The Draft Genome Sequence of European Pear (Pyrus communis L. ‘Bartlett’)

We present a draft assembly of the genome of European pear (Pyrus communis) ‘Bartlett’. Our assembly was developed employing second generation sequencing technology (Roche 454), from single-end, 2 kb, and 7 kb insert paired-end reads using Newbler (version 2.7). It contains 142,083 scaffolds greater than 499 bases (maximum scaffold length of 1.2 Mb) and covers a total of 577.3 Mb, representing most of the expected 600 Mb Pyrus genome. A total of 829,823 putative single nucleotide polymorphisms (SNPs) were detected using re-sequencing of ‘Louise Bonne de Jersey’ and ‘Old Home’. A total of 2,279 genetically mapped SNP markers anchor 171 Mb of the assembled genome. Ab initio gene prediction combined with prediction based on homology searching detected 43,419 putative gene models. Of these, 1219 proteins (556 clusters) are unique to European pear compared to 12 other sequenced plant genomes. Analysis of the expansin gene family provided an example of the quality of the gene prediction and an insight into the relationships among one class of cell wall related genes that control fruit softening in both European pear and apple (Malus×domestica). The ‘Bartlett’ genome assembly v1.0 (http://www.rosaceae.org/species/pyrus/pyrus_communis/genome_v1.0) is an invaluable tool for identifying the genetic control of key horticultural traits in pear and will enable the wide application of marker-assisted and genomic selection that will enhance the speed and efficiency of pear cultivar development.     

Organogenesis from ‘Passe Crassane’ and ‘Old Home’ pear (Pyrus communis L.) protoplasts and isoenzymatic trueness-to-type of the regenerated plants

Summary Large numbers of highly viable mesophyll protoplasts were isolated from shoot cultures of the scion cv Passe Crassane and the rootstock genotype Old Home of common pear (Pyrus communis L.). Protoplasts were cultured for both genotypes either as liquid layers or as liquid-over-agar cultures, in ammonium-free MS medium with 0.5 M mannitol, 50 mg/l casein enzymatic hydrolysate (CEH), 2.0 mg/l NAA and 1.0 mg/l BAP, plus either 0.5 mg/l IAA (for Old Home) or 2.0 mg/l IAA (for Passe Crassane). Protoplast microcalli, obtained by day 60 (Passe Crassane) or day 80 (Old Home), were transferred for further growth to ammonium-free MS medium with 2.0 mg/l NAA and 1.0 mg/l BAP. Shoot bud regeneration from the protoplastderived callus was first attempted between 100 (Passe Crassane) and 120 (Old Home) days after protoplast isolation. For Passe Crassane, shoot buds were regenerated (day 130) on a half-strength MS medium with 0.1 mg/l IBA, 0.5 mg/l BAP, 50 mg/l CEH and 20 mg/l Ca-panthotenate. For Old Home, shoot but regeneration only occurred 30 days later and on the same medium as above, which was additionally supplemented with double the concentration of the group B vitamins found in the original MS formulation and 0.05 mg/l GA₃. Following micropropagation and in vitro rooting of shoots, the plants were transferred to soil following standard procedures. Trueness-to-type of the regenerated plants was assessed by analysing their leaf isozyme banding profiles (for EST, AP, PRX, SOD, ENP, LAP, PGI, AAT, ADH, MDH and PGM) and comparing them to those corresponding to the original shoots that provided the protoplasts. No differences between the mother shoots and the protoclones were observed for any one of the 11 isozyme systems studied.     

Agrobacterium-mediated transformation of Solanum tuberosum L. cv. ‘Russet Burbank’

Stem sections from shoot cultures maintained in vitro were used to produce transgenic plants of the potato, Solanum tuberosum L. cv. Russet Burbank. Stem internode pieces inoculated with Agrobacterium tumefaciens containing coat protein genes from potato virus X and potato virus Y, produced shoots with a frequency of 60% in the absence of selection and 10% on medium containing 100 mg/l kanamycin monosulfate. Regenerated shoots were assayed for kanamycin resistance by placing stem segments on callus induction medium containing an increased level of kanamycin. Of a total 255 regenerated shoots, 47 (18%) were kanamycin resistant. Of the kanamycin resistant shoots, 25 (53%) expressed the PVX or PVY coat protein genes as assayed by enzyme-linked immunosorbent assay or Western immunoblot analysis.     

Efficient somatic embryogenesis and Agrobacterium-mediated transformation of pothos (Epipremnum aureum) ‘Jade’

Leaf and petiole explants of monocotyledonous pothos (Epipremnum aureum) ‘Jade’ were cultured on Murashige and Skoog basal medium supplemented with N-(2-chloro-4-pyridl)-N′-phenylurea (CPPU) or N-phenyl-N′-1,2,3-thiadiazol-5-ylurea (TDZ) with α-naphthalene acetic acid (NAA). Somatic embryos appeared directly from explants after 4–8 weeks of culture; 9.1 μM TDZ with 1.1 μM NAA induced 61.1 % leaf discs and 94.4 % of petiole segments to produce plantlets through embryo conversion. Using this established regeneration method and an enhanced green fluorescent protein (GFP) gene (egfp) as a reporter marker, an Agrobacterium-mediated transformation procedure was developed. Leaf discs and petiole segments were inoculated with Agrobacterium tumefaciens strain EHA105 harboring a binary vector pLC902 that contains novel bi-directional duplex promoters driving the egfp gene and hygromycin phosphotransferase gene (hpt), respectively. The explants were co-cultivated with strain EHA105 for 3, 5, and 7 days, respectively prior to selective culture with 25 mg l^?1 hygromycin. A 5-day co-cultivation led to 100 % of leaf discs to show transient GFP expression and 23.8 % of the discs to produce stable GFP-expressing somatic embryos. A 7-day co-cultivation of petiole explants resulted in the corresponding responses at 100 and 14.3 %, respectively. A total of 237 transgenic plants were obtained, and GFP fluorescence was observed in all plant organs. Regular PCR and quantitative real-time PCR analyses confirmed the presence of 1 or 2 copies of the egfp gene in analyzed plants. The highly efficient regeneration and transformation systems established in this study may enable genetic improvement of this vegetatively propagated species through biotechnological means.     

Isoforms of green fluorescent protein differ from each other in solvent molecules ‘trapped’ inside this protein

Green fluorescent protein (GFP) has been studied quite thoroughly, however, up to now some experimental data have not been explained explicitly. For example, under native conditions this protein can have two isoforms differing in their mobility in gel. In this case, no differences between the isoforms are revealed under denaturing conditions. In order to understand the difference in the isoforms of this protein, we have investigated GFP-cycle3 using mass spectrometry, gel electrophoresis, size exclusion chromatography, microcalorimetry, and spectroscopy methods under varying conditions. We have also designed and studied three mutant forms of this protein with substitutions of amino acid residues inside the GFP barrel. The mutations have allowed us to influence the formation of different GFP isoforms. Each of the mutant proteins has predominantly only one isoform. As a result of the performed research, it can be concluded that most likely the GFP isoforms differ in the solvent molecules ‘trapped’ inside the GFP barrel. In their turn, these molecules have an effect on the protein charge and consequently on its mobility at electrophoresis under native conditions.     

Agrobacterium tumefaciens-mediated transformation of safflower (Carthamus tinctorius L.) cv. ‘Centennial’

Efficient callus formation was achieved from cotyledon, stem, and leaf expiants of the domestic safflower cultivar Centennial on MS salts medium containing 1 mg/L BAP and 1 mg/L NAA. Shoot buds were regenerated from 26% of leaf-derived calli on callus induction medium, although attempts to root regenerated shoots were not successful. Centennial expiants inoculated with Agrobacterium tumefaciens containing NPT II and GUS genes produced kanamycin-resistant calli from which buds were regenerated. Transformation and stable integration of transgenes was confirmed by GUS assay and DNA hybridization in kanamycin-resistant calli, and GUS assay in regenerated shoots.Abbreviations BAP 6-benzylaminopurine - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) - GUS -glucuronidase - IAA indole-3-acetic acid - NPT II neomycin phosphotransferase II     

The ‘A’ genome donor of Eleusine coracana (L.) Gaertn. (Gramineae)

Summary In an attempt to discover A and B genome donor(s) to finger millet, Eleusine coracana, or its progenitor species, E. africana (both allotetraploid 2n=4x=36), five diploid species, E. Indica, E. Floccifolia, E. multiflora, E. tristachya and E. intermedia, were crossed to finger millet and its progenitor taxon. Crosses were successful only with E. coracana. Three combinations of triploid hybrids E. coracana x E. indica, E. coracana x E. floccifolia, and E. coracana x E. multiflora were obtained and analysed. Meiotic behaviour was perfectly normal in parental species. The regular number of 18 bivalents in E. coracana, 9 bivalents in E. indica, E. intermedia, E. tristachya and E. floccifolia and 8 bivalents in E. multiflora were invariably noticed. In E. coracana x E. indica hybrids a mean chromosome pairing of 8.84_I+8.80_II+0.03_III+0.10_IV per cell was found. About 86.5% of the cells showed the typical 9_I+9_II configuration, suggesting that E. indica (AA) is one of the diploid genome donors to cultivated species E. coracana. A mean chromosome pairing of 11.08_I+7.63_II+0.16_III+0.04_IV per cell was found in E. coracana x E. floccifolia hybrids. Two to ten bivalents and varying numbers of univalents were seen in 55% of the cells. About 45% of the cells showed the 9_I+9_II configuration. Various evidence suggests that perennial E. floccifolia is a primitive member of the A genome group of Eleusine species, and it may not be a genome donor to E. coracana. In E. coracana x E. multiflora hybrids (2n=26) mean chromosome pairing of 21.45_I+1.97_II+0.13_III+0.04_IV per cell was found. About 91% of the cells were observed to have 20–26 univalents. Only a small percentage of the cells contained bivalents or multivalents. This pairing behaviour indicates that E. multiflora lacks genomic homology with the A or B genome of E. coracana. Genomically E. multiflora is a distinct species and a genomic symbol of C is assigned to it. Identification of the B genome donor species to cultivated millet. E. coracana remains elusive.     

The green fluorescent protein as an efficient selection marker for Agrobacterium tumefaciens-mediated transformation in Hevea brasiliensis (Müll. Arg)

An efficient genetic transformation procedure using a recombinant green fluorescent protein (GFP) has been developed in Hevea brasiliensis clone PB260. Transformation experiments have been performed using an Agrobacterium tumefaciens binary vector harbouring both uidA and S65T-GFP reporter genes in order to compare selection methods using glucuronidase assay (GUS activity) and paromomycin resistance, GFP activity and paromomycin resistance, or GFP activity only. At transient level, the number of spots showing GUS or GFP activities was similar for 4 and 5 days after coculture. After selection, stable transformation events were observed and led to the establishment of transgenic callus lines. A higher number of lines were generated with GFP selection compared to the GUS one. GFP selection is less time-consuming in terms of callus subculturing, and offers the possibility of producing antibiotic resistance marker-free transgenic plants.     

低温对梨(Pyrus communis L.)果实后熟过程中乙烯合成和反应的影响(英文)

Passe Crassane梨果实采后需经过 6 0～ 80d的低温处理才能正常后熟。为了明确低温促进果实成熟的机理 ,对果实进行了低温和低温结合 1 MCP(1 甲基环丙烯 ,乙烯作用抑制剂 )和丙烯 (乙烯类似物 )处理。研究发现 :果实经低温处理后 ,乙烯合成前体———ACC含量大幅度升高 ,而未经低温处理的果实 ,无论贮藏在空气中或用丙烯 1 0 0 0μl/L处理 ,果实中ACC、M ACC含量均保持较低水平。但冷藏前用 1 MCP处理可抑制冷藏果实或冷藏后升温的果实ACC含量的增高。这说明果实的后熟过程与低温和依赖乙烯的ACC合成酶的活性和基因的表达密切相关。未经冷藏的果实于 2 0℃下用丙烯处理 ,果实不能自发合成乙烯 ,但当果实经过冷藏后再用丙烯处理 ,则果实对丙烯的反应能力随冷藏时间延长而增强。为了进一步了解低温诱导的乙烯反应过程。我们对乙烯受体基因进行了研究。定量PCR分析结果表明 ,与拟南芥ETR1同源的基因的表达不受低温的调节。但冷藏后升温 ,或在升温后用丙烯处理时 ,mRNA含量降低。这些结果说明 ,低温可能是通过影响乙烯信号转导途径下游的其它因子而调节依赖乙烯的ETR1基因和ACC合成酶基因的表达 ,从而影响果实的成熟过程     

Genetic Features of the Spontaneous Self-Compatible Mutant, ‘Jin Zhui’ (Pyrus bretschneideri Rehd.)

‘Jin Zhui’ is a spontaneous self-compatible mutant of ‘Ya Li’ (Pyrus bretschneideri Rehd. S₂₁S₃₄), the latter displaying a typical S-RNase-based gametophytic self-incompatibility (GSI). The pollen-part mutation (PPM) of ‘Jin Zhui’ might be due to a natural mutation in the pollen-S gene (S₃₄ haplotype). However, the molecular mechanisms behind these phenotypic changes are still unclear. In this study, we identified five SLF (S-Locus F-box) genes in ‘Ya Li’, while no nucleotide differences were found in the SLF genes of ‘Jin Zhui’. Further genetic analysis by S-RNase PCR-typing of selfed progeny of ‘Jin Zhui’ and ‘Ya Li’ × ‘Jin Zhui’ progeny showed three progeny classes (S₂₁S₂₁, S₂₁S₃₄ and S₃₄S₃₄) as opposed to the two classes reported previously (S₂₁S₃₄ and S₃₄S₃₄), indicating that the pollen gametes of ‘Jin Zhui’, bearing either the S₂₁- or S₃₄-haplotype, were able to overcome self-incompatibility (SI) barriers. Moreover, no evidence of pollen-S duplication was found. These findings support the hypothesis that loss of function of S-locus unlinked PPM expressed in pollen leads to SI breakdown in ‘Jin Zhui’, rather than natural mutation in the pollen-S gene (S₃₄ haplotype). Furthermore, abnormal meiosis was observed in a number of pollen mother cells (PMCs) in ‘Jin Zhui’, but not in ‘Ya Li’. These and other interesting findings are discussed.     

Sequence comparison of the 5’ flanking regions of Japanese pear (Pyrus pyrifolia) S-RNases associated with gametophytic self-incompatibility

Genomic clones of 2.8 kb, 4.3 kb and 6.5 kb for the S₂-, S₃- and S₅-RNases of Japanese pear(Pyrus pyrifolia), respectively, were isolated and sequenced. Comparison of the 5’-flanking regions of these genes with the same region of the S₄-RNase gene indicated that a highly similar region of approximately 200 bp exists in the regions just upstream of the putative TATA boxes of the four Japanese pear S-RNase genes. This suggests the presence of cis-regulatory element(s) in this region. Received: 5 October 2000 / Revision accepted: 2 January 2001     

Expression of a cholera toxin B subunit in transgenic lettuce (Lactuca sativa L.) using Agrobacterium-mediated transformation system

To increase expression level of cholera toxin B subunit (CTB) in lettuce plants, synthetic CTB (sCTB) gene based on the optimized codon usage was fused with an endoplasmic reticulum retention signal, KDEL. The sCTB gene was introduced into a plant expression vector and transformed to lettuce plants using Agrobacterium-mediated transformation system. As a selection marker, a bialaphos resistance (bar) gene that encodes phosphinothricin acetyltransferase (PAT), conferring tolerance to the herbicide phosphinothricin (PPT), was used. PCR amplification of genomic DNA confirmed the presence of the sCTB gene in the transgenic lettuce plants. Expressions of mRNA and protein of sCTB were observed by Northern and Western blot analyses, respectively. The sCTB synthesized in the transgenic lettuce showed strong affinity for GM₁-ganglioside suggesting that the sCTB conserved the antigenic sites for binding and proper folding of pentameric CTB structure. The expression level of CTB was relatively high, reaching total soluble protein (TSP) levels of 0.24% in transgenic lettuce.