Gene mining in halophytes: functional identification of stress tolerance genes in Lepidium crassifolium

Extremophile plants are valuable sources of genes conferring tolerance traits, which can be explored to improve stress tolerance of crops. Lepidium crassifolium is a halophytic relative of the model plant Arabidopsis thaliana, and displays tolerance to salt, osmotic and oxidative stresses. We have employed the modified Conditional cDNA Overexpression System to transfer a cDNA library from L. crassifolium to the glycophyte A. thaliana. By screening for salt, osmotic and oxidative stress tolerance through in vitro growth assays and non‐destructive chlorophyll fluorescence imaging, 20 Arabidopsis lines were identified with superior performance under restrictive conditions. Several cDNA inserts were cloned and confirmed to be responsible for the enhanced tolerance by analysing independent transgenic lines. Examples include full‐length cDNAs encoding proteins with high homologies to GDSL‐lipase/esterase or acyl CoA‐binding protein or proteins without known function, which could confer tolerance to one or several stress conditions. Our results confirm that random gene transfer from stress tolerant to sensitive plant species is a valuable tool to discover novel genes with potential for biotechnological applications.     

Plant abiotic stress: deciphering remedial strategies for emerging problem

Growing in their natural environment, plants often encounter unfavorable environmental conditions that interrupt normal plant growth and productivity. Drought, high/low temperature and saline soils are the most common abiotic stresses that plants encounter in their natural environments. Molecular and genomic analyses have facilitated gene discovery and enabled genetic engineering using several functional or regulatory genes that are known to be involved in stress response and preliminary tolerance, to activate specific or broad pathways related to abiotic stress tolerance in plants. Through the use of transgenic technology, goals such as production of plants with desired traits that were unattainable with traditional selection programs are achieved. This review deals with recent advancement in understanding the role of various stress responsive genes and their critical importance for explaining the control mechanism of abiotic stress tolerance and engineering stress tolerant crops based on the expression of specific stress related genes.     

Plant salt-tolerance mechanism: A review

Almost all crops that are important to humans are sensitive to high salt concentration in the soil. The presence of salt in soil is one of the most significant abiotic stresses in farming. Therefore, improving plant salt tolerance and increasing the yield and quality of crops in salty land is vital. Transgenic technology is a fast and effective method to obtain salt-tolerant varieties. At present, many scholars have studied salt damage to plant and plant salt-tolerance mechanism. These scholars have cloned a number of salt-related genes and achieved high salt tolerance for transgenic plants, thereby showing attractive prospects.In this paper, the salt-tolerance mechanism of plants is described from four aspects: plant osmotic stress, ion toxicity, oxidative stress, and salt tolerance genes. This review may help in studies to reveal the mechanism of plant salt tolerance, screen high efficiency and quality salt tolerance crops.     

Molecular Strategies for Addressing Gene Flow Problems and Their Potential Applications in Abiotic Stress Tolerant Transgenic Plants

Transgenic plant technology provides a powerful tool to improve abiotic stress tolerance of crop plants. However, introgression of stress tolerance genes into weedy relatives may increase the potential for persistence and invasiveness, resulting in undesirable ecological consequences. A variety of gene confinement strategies have been developed to reduce unwanted transgene movement. In this review, we discuss some of these strategies, such as male and female sterility, GeneSafe?, parthenocarpy, chloroplast transformation and gene deletor technologies. In the case of the gene deletor technology, all transgenes from pollen, seeds, fruits or other organs may be eliminated once the transgene functions are no longer needed at the stage when the presence of the transgene becomes a cause for ecological or public concern. The gene deletor and other technologies can be useful to reduce unintended dispersal of stress tolerance genes and thus may facilitate commercialization of transgenic crops with enhanced tolerance to abiotic stresses.     

A selection strategy in plant transformation based on antisense oligodeoxynucleotide inhibition

Antisense oligodeoxynucleotide (asODN) inhibition was developed in the 1970s, and since then has been widely used in animal research. However, in plant biology, the method has had limited application because plant cell walls significantly block efficient uptake of asODN to plant cells. Recently, we have found that asODN uptake is enhanced in a sugar solution. The method has promise for many applications, such as a rapid alternative to time‐consuming transgenic studies, and high potential for studying gene functionality in intact plants and multiple plant species, with particular advantages in evaluating the roles of multiple gene family members. Generation of transgenic plants relies on the ability to select transformed cells. This screening process is based on co‐introduction of marker genes into the plant cell together with a gene of interest. Currently, the most common marker genes are those that confer antibiotic or herbicide resistance. The possibility that traits introduced by selectable marker genes in transgenic field crops may be transferred horizontally is of major public concern. Marker genes that increase use of antibiotics and herbicides may increase development of antibiotic‐resistant bacterial strains or contribute to weed resistance. Here, we describe a method for selection of transformed plant cells based on asODN inhibition. The method enables selective and high‐throughput screening for transformed cells without conferring new traits or functions to the transgenic plants. Due to their high binding specificity, asODNs may also find applications as plant‐specific DNA herbicides.     

Recent advances in rice biotechnology--towards genetically superior transgenic rice

Rice biotechnology has made rapid advances since the first transgenic rice plants were produced 15 years ago. Over the past decade, this progress has resulted in the development of high frequency, routine and reproducible genetic transformation protocols for rice. This technology has been applied to produce rice plants that withstand several abiotic stresses, as well as to gain tolerance against various pests and diseases. In addition, quality improving and increased nutritional value traits have also been introduced into rice. Most of these gains were not possible through conventional breeding technologies. Transgenic rice system has been used to understand the process of transformation itself, the integration pattern of transgene as well as to modulate gene expression. Field trials of transgenic rice, especially insect-resistant rice, have recently been performed and several other studies that are prerequisite for safe release of transgenic crops have been initiated. New molecular improvisations such as inducible expression of transgene and selectable marker-free technology will help in producing superior transgenic product. It is also a step towards alleviating public concerns relating to issues of transgenic technology and to gain regulatory approval. Knowledge gained from rice can also be applied to improve other cereals. The completion of the rice genome sequencing together with a rich collection of full-length cDNA resources has opened up a plethora of opportunities, paving the way to integrate data from the large-scale projects to solve specific biological problems.     

Problems in monitoring horizontal gene transfer in field trials of transgenic plants

Transgenic crops are approved for release in some countries, while many more countries are wrestling with the issue of how to conduct risk assessments. Controls on field trials often include monitoring of horizontal gene transfer (HGT) from crops to surrounding soil microorganisms. Our analysis of antibiotic-resistant bacteria and of the sensitivity of current techniques for monitoring HGT from transgenic plants to soil microorganisms has two major implications for field trial assessments of transgenic crops: first, HGT from transgenic plants to microbes could still have an environmental impact at a frequency approximately a trillion times lower than the current risk assessment literature estimates the frequency to be; and second, current methods of environmental sampling to capture genes or traits in a recombinant are too insensitive for monitoring evolution by HGT. A model for HGT involving iterative short-patch events explains how HGT can occur at high frequencies but be detected at extremely low frequencies.     

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.     

An efficient and novel plant selectable marker based on organomercurial resistance

A selectable marker gene facilitates the detection of genetically modified plant cells during transformation experiments. So far, these marker genes are almost exclusively of two types, conferring either antibiotic resistance or herbicide tolerance. However, more selectable markers must be developed as additional transgenic traits continue to be incorporated into transgenic plants. Here, we used mercury resistance, conferred by the organomercurial lyase gene, as a selectable marker for transformation. The merB gene fromStreptococcus aureus was modified for plant expression and transferred to a hybrid poplar(Populus alba xPopulus glandulosa), using the stem segment-agrobacteria co-cultivation method. The transformed cells were selected on a callus-inducing medium containing as little as 1 μM methylmercury. Subsequent plant regeneration was done in the presence of methylmercury. Resistance to Hg was stably maintained in mature plants after two years of growth in the nursery. We suggest that this gene could serve as an excellent selectable marker for plant transformation.     

Rice A20/AN1 zinc-finger containing stress-associated proteins (SAP1/11) and a receptor-like cytoplasmic kinase (OsRLCK253) interact via A20 zinc-finger and confer abiotic stress tolerance in transgenic Arabidopsis plants

? The inbuilt mechanisms of plant survival have been exploited for improving tolerance to abiotic stresses. Stress-associated proteins (SAPs), containing A20/AN1 zinc-finger domains, confer abiotic stress tolerance in different plants, however, their interacting partners and downstream targets remain to be identified. ? In this study, we have investigated the subcellular interactions of rice SAPs and their interacting partner using yeast two-hybrid and fluorescence resonance energy transfer (FRET) approaches. Their efficacy in improving abiotic stress tolerance was analysed in transgenic Arabidopsis plants. Regulation of gene expression by genome-wide microarray in transgenics was used to identify downstream targets. ? It was found that the A20 domain mediates the interaction of OsSAP1 with self, its close homolog OsSAP11 and a rice receptor-like cytoplasmic kinase, OsRLCK253. Such interactions between OsSAP1/11 and with OsRLCK253 occur at nuclear membrane, plasma membrane and in nucleus. Functionally, both OsSAP11 and OsRLCK253 could improve the water-deficit and salt stress tolerance in transgenic Arabidopsis plants via a signaling pathway affecting the expression of several common endogenous genes. ? Components of a novel stress-responsive pathway have been identified. Their stress-inducible expression provided the protection against yield loss in transgenic plants, indicating the agronomic relevance of OsSAP11 and OsRLCK253 in conferring abiotic stress tolerance.     

Targeting metabolic pathways for genetic engineering abiotic stress-tolerance in crops

Abiotic stress conditions are the major limitations in modern agriculture. Although many genes associated with plant response(s) to abiotic stresses have been indentified and used to generate stress tolerant plants, the success in producing stress-tolerant crops is limited. New technologies are providing opportunities to generate stress tolerant crops. Biotechnological approaches that emphasize the development of transgenic crops under conditions that mimic the field situation and focus on the plant reproductive stage will significantly improve the opportunities of producing stress tolerant crops. Here, we highlight recent advances and discuss the limitations that hinder the fast integration of transgenic crops into agriculture and suggest possible research directions. This article is part of a Special Issue entitled: Plant gene regulation in response to abiotic stress.     

RNA‐directed DNA methylation and seed development: an unexpected difference between Arabidopsis thaliana and Brassica rapa

Biotechnology provides a means for the rapid genetic improvement of plants. Although single genes have been important in engineering herbicide and pest tolerance traits in crops, future improvements of complex traits like yield and nutritional quality will likely require the introduction of multiple genes. This research reports a system (GAANTRY; Gene Assembly in Agrobacterium by Nucleic acid Transfer using Recombinase technologY) for the flexible, in vivo stacking of multiple genes within an Agrobacterium virulence plasmid Transfer‐DNA (T‐DNA). The GAANTRY system utilizes in vivo transient expression of unidirectional site‐specific recombinases and an alternating selection scheme to sequentially assemble multiple genes into a single transformation construct. To demonstrate GAANTRY's capabilities, 10 cargo sequences were sequentially stacked together to produce a 28.5‐kbp T‐DNA, which was used to generate hundreds of transgenic events. Approximately 90% of the events identified using a dual antibiotic selection screen exhibited all of the introduced traits. A total of 68% of the tested lines carried a single copy of the selection marker transgene located near the T‐DNA left border, and only 8% contained sequence from outside the T‐DNA. The GAANTRY system can be modified to easily accommodate any method of DNA assembly and generate high‐quality transgenic plants, making it a powerful, yet simple to use tool for plant genetic engineering.     

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.     

Molecular plant breeding: achievements in green biotechnology and future perspectives

Since one decade ago, transgenic crop plants are globally grown; in 2004, it was estimated to cover a total of 81 Mio ha in 17 countries. At present, four plant species (soybean, maize, cotton and rapeseed) dominate with two traits (herbicide tolerance and insect resistance). The traits on which research concentrates and the constructs which might come next onto the market are outlined. The procedure on how to clone such genes of interest, e.g. via map-based cloning, and some other helpful approaches of green biotechnology, like high throughput techniques and functional markers, are summarised, and a rough calculation about the market value of transgenic crops in US dollars is quoted.     

Modification of plant hormone levels and signaling as a tool in plant biotechnology

Plant hormones are signal molecules, present in trace quantities, that act as major regulators of plant growth and development. They are involved in a wide range of processes such as elongation, flowering, root formation and vascular differentiation. For many years, agriculturists have applied hormones to their crops to either increase the yield, or improve the quality of the commercial product. Nowadays, the knowledge of hormone biosynthesis, degradation and signaling pathways has allowed the utilization of biotechnological tools to further improve the main agricultural crops. Natural or artificial mutants, with impaired functioning of the corresponding genes, have been adopted because of their superior phenotype in specific agricultural traits. In addition, transgenic plants have been generated to regulate internal hormone levels, or their signaling pathways, resulting in some crops that have revolutionized agriculture.     

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.     

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.