Selectable marker genes from plants: reliability and potential

Selectable marker genes (SMGs) are still useful to efficiently obtain transgenic plants, although marker-free techniques are available, but with limitations. The presence of SMGs, especially bacterial antibiotic resistance genes, in transgenic crops is criticized. Fortunately, several genes isolated from plants are available that can serve as SMGs. Here, I review the plant genes reported to have been used as SMGs. Some are wild-type genes that, when overexpressed, confer a selective advantage during in vitro plant regeneration, whereas some are mutated genes encoding enzymes resistant to inhibitory chemicals. Most of the genes have not yet been tested in a significant number of species. The effect of SMGs expression on the phenotype has often been superficially examined and should be better characterized. The sequence conservation of some SMGs could allow derivation of a SMGs from any plant species, if an intragenic or cisgenic approach to genetic engineering is preferred. I conclude that several promising SMGs have been isolated from plants, allowing avoidance of bacterial genes for transformation, transgene stacking, and intragenic or cisgenic engineering approaches. Nonetheless, further testing in more plant species would be useful to fully assess phenotypic neutrality, efficiency, and versatility. Patent rights restrict the immediate use of most plant SMGs for commercial applications, but freely available marker systems do exist.     

Mercury detoxification with transgenic plants and other biotechnological breakthroughs for phytoremediation

Summary Phytoremediation, or the use of plants for removal and detoxification of environmental pollutants, has garnered great attention in recent years. This heightened interest is both scientifically, due the fascinating processes utilized by plants for tolerance and removal of harmful compounds, and commercially, as plants represent a more environmentally compatible and less expensive method of site remediation compared to standard approaches. The majority of phytoremediation studies have been with naturally occurring plant species after empirical discovery of their exceptional abilities for such applications. This has led to a growing body of literature and wider acceptance for plants in many aspects of environmental rehabilitation. However, this has occurred with little understanding of their basic biological mechanisms of action or investigation of alternative strategies for enhancing the capabilities of these extraordinary plants. Better understanding of plant physiology, biochemistry and molecular biology in response to specific contaminants is critical for optimization and advancement of phytoremediation. By applying the tools of biotechnology, the potential for plants as an aggressive method of environmental decontamination may be realized. This paper will serve as an introduction to the first Symposium assembled exclusively to review the use of molecular genetic and biotechnological methods for improvement of plants for phytoremediation. After a brief review of the other invited speakers' works (with more extensive papers following), the pioneering work using bacterial genes expressed in plants for removal of mercurial compounds will be surveyed.     

Assessment of potential indigenous plant species for the phytoremediation of arsenic-contaminated areas of Bangladesh

Soil and water contaminated with arsenic (As) pose a major environmental and human health problem in Bangladesh. Phytoremediation, a plant-based technology, may provide an economically viable solution for remediating the As-polluted sites. The use of indigenous plants with a high tolerance and accumulation capacity for As may be a very convenient approach for phytoremediation. To assess the potential of native plant species for phytoremediation, plant and soil samples were collected from four As-contaminated (groundwater) districts in Bangladesh. The main criteria used for selecting plants for phytoremediation were high bioconcentration factors (BCFs) and translocation factors (TFs) of As. From the results of a screening of 49 plant species belonging to 29 families, only one species of fern (Dryopteris filix-mas), three herbs (Blumea lacera, Mikania cordata, and Ageratum conyzoides), and two shrubs (Clerodendrum trichotomum and Ricinus communis) were found to be suitable for phytoremediation. Arsenic bioconcentration and translocation factors > 1 suggest that these plants are As-tolerant accumulators with potential use in phytoextraction. Three floating plants (Eichhornia crassipes, Spirodela polyrhiza, and Azolla pinnata) and a common wetland weed (Monochoria vaginalis) also showed high BCF and TF values; therefore, these plants may be promising candidates for cleaningup As-contaminated surface water and wetland areas. The BCF of Oryza sativa, obtained from As-contaminated districts was > 1, which highlights possible food-chain transfer issues for As-contaminated areas in Bangladesh.     

Phytoremediation of organic contaminants in soils

Soil pollution, a very important environmental problem, has been attracting considerable public attention over the last decades. Unfortunately, the enormous costs associated with the removal of pollutants from soils by means of traditional physicochemical methods have been encouraging companies to ignore the problem. Phytoremediation is an emerging technology that uses plants to clean up pollutants in the environment. As overwhelmingly positive results have become available regarding the ability of plants to degrade certain organic compounds, more and more people are getting involved in the phytoremediation of organic contaminants. Phytoremediation of organics appears a very promising technology for the removal of these contaminants from polluted sites.     

Transgenic plants for enhanced biodegradation and phytoremediation of organic xenobiotics

Phytoremediation — the use of plants to clean up polluted soil and water resources — has received much attention in the last few years. Although plants have the inherent ability to detoxify xenobiotics, they generally lack the catabolic pathway for the complete degradation of these compounds compared to microorganisms. There are also concerns over the potential for the introduction of contaminants into the food chain. The question of how to dispose of plants that accumulate xenobiotics is also a serious concern. Hence the feasibility of phytoremediation as an approach to remediate environmental contamination is still somewhat in question. For these reasons, researchers have endeavored to engineer plants with genes that can bestow superior degradation abilities. A direct method for enhancing the efficacy of phytoremediation is to overexpress in plants the genes involved in metabolism, uptake, or transport of specific pollutants. Furthermore, the expression of suitable genes in root system enhances the rhizodegradation of highly recalcitrant compounds like PAHs, PCBs etc. Hence, the idea to amplify plant biodegradation of xenobiotics by genetic manipulation was developed, following a strategy similar to that used to develop transgenic crops. Genes from human, microbes, plants, and animals are being used successfully for this venture. The introduction of these genes can be readily achieved for many plant species using Agrobacterium tumefaciens-mediated plant transformation or direct DNA methods of gene transfer. One of the promising developments in transgenic technology is the insertion of multiple genes (for phase 1 metabolism (cytochrome P450s) and phase 2 metabolism (GSH, GT etc.) for the complete degradation of the xenobiotics within the plant system. In addition to the use of transgenic plants overexpressed with P450 and GST genes, various transgenic plants expressing bacterial genes can be used for the enhanced degradation and remediation of herbicides, explosives, PCBs etc. Another approach to enhancing phytoremediation ability is the construction of plants that secrete chemical degrading enzymes into the rhizosphere. Recent studies revealed that accelerated ethylene production in response to stress induced by contaminants is known to inhibit root growth and is considered as major limitation in improving phytoremediation efficiency. However, this can be overcome by the selective expression of bacterial ACC deaminase (which regulates ethylene levels in plants) in plants together with multiple genes for the different phases of xenobiotic degradation. This review examines the recent developments in use of transgenic-plants for the enhanced metabolism, degradation and phytoremediation of organic xenobiotics and its future directions.     

Marker-free transformation: increasing transformation frequency by the use of regeneration-promoting genes

The generation of transgenic plants free of antibiotic resistance markers is a major challenge to plant biologists and plant breeders. Currently, there are two main strategies to achieve this goal: one approach is to excise or segregate marker genes from the host genome after regeneration of transgenic plants, and the second is based on so-called 'marker-free' transformation. Marker-free transformation has been successfully demonstrated by the use of several plant and non-plant genes that are capable of promoting explant regeneration. This approach appears not only to be effective for the generation of marker-free transgenic plants, but also has great potential to improve the transformation frequency of recalcitrant species.     

花卉植物应用于污染土壤修复的可行性研究

植物修复是解决污染土壤问题的有效途径之一,而已报道的超积累植物的种类非常有限.如果能从物种繁多的花卉植物中筛选出修复植物,不但能够弥补这一不足,而且还能在美化环境的同时,产生一定的经济效益.从植物修复的重要性和修复植物的筛选出发,概括了修复植物的判断标准及基本特征.通过描述花卉植物资源及其在环境保护中的作用,列举花卉植物与其它植物相比的优势,分析花卉植物的耐性、积累性和修复类型,探讨花卉植物应用于污染土壤修复实践的可行性.从花卉中筛选超积累植物,将为污染土壤的修复工作提供的生物材料.     

Analysis of differentially expressed genes in abiotic stress response and their role in signal transduction pathways

The study of abiotic stress response of plants is important because they have to cope with environmental changes to survive. The plant genomes have evolved to meet environmental challenges. Salt, temperature, and drought are the main abiotic stresses. The tolerance and response to stress vary differently in plants. The idea was to analyze the genes showing differential expression under abiotic stresses. There are many pathways connecting the perception of external stimuli to cellular responses. In plants, these pathways play an important role in the transduction of abiotic stresses. In the present study, the gene expression data have been analyzed for their involvement in different steps of signaling pathways. The conserved genes were analyzed for their role in each pathway. The functional annotations of these genes and their response under abiotic stresses in other plant species were also studied. The enzymes of signal pathways, showing similarity with conserved genes, were analyzed for their role in different abiotic stresses. Our findings will help to understand the expression of genes in response to various abiotic stresses. These genes may be used to study the response of different abiotic stresses in other plant species and the molecular basis of stress tolerance.     

Genetic transformation technology: Status and problems

Summary Transfer of genes from heterologous species provides the means of selectively introducing new traits into crop plants and expanding the gene pool beyond what has been available to traditional breeding systems. With the recent advances in genetic engineering of plants, it is now feasible to introduce into crop plants, genes that have previously been inaccessible to the conventional plant breeder, or which did not exist in the crop of interest. This holds a tremendous potential for the genetic enhancement of important food crops. However, the availability of efficient transformation methods to introduce foreign DNA can be a substantial barrier to the application of recombinant DNA methods in some crop plants. Despite significant advances over the past decades, development of efficient transformation methods can take many years of painstaking research. The major components for the development of transgenic plants include the development of reliable tissue culture regeneration systems, preparation of gene constructs and efficient transformation techniques for the introduction of genes into the crop plants, recovery and multiplication of transgenic plants, molecular and genetic characterization of transgenic plants for stable and efficient gene expression, transfer of genes to elite cultivars by conventional breeding methods if required, and the evaluation of transgenic plants for their effectiveness in alleviating the biotic and abiotic stresses without being an environmental biohazard. Amongst these, protocols for the introduction of genes, including the efficient regeneration of shoots in tissue cultures, and transformation methods can be major bottlenecks to the application of genetic transformation technology. Some of the key constraints in transformation procedures and possible solutions for safe development and deployment of transgenic plants for crop improvement are discussed.     

Factors influencing <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of monocotyledonous species

Summary Since the success of Agrobacterium-mediated transformation of rice in the early 1990s, significant advances in Agrobacterium-mediated transformation of monocotyledonous plant species have been achieved. Transgenic plants obtained via Agrobacterium-mediated transformation have been regenerated in more than a dozen monocotyledonous species, ranging from the most important cereal crops to ornamental plant species. Efficient transformation protocols for agronomically important cereal crops such as rice, wheat, maize, barley, and sorghum have been developed and transformation for some of these species has become routine. Many factors influencing Agrobacterium-mediated transformation of monocotyledonous plants have been investigated and elucidated. These factors include plant genotype, explant type, Agrobacterium strain, and binary vector. In addition, a wide variety of inoculation and co-culture conditions have been shown to be important for the transformation of monocots. For example, antinecrotic treatments using antioxidants and bactericides, osmotic treatments, desiccation of explants before or after Agrobacterium infection, and inoculation and co-culture medium compositions have influenced the ability to recover transgenic monocols. The plant selectable markers used and the promoters driving these marker genes have also been recognized as important factors influencing stable transformation frequency. Extension of transformation protocols to elite genotypes and to more readily available explants in agronomically important crop species will be the challenge of the future. Further evaluation of genes stimulating plant cell division or T-DNA integration, and genes increasing competency of plant cells to Agrobacterium, may increase transformation efficiency in various systems. Understanding mechanisms by which treatments such as desiccation and antioxidants impact T-DNA delivery and stable transformation will facilitate development of efficient transformation systems.     

Genetic Engineering of Plants to Enhance Selenium Phytoremediation

Referee: Dr. Dean A. Martens, USDAARS Southwest Watershed, Research Center, 200 E. Allen Road, Tucson, AZ 85719 Phytoremediation is the use of plants to remove, contain, or render harmless environmental pollutants. In recent years, much attention has been focused on the improvement of such technologies for this purpose. In this review, we introduce selenium phytoremediation and describe the attempts made to enhance it through genetic engineering. Initial efforts have taken advantage of the knowledge of the enzymatic pathways for selenium assimilation and volatilization, especially by overexpressing genes of rate-limiting enzymes in plants. Another possible approach is to introduce additional metabolic pathways from selenium hyperaccumulators or organisms other than plants that can help detoxify selenium compounds. In this way the capacity of plants to take up, accumulate, and volatilize compounds can be increased beyond that of any naturally occurring plant species. Here we report on the progress that has been made in overexpressing potentially important enzymes involved in the selenium/sulfur pathways and discuss possible future directions in the enhancement of phytoremediation through genetic engineering.     

Plant mutant tubulin genes as marker selective genes for genetic engineering

The approaches for new marker genes usage in selection of transformed plant cells, which are based on using mutant tubulin genes from natural plant biotypes and, in perspective, from induced plant mutants have been considered. The results of investigations of plant (biotypes, mutants) resistance to herbicides with antimicrotubular mode of action on molecular and cellular levels have been summarized. The reports dealing with study the transferring and the expression of mutant tubulin genes conferring resistance to amiprophosmethyl (phosphorothioamidate herbicide) and to trifluralin (dinitroaniline herbicide) from corresponding N. plumbaginifolia mutants into related and remote plant species by somatic hybridisation methods have been analyzed. The results of experiments on monocotyledonous and dicotyledonous. plant transformation by mutant alpha-tubulin gene conferring resistance to dinitroanilines are described to test the possibility of its using as a marker gene with obtaining, at the same time, a dinitroaniline-resistant plants.     

Microalgae engineering toolbox: Selectable and screenable markers

Engineering microalgae has opened a new era for plant biologists and biotechnologists. Microalgae had been proved as a promising candidate for the production of biopharmaceuticals, nutraceuticals, antioxidants, antimicrobial and antiviral compounds, in dyeing and food industry as well for biofuel production. Genetic transformation of some important microalgae has been successful, but several other potential microalgae species still need scientific attention. The success of the genetic transformation depends mainly on the utilization of the selectable and screenable markers. Like for other higher crop plants, several useful markers have been reported for microalgae transformation. In this follow-up, we compared different marker genes for genetic engineering of approximately all the industrially important microalgae. We have discussed the expression host, the targeted genome, appropriate selection agent, as well as the transformation method. Genetic transformation is an expensive and labor intensive process and this review will aid to shorten the time span by providing a database of appropriate markers for microalgae research which could serve as a guide for those involved in the genetic engineering of microalgae.     

基因工程改良植物重金属抗性与富集能力的研究进展

基于分子水平上对植物吸收、解毒、忍耐以及超富集重金属的几个关键步骤的认识,以及一些功能基因相继在细菌、酵母、植物和动物中被分离、鉴定,近年来,人们利用转基因技术提高植物重金属抗性和富集能力方面已获得进展, 一些功能基因（如gsh1, MerA和ArsC)及其工程植物已显示出植物修复产业化潜力。特别对转基因技术所采取的分子生物学途径、达到的效果以及存在的问题进行了详述,对今后研究的重点和策略进行了探讨。     

Genetic transformation in higher plants

Successful transformation of plant cells has been obtained utilizing vectors and DNA delivery methods derived from the plant pathogen, Agrobacterium tumefaciens. This soil bacterium is capable of transferring a DNA segment (T‐DNA), located between specific nucleotide border sequences, from its large tumor inducing (Ti) plasmid into the nuclear DNA of infected plant cells. The exploitation of the Agrobacterium/Ti plasmid system for plant cell transformation has been facilitated by (1) the construction of modified Agrobacterium strains in which the genes responsible for pathogenicity have been deleted; (2) the design of intermediate vectors containing selectable drug markers for introducing foreign genes into the Ti plasmid and subsequently into plant cells; and (3) the development of efficient in vitro methods for transforming plant cells and tissues with engineered Agrobacterium strains. These modifications have led to the development of a simple, efficient, and reproducible transformation system from which morphologically normal transformed plants can be readily regenerated. The foreign genes are stably maintained and expressed in the resulting plants and are inherited by progeny as typical Mendelian traits. The availability of transformation systems has already facilitated numerous studies on gene expression and regulation in plants and should eventually allow for the modification of various crop species in an agronomically significant manner. The needs and possibilities for the development of alternate vectors and transformation procedures will be discussed.     

Developing salt tolerant plants in a new century: a molecular biology approach

Soil salinity is a major abiotic stress in plant agriculture strongly, influencing plant productivity world-wide. Classical breeding for salt tolerance in crop plants has been attempted to improve field performance without success. Therefore, an alternative strategy is to generate salt tolerant plants through genetic engineering. Several species and experimental approaches have been used in order to identify those genes that are important for salt tolerance. Due to high level of salt tolerance, halophytes are good candidates to identify salt tolerance genes. However, other species such as yeast and glycophytes have also been employed. Three approaches are commonly used to identify genes important for salt tolerance. The first approach is to identify genes involved in processes known to be critical for salt tolerance (osmolyte synthesis, ion homeostasis, etc.). The second approach is to identify genes whose expression is regulated by salt stress. This is relatively simply and applicable to any plant species. Genetic amenability of some species allows the third approach, which consists in the identification of salt tolerance determinants based on functionality. At the moment, there is a large number of reports in the literature claiming that plants with increased salt tolerance have been obtained. The main problem is that different plant species, stage of development, organs, promoters and salt conditions used it is difficult to compare the degree of salt tolerance conferred by different genes. In this review, we discuss progress made towards understanding the molecular elements involved in salt stress responses that have been used in transgenic approaches to improve salt tolerance.     

Bioplastik aus Nutzpflanzen: Palette der nachwachsenden Rohstoffe erweitert

Crop plants are ultivated for producing starch, oils, proteins, sugar, fibres and other products. An increasing amount of these products is used as renewable resources for industrial purposes. But there is still a demand for new products with improved properties such as biodegradable plastics like polyhydroxyfatty acids (PHF) which cannot be found in the plant kingdom. PHF are linear polymers found as a major storage component in many bacterial species. Polyhydroxy‐butyric acid, poly(3HB), is the most abundant representative of this class of polymers. Three enzymes have been identified from the bacterium Ralstonia eutropha responsible for poly(3HB)‐synthesis. These enzymes are encoded by three different genes. PHF can be utilized by many microorganisms as carbon‐ and energy‐source with the help of certain depo lymerases. PHF‐storing plants fulfill the criteria as renewable resource for biodegradable plastics. Using Arabidopsis thaliana as a model plant, the poly(3HB)‐metabolism has been established in plants after transformation with the three bacterial genes under the control of plant promotors. Transgenic plants have been selected accumulating up to 40% poly(3HB) dry weight. Recent data demonstrate that high amounts of poly(3HB) can also be stored in rape seed, the main oil producing rop for moderate limates. This offers the possibility to breed poly(3HB)‐producing rop plants with full agronomical performance.     

草坪草生物技术研究进展

草坪草转基因方法主要有原生质体融合法,基因枪法和农杆菌介导法。抗生素（潮霉素,G418)和除草剂（bialaphos,ppt)都可用于草坪草也可能存在生态风险性。     

Effect of genotype and environment on branching in weedy green millet (Setaria viridis) and domesticated foxtail millet (Setaria italica) (Poaceae)

Many domesticated crops are derived from species whose life history includes weedy characteristics, such as the ability to vary branching patterns in response to environmental conditions. However, domesticated crop plants are characterized by less variable plant architecture, as well as by a general reduction in vegetative branching compared to their progenitor species. Here we examine weedy green millet and its domesticate foxtail millet that differ in the number of tillers (basal branches) and axillary branches along each tiller. Branch number in F(2:3) progeny of a cross between the two species varies with genotype, planting density, and other environmental variables, with significant genotype-environment interactions (GEI). This is shown by a complex pattern of reaction norms and by variation in the pattern of significant quantitative trait loci (QTL) amongst trials. Individual and joint analyses of high and low density trials indicate that most QTL have significant GEI. Dominance and epistasis also explain some variation in branching. Likely candidate genes underlying the QTL (based on map position and phenotypic effect) include teosinte branched1 and barren stalk1. Phytochrome B, which has been found to affect response to shading in other plants, explains little or no variation. Much variation in branching is explained by QTL that do not have obvious candidate genes from maize or rice.     

Assessing Plant Phytoextraction Potential Through Mathematical Modeling

One of the most serious and long-term consequences of environmental pollution is heavy metal contamination of soils. Elements such as zinc, cadmium, lead, nickel, and chromium are being released into the environment by many industrial processes and have now reached concentrations that are of concern. Phytoremediation is a new, low-cost, and environmentally friendly technique that relies on the natural properties of some plants to clean-up the ground through their ability to take up metals from the soil. Hyperaccumulator plants, capable of accumulating metals far in excess of any normal physiological requirement, represent a most promising tool for metal phytoextraction, but the in field establishment of their conditions for utilization needs a long period because of the plant life-cycle. The use of a mathematical model is proposed to process growth and uptake data from in vitro experiments for a rapid assessment of the time and concentration parameters for the deployment of hyperaccumulator plants for phytoextraction purposes. This preliminary research has been carried out using Alyssum bertolonii Desv., a nickel hyperaccumulator endemic to Italian serpentine soils.