Strategies for transgenic nematode control in developed and developing world crops

Nematodes cause an estimated $118b annual losses to world crops and they are not readily controlled by pesticides or other control options. For many crops natural resistance genes are unavailable to plant breeders or progress by this approach is slow. Transgenic plants can provide nematode resistance for such crops. Two approaches have been field trialled that control a wide range of nematodes by either limiting use of their dietary protein uptake from the crop or by preventing root invasion without a direct lethality. In addition, RNA interference increasingly in tandem with genomic studies is providing a range of potential resistance traits that involve no novel protein production. Transgenic resistance can be delivered by tissue specific promoters to just root tissues where most economic nematodes invade and feed rather than the harvested yield. High efficacy and durability can be provided by stacking nematode resistance traits including any that natural resistance provides. The constraints to uptake centre on market acceptance and not the availability of appropriate biotechnology. The need to deploy nematode resistance is intensifying with loss of pesticides, an increased need to protect crop profit margins and in many developing world countries where nematodes severely damage both commodity and staple crops.     

Engineering photoassimilate partitioning in tobacco plants improves growth and productivity and provides pathogen resistance

Expression of pathogenesis-related (PR) genes is part of the plant's natural defense response against pathogen attack. To study the in vivo role and function of the maize PRms protein, tobacco plants were transformed with the PRms cDNA under the control of the CaMV35S promoter. Transgenic tobacco plants grow faster and yield more leaf and seed biomass. By using immunoelectron microscopy, we found that PRms is associated with plasmodesmata in leaves of transgenic tobacco plants. Furthermore, we found that activation of sucrose efflux from photosynthetically active leaves and accumulation of higher levels of sucrose in leaf tissues are characteristic features of PRms tobacco plants. This, in turn, results in the constitutive expression of endogenous tobacco PR genes and resistance to phytopathogens. The expression of multiple plant defense genes can then be achieved by using a single transgene. These data provide a new approach for engineering disease-resistant plants while simultaneously improving plant yield and productivity through the modification of photoassimilate partitioning.     

融合基因在转基因植物中的应用

目前双基因和多基因转基因植物已经商品化,并展现了广泛的应用前景。但在转基因植物研究中,使多个基因同时在植物体中表达调控依然很难实现,是植物基因工程和生物技术发展中的难点。融合基因表达载体作为一种新型的方法,弥补了获得双价或多价转基因植物传统方法的缺点,具有更高的应用价值。本文对目前构建融合基因的方法作了评述,并对比较新颖的连接肽2A和LP4做了详细介绍。     

植物转基因沉默与消除

植物基因工程研究是希望获得高稳定表达的转基因植株,而转基因沉默现象却限制了转基因植物的应用前景。基因沉默的机制是多方面的,包括转基因多拷贝之间的异位配对,转基因序列的甲基化,插入位点在染色体结构上的改变及转录后的衰退调控等。研究外源基因的失活原因及寻找相应的策略控制失活,对于植物基因工程的发展有着重要的意义。     

植物转基因沉默与消除

植物基因工程研究是希望获得高稳定表达的转基因植株,而转基因沉默现象却限制了转基因植物的应用前景,基因沉默的机制是多方面的,包括转基因多拷贝之间的异位配对,转基因序列的甲基化,插入位点在染色体结构上的改变及转录后的衰退调控等,研究外源基因的失活原因及寻找相应的策略控制失活,对于植物基因工程的发展有着重要的意义。     

Transgenic organisms in evolutionary ecology

Evolutionary ecology aims to understand how phenotypes are designed for reproductive success and survival. Perhaps the most powerful approach towards this goal is to alter a character genetically and observe the resulting change in reproduction, survival, growth, defense or competitive ability. Until recently, this strategy was not practical. Transgenic manipulation now offers a solution - novel genes are introduced into the germ line and are then expressed in the developing organism. This technique is already available in model and agricultural organisms. The challenge for molecular evolutionary ecologists is to find ways to adopt these powerful systems to understand the mechanisms underlying adaptive traits and their evolution.     

Plastid transformation in the monocotyledonous cereal crop, rice (Oryza sativa) and transmission of transgenes to their progeny

The plastid transformation approach offers a number of unique advantages, including high-level transgene expression, multi-gene engineering, transgene containment, and a lack of gene silencing and position effects. The extension of plastid transformation technology to monocotyledonous cereal crops, including rice, bears great promise for the improvement of agronomic traits, and the efficient production of pharmaceutical or nutritional enhancement. Here, we report a promising step towards stable plastid transformation in rice. We produced fertile transplastomic rice plants and demonstrated transmission of the plastid-expressed green fluorescent protein (GFP) and aminoglycoside 3'-adenylyltransferase genes to the progeny of these plants. Transgenic chloroplasts were determined to have stably expressed the GFP, which was confirmed by both confocal microscopy and Western blot analyses. Although the produced rice plastid transformants were found to be heteroplastomic, and the transformation efficiency requires further improvement, this study has established a variety of parameters for the use of plastid transformation technology in cereal crops.     

Genome wide association studies for milk production traits in Chinese Holstein population

Genome-wide association studies (GWAS) based on high throughput SNP genotyping technologies open a broad avenue for exploring genes associated with milk production traits in dairy cattle. Motivated by pinpointing novel quantitative trait nucleotide (QTN) across Bos Taurus genome, the present study is to perform GWAS to identify genes affecting milk production traits using current state-of-the-art SNP genotyping technology, i.e., the Illumina BovineSNP50 BeadChip. In the analyses, the five most commonly evaluated milk production traits are involved, including milk yield (MY), milk fat yield (FY), milk protein yield (PY), milk fat percentage (FP) and milk protein percentage (PP). Estimated breeding values (EBVs) of 2,093 daughters from 14 paternal half-sib families are considered as phenotypes within the framework of a daughter design. Association tests between each trait and the 54K SNPs are achieved via two different analysis approaches, a paternal transmission disequilibrium test (TDT)-based approach (L1-TDT) and a mixed model based regression analysis (MMRA). In total, 105 SNPs were detected to be significantly associated genome-wise with one or multiple milk production traits. Of the 105 SNPs, 38 were commonly detected by both methods, while four and 63 were solely detected by L1-TDT and MMRA, respectively. The majority (86 out of 105) of the significant SNPs is located within the reported QTL regions and some are within or close to the reported candidate genes. In particular, two SNPs, ARS-BFGL-NGS-4939 and BFGL-NGS-118998, are located close to the DGAT1 gene (160bp apart) and within the GHR gene, respectively. Our findings herein not only provide confirmatory evidences for previously findings, but also explore a suite of novel SNPs associated with milk production traits, and thus form a solid basis for eventually unraveling the causal mutations for milk production traits in dairy cattle.     

Advances in Transgenic Rice Biotechnology

Rice is the most amenable crop plant for genetic manipulation amongst monocots due to its small genome size, enriched genetic map, availability of entire genome sequence, and relative ease of transformation. Improvement in agronomic traits of rice is bound to affect a sizeable population since it is a primary source of sustenance. Recent advances like use of ‘clean gene’ technology or matrix attachment regions would help improve rice transformation. Function of several novel genes and their promoters has been analyzed in transgenic rice. Significant progress has been made in introducing traits like herbicide, biotic stress and abiotic stress tolerance. Attempts also have been made to enhance nutritional characteristics of the grain and yield. Identification of genes controlling growth and development can be used to modify plant architecture and heading period. Transgenic rice can serve as a biofactory for the production of molecules of pharmaceutical and industrial utility. The drive to apply transgenic rice for public good as well as commercial gains has fueled research to an all time high. Successful field trials and biosafety of transgenic rice have been reported. This would act as a catalyst for greater acceptance of genetically modified food crops. The lessons learnt from rice can be extended to other cereals thereby opening new opportunities and possibilities.     

Systems metabolic engineering: Genome-scale models and beyond

The advent of high throughput genome-scale bioinformatics has led to an exponential increase in available cellular system data. Systems metabolic engineering attempts to use data-driven approaches – based on the data collected with high throughput technologies – to identify gene targets and optimize phenotypical properties on a systems level. Current systems metabolic engineering tools are limited for predicting and defining complex phenotypes such as chemical tolerances and other global, multigenic traits. The most pragmatic systems-based tool for metabolic engineering to arise is the in silico genome-scale metabolic reconstruction. This tool has seen wide adoption for modeling cell growth and predicting beneficial gene knockouts, and we examine here how this approach can be expanded for novel organisms. This review will highlight advances of the systems metabolic engineering approach with a focus on de novo development and use of genome-scale metabolic reconstructions for metabolic engineering applications. We will then discuss the challenges and prospects for this emerging field to enable model-based metabolic engineering. Specifically, we argue that current state-of-the-art systems metabolic engineering techniques represent a viable first step for improving product yield that still must be followed by combinatorial techniques or random strain mutagenesis to achieve optimal cellular systems.     

Flower Color Modification by Engineering of the Flavonoid Biosynthetic Pathway: Practical Perspectives

The status quo of flavonoid biosynthesis as it relates to flower color is reviewed together with a success in modifying flower color by genetic engineering. Flavonoids and their colored class compounds, anthocyanins, are major contributors to flower color. Many plant species synthesize limited kinds of flavonoids, and thus exhibit a limited range of flower color. Since genes regulating flavonoid biosynthesis are available, it is possible to alter flower color by overexpressing heterologous genes and/or down regulating endogenous genes. Transgenic carnations and a transgenic rose that accumulate delphinidin as a result of expressing a flavonoid 3′,5′-hydroxylase gene and have novel blue hued flowers have been commercialized. Transgenic Nierembergia accumulating pelargonidin, with novel pink flowers, has also been developed. Although it is possible to generate white, yellow, and pink-flowered torenia plants from blue cultivars by genetic engineering, field trial observations indicate difficulty in obtaining stable phenotypes.     

鱼和熊掌的选择:反向重复序列变异介导的玉米环境适应与产量平衡

作物育种的目标是找到产量和抗性的最佳平衡点,其中涉及“鱼和熊掌”二者兼得的选择策略。哪些逆境负调控位点影响产量性状,以及如何调控等是突破育种瓶颈的重要科学问题。近百年来,高产玉米(Zeamays)育种使玉米单产不断提高,同时现代玉米品种对干旱的敏感性也呈现出增强趋势,故而存在高产稳产的潜在风险。可对于这一现象背后确切的遗传机制却知之甚少,从而限制了既高产又高抗玉米新品种的培育。玉米的非生物胁迫抗性与产量性状均为多基因控制的复杂数量性状,涉及全基因组范围内大量基因的表达与调控。玉米基因组内存在大量的小RNA (sRNA),其对基因表达起精细调控作用,但人们对sRNA调控作物环境胁迫应答与产量性状机制的理解仍然有限。近日,华中农业大学代明球课题组与李林和李峰两个课题组合作,基于对338份玉米关联群体在不同环境下的sRNA表达组分析,鉴定到大量干旱应答的sRNA,以及调控这些sRNA表达的遗传位点(eQTL);并克隆了8号染色体上1个干旱特异性eQTL热点DRESH8。生物信息学分析显示, DRESH8是1个由转座子组成的长度约为21.4 kb的反向重复序列(TE-IR)。DRESH8通过产...     

Genetic basis of yield as viewed from a crop physiologist's perspective

The final yield of a crop is the product of growth during the growing season and a number of developmental processes occurring throughout the life cycle of a crop, with most genes influencing the final outcome to a degree. However, recent advances in molecular biology have developed the potential to identify and map many genes or QTLs related to various important traits, including yield, plant adaptation and tolerance to stresses. Significant G×E interactions for yield have been identified, as have interactions associated with QTLs for yield. However, there is little evidence available to confirm that a QTL for yield from a parental line in one mapping population may improve yield when transferred into an adapted, high‐yielding line of another population. In order to narrow the apparent gap between the genotype and the phenotype with regard to yield, it is important to identify key traits related to yield and then attempt to identify and locate the genes controlling them. The partitioning of the developmental time to anthesis into different phases: from sowing to the onset of stem elongation and from then to anthesis, as a relatively simple physiological attribute putatively related to yield, is discussed. If the relationship holds in a wider range of conditions and the genetic factors responsible are located then the genetic basis of yield should be identified. There has also been significant progress in crop simulation modelling. Using knowledge of crop physiology and empirical relationships these models can simulate the performance of crops, including the G×E interactions. Such models require information regarding the genetic basis of yield, which are included in the form of genetic coefficients. Essentially models are constructed as decision‐making tools for management but may be of use in detecting prospective traits for selection within a breeding programme. Problems associated with this approach are discussed. This review discusses the need to use crop physiology approaches to analyse components of yield in order to reliably identify the genetic basis of yield.     

Genetic engineering of a mouse: Dr. Frank Ruddle and somatic cell genetics

Genetic engineering is the process of modifying an organism's genetic composition by adding foreign genes to produce desired traits or evaluate function. Dr. Jon W. Gordon and Sterling Professor Emeritus at Yale Dr. Frank H. Ruddle were pioneers in mammalian gene transfer research. Their research resulted in production of the first transgenic animals, which contained foreign DNA that was passed on to offspring. Transgenic mice have revolutionized biology, medicine, and biotechnology in the 21st century. In brief, this review revisits their creation of transgenic mice and discusses a few evolving applications of their transgenic technology used in biomedical research.     

Genetic transformation of cowpea (Vigna unguiculata L.) and stable transmission of the transgenes to progeny

Cowpeas are nutritious grains that provide the main source of protein, highly digestible energy and vitamins to some of the world's poorest people. The demand for cowpeas is high but yields remain critically low, largely because of insect pests. Cowpea germplasm contains little or no resistance to major insect pests and a gene technology approach to adding insect protection traits is now a high priority. We have adapted features of several legume and other transformation systems and reproducibly obtained transgenic cowpeas that obey Mendelian rules in transmitting the transgene to their progeny. Critical parameters in this transformation system include the choice of cotyledonary nodes from developing or mature seeds as explants and a tissue culture medium devoid of auxins in the early stages, but including the cytokinin BAP at low levels during shoot initiation and elongation. Addition of thiol-compounds during infection and co-culture with Agrobacterium and the choice of the bar gene for selection with phosphinothricin were also important. Transgenic cowpeas that transmit the transgenes to their progeny can be recovered at a rate of one fertile plant per thousand explants. These results pave the way for the introduction of new traits into cowpea and the first genes to be trialled will include those with potential to protect against insect pests.     

Prospects of genetic engineering of plants for phytoremediation of toxic metals

Bioremediation is gaining a lot of importance in recent times as an alternate technology for removal of elemental pollutants in soil and water, which require effective methods of decontamination. Phytoremediation--the use of green plants to remove, contain or render harmless environmental pollutants--may offer an effective, environmentally nondestructive and cheap remediation method. The use of genetic engineering to modify plants for metal uptake, transport and sequestration may open up new avenues for enhancing efficiency of phytoremediation. Metal chelator, metal transporter, metallothionein (MT), and phytochelatin (PC) genes have been transferred to plants for improved metal uptake and sequestration. Transgenic plants, which detoxify/accumulate cadmium, lead, mercury, arsenic and selenium have been developed. A better understanding of the mechanisms of rhizosphere interaction, uptake, transport and sequestration of metals in hyperaccumulator plants will lead to designing novel transgenic plants with improved remediation traits. As more genes related to metal metabolism are discovered, facilitated by the genome sequencing projects, new vistas will be opened up for development of efficient transgenic plants for phytoremediation.     

The intragenic approach as a new extension to traditional plant breeding

The novel intragenic approach to genetic engineering improves existing varieties by eliminating undesirable features and activating dormant traits. It transforms plants with native expression cassettes to fine-tune the activity and/or tissue specificity of target genes. Any intragenic modification of traits could, at least in theory, also be accomplished by traditional breeding and transgenic modification. However, the new approach is unique in avoiding the transfer of unknown or foreign DNA. By consequently eliminating various potential risk factors, this method represents a relatively safe approach to crop improvement. Therefore, we argue that intragenic crops should be cleared through the regulatory process in a timely and cost-effective manner.     

Genetically pyramiding protease-inhibitor genes for dual broad-spectrum resistance against insect and phytopathogens in transgenic tobacco

Protease inhibitors provide a promising means of engineering plant resistance against attack by insects and pathogens. Sporamin (trypsin inhibitor) from sweet potato and CeCPI (phytocystatin) from taro were stacked in a binary vector, using pMSPOA (a modified sporamin promoter) to drive both genes. Transgenic tobacco lines of T0 and T1 generation with varied inhibitory activity against trypsin and papain showed resistance to both insects and phytopathogens. Larvae of Helicoverpa armigera that ingested tobacco leaves either died or showed delayed growth and development relative to control larvae. Transgenic tobacco-overexpressing the stacked genes also exhibited strong resistance against bacterial soft rot disease caused by Erwinia carotovora and damping-off disease caused by Pythium aphanidermatum. Thus, stacking protease-inhibitor genes, driven by the wound and pathogen responsive pMSPOA promoter, is an effective strategy for engineering crops to resistance against insects and phytopathogens.     

Inducing drought tolerance in plants: Recent advances

Undoubtedly, drought is one of the prime abiotic stresses in the world. Crop yield losses due to drought stress are considerable. Although a variety of approaches have been used to alleviate the problem of drought, plant breeding, either conventional breeding or genetic engineering, seems to be an efficient and economic means of tailoring crops to enable them to grow successfully in drought-prone environments. During the last century, although plant breeders have made ample progress through conventional breeding in developing drought tolerant lines/cultivars of some selected crops, the approach is, in fact, highly time-consuming and labor- and cost-intensive. Alternatively, marker-assisted breeding (MAB) is a more efficient approach, which identifies the usefulness of thousands of genomic regions of a crop under stress conditions, which was, in reality, previously not possible. Quantitative trait loci (QTL) for drought tolerance have been identified for a variety of traits in different crops. With the development of comprehensive molecular linkage maps, marker-assisted selection procedures have led to pyramiding desirable traits to achieve improvements in crop drought tolerance. However, the accuracy and preciseness in QTL identification are problematic. Furthermore, significant genetic × environment interaction, large number of genes encoding yield, and use of wrong mapping populations, have all harmed programs involved in mapping of QTL for high growth and yield under water limited conditions. Under such circumstances, a transgenic approach to the problem seems more convincing and practicable, and it is being pursued vigorously to improve qualitative and quantitative traits including tolerance to biotic and abiotic stresses in different crops. Rapid advance in knowledge on genomics and proteomics will certainly be beneficial to fine-tune the molecular breeding and transformation approaches so as to achieve a significant progress in crop improvement in future. Knowledge of gene regulation and signal transduction to generate drought tolerant crop cultivars/lines has been discussed in the present review. In addition, the advantages and disadvantages as well as future prospects of each breeding approach have also been discussed.