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.     

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     

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     

Optimisation of somatic embryogenesis in fourteen cultivars of sweet potato [Ipomoea batatas (L.) Lam.]

Culture procedures have been developed to facilitate the induction and maintenance of somatic embryogenic tissues in 14 out of 16 tested cultivars of sweet potato [Ipomoea batatas (L.) Lam]. Both the size of the axillary bud explant and the type of auxin were found to be critical for the successful induction of somatic embryogenesis. Of the five auxins screened 2,4-dichlorophenoxyacetic acid 2,4-D and 2,4,5-trichlorophenoxyacetic acid were the most effective, with use of the latter inducing the production of embryogenic tissues in 7 cultivars which responded poorly or not at all to 2,4-D. Procedures for secondary/cyclic embryogenesis, formation of mature embryos and their conversion to plants are also described. Received: 24 September 1996 / Revision received: 16 December 1996 / Accepted 27 January 1997     

Touch-induced gene (IbTCH1) from sweet potato [Ipomoea batatas (L.) Lam.]: molecular cloning and functional analysis

The cDNA of the touch-induced genes (TCH) of the sweet potato [Ipomoea batatas (L.) Lam.] has been cloned and analyzed. IbTCH1, which exists as at least two-copy genes in the genome of the sweet potato, encodes for 148-amino acid polypeptides, and harbors four conversed Ca²⁺-binding motif EF-hands. IbTCH1 was shown to be expressed in the flower, leaf, thick pigmented root, and particularly in the white fibrous root, but expressed only weakly in the petiole. IbTCH1 is upregulated upon exposure to environmental stresses, dehydration, and jasmonic acid. Furthermore, IbTCH1 is developmentally regulated in the leaf and root. These results strongly indicate that the gene performs functions in both plant development and in defense/stress-signaling pathways.     

A dithiol glutaredoxin cDNA from sweet potato (Ipomoea batatas [L.] Lam): enzyme properties and kinetic studies

Glutaredoxins (Grx) play an important role in reduction of protein glutathione mixed disulphides. An IbGrx cDNA (561 bp, EF362614 ) encoding a putative dithiol Grx was cloned from sweet potato (Ipomoea batatas [L.] Lam). The deduced amino acid sequence is conserved among the reported dithiol Grx, having a CGYC dithiol motif at the active site. A 3‐D structural model was created based on the known crystal structure of a poplar Grx (GrxC1). To characterise the IbGrx protein, the coding region was subcloned into an expression vector and transformed into Escherichia coli. The recombinant His₆‐tagged IbGrx was expressed and purified by metal affinity chromatography. The purified enzyme showed a monomeric band, as demonstrated with 15% SDS‐PAGE. The Michaelis constant (K_M) for ß‐hydroxyethyl disulphide (HED) was 0.50 ± 0.08 Mm . The enzyme retained 60% activity at 80 °C for 16 min. The enzyme was active over a broad pH range from 6.0 to 11.0, and in the presence of imidazole up to 0.4 M . The enzyme was susceptible to protease.     

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。     

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%.     

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.     

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

 Routine cryopreservation of shoot tips from sweet potato [Ipomoea batatas (L.) Lam] has been hampered by their survival variability after cryogenic exposure. We examined the effects of light conditions on stock plants, sucrose preculture and cryoprotectant loading on survival after vitrification using PVS2 solution. The survival of vitrified sweet potato shoot tips cooled to approximately –208  °C was increased by preculturing with 0.3 M sucrose for 24 h at 22  °C. Survival was also enhanced by excising shoot tips immediately after the 8-h dark photoperiod. The best survival after cryogenic exposure was obtained using 2 M glycerol +0.4 M sucrose for 1 h at 22  °C followed by dehydration with PVS2 for 16 min at 22  °C. Rapid cooling was used and achieved by the immersion of foil strips into partially solidified nitrogen. Successfully vitrified and warmed shoot tips directly developed shoots on a medium containing 1 μM NAA, 0.5 μM BA and 0.1 μM kinetin with only minimum callus formation. Shoot formation occurred in all surviving shoot tips. This procedure shows promise for cryopreserving sweet potato shoot tips. Received: 2 March 1999 / Revision received: 21 September 1999 / Accepted: 29 September 1999     

Sequence polymorphism and chromosomal localization of 5S rDNA of three cultivated varieties of sweetpotato (Ipomoea batatas (L.) Lam.)

The 5S rDNA of plant is organized into clusters of tandem repeat units which include a coding region of 5S rRNA gene and variable sequences of nontranscribed spacer (NTS). In this study, we investigated sequence polymorphism and chromosomal localization of 5S rDNA in three cultivated varieties of sweet potato (Ipomoea batatas Lam.). Two different PCR products of 5S rDNA were amplified from all three varieties, as approximately 0.25 kb and 0.34 kb with multiples. In sequence analysis, the 5S rDNA ofI. batatas were discriminated from four consensus sequences by in reasonable sizes and molecular informative factors. Four consensus sequences were divided into three short sequences, including 263, 253, and 243 – 283 bp by sequence variation between 160 and 186 bp in NTS region, and one long sequence with 340 bp. To identify molecular relationship among varieties, phylogenetic analysis was applied. A total of 35 sequenced clones in this study were classified into four groups in phylogenetic tree. Interestingly, two varieties included all four groups, but one variety only two groups. To localize the physical map of 5S rDNA, fluorescencein situ hybridization (FISH) was performed in metaphase chromosomes of each varieties. In 90 chromosomes ofI. batatas, 6 loci of 5S rDNA were detected in chromosomes for all varieties. Our results will help to further more understand the genomic relationship inI. batatas, to investigate molecular relationship among varieties.     

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。     

Computer vision analysis of somatic embryos of sweet potato [Ipomoea batatas (L.) Lam.] for assessing their ability to convert to plants

Apical and axial shoot tips of sweet potato were cultured to produce somatic embryos that mature and develop into plants in basal nutrient medium. However, the lack of high regeneration efficiency is an impediment to the use of somatic embryos to produce synthetic seeds. Conversion experiments with mature embryos over a 20-day period revealed that 80–90% of the embryos formed roots but only 40–50% formed shoots. Using computer vision and canonical or Fisher discriminant function (CDA) analysis along with conversion results, it was possible to correctly classify competent embryos 40–50% of the time based on size features, 50–60% of the time based on shape features, and 55–60% of the time based on color features. Non-competent embryos were correctly classified 65–75%, 55–60%, and 70–75% of the time based on size, shape, and color, respectively. These results can be used effectively to identify and select competent embryos for improved regeneration efficiency. Received: 2 January 1997 / Revision received: 21 January 1998 / Accepted: 12 February 1998     

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.     

A comparison of shoot-forming and non-shoot-forming somatic embryos of sweet potato [Ipomoea batatas (L.) Lam.] using computer vision and histological analyses

Diagnostic structural features for competence to form shoots were tested among sweet potato embryos by combining morphological image capture (using a computer vision system) with anatomical analyses (using light microscopy). Five major morphological variants (`perfect', `near perfect', `limited/no meristematic activity', `disrupted internal anatomy', and `proliferating') were identified among torpedo- and cotyledonary-stage embryos. Among these, only the first two were found to be competent for conversion into plantlets. Lack of organized shoot development in somatic embryos of sweet potato was associated with the following abnormalities: lack of an organized apical meristem, sparcity of dividing cells in the apical region, flattened apical meristem, and multiple meristemoids and/or diffuse meristematic activity throughout the embryo. Diagnostic separation of most shoot-forming and non-shoot-forming torpedo and cotyledonary embryo variants was achieved. Received: 27 January 1997 / Revision received: 28 January 1998 / Accepted: 12 February 1998     

Regeneration of plants from Ipomoea cairica L. protoplasts and production of somatic hybrids between I. cairica L. and sweetpotato, I. batatas (L.) Lam.

Plants were regenerated from mesophyll protoplasts of Ipomoea cairica L., a wild relative of sweetpotato (Ipomoea batatas (L.) Lam.), and somatic hybrids between I. cairica L. and sweetpotato cv. Xushu 18 were obtained by PEG-mediated method. I. cairica L. protoplasts were isolated from the leaves of in vitro grown plants and cultured in a modified MS medium containing 0.05 mg l⁻¹ 2,4-D and 0.5 mg l⁻¹ kinetin. Nine weeks after plating, the obtained small calluses up to about 2 mm in diameter were transferred to solid MS medium supplemented with 0.05 mg l⁻¹ 2,4-D and 0.5 mg l⁻¹ kinetin for callus proliferation. Three weeks after transfer, the calluses were transferred to MS medium supplemented with 0–1.0 mg l⁻¹ IAA and 1.0–3.0 mg l⁻¹ BAP and further to hormone-free MS medium for plant regeneration. The frequencies of calluses forming plants ranged from 6.0% to 41.3% based on the different concentrations of IAA and BAP, and 2.0 mg l⁻¹ BAP gave the highest regeneration frequency of protoplast-derived calluses in I. cairica L.. The regenerated plants, when transferred to soil, showed 100% survival. No morphological variations were observed. Mesophyll protoplasts of I. cairica L. were fused with protoplasts isolated from embryogenic suspension cultures of Xushu 18 by PEG-mediated method. The fused products were cultured with the best protoplast culture system of I. cairica L.. Finally, 114 plants were produced from 63 of the 182 calluses derived from the fused protoplasts, and 46 plants of them were confirmed to be somatic hybrids through peroxidase isozyme, RAPD, morphological and cytological analyses.