Production of transgenic sweetpotato plants resistant to stem nematodes using oryzacystatin-I gene

Stem nematode (Ditylenchus destructor Thorne) is one of most serious diseases limiting sweetpotato (Ipomoea batatas (L.) Lam.) production, and it is urgent to develop sweetpotato varieties resistant to this disease. In present study, we have developed transgenic sweetpotato (cv. Xushu 18) plants resistant to stem nematodes using oryzacystatin-I (OCI) gene with Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain EHA105 harbors a binary vector pCAMBIA1301 with OCI gene, uidA gene and hptII gene. Selection culture was conducted using 7 mg/l hygromycin. A total of 2119 plants were produced from the inoculated 1710 cell aggregates of Xushu 18 via somatic embryogenesis. GUS assay and PCR analysis of the regenerated plants randomly sampled showed that 92.8% of the regenerated plants were transgenic plants. Transgenic plants exhibited enhanced resistance to stem nematodes compared to the untransformed control plants by the field evaluation and the inoculation test with stem nematodes and stem nematode-resistant plants were selected from the transgenic plants. Stable integration of the OCI gene into the genome of resistant transgenic plants was confirmed by Southern blot analysis, and the copy number of integrated OCI gene ranged from 1 to 3. High level of OCI gene expression in the resistant transgenic plants was demonstrated by real-time quantitative PCR analysis. This study provides a way for improving stem nematode resistance of sweetpotato.     

Efficient production of transgenic plants using the bar gene for herbicide resistance in sweetpotato

Efficient production of transgenic sweetpotato (Ipomoea batatas (L.) Lam.) plants using the bar gene for herbicide resistance was achieved through the use of embryogenic suspension cultures and Agrobacterium tumefaciens-mediated transformation. Cell aggregates from embryogenic suspension cultures of sweetpotato cv. Lizixiang were cocultivated with A. tumefaciens strain EHA 105 harboring a binary vector pCAMBIA3300 with the bar gene and uidA gene. Selection culture was conducted using 0.5 mg/l PPT. A total of 1431 plants were produced from the inoculated 870 cell aggregates via somatic embryogenesis. GUS assay and PCR analysis of the regenerated plants randomly sampled showed that 86.5% of the regenerated plants were transgenic plants. Stable integration of the bar gene into the genome of transgenic plants was confirmed by Southern blot analysis and transgene expression was demonstrated by Northern blot analysis. The copy number of integrated bar gene ranged from 1 to 3. Transgenic plants exhibited functional expression of the bar gene by in vivo assay for herbicide resistance. This study also provides a simple and efficient transformation system of sweetpotato based on the use of bar gene as a selectable marker gene, which can be combined with other agronomically important genes for the improvement of sweetpotato.     

Development of a simple thermal time method for describing the onset of morpho-anatomical features related to sweetpotato storage root formation

A thermal time-based approach using growing degree days was used to describe the onset of morpho-anatomical features associated with storage root developmental stages in the sweetpotato cultivar ‘Beauregard.’ Initially, we calculated accumulated growing degree days corresponding to adventitious root initiation, the onset of storage root formation (appearance of anomalous cambium), and appearance of visible storage roots with localized thickening and pigmentation in sweetpotatoes grown under greenhouse conditions. Subsequently, we performed field calibration of this approach to verify the timing of these storage root developmental stages under natural growing conditions. Under field conditions, transplants required relatively fewer calendar days to achieve the thermal time corresponding to each developmental stage in comparison to greenhouse-grown plants. Further calibration of this approach for ‘Beauregard’ and other sweetpotato varieties grown in diverse environments will help enhance our understanding of the effects of management and agroclimatic variables on sweetpotato yield determination. This study demonstrates the advantage of using thermal time vs. calendar days in estimating storage root development in sweetpotatoes.     

Biocompatibility of sweetpotato and peanut in a hydroponic system

'Georgia Red' peanut (Arachis hypogaea L.) and TU-82-155 sweetpotato [Ipomoea batatas (L.) Lam] were grown in monocultured or intercropped recirculating hydroponic systems in a greenhouse using the nutrient film technique (NFT). The objective was to determine whether growth and subsequent yield would be affected by intercropping. Treatments were sweetpotato monoculture (SP), peanut monoculture (PN), and sweetpotato and peanut grown in separate NFT channels but sharing a common nutrient solution (SP-PN). Greenhouse conditions ranged from 24 to 33 degrees C, 60% to 90% relative humidity (RH), and photosynthetic photon flux (PPF) of 200 to 1700 micromoles m-2 s-1. Sweetpotato cuttings (15 cm long) and 14-day-old seedlings of peanuts were planted into growth channels (0.15 x 0.15 x 1.2 m). Plants were spaced 25 cm apart within and 25 cm apart between growing channels. A modified half-Hoagland solution with a 1 N: 2.4 K ratio was used. Solution pH was maintained between 5.5 and 6.0 for treatments involving SP and 6.4 and 6.7 for PN. Electrical conductivity (EC) ranged between 1100 and 1200 microS cm-1. The number of storage roots per sweetpotato plant was similar for both SP and SP-PN. Storage root fresh and dry mass were 29% and 36% greater, respectively, for plants in the SP-PN treatment than for plants in the SP treatment. The percent dry mass of the storage roots, dry mass of fibrous and pencil roots, and the length-to-diameter ratio of storage roots were similar for SP and SP-PN sweetpotato plants. Likewise, foliage fresh and dry mass and harvest index were not significantly influenced by treatment. Total dry mass was 37% greater for PN than for SP-PN peanut plants, and pod dry mass was 82% higher. Mature and total seed dry mass and fibrous root dry mass were significantly greater for PN than for SP-PN plants. Harvest index (HI) was similar for both treatments. Root length tended to be lower for seedlings grown in the nutrient solution from the SP-PN treatment.     

Mapping of quantitative trait loci for carotenoid pigmentation in flower tepals of Asiatic hybrid lily

Flower pigmentation is one of the most important traits for ornamental plants. To clarify the genetic basis for carotenoid pigmentation in flower tepals of Asiatic hybrid lily (Lilium sp.), we evaluated the segregation of a tepal-carotenoid content among F₁ plants derived from a cross between ‘Montreux’ (having a small amount of carotenoids) and ‘Connecticut King’ (having a large amount of carotenoids), and mapped genetic loci for the carotenoid pigmentation onto the molecular linkage maps of ‘Montreux’ and ‘Connecticut King’ that we constructed previously. The tepal-carotenoid content among the F₁ plants showed continuous segregation, indicating that several genes are associated with this trait. Quantitative trait loci (QTL) analysis identified one QTL, qCARmon6, on the sixth linkage group of the ‘Montreux’ map. qCARmon6 explained 58.2% of the total phenotypic variation, that is, this locus had a large effect on the carotenoid accumulation. The result that qCARmon6 was mapped on the linkage group of ‘Montreux’ which has a small amount of carotenoid pigments in tepals indicates that this locus has a dominant negative effect on carotenoid pigmentation.     

Development of cleaved amplified polymorphic sequence (CAPS)-based markers for identification of sweetpotato cultivars

To develop a simple and reliable method to identify sweetpotato cultivars, we designed cleaved amplified polymorphic sequence (CAPS)-based markers and used them to perform genotyping of Japanese sweetpotato cultivars. In order to screen the CAPS-based markers, 13 primer pairs were designed from the exon sequences of 11 sweetpotato genes to amplify fragments containing an intron. By digesting the amplified products with 8 restriction enzymes having different recognition sites, a total of 27 polymorphic marker fragments were obtained. Genotyping of 60 Japanese sweetpotato cultivars using these markers suggested that the markers can effectively distinguish sweetpotato cultivars. Among the genes used for primer design, the gene encoding the dihydroflavonol 4-reductase (DFR) showed the largest degree of polymorphism. To our knowledge, this is the first report on the development of CAPS-based markers in sweetpotato.     

Expression of carotenogenic genes during the development and ripening of watermelon fruit

Five watermelon varieties with different flesh colors: “ZAOHUA” (red); “96B41” (pink); “307CHAOFENG” (yellow); “UNKNOWN” (orange); and “SANBAI” (white) were used to investigate the relationship between carotenoid profiles and expression of carotenoid-related genes during fruit development and ripening. In mature fruit, “ZAOHUA” and “96B41” accumulated high level of lycopene, “SANBAI” contained trace amounts of carotenoids. Through HPLC and dot blot hybridization, carotenoid content was analyzed and compared to the expression of carotenoid-related genes including Psy, Pds, Zds, CrtIso, Lcyb, Chyb, Nced1, Nced2, Nced3 during fruit development. All genes examined were expressed in all five watermelon varieties. In red and pink color varieties, the decrease of gene expression for Lcyb and Chyb at stage C (30 d after pollination) may be consistent with the high accumulation of lycopene and β-carotene. In the yellow variety, expression of the detected genes increased markedly with the onset of maturation, but little carotenoid accumulation was detected, which may be due to high level of carotenoid cleavage dioxygenases. Expression of all detected genes decreased rapidly in the white color variety upon fruit ripening, which may be attributed to a reduction in metabolic flux in the carotenoid pathway. These results imply the complex regulation of the carotenoid biosynthetic pathway in watermelon.     

Enlargement of the variances in amount and composition of anthocyanin pigments in sweetpotato storage roots and their effect on the differences in DPPH radical-scavenging activity

To investigate the effect of differences in anthocyanin composition on radical-scavenging activity in the purple-fleshed sweetpotato storage root, 124 sweetpotato clones with various compositions of anthocyanin were produced by crosses among superior sweetpotato genetic resources, and were analyzed for anthocyanin composition and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity (RSA). The highest content of anthocyanin among the clones was twofold higher than the content of cv. Ayamurasaki, which has been released for use in industrial pigment production in Japan. The range of variance in anthocyanin composition was very wide, as the ratio of peak areas of cyanidin-based anthocyanins to total anthocyanin peak areas ranged from 85.7% to 6.0%. The relationship between anthocyanin content and RSA was positively correlated. Correlation coefficients between RSA and the HPLC peak areas of cyanidin-based anthocyanins ranged from 0.606 to 0.687, whereas those of peonidin-based anthocyanins were 0.124–0.271. These results clearly demonstrated that cyanidin-based anthocyanins are closely related to DPPH RSA in sweetpotato storage roots.     

Effects of ages of plug transplants and planting depths on the growth and yield of sweetpotato

Sweetpotato [Ipomoea batatas (L.) Lam. cv. Beniazuma] plug transplants produced from single node leafy cuttings under artificial light in a closed-type growth chamber were planted with roots and substrate of 11- and 15-day old (ca. two to three unfolded leaves with 0.08 m shoot length and three to four unfolded leaves with 0.11 m shoot length, respectively). The plug transplants of both the 11- and 15-day old were planted with one and three nodes depth (ca. 4 and 25 mm deep, respectively) inside the soil ridges (called one- and three-node depth, respectively, hereafter). The conventional vine cuttings (ca. 0.3 m long with seven to eight unfolded leaves) without roots were planted as Control treatment to compare the growth and yield of sweetpotato with each of the treatments of plug transplant. The main objectives of the study were to see the effects of ages of plug transplants and depths of planting for greater growth and yield of sweetpotato in the field. The yield of storage roots 115 days after planting in the field was 33 t ha⁻¹ when using 15-day old plug transplants planted with three-node depth and was 10 t ha⁻¹ greater than that in the Control. The mean storage root length was about 259 mm when using 11-day old plug transplants planted with three-node depth and was 33 mm greater than that in the Control. The mean diameter of storage roots was 70 mm when using 11- and 15-day old plug transplants planted with one-node depth and was 21 mm greater than that in the Control. The plug transplants planted either 11- or 15-day old showed greater overall performances than those of the conventional cuttings. The plug transplants planted with three-node depth showed greater performances than did the plug transplants planted with one-node depth.     

Diversity of nitrate,oxalate, vitamin C and carotenoid contents in different spinach accessions and their correlation with various morphological traits

Spinach (Spinacia oleracea L.) accumulates high amounts of carotenoids, vitamin C, nitrate and oxalate. Breeding to reduce nitrate and oxalate levels and increase vitamin C and carotenoid levels is a major target in spinach nutritional quality improvement. Here we investigate natural variations in the levels of nitrate, oxalate, vitamin C, and total carotenoid in 98 spinach accessions, and correlations among these four nutritional components and 12 important morphological traits. Our results showed that the concentrations of nitrate, oxalate, vitamin C, and total carotenoid ranged from 0.21–3.83, 2.38–34.72, 0.51–1.30, and 0.18–0.58 mg g^?1 fresh weight (FW), respectively. Moderate positive correlations were observed between the levels of oxalate and vitamin C, and between vitamin C and total carotenoid, whereas the levels of nitrate were correlated weakly and negatively with those of vitamin C and total carotenoid. The nitrate contents of crinkle-leaf accessions were significant higher than those of smooth-leaf accessions, and the smooth-leaf accessions had significantly higher concentrations of oxalate and vitamin C than crinkle-leaf accessions. Furthermore, hierarchical clustering analysis identified eight spinach accessions that could be served as starting materials for breeding new spinach cultivars with increased levels of vitamin C and carotenoids and simultaneously decreased accumulation of nitrate and oxalate.     

Quantification of phenolic acids and antioxidant activity in sweetpotato genotypes

Phenolic acid composition and antioxidant activity in roots of 14 commercially important sweetpotato genotypes were evaluated. Significant differences in total phenolics, individual phenolic acids, and antioxidant activity were found among the different sweetpotato genotypes. Total phenolic content, expressed in terms of chlorogenic acid equivalent, in different genotypes ranged from 1.4 to 4.7 mg g⁻¹ dry weight (DW). Antioxidant activity was evaluated as Trolox equivalent, ranging from 1.3 to 4.6 mg g⁻¹ DW. The highest total phenolic content and antioxidant activity were observed in a purple-fleshed genotype. Chlorogenic acid and 3,5-dicaffeoylquinic acid were the predominant phenolic acids, while caffeic acid was the least abundant in most genotypes. The highest content of chlorogenic acid (422.4 μg g⁻¹ DW) was present in a white-fleshed cultivar ‘Quarter Million’ imported from Jamaica. However, a purple-fleshed genotype had the highest amounts of 3,5-dicaffeoylquinic (485.6 μg g⁻¹ DW), 3,4-dicaffeoylquinic (125.6 μg g⁻¹ DW), 4,5-dicaffeoylquinic (284.4 μg g⁻¹ DW), and caffeic (20.5 μg g⁻¹ DW) acids.     

Vine growth and nitrogen metabolism of ‘Fujiminori’ grapevines in response to root restriction

Two-year-old ‘Fujiminori’ grapevines (Vitis Venifera × V. Labrasca) planted in plastic pots (10 L) were used to evaluate vine growth and nitrogen metabolism in response to root restriction. Results show that root restriction reduced shoot growth and photosynthetic rate, but promoted root growth in vines. NO₃⁻-N concentration in all plant parts, and total N concentrations in brown roots and new leaves were decreased by root restriction, and chlorophyll and carotenoid concentrations in mature leaves were also reduced. Nitrate and nitrite reductase activities in brown roots and mature leaves were significantly reduced in root-restricted vines. The results suggest that the reduction of nitrate and nitrite reductase activities caused the inhibition of nitrogen assimilation, and this might be an important reason for root restriction inhibiting shoot growth.     

宫内伊予柑果实发育期间色泽和色素的变化

 以宫内伊予柑为试材, 对果实发育期间色泽、色素种类及含量, 尤其是类胡萝卜素组分进行了研究。结果表明, 幼果中高含量的叶绿素使果实呈现青绿色, 采前8周起类胡萝卜素开始积累, 采前4周起随叶绿素含量下降黄色得以显现。果实类胡萝卜素主要存在于果皮中, 成熟果实果皮类胡萝卜素含量达囊瓣的16.62倍, 总量占整果的83.62%。用高效液相色谱—二极管阵列检测(HPLC - PDAD) 技术,对采前12周至采收期的果皮类胡萝卜素组成进行了分析, 共分离出类胡萝卜素组分23种, 其中7种得到鉴定。在采收期果皮中, 已鉴定的类胡萝卜素中以β - 隐黄质和玉米黄素较丰富, 占总类胡萝卜素的8.00%和6.78%; β - 胡萝卜素不足总类胡萝卜素的1% , 没有检测出α - 胡萝卜素和番茄红素。果实成熟过程中β - 隐黄质及玉米黄素含量上升, 并伴随α - 胡萝卜素、叶黄质和β - 胡萝卜素含量下降, 暗示类胡萝卜素的β主链合成能力和β环羟化活性急剧增强, 与此同时, 阿朴类胡萝卜素的出现则表明类胡萝卜素的裂解能力随果实成熟而上升。     

植物类胡萝卜素分析方法研究进展

类胡萝卜素是广泛分布于动物、植物和微生物中一类色素,对于植物的外观色泽、商品性和人类健康起着十分重要的作用。文章详细综述了植物类胡萝卜素的分析方法,以期为植物类胡萝卜素的合理开发提供参考。     

Experiments on the Control of Brown Rot of Apples and Plums: I. Laboratory Tests

The symptoms and some properties of five viruses isolated from celery crops in Britain are described. Cucumber mosaic and lucerne mosaic viruses were found to be prevalent in celery and other crops. Tomato aspermy virus, though widespread in chrysanthemum and occasional in tomato crops in England, and economically damaging in Continental celery crops, has not so far been reported in celery in Britain. British celery varieties tested were resistant to experimental infection with tomato aspermy virus. These three viruses were readily isolated, purified and identified.

Three viruses, designated celery yellow spot, celery yellow vein and celery ringspot, isolated from affected celery plants, were less readily transmitted from celery to celery, and could not be purified by techniques that succeed with many viruses.

Celery yellow spot was transmitted by sap-rubbing inoculation only when cucumber mosaic virus was also present ; it was tentatively identified with the celery yellow spot virus reported from the U.S.A. Celery ringspot virus showed some similarities to the poison hemlock ringspot virus found in the U.S.A., but none of the critical tests for virus identification could be applied to either of these two viruses. Celery yellow net virus differed from all the viruses so far recorded in celery. The chronic symptoms of the different viruses in celery were often similar, and therefore unreliable for diagnosis. The initial or “ shock ” symptoms, however, were usually distinctive.     

The carotenoid pigments of the date

The carotenoid pigments of the date (Phoenix dactylifera) were investigated in 2 soft cultivars, ‘Hayany’ and ‘Barhee’, and in the semi-dry ‘Deglet Noor’. The total carotenoid content in the ripe fruit was 10–12 μg/g fresh weight. The carotenoid pattern in all investigated fruits was that of the chloroplast type, with only slight differences between the cultivars. Changes in the carotenoid pattern during ripening were followed in ‘Hayany’ and ‘Deglet Noor’. The total carotenoid content decreased drastically from 36.3 to 21.2 μg/g dry weight in the soft dates, and from 23.2 to 12.0 μg/g dry weight in the semi-dry cultivar. The carotenoid degradation may be due primarily to the loss of moisture during maturation, and is probably unrelated to the gradual darkening of the ripening fruit. The pattern of the retained carotenoids remained essentially similar.The vitamin A values of the 3 cultivars, expressed as retinol equivalents, were calculated.     

常见桑科榕属植物叶片类胡萝卜素含量的比较分析

采用酒精-丙酮(1∶1)进行萃取,用分光光度法测定常见桑科榕属植物小叶榕、黄金榕和花叶榕的胡萝卜素、叶黄素和总类胡萝卜素的含量。结果表明,胡萝卜素含量为:小叶榕>黄金榕>花叶榕;叶黄素含量是:黄金榕>花叶榕>小叶榕;总类胡萝卜素含量为:小叶榕>花叶榕>黄金榕。可见,黄金榕叶黄素含量最高,小叶榕的最低,花叶榕的居中;而小叶榕的胡萝卜素含量最高,花叶榕的最低,黄金榕的居中。由于这3种植物各种色素的含量差异,使得这3种植物的叶片呈现出不同的颜色。     

‘中油桃9号’及其黄肉突变体花和叶片中类胡萝卜素代谢及相关基因的时空表达

以‘中油桃9号’及其黄肉芽变的花和叶片为试材,采用HPLC法对类胡萝卜素的积累水平进行定性和定量分析,实时荧光定量PCR法对类胡萝卜素代谢关键基因的表达水平进行分析。结果表明:在花萼中,黄肉突变体的总类胡萝卜素水平为‘中油桃9号’的5.7倍,其中的β–胡萝卜素为‘中油桃9号’的19.6倍;除DXS和LCY-E外,突变体花萼中无论是合成类胡萝卜素的基因,还是降解类胡萝卜素基因的表达水平均显著高于‘中油桃9号’。在花瓣中,两个材料的类胡萝卜素总量无显著差异,但各类胡萝卜素成分含量存在显著差异;突变体DXS、HDR、PDS、LCY-E、CHY-B和CCD1的表达量显著高于‘中油桃9号’,ZDS、NCED1和CCD4的表达量显著低于‘中油桃9号’。在叶片中,‘中油桃9号’和黄肉突变体均以β–胡萝卜素、紫黄质、9–顺式–紫黄质和叶黄质为主,幼果期时两个材料的类胡萝卜素总量无显著差异,果实成熟后突变体的类胡萝卜素总量呈上升趋势,显著高于‘中油桃9号’;自花后55～80 d,仅CCD4的表达模式与类胡萝卜素的积累一致,因而认为CCD4的表达差异是两个材料后期叶片类胡萝卜素含量差异的主要原因。     

胡萝卜根中主要类胡萝卜素含量相关QTL的精细定位

以白色的野生胡萝卜‘松滋野生’(Ws)和橘色的栽培胡萝卜品种‘Amsterdam’(Af)为亲本构建的回交重组自交系(BIL)为试材,基于低倍重测序技术开发SNP标记,构建了由1 976个Bin标记组成,包含29 435个SNP标记的遗传图谱。图谱总距离834.28 c M,平均图距0.42 c M。通过对胡萝卜肉质根中类胡萝卜素含量相关QTL分析,在连锁群LG04和LG08中检测到调控α–胡萝卜素、β–胡萝卜素、ζ–胡萝卜素、叶黄素、玉米黄质和总类胡萝卜素含量的主效QTL(M-QTL)2、2、3、2、2和2个,表型贡献率为11.47%~19.18%;另检测到调控α–胡萝卜素、β–胡萝卜素、ζ–胡萝卜素、玉米黄质和总类胡萝卜素含量的上位性QTL(E-QTL)1、1、2、1和1个,表型贡献率为2.50%~3.66%。在M-QTL显著区间内共检索到36个有功能注释的预测基因,其中Dck018297为ζ–胡萝卜素脱氢酶2基因,与调控β–胡萝卜素合成和总类胡萝卜素含量有关;Dck008006为乙烯响应因子2.2的同源基因,与调控α–胡萝卜素、ζ–胡萝卜素合成有关;Dck029898为转录因子b HLH135的同源基因,与调控玉米黄质合成有关。     

Development,and genetic and metabolic characterization of new tomato mutants with enhanced and deficient carotenoid content

Tomato (Solanum lycopersicum) is a valuable vegetable crop rich in health-protective carotenoids, but breeding improvements are limited by its narrow genetic diversity. New mutants with enhanced and deficient carotenoid content in a single genetic background of tomato cv. MicroTom were developed via chemical mutagenesis. Genetic and metabolic analyses showed that mutant DC260, which exhibited fruit color alteration from red to deep red interlaced with orange color, had significant (P < 0.05) increases of lycopene (up to 42.8%) and ß-carotene (up to 61.5%) compared with control plants. Pearson correlation analysis of M1 and M2 generations in DC260 revealed that fruit color alteration was significantly (P < 0.05) correlated with lycopene (coefficient = 0.55) and ß-carotene content (coefficient = 0.63). The fruit color alteration of DC260 was controlled by a single gene at a heterozygous locus. In contrast, mutant DC107 and DC624, which exhibited fruit color alteration from red to orange-yellow, was significantly (P < 0.05) carotenoid-deficient with up to 346.3-, 10.8-, and 185.2- fold reductions of lycopene, ß-carotene, and total carotenoids, respectively, compared with the control plants. Carotenoid deficiency in DC170 and DC624 was responsible for the fruit color alteration and was controlled by a dominant gene at a homozygous locus.