Genetic Transformation Technology Research Articles

Utilization of sucrose during cocultivation positively affects Agrobacterium-mediated transformation efficiency in sugar beet (Beta vulgaris L.)

Sugar beet (Beta vulgaris L.) is one of the most important industrial crops throughout world. With the availability of suitable genetic transformation technologies, the yield, quality, and stress tolerance of sugar beet could be improved significantly. However, low transformation efficiencies seriously limit the application of molecular technologies to the genetic improvement of sugar beet. With the aim of improving gene transfer techniques for sugar beet, the effect of different sucrose concentrations during cocultivation on the initial Agrobacterium-mediated transformation efficiencies in sugar beet was tested. To develop an efficient experimental system through which the effect of sucrose could be tested, first, a prolific regeneration system was optimized by testing the effect of different plant growth regulators on in vitro regeneration and rooting efficiencies from sugar beet cotyledonary node explants. The highest mean number of regenerated shoots per explant was obtained when the cotyledonary node explants excised from young seedlings were grown on MS medium supplemented with 1.0 mg/L 6-benzylaminopurine. Using this regeneration system, the effect of different concentrations of sucrose included in the cocultivation medium on the initial genetic transformation efficiencies observed in T0 plants was tested using an Agrobacterium tumefaciens strain carrying the pBin19/35S:GUS-INT construct. The inclusion of 4.5% sucrose in the cocultivation medium resulted in significantly higher transformation (34.09%) and expression efficiencies (22.72%), confirmed by polymerase chain reaction and β-glucuronidase assays, respectively, in regenerated T0 seedlings. If translated into stably inherited transformation efficiencies, these findings could contribute to the success of genetic transformation studies in sugar beet and other crops recalcitrant to Agrobacterium-mediated transformation.

Recent developments and applications of genetic transformation and genome editing technologies in wheat.

Wheat (Triticum aestivum) is a staple crop across the world and plays a remarkable role in food supplying security. Over the past few decades, basic and applied research on wheat has lagged behind other cereal crops due to the complex and polyploid genome and difficulties in genetic transformation. A breakthrough called as PureWheat was made in the genetic transformation of wheat in 2014 in Asia, leading to a noticeable progress of wheat genome editing. Due to this great achievement, it is predicated that wheat biotechnology revolution is arriving. Genome editing technologies using zinc finger nucleases, transcription activator-like effector nuclease, and clustered regularly interspaced short palindromic repeats-associated endonucleases (CRISR/Cas) are becoming powerful tools for crop modification which can help biologists and biotechnologists better understand the processes of mutagenesis and genomic alteration. Among the three genome editing systems, CRISR/Cas has high specificity and activity, and therefore it is widely used in genetic engineering. Generally, the genome editing technologies depend on an efficient genetic transformationsystem. In this paper, we summarize recent progresses and applications on genetic transformation and genome editing in wheat. We also examine the future aspects of genetic transformation and genome editing. We believe that the technologies for wheat efficient genetic engineering and functional studies will become routine with the emergence of high-quality genomic sequences.

Molecular tagging of biocontrol fungus Trichoderma asperellum and its colonization in soil.

To conduct molecular tagging of the biocontrol fungus Trichoderma asperellum strain T4 and elucidate its colonization patterns in soil. We constructed an expression vector harbouring a hygromycin B-resistant gene (hph) and an efficient green fluorescent protein (egfp) gene. By applying Agrobacterium AGL-1-mediated genetic transformation technology, we conducted molecular tagging of T. asperellum and monitored the colonization dynamics of T. asperellum in soil. The results of tracking five independent transformants of T. asperellum indicated that its expansion rates ranged from 4·7 to 6·8cmweek-1 . After inoculation in soil, the quantities of T. asperellum could be maintained at over 10×104 CFU per gram soil in the first year. In the third year after inoculation, the quantities of T. asperellum in soil were still higher than 1×103 CFU per gram soil. In addition, molecularly tagged T. asperellum in soil in the second year (i.e. 12months) after inoculation could still reach the biocontrol effect on cucumber Rhizoctonia rot by more than 74%. Trichoderma asperellum strain T4 is capable of effectively colonizing in soil and surviving for more than 1year. This study has provided the scientific basis for applying T. asperellum as the biocontrol fungus for prevention and control of plant diseases.

Development of a Gateway-compatible two-component expression vector system for plants.

Genetic transformation of plants offers the possibility of functional characterization of individual genes and the improvement of plant traits. Development of novel transformation vectors is essential to improve plant genetic transformation technologies for various applications. Here, we present the development of a Gateway-compatible two-component expression vector system for Agrobacterium-mediated plant transformation. The expression system contains two independent plasmid vector sets, the activator vector and the reporter vector, based on the concept of the GAL4/UAS trans-activation system. The activator vector expresses a modified GAL4 protein (GAL4-VP16) under the control of specific promoter. The GAL4-VP16 protein targets the UAS in the reporter vector and subsequently activates reporter gene expression. Both the activator and reporter vectors contain the Gateway recombination cassette, which can be rapidly and efficiently replaced by any specific promoter and reporter gene of interest, to facilitate gene cloning procedures. The efficiency of the activator-reporter expression system has been assessed using agroinfiltration mediated transient expression assay in Nicotiana benthamiana and stable transgenic expression in Arabidopsis thaliana. The reporter genes were highly expressed with precise tissue-specific and subcellular localization. This Gateway-compatible two-component expression vector system will be a useful tool for advancing plant gene engineering.

Genetic transformation of the dinoflagellate chloroplast

Coral reefs are some of the most important and ecologically diverse marine environments. At the base of the reef ecosystem are dinoflagellate algae, which live symbiotically within coral cells. Efforts to understand the relationship between alga and coral have been greatly hampered by the lack of an appropriate dinoflagellate genetic transformation technology. By making use of the plasmid-like fragmented chloroplast genome, we have introduced novel genetic material into the dinoflagellate chloroplast genome. We have shown that the introduced genes are expressed and confer the expected phenotypes. Genetically modified cultures have been grown for 1 year with subculturing, maintaining the introduced genes and phenotypes. This indicates that cells continue to divide after transformation and that the transformation is stable. This is the first report of stable chloroplast transformation in dinoflagellate algae.

Arabidopsis LEC1 and LEC2 Orthologous Genes Are Key Regulators of Somatic Embryogenesis in Cassava

High genotype-dependent variation in friable embryogenic callus (FEC) induction and subsequent somaclonal variation constitute bottlenecks for the application and scaling of genetic transformation (GT) technology to more farmer- and industry-preferred cassava varieties. The understanding and identification of molecular factors underlying embryogenic development in cassava may help to overcome these constraints. Here, we described the Arabidopsis thaliana LEAFY COTYLEDON (LEC) LEC1 and LEC2 orthologous genes in cassava, designated as MeLEC1 and MeLEC2, respectively. Expression analyses showed that both, MeLEC1 and MeLEC2, are expressed at higher levels in somatic embryogenic (SE) tissues in contrast with differentiated mature tissues. The rapid expression increase of MeLEC genes at early SE induction times strongly suggests that they are involved in the transition from a somatic to an embryonic state, and probably, in the competence acquisition for SE development in cassava. The independent overexpression of the MeLEC genes resulted in different regenerated events with embryogenic characteristics such as MeLEC1OE plants with cotyledon-like leaves and MeLEC2OE plants with somatic-like embryos that emerged over the surface of mature leaves. Transcript increases of other embryo-specific regulating factors were also detected in MeLECOE plants, supporting their mutual interaction in the embryo development coordination. The single overexpression of MeLEC2 was enough to reprogram the vegetative cells and induce direct somatic embryogenesis, which converts this gene into a tool that could improve the recovery of transformed plants of recalcitrant genotypes. The identification of MeLEC genes contributes not only to improve our understanding of SE process in cassava, but also provides viable alternatives to optimize GT and advance in gene editing in this crop, through the development of genotype-independent protocols.

Sweetcane (Erianthus arundinaceus) as a native bioenergy crop with environmental remediation potential in southern China: A review

AbstractSweetcane (Erianthus arundinaceus [Retzius] Jeswiet) is an ecologically dominant warm‐season perennial grass native to southern China. It traditionally plays an important role in sugarcane breeding due to its excellent biological traits and genetic relatedness to sugarcane. Recent studies have shown that sweetcane has a great potential in bioenergy and environmental remediation. The objective of this paper is to review the current research on sweetcane biology, phenology, biogeography, agronomy, and conversion technology, in order to explore its development as a bioenergy crop with environmental remediation potential. Sweetcane is resistant to a variety of stressors and can adapt to different growth environments. It can be used for ecological restoration, soil and water conservation, contaminated land repairing, nonpoint source pollutants barriers in buffer strips along surface waters, and as an ornamental and remediation plant on roadsides and in wetlands. Sweetcane exhibits higher biomass yield, calorific value and cellulose content than other bioenergy crops under the same growth conditions, thereby indicating its superior potential in second‐generation biofuel production. However, research on sweetcane as a bioenergy plant is still in its infancy. More works need be conducted on breeding, cultivation, genetic transformation, and energy conversion technologies.

Application of fluorescent and UV-Vis detection methods to profile antimicrobial activity of cassava tissues for an efficient Agrobacterium-mediated transformation.

The majority of tissue culture and transformation studies in cassava (Manihot esculenta Crantz) focus on the modification of conditions in order to establish a better protocol. Although this is a standard approach for making progress in genetic transformation technology for a target plant variety, serious difficulty still remains due to the limited applicability and adaptability of the given protocol. In the present study, we aim to develop a new concept that focuses on the development of simple but adaptable genetic transformation technology in cassava. In order to establish an efficient transformation protocol, two local edible cassava varieties, R-type, with a broad leaf with reddish petiole, and S-type, with a thin leaf with shiny greenish petiole, were obtained from Okinawa, Japan. Three detection methods, i.e., fluorescence microscopy, thin-layer chromatography (TLC) with detection under an ultraviolet (UV) illumination (254 nm) and light emitting diode (LED) illuminations (365 nm and 500 nm) without any staining, and a spectrum scanning (250-700 nm) by a microplate reader system were employed to identify a series of unique features of the petioles and leaves. Antimicrobial activity of methanol extracts from the tissues was also assayed. We succeeded in the transient expression of the GUS gene using cassava leaves and also established stable introduction of the GUS gene into three organogenic cassava calli by adapting Agrobacterium-mediated transformation. With all the findings, we have identified a flexible tool to create a better match between explants and Agrobacterium strains.

Exploring factors affecting anther culture in rice (Oryza sativa L.)

Anther culture is an important in vitro culture technique for production of double haploids. It is largely species and genotype-specific. Asian cultivated rice (O. sativa, ssp. indica) is recalcitrant to anther culture which limits its practical application in rice breeding. Several researchers tried to optimize the media recipes and culture techniques for callus induction and plantlet regeneration. Negligible response to callus induction and recovery of higher frequency of albino plants are the major hindrance for use of the technique in crop improvement. Shortening the culture period and sexual hybridization of rice sub-groups (japonica/indica) may be adopted to improve green plant regeneration from anther-derived callus in indica rice. However, genetic transformation technology using individual genes related to different aspects of anther culture could be a more direct approach. Besides, the role of genotypes, physiological status of donor plant, developmental stage of pollen, pre-treatment, culture media, phytohormones and culture conditions for successful anther culture have been discussed. Key words: Anther culture, factors for anther culture, double haploids, genetic improvement, rice.

Anther culture in rice: Progress and breeding perspective

Anther culture is an important biotechnological tool for the rapid recovery of fixed breeding lines in rice. The method is largely species- and genotype-specific. Indica rice is known to have a recalcitrant genetic background. Oryza glaberrima responds better to the anther culture than O. sativa while japonica sub-group is more responsive to microspore embryogenesis than Indica types. Media recipes and culture techniques have been standardized for callus induction and green plantlet regeneration using Indica and Japonica varieties as well as their inter se hybrids. Additive effects seem more important than dominant effects of the genes concerned for callus induction while preponderance of dominant effects is reported for regeneration response. Sexual hybridization of rice sub-groups (Japonica x Indica) followed by the selection of favourable QTL for green plant regeneration may be adopted to improve plant regeneration from anther-derived callus in Indica rice. However, genetic transformation technology using individual genes related to different aspects of anther culture could be a more direct approach. This paper reviews the progress and breeding perspective of anther culture in rice for genetic improvement of agronomic- and stress-related traits using double haploid breeding.

Cold Hardiness in Trees: A Mini-Review.

Significant advances have been made in our understanding of the regulation of cold hardiness. The existence of numerous biophysical and biochemical adaptive mechanisms in perennial woody plants and the complexity their regulation has made the development of methods for managing and improving cold hardiness in perennial woody plants has been very difficult. This may be partially attributed to viewing cold hardiness as a single dimensional response, rather than as a complex phenomenon, involving different mechanisms (avoidance and tolerance), different stages (mid-winter vs. late winter), and having an intimate overlap with the genetic regulation of dormancy. In particular separating the molecular regulation of cold hardiness from growth processes has been challenging. ICE and C-repeat binding factor (CBF), transcription factors (Inducer of CBF expression and CRT-binding factor) have been shown to be an important aspect in the regulation of cold-induced gene expression. Evidence has emerged, however, that they are also intimately involved in the regulation of growth, flowering, dormancy, and stomatal development. This evidence includes the presence of CBF binding motifs in genes regulating these processes, or through cross-talk between the pathways that regulate them. Recent changes in climate that have resulted in erratic episodes of unseasonal warming followed by more seasonal patterns of low temperatures has also highlighted the need to better understand the genetic and molecular regulation of deacclimation, a topic of research that is only more recently being addressed. Environmentally-induced epigenetic regulation of stress responses and seasonal processes such as cold acclimation, deacclimation, and dormancy have been documented but are still poorly understood. Advances in the ability to efficiently generate large DNA and RNA datasets and genetic transformation technologies have greatly increased our ability to explore the regulation of gene expression and explore genetic diversity. Greater knowledge of the interplay between epigenetic and genetic regulation of cold hardiness, along with the application of advanced genetic analyses, such as genome-wide-association-studies (GWAS), are needed to develop strategies for addressing the complex processes associated with cold hardiness in woody plants. A cautionary note is also indicated regarding the time-scale needed to examine and interpret plant response to freezing temperatures if progress is to be made in developing effective approaches for manipulating and improving cold hardiness.

Agrobacterium-Mediated Transformation of Oilseed Rape (Brassica napus).

Oilseed rape (Brassica napus) is a commercially important member of the Brassicacea family. It is grown for its edible and industrial oils as well as for animal feed. Genetic transformation technology has been used to study gene function and produce oilseed rape with improved agronomic characteristics. This protocol describes a method for the Agrobacterium tumefaciens-mediated transformation of oilseed rape cotyledonary petioles. The method is reproducible and has been used to transform both spring and winter cultivars. Modifications have been made to the rooting stage, which have reduced the vitrification of shoots. This has not only increased the number of phenotypically normal shoots but has also resulted in an increase in transformation efficiency, concomitant with a dramatic reduction in the number of escapes regenerated. Transformation frequencies typically range from 5% to 10%, with an average of 12% using doubled haploid model varieties, but a maximum efficiency of 20% has been achieved. © 2017 by John Wiley & Sons, Inc.

In Vitro Plant Regeneration from Commercial Cultivars of Soybean.

Soybean, a major legume crop, is the source of vegetable oil and protein. There is a need for transgenic approaches to breeding superior soybean varieties to meet future climate challenges. Efficient plant regeneration is a prerequisite for successful application of genetic transformation technology. Soybean cultivars are classified into different maturity groups based on photoperiod requirements. In this study, nine soybean varieties belonging to different maturity group were regenerated successfully from three different explants: half split hypocotyl, complete hypocotyl, and cotyledonary node. All the genotypes and explant types responded by producing adventitious shoots. Shoot induction potential ranged within 60–87%, 50–100%, and 75–100%, and regeneration rate ranged within 4.2–10, 2.7–4.2, and 2.6–10.5 shoots per explant using half split hypocotyl, complete hypocotyl, and cotyledonary explants, respectively, among all the tested genotypes. Bunya variety showed the best regeneration response using half split and complete hypocotyl explants and the PNR791 with cotyledonary node. The regenerated shoots were successfully rooted and acclimatized to glasshouse conditions. This study shows that commercial varieties of soybean are amenable to shoot regeneration with high regeneration frequencies and could be exploited for genetic transformation. Further, our results show no correlation between shoots regeneration capacity with the maturity grouping of the soybean cultivars tested.

Enhanced green fluorescent protein (egfp) gene expression in Tetraselmis subcordiformis chloroplast with endogenous regulators.

On the basis of fundamental genetic transformation technologies, the goal of this study was to optimize Tetraselmis subcordiformis chloroplast transformation through the use of endogenous regulators. The genes rrn16S, rbcL, psbA, and psbC are commonly highly expressed in chloroplasts, and the regulators of these genes are often used in chloroplast transformation. For lack of a known chloroplast genome sequence, the genome-walking method was used here to obtain full sequences of T. subcordiformis endogenous regulators. The resulting regulators, including three promoters, two terminators, and a ribosome combination sequence, were inserted into the previously constructed plasmid pPSC-R, with the egfp gene included as a reporter gene, and five chloroplast expression vectors prepared. These vectors were successfully transformed into T. subcordiformis by particle bombardment and the efficiency of each vector tested by assessing EGFP fluorescence via microscopy. The results showed that these vectors exhibited higher efficiency than the former vector pPSC-G carrying exogenous regulators, and the vector pRFA with Prrn, psbA-5'RE, and TpsbA showed the highest efficiency. This research provides a set of effective endogenous regulators for T. subcordiformis and will facilitate future fundamental studies of this alga.

Applications of tissue culture to the genetic improvement of grapevines

The grapevine was among the first plants to be cultured in vitro (1944). Regeneration by somatic embryogenesis and organogenesis was reported in the 1970s and plantlet production from cell suspensions or callus is now a routine procedure in many laboratories. Methods for isolating grapevine protoplasts have yet to be achieved. The fragmented apex technique, involving high-frequency adventitious bud formation, is a novel and efficient method for rapid multiplication of grapevines but culture of anthers and pollen has been generally unsuccessful. Micropropagation procedures for vinifera grapes, Vitis species and interspecific hybrids, including rootstocks, are all available. Seedless-seedless hybridization, involving embryo rescue in crosses with stenospermocarpic female parents, is of major significance in breeding seedless table grapes. There has been substantial progress in protoplast cell, tissue and organ culture of grapevines, but this technology is still less well developed than with some other fruit crops (notably citrus and apples). So far, tissue culture has little impact on genetic improvement. Exploitation of somaclonal variation for clonal selection is an attractive option for premium wine cultivars. There is evidence of somaclonal variation in vitro but the usefulness of this random genetic variation in viticulture is still uncertain. To date, results of field trials with vines from somatic embryos have been disappointing. The grapevine is proving to be a difficult subject for Agrobacterium -mediated genetic transformation ( A. tumefaciens and A. rhizogenes ) and microprojectile technology is another option which is being investigated.

Ecologically driven competence for exogenous DNA uptake in yeast.

Unlike prokaryotes, eukaryotic organisms do not seem to be equipped with natural cell process(es) designated for exogenous DNA uptake. However, it is barely known that under laboratory circumstances resembling wild fungal environment(s), at least some lower eukaryotes could become naturally competent for exogenous DNA uptake. Thus, apart from the known fact that non-manipulated cells of yeast Saccharomyces cerevisiae take exogenous DNA by conjugation with certain bacteria, there are also mechanical and physiological mechanisms enabling their transformation under environmental conditions. This clearly shows that lower eukaryotes are amenable to transformation without applying man-made technology (i.e., naturally). However, this topic failed to raise critical scientific interest. Therefore, this review aims to scrutinize the overall implication of the phenomenon stressing its fundamental and applicable importance. It also summarizes all axiomatic laboratory circumstances/vehicles hitherto known to provoke yeast competence naturally and critically discusses plausible mechanisms behind. Possible pathways underlying the phenomenon are emphasized and a unifying model is proposed. This story potentially spans several different research fields, from evolutionary genetics to genetic transformation technology.

Functional characterization of the vitellogenin promoter in the silkworm, Bombyx mori.

Genetic transformation and genome editing technologies have been successfully established in the lepidopteran insect model, the domesticated silkworm, Bombyx mori, providing great potential for functional genomics and practical applications. However, the current lack of cis-regulatory elements in B. mori gene manipulation research limits further exploitation in functional gene analysis. In the present study, we characterized a B. mori endogenous promoter, Bmvgp, which is a 798-bp DNA sequence adjacent to the 5'-end of the vitellogenin gene (Bmvg). PiggyBac-based transgenic analysis shows that Bmvgp precisely directs expression of a reporter gene, enhanced green fluorescent protein (EGFP), in a sex-, tissue- and stage-specific manner. In transgenic animals, EGFP expression can be detected in the female fat body from larval-pupal ecdysis to the following pupal and adult stage. Furthermore, in vitro and in vivo experiments revealed that EGFP expression can be activated by 20-hydroxyecdysone, which is consistent with endogenous Bmvg expression. These data indicate that Bmvgp is an effective endogenous cis-regulatory element in B. mori.

The genetic basis of plasmid tropism betweenChlamydia trachomatisandChlamydia muridarum

The development of genetic transformation technology for Chlamydia trachomatis using its endogenous plasmid has recently been described. Chlamydia muridarum cannot be transformed by the C. trachomatis plasmid, indicating a barrier between chlamydial species. To determine which regions of the plasmid conferred the species specificity, we used the novel approach of transforming wild-type C. muridarum carrying the endogenous plasmid pNigg and forced recombination with the C. trachomatis vector pGFP::SW2 which carries the complete C. trachomatis plasmid (pSW2). Penicillin and chloramphenicol-resistant transformants expressing the green fluorescent protein were selected. Recovery of plasmids from these transformants showed they were recombinants. The differences between the pSW2 and pNigg allowed identification of the recombination breakpoints and showed that pGFP::SW2 had exchanged a ∼ 1 kbp region with pNigg covering CDS 2. The recombinant plasmid (pSW2NiggCDS2) is maintained under antibiotic selection when transformed into plasmid-cured C. muridarum. The ability to select for recombinants in C. muridarum shows that the barrier is not at transformation, but at the level of plasmid replication or maintenance. Our studies show that CDS 2, together with adjoining sequences, is the main determinant of plasmid tropism.

Intrasperm vertical symbiont transmission.

Symbiotic bacteria are commonly associated with cells and tissues of diverse animals and other organisms, which affect hosts' biology in a variety of ways. Most of these symbionts are present in the cytoplasm of host cells and maternally transmitted through host generations. The paucity of paternal symbiont transmission is likely relevant to the extremely streamlined sperm structure: the head consisting of condensed nucleus and the tail made of microtubule bundles, without the symbiont-harboring cytoplasm that is discarded in the process of spermatogenesis. Here, we report a previously unknown mechanism of paternal symbiont transmission via an intrasperm passage. In the leafhopper Nephotettix cincticeps, a facultative Rickettsia symbiont was found not only in the cytoplasm but also in the nucleus of host cells. In male insects, strikingly, most sperm heads contained multiple intranuclear Rickettsia cells. The Rickettsia infection scarcely affected the host fitness including normal sperm functioning. Mating experiments revealed both maternal and paternal transmission of the Rickettsia symbiont through host generations. When cultured with mosquito and silkworm cell lines, the Rickettsia symbiont was preferentially localized within the insect cell nuclei, indicating that the Rickettsia symbiont itself must have a mechanism for targeting nucleus. The mechanisms underlying the sperm head infection without disturbing sperm functioning are, although currently unknown, of both basic and applied interest.

Genomics of Cold Hardiness in Woody Plants

The term cold hardiness or freezing tolerance is used to represent, in a general sense, the ability of plants to adapt to and withstand freezing temperatures. It is a complex, multigenic trait that is too often viewed as a single entity when in fact it is composed of many aspects, all of which can be to some extent viewed as genetically distinct. Advances in molecular biology and genomics have provided significant advances in understanding how plants respond to low temperature and acquire freezing tolerance. Among the most important discoveries has been the identification of the CBF/DREB transcription factor. This transcription factor, along with its regulators such as ICE transcription factors, play a major role in sensing low temperature, initiating the process of cold acclimation, and inducing the expression of a large set of cold-regulated genes. These latter genes are presumed to ameliorate injury to plant cells as a result of freeze-induced desiccation and the presence of extracellular ice. The present review provides a comprehensive overview of CBF and ICE genes in a number of woody plants whose genomes have been sequenced and provides information on the attempts to identify genetic markers for use in marker-assisted selection (MAS) or to improve cold hardiness using genetic transformation technologies. Functional studies of CBF genes in woody plants have indicated that their regulation and impact on abiotic stress resistance are more complex than in herbaceous plants. In particular, the possible relationship of CBF to dormancy is highlighted. Cold hardiness is a complex trait and the challenge in the future will be to use the molecular and genetic tools that are being developed, as well as new developments in bioinformatics, to integrate complex sets of data into a systems view of plant biology. This approach holds the best promise for developing the ability to significantly improve cold hardiness in economically important crops while still maintaining high levels of plant productivity and yield.