Morphohistobiochemical characteristics of embryogenic and nonembryogenic callus cultures of sweet potato (Ipomoea batatas L.)

Ipomoea batatas callus culture raised in a medium supplemented with 2,4-D (2,4-dichlorophenoxy acetic acid) alone or 2,4-D in combination with benzyl adenine, were found to be embryogenic. Supplementation of exogenous chemicals, such as 5 g/l NaCI or 0.7 g/l proline together with a mild dose of 0.2 mg/l 2,4-D, enhanced somatic embryogenesis significantly in all the genotypes tested. Morphological, growth, physiological, histological, and biochemical characteristics of the embryogenic callus were different from the nonembryogenic callus. The former was compact, slow growing, and nodular compared with the fast growing, fragile, nonembryogenic callus. The embryogenic callus tissue had more dry matter, protein and reducing sugar contents compared with the less embryogenic callus. The somatic embryogenic response remained steady in the cultures for up to 96 weeks.     

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。     

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.     

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。     

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.     

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.     

Carotenoids gene markers for sweetpotato (Ipomoea batatas L. Lam): applications in genetic mapping,diversity evaluation and cross-species transference

Carotenoids play essential biological roles in plants, and genes involved in the carotenoid biosynthesis pathway are evolutionarily conserved. Orange sweetpotato is an important source of β-carotene, a precursor of vitamin A. In spite of this, only a few research studies have focussed on the molecular aspects of carotenoid genes regarding their specific sequence and structure. In this study, we used published carotenoid gene sequences from Ipomoea and other species for “exon-primed intron-crossing” approaches. Fifteen pairs of primers representing six carotenoid genes were designed for different introns, eleven of which amplified scorable and reproducible alleles. The sequence of PCR products showed high homology to the original ones. Moreover, the structure and sequence of the introns and exons from five carotenoid structural genes were partially defined. Intron length polymorphism and intron single nucleotide polymorphisms were detected in amplified sequences. Marker dosages and allelic segregations were analysed in a mapping population. The developed markers were evaluated in a set of Ipomoeas batatas accessions so as to analyse genetic diversity and conservation applicability. Using CG strategy combined with EPIC-PCR technique, we developed carotenoid gene markers in sweetpotato. We reported the first set of polymorphic Candidate Gene markers for I. batatas, and demonstrated transferability in seven wild Ipomoea species. We described the sequence and structure of carotenoid genes and introduced new information about genomic constitution and allele dosage.     

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.     

甘薯基因组 TCP转录因子鉴定与逆境胁迫表达分析

基于甘薯( Ipomoea batatas ( L.) Lam.)全基因组序列, 利用生物信息学方法鉴定筛选了全基因组中的TCP( teosinte branched1/cincinnata/proliferating cell factor)转录因子, 并分析了甘薯苗期在蔓割病菌胁迫及块根储藏期低温胁迫下TCP基因的...     

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.     

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.     

Amylase secretion by sweet potato,Ipomoea batatas (L.) Lam., cell cultures grown on various carbon sources

Cell cultures of sweet potato grown in media containing sucrose, glucose, maltose, or starch secreted amylase into the growth medium. The growth rate of cells was not appreciably affected by the carbon source employed for growth, although cells grown on sucrose had a slightly longer lag period before exponential growth occurred. Amylase levels inside the cells were not affected by carbon source, but the amount of amylase released into the medium was drastically affected. Maltose-grown cells released the most amylase while sucrose-grown cells released the least. Cells grown in the light released about twice as much amylase as cells grown in the dark when grown on glucose, maltose, or starch.Three amylase electrophoretic forms were found in the storage root tissue from which all cultures were derived. Cells grown in culture exhibited either two or three amylase forms, depending on the carbon source. The slowest migrating root amylase was found only in cells grown on starch. The root amylase having intermediate mobility was present in all cultures, as was a form having higher mobility than the most mobile root form. The fastest migrating electrophoretic form from the root was not present in any of the cells.Paper No. 8466 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of products named, nor criticism of similar ones not mentioned.     

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.     

Anomalous Growth in the Stem of Ipomoea batatas (L.) Lam. Infested with Megastes Larvae

The stem of Ipomoea batatas (L.) Lam. is characterized by thepossession of a ring of bicollateral, leaftrace bundles. Lacticifersoccur in the pith, in the parenchyma between neighbouring islandsof medullary phloem, and in the cortex. The xylem groups become united by the activity of the inter-fascicularcambium. The production of a certain amount of secondary xylemtakes place before the production of secondary phloem begins.The former is produced more extensively in some areas than inothers, so that the original symmetry of the vascular cylinderis lost. The phellogen originates in the cells of the epidermis. When the stem is attacked by the larvae of Megastes grandalisGuen., which remove most of the internal tissues, anomalousgrowth takes place as a result of the activity of accessorycambia, which develop in the primary cortex, the secondary phloem,and the phelloderm. Residual parenchyma of the pith and/or xylemundergoes hyperplasia to produce a callus tissue which linesthe cavity made by the larvae.     

Antimutagenicity of mono-, di-, and tricaffeoylquinic acid derivatives isolated from sweetpotato (Ipomoea batatas L.) leaf

The caffeoylquinic acid derivatives, 3-mono-O-caffeoylquinic acid (chlorogenic acid, ChA), 3,4-di-O-caffeoylquinic acid (3,4-diCQA), 3,5-di-O-caffeoylquinic acid (3,5-diCQA), 4,5-di-O-caffeoylquinic acid (4,5-diCQA) and 3,4,5-tri-O-caffeoylquinic acid (3,4,5-triCQA), and caffeic acid (CA) were isolated from the sweetpotato (Ipomoea batatas L.) leaf. We examined the antimutagenicity of these caffeoylquinic acid compounds to promote new uses of the sweetpotato leaf. These caffeoylquinic acid derivatives effectively inhibited the reverse mutation induced by Trp-P-1 on Salmonella typhimurium TA 98. The antimutagenicity of these derivatives was 3,4,5-triCQA > 3,4-diCQA = 3,5-diCQA = 4,5-diCQA > ChA in this order. There was no difference in the antimutagenicity of all dicaffeoylquinic acid derivatives. A comparison of the activities and structures of these compounds suggested that the number of caffeoyl groups bound to quinic acid played a role in the antimutagenicity of the caffeoylquinic acid derivatives. The sweetpotato leaves contained distinctive polyphenolic components with a high content of mono-, di-, and tricaffeoylquinic acid derivatives and could be a source of physiological functions.