Plant Genetic Engineering: Technological Pathways, Application Scenarios, and Future Directions.

BackgroundBiomass recalcitrance and plant lodging are two complex traits that tightly associate with plant cell wall structure and features. Although genetic modification of plant cell walls can potentially reduce recalcitrance for enhancing biomass saccharification, it remains a challenge to maintain a normal growth with enhanced biomass yield and lodging resistance in transgenic plants. Sucrose synthase (SUS) is a key enzyme to regulate carbon partitioning by providing UDP-glucose as substrate for cellulose and other polysaccharide biosynthesis. Although SUS transgenic plants have reportedly exhibited improvement on the cellulose and starch based traits, little is yet reported about SUS impacts on both biomass saccharification and lodging resistance. In this study, we selected the transgenic rice plants that expressed OsSUS3 genes when driven by the AtCesA8 promoter specific for promoting secondary cell wall cellulose synthesis in Arabidopsis. We examined biomass saccharification and lodging resistance in the transgenic plants and detected their cell wall structures and wall polymer features.ResultsDuring two-year field experiments, the selected AtCesA8::SUS3 transgenic plants maintained a normal growth with slightly increased biomass yields. The four independent transgenic lines exhibited much higher biomass enzymatic saccharification and bioethanol production under chemical pretreatments at P < 0.01 levels, compared with the controls of rice cultivar and empty vector transgenic line. Notably, all transgenic lines showed a consistently enhanced lodging resistance with the increasing extension and pushing forces. Correlation analysis suggested that the reduced cellulose crystallinity was a major factor for largely enhanced biomass saccharification and lodging resistance in transgenic rice plants. In addition, the cell wall thickenings with the increased cellulose and hemicelluloses levels should also contribute to plant lodging resistance. Hence, this study has proposed a mechanistic model that shows how OsSUS3 regulates cellulose and hemicelluloses biosyntheses resulting in reduced cellulose crystallinity and increased wall thickness, thereby leading to large improvements of both biomass saccharification and lodging resistance in transgenic rice plants.ConclusionsThis study has demonstrated that the AtCesA8::SUS3 transgenic rice plants exhibited largely improved biomass saccharification and lodging resistance by reducing cellulose crystallinity and increasing cell wall thickness. It also suggests a powerful genetic approach for cell wall modification in bioenergy crops.

The adoption of pest-resistant transgenic plants to reduce yield loss and pesticide utilization has been successful in the past three decades. Recently, transgenic plant expressing double-stranded RNA (dsRNA) targeting pest genes emerges as a promising strategy for improving pest resistance in crops. The steroid hormone, 20-hydroxyecdysone (20E), predominately controls insect molting via its nuclear receptor complex, EcR-USP. Here we report that pest resistance is improved in transgenic tobacco plants expressing dsRNA of EcR from the cotton bollworm, Helicoverpa armigera, a serious lepidopteran pest for a variety of crops. When H. armigera larvae were fed with the whole transgenic tobacco plants expressing EcR dsRNA, resistance to H. armigera was significantly improved in transgenic plants. Meanwhile, when H. armigera larvae were fed with leaves of transgenic tobacco plants expressing EcR dsRNA, its EcR mRNA level was dramatically decreased causing molting defects and larval lethality. In addition, the transgenic tobacco plants expressing H. armigera EcR dsRNA were also resistant to another lepidopteran pest, the beet armyworm, Spodoptera exigua, due to the high similarity in the nucleotide sequences of their EcR genes. This study provides additional evidence that transgenic plant expressing dsRNA targeting insect-associated genes is able to improve pest resistance.

Switching on Plant Immune Signaling Systems Using Microbe-Associated Molecular Patterns

Simultaneous overexpression of both CuZn superoxide dismutase and ascorbate peroxidase in transgenic tall fescue plants confers increased tolerance to a wide range of abiotic stresses

Osmotin and osmotin-like proteins are stress proteins belonging to the plant PR-5 group of proteins induced in several plant species in response to various types of biotic and abiotic stresses. We report here the overexpression of tobacco osmotin in transgenic mulberry plants under the control of a constitutive promoter (CaMV 35S) as well as a stress-inducible rd29A promoter. Southern analysis of the transgenic plants revealed the stable integration of the introduced genes in the transformants. Real-time PCR analysis provided evidence for the expression of osmotin in the transgenic plants under both the constitutive and stress-inducible promoters. Transgenic plants with the stress-inducible promoter were observed to better tolerate salt and drought stress than those with the constitutive promoter. Transgenic plants when subjected to simulated salinity and drought stress conditions showed better cellular membrane stability (CMS) and photosynthetic yield than non-transgenic plants under conditions of both salinity and drought stress. Proline levels were very high in transgenic plants with the constitutive promoter relative to those with the stress-inducible promoter. Fungal challenge undertaken with three fungal species known to cause serious losses to mulberry cultivation, namely, Fusarium pallidoroseum, Colletotrichum gloeosporioides and Colletotrichum dematium, revealed that transgenic plants with osmotin under control of the constitutive promoter had a better resistance than those with osmotin under the control of the stress-inducible promoter. Evaluation in next generation was undertaken by studying bud break in transgenic and non-transgenic plants under simulated drought (2% polyethylene glycol) and salt stress (200mM NaCl) conditions. The axillary buds of the selected transgenic lines had a better bud break percentage under stressed conditions than buds from non-transgenic mulberry lines. A biotic assay with Bombyx mori indicated that osmotin protein had no undesirable effect on silkworm rearing and feeding. We therefore conclude that 35S transgenic plants are better suited for both abiotic stress also biotic challenges (fungal), while the rd29A transgenic plants are more responsive to drought.

Seven kinds of transgenic tobacco plants transformed with combinations of three FBE genes were obtained. The transgenic plants transformed with Ta1-SST + Ta6-SFT genes appeared to have the highest fructan or soluble sugar content and the strongest salt tolerance. Fructan is thought to be one of the important regulators involved in plant tolerance to various abiotic stresses. In this study, wheat-derived genes, Ta1-SST, Ta6-SFT, and Ta1-FFT, encoding fructan biosynthesis enzymes (FBE) were isolated and cloned into vectors modified pBI121 or pZP211. Seven different combinations of the three target genes were transformed into tobacco plants through an Agrobacterium-mediated approach, and transgenic tobacco plants were identified by PCR, ELISA, and Southern blotting. Compared with tobacco plants transformed with other six combinations of the three target genes and with wild-type plants, the transgenic plants transformed with Ta1-SST + Ta6-SFT genes contained the highest fructan and soluble sugar content. All seven types of transgenic tobacco plants displayed a much higher level of tolerance to drought, low temperature, and high salinity compared with the wild type. Differences of drought and low temperature tolerance between the transgenic plants containing a single FBE gene and those harboring two or three FBE genes were not significant, but the salt tolerance level of the transgenic plants with different FBE gene combinations from high to low was: Ta1-SST + Ta6-SFT > Ta1-SST + Ta6-SFT + Ta1-FFT > Ta1-SST + Ta1-FFT > Ta1-SFT + Ta1-FFT > single FBE gene. These results indicated that the tolerances of the transgenic tobacco plants to various abiotic stresses were associated with the transformed target gene combinations and the contents of fructan and soluble sugar contained in the transgenic plants.

Antioxidant enzymes play an important role in conferring abiotic stress tolerance. Superoxide dismutase (SOD) is the first enzyme in the enzymatic antioxidative pathway. Halophytic plants like mangroves have been reported to have a high level of SOD activity, which plays a major role in defending the mangrove species against severe abiotic stresses. We had previously reported the isolation of Sod1, a cDNA encoding a cytosolic copper zinc superoxide dismutase from the mangrove plant Avicennia marina and its mRNA expression pattern during various oxidative and abiotic stresses. The present study is an extension of the previous study in further characterizing the Sod1 cDNA by transforming it into rice and analysing the transgenic plants for abiotic stress tolerance. Southern hybridization of A. marina genomic DNA using Sod1, revealed that this gene in A. marina genome is present as a single copy. The cDNA was cloned into a binary vector (pCAMBIA 1300) and transformed into indica rice var Pusa Basmati-1. Southern hybridization analysis of transgenic rice plants revealed stable integration of the Sod1 transgene in the rice genome. The mRNA transcript of Sod1 was detected by Northern hybridisation in the transgenic rice plants. SOD isozyme assay of the transgenic rice plants revealed the stable expression of the transgenic Sod1 protein. The transgenic plants were more tolerant to methyl viologen mediated oxidative stress in comparison to the untransformed control plants. The transgenic plants also withstood salinity stress of 150 mM of NaCl for a period of eight days while the untransformed control plants wilted at the end of the stress treatment in hydroponics. Pot grown transgenic plants could also tolerate salinity stress better than the untransformed control plants, when irrigated with saline water. The transgenic plants also revealed better tolerance to drought stress in comparison to untransformed control plants.

Current and predicted climatic conditions, such as prolonged drought and heat episodes affect plant growth and yield, cause annual losses estimated at billions of dollars and pose a serious challenge for agricultural production worldwide, (Boyer 1982, Mittler 2006). Progress in generating transgenic crops with enhanced tolerance to abiotic stress has nevertheless been slow. The complex field environment with its heterogenic conditions, abiotic stress combinations, and global climatic changes are but a few of the challenges of modern agriculture. A combination of approaches has contributed significantly towards improving abiotic stress tolerance in both the greenhouse and the field (Hirayama and Shinozaki 2010). However, the increasing unpredictability and vagaries of rainfall and temperature variations consequent to global climate change, the dwindling availability of irrigation water and phosphate fertilizer, the escalating population in developing countries indicate that new ways of improving maize yield stability and stress tolerance under suboptimal conditions must be found and employed. A novel strategy to improve abiotic stress tolerance in Arabidopsis and Brassica has been reported by De Block et al., (2005). In their study, poly(ADP-ribosyl) ation activity was downregulated and transgenic plants became tolerant to a broad range of abiotic stresses such as drought, high light and heat. The researchers noted that stress tolerance was obtained by maintaining energy homeostasis due to reduced stress-induced energy consumption by prevention of NADP breakdown. Under abiotic stress conditions, plants overexpressing hairpin RNAi against Arabidopsis APP gene preserve their energy homeostasis without an overactivation of the mitochondrial respiration and avoiding the production of reactive oxygen species. Hence, plants with a lowered PARP activity appear tolerant to multiple stresses. Furthermore, a genome-wide transcript analysis of stressed anti PARP2 transgenic Arabidopsis (hpAt-PARP2) plants revealed that the induction of specific ABA signalling pathways steered increased levels of the cyclic nucleotide ADP-ribose (cADPR) thereby contributing towards tolerance to abiotic stresses (Vanderauwera et al., 2007). Indeed, modulation of poly(ADPribosyl) ation (PAR) reaction by an Arabidopsis thaliana ADP-ribose/NADH pyrophosphohydrolase, AtNUDX7 led to plants becoming tolerant to oxidative stress (Ogawa et al., 2009). Under oxidative stress, AtNUDX7 serves to maintain NAD+ levels by supplying ATP via nucleotide recycling from free ADP-ribose molecules and thus regulates the defence mechanisms against oxidative DNA damage via modulation of the PAR reaction. Furthermore, energy use efficiency is characterized by an epigenetic component that can be directed through selection to increase yield on top of heterosis (Hauben et al., 2009). Arabidopsis findings have previously been found to be directly applicable to commercial crop improvement (Nelson et al., 2005). Therefore, engineering crop plants for high NAD+ regeneration by an efficient 8 upregulation of the NAD+ salvage pathway or by a reduced NAD+ consumption under stress conditions, is a valuable approach to enhance overall stress tolerance in crops. The aim of this study was to assess a similar approach in the model species Zea mays through silencing maize PARP1 gene, a homolog of the Arabidopsis APP gene. This was achieved by establishing tropical maize regeneration system from immature embryo through somatic embryogenesis as a prerequisite for effective genetic transformation mediated by Agrobacterium. Our next goal was to engineer two hairpin constructs targeting maize PARP1 gene within the specific region at the 5’-end of the gene because silencing of the Arabidopsis APP gene using hairpin RNA constructs proved to be a valuable approach to obtain drought tolerance in Arabidopsis and canola (De Block et al., 2005). However, hairpin constructs are not so stable in extreme temperatures in the greenhouse which results in problems with stability in subsequent progenies (Szittya et al., 2003). Therefore, we aimed at evaluating the utility of artificial micro- RNAs (amiRNA) to silence the maize PARP1 gene. This technology is expected to be more effective and stable than the hairpin RNA approach because temperature does not affect the accumulation of microRNA (Szittya et al., 2003). Three amiRNAs were designed targeting maize PARP1 gene. As a complementary approach to the amiRNA and RNAi transgene technology we sought for mutants in the maize PARP1 gene in the Uniform Mu collections mutagenized by the transposon Mutator. Transgene technology and efficient transformation are important to fully exploit a species as a model for functional genomics studies. We developed an efficient and standardized maize transformation method based on the following criteria: (1) Agrobacterium tumefaciens co-cultivation was preferred over particle bombardment because usually intact T-DNA’s are transferred at low copy number, (2) a maize inbred line was favoured over a hybrid line because homogeneous progenies allow transgene testing already in T1 or T2, (3) Gateway vectors were optimized for use in monocots and provide a toolbox for rapid gene cloning. And finally, the transgenic maize plants overexpressing amiRNA constructs against maize PARP1 gene were evaluated for drought and Methyl Viologen-induced oxidative stress tolerance.

Plants in the juvenile state are more tolerant to adverse conditions. Constitutive expression of MicroRNA156 (miR156) prolonged the juvenile phase and increased resistance to abiotic stress, but also affected the architecture of transgenic plants. In this study, we investigated the possibility of subtle manipulation of miR156 expression in flowering plants, with the goal to increase tolerance to abiotic stress without altering the normal growth and development of transgenic plants. Transgenic tobacco plants expressing ZmmiR156 from maize were generated, driven either by the cauliflower mosaic virus (CaMV) 35S promoter or the stress-inducible ZmRab17 promoter. Expression of ZmmiR156 led to improved drought and salt tolerance in both 35S::MIR156 and Rab17::MIR156 transgenic plants, as shown by more vigorous growth, greater biomass production and higher antioxidant enzyme expression after a long period of drought or salt treatment, when compared to wild type and transgenic vector control plants. However, constitutive expression of ZmmiR156 also resulted in retarded growth, increased branching and delayed flowering of transgenic plants. These undesirable developmental changes could be mitigated by using the stress-inducible ZmRab17 promoter. Furthermore, under drought or salt stress conditions, expression of ZmmiR156 reduced the transcript level of NtSPL2 and NtSPL9, the genes potentially targeted by ZmmiR156, as well as that of CP1, CP2, and SAG12, the senescence-associated genes in tobacco. Collectively, our results indicate that ZmmiR156 can be temporally manipulated for the genetic improvement of plants resistant to various abiotic stresses.

The colour of crop improvement

Trehalose is a non-reducing disaccharide of glucose that confers tolerance against abiotic stresses in many diverse organisms, including higher plants. It was previously reported that overexpression of the yeast trehalose-6-phosphate synthase gene in tomato results in improved tolerance against abiotic stresses. However, these transgenic tomato plants had stunted growth and pleiotropic changes in appearance. In this study, transgenic tomato plants were generated by the introduction of a gene encoding a bifunctional fusion of trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase genes from Escherichia coli under the control of the CaMV35S promoter. Transgenic plants accumulated higher levels of trehalose in their leaves and exhibited enhanced drought and salt tolerance and photosynthetic rates under salt stress conditions than wild-type plants. All of the transgenic plants had normal growth patterns and appearances. Therefore, the system described in this study can be used for practical application of the gene in crop improvement.

: Trehalose is a non-reducing disaccharide of glucose that functions as a protectant in the stabilization of biological structures and enhances the tolerance of organisms to abiotic stress. In the present study, we report on the expression of the Grifola frondosa Fr. trehalose synthase (TSase) gene for manipulating abiotic stress tolerance in tobacco (Nicotiana tabaccum L.). The expression of the transgene was under the control of two tandem copies of the CaMV3 5 S promoter and was transferred into tobacco by Agrobacterium tumefaciens EHA105. Compared with non-transgenic plants, transgenic plants were able to accumulate high levels of products of trehalose, which were increased up to 2.126–2.556 mg/g FW, although levels were undetectable in non-transgenic plants. This level of trehalose in transgenic plants was 400-fold higher than that of transgenic tobacco plants cotransformed with Escherichia coli TPS and TPP on independent expression cassettes, twofold higher than that of transgenic rice plants transformed with a bi functional fusion gene (TPSP) of the trehalose-6-phosphate (T-6-P) synthase (TPS) and T-6-P phosphatase (TPP) of E. coli, and 12-fold higher than that of transgenic tobacco plants transformed the yeast TPS1 gene. It has been reported that transgenic plants with E. coli TPS and/or TPP were severely stunted and had morphological alterations of their roots. Interestingly, our transgenic plants have obvious morphological changes, including thick and deep-coloured leaves, but show no growth inhibition; moreover, these morphological changes can restore to normal type in T2 progenies. Trehalose accumulation in 35S–35S:TSase plants resulted in increased tolerance to drought and salt, as shown by the results of tests on drought, salt tolerance, and drought physiological indices, such as water content in excised leaves, malondialdehyde content, chlorophyll a and b contents, and the activity of superoxide dismutase and peroxidase in excised leaves. These results suggest that transgenic plants transformed with the TSase gene can accumulate high levels of trehalose and have enhanced tolerance to drought and salt. (Managing editor: Li-Hui ZHAO)

The diamine putrescine and the polyamines (PAs), spermidine (Spd) and spermine (Spm), are ubiquitously occurring polycations associated with several important cellular functions, especially antisenescence. Numerous studies have reported increased levels of PA in plant cells under conditions of abiotic and biotic stress such as drought, high salt concentrations, and pathogen attack. However, the physiological mechanism of elevated PA levels in response to abiotic and biotic stresses remains undetermined. Transgenic plants having overexpression of SAMDC complementary DNA and increased levels of putrescine (1.4-fold), Spd (2.3-fold), and Spm (1.8-fold) under unstressed conditions were compared to wild-type (WT) plants in the current study. The most abundant PA in transgenic plants was Spd. Under salt stress conditions, enhancement of endogenous PAs due to overexpression of the SAMDC gene and exogenous treatment with Spd considerably reduces the reactive oxygen species (ROS) accumulation in intra- and extracellular compartments. Conversely, as compared to the WT, PA oxidase transcription rapidly increases in the S16-S-4 transgenic strain subsequent to salt stress. Furthermore, transcription levels of ROS detoxifying enzymes are elevated in transgenic plants as compared to the WT. Our findings with OxyBlot analysis indicate that upregulated amounts of endogenous PAs in transgenic tobacco plants show antioxidative effects for protein homeostasis against stress-induced protein oxidation. These results imply that the increased PAs induce transcription of PA oxidases, which oxidize PAs, which in turn trigger signal antioxidative responses resulting to lower the ROS load. Furthermore, total proteins from leaves with exogenously supplemented Spd and Spm upregulate the chaperone activity. These effects of PAs for antioxidative properties and antiaggregation of proteins contribute towards maintaining the physiological cellular functions against abiotic stresses. It is suggested that these functions of PAs are beneficial for protein homeostasis during abiotic stresses. Taken together, these results indicate that PA molecules function as antisenescence regulators through inducing ROS detoxification, antioxidative properties, and molecular chaperone activity under stress conditions, thereby providing broad-spectrum tolerance against a variety of stresses.

A novel Medicago truncatula HD-Zip gene, MtHB2, is involved in abiotic stress responses

When will agricultural biotechnologies, such as genetically modified (GM) crops, reach Europe? This was the main question at the Agricultural Biotechnology International Conference (ABIC)—the largest of its kind—that took place in September this year in Cologne, Germany. Given that the ABIC was accompanied by a parallel conference organized by critics of GM crops and foods, this is an appropriate question. Most of the European Union (EU) member states have not yet approved the GM crops that are used widely and safely elsewhere in the world. Moreover, although the EU has finally lifted its moratorium on GM crops, and has passed new regulations for growing and marketing GM foods, national politics, legislation and ideological views about consumer and environmental protection have further hampered their use. European consumers remain wary of agricultural biotechnology and its products, as they do not see any direct benefits from GM crops and are, therefore, understandably reluctant to accept them. But it is only a matter of time before GM foods arrive on supermarket shelves across Europe, predicts Ashley O'Sullivan, President and CEO of Ag‐West Bio Inc. (Saskatoon, Saskatchewan, Canada). “The reality for legislation to regulate agricultural biotechnology is that the train has left the station and there is no way of going back,” he added. > …to convince the cautious European public, agricultural biotechnology still has to […] offer products that directly benefit consumers But to convince the cautious European public, agricultural biotechnology still has to show that it can do more than increase the returns to farmers, and offer products that directly benefit consumers. The next wave of GM plants, which are currently being developed and tested in academic and industry laboratories around the world, including Europe, may soon do this. A range of new GM crops in the research pipeline will offer direct benefits to …