An efficient selection and regeneration protocol for Agrobacterium-mediated transformation of oriental melon (Cucumis melo L. var. makuwa)

An efficient selection and plant regeneration protocol for Agrobacterium-mediated transformation using cotyledon explants of oriental melon (Cucumis melo L. var. makuwa) has been developed. All six oriental melon cultivars evaluated in the study showed a >90?% shoot regeneration frequency and produced 1.8?C3.6 shoots per cotyledon explant when cultured on Murashige and Skoog (MS) medium supplemented with 1.0?mg?L^?1 benzyladenine and 0.01?mg?L^?1 indoleacetic acid. Kanamycin (Km) and geneticin (Gt) in the shoot induction medium (SIM) were compared both qualitatively and quantitatively for their efficiency as a selection agent for the selection and regeneration of transgenic plants after Agrobacterium-mediated transformation. Shoot formation was completely inhibited at 50?mg?L^?1 Km and 10?mg?L^?1 Gt. Relatively high concentrations of both Gt and Km (>100?mg?L^?1 Km and >25?mg?L^?1 Gt) were necessary because large numbers of non-transgenic shoots survived during the selection process. The incorporation of a selectable marker (neomycin phosphotransferase II) into the genome of transgenic plants was confirmed using ??-glucuronidase (GUS), PCR and Southern blot analysis. Shoot regeneration frequencies were 41.2?% at 100?mg?L^?1 Km and 15.2?% at 30?mg?L^?1 Gt 8?weeks after transformation, whereas the transformation frequencies based on the PCR were 2.9 and 7.1?%, respectively, 16?weeks after transformation. These results demonstrate that a large portion of the regenerated shoots on SIM supplemented with 100?mg?L^?1 Km consisted of non-transformed or escaped shoots, indicating that 30?mg?L^?1 Gt is the more suitable for the selection and regeneration of transgenic plants in oriental melon.     

Heterologous Expression and Biochemical Characterization of Two Lipoxygenases in Oriental Melon,Cucumis melo var. makuwa Makino

Lipoxygenases (LOXs) are a class of non-heme iron-containing dioxygenases that catalyse oxidation of polyunsaturated fatty acids to produce hydroperoxidation that are in turn converted to oxylipins. Although multiple isoforms of LOXs have been detected in several plants, LOXs in oriental melon have not attracted much attention. Two full-length LOX cDNA clones, CmLOX10 and CmLOX13 which have been isolated from oriental melon (Cucumis melo var. makuwa Makino) cultivar “Yumeiren”, encode 902 and 906 amino acids, respectively. Bioinformatics analysis showed that CmLOX10 and CmLOX13 included all of the typical LOX domains and shared 58.11% identity at the amino acid level with each other. The phylogenetic analysis revealed that CmLOX10 and CmLOX13 were members of the type 2 13-LOX subgroup which are known to be involved in biotic and abiotic stress. Heterologous expression of the full-length CmLOX10 and truncated CmLOX13 in Escherichia coli revealed that the encoded exogenous proteins were identical to the predicted molecular weights and possessed the lipoxygenase activities. The purified CmLOX10 and CmLOX13 recombinant enzymes exhibited maximum activity at different temperature and pH and both had higher affinity for linoleic acid than linolenic acid. Chromatogram analysis of reaction products from the CmLOX10 and CmLOX13 enzyme reaction revealed that both enzymes produced 13S-hydroperoxides when linoleic acid was used as substrate. Furthermore, the subcellular localization analysis by transient expression of the two LOX fusion proteins in tobacco leaves showed that CmLOX10 and CmLOX13 proteins were located in plasma membrane and chloroplasts respectively. We propose that the two lipoxygenases may play different functions in oriental melon during plant growth and development.     

新疆甜瓜地方种质资源遗传多样性的SRAP分析

为研究我国新疆甜瓜地方种质资源亲缘关系及其分类,充分高效的利用种质资源,利用SRAP(sequence-related amplified polymorphism technique)标记对117份中国新疆甜瓜地方品种和28份国内外对照材料进行亲缘关系和遗传多样性分析。结果表明,20对SRAP引物共扩增出224个带,其中多态性谱带216个,多态性比率达96%,平均每对引物扩增的带数和多态性带数分别为11.2个和10.8个,每对引物的多态性信息含量PIC值为0.73~0.94,平均为0.85;不同生态区域供试材料的Nei's基因多样性指数(H)和Shannon's信息指数(I)分别为0.1075~0.2560和0.1569~0.4061,中国新疆的南疆、东疆和北疆均高于其他生态区域供试材料,且以南疆最高,具有非常丰富的遗传多样性;不同生态区域甜瓜种质资源的遗传一致度和遗传距离分别为0.6384~0.9919和0.0081~0.4488,其中南疆、东疆和北疆两两之间的遗传一致度均在0.95以上,遗传距离均在0.04以下,三者之间遗传分化较小;中国新疆甜瓜与印度、西亚、西班牙的甜瓜种质资源亲缘关系较近,与韩国、日本、美国和前苏联的甜瓜种质资源亲缘关系较远。聚类分析结果表明,以遗传相似系数0.548为阈值,145份种质材料可分为3大类群;厚皮甜瓜与薄皮甜瓜间在分子水平上没有严格的界限,两者之间亲缘关系的远近在不同的种质材料间差异很大;117份中国新疆甜瓜地方种质资源可分为A(Ⅰ-1)、B(Ⅰ-2、Ⅰ-3、Ⅰ-5)、C(Ⅰ-6)、D(Ⅱ)等4大类6个亚类群,与传统4个变种10个品种群分类结果不同,但在每个大类或亚类群中属于同一变种或品种群的材料倾向于聚在一起。     

In vitro morphogenesis of Cucumis melo var. inodorus

In vitro morphogenesis of C. melo L. var. inodorus was studied by the induction of adventitious buds and somatic embryos. Organogenesis was obtained from cotyledon segments and leaf discs in culture medium supplemented with benzylaminopurine (1 mg l⁻¹) and somatic embryogenesis was induced in medium containing 2,4-dichlorophenoxyacetic acid (5 mg l⁻¹) + thidiazuron (1 mg l⁻¹). Through histological analysis it was possible to verify that in cotyledonary explants, protuberances that do not develop into well-formed shoot buds and leaf primordia are more frequently formed than complete shoot buds, resulting in a low frequency of plant recovery in the organogenic process. A high percentage of explants responded with the formation of somatic embryos; the microscopical analysis showed that the somatic embryos lacking well developed apical meristems had a low conversion rate into plants. Plant recovery was not obtained from leaf-disc explants, with high rates of contamination and formation of protuberances which did not develop into shoot buds. Histological sections showed the development of epidermis and leaf hairs, indicating those structures could be leaf primordia; however, these were not associated with a shoot apical meristem. This revised version was published online in June 2006 with corrections to the Cover Date.     

Phenolic glycosides from Cucumis melo var. inodorus seeds

A new phenolic glycoside (E)-4-hydroxycinnamyl alcohol 4-O-(2′-O-β-d-apiofuranosyl)(1″ → 2′)-β-d-glucopyranoside (1) was isolated and identified from Cucumis melo seeds together with benzyl O-β-d-glucopyranoside (2), 3,29-O-dibenzoylmultiflor-8-en-3α,7β,29-triol (3) and 3-O-p-amino-benzoyl-29-O-benzoylmultiflor-8-en-3α,7β,29-triol (4). Their structures were elucidated by extensive NMR experiments including ¹H–¹H (COSY, TOCSY, ROESY) and ¹H–¹³C (HSQC and HMBC) spectroscopy and chemical evidence. The multiflorane triterpene esters were identified as new melon constituents.     

哈密瓜组织培养和离体快繁

1　植物名称　哈密瓜 (Cucumismelovar.sacchar inus)品种金皇后。2　材料类别　子叶。3　培养条件　以MS为基本培养基。 ( 1 )种子发芽培养基 :1 /2MS ;( 2 )愈伤组织诱导培养基 :MS + 6 BA 0 .5mg·L- 1 (单位下同 ) +IBA 0 .1 ;( 3)不定芽诱导培养基 :MS + 6 BA 1 .0 ;( 4 )不定芽伸长培养基 :MS + 6 BA 1 .0 +GA30 .5 ;( 5 )生根培养基 :1 /2MS +IBA 2 .0。以上培养基均附加 0 .8%琼脂、3%蔗糖 ,pH 5 .8。培养温度( 2 6± 1 )℃ ,光照 1 6h·d- 1 ,光照度 2 0 0 0lx。4　生长与分化情况4.1　无菌材料的获得　金皇后种子去皮…     

厚皮甜瓜(Cucumis melo var. reliculatus)的快速繁殖

以厚皮甜瓜 (Cucumismelovar.reliculatus)西薄洛托带腋芽茎段为外植体进行离体快速繁殖研究。结果表明 :在MS BA 0 .5～ 1 .0mg/L IAA 0 .1mg/L的培养基上利于诱导形成丛生芽 ,芽的月增殖系数达到 1 1以上 ;在 1 /2MS IAA 0 .5mg/L培养基上并经暗处理 3d最易生根 ,生根率 90 % ;在蛭石 :草炭土 =1 :1 (体积比 )基质中移栽驯化效果好。试管植株定植大田后种性不变 ,生长和结果习性优于种子苗。     

厚皮甜瓜（Cucumis melo var. reliculatus）的快速繁殖

以厚皮甜瓜（Cucumis melo var. reliculatus）西薄洛托带腋芽茎段为外植体进行离体快速繁殖研究。结果表明：在MS+BA 0.5～1.0 mg/L+IAA 0.1 mg/L 的培养基上利于诱导形成丛生芽, 芽的月增殖系数达到11以上; 在1/2 MS+IAA 0.5 mg/L培养基上并经暗处理3 d最易生根,生根率90%;在蛭石：草炭土=1：1(体积比)基质中移栽驯化效果好。试管植株定植大田后种性不变,生长和结果习性优于种子苗。     

甜瓜(Cucumis melo L.)AFLP标记的多态性及聚类分析研究

利用 AFLP 技术对30个甜瓜材料进行多态性和聚类分析研究。从120对 MseI 和 PstI 引物中筛选出25对扩增效果好的引物,共扩增出262条多态性带。聚类分析结果新疆甜瓜中的夏甜瓜类型,新疆甜瓜中的冬甜瓜类型、美国粗皮甜瓜类型、日本甜瓜类型、梨瓜类型各聚为一组。上述材料的聚类分析结果与依据生态类型和地理起源的分类结果基本吻合,表明 AFLP 用于甜瓜种内不同材料间的遗传变异性分析是可行的。     

Development of EST-derived SSR markers using next-generation sequencing to reveal the genetic diversity of 50 chrysanthemum cultivars

Chrysanthemum plants are popular worldwide as cut flowers, potted, and in gardens. Several hundred cultivars have been commercialized, indicating that there is substantial genetic variations that can be manipulated under cultivations to produce a wide array of phenotypic variation. To study the genetic diversity of chrysanthemum cultivars in Korea, we first identified simple sequence repeats from chrysanthemum expressed sequence tags generated by FLX 454 sequencing. A total of 1109 ESTs out of 18,226 chrysanthemum ESTs were identified to carry SSRs. A total of 16 out of 46 primer pairs exhibited several polymorphisms among 50 chrysanthemum cultivars. The number of alleles per locus varied from 1 to 15, with an average of 6.25 alleles. The expected heterozygosity ranged from 0 to 0.8958, whereas polymorphism information content ranged from 0 to 0.8872. Based on polymorphisms using 16 SSR markers, a phylogenetic tree was generated revealing four groups within the 50 cultivars showing various levels of genetic diversity. The 16 polymorphic chrysanthemum SSR markers generated in this study would be useful for studies of the genetic conservation, diversity, and population structure of commercial chrysanthemum cultivars as well as closely related species.     

ACC脱氨酶基因转化白兰瓜的初步研究

用带有ACC脱氨酶基因和卡那霉素抗性基因（NptⅡ,作为报告基因）的工程根癌农杆菌转化白兰瓜子叶。通过组织培养,得到具有卡那霉素（Km)抗性的再生小苗,经NptⅡ报告基因的PCR扩增,ACC脱氨酶基因的Southern点杂交以及ACC脱氨酶活性的生理生化检测可知,ACC脱氨酶基因已成功转入白兰瓜子叶再生小苗,而且不同植株的真叶中表现出不同水平的ACC脱氨酶活性。     

Development of loop-mediated isothermal amplification (LAMP)-based SNP markers for shelf-life in melon (Cucumis melo L.)

In this study, LAMP markers linked to shelf-life in melon (Cucumis melo L.) were developed by converting a cleaved amplified polymorphic sequences (CAPS) marker (C2). The CAPS-PCR fragments from the long-shelf-life melon (O-3) and short-shelf-life melon (Nat-2) were cloned and sequenced to construct LAMP primers. A single nucleotide polymorphism (SNP) was identified between O-3 and Nat-2. LAMP primers were designed to detect the SNP. In the LAMP reaction to detect long-shelf-life melon, the turbidity of the templates using O-3, F1, homozygous long-shelf-life F2 lines and heterozygous long-shelf-life F2 lines started to increase after 40 min. In contrast, the turbidity of Nat-2 and homozygous short-shelf-life F2 lines did not increase even after 90 min. In the LAMP reaction to detect short-shelf-life melon, the turbidity of the templates using Nat-2, F1, homozygous short-shelf-life F2 lines and heterozygous long-shelf-life F2 lines started to increase after 40 min. But the turbidity of O-3 and homozygous long-shelf-life F2 lines did not increase after 90 min. This attests to the high reliability and usefulness of LAMP for marker-assisted selection.     

Development of genomic simple sequence repeat markers in faba bean by next-generation sequencing

Faba bean (Vicia faba L.) is an important food legume crop with a huge genome. Development of genetic markers for faba bean is important to study diversity and for molecular breeding. In this study, we used Next Generation Sequencing (NGS) technology for the development of genomic simple sequence repeat (SSR) markers. A total of 14,027,500 sequence reads were obtained comprising 4,208 Mb. From these reads, 56,063 contigs were assembled (16,367 Mb) and 2138 SSRs were identified. Mono and dinucleotides were the most abundant, accounting for 57.5 % and 20.9 % of all SSR repeats, respectively. A total of 430 primer pairs were designed from contigs larger than 350 nucleotides and 50 primers pairs were tested for validation of SSR locus amplification. Nearly all (96 %) of the markers were found to produce clear amplicons and to be reproducible. Thirty-nine SSR markers were then applied to 46 faba bean accessions from worldwide origins, resulting in 161 alleles with 87.5 % polymorphism, and an average of 4.1 alleles per marker. Gene diversity (GD) of the markers ranged from 0 to 0.48 with an average of 0.27. Testing of the markers showed that they were useful in determining genetic relationships and population structure in faba bean accessions.     

Development of genomic simple sequence repeat markers in opium poppy by next-generation sequencing

Opium poppy (Papaver somniferum L.) is an important pharmaceutical crop with very few genetic marker resources. To expand these resources, we sequenced genomic DNA using pyrosequencing technology and examined the DNA sequences for simple sequence repeats (SSRs). A total of 1,244,412 sequence reads were obtained covering 474 Mb. Approximately half of the reads (52 %) were assembled into 166,724 contigs representing 105 Mb of the opium poppy genome. A total of 23,283 non-redundant SSRs were identified in 18,944 contigs (11.3 % of total contigs). Trinucleotide and tetranucleotide repeats were the most abundant SSR repeats, accounting for 49.0 and 27.9 % of all SSRs, respectively. The AAG/TTC repeat was the most abundant trinucleotide repeat, representing 19.7 % of trinucleotide repeats. Other SSR repeat types were AT-rich. A total of 23,126 primer pairs (98.7 % of total SSRs) were designed to amplify SSRs. Fifty-three genomic SSR markers were tested in 37 opium poppy accessions and seven Papaver species for determination of polymorphism and transferability. Intraspecific polymorphism information content (PIC) values of the genomic SSR markers were intermediate, with an average 0.17, while the interspecific average PIC value was slightly higher, 0.19. All markers showed at least 88 % transferability among related species. This study increases sequence coverage of the opium poppy genome by sevenfold and the number of opium poppy-specific SSR markers by sixfold. This is the first report of the development of genomic SSR markers in opium poppy, and the genomic SSR markers developed in this study will be useful in diversity, identification, mapping and breeding studies in opium poppy.     

Microsatellite markers from Ceanothus roderickii (Rhamnaceae) using next-generation sequencing technology

? Premise of the study: Ceanothus roderickii is an endangered shrub endemic to California. To investigate the population genetics of this species, including the genetic consequences of population fragmentation and hybridization, 10 microsatellite markers were developed. ? Methods and Results: Using next-generation sequencing (454) data from a single C. roderickii individual, 10 microsatellite markers were developed. A group of 12 individuals representing all of the major C. roderickii populations were analyzed. All loci were found to be polymorphic, with a range from two to 12 alleles per locus. Observed heterozygosity ranged from 0.08 to 0.83 across loci. All 10 loci were also amplifiable in at least one other Ceanothus species. ? Conclusions: Results presented here indicate the utility of our new microsatellite primers in ongoing and future studies concerning population genetics and gene flow in C. roderickii, as well as the potential applicability of these primers in similar studies on other Ceanothus species.     

Regeneration and characterisation of Cucumis melo L. (+) Cucumis anguria L. var. longipes (Hook. fil.) Meeuse somatic hybrids

Cotyledon protoplasts of an albino Cucumis melo L. ‘Cantaloup Charentais’ somaclonal variant were electrofused with protoplasts of the wild species Cucumis anguria L. var. longipes (Hook. fil.) Meeuse. Selection of putative somatic hybrids was based on competence of the albino melon to grow and regenerate shoots together with the ability of the wild species to synthesise chlorophyll. The ITS region of C. anguria ribosomal DNA was sequenced to design species-specific primers, which allowed us to distinguish between parental lines and fusion products by PCR amplification. By using this method, all the organogenic lines characterised proved to be somatic hybrids. Three of sixteen selected lines produced shoots with albino and green sectors. Eleven lines remained green but shoots developed abnormally and did not produce roots in vitro. Two hybrid lines regenerated normal shoots but with a limited ability to produce roots in vitro and in vivo. Applicability of molecular characterisation to optimise the quick recovery of fusion products is discussed. This revised version was published online in June 2006 with corrections to the Cover Date.