Chemical and environmental growth regulation of sweetpotato (Ipomoea batatas (L.) Lam.) in vitro

The need for conservation of biotic diversity is well recognized. However, improved techniques for the efficient, cost effective-preservation of plant germplasm are needed. The conservation and distribution of plant germplasm in vitro is gaining acceptance. However, increased usage is dependent upon the ability of curators to minimize culture maintenance requirements. This report examines the effect of various levels of sucrose, photoperiod, temperature, sorbitol and mannitol on minimal growth storage of Ipomoea batatas (L.) Lam. Growth was reduced 50% with a temperature reduction of from 21.1 to 15.6°C. Sucrose concentrations of 15 and 20 g l^-1 resulted in reduced plant stature with few adverse effects on plantlet viability or morphology. Reduction of photoperiod from 16 to 4 h produced smaller, slightly chlorotic, but otherwise normal plants. The addition of sorbitol or mannitol to culture media generally produced undesirable effects on gross plant morphology and loss of apical dominance. Genotype x growth retarding treatment interactions were observed for all variables examined.Abbreviations PL plant introduction - f.w. fresh weight - SE standard error     

Anthocyanin compositions in sweetpotato (Ipomoea batatas L.) leaves

The anthocyanin composition of three varieties, Simon No. 1, Kyushu No. 119, and Elegant Summer, in sweetpotato (Ipomoea batatas L.) leaves was examined for promoting new uses. Fifteen anthocyanin compounds were identified and measured. HPLC clearly showed quantitative differences, but not qualitative ones. The anthocyanins were acylated cyanidin and peonidin type. The result suggests that the major anthocyanin composition of sweetpotato leaves is cyanidin type.     

A genetic linkage map of sweetpotato [Ipomoea batatas (L.) Lam.] based on AFLP markers

Amplified Fragment Length Polymorphism (AFLP) based genetic linkage maps were developed for hexaploid sweetpotato (Ipomoea batatas (L.) Lam., 2n = 6x = 90) using a segregating population derived from a biparental cross between the cultivars 'Tanzania' and 'Bikilamaliya'. A total of 632 ('Tanzania') and 435 ('Bikilamaliya') AFLPs could be ordered in 90 and 80 linkage groups, respectively. Total map lengths were 3655.6 cM and 3011.5 cM, respectively, with an average distance of 5.8 cM between adjacent markers. The genetic linkage analysis was performed in two steps. First a framework map was elaborated from the single dose markers. Interspersed duplex and double-simplex markers were used to detect homologous groups within and corresponding linkage groups among the parental maps. The type of polyploidy (autopolyploidy vs. allopolyploidy) was examined using the ratio of linkage in coupling phase to linkage in repulsion phase and the ratio of non-simplex to simplex markers. Our data support the predominance of polysomic inheritance with some degree of preferential pairing.     

Auxin-regulated gene expression and embryogenic competence in callus cultures of sweetpotato, Ipomoea batatas (L.) Lam.

High levels of 2,4-dichlorophenoxyacetic acid (2,4-D, 10 µM) and sucrose (3%) are required for both the induction and maintenance of callus for somatic embryogenesis in sweetpotato. Newly inducted embryogenic callus lines in sweetpotato cv. White Star produce competent embryos that convert readily into plantlets. With age, these embryogenic callus lines produce a greater proportion of incompetent embryos with poor conversion potential. One hypothesis for this change is that auxin- and/or sugar-responsiveness may be altered with aging. A comparison of two older embryogenic lines (K592 and M892) to two new lines (K1194 and K195) addressed the relationship between extent of embryo conversion and relative abundance of mRNAs hybridizing with heterologous auxin- or sugar-responsive gene probes. The respective cDNAs utilized were the auxin-responsive pJCW1 and pJCW2 and the sugar-responsive Ivr2 and Sh1. Embryos from new callus lines formed more shoots, roots, and viable plantlets than embryos from older callus lines. In addition, new callus lines had greater relative levels of mRNAs hybridizing to auxin-responsive cDNAs. These sweetpotato mRNAs were themselves found to be 2,4-D-responsive in dosage analyses. In contrast, differences between young and old cultures were not evident for mRNAs hybridized to sugar-regulated genes. Our results support the suggestion that desensitization of auxin-responsiveness is a central feature of reduced embryogenic competence in callus lines following prolonged exposure to 2,4-D and elevated sucrose levels.     

甘薯NBS类抗病基因类似物的分离与序列分析

利用已克隆植物抗病基因NBS（Nucleotide binding site）序列中的保守模体（motif）“P-loop”和“GLPL”合成简并引物,以甘薯（Ipomoea batatas）栽培品种青农2号基因组DNA为模板进行PCR扩增,通过T/A克隆、测序和序列分析,共得到15条具有连续ORF的抗病基因类似物（Resistance gene analogues,RGAs）序列,它们之间核苷酸序列间的相似性系数在41.2%-99.4%之间,而相应推测的氨基酸序列间的相似性系数在20.6%-100%之间,同时对分离的RGAs的核苷酸和氨基酸序列进行系统发育树分析,表明甘薯RGAs可分为TIR（Drosophila Toll or human interleukin receptor-like）和nonTIR两类.对甘薯RGAs和5个已克隆植物NBS的氨基酸序列进行结构分析表明,它们包括“P-loop”、“Kinase-2”、“Kinase-3a”、“GLPL”4个抗病基因所共有的保守模体.这些表明甘薯与其它物种的NBS类RGAs可能具有同样的起源和进化机制.     

甘薯与牵牛EST资源的SSR信息分析

为挖掘番薯（Ipomoea）属EST-SSR资源,从NCBI数据库下载23406条甘薯（Ipomoea batatas (L.) Lam.）EST和62282条牵牛（Ipomoea nil (L.) Roth）EST,利用生物信息学软件预处理、去冗余、拼接处理后得到12812条无冗余的甘薯EST（6.70 Mb）和28422条牵牛唯一序列（17.19 Mb）。对这些序列进行SSR搜索,在甘薯上获得328个SSR位点,发生频率为2.56%;牵牛上筛选到962个SSR位点,出现频率为3.38%。甘薯和牵牛EST-SSR具有多个共同特征：在SSR位点中,主要是二核苷酸重复类型,其次是三核苷酸重复;在二核苷酸重复中,出现最多的重复基序为AG/CT,其次是AT/AT;在三核苷酸重复中,主要基序是AAG/CCT;SSR位点的长度主要集中在20~22 bp。结果表明,这些搜索出的EST-SSR重复基序类型丰富、多态性潜能高,具有较高的开发和利用价值。     

甘薯谷胱甘肽-S-转移酶基因在胁迫条件下的表达分析

成功地构建了甘薯谷胱甘肽-S-转移酶基因IBGSTU1 的原核表达质粒pET-IbGST, 并在大肠杆菌 BL21(DE3)中进行IPTG 诱导表达。重组蛋白部分以包涵体形式存在, 部分以可溶性蛋白形式存在。酶活性测定表明可溶的重组蛋白具有GST的活性。纯化的重组蛋白质用于多克隆抗体的制备。半定量RT-PCR 和 Western blotting 分析结果显示, 在正常的生长条件下, 甘薯组织不启动IBGSTU1 基因的转录和翻译, 但是在冷胁迫或重金属离子等的作用下, 可以检测到该基因的mRNA和编码的蛋白质, 表明该基因在甘薯的胁迫耐受中行使重要功能, 并发现该基因的表达具有组织特异性。     

The effect of replicate number and image analysis method on sweetpotato [Ipomoea batatas (L.) Lam.] cDNA microarray results

Microarray analysis makes it possible to determine the relative expression of thousands of genes simultaneously. It has gained popularity at a rapid rate, but many caveats remain. In an effort to establish reliable microarray protocols for sweetpotato [Ipomoea batatas (L.) Lam.], we compared the effect of replication number and image analysis software with results obtained by quantitative rela-time PCR (Q-RT-PCR). Sweetpotato storage root development is the most economically important process in sweetpotato. In order to identify genes that may play a role in this process, RNA for microarray analysis was extracted from sweetpotato fibrous and storage roots. Four data sets, Spot4, Spot6, Finder4 and Finder6, were created using 4 or 6 replications, and the image analysis software of UCSF Spot or TIGR Spotfinder were used for spot detection and quantification. The ability of these methods to identify significant differential expression between treatments was investigated. The data sets with 6 replications were better at identifying genes with significant differential expression than the ones of 4 replications. Furthermore when using 6 replicates, UCSF Spot was superior to TIGR Spotfinder in identifying genes differentially expressed (18 out of 19) based on Q-RT-PCR. Our study shows the importance of proper replication number and image analysis for microarray studies.     

Effects of shading on the photosynthetic capacity,endogenous hormones and root yield in purple-fleshed sweetpotato (Ipomoea batatas (L.) Lam)

A field experiment was conducted to examine the effects of shading on the photosynthetic capacity, endogenous hormones and root yield in purple-fleshed sweetpotato [Ipomoea batatas L. cv. Jishu18 and Ayamuraski (Aya)]. Sweetpotato plants were treated with two shading levels, 40 and 70 % shading, with full radiation used as a control. The results showed that the photosynthetic rate, adenosine triphosphatase activity, Ribulose 1,5-bisphosphate carboxylase activity and soluble sugar content decreased under both shading treatments. Leaf indole-3-acetic acid (IAA) and abscisic acid content increased, whereas leaf gibberellic acid content, zeatin riboside (ZR) content, root IAA, and ZR content decreased in the plants under both shading treatments. Shading also altered the production of sweetpotato storage root, including reductions in the root yield and dry matter accumulation, increase in the top/root (T/R) ratio, and the difference between the treatments and control for the T/R value and storage root yield was significant. Therefore, the responses of the photosynthetic parameters and endogenous hormones to shading were closely correlated with the variation in the storage root yield of the different cultivars. In response to shading, the reduction of root ZR contents, the fresh dry weight of the above-ground parts and the root yield for Jishu18 were higher than that for cv. Aya, indicating that cv. Jishu18 might be more sensitive to weak light than cv. Aya.     

RNA silencing-mediated resistance to a crinivirus (Closteroviridae) in cultivated sweetpotato (Ipomoea batatas L.) and development of sweetpotato virus disease following co-infection with a potyvirus

Sweetpotato chlorotic stunt virus (SPCSV; genus Crinivirus , family Closteroviridae) is one of the most important pathogens of sweetpotato ( Ipomoea batatas L.). It can reduce yields by 50% by itself and cause various synergistic disease complexes when co-infecting with other viruses, including sweetpotato feathery mottle virus (SPFMV; genus Potyvirus , family Potyviridae). Because no sources of true resistance to SPCSV are available in sweetpotato germplasm, a pathogen-derived transgenic resistance strategy was tested as an alternative solution in this study. A Peruvian sweetpotato landrace 'Huachano' was transformed with an intron-spliced hairpin construct targeting the replicase encoding sequences of SPCSV and SPFMV using an improved genetic transformation procedure with reproducible efficiency. Twenty-eight independent transgenic events were obtained in three transformation experiments using a highly virulent Agrobacterium tumefaciens strain and regeneration through embryogenesis. Molecular analysis indicated that all regenerants were transgenic, with 1–7 transgene loci. Accumulation of transgene-specific siRNA was detected in most of them. None of the transgenic events was immune to SPCSV, but ten of the 20 tested transgenic events exhibited mild or no symptoms following infection, and accumulation of SPCSV was significantly reduced. There are few previous reports of RNA silencing-mediated transgenic resistance to viruses of Closteroviridae in cultivated plants. However, the high levels of resistance to accumulation of SPCSV could not prevent development of synergistic sweet potato virus disease in those transgenic plants also infected with SPFMV.     

甘薯叶绿体rbcL基因的克隆与序列分析

根据烟草、水稻和菠菜叶绿体的全基因组序列设计引物,以甘薯的叶绿体基因组DNA为模板,PCR扩增包舍甘暑叶绿体rbcL完整基因(GenBank登录号为AY942199)在内的一段序列.序列分析表明:此片段的全长为1 627 bp,包括1 443bp的编码区序列在内.推测编码480个氨基酸,同时构建了此片段的限制性酶切图谱.相似性比较显示,此基因编码区序列与烟草、菠菜、小麦、水稻、玉米、矮牵牛、紫花苜蓿、拟南芥、莨菪、葡萄以及甜菜的rbcL基因核苷酸的同源性为85%～98%,氨基酸的同源性为92%～95%.     

Antimutagenicity of sweetpotato (Ipomoea batatas) roots

Antimutagenicity of the water extracts prepared from the storage roots of four varieties of sweetpotato with different flesh colors was investigated using Salmonella typhimurium TA 98. The extract from the whole roots of the purple-colored Ayamurasaki variety effectively decreased the reverse mutation induced not only by Trp-P-1, Trp-P-2, IQ, B[a]P, and 4-NQO but also by dimethyl sulfoxide extracts of grilled beef. Comparison of the inhibitory activity of the extracts from the normal Ayamurasaki and its anthocyanin-deficient mutant one suggested that the anthocyanin pigment in the flesh decreases the mutagenic activity of the mutagens as heterocyclic amines. Two anthocyanin pigments purified from purple-colored sweet-potato, 3-(6,6'-caffeylferulylsophoroside)-5-glucoside of cyanidin (YGM-3) and peonidin (YGM-6) effectively inhibited the reverse mutation induced by heterocyclic amines, Trp-P-1, Trp-P-2, and IQ in the presence of rat liver microsomal activation systems.     

Isozyme modifications and plant regeneration through somatic embryogenesis in sweet potato (Ipomoea batatas (L.) Lam.)

Summary The potential of somatic embryogenesis was evaluated for 10 cultivars of sweet potato through extensive embryogenic response and isozyme analysis. Embryogenic callus was induced by incubating lateral buds on Murashige and Skoog medium containing 10 M 2,4-dichlorophenoxyacetic acid for 6–8 weeks. The frequency of embryogenic response was low, and varied with genotypes, ranging from 0 to 17%. Embryo to plantlet formation could be enhanced by the use of the combination of 2,4-dichlorophenoxyacetic acid with kinetin, both used at 0.01 M. Embryogenic callus with its potential of plantlet formation has constantly been maintained for over two years. However, after several subcultures, 0.5 to 12% of embryogenic callus reverted irreversibly into friable fast-growing non-embryogenic callus whose ability to regenerate shoots was then definitively lost. The isozymes of esterase, peroxidase, glutamate oxaloacetate transaminase and acid phosphatase investigated in this study were found appropriate to distinguish compact embryogenic from friable non-embryogenic callus in sweet potato. In fact, the callus reversion was associated with a loss of bands or a decline in isozyme activity. On the contrary, very small changes in isozyme activity or no specific changes at all were observed during the differentiation of embryogenic callus into globular embryos.Abbreviations Acp acid phosphatase - BAP 6-benzylaminopurine - cv cultivar - df degree of freedom - 2,4-D 2,4-dichlorophenoxyacetic acid - Est esterase - Got glutamate oxaloacetate transaminase - IAA indole-3-acetic acid - MS Murashige and Skoog (1962) medium - Prx peroxidase - Tris tris(hydroxymethyl)aminomethane     

Cryopreservation of sweet potato (Ipomoea batatas [L.] Lam.) shoot tips by vitrification

Summary Vitrification is a technically simple method for cryopreserving plant germplasm, requiring only the application of suitable cryoprotectants and rapid cooling rates. Sweetpotato (Ipomoea batatas [L.] Lam.) shoot tips obtained from in vitro plants survived liquid nitrogen (–196°C) exposure following a vitrification-inducing pretreatment. Shoot tips were treated in a stepwise manner with a vitrification solution containing 30% glycerol, 15% ethylene glycol and 15% dimethylsulfoxide in growth medium. Incubation of shoot tips for 1 to 2 h in low concentrations of the vitrification solution enhanced survival. Most surviving shoot tips developed callus, and a variable percentage subsequently formed shoots. Survival was not achieved using two-step cooling procedures. The percentage of shoot tips surviving vitrification and those subsequently forming a shoot varied widely among replications.Abbreviations BA N⁶-benzyladenine - IBA indole-3-butyric acid - EG ethylene glycol - DMSO dimethylsulfoxide - MS Murashige and Skoog (1962) minerals and vitamins - LN liquid nitrogen - PI plant introduction     

Phosphate fertiliser alters carboxylates and bacterial communities in sweet potato (Ipomoea batatas (L.) Lam.) rhizosheaths

Plant and Soil - Selenium (Se) is an essential element for mammals and its deficiency in the diet is a global problem. Plants accumulate Se and thus represent a major source of Se to consumers....     

Effect of Light and NaCI Salinity on the Growth of Callus Cultures of Ipomoea pes-caprae (L.) R. Brown and Ipomoea batatas (L.) Lam

Callus cultures of Ipomoea pes-caprae and I. batatas were establishedon MS medium containing 10^–5 M 2,4-D and 10^–8 Mbenzyladenine. Ipomoea pes-caprae calli exhibited green pigmentationand grew better in the light than in darkness. Callus tissuesof I. batatas showed a pale-yellow colouration and they grewat the same rate in light as in dark conditions. I. pes-capraeand I. batatas callus cultures were sensitive to the presenceof 60 mM NaCl in the culture medium, the growth of the formerbeing more sensitive in light than in darkness. The significanceof the responses of I. pes-caprae callus cultures in relationto the mechanism of salt tolerance is discussed. Ipomoea batatas, Ipomoea pes-caprae, sweet potato, railroad vine, callus cultures, salinity, light     

A proteomic analysis of storage stress responses in Ipomoea batatas (L.) Lam. tuberous root

During post-harvest storage, tuberous roots of sweet potato (Ipomoea batatas L. Lam.) usually undergo a biotic and abiotic stress influencing protein expression pattern and substance contents. This research compared the change of total proteins and carbohydrate content in tuberous roots of sweet potato during the storage period. The result of the two-dimensional electrophoresis analysis demonstrated that there were 25 differentially expressed proteins between day 0 and day 75 during the storage. Among these proteins, 11 proteins were down-regulated and the other 14 were up-regulated. The results from MALDI-TOF-TOF/MS analyses and mascot database searching showed that 11 of the 25 differentially expressed proteins were identified as store-stress regulated proteins. It was also found that the proteins involved in the energy metabolism and the stress-response were drastically up-regulated, whereas those in biomacromolecule synthesis were markedly down-regulated. Meanwhile, under the experimental conditions, the content of the starch and the cellulose was decreased by more than a quarter and the amylase activity was increased moderately.     

Plant regeneration from protoplast culture of sweet potato (Ipomoea batatas Lam.)

This is the first report on successful plant regeneration from protoplasts of sweet potato. Two cultivars (Guyana and Duclos XI) of sweet potato plants propagated under in vitro conditions were used as the source of protoplasts. Green compact calli with meristematic areas were induced in the medium supplemented with 2mg1^–1 zeatin, and plant regeneration occurred when these calli were transferred onto the medium with zeatin level reduced to 0.25mg1^–1. Plant regeneration was found to be genotype-dependent, since it was only obtained for cultivar Duclos XI.Abbreviations MS Murashige and Skoog basal medium - IAA Indol-3-acetic acid - NAA naphthaleneacetic acid - 2,4-D dichlorophenoxyacetic acid - Mes 2-(N-morpholino)-ethanesulfonic acid - Cpw cell and protoplast washing solution