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Jitao Li, Nian Wang, Lina Wang, Haiping Xin, and Shaohua Li

Cold stress is an important factor that limits grape (Vitis sp.) production around the world. The high expression of osmotically responsive genes 1 (HOS1) protein acts as a repressor of cold-responsive genes in plants. To increase understanding of mechanism regulating cold tolerance in grape, we isolated and characterized a novel HOS1 gene, designated VvHOS1 from ‘Muscat Hamburg’ grapevine (Vitis vinifera). Real-time polymerase chain reaction (PCR) analysis revealed that the expression of VvHOS1 could be induced by the application of exogenous abscisic acid and various abiotic environmental conditions such as low temperature, drought, and salinity. Moreover, VvHOS1 expression could also be induced by cold plus drought conditions (4 °C, 10% polyethylene glycol 6000). In addition, overexpression of VvHOS1 in arabidopsis (Arabidopsis thaliana) decreased the plants’ tolerance to cold, drought, and salt as well as negatively regulated the expression level of two stress-responsive genes, AtRD29A and AtCOR47. The results obtained in this study should help us to elucidate the function of VvHOS1 and understand the cold-responsive pathway in grapevine.

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Shu’an Wang, Rutong Yang, Peng Wang, Qing Wang, Linfang Li, Ya Li, and Zengfang Yin

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Xiaoming Wang, Jianjun Chen, Yongxin Li, Qiying Nie, and Junbin Li

‘Jincuilei’ is a mutant selected from Lonicera macranthoides Hand.-Mazz. It produces abundant flowers that never open with a chlorogenic acid (CGA) content up to 6.0%. Propagation through rooting or grafting has only a 30% survival rate. This study was undertaken to establish an efficient protocol for rapidly regenerating this mutant. Leaf explants were inoculated on Gamborg's B5 medium supplemented with different concentrations of 6-benzyladenine (BA) and 2,4-dichlorophenozyacetic acid (2,4-D). The optimal combination for callus induction was 4.4 μm BA with 2.26 μm 2,4-D, which resulted in 86.7% of leaf explants producing calluses in 4 weeks. Calluses produced from this optimal medium were cultured on B5 medium containing different concentrations of kinetin (KT) and α-naphthalene acetic acid (NAA). The best formulation for shoot induction was B5 medium containing 0.9 μm KT and 5.4 μm NAA in which 73.4% of cultured calluses produced shoots in 8 weeks, and shoot numbers ranged from three to six per callus piece (1 cm3). Adventitious shoots were cut and rooted in half-strength Murashige and Skoog medium supplemented with 14.8 μm 3-indolebutyric acid. Roots initiated 10 d after culture, and rooting percentages ranged from 98% to 100%. Plantlets grown in a container substrate in a shaded greenhouse had over a 95% survival rate. During the last 6 years, over four million plantlets were regenerated using this established procedure, and there was no somaclonal variation. Fresh and dry weights of 1000 flowers, CGA contents, and dry flower yields of the regenerated plants were not significantly different from those of the stock ‘Jincuilei’ propagated by cutting, indicating that plants regenerated from this established procedure were stable. This established in vitro culture method has led to rapid commercial production of this medicinal plant on more than 1500 ha of production field.

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Jing Ma, Zheng Li, Bin Wang, Shunzhao Sui, and Mingyang Li

Expansins are extracellular proteins that are involved in cell wall modifications such as cell wall disassembly, cell separation, and cell expansion. Little is known about expansin gene expression during flower development of wintersweet (Chimonanthus praecox). In the present study, an expansin gene, CpEXP1, was isolated from the wintersweet flower cDNA library through random sequencing; this gene encodes a putative protein of 257 amino acids with the essential features conserved, like in other alpha expansins. The CpEXP1 gene exhibited different transcription levels in different tissues and had a significantly higher expression in flowers than other tissues. It is strongly correlated with the development of the flower. The expression of CpEXP1 increased in the flower buds or whole flowers from Stage 1 to 4 and decreased from Stage 5 to 6 during natural opening. Ethephon (an ethylene releaser) treatment promoted cut flower senescence, whereas 1-methylcyclopropene (1-MCP) (an ethylene perception inhibitor) delayed the process of flower wilting. This result is associated with the concomitant lower transcript levels of CpEXP1 in the ethephon-treated samples as well as the steady expression in the 1-MCP-treated samples compared with that in control flowers. The studies show the interesting observation that the expression of an expansin gene CpEXP1 is correlated with the development of Chimonanthus praecox flowers, the upregulation during flower opening vs. the downregulation during senescence.

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Jianjun Chen, Lijia Li, and Ying Wang

Epimedium species are traditional Chinese medicinal plants as well as potential groundcover and ornamental plants. In this study, genome size and genome structures of Epimedium species were investigated using flow cytometric and fluorescence in situ hybridization (FISH). The nuclear DNA content of Epimedium species ranged from 8.42 pg/2C (8230.7 Mbp) to 9.97 pg/2C (9752.8 Mbp). The pairwise nucleotide diversity (π) of the fragments of the genes for reverse transcriptase (rt) of Ty1-copia retrotransposon within a species of rt fragments ranged from 0.251 to 0.428 in 10 Epimedium species. Phylogenetic analysis of the sequences revealed four major clades with the largest subclade containing 72 sequences of relatively low nucleotide diversity. FISH indicated that Ty1-copia retrotransposons are distributed unevenly along the pachytene chromosomes of E. wushanense and E. sagittatum, mostly associated with the pericentromeric and terminal heterochromatin. The relatively low sequence heterogeneity of Ty1-copia rt sequences implies that the Epimedium genomes have experienced a few relatively large-scale proliferation events of copia elements, which could be one of the major forces resulting in the large genome size of Epimedium species.

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Qingren Wang, Yuncong Li, and Waldemar Klassen

A pot experiment with summer cover crops and soil amendments was conducted in two consecutive years to elucidate the effects of these cover crops and soil amendments on `Clemson Spineless 80' okra (Abelmoschus esculentus) yields and biomass production, and the uptake and distribution of soil nutrients and trace elements. The cover crops were sunn hemp (Crotalaria juncea), cowpea (Vigna unguiculata), velvetbean (Mucuna deeringiana), and sorghum sudangrass (Sorghum bicolor × S. bicolor var. sudanense) with fallow as the control. The organic soil amendments were biosolids (sediment from wastewater plants), N-Viro Soil (a mixture of biosolids and coal ash, coal ash (a combustion by-product from power plants), co-compost (a mixture of 3 biosolids: 7 yard waste), and yard waste compost (mainly from leaves and branches of trees and shrubs, and grass clippings) with a soil-incorporated cover crop as the control. As a subsequent vegetable crop, okra was grown after the cover crops, alone or together with the organic soil amendments, had been incorporated. All of the cover crops, except sorghum sudangrass in 2002-03, significantly improved okra fruit yields and the total biomass production (i.e., fruit yields were enhanced by 53% to 62% in 2002-03 and by 28% to 70% in 2003-04). Soil amendments enhanced okra fruit yields from 38.3 to 81.0 g/pot vs. 27.4 g/pot in the control in 2002-03, and from 59.9 to 124.3 g/pot vs. 52.3 g/pot in the control in 2003-04. Both cover crops and soil amendments can substantially improve nutrient uptake and distribution. Among cover crop treatments, sunn hemp showed promising improvement in concentrations of calcium (Ca), zinc (Zn), copper (Cu), iron (Fe), boron (B), and molybdenum (Mo) in fruit; magnesium (Mg), Zn, Cu, and Mo in shoots; and Mo in roots of okra. Among soil amendments, biosolids had a significant influence on most nutrients by increasing the concentrations of Zn, Cu, Fe, and Mo in the fruit; Mg, Zn, Cu, and Mo in the shoot; and Mg, Zn, and Mo in the root. Concentrations of the trace metal cadmium (Cd) were not increased significantly in either okra fruit, shoot, or root by application of these cover crops or soil amendments, but the lead (Pb) concentration was increased in the fruit by application of a high rate (205 g/pot) of biosolids. These results suggest that cover crops and appropriate amounts of soil amendments can be used to improve soil fertility and okra yield without adverse environmental effects or risk of contamination of the fruit. Further field studies will be required to confirm these findings.

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Xiaoli Wang, Zhiyong Wang, Li Liao, Xinyi Zhang, and Changjun Bai

Carpetgrass [Axonopus compressus (Sw.) Beauv.] is an important warm-season perennial turfgrass that is widely used in tropical and subtropical areas. The genetic diversity of 63 carpetgrass accessions in China was studied using simple sequence repeat (SSR) markers. Fourteen SSR primer combinations generated a total of 49 distinct bands, 48 (97.96%) of which were polymorphic. The number of observed alleles ranged from 2 to 6, with an average of 3.5. Coefficients of genetic similarity among the accessions ranged from 0.24 to 0.98. Unweighted pair-group method with arithmetic means (UPGMA) clustered the 63 accessions into three groups, and not all samples from the same region belonged to the same group. SSR markers will promote marker-assisted breeding and the assessment of genetic diversity in wild germplasm resources of carpetgrass.

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Yuyu Wang, Faju Chen, Yubing Wang, Xiaoling Li, and Hongwei Liang

High-frequency somatic embryogenesis and plant regeneration were achieved from immature cotyledonary-stage embryos in the endangered plant, Tapiscia sinensis Oliv. Plant growth regulators with different concentrations and combinations on embryogenesis capacity were studied. The optimal explants for in vitro somatic embryogenesis were immature embryos in T. sinensis. A high callus induction rate of 100% was achieved on Murashige and Skoog (MS) basal medium supplemented with 1.0 mg·Ll−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5% (w/v) activated charcoal. Alternatively, a high induction rate (96.16%) of somatic embryogenesis was obtained on MS basal medium supplemented with the combination of 0.05 mg·L−1 α-naphthaleneacetic acid (NAA) and 0.2 mg·L−1 6-benzylaminopurine (6-BA), and somatic embryos proliferated fastest on the mentioned medium supplemented with 0.5% (w/v) activated charcoal and 3% (w/v) sucrose, inoculation of explants proliferating 21 times in the 23-day subculture. Of the 100 plantlets transferred to field after the acclimation, 95 (95%) survived. Based on the histocytological observations, the development of somatic embryos was similar to that of zygotic embryos. There were two accumulation peaks of starch grains in the embryogenic calli and in the globular-stage embryos, both closely related to the energy supply, and the embryoids were of multicelluar origin.

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Aoxue Wang, Fanjuan Meng, Xiangyang Xu, Yong Wang, and Jingfu Li

Leaf mold, caused by the fungus Cladosporium fulvum, is a serious disease of tomato. In the current study, the main physiological races of C. fulvum collected from three northeastern provinces of China were identified using a set of identification hosts. The results showed that the prevalent pathogenic physiological races were 1.2.3, 1.3, 3, 1.2.3.4, and 1.2.4. F1, F2, and BC1 tomato plants were obtained by crossing C. fulvum-resistant cultivar 03748 carrying the Cf-6 gene and susceptible cultivar 03036. Three 10-mer oligonucleotide random amplified polymorphic DNA (RAPD) primers and two simple sequence repeat (SSR) primers were selected for the further molecular marking analysis after 210 RAPD primers and 50 SSR primers were screened using the bulked segregate analysis method. The polymorphic DNA bands were amplified among parents, 10 F1 plants, 184 F2 plants including 145 resistant plants and 39 sensitive plants using three RAPD primers and two SSR primers so that three RAPD molecular markers and two SSR molecular markers linked to the Cf-6 loci were identified. Three RAPD markers were linked to the Cf-6 resistant locus separated with 8.7 cM, 20.3 cM, and 33.4 cM. Also, one RAPD codominant marker S374619/559 was found. The locations of the two SSR markers were 12.6 cM and 9.7 cM away from the Cf-6 locus. After cloning and sequencing two specific DNA fragments closely connected to the Cf-6 resistant and susceptible alleles respectively, in the RAPD codominant marker S374619/559 and one codominant sequence characterized amplified region marker S674619/559 was converted from RAPD marker S374619/559. In the RAPD marker S374619/559, the length difference of two specific fragments, 619-bp fragment and 559-bp fragment, is the result of one insertion (60 bp) in the 619-bp fragment. These markers will facilitate the selection of resistant tomato germplasm containing the Cf-6 gene and cloning of Cf-6 to breed new C. fulvum resistant tomato cultivars.

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Fanjuan Meng, Ruoding Wang, Mu Peng, Chao Wang, Zhongkui Wang, Fachun Guan, and Yajun Li

Inter simple sequence repeat (ISSR) were used to evaluate the genetic diversity of Kongpo Monkshood (Aconitum kongboense L.) in Motuo, Tibet Plateau. From 70 accessions of three populations, 10 out of 100 informative ISSR primers were chosen for polymorphism analysis. Percentage of polymorphic bands was 50% to 66.67% with a mean of 58.42%. The effective number of alleles (Ne) was between 1.545 (population 3) and 1.586 (population 2), and the mean value was 1.564; the Nei’s gene diversity (h) ranged from 0.315 to 0.327 with the average value of 0.320; the value of Shannon’s information index (I) ranged from 0.459 to 0.478, with the mean of 0.469. Based on molecular data, cluster analysis classified the 70 cultivars into three groups. Most accessions were related to the geographical origin and their genetic backgrounds. Bayesian structure and PCoA analysis were consistent with the dendrogram result. Based on the analysis, it will provide a reference for Kongpo Monkshood breeding purposes and contribute to identification, rational exploitation, and conservation of germplasms.