转基因玉米的研究进展和食用安全性评价

玉米是我国重要的粮食作物和饲料作物。目前转基因技术作为生物育种技术的代表,已成为国际上育种的前沿和核心技术。世界各国利用转基因技术相继研发了具有多种优良性状的转基因玉米,创造了巨大的经济效益;同时统一、有效的监管措施是转基因玉米研发、推广和商业化的重要基础。食用安全性评价是有效监管的前提。针对转基因玉米的商业化和控制玉米重要性状基因的研究进展进行了综述,并对转基因玉米的食用安全性评价进行归纳分析,以期为我国转基因玉米的研发、管理和推广提供理论参考。     

Sugarcane (Saccharum X officinarum): A Reference Study for the Regulation of Genetically Modified Cultivars in Brazil

Global interest in sugarcane has increased significantly in recent years due to its economic impact on sustainable energy production. Sugarcane breeding and better agronomic practices have contributed to a huge increase in sugarcane yield in the last 30?years. Additional increases in sugarcane yield are expected to result from the use of biotechnology tools in the near future. Genetically modified (GM) sugarcane that incorporates genes to increase resistance to biotic and abiotic stresses could play a major role in achieving this goal. However, to bring GM sugarcane to the market, it is necessary to follow a regulatory process that will evaluate the environmental and health impacts of this crop. The regulatory review process is usually accomplished through a comparison of the biology and composition of the GM cultivar and a non-GM counterpart. This review intends to provide information on non-GM sugarcane biology, genetics, breeding, agronomic management, processing, products and byproducts, as well as the current technologies used to develop GM sugarcane, with the aim of assisting regulators in the decision-making process regarding the commercial release of GM sugarcane cultivars.     

Sugarcane improvement: how far can we go?

In recent years, efforts to improve sugarcane have focused on the development of biotechnology for this crop. It has become clear that sugarcane lacks tools for the biotechnological route of improvement and that the initial efforts in sequencing ESTs had limited impact for breeding. Until recently, the models used by breeders in statistical genetics approaches have been developed for diploid organisms, which are not ideal for a polyploid genome such as that of sugarcane. Breeding programs are dealing with decreasing yield gains. The contribution of multiple alleles to complex traits such as yield is a basic question underlining the breeding efforts that could only be addressed by the development of specific tools for this grass. However, functional genomics has progressed and gene expression profiling is leading to the definition of gene networks. The sequencing of the sugarcane genome, which is underway, will greatly contribute to numerous aspects of research on grasses. We expect that both the transgenic and the marker-assisted route for sugarcane improvement will contribute to increased sugar, stress tolerance, and higher yield and that the industry for years to come will be able to rely on sugarcane as the most productive energy crop.     

Recombinant Cellulase Accumulation in the Leaves of Mature,Vegetatively Propagated Transgenic Sugarcane

The cost of enzymes that hydrolyse lignocellulosic substrates to fermentable sugars needs to be reduced to make cellulosic ethanol a cost-competitive liquid transport fuel. Sugarcane is a perennial crop and the successful integration of cellulase transgenes into the sugarcane production system requires that transgene expression is stable in the ratoon. Herein, we compared the accumulation of recombinant fungal cellobiohydrolase I (CBH I), fungal cellobiohydrolase II (CBH II), and bacterial endoglucanase (EG) in the leaves of mature, initial transgenic sugarcane plants and their mature ratoon. Mature ratoon events containing equivalent or elevated levels of active CBH I, CBH II, and EG in the leaves were identified. Further, we have demonstrated that recombinant fungal CBH I and CBH II can resist proteolysis during sugarcane leaf senescence, while bacterial EG cannot. These results demonstrate the stability of cellulase enzyme transgene expression in transgenic sugarcane and the utility of sugarcane as a biofactory crop for production of cellulases.     

Generation of a selectable marker free,highly expressed single copy locus as landing pad for transgene stacking in sugarcane

Key message

A selectable marker free, highly expressed single copy locus flanked by insulators was created as landing pad for transgene stacking in sugarcane. These events displayed superior transgene expression compared to single-copy transgenic lines lacking insulators. Excision of the selectable marker gene from transgenic sugarcane lines was supported by FLPe/FRT site-specific recombination.

Abstract

Sugarcane, a tropical C4 grass in the genus Saccharum (Poaceae), accounts for nearly 80% of sugar produced worldwide and is also an important feedstock for biofuel production. Generating transgenic sugarcane with predictable and stable transgene expression is critical for crop improvement. In this study, we generated a highly expressed single copy locus as landing pad for transgene stacking. Transgenic sugarcane lines with stable integration of a single copy nptII expression cassette flanked by insulators supported higher transgene expression along with reduced line to line variation when compared to single copy events without insulators by NPTII ELISA analysis. Subsequently, the nptII selectable marker gene was efficiently excised from the sugarcane genome by the FLPe/FRT site-specific recombination system to create selectable marker free plants. This study provides valuable resources for future gene stacking using site-specific recombination or genome editing tools.

     

Transgene stability and dispersal in forest trees

Transgenics from several forest tree species, carrying a number of commercially important recombinant genes, have been produced, and are undergoing confined field trials in a number of countries. However, there are questions and issues regarding stability of transgene expression and transgene dispersal that need to be addressed in long-lived forest trees. Variation in transgene expression is not uncommon in the primary transformants in plants, and is undesirable as it requires screening a large number of transformants in order to select transgenic lines with acceptable levels of transgene expression. Therefore, the current focus of plant transformation is toward fine tuning of transgene expression and stability in the transgenic forest trees. Although a number of studies have reported a relatively stable transgene expression for several target traits, including herbicide resistance, insect resistance, and lignin modification, there was also some unintended transgene instability in the genetically modified (GM) forest trees. Transgene dispersal from GM trees to feral forest populations and their containment remain important biological and regulatory issues facing commercial release of GM trees. Containment of transgenes must be in place to effectively prevent escape of transgenic pollen, seed, and vegetative propagules in economically important GM forest trees before their commercialization. Therefore, it is important to devise innovative technologies in genetic engineering that lead to genetically stable transgenic trees not only for qualitative traits (herbicide resistance, insect resistance), but also for quantitative traits (accelerated growth, increased height, increased wood density), and also prevent escape of transgenes in the forest trees.     

Field performance of transgenic sugarcane produced using Agrobacterium and biolistics methods

Future genetic improvement of sugarcane depends, in part, on the ability to produce high‐yielding transgenic cultivars with improved traits such as herbicide and insect resistance. Here, transgenic sugarcane plants generated by different transformation methods were assessed for field performance over 3 years. Agrobacterium‐mediated (Agro) transgenic events (35) were produced using four different Agrobacterium tumefaciens strains, while biolistic (Biol) transgenic events (48) were produced using either minimal linearized DNA (LDNA) transgene cassettes with 5′, 3′ or blunt ends or whole circular plasmid (PDNA) vectors containing the same transgenes. A combined analysis showed a reduction in growth and cane yield in Biol, Agro as well as untransformed tissue culture (TC) events, compared with the parent clone (PC) Q117 (no transformation or tissue culture) in the plant, first ratoon and second ratoon crops. However, when individual events were analysed separately, yields of some transgenic events from both Agro and Biol were comparable to PC, suggesting that either transformation method can produce commercially suitable clones. Interestingly, a greater percentage of Biol transformants were similar to PC for growth and yield than Agro clones. Crop ratoonability and sugar yield components (Brix%, Pol%, and commercial cane sugar (CCS)) were unaffected by transformation or tissue culture. Transgene expression remained stable over different crop cycles and increased with plant maturity. Transgene copy number did not influence transgene expression, and both transformation methods produced low transgene copy number events. No consistent pattern of genetic changes was detected in the test population using three DNA fingerprinting techniques.     

Rapid production of transgenic sugarcane with the introduction of simple loci following biolistic transfer of a minimal expression cassette and direct embryogenesis

A protocol is described that supports the production of transgenic sugarcane plants ready for transfer to soil within 3 mo from culture initiation. Biolistic gene transfer into cross-sections of immature leaf whorl explants followed by direct somatic embryogenesis resulted in the stable genetic transformation of the commercially important sugarcane cultivar CP 88-1762. Accelerating the production of transgenic sugarcane plants not only saves time and effort but will likely also minimize somaclonal variation. Southern blot analysis revealed simple transgene integration patterns ranging from one to five hybridization products. NPTII-ELISA confirmed that most of the transgenic plants expressed the transgene stably in vegetative progeny. Using a minimal, linear expression cassette (MC) without vector backbone sequences for the biolistic gene transfer and reducing the amount of MC to 10 ng per shot may have led to simple transgene integration and stable transgene expression. Therefore, this protocol has great potential for the generation of commercial transgenic sugarcane events.     

Improving crop productivity and resource use efficiency to ensure food security and environmental quality in China

In recent years, agricultural growth in China has accelerated remarkably, but most of this growth has been driven by increased yield per unit area rather than by expansion of the cultivated area. Looking towards 2030, to meet the demand for grain and to feed a growing population on the available arable land, it is suggested that annual crop production should be increased to around 580 Mt and that yield should increase by at least 2% annually. Crop production will become more difficult with climate change, resource scarcity (e.g. land, water, energy, and nutrients) and environmental degradation (e.g. declining soil quality, increased greenhouse gas emissions, and surface water eutrophication). To pursue the fastest and most practical route to improved yield, the near-term strategy is application and extension of existing agricultural technologies. This would lead to substantial improvement in crop and soil management practices, which are currently suboptimal. Two pivotal components are required if we are to follow new trajectories. First, the disciplines of soil management and agronomy need to be given increased emphasis in research and teaching, as part of a grand food security challenge. Second, continued genetic improvement in crop varieties will be vital. However, our view is that the biggest gains from improved technology will come most immediately from combinations of improved crops and improved agronomical practices. The objectives of this paper are to summarize the historical trend of crop production in China and to examine the main constraints to the further increase of crop productivity. The paper provides a perspective on the challenge faced by science and technology in agriculture which must be met both in terms of increased crop productivity but also in increased resource use efficiency and the protection of environmental quality.     

Use of a Mutated Protoporphyrinogen Oxidase Gene as an Effective In Vitro Selectable Marker System that Also Conveys <Emphasis Type="Italic">in planta</Emphasis> Herbicide Resistance in Sugarcane

Genetic engineering can be used to introduce economically important traits in sugarcane cultivars. Part of any transformation process involves the selection of genetically transformed cells. In this study, an efficient sugarcane in vitro selection system was developed using mutated protophorhyrinogen oxidase (PPO) genes as selectable markers. Two PPO genes, that encode proteins targeted either to the mitochondria or plastid, were isolated from tobacco and maize. Site-directed mutagenesis was used to alter the nucleotide sequence of these genes so that the resulting proteins are less sensitive to diphenylether type herbicides. Sugarcane callus was genetically transformed through particle bombardment with constructs allowing expression of either transgene, and putative transgenic calli were selected on fomesafen. It took approximately 4 weeks to select herbicide resistant calli clones on 10 mg/l fomesafen in the presence of light, which increased the selection pressure, and a further 8 weeks to regenerate resistant plantlets. PCR analysis confirmed that all regenerated putative transgenic sugarcane plants contained the transgene. All transgenic plants showed levels of herbicide resistance when planted in soil.     

Alleviation of detrimental effects of NaCl by silicon nutrition in salt-sensitive and salt-tolerant genotypes of sugarcane (Saccharum officinarum L.)

Salt stress is a major environmental factor which adversely affects the crop yield and quality. However, adequate regulation of mineral nutrients may ameliorate the deleterious effects of salts and help to sustain crop productivity under salt stress. Salt-sensitive (SPF 213) and salt-tolerant (HSF 240) sugarcane genotypes were grown in gravel at 0 and 100 mM NaCl by supplying 0, 1.4 mM, 2.1 mM and 2.8 mM of Si as calcium silicate. Results revealed that plants treated with NaCl alone showed a significant (P?≤?0.05) reduction in dry matter production, K⁺ concentration, cane yield and juice quality in both genotypes but the magnitude of reduction was relatively more in salt-sensitive genotype than salt-tolerant. Addition of Si significantly (P?≤?0.05) reduced the uptake and translocation of Na⁺ but increased K⁺ concentrations particularly in shoots of both sugarcane genotypes. Cane yield and yield attributes were significantly (P?≤?0.05) higher where Si was added. Juice quality characteristics were significantly (P?≤?0.05) improved in salt-sensitive and salt-tolerant sugarcane genotypes with the application of Si. The results suggested that added Si interacted with Na⁺, reduced its uptake and transport to shoots and consequently improved cane yield and juice quality in salt-sensitive and salt-tolerant sugarcane genotypes under salt stress.     

Evaluating genetic containment strategies for transgenic plants

One of the primary concerns about genetically engineered crop plants is that they will hybridize with wild relatives, permitting the transgene to escape into the environment. The likelihood that a transgene will spread in the environment depends on its potential fitness impact. The fitness conferred by various transgenes to crop and/or wild-type hybrids has been evaluated in several species. Different strategies have been developed for reducing the probability and impact of gene flow, including physical separation from wild relatives and genetic engineering. Mathematical models and empirical experimental evidence suggest that genetic approaches have the potential to effectively prevent transgenes from incorporating into wild relatives and becoming established in wild populations that are not reproductively isolated from genetically engineered crops.     

Effects of biocides and rotation breaks on soil organisms associated with the poor early growth of sugarcane in continuous monoculture

Glasshouse and field experiments were conducted to determine the effects of biocides and rotation breaks on deleterious soil organisms associated with the poor early growth and subsequent yield decline of sugarcane grown in continuous monoculture. Fumigation of a soil that had been under sugarcane monoculture with minimal breaks for more than 30 years markedly improved the health and growth of the sugarcane sett and shoot root systems, increased the growth of the primary shoot and stimulated more and larger secondary shoots. It also reduced populations of culturable fungi in the rhizosphere of the sett roots and reduced colonization of the sett and shoot roots by lesion nematode (Pratylenchus zeae). Exposure of the developing sett root system for 14 days to mono-cultured sugarcane soil was sufficient to significantly retard subsequent plant growth. In field experiments, fungicide and nematicide (mancozeb + aldicarb), when applied together to land under sugarcane monoculture, was as effective as fumigation in improving early sugarcane growth and increasing sugarcane yields. Rotation breaks (alternate crops, sown pasture, bare fallow) that were in place for 54 months, increased sugarcane establishment and increased sugarcane yields to levels similar to that obtained following fumigation of land under sugarcane monoculture. Fumigation of land that had been under the rotation breaks gave plant growth responses that were in addition to that achieved by the breaks alone. A mancozeb + aldicarb treatment was as effective as fumigation in increasing sugarcane yields after a bare fallow break but accounted for only a portion of the fumigation response following the crop and pasture breaks. Improved plant nutrition may be a factor in the fumigation response following the crop and pasture breaks. Plant growth responses to fumigation and the manocozeb + aldicarb treatments that were manifested in final sugarcane yields (after one years growth) were evident as plant growth responses (sett root, shoot root and primary shoot dry weight) measured 54 days after planting. The experimental results support the concept that when sugarcane is grown as a monoculture, deleterious fungi and nematodes retard plant establishment and early plant growth and that this leads to reduced sugarcane yields.     

Stress-induced Δ1-pyrroline-5-carboxylate synthetase (P5CS) gene confers tolerance to salt stress in transgenic sugarcane

High salinity interferes in sugarcane growth and development, affecting not only crop yield but also reducing sucrose concentration in culms. Sugarcane plants submitted to salt stress can accumulate compatible solutes, such as proline, which may counteract the effects of salt accumulation in the vacuole and scavenge reactive oxygen species. The objective of this study was to evaluate the response to salt stress of sugarcane plants transformed with the Vigna aconitifolia P5CS gene, which encodes ?1-pyrroline-5-carboxylate synthetase, under the control of a stress-induced promoter AIPC (ABA-inducible promoter complex). For this, 4-month-old clonally multiplied sugarcane plants from two transformation events were irrigated every 2 days with 1/10 Hoagland’s solution supplemented with 100, 150 and 200 NaCl, progressively, during 28 days. Transgenic lines showed increased transgene expression in 3.75-fold when compared with the control plants after 9 days of irrigation with saline water, which can explain the higher proline concentration found in these plants. At the end of the experiment (day 28), the transgenic lines accumulated up to 25 % higher amounts of proline when compared with non-transformed control plants. Stress response in transgenic plants was also accompanied by a reduction of malondialdehyde (MDA) derived from cellular lipid peroxidation in leaves, lower Na⁺ accumulation in leaves and maintenance of photochemical efficiency of PSII. Thus, proline contributed to the protection of the photosynthetic apparatus and the prevention of oxidative damage in transgenic sugarcane under salt stress.     

Control of Diatraea saccharalis by the endophytic Pantoea agglomerans 33.1 expressing cry1Ac7

Despite the fact that Bacillus thuringiensis (Bt) is found in more than 90 % of the products used against insects, it has some difficulty reaching the internal regions where the larvae feed. To solve this problem, many genetically modified microorganisms that colonize the same pests have been developed. Thus, the endophytic bacterium Pantoea agglomerans (33.1), which has been recently described as a promising sugarcane growth promoter, was genetically modified with the pJTT vector (which carries the gene cry1Ac7) to control the sugarcane borer, Diatraea saccharalis. Firstly, the bioassays for D. saccharalis control by 33.1:pJTT were conducted with an artificial diet. A new in vivo methodology was also developed, which confirmed the partial control of larvae by 33.1:pJTT. The 33.1:pJTT strain was inoculated into sugarcane stalks containing the D. saccharalis larvae. In the sugarcane stalks, 33.1:pJTT was able to increase the mortality of D. saccharalis larvae, impair larval development and decrease larval weight. Sugarcane seedlings were inoculated with 33.1:pJTT, and re-isolation confirmed the capacity of 33.1:pJTT to continuously colonize the sugarcane. These results prove that P. agglomerans (33.1), a sugarcane growth promoter, can be improved by expressing the Cry protein, and the resulting strain is able to control the sugarcane borer.     

Response of eight sugarcane cultivars to glyphosine and glyphosate ripeners

Studies were conducted on eight sugarcane (Saccharum spp. hydrid) cultivars during the 1982–83 (plant crop) and 1983–84 (ratoon crop) growing seasons to determine the effects of glyphosine (Polaris) (N,N-bis (phosphonomethyl) glycine) and glyphosate (Polado) (sodium-N-(phosphonomethyl) glycine) on stalk sucrose content and yield. Difference due to crops (plant vs. ratoon) for sugarcane quality, kilograms of sugar per ton of cane (S/T), sugarcane yield, tons of cane per hectare (TCH), and sugar yield, tons of sugar per hectare (TSH) were significant. Significant differences were found in quality for the ratoon crop and cane and sugar yield in both crops due to ripener treatment. Cultivars in both crops differed significantly in quality and yield. Harvest dates were significantly different for all plant characteristics. Interactions of cultivar by treatment for the plant crop, harvest date by treatment for the ratoon crop, and cultivar by harvest date for both crops for cane quality also were significant. Time from ripener application to achievement of maximum sugar concentration also depended on cultivar. This is important in determining the economic benefits of a ripener treatment. Climatic conditions may also affect the benefits of such applications.     

Field testing and exploitation of genetically modified cassava with low-amylose or amylose-free starch in Indonesia

The development and testing in the field of genetically modified -so called- orphan crops like cassava in tropical countries is still in its infancy, despite the fact that cassava is not only used for food and feed but is also an important industrial crop. As traditional breeding of cassava is difficult (allodiploid, vegetatively propagated, outbreeding species) it is an ideal crop for improvement through genetic modification. We here report on the results of production and field testing of genetically modified low-amylose transformants of commercial cassava variety Adira4 in Indonesia. Twenty four transformants were produced and selected in the Netherlands based on phenotypic and molecular analyses. Nodal cuttings of these plants were sent to Indonesia where they were grown under biosafety conditions. After two screenhouse tests 15 transformants remained for a field trial. The tuberous root yield of 10 transformants was not significantly different from the control. Starch from transformants in which amylose was very low or absent showed all physical and rheological properties as expected from amylose-free cassava starch. The improved functionality of the starch was shown for an adipate acetate starch which was made into a tomato sauce. This is the first account of a field trial with transgenic cassava which shows that by using genetic modification it is possible to obtain low-amylose cassava plants with commercial potential with good root yield and starch quality.