Genetic modification of cassava for enhanced starch production

To date, transgenic approaches to biofortify subsistence crops have been rather limited. This is particularly true for the starchy root crop cassava ( Manihot esculenta Crantz). Cassava has one of the highest rates of CO₂ fixation and sucrose synthesis for any C3 plant, but rarely reaches its yield potentials in the field. It was our hypothesis that starch production in cassava tuberous roots could be increased substantially by increasing the sink strength for carbohydrate. To test this hypothesis, we generated transgenic plants with enhanced tuberous root ADP-glucose pyrophosphorylase (AGPase) activity. This was achieved by expressing a modified form of the bacterial glgC gene under the control of a Class I patatin promoter. AGPase catalyses the rate-limiting step in starch biosynthesis, and therefore the expression of a more active bacterial form of the enzyme was expected to lead to increased starch production. To facilitate maximal AGPase activity, we modified the Escherichia coli glgC gene (encoding AGPase) by site-directed mutagenesis (G336D) to reduce allosteric feedback regulation by fructose-1,6-bisphosphate. Transgenic plants (three) expressing the glgC gene had up to 70% higher AGPase activity than control plants when assayed under conditions optimal for plant and not bacterial AGPase activity. Plants having the highest AGPase activities had up to a 2.6-fold increase in total tuberous root biomass when grown under glasshouse conditions. In addition, plants with the highest tuberous root AGPase activity had significant increases in above-ground biomass, consistent with a possible reduction in feedback inhibition on photosynthetic carbon fixation. These results demonstrate that targeted modification of enzymes regulating source–sink relationships in crop plants having high carbohydrate source strengths is an effective strategy for increasing carbohydrate yields in sink tissues.     

Metabolic profiles of six African cultivars of cassava (Manihot esculenta Crantz) highlight bottlenecks of root yield

Cassava is an important staple crop in sub‐Saharan Africa, due to its high productivity even on nutrient poor soils. The metabolic characteristics underlying this high productivity are poorly understood including the mode of photosynthesis, reasons for the high rate of photosynthesis, the extent of source/sink limitation, the impact of environment, and the extent of variation between cultivars. Six commercial African cassava cultivars were grown in a greenhouse in Erlangen, Germany, and in the field in Ibadan, Nigeria. Source leaves, sink leaves, stems and storage roots were harvested during storage root bulking and analyzed for sugars, organic acids, amino acids, phosphorylated intermediates, minerals, starch, protein, activities of enzymes in central metabolism and yield traits. High ratios of RuBisCO:phosphoenolpyruvate carboxylase activity support a C₃ mode of photosynthesis. The high rate of photosynthesis is likely to be attributed to high activities of enzymes in the Calvin–Benson cycle and pathways for sucrose and starch synthesis. Nevertheless, source limitation is indicated because root yield traits correlated with metabolic traits in leaves rather than in the stem or storage roots. This situation was especially so in greenhouse‐grown plants, where irradiance will have been low. In the field, plants produced more storage roots. This was associated with higher AGPase activity and lower sucrose in the roots, indicating that feedforward loops enhanced sink capacity in the high light and low nitrogen environment in the field. Overall, these results indicated that carbon assimilation rate, the K battery, root starch synthesis, trehalose, and chlorogenic acid accumulation are potential target traits for genetic improvement.     

The Cassava Source–Sink project: opportunities and challenges for crop improvement by metabolic engineering

Cassava (Manihot esculenta Crantz) is one of the important staple foods in Sub‐Saharan Africa. It produces starchy storage roots that provide food and income for several hundred million people, mainly in tropical agriculture zones. Increasing cassava storage root and starch yield is one of the major breeding targets with respect to securing the future food supply for the growing population of Sub‐Saharan Africa. The Cassava Source–Sink (CASS) project aims to increase cassava storage root and starch yield by strategically integrating approaches from different disciplines. We present our perspective and progress on cassava as an applied research organism and provide insight into the CASS strategy, which can serve as a blueprint for the improvement of other root and tuber crops. Extensive profiling of different field‐grown cassava genotypes generates information for leaf, phloem, and root metabolic and physiological processes that are relevant for biotechnological improvements. A multi‐national pipeline for genetic engineering of cassava plants covers all steps from gene discovery, cloning, transformation, molecular and biochemical characterization, confined field trials, and phenotyping of the seasonal dynamics of shoot traits under field conditions. Together, the CASS project generates comprehensive data to facilitate conventional breeding strategies for high‐yielding cassava genotypes. It also builds the foundation for genome‐scale metabolic modelling aiming to predict targets and bottlenecks in metabolic pathways. This information is used to engineer cassava genotypes with improved source–sink relations and increased yield potential.     

Development and application of transgenic technologies in cassava

The capacity to integrate transgenes into the tropical root crop cassava (Manihot esculenta Crantz) is now established and being utilized to generate plants expressing traits of agronomic interest. The tissue culture and gene transfer systems currently employed to produce these transgenic cassava have improved significantly over the past 5 years and are assessed and compared in this review. Programs are underway to develop cassava with enhanced resistance to viral diseases and insects pests, improved nutritional content, modified and increased starch metabolism and reduced cyanogenic content of processed roots. Each of these is described individually for the underlying biology the molecular strategies being employed and progress achieved towards the desired product. Important advances have occurred, with transgenic plants from several laboratories being prepared for field trails.     

Improved Cassava Starch by Antisense Inhibition of Granule-bound Starch Synthase I

Cassava is a poor man's crop which is mainly grown as a subsistence crop in many developing countries. Its commercial use was first as animal feed (also known as tapioca), but has shifted since the late sixties to a source of native starch. The availability of native starches, which on the one hand do not require substantial chemical derivatisation and on the other hand have improved properties, would make cassava also for small farmers a potentially attractive cash crop. Since breeding is difficult in this polyploid, vegetatively propagated, crop a transgenic approach would be ideal to improve certain characteristics. We have created a cassava genotype producing amylose-free starch by genetic modification. The absence of amylose increased the clarity and stability of gels made with the transgenic starch, without requiring treatment with environment-unfriendly chemicals such as epoxides (propylene oxide, ethylene oxide) and acetic anhydride, which are normally used to improve stability. The amylose-free starch showed no changes in particle size distribution, chain length distribution or phosphorous content when compared to amylose-containing starch, but the granule melting temperature was increased by almost 2°C. Furthermore, the amylose-free cassava starch shows enhanced clarity and stability properties. These improved functionalities are desired in technical applications in paper and textile manufacturing, but also in the food industry for the production of sauces, dairy products and noodles.     

玉米遗传转化与商业化转基因玉米开发*

玉米是世界上种植面积最大的粮食作物,为了提高玉米产量和满足人类需求,转基因育种已经成为改良玉米性状的有效手段。自1996年美国种植商业化转基因玉米以来,利用玉米遗传转化技术开发商业化转基因玉米已取得巨大成功。综述了玉米遗传转化体系优化的重要步骤,系统总结了已开发的商业化转基因玉米的种类,并对玉米遗传转化未来发展方向进行了展望。     

Plant biotechnology: ecological case studies on herbicide resistance

The emerging field of molecular ecology aims to improve the ecological predictability of transgenic crop plants. The most widely cultivated lines are Roundup-Ready plants, which are genetically modified to be resistant to the broad-spectrum herbicide glyphosate. Recent publications demonstrate two ecological effects that were not anticipated: the widespread emergence of glyphosate-resistant weed biotypes and the formation of a metabolic herbicidal residue. Both effects appear to be due to the increased use of glyphosate rather than the genetic modification in the transgenic crop plant. With one prominent exception, opinions collected from the literature point towards a certain degree of resistance mismanagement and an inadequate testing of the ecological effects of extensive glyphosate use.     

木薯分子育种中若干基本科学问题的思考与研究进展

木薯作为全球重要的薯类作物,既是热带地区粮食安全的保障,也是重要的淀粉工业原材料,保障其稳产、高产、优质一直是育种家不变的研究主题．当前,木薯品种选育正处在从杂交育种转向分子育种的发展阶段,深入解析木薯特有的经济性状和生物学特点是利用生物技术进行遗传改良的基础．不同于谷物类作物,木薯光合同化物的转运和库源分配的调控机制必有其独特之处;同时,储藏根的“库容”直接影响其产量．作为热带作物,了解木薯对低温和干旱的响应可为改良其抗逆境能力提供理论依据．不同于其他薯类作物,木薯储藏根特有的“采后生理性变质”问题亟待解决,其发生和调控机制的解析对延长木薯货架期意义重大．随着分子生物学的发展,针对上述各方面的研究日益深入,不仅激发了感兴趣的公众对这些问题的认知和思考,也激励了科研人员不断努力寻找解析相关机制的方法,为最终通过分子育种手段改良木薯提供思路和技术方案,揭开木薯的层层“面纱”,推动木薯分子育种的发展．     

Field testing of virus resistant transgenic plants

A number of crop plants have been genetically modified for the purpose of resisting virus infection. Different resistance types have been observed in transgenic crops. The practical value of genetically modified, virus resistant, economically important crops can be evaluated only by field testing. The criteria for effective field resistance to viral disease can vary significantly depending on the crop and the virus. Furthermore, field testing is required to determine whether important agronomic properties of modified crops were changed by plant transformation and to confirm that the resistance observed under controlled environment is effective also under natural field conditions and to demonstrate the economical value of virus resistant, transgenic plants.     

Genetically modified sugarcane for bioenergy generation

Sugarcane breeding has significantly progressed over the past 30 years, but attempts to further increase crop yield have been limited due to the complexity of the sugarcane genome. An alternative to boost the crop yield is the introduction of genes encoding desirable traits in the elite sugarcane cultivars. Genetically modified sugarcane with increased yield and pest and disease resistance has already proven its value not only by the increased sugar content but also for the improvement of the crop performance. However, transgene stability is still a challenge since transgene silencing seems to occur in a large proportion of genetically modified sugarcane plants. In addition, regulatory issues associated with the crop propagation model will also be a challenge to the commercial approval of genetically modified sugarcane.     

代谢基因工程提高作物产量的分子靶标

应用基因工程技术对植物细胞内的代谢途径进行遗传修饰,已成功地使细胞代谢发生改变或合成新的化合物。光合作用,淀粉合成,氮素同化和水分利用等是形成作物产量的基础代谢。对这些代谢途径中的关键步骤和靶分子进行基因修饰以提高作物产量的研究已取得长足的进展,并正在发展成为提高作物产量的新途径。本文着重论述应用代谢基因工程提高作物产量的技术策略,研究现状,存在的问题,所面临的挑战和应用前景。     

Suitability of non-lethal marker and marker-free systems for development of transgenic crop plants: present status and future prospects

Genetically modified crops are one of the prudent options for enhancing the production and productivity of crop plants by safeguarding from the losses due to biotic and abiotic stresses. Agrobacterium-mediated and biolistic transformation methods are used to develop transgenic crop plants in which selectable marker genes (SMG) are generally deployed to identify 'true' transformants. The commonly used SMG obtained from prokaryotic sources when employed in transgenic plants pose risks due to their lethal nature during selection process. In the recent past, some non-lethal SMGs have been identified and used for selection of transformants with increased precision and high selection efficiency. Considering the concerns related to bio-safety of the environment, it is desirable to remove the SMG in order to maximize the commercial success through wide adoption and public acceptance of genetically modified (GM) food crops. In this review, we examine the availability, and the suitability of wide range of non-lethal selection markers and elimination of SMG methods to develop marker-free transgenics for achieving global food security. As the strategies for marker-free plants are still in proof-of-concept stage, adaptation of new genomics tools for identification of novel non-lethal marker systems and its application for developing marker-free transgenics would further strengthen the crop improvement program.     

Simultaneous boosting of source and sink capacities doubles tuber starch yield of potato plants

An important goal in biotechnological research is to improve the yield of crop plants. Here, we genetically modified simultaneously source and sink capacities in potato (Solanum tuberosum cv. Desirée) plants to improve starch yield. Source capacity was increased by mesophyll‐specific overexpression of a pyrophosphatase or, alternatively, by antisense expression of the ADP‐glucose pyrophosphorylase in leaves. Both approaches make use of re‐routing photoassimilates to sink organs at the expense of leaf starch accumulation. Simultaneous increase in sink capacity was accomplished by overexpression of two plastidic metabolite translocators, that is, a glucose 6‐phosphate/phosphate translocator and an adenylate translocator in tubers. Employing such a ‘pull’ approach, we have previously shown that potato starch content and yield can be increased when sink strength is elevated. In the current biotechnological approach, we successfully enhanced source and sink capacities by a combination of ‘pull’ and ‘push’ approaches using two different attempts. A doubling in tuber starch yield was achieved. This successful approach might be transferable to other crop plants in the future.     

Provitamin A accumulation in cassava (Manihot esculenta) roots driven by a single nucleotide polymorphism in a phytoene synthase gene

Cassava (Manihot esculenta) is an important staple crop, especially in the arid tropics. Because roots of commercial cassava cultivars contain a limited amount of provitamin A carotenoids, both conventional breeding and genetic modification are being applied to increase their production and accumulation to fight vitamin A deficiency disorders. We show here that an allelic polymorphism in one of the two expressed phytoene synthase (PSY) genes is capable of enhancing the flux of carbon through carotenogenesis, thus leading to the accumulation of colored provitamin A carotenoids in storage roots. A single nucleotide polymorphism present only in yellow-rooted cultivars cosegregates with colored roots in a breeding pedigree. The resulting amino acid exchange in a highly conserved region of PSY provides increased catalytic activity in vitro and is able to increase carotenoid production in recombinant yeast and Escherichia coli cells. Consequently, cassava plants overexpressing a PSY transgene produce yellow-fleshed, high-carotenoid roots. This newly characterized PSY allele provides means to improve cassava provitamin A content in cassava roots through both breeding and genetic modification.     

Influence of crop rotation and intercropping of cassava with legumes on VA mycorrhizal symbiosis of cassava

Summary In comparison to cassava grown in monoculture the root infection of cassava with vesicular-arbuscular mycorrhiza was increased by crop rotation with grain legumes in the field. This was also found when cassava was intercropped with legumes and fertilized. A possible specificity of mycorrhizal fungi to increase the yield of one species more than the other when grown in association, is discussed.     

Genetic Modification in Floriculture

An important driving force for the floriculture industry is the development of novel plants and flowers. New varieties provide marketing opportunities for retailers and judicious selection can increase productivity for growers, as well as improving the quality of the final product in the consumer's hands. While plant exploration and conventional breeding programs have been very successful in achieving these goals, genetic modification offers additional routes for the generation of new varieties of important floricultural plants. This can be achieved by the incorporation of genes from outside of the normally available gene pool. This paper provides a summary of the potential applications of gene technology in floriculture and reviews progress to date, with a particular emphasis on the manipulation of flower color. The manipulation of the anthocyanin biosynthesis pathway in carnation to produce novel-colored flowers is so far the only commercial application of genetic modification in floriculture. This progress is in stark contrast to the widespread cultivation of genetically modified broad-acre crops. The commercial use of gene technology requires adherence to regulatory regimes specific to genetically modified plants, and compliance with intellectual property laws. These added complexities are a significant cost, which may be hampering the use of gene technology by breeders of floricultural crops. Another factor may be a perception that the public and retail trade may not accept genetically modified floricultural products. Experience in the real marketplace with the Florigene Moon-series? of genetically modified carnation suggests that these concerns are unwarranted.     

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.     

Cassava genetic transformation and its application in breeding

As a major source of food, cassava (Manihot esculenta Crantz) is an important root crop in the tropics and subtropics of Africa and Latin America, and serves as raw material for the production of starches and bioethanol in tropical Asia. Cassava improvement through genetic engineering not only overcomes the high heterozygosity and serious trait separation that occurs in its traditional breeding, but also quickly achieves improved target traits. Since the first report on genetic transformation in cassava in 1996, the technology has gradually matured over almost 15 years of development and has overcome cassava genotype constraints, changing from mode cultivars to farmer-preferred ones. Significant progress has been made in terms of an increased resistance to pests and diseases, biofortification, and improved starch quality, building on the fundamental knowledge and technologies related to planting, nutrition, and the processing of this important food crop that has often been neglected. Therefore, cassava has great potential in food security and bioenergy development worldwide.     

The VIRCA Project: virus resistant cassava for Africa

The VIRCA (Virus Resistant Cassava for Africa) project is a collaborative program between the Donald Danforth Plant Science Center, USA the National Crops Resources Research Institute, Uganda and the Kenya Agricultural Research Institute, Kenya. VIRCA is structured to include all aspects of the intellectual property, technology, regulatory, biosafety, quality control, communication and distribution components required for a GM crop development and delivery process. VIRCA's goal is to improve cassava for resistance to the viral diseases cassava brown streak disease (CBSD) and cassava mosaic disease (CMD) using pathogen-derived RNAi technology, and to field test, obtain regulatory approval for and deliver these products to small landholder farmers. During Phase I of the project, proof of concept was achieved by production and testing of virus resistant plants under greenhouse and confined field trials in East Africa. In VIRCA Phase II, two farmer-preferred varieties will be modified for resistance to CBSD and CMD, and lead events identified after molecular and field screening. In addition to delivery of royalty-free improved planting materials for farmers, VIRCA capacity building activities are enhancing indigenous capability for crop biotechnology in East Africa.