Agrobacterium-mediated transformation of the CrDAT gene and selection of transgenic periwinkle lines with a high vincristine accumulation

Catharanthus roseus contains vincristine and vinblastine, which are outstanding drugs for cancer. In the biosynthetic pathways of terpenoid indole alkaloids (TIAs) in C. roseus, deacetylvindoline 4-O-acetyltransferase (DAT) is a key enzyme that catalyses the last reaction of vindoline biosynthesis to form vinblastine and vincristine. In this study, the CrDAT transgene was transferred into the periwinkle by Agrobacterium-mediated transformation and generated transgenic periwinkle lines with an increase in vincristine accumulation. The C. roseus DAT gene was introduced into C. roseus plants and it was confirmed that CrDAT was successfully transferred into the genome of periwinkle plants and efficiently translated to synthesise recombinant DAT protein. Four transgenic periwinkle lines in T1 generation, T1-1, T1-3, T1-6, and T1-7, expressed recombinant DAT protein with the total protein content in the range of 2.86 μg.mg^?1 to 5.12 μg.mg^?1. Moreover, the vincristine contents of four transgenic lines increased by 1.63?2.48-fold compared to non-transgenic plants, ranging from 6.91 µg.g^?1 (fresh weight) to 10.53 µg.g^?1 (fresh weight). The T1-1 line had the highest vincristine content. Hence, the overexpression of the recombinant DAT protein can improve the vincristine accumulation of transgenic C. roseus plants.

Abbreviation: CrDAT - Catharanthus roseus Deacetylvindoline-4-O-Acetyl Transferase; D4H - Deacetoxyvindoline 4-hydroxylase; ELISA - Enzyme-Linked Immunosorbent Assay Monoterpene indole alkaloid; T0, T1 - Generations of transgenic plants; TIAs - Terpenoid indole alkaloids; WT- The wild-type tobacco plants (non transgenic plant); 35S - Cauliflower mosaic virus 35S promoter     

Are Phenological Observations Sufficient to Estimate the Quality of Seed Crops from a <Emphasis Type="Italic">Eucalyptus grandis</Emphasis> Open-pollinated Seed Orchard? Consequences for Seed Collections

Reproductive phenology was observed over three years in a Eucalyptus grandis seed orchard in Madagascar to determine the impact of geographic differences of parental selections on the expected genetic composition of seed crops. Pollen flow (observed pollen cloud) was deduced from a paternity assignment based on sampling from one year’s seed production. The two approaches were used to verify whether knowledge of phenology is sufficient to predict the genetic quality of the seeds collected. Despite the high flowering level, with a constant cycle over three years, the results demonstrated wide reproductive phenological differences associated with the parents’ origin, suggesting putative pollination disequilibrium. From both observed and expected pollen clouds, the results showed preferential mating among different provenances, which has consequences for seed crop composition. Phenological observations, which can be made inexpensively, can be used to promote methods of effective seed orchard management in order to improve the genetic quality of seeds.     

马达加斯加的热带森林

马达加斯加是非洲大陆的离岛。天然森林类型复杂多样。原生性的森林群落主要为常绿林，落叶林以及多刺、肉质的落叶灌木丛。在不同类型的岩石露头发育着岩生矮林；高山上可见山顶矮林，滨海湿地则为红树林。马达加斯加的森林有着独特的植物和动物种类，有着巨大的生物多样性。但由于人类长期对其生境的破坏，不少动物种类已处于濒危状态。到目前为止，60％－85％的森林植被已遭到破坏。非持续的农业轮作是森林植被完整性的主要威胁。在某些地区，火烧林地建立牧场业是引起森林面积减少的主要因素。     

Glomus mosseae bioprotection against aster yellows phytoplasma (16srI-B) and Spiroplasma citri infection in Madagascar periwinkle

The bio-control potential of arbuscular mycorrhizal fungus Glomus mosseae against two pathogenic microorganisms aster yellows (AY) phytoplasma and Spiroplasma citri has been examined in Madagascar periwinkle (Catharanthus roseus). G. mosseae had a positive influence on healthy C. roseus plants and S. citri infection. It provided bioprotection against S. citri pathogen and induced significant degree of resistance to spiroplasma infection. Besides, symptom expression significantly reduced and shoot height, leaves number, root fresh and dry weight increased in spiroplasma-infected plants treated with mycorrhiza fungus. Although, G. mosseae had no positive effect on phytoplasma disease. The root architectures were affected by the phytoplasma pathogen, and the root surface area dramatically decreased in G. mosseae treated AY-infected periwinkles compared with the control. Nitrogen and Phosphorus concentrations notably increased in spiroplasma + G. mosseae compared with control plants. Potassium concentration did not differ significantly in all mycorrhizal treated and untreated infected plants except in G. mosseae treated healthy plants. The spore density and root colonization rate did not vary in both pathogen treatments G. mosseae + spiroplasma and G. mosseae + phytoplasma. To our knowledge, this is the first report showing the bioprotective effect of G. mosseae on S. citri. The possible mechanisms involved in complex interaction between plants, cell wall-less bacteria and arbuscular mycorrhizal fungi (AMF) are discussed and the underlying mechanisms for the functioning of AMF are hypothesized.     

长春花感染柑橘黄龙病菌微波热处理防控效果

为探索黄龙病防治途径,开展微波热处理对黄龙病的防控效果研究。该研究搭建了微波热处理平台,对长春花微波热处理参数（转盘转速、单个磁控管的微波功率、磁控管数量）进行优化,并用优化后的参数组合对感病长春花进行热处理,处理后90 d内跟踪检测长春花叶片中黄龙病菌的浓度、淀粉和类黄酮的含量。结果表明,长春花微波热处理的最优参数组合为单个磁控管的微波功率150 W、3个磁控管、转速15 r/min;热处理90 d后,感病长春花叶片中的黄龙病菌Ct值（阈值循环数,该值低于32时为阳性）由阳性(17.01±0.97)转至近阴性（31.91±2.35）,病菌浓度下降了99.98%,淀粉含量和类黄酮含量恢复至正常水平,植株的活性恢复较好。研究表明,微波热处理有效抑制植物体内的黄龙病菌,可为黄龙病的规模化防治提供新思路。     

长春花ISSR-PCR 反应体系的正交优化

[目的]筛选最适的长春花ISSR-PCR反应体系。[方法]采用正交试验方法,对影响长春花ISSR-PCR反应体系的引物浓度、dNTP浓度、Mg2＋浓度、TaqDNA聚合酶浓度和模板DNA浓度5个因素在4个水平上进行正交试验,建立适合长春花ISSR-PCR的反应体系。[结果]长春花ISSR-PCR反应体系的最佳条件：引物浓度0.50μmol/L,dNTP浓度0.10 mmol/L,Mg2＋浓度3.00 mmol/L,Taq酶浓度0.75U/25μl,DNA模板浓度350 ng/25μl。采用该反应体系筛选出12对适合于长春花ISSR-PCR反应的引物。[结论]利用优化系统进行长春花的ISSR-PCR反应,可获得稳定性高、重复性好、背景清晰的电泳结果。     

杂交水稻在马达加斯加的推广前景

马达加斯加为印度洋岛国,粮食不能自给.为了解决马达加斯加的粮食危机,推广中国杂交水稻技术,实现粮食产量翻番的目标,介绍了马达加斯加水稻生产现状,并对在马达加斯加推广杂交水稻面临的机遇及困难进行了分析.     

PHYSIOLOGICAL AND BIOCHEMICAL CHANGES IN CATHARANTHUS ROSEUS L. IN RESPONSE TO BORON NUTRITION

Catharanthus roseus L., a medicinally important plant was grown till maturity at varying levels of boron (0.033, 0.066, 0.33 and 3.3 mg B L^?1) supply. Optimum yield was observed in plants receiving 0.33 mg B L^?1. Plants receiving deficient boron showed growth reduction and visual symptoms such as chlorosis and cupping of young emerging leaves and apical tip necrosis. The number and size of the flowers, pods and seeds formed and the pollen viability of the boron deficient plants was markedly reduced. The threshold values for deficiency and toxicity were 57 and 79 μg B g^?1 dry weight for vegetative growth and 60.4 and 68.9 μg B g^?1 dry weight for reproductive growth, respectively, and is reported for the first time in periwinkle. An increase was observed in reducing and non-reducing sugars and in activity of acid phosphatase and ribonuclease in boron stressed plants.     

Seasonal dependence of rooting success in cuttings from natural forest trees in Madagascar

Four ligneous species from the tropical forest in the east of Madagascar, with a proven or potentially high economic value, were subject to ‘low-tech’ vegetative propagation tests from stem cuttings. The species concerned were Aphloia theiformis, Ilex mitis, Prunus africana and Ravensara aromatica. The cuttings were three-node segments of stems on which one leaf was retained. All the species proved amenable to rooting. The maximum percentage of rooting ranged from 33% for P. africana to 60% for I. mitis. Rooting success was dependant on the season of cutting (high in the hot season, from October to May, and null in cold season). This study is the first successful attempt at propagating cuttings from Malagasy forest species. This result is of particular importance to P. africana, threatened by destructive exploitation in Madagascar. It goes a step further in the domestication of this species by demonstrating the ability of cutting from 10 year old ortets collected in natural forest to root as it offers the possibility of a reliable and effective method of reintroduction for the species in overexploited zones.