Biotechnological aspects of plum pox virus

Plum pox potyvirus (PPV), the causal agent of a devastating disease that affects stone fruit trees, is becoming a target of intense studies intended both to fight against viral infection and to develop practical applications based on the current knowledge of potyvirus molecular biology. This review focuses on biotechnological aspects related to PPV, such as novel diagnostic techniques that facilitate detection and typing of virus isolates, strategies to implement pathogen-derived resistance through plant transformation, the potential use of genetic elements derived from the virus, and the recent development of PPV-based expression vectors.     

化学诱导表达系统及其在植物中的应用

化学诱导启动子可以在特定时间和部位激活或抑制目的基因的表达。目前,已经建立了多种化学诱导表达系统,用于基因功能分析、无标记植物转化、特定位点DNA切除、育性恢复和RNA沉默等方面的研究。化学诱导表达系统为基础分子生物学研究和生物技术应用提供了强有力的工具,将大大加快植物转基因技术的应用。     

Forest biotechnology: Innovative methods, emerging opportunities

Summary The productivity of plantation forests is essential to meet the future world demand for wood and wood products in a sustainable fashion and in a manner that preserves natural stands and biodiversity. Plantation forestry has enormously benefited from development and implementation of improved silvicultural and forest management practices during the past century. A second wave of improvements has been brought about by the introduction of new germplasm developed through genetics and breeding efforts for both hardwood and conifer tree species. Coupled with the genetic gains achieved through tree breeding, the emergence of new biotechnological approaches that span the fields of plant developmental biology, genetic transformation, and discovery of genes associated with complex multigenic traits have added a new dimension to forest tree improvement programs. Significant progress has been made during the past five years in the area of plant regeneration via organogenesis and somatic embryogenesis (SE) for economically important tree species. These advances have not only helped the development of efficient gene transfer techniques, but also have opened up avenues for deployment of new high-performance clonally replicated planting stocks in forest plantations. One of the greatest challenges today is the ability to extend this technology to the most elite germplasm, such that it becomes an, economically feasible means for large-scale production and delivery of improved planting stock. Another challenge will be the ability of the forestry research community to capitalize rapidly on current and future genomics-based elucidation of the underlying mechanisms for important but complex phenotypes. Advancements in gene cloning and genomics technology in forest trees have enabled the discovery and introduction of value-added traits for wood quality and resistance to biotic and abiotic stresses into improved genotypes. With these technical advancements, it will be necessary for reliable regulatory infrastructures and processes to be in place worldwide for testing and release of trees improved through biotechnology. Commercialization of planting stocks, as new varieties generated through clonal propagation and advanced breeding programs or as transgenic trees with high-value traits, is expected in the near future, and these trees will enhance the quality and productivity of our plantation forests.     

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.     

Biotechnology: Genetic improvement of sorghum (Sorghum bicolor (L.) Moench)

Summary This report reviews the contributions to the improvement of sorghum (Sorghum bicolor (L.) Moench) through traditional approaches with emphasis on the application of biotechnological methods. Strategies include breeding for higher yield, improved grain quality, and biotic and abiotic stress tolerance. Hybrid development and polyploidy breeding are also discussed. Plant breeders, working in concert with biotechnologists, have developed new powerful tools for plant genetic manipulation and genotype evaluation that will significantly improve the efficiency of plant breeding. Improving sorghum through biotechnology is the latest in a long series of technologies that have been applied to this crop. Five basic tools of technology have been developed for sorghum improvement: (1) in vitro protocols for efficient plant regeneration; (2) molecular markers; (3) gene identification and cloning; (4) genetic engineering and gene transfer technology to integrate desirable traits into the sorghum genome; and (5) genomics and germplasm databases. Reports on studies involving the problems, progress, and prospects for utilizing the biotechnological methods for sorghum improvement are discussed.     

Breeding and biotechnology for improving berry nutritional quality

Breeding and biotechnological approaches are currently used to increase the content of specific bioactive components of plants, but the manipulation of plant metabolism is still not easy to address. There is an increasing awareness that multiple genetic and environmental factors affect production and accumulation of bioactive compounds, but these factors are rarely taken into account when fruit is marketed. Rigorous and unprejudiced evaluation of scientific evidence requires a defined set of criteria and methods of evaluation, particularly when breeding and biotech programs are aimed of producing new varieties with improved nutritional values combined with high plant production efficiency and fruit quality. In order to develop new genotypes and commercial cultivars the availability of new sources of Quality Attributes (QA) and Nutritional Attributes (NA) should be explored. In the strawberry, wild species such as F. virginiana glauca and F. vesca are good sources of bioactive compounds, but in raspberries the introduction of the wild germplasm (R. parvifolium) did not improve the nutritional quality of fruit. The methods available for detecting fruit TAC, combined with TPH and other quality parameters such as sugars, total acidity and fruit color, can be proposed as excellent tools for developing a fast and reliable program for screening large breeding populations for high nutritional quality genotypes. Furthermore, NA can represent a useful tool to facilitate analysis of "substantial equivalence" of transgenic and control derived fruit.     

In vitro culture and genetic engineering of Populus spp.: synergy for forest tree improvement

Populus species and hybrids are intensively cultivated as sources of woody biomass for the forest products industry and for reforestation of lowlands in temperate regions of the world. However, the long generation time of trees, the presence of seasonal dormancy and the prolonged period required for evaluation of mature traits are strong limitations for classical breeding and selection. The development of methods for in vitro culture and genetic engineering has increased the possibility of producing poplar genotypes improved in insect pest resistance, herbicide tolerance, growth rate and wood quality, or reduction in undesirable traits. Poplar has become a model system in forest tree biotechnology due to several useful features: small genome size, short rotation cycle, rapid growth rate and ease of vegetative propagation. The combination of molecular techniques and classical breeding will help create forest trees with positive effects on the environment. However, risks associated with the biotechnological applications (concerning the impact on biodiversity, long-term adaptation, transgene inheritance and stability) should be carefully evaluated and field tests performed with transgenic poplar.     

A new versatile database created for geneticists and breeders to link molecular and phenotypic data in perennial crops: the AppleBreed DataBase

OBJECTIVE: AppleBreed DataBase (DB) aims to store genotypic and phenotypic data from multiple pedigree verified plant populations (crosses, breeding selections and commercial cultivars) so that they are easily accessible for geneticists and breeders. It will help in elucidating the genetics of economically important traits, in identifying molecular markers associated with agronomic traits, in allele mining and in choosing the best parental cultivars for breeding. It also provides high traceability of data over generations, years and localities. AppleBreed DB could serve as a generic database design for other perennial crops with long economic lifespans, long juvenile periods and clonal propagation. RESULTS: AppleBreed DB is organized as a relational database. The core element is the GENOTYPE entity, which has two sub-classes at the physical level: TREE and DNA-SAMPLE. This approach facilitates all links between plant material, phenotypic and molecular data. The entities TREE, DNA-SAMPLE, PHENOTYPE and MOLECULAR DATA allow multi-annual observations to be stored as individual samples of individual trees, even if the nature of these observations differs greatly (e.g. molecular data on parts of the apple genome, physico-chemical measurements of fruit quality traits, and evaluation of disease resistance). AppleBreed DB also includes synonyms for cultivars and pedigrees. Finally, it can be loaded and explored through the web, and comes with tools to present basic statistical overviews and with validation procedures for phenotypic and marker data to certify data quality. AppleBreed DB was developed initially as a tool for scientists involved in apple genetics within the framework of the European project, 'High-quality Disease Resistance in Apples for Sustainable Agriculture' (HiDRAS), but it is also applicable to many other perennial crops.     

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.     

蔷薇科果树类受体激酶的研究进展

类受体激酶(receptor like kinase,RLK)参与调控植物几乎所有的生命活动,是植物生长发育和环境适应的“中央处理器”。该文对近年来国内外有关蔷薇科果树RLK基因鉴定、进化特征及其在各器官生长发育、非生物和生物逆境中的作用及调控机制等方面的研究进展进行了综述。蔷薇科果树基因组中存在数目庞大的RLKs,不同树种间的RLK数目和各亚家族成员数目都存在较大差异,而且蔷薇科果树RLK存在极为普遍的部分重复和串联重复现象,是导致家族成员迅速变化的重要原因。有研究发现,一些RLKs调控蔷薇科果树器官发育和对环境的适应性。在器官发育方面,LRR RLK亚家族成员调控根系发育,CrRLK1L、LysM RLK和LRR RLK亚家族部分成员参与调控果实发育,CrRLK1L亚家族成员参与调控花粉管发育,LRR RLK、LysM RLK、L LEC RLK和B Lectin RLK亚家族部分成员调控蔷薇科果树对生物逆境的适应。今后RLK功能研究可侧重于蔷薇科果树特色性状,通过提高目标基因的筛选和验证的效率,加速主效RLKs的筛选进程,并通过筛选主效RLKs诱导方式和加速分子育种进程等途径,将研究成果应用于实际生产。     

Transformation of fruit trees. Useful breeding tool or continued future prospect?

Regeneration and transformation systems using mature plant material of woody fruit species have to be achieved as a necessary requirement for the introduction of useful genes into specific cultivars and the rapid evaluation of resulting horticultural traits. Although the commercial production of transgenic annual crops is a reality, commercial genetically-engineered fruit trees are still far from common. In most woody fruit species, transformation and regeneration of commercial cultivars are not routine, generally being limited to a few genotypes or to seedlings. The future of genetic transformation as a tool for the breeding of fruit trees requires the development of genotype-independent procedures, based on the transformation of meristematic cells with high regeneration potential and/or the use of regeneration-promoting genes. The public concern with the introduction of antibiotic resistance into food and the restrictions due to new European laws that do not allow deliberate release of plants transformed with antibiotic-resistance genes highlight the development of methods that avoid the use of antibiotic-dependent selection or allow elimination of marker genes from the transformed plant as a research priority in coming years     

基于RNAi技术的转基因植物监管现状及其面临的育种领域的挑战

RNA干扰（RNA interference,RNAi）在植物、动物和真菌的生长、发育、病毒防御和转座子失活中起着至关重要的作用。目前已成功利用RNAi技术培育出抗病虫和品质改良等具有优良性状的生物技术产品,为农业绿色发展提供了强有力的支撑。然而,目前RNAi的相关机制尚未完全明确,基于RNAi技术的转基因植物面临着一些亟待解决的问题,同时,对于RNAi转基因植物应用的安全高效监管也需进一步完善。基于此,对RNAi转基因植物的监管现状及其面临的育种上的挑战进行了综述,并对存在的问题提出了解决建议,以期为RNAi技术进一步应用于农业植物改良育种提供新思路,并为其监管评价提供依据。     

Advances in breeding and biotechnology of legume crops

Legume crops are relevant globally to the feeding and the nutrition of humans and animals because of their relatively high seed content of protein and essential amino acids. Additionally, they are related to sustainable agriculture, considering their ability to associate with atmospheric nitrogen fixing bacteria (Rhizobia). Despite this, several technical constraints of legumes crops have maintained their worldwide production far behind from cereals. This review article focuses in current information about recent advances in breeding and biotechnology of the major leguminous crops. Conventional breeding has mainly focused in improving multiple vegetative and reproductive traits that have associated to distinct heritability values, which reflects how amenable each character is for genetic improvement. Legumes have strongly entered into the genomics era through the complete genome sequencing of several species in the last decade. Moreover, a wealth of tools and techniques of Fabaceae genomics are now available and discussed throughout this article. In addition, there is an increasing amount of quantitative trait loci, candidate genes, and genes associated to abiotic and biotic resistance and to agronomic traits that have been reported, which will potentially allow more rapid progress of legume genetic improvement. Two successful examples of genetically modified legume crops are examined in this paper: glyphosate-resistant transgenic soybean and transgenic common bean resistant to Bean golden mosaic virus. Finally, legumes genomics and breeding programs, using classical breeding methods, marker-assisted selection, and biotechnological tools face a promising momentum for further application of technology and information that could boost their global production.     

果树成花转变途径与调控研究进展

植物成花转变是营养生长向生殖生长转变的过程,木本果树过长的童期严重制约了育种的进程。相对于模式植物,目前对果树成花转变与调控的研究相对较少。因此,了解并掌握果树成花转变的途径及调控方法,对于缩短果树童期、调控开花,加速果树育种具有重要意义。基于近年来国内外相关研究,本文系统总结了果树的成花途径,阐述了果树栽培措施、植物生长调节剂等成花调控方法,以及果树中成花调控的相关基因及网络机制。最后,本文还对以修饰组学为主的多组学以及嫁接和植物生长调节剂在果树成花调控中的研究前景进行了展望。     

Fruit tree genetics at a turning point: the almond example

 The large size and the long generation time of fruit trees generally reduce the possibilities of obtaining genetic information on the transmission and heritability of useful agronomic traits in these species. However, from breeding work carried out with fruit trees, an important amount of data is now available, although large differences are apparent among the different species. There is not much information known about almond compared to what is available on other Prunus fruit species, but more data have been accumulated on it than on most of the other nut trees, thus making almond special among all the temperate fruit and nut species. Only five qualitative traits have been described in almond, with an additional two also possibly qualitative. Heritabilities have been estimated for an important number of quantitative traits, mainly phenological times and fruit characters. Important information is available on molecular markers, including enzymes, RFLPs, RAPDs and other recently developed markers. Linkages, however, have only been established among molecular markers, allowing accurate genetic maps to be built but not yet enabling agronomical characters to be located in these maps, probably because the latter have not been sufficiently studied. The effectiveness of the application of genetic maps in plant breeding will depend on the accuracy of the study of different agronomic traits and their expression, implying more field work and recognition of this work. Ultimately, any new fruit cultivar has to be grown in the field and has to allow the grower to make a profit. Received: 1 June 1997 / Accepted: 1 September 1997     

Restoration of threatened species: a noble cause for transgenic trees

Some of the first applications of transgenic trees in North America may be for the conservation or restoration of threatened forest trees that have been devastated by fungal pathogens or insect pests. In some cases, where resistance has yet to be found in the natural population of a tree species, incorporating genes from other organisms may offer the only hope for restoration. In others, transgenics may play a role as part of an integrated approach, along with conventional breeding or biocontrol agents. American chestnut (Castanea dentata) was wiped out as a canopy species by a fungal disease accidentally introduced into the United States around 1900. Similarly, American elm (Ulmus americana) virtually disappeared as a favored street tree from Northeastern U.S. cities after the introduction of the Dutch elm disease fungus in the 1940s. In both cases, progress has been made toward restoration via conventional techniques such as selection and propagation of tolerant cultivars (American elm) or breeding with a related resistant species (American chestnut). Recently, progress has also been made with development of systems for engineering antifungal candidate genes into these “heritage trees.” An Agrobacterium-leaf disk system has been used to produce transgenic American elm trees engineered with an antimicrobial peptide gene that may enhance resistance to Dutch elm disease. Two gene transfer systems have been developed for American chestnut using Agrobacterium-mediated transformation of embryogenic cultures, setting the stage for the first tests of potential antifungal genes for their ability to confer resistance to the chestnut blight fungus. Despite the promise of transgenic approaches for restoration of these heritage trees, a number of technical, environmental, economic, and ethical questions remain to be addressed before such trees can be deployed, and the debate around these questions may be quite different from that associated with transgenic trees developed for other purposes.     

Field performance of transgenic citrus trees: Assessment of the long-term expression of uidA and nptII transgenes and its impact on relevant agronomic and phenotypic characteristics

ABSTRACT: BACKGROUND: The future of genetic transformation as a tool for the improvement of fruit trees depends on the development of proper systems for the assessment of unintended effects in field-grown GM lines. In this study, we used eight transgenic lines of two different citrus types (sweet orange and citrange) transformed with the marker genes beta-glucuronidase (uidA) and neomycin phosphotransferase II (nptII) as model systems to study for the first time in citrus the long-term stability of transgene expression and whether transgene-derived pleiotropic effects occur with regard to the morphology, development and fruit quality of orchard-grown GM citrus trees. RESULTS: The stability of the integration and expression of the transgenes was confirmed in 7-year-old, orchard-grown transgenic lines by Southern blot analysis and enzymatic assays (GUS and ELISA NPTII), respectively. Little seasonal variation was detected in the expression levels between plants of the same transgenic line in different organs and over the 3 years of analysis, confirming the absence of rearrangements and/or silencing of the transgenes after transferring the plants to field conditions. Comparisons between the GM citrus lines with their non-GM counterparts across the study years showed that the expression of these transgenes did not cause alterations of the main phenotypic and agronomic plant and fruit characteristics. However, when comparisons were performed between diploid and tetraploid transgenic citrange trees and/or between juvenile and mature transgenic sweet orange trees, significant and consistent differences were detected, indicating that factors other than their transgenic nature induced a much higher phenotypic variability. CONCLUSIONS: Our results indicate that transgene expression in GM citrus remains stable during long-term agricultural cultivation, without causing unexpected effects on crop characteristics. This study also shows that the transgenic citrus trees expressing the selectable marker genes that are most commonly used in citrus transformation were substantially equivalent to the non-transformed controls with regard to their overall agronomic performance, as based on the use of robust and powerful assessment techniques. Therefore, future studies of the possible pleiotropic effects induced by the integration and expression of transgenes in field-grown GM citrus may focus on the newly inserted trait(s) of biotechnological interest.     

复合叶面肥对油桃和葡萄生长生理及果树品质的影响

我国北方一些地区因土壤pH高,导致其中有效性微量元素含量很低而不能满足果树植物的正常生长发育,进而导致严重的缺素症。通过土壤直接施肥或通过灌溉水将化肥施入土壤,肥料中的微量元素很快会被土壤固定,因而这些方法不能有效地解决缺素症问题。作者用5年时间研制成功了应用果树植物的系列复合叶面肥,这些叶面肥不仅含有植物所需的大量元素,而且含有微量元素、有机营养及植物生长调节剂;叶面肥的配方也因果树植物种类生长习性的不同而异。以油桃和葡萄为试材,喷施复合叶面肥72h后,叶片中叶绿体含量、硝酸还原酶活性、铁和锌含量显著提高;叶面肥还能增进油桃和葡萄果实品质,促进葡萄枝芽增重,但不会引起葡萄树体徙长。整个生长季节喷施复合叶面肥对树体均有益,但喷施间隔时间不应少7-10d。