-benzyl amino purine - 2iP 6-(3-methyl-2 butenyl 1-amino)purine - GA₃ gibberellic acid     

Sequence and evolution of the gene for the monomeric globin I and its linkage to genes coding for dimeric globins in the insect Chironomus thummi

We isolated genomic clones containing sequences encoding globins I and IA from a Chironomus thummi thummi genomic library. Three clones contain globin IA (ctt-1A) genes, while one contains a globin I (ctt-1) gene. The coding regions of the four genes are identical except for the single base substitution accounting for the globin I/IA polymorphism. The noncoding DNA flanking the coding region is more than 98% similar, confirming a previous hypothesis that the globin ctt-1 and ctt-1A genes are alleles. Hemoglobins I and IA are monomeric in the insect hemolymph. Earlier in situ hybridization studies suggested that monomeric and dimeric globin genes are clustered at different chromosomal loci. In situ hybridization of ctt-1 DNA to polytene salivary gland chromosomes places the ctt-1 gene on the same band as genes for the dimeric globins II and VIIB, forcing revision of the earlier hypothesis that genes for monomeric and dimeric globin genes are at different loci. The evolution of the ctt-1 and ctt-1A alleles and of the two globin gene loci are discussed. Correspondence to: G. Bergtrom     

Characterization of the KNOX class homeobox genes Oskn2 and Oskn3 identified in a collection of cDNA libraries covering the early stages of rice embryogenesis

For identification of genes involved in embryogenesis in the model cereal rice, we have constructed a collection of cDNA libraries of well-defined stages of embryo development before, during and after organ differentiation. Here, we focus on the possible role of KNOX (maize Knotted1-like) class homeobox genes in regulation of rice embryogenesis. Three types of KNOX clones were identified in libraries of early zygotic embryos. Two of these, Oskn2 and Oskn3, encode newly described KNOX genes, whereas the third (Oskn1) corresponds to the previously described OSH1 gene. In situ hybridizations showed that during the early stages of embryo development, all three KNOX genes are expressed in the region where the shoot apical meristem (SAM) is organizing, suggesting that these genes are involved in regulating SAM formation. Whereas OSH1 was previously proposed to function also in SAM maintenance, Oskn3 may be involved in patterning organ positions, as its expression was found to mark the boundaries of different embryonic organs following SAM formation. The expression pattern of Oskn2 suggested an additional role in scutellum and epiblast development. Transgenic expression of Oskn2 and Oskn3 in tobacco further supported their involvement in cell fate determination, like previously reported for Knotted1 and OSH1 ectopic expression. Whereas Oskn3 transformants showed the most pronounced phenotypic effects during vegetative development, Oskn2 transformants showed relatively mild alterations in the vegetative phase but a more severly affected flower morphology. The observation that the KNOX genes produce similar though distinct phenotypic reponses in tobacco, indicates that their gene products act on overlapping but different sets of target genes, or that cell-type specific factors determine their precise action.     

Production of fertile tobacco pollen from microspores in suspension culture and its storage for in situ pollination

Summary A simple procedure is described for the in vitro production of tobacco (Nicotiana tabacum L.) pollen from microspores isolated just before entering mitosis. During a 3-day culture period in a liquid medium containing pyrimidine nucleosides these microspores develop into young pollen grains to the stage of starch deposition. Pollen maturation and transition to dormancy is achieved during a further 2- to 3-day culture period in the same medium stepwise supplemented by a concentrated solution of sucrose and l-proline. Upon transfer of the pollen to a simple germination medium containing sucrose and boric acid, up to 40% of the grains were observed to produce relatively long tubes. The in vitro-matured pollen grains can be stored at-20° C either suspended in 1.17 M sucrose and 100 mM l-proline or separated from the medium on filter paper discs. The stored pollen germinated both in vitro and on the stigma, the pollen tubes grew through the style into the ovary and pollination produced up to 300 viable seeds per pod. The procedure is of interest for pollen developmental studies and various fields of pollen manipulation, such as in vitro pollen selection.     

Differential localization of the LIM domain protein PLIM-1 in microspores and mature pollen grains from sunflower

 PLIM-1 is a LIM domain protein specifically expressed in pollen grains. Using two PLIM-1-specific monoclonal antibodies we studied its expression and intracellular location at various developmental stages of sunflower (Helianthus annuus L.) pollen. Our studies show that the protein appears at the microspore stage in a limited number of cytoplasmic bodies, becomes undetectable in bicellular pollen, and reappears in tricellular pollen grains in cortical patches particularly concentrated in the F-actin-enriched germination cones of the vegetative cell. The developmental stage-dependent, different location of the protein suggests a dual function during pollen development. While this function in microspore development remains obscure, the high concentration of PLIM-1 in the germination cones of mature pollen suggests that it participates in the germination process as well as in pollen tube growth. Received: 11 August 1998 / Revision accepted: 15 December 1998     

普通小麦与钩刺山羊草杂交F1的农艺性状及细胞遗传学的观察

为有效地利用钩刺山羊草（Ａｅｇｉｌｏｐｓ ｔｒｉｕｎｃｉａｌｉｓ Ｌ．）的抗白粉病基因对小麦（Ｔｒｉｔｉｃｕｍ ａｅｓｔｉｖｕｍ Ｌ．）进行遗传改良,了解两者杂交后杂种Ｆ１的遗传机制是十分必要的。对Ｆ１杂种的研究表明,二价体频率高于理论值,是分别存在于钩刺山羊草Ｃ和Ｕ基因组中的小麦５Ｂ染色体上Ｐｈ基因抑制因子联合作用的结果。以尾状山羊草（Ａｅｇｉｌｏｐｓ ｃａｕｄａｔａ Ｌ．）Ｃ基因组特异重复序列     

Direct proof for the presence and expression of two 5% different 16S rRNA genes in individual cells of Haloarcula marismortui

A single-cell-derived pure culture of Haloarcula marismortui was obtained using the optical tweezers technique. Analysis of the 16S rRNA genes unambiguously demonstrated two 5% different 16S rRNA sequences. In addition, expression of both operons in the same cell was shown by fluorescence in situ hybridization with operon-specific probes. Received: September 19, 2000 / Accepted: October 2, 2000     

Genome analysis of intergeneric hybrids of apomictic and sexual Australian Elymus species with wheat,barley and rye: implication for the transfer of apomixis to cereals

Summary Four hybrids were obtained between three Australian Elymus taxa and three cereal grains: wheat, rye, and barley. Mean meiotic metaphase-I configurations were 41.14 I, 0.42 rod II, 0.003 ring II, and 0.01 III for E. scabrus var plurinervis x Triticum aestivum (1 hybrid plant), 22.27 I, 2.63 rod II, 0.06 ring II, and 0.12 III for E. scabrus var scabrus x Secale cereale (4 hybrid plants), and 26.65 I, 0.66 rod II, 0.00 ring II, and 0.01 III for E. scabrus var plurinervis x Hordeum vulgare (13 hybrid plants). The I genome of barley also paired very little in a B_III hybrid of apomictic E. rectisetus x H. vulgare (2 hybrid plants). Megasporogenesis in this B_III hybrid was at least facultatively apomeiotic, with the same sort of nuclear elongation, apomeiotic division, and dyad formation seen previously in E. rectisetus itself. All four hybrid combinations were sterile. While spike morphology in the E. scabrus x T. aestivum and E. scabrus x H. vulgare hybrids were intermediate to their parents, E. scabrus x S. cereale and E. rectisetus x H. vulgare looked like their maternal parents.     

Sequential solvent extraction and structural characterization of polysaccharides from the endosperm cell walls of barley grown in different environments

The objective of this study was to examine the composition and molecular structure of the endosperm cell walls (CW) derived from barley grain grown in three environments in Canada, and differing in grain hardness, protein, and total β-glucan contents. The endosperm CW were isolated from barley, cv. Metcalfe, grown in Davidson, SK (Sample A), Hythe, AB (sample B), and Hamiota, MB (sample C). The CW were sequentially extracted with water at 65 ^oC, saturated Ba(OH)₂, again with water at 25 ^oC, and 1 M NaOH, resulting in fractions designated WE65, BaE, Ba/WE, and NaE, respectively. The monosaccharide analysis indicated the presence of β-glucans, arabinoxylans, and small amounts of arabinogalactans, glucomannans, and xyloglucans. Cellulose was detected in the CW remnants. The CW of sample A, exhibiting a lower grain hardness than sample B, contained the lowest amount of β-glucans, but the highest amount of arabinoxylans and the mannose-containing polysaccharides. The CW of sample C, characterized by very high protein content in the grain, contained the highest amount of β-glucans and the lowest amount of other polysaccharides. Polysaccharides in the CW of sample B, exhibiting the highest grain hardness, were characterized by the highest weight average molecular weights (M_w). β-Glucans in the CW of Sample B showed the highest ratio of DP3/DP4 and the longest cellulosic fragments in the polymeric chains. Of the three barley samples, arabinoxylans in the endosperm CW of sample A exhibited the lowest degree of branching, the highest amount of unsubstituted Xyl residues, and the highest ratio of singly to doubly substituted Xylp. The highest water solubility of the CW of sample C was associated with the highest concentration of β-glucans, the lowest DP3/DP4 ratio, and the lowest M_w of the polymeric constituents. Arabinoxylans with the lowest amount of doubly substituted but the highest amount of unsubstituted xylose residues and long sequences of unsubstituted xylan regions were found in the NaE fractions. The NaE fractions showed a high ratio of →4)-Glcp-(1→ to →3)-Glcp-(1→ linkages and some →4)-Manp-(1→ linkages, indicating a high level of long cellulosic regions in β-glucan chains and the presence of glucomannans.     

Regeneration from intact and sectioned immature embryos of barley (Hordeum vulgare L.): the scutellum exhibits an apico-basal gradient of embryogenic capacity

Immature zygotic embryos from spring barley cv. Dissa were used to induce somatic embryogenenesis. Up to 158 germinated somatic embryos could be recovered per plated zygotic embryo. Critical factors for obtaining a high yield of regenerants were the size of the explant, the level of 2,4-D used for callus induction and the careful division of callus at each subculture. Use of microsections of immature embryos as explants revealed a pronounced gradient of callus formation and embryogenic response across the scutellum. Sections from the scutellar tissue at the coleoptilar end of the embryo gave the most callus and were highly embryogenic. The regeneration response of sectioned explants was comparable to that recovered from intact embryos of similar size.     

Physical mapping of three fruit ripening genes：Endopolygalacturonase,ACC oxidase and ACC synthase from apple（Malus x domestica） in an apple rootstock A106（Malus sieboldii

The apple rootstock,A106(Malus sieboldii),had 17 bivalents in pollen mother cells at meiotic metaphase 1,and 17 chromosomes in a haploid pollen cell.Karyotypes were prepared from root-tip cells with 2n=34 chromosomes,Seven out of 82 karyotypes(8.5%) showed one pari of satellites at the end of the short arm of chromosome 3.C-bands were shown on 6 pairs of chromosomes 2,4,6,8,14,and 16 near the telomeric regions of short arms.Probes for three ripening-related genes from Malus x domestica:endopolygalacturonase(EPG,0.6kb),ACC oxidase(1.2kb),and ACC synthase(2kb)were hybridized in situ to metaphase chromosomes of A106.Hybridization sites for the EPG gene were observed on the long arm of chromosome 14 in 15 out of 16 replicate spreads and proximal to the centromere of chromosomes 6 and 11.For the ACC oxidase gene,hylridization sites were observed in the telomeric region of the short arm of chromosomes 5 and 11 in 87% and 81% of 16 spreads respectively,proxiaml to the centromere of chromosome 1 in 81% of the spreads,and on the long arm of chromosome 13 in 50% of the spreads. Physical mapping of three fruit ripening genes in an apple rootstock A106.Twenty five spreads were studied for the ACC synthase gene and hybridization sites were observed in the telomeric region of the short arm of chromosome 12 in 96% of the spreads.chromosomes 9 and 10 in 76% of the spreads,and chromosome 17 in 56% of the spreads.     

The distribution, organization and evolution of two abundant and widespread repetitive DNA sequences in the genus Hordeum

The genomic organization and chromosomal distributions of two abundant tandemly repeated DNA sequences, dpTa1 and pSc119.2, were examined in six wild Hordeum taxa, representing the four basic genomes of the genus, by Southern and fluorescence in situ hybridization. The dpTa1 probe hybridized to between 30 and 60 sites on the chromosomes of all five diploid species studied, but hybridization patterns differed among the species. Hybridization of the pSc119.2 sequence to the chromosomes and Southern blots of digested DNA detected signals in Hordeum bulbosum, Hordeum chilense, Hordeum marinum and Hordeum murinum 4x, but not in Hordeum murinum 2x and Hordeum vulgare ssp. spontaneum. A maximum of one pSc119.2 signal was observed in the terminal or subterminal region of each chromosome arm in the species carrying this sequence. The species carrying the same I-genome differed in the presence (Hordeum bulbosum) or absence (Hordeum spontaneum) of pSc119.2. The presence of pSc119.2 in the tetraploid cytotype of Hordeum murinum, but its absence in the diploid cytotype, suggests that the tetraploid is not likely to be a simple autotetraploid of the diploid. Data about the inter- and intra-specific variation of the two independent repetitive DNA sequences give information about both the interrelationships of the species and the evolution of the repetitive sequences. Received: 17 March 1999 / Accepted: 16 June 1999