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The Sequence and Detection of the cry1Aa Gene Expression Cassette in Transgenic Bt Cotton
SUN Yao, XIE Jia-Jian, FAN Yin-Yin, LI Rui, PENG Yu-Fa
Cotton Science    2011, 23 (3): 224-227.  
Abstract2412)      PDF(pc) (529KB)(2500)       Save
A new Bt gene expression cassette cry1Aa gene expression cassette was identified from transgenic insect-resistance cotton "Nannong 6 F1" in this study. The difference was found located at the connection region between Bt gene and 7S UTR terminator while being compared with the cry1Ac gene and cry1Ab/Ac gene expression cassettes reported previous. Primer pair cry1Aa-F/cry1Aa-R was designed and construct-specific detection method of cry1Ab/Aa gene expression cassette was established based on the difference. Eight of eighteen cotton varieties from Jiangsu province were identified to have cry1Aa gene expression cassette using this methods. The method established was useful for identification Bt gene expression cassette and analysis of transgenic lineages.
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Molecular Cloning and Characterization Analysis of GhWRKY4, a Transcription Factor Gene in Cotton (Gossypium hirsutum L.)
LI Guang-Lei, FAN Shu-Li, SONG Mei-Zhen, PANG Chao-You, WEI Heng-Ling, YU Shu-Xun-
Cotton Science    2013, 25 (3): 205-210.  
Abstract1106)      PDF(pc) (767KB)(2353)       Save
In the present study, a WRKY gene, GhWRKY4, was cloned from cotton(Gossypium hirsutum L.). The full-length cDNA of GhWRKY4 was 1281 bp long and contained a 1083-bp open reading frame (ORF) encoding a putative group-IIc WRKY protein of 360 amino acids. Bioinformatic analysis of the GhWRKY4 gene revealed that GhWRKY4 consists of three exons and two introns. A subcellular localization assay confirmed that the protein product was targeted in onion epidermal cell nuclei. Quantitative RT-PCR indicated that GhWRKY4 is constitutively expressed in cotton roots, stems, leaves, and flowers. The 5' flanking region of GhWRKY4 was obtained by genome walking. Bioinformatic analysis revealed that GhWRKY4 contains a series of putative cis-acting elements involved in stress regulation. Further stress treatments combined with gene expression analysis indicated that GhWRKY4 is involved in regulation of salt and cold stress.
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Advances of Agrobacterium-mediated Transformation and Regeneration System of Upland Cotton
JIE Xiao-Hong, ZHANG Huan-Yang, LI Jing, JIAO Gai-Li, WU Shen-Jie
Cotton Science    2011, 23 (4): 379-384.  
Abstract2422)      PDF(pc) (785KB)(2260)       Save
Advance of Agrobacterium-mediated transformation and regeneration system of cotton(Gossypium hirsutum L.) is summarized. Genotype-dependent, long transformation period, low frequency of embryogenesis, and high rates of abnormal embryos are main bottlenecks of Agrobacterium-mediated transformation of cotton via somatic embryogenesis. To solve these problems, the researchers optimized traditional transformation systems, expanded recipient genotypes, screened high-frequency regeneration lines, selected different organs or tissues as recipient explants, and established new methods of Agrobacterium-mediated cotton transformation which is tissue culture- and genotypes-independent.
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Molecular Marker of QTL for Fiber Quality Traits in Upland Cotton with Elite Fiber Quality
WANG Yi-Qing, LI Jun-Wen, SHI Yu-Zhen, LIU Ai-Ying, SHANG Hai-Hong, GONG Ju-Wu, WANG  Tao, GONG Wan-Kui, YUAN You-Lu
Cotton Science    2010, 22 (6): 533-538.  
Abstract2881)      PDF(pc) (605KB)(2101)       Save
In order to detect and exploit the QTLs related to superior fiber qualities in upland cotton (Gossypium hirsutum) accession NM 03102,we constructed an F2 and its corresponding F2:3 populations by crossing Lumianyan 21 to NM 03102 and employed 7892 simple sequence repeats (SSR) primer pairs to screen the polymorphism between the two parents, 222 polymorphic primer pairs were obtained for genotyping the 195 individual plants of F2 population, and 242 loci were obtained. The linkage test indicated that 182 loci could be mapped to 37 linkage groups, 35 of them could be assigned to 20 chromosomes. The linkage map covered 1661.6 cM , about 37.34% of the whole genome, and the average distance between two loci was 9.1 cM.  20 QTLs were detected based on Composite Interval Mapping (CIM) method, 4 for fiber length, 4 for fiber strength, 5 for fiber micronaire value, 4 for fiber uniformity ration and 3 for fiber elongation, respectively. These QTLs could explain 5.10%~ 28.49% of the corresponding phenotypic variations. QTLs qFS-5-1 and qFU-24-1 were consistent with previous reports in different mapping populations. And 2 QTLs (qFS-5-1 and qFMIC-1-1) could be detected in both F2 and F2:3 generations. These stable QTL might be utilized to improve the fiber qualities in the molecular markers assisted selection(MAS) breeding programs.
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RNA Interference and Its Applications on Silencing of Insecticide-resistant Genes in Insects
TANG  Tao, LIU Xue-Yuan, QIU Li-Hong
Cotton Science    2010, 22 (6): 617-624.  
Abstract3413)      PDF(pc) (1103KB)(2082)       Save
There have been enormous economical losses in agriculture caused by devastating insect pests, such as cotton bollworm Helicoverpa armigera (Hübner) and diamondback moth Plutella xylostella (L.), etc. The pests could be effectively controlled by insecticides, however many kinds of the pests have showed serious resistance to different insecticides. Studies have proved that insect resistance related to the detoxification enzymes or receptors including cytochrome P450 enzymes, esterases and cadherin proteins, etc. Knockout of genes related with resistance in insect pests has been carried out by RNA interference (RNAi) recently, which is a powerful tool in molecular biology for researches on functional genes and genomes. This review focuses on the mechanisms of RNAi and its applications in silencing resistance-related genes in insects in vivo, aiming at providing novel ideas and approaches for insect controlling and resistant management in agricultural pests.
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The Molecular Mechanism of Pathogenicity for Verticillium dahliae Kleb
WANG Min, JIAO Rui, XING Xiao-Ping, FANG Wei-Ping, LI Hong-Lian
Cotton Science    2011, 23 (3): 272-278.  
Abstract2989)      PDF(pc) (987KB)(1909)       Save
In this review, we elucidated the role of cell-wall-degrading enzymes, proteases, phytotoxins produced by Verticillium dahliae Kleb in pathogenicity and the mechanism of microsclerotial development. Recent advances in fungi functional genomics technologies such as genomics methods, RNA interference(RNAi) and Agrobacterium tumefaciens-mediated transformation(ATMT)applied for researching on the pathogenisis molecular mechanism of V. dahliae were also reviewed.
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Identification of the Pathogenicity-related Gene DVK1 in Verticillium dahliae
GAO  Feng, PENG  Shan, PENG Xiao-Ling, LI  Hui, LI Guo-Ying
Cotton Science    2011, 23 (1): 64-68.  
Abstract2926)      PDF(pc) (806KB)(1758)       Save
In this study, we carried out T-DNA insertional mutagenesis to identify mutants, which are effective in pathogenicity. The highly virulent, defoliating strain was mutated through Agrobacterium tumefaciens-mediated transformation, and 5000 transformants were obtained. Pathogenicity test of randomly selected 1000 transformants found that five mutants lost their virulence on a susceptible cotton cultivar. A new mutant, d1, was unable to cause full disease on cotton. Analysis of the mutation using Thermal Asymmetric Interlaced PCR(TAIL-PCR) confirmed an insertion into a gene DVK1. The full length of DVK1 genomic DNA and cDNA sequences were obtained using TAIL-PCR and RT-PCR methods. DVK1 has an open reading frame of 3325 bp interrupted by two introns with 52 bp and 36 bp, respectively, and putatively encodes a 1079 aa protein. The reduced pathogenic phenotype of d1 was fully complemented by reintroduction of the gene, indicating DVK1 is essential for pathogenicity in V. dahliae.
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Dynamics Analysis of Culturable Soil Microflora and Microbial Activity in Continuous and Rotation Cropping Systems of Xinjiang Cotton
HAN  Jian, ZHANG Jing-Wen, XU Wen-Xiu, LUO  Ming, WU Li-Li
Cotton Science    2011, 23 (1): 69-74.  
Abstract3164)      PDF(pc) (554KB)(1637)       Save
Variation of culturable soil microflora and microbial activity were investigated in continuous and rotation cropping cotton field in Xinjiang. The results showed that culturable microbial population gradually decreased with long-term continuous cropping of cotton. Compared with 5 years continous cropping, the total quantity of soil microbes in 6~8 years, 9~12 years and more than 13 years continuous cropping, decreased by 40.2%, 46.7%, 52.4%,respectively. After more than  5 years continuous cropping, the structure of the soil microbial community transformed from rich nutrition bacteria type to lower nutrition fungi type, the ratio of bacteria to fungi and actinomycetes to fungi decreased significantly. The amount of nitrogen physiological communities such as ammonifying bacteria, nitrobacteria and aerobic nitrogen-fixing bacteria decreased, while denitrifying bacteria increased. Moreover, continuous cropping resulted in soil respiration intensity and cellulolytic activity reducing. Contrary to continuous cropping, under the cotton/melilotus suavena, tomato, spring wheat or corn rotation systems were  most beneficial for increasing the total quantity of soil micro-organism, improving  the capability of soil microbial activity, adjusting the balance of microbial community. Also there was substantial increasement in the number of azotobacteria. The effects of different rotation modes were different, the benefits of cotton-tomato and cotton-melilotus suavena rotation were more obvious.
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The Identification and Analysis of RNA Editing Sites of 10 Chloroplast Protein-coding Genes from Virescent Mutant of Gossypium hirsutum
JIANG  Yuan, HE  Jun, FAN Shu-Li, YU Jia-Ning, SONG Mei-Zhen
Cotton Science    2011, 23 (1): 1-9.  
Abstract2276)      PDF(pc) (825KB)(1614)       Save
RNA editing is one of the post-transcriptional regulation mechanisms of gene expression in the chloroplast of land plants. Recently, studies have shown that albino or yellow phenotype of high plants may be relevant to chloroplast RNA editing. In this paper, we investigated the RNA editing sites of 10 plasmid protein-coding genes from cotyledons and leaves of virescent mutant V1, Gossypium hirsutum, using PCR, RT-PCR and sequencing methods. There were 34 editing sites in both of them, but the editing efficiency was different in 6 sites of 34. Compared all editing sites between V1 and Coker310FR, we found that V1 had 5 novelty editing sites in accD-109, accD-468, ndhD-347, rpoA-69, and rpoA-279. Analyzed all the editing sites by using bioinformatics, the results showed that 13 sites, accD-109, clpP-187, ndhB-50, ndhB-196, ndhD-128, ndhD-225 and so on, would affect their protein secondary structures or three-dimensional structures. All the results indicate that the above-mentioned sites may play important roles in proteins assemble correctly and execute their function effectively.
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Cloning, Sequence and Expression Analysis of Gossypium barbadense L. pepc Gene(in English)
WANG De-Long, YU Ji-Wen, YU Shu-Xun, DI Hong-Hong, FAN Shu-Li, SONG Mei-Zhen, ZHANG Jin-Fa
Cotton Science    2011, 23 (1): 80-89.  
Abstract2568)      PDF(pc) (1344KB)(1582)       Save
According to published EST sequences, an new pepc(Phosphoenolpyruvate carboxylase) gene was cloned from Gossypium barbadense L. cultivar 7124 by the utilization of the RACE and genome walking technology. The gene was named Gb.pepc3. It consists of 3259 bps, contains an open reading frame for 2910 bps, and encodes 969 amino acids with a deduced molecular weight of 110.7 kd and an isoelectric point of 6.08 I. Homology and phylogenetic trees analysis showed high similarity between Gb.pepc3 and pepcs in other reported plants, and all belong to C3 type pepc. The result of the fluorescence quantitative PCR displayed that Gb.pepc3 existed in all tissues of cotton. It expressed highly in the embryo but low in fiber. The expression quantity of Gb.pepc3 varied in different stages of the development of cotton. The expression of Gb.pepc3 reached the highest level at 15 days after blossoming for Gossypium barbadense L. while 20 days after blossoming for G. hirsutum, and there was significant difference in the expression quantities between two species.
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Molecular Cloning and Expression Analysis of a Receptor-like Protein Kinase Gene in Upland Cotton
YANG Yu-Wen, HE  Bing, ZHANG Bao-Long, NI Wan-Chao
Cotton Science    2011, 23 (1): 15-21.  
Abstract2559)      PDF(pc) (948KB)(1562)       Save
The cDNA encoding the full length of a receptor-like protein kinase of cotton was cloned through degenerate primers amplification, genome walking and RT-PCR. Sequence analysis showed that introns were absent in the gene. It contains a 2964 bp open reading frame, encodes a deduced protein of 988 amino acid residues, and shows 60% homology to HAESA-like2, which regulates floral organ abscission in Arabidopsis. This gene was tentatively designated as GRLK5.  GRLK5 expresses higher in seed, stem bark, root, and fibre than in other organs. The ABA treatment induce the expression of GRLK5. The over expression vector was constructed by inserting GRLK5 into the pCAMBIA2301, tobacco plants were transformed by co-cultivating leaves method via Agrobacterium mediation.The object gene was verified to have been integrated into the genome of tobacco by PCR.
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Population Structure and Linkage Disequilibrium Analysis of Germplasm Resources in Upland Cotton
ZHANG You-Chang, BIE Shu, YI Xian-Da, ZHANG Cheng, LI Cheng-Qi, QIN Hong-De
Cotton Science    2011, 23 (6): 500-506.  
Abstract2195)      PDF(pc) (633KB)(1547)       Save
Population structure and LD(Linkage disequilibrium)of 204 upland cotton accessions were analyzed with 79 SSR (Simple sequence repeat) markers located on nine chromosomes.  Analysis of population genetic structure based on SSR data revealed that this population could be divided into three groups, two out of which were composed of three subgroups, respectively. 47% of the SSR loci pairs showed LD at significant level of P≤0.05. The maximum genetic distance of LD could be observed extended to 120 cM. The LD average decay distance was 29.7 cM at r2≤0.05. Genome wide LD reduced to 3.4 cM at r2≥0.1, providing evidence of the potential for association mapping of important traits in cotton breeding program.
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Construction of a Fosmid Library of Chloroplast Genome in Gossypium raimondii
LI Peng-Bo, XUE Long-Fei, WANG Yan-Xia, ZHANG  Xi, LI Zhao-Hu, HUA Jin-Ping
Cotton Science    2011, 23 (1): 10-14.  
Abstract2236)      PDF(pc) (728KB)(1545)       Save
In this paper, a Fosmid library of G. raimondii chloroplast genome was constructed. The chloroplast DNA was isolated by high ionic strength and low pH buffer method. The DNA was randomly sheared and cloned into pCC1FOS vector. Recombinant DNA was packaged with the Lambda Packaging Extracts, then transfected into E. coli strain EPI300. The best sheared parameter employed in the study was 18 times with middle speed using a 1 mL injector. The library of chloroplast genome (titer: 1×104 cfu·mL-1) was obtained in which the average inserted DNA fragment was 38 kb. Thirty-nine clones covering 9.2 fold the chloroplast genome were selected by selection marker to be further analyzed. Six clones, F66, F46, F28, F8, F55, and F3, which could span G. raimondii complete genome, were screened out by cotton chloroplast markers. The library would be a valuable resource for study on genome structure and functional genes investigation in cotton.
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Preliminary Study on Multiplex PCR Technique and Its Application in Hybrid Cotton Seed Purity Test
CHEN Hao-Dong, LIU Fang, WANG Wei, XIAO Cai-Sheng, LI Xiang, WANG Lin, LI Yu-Qiang, WANG Kun-Bo
Cotton Science    2011, 23 (1): 22-27.  
Abstract2991)      PDF(pc) (625KB)(1543)       Save
A series of stable polymorphism SSR primers in Xiangzamian series cultivars were used to construct various primer combinations according to the difference of their amplified band’s size for multiplex PCR amplification of cotton. Eighty percent of double PCR amplified normally based on the principle of amplified fragments size difference under the same conditions as their single PCR reactions. Besides, triplex and quadruple PCR were also amplified normally using primers of double PCR with good results. So we proposed a simplified protocol of multiplex PCR assay in cotton. The multiplex PCR technology was used to detect the purity of hybrid cotton seeds which clearly identified impurity and blend of female parent seeds and also other hybrid cotton seeds. It can be used as a reference for the rapid and accurate identification of cotton hybrids.
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Verticillium Wilt Resistance of  A Transgenic Cotton Line with Chitinase and Glucanase Genes
ZHU He-Qin, FENG Zi-Li, LI Zhi-Fang, ZHAO Li-Hong, SHI Yong-Qiang, YIN Zhi-Xin
Cotton Science    2011, 23 (1): 58-63.  
Abstract3084)      PDF(pc) (623KB)(1531)       Save
Artificial disease nursery, severe disease field and greenhouse were jointly used for Verticillium wilt disease resistance identification of a transgenic cotton line 61217-1 into which chitinase and glucanase genes were transferred. Results showed that transgenic line 61217-1 exhibited high resistant level to Verticillium wilt disease in artificial nursery during successive three years. Average disease index of line 61217-1 was 17.9, which was significantly lower than that of the acceptor CCRI 24. In 2009, in the severe disease fields, the average disease indexes of this transgenic line were 18.7, 18.1 and 16.7 in Anyang, Henan Province, Dafeng, Jiangsu Province, and Shihezi, Xinjiang, respectively. These indexes were obviously lower than those of the acceptor CCRI 24, respectively. Furthermore, this chitinase-glucanase transgenic cotton line 61217-1 also showed higher resistant to strong virulent defoliating Verticillium dahliae, which was isolated from Hebei, Hubei and Xinjiang, respectively. And their average disease indexes were 16.4, 16.8 and 15.6, which were significantly lower than that of CCRI 24, respectively. The results indicated that the transgenic cotton line 61217-1 (Chi+Glu) exhibited excellent and stable resistance to Verticillium wilt disease.
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Functional Analysis of the SUPERMAN-like Zinc Finger Protein Gene GZFP and Its Promoter
YANG Yu-Wen, ZHOU Jian-Wu, ZHANG Bao-Long, FAN Xiao-Hui, REN Yong-Zhe, CHEN Tian-Zi
Cotton Science    2011, 23 (6): 483-489.  
Abstract2130)      PDF(pc) (983KB)(1524)       Save
GZFP, derived from Gossypium hirsutum L, belongs to the SUPERMAN-like zinc finger protein. To further analyze the function of GZFP, the promoter region of GZFP was obtained by genome walking. The recombination vector pBI-pGZFP-5::GUS containing the promoter was constructed and transformed to Arabidopsis by Agrobacterium-mediated method. GUS staining showed GZFP promoter driven GUS activity was specifically detected in roots and flowers and the activity was very low in leaves. In addition, the GUS activity in roots was very strong throughout the whole growth period. By semi-quantitative RT-PCR, we have found the expression of GZFP was induced by ABA, drought and salt. The over expression vector was constructed by inserting GZFP into the pCAMBIA2301, tobacco plants were transformed by co-cultivating leaves method via Agrobacterium mediation.The object gene was verified to have been integrated into the genome of tobacco by PCR. The phenotype of transgenic plants was normal compared to the wild type, but the expression of NtOPBP1 and NtERD10 was relatively higher than the wild type. As the two genes involved in the stress reaction, GZFP may have some relevance to the plant stress reaction.
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Effect of Regulation of Water and Nitrogen on Cotton Yield and Water Use Efficiency under Different Furrow Irrigation Patterns
LI Pei-Ling, ZHANG Fu-Cang
Cotton Science    2011, 23 (1): 28-33.  
Abstract2219)      PDF(pc) (542KB)(1520)       Save
By setting the alternative furrow irrigation, conventional furrow irrigation, and fixed every-other furrow irrigation, the amount of nitrogen and irrigation design using two general rotation experiment conducted to study the regulation effect of water and nitrogen on cotton yield and water use efficiency. The results showed that: cotton yield was significantly positively correlative, with nitrogen applied amount in the range of 56.2 ~ 95.2 kg·hm-2, and same as cotton yield and water within 37.52 ~ 160.00 mm; Cotton yield was not significant different under the same water and nitrogen treatments between alternative furrow irrigation and conventional furrow irrigation, cotton yield of conventional furrow irrigation was higher on average than the fixed every-other furrow irrigation by 9.15%. Cotton water use efficiency and nitrogen within 56.2 ~ 122.8 kg·hm-2 was a significant positive correlation, and was significantly negatively correlated with irrigation within 37.52 ~160.00 mm. Cotton water use efficiency was no significant difference under the same water and nitrogen treatments between conventional furrow irrigation and alternative furrow irrigation, and conventional furrow irrigation was higher on average than the fixed every-other furrow irrigation in cotton water use efficiency by 9.01%. Therefore, alternative furrow irrigation can increase the cotton yield and water use efficiency.
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Using RENI to Study the Distribution Characteristics of Cotton Roots in Different Amounts of Drip Irrigation under Film
LIU Feng-Shan, ZHOU Zhi-Bin, CHEN Xiu-Long, GAN Yong-De
Cotton Science    2011, 23 (1): 39-43.  
Abstract3187)      PDF(pc) (452KB)(1496)       Save
The cotton root distribution characteristics under different amounts of drip irrigation was studied using root ecological niche index(RENI) which was compared with root parameters(root length, root dry weight, root volume, root surface area) ahead. And the results surely will be contributed to describing root distribution traits and root research methodology. The results showed: (1) RENI was well correlated with other root parameters and so can reflect root distribution characteristics. The contribution rate of root dry weight to RENI was of maximum under sufficient irrigation, and was of minimum under insufficient irrigation. However, root volume was the least influence factor of RENI under sufficient irrigation, and was the most important influence factor under insufficient irrigation. (2)Shown as skewed normal distribution, the RENI of cotton in the upper layer  soil above 40 cm depth accounted for 86.8% of the total RENI. (3) Roots developed in film-uncovered land were centrally impacted by the distance to the cotton plant,i.e., the closer of the distance,the higher of the RENI.
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Effect of Chemical Detopping on the Canopy and Yield of Cotton(Gossypium hirsutum) in South Xinjiang
ZHAO Qiang, ZHOU Chun-Jiang, ZHANG Ju-Song, LI Song-Lin, YUN You-Lan, TIAN Xiao-Li
Cotton Science    2011, 23 (4): 329-333.  
Abstract3242)      PDF(pc) (514KB)(1480)       Save
Field experiment about cotton chemical and manual detopping was conducted in Akesu (41°17′N, 86°26′E), Xinjiang, with cultivar CCRI 49 as material, from 2008 to 2010. The chemical detopping agent contained slow-released mepiquat chloride and additives, and was applied at the same time as the manual detopping in cotton. The results indicated that the plants detopped by chemical agent were taller and narrower than those detopped by hands, which resulted in more light transmittance in middle and lower layer of cotton canopy. Additionally, chemical detopping did not alter the boll weight, but reduced the lint percent slightly. The boll number per plant and yield for chemical detopping were greater than those for manual detopping in 2010, but the same as the latter in 2009. There were no significant difference in comprehensive fiber quality between chemical detopping and manual detopping. It was concluded that the chemical detopping is a potential practice to replace the manual detopping in cotton in Xinjiang.
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Study on Cold Flow Properties of Biodiesel Derived from Cottonseed Oil
CHEN  Xiu, LAI Yong-Bin
Cotton Science    2011, 23 (1): 75-79.  
Abstract2140)      PDF(pc) (474KB)(1431)       Save
 The chemical compositions of biodiesel derived from cottonseed oil(CME) were analyzed by GC-MS. The cold flow properties of CME were studied by cold filer plugging point tester and crystallization mechanism of biodiesel, three approaches for improving cold flow properties of CME were put forward: (i) crystallization fractionation; (ii) blending with winter petrodiesel; and (iii) treating with cold flow improver additives. A good correlation model was proposed for predicting cold fitler plugging point(CFPP) by CME blending ratio. The study shows that the CME is mainly composed of saturated fatty acid methyl esters (C14:0~C24:0) and unsaturated fatty acid methyl esters (C16:1~C22:1, C18:2 and C18:3). The mass fraction of SFAME and UFAME is 32.12% and 66.19%, respectively. The CFPP of CME is 6℃. Crystallization fractionation and blending with -10PD decreased the CFPP of CME to -1℃ and -12℃, respectively. Adding not more than 1.5%(volume fraction) of Flow Fit, Flow Fit K and T818 additives decreased the CFPP of CME and CME/-10PD to 0℃ and -26℃, respectively. This study has effectively improved cold flow properties of CME and provides technical support for using CME.
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