Volume 1 Number 1 2007

CONTENTS AND ABSTRACTS

L. Alexander Lyznik, William Gordon-Kamm, Huirong Gao, Christopher Scelonge (USA) Application of Site-Specific Recombination Systems for Targeted Modification of Plant Genomes (pp 1-9)

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Special Feature: Biotechnology has already become a major driving force in the creation of new agricultural products. Transgenic plants bring benefits to farmers, consumers, and reduce a heavy toll inflicted on the environment by conventional agricultural practices. Yet debate continues around the issues related to the production of transgenic organisms, including plants. New transformation technologies being developed, such as site-specific recombination systems, may address some concerns and, at the same time, streamline procedures utilized in the production of transgenic crops. Here, we review progress in the implementation of emerging technologies based on site-specific recombination for plant transgenic research. We also comment on potential improvements that can make such technologies increasingly applicable for transgenic plant production.

Chongmei Dong, Peter Sharp (Australia) Oligonucleotide-directed Gene Repair: Promises and Limitations for Plant Gene Modification (pp 10-16)

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Invited Mini-Review: As a new technology for specific mutation induction, oligonucleotide-directed gene repair was investigated for the last decade, in addition to traditional gene transformation and gene targeting based on homologous recombination. This targeted gene repair relies on induction of cellular endogenous DNA repair mechanisms to produce predicted gene alterations, so that the altered gene is expressed under the control of its native promoter. Early successful application of this technology in cultured animal and human cells, and in tobacco and maize cells, promised a new approach for human gene therapy, crop improvement and functional genomics. However, large variations in the repair frequencies and a lack of reproducibility of the early experiments made the technology controversial. Several factors, such as the quality and the delivery of oligonucleotides into cells, were identified as influencing the frequency of repair, while the main obstacle of the technology is a lack of detailed knowledge of gene repair mechanisms at the molecular level. Much research has recently focused on the mechanisms, such as identifying specific DNA repair pathways, and assessing the influence of the cell cycle in the regulation of the repair process. Before the mechanisms are demonstrated and a high repair frequency can be achieved consistently, oligonucleotide-directed gene repair is unlikely to be a favored method for crop improvement and functional genomics in plants. Emerging technologies such as zinc-finger nuclease assisted gene targeting and TILLING may be more efficient methods for crop improvement and functional genomics.

Irina E. Dodueva, Nadezhda V. Frolova, Ludmila A. Lutova (Russia) Plant Tumorigenesis: Different Ways for Shifting Systemic Control of Plant Cell Division and Differentiation (pp 17-38)

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Invited Review: Investigation of plant tumorigenesis is a way to clarify the mechanisms of systemic control of plant cell division and differentiation. Two spacious groups of plant tumors are exist. The first are tumors which are induced by different pathogens. Most known tumorigenic plant pathogens are the representatives of the Agrobacterium genus which use a specific way to genetically colonize and insert a plasmid DNA fragment (T-DNA) into the host genome. The second group includes spontaneous tumors that form on plants with certain genotypes: interspecific hybrids in some genera (most known of them are Nicotiana interspecific hybrids), inbred lines of some cross-pollinating species (e.g. tumorous lines of radish Raphanus sativus var. radicula Pers.), tumorous mutants and transgenic plants (e.g. pas and tsd mutants of Arabidopsis thaliana, CHRK1-supressed transgenic Nicotiana tabacum plants). The cause for all types of plant tumorigenesis is deviations in the metabolism and/or signaling of two main groups of phytohormones - cytokinins and auxins. These hormones take part in the control of the plant cell cycle via regulation of cyclins and cyclin-dependent kinase gene expression. In this review we examine different ways in which the cytokinin/auxin balance shifts for some types of plant tumors.

André M. Murad, Patrícia B. Pelegrini, Simone M. Neto, Octavio L. Franco (Brazil) Novel Findings of Defensins and their Utilization in Construction of Transgenic Plants (pp 39-48)

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Invited Review: Defensins are small peptides with a common structure, the cysteine-stabilized a/b motif. These molecules are found in diverse organisms, such as plants, mammals and insects and can be active against a wide range of pathogens, including fungi and bacteria. Moreover, defensins are also able to inhibit digestive enzymes, protein synthesis and the activity of ion channels. In this field, defensins have also become an alternative strategy for pest biocontrol, being active against several insects such as bean bruchids. Hence, the development of biotechnology has led to the application of defensins in plant improvement, enhanced pest resistance and increased crop production. Furthermore, they are also being used in the pharmaceutical field for development of novel antibiotics and fungicides active against pathogenic microorganims. This review describes the latest information in defensin structure and function, and also brings insights for this peptide’s biotechnological use, through transgenic strategies, in agriculture and pharmacy.

Antanas V. Spokevicius, Josquin F. G. Tibbits, Gerd Bossinger (Australia) Whole Plant and Plant Part Transgenic Approaches in the Study of Wood Formation - Benefits and Limitations (pp 49-59)

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Invited Review: Studying the molecular basis of wood formation (secondary xylogenesis) in trees is complicated by a suite of biological factors that place trees on top of the list of ‘most cumbersome study organisms’. The identification of genes suspected to be involved in wood formation has advanced rapidly through the application of modern genomics tools such as large-scale gene expression studies and, to a lesser extent, through studies which aim to associate DNA sequence polymorphism with phenotypic variation. Transgenesis provides a powerful complementary approach enabling specific functional gene assessment at specific stages of wood formation. Genetic transformation as a tool to understand plant development has been widely used in annual plant species and, with the development of new molecular and culturing techniques, is becoming increasingly available for the study of woody perennials. The generation of full transgenic trees allows the study of individual gene effects on a whole tree basis, albeit only in few individuals, while new plant part methods for in vitro culturing of specific cells or tissue types enables analysis during defined developmental stages in isolation. Also, new plant part in vivo transformation methods that directly target cambial tissue in tree stems and developing vascular tissue in dormant lateral buds allow the analysis of independent transformed somatic wood sectors against surrounding control tissue within a single plant, and within short time frames. Here we review the more traditional whole plant transgenic systems and the recently developed plant part transgenic systems with the aim of identifying benefits and limitations of each approach in the study of wood formation.

M. I. Chumakov (Russia) Agrobacterium-Mediated Plant Transformation under in Planta Conditions (pp 60-65)

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Invited Mini-Review: This review describes methods of Agrobacterium-mediated T-DNA transfer to plant vegetative and generative cells under in planta, ex planta and “floral dip” conditions, including designing and testing methods that require the in vitro cultivation of transgenic plant cells and tissues. At present, most methods of Agrobacterium-mediated transformation are based on the coincubation of plant vegetative organs and tissues (leaves, roots, stems, or meristems) with bacterial cell suspensions. Adult plants are then regenerated from the cultivated cells or tissues. This approach gives rise to chimeric transformants and bottlenecks with the regeneration from the cultivated cells or tissues of some monocotyledonous plants. Alternatively, T-DNA integrates into the plant genome as a result of treatment of the male and female plant gametophytes with Agrobacterium cells containing activated vir genes by using the “floral dip” method and its variations. Since the transformation frequency is not sufficiently high, especially for monocotyledonous plants, factors affecting the transformation frequency and the Agrobacterium-mediated T-DNA transfer mechanism have been analyzed.

Eiichiro Ono, Toru Nakayama (Japan) Molecular Breeding of Novel Yellow Flowers by Engineering the Aurone Biosynthetic Pathway (pp 66-80)

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Invited Review: Aurone flavonoids confer a bright yellow color to flowers, such as snapdragon (Antirrhinum majus). A. majus aureusidin synthase (AmAS1), a polyphenol oxidase, was identified as the key enzyme catalyzing the oxidative formation of aurones from chalcones. To date, all known PPOs have been found to be localized in plastids, whereas flavonoid biosynthesis is thought to take place on the cytoplasmic surface of the endoplasmic reticulum. Interestingly, AmAS1 is transported to the vacuole lumen, but not to the plastid, via ER-to-Golgi trafficking. A sequence-specific vacuolar sorting determinant is encoded in the 53-residue N-terminal sequence of the precursor, demonstrating the first example of the biosynthesis of a flavonoid skeleton in vacuoles. Transgenic flowers overexpressing AmAS1, however, failed to produce aurones. The identification of A. majus chalcone 4’-O-glucosyltransferase (UGT88D3) showed that the glucosylation of chalcone by cytosolic UGT88D3 followed by oxidative cyclization by vacuolar aureusidin synthase is the biochemical basis of the formation of aurone 6-O-glucosides in vivo. Co-expression of the UGT88D3 and AmAS1 genes was sufficient for the accumulation of aurone 6-O-glucoside in transgenic flowers, suggesting that glucosylation facilitates vacuolar transport of chalcones. Furthermore, their co-expression, combined with the knockdown of anthocyanin biosynthesis by RNAi, increased aurones and decreased other flavonoids, resulting in yellow flowers. These findings not only demonstrate that aurones, flavones, and anthocyanins are derived from the same chalcone pool but also open transgenic strategies to generate yellow flowers for major ornamental species lacking this color variant, such as geraniums, cyclamens, campanulas, and saintpaulias, beyond genetic constraints.

Armin Hallmann (Germany) Algal Transgenics and Biotechnology (pp 81-98)

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Invited Review: Transgenesis in algae is a complex and fast-growing technology. Selectable marker genes, promoters, reporter genes, transformation techniques, and other genetic tools and methods are already available for various species and currently about 25 species are accessible to genetic transformation. Fortunately, large-scale sequencing projects are also planned, in progress, or completed for several of these species; the most advanced genome projects are those for the red alga Cyanidioschyzon merolae, the diatom Thalassiosira pseudonana, and the three green algae Chlamydomonas reinhardtii, Volvox carteri and Ostreococcus tauri. The vast amount of genomic and EST data coming from these and a number of other algae has the potential to dramatically enlarge not only the algae’s molecular toolbox. A powerful driving force in algal transgenics is the prospect of using genetically modified algae as bioreactors. In general, today’s non-transgenic, commercial algal biotechnology produces food additives, cosmetics, animal feed additives, pigments, polysaccharides, fatty acids, and biomass. But recent progress in algal transgenics promises a much broader field of application: molecular farming, the production of proteins or metabolites that are valuable to medicine or industry, seems to be feasible with transgenic algal systems. Indeed, the ability of transgenic algae to produce recombinant antibodies, vaccines, insecticidal proteins, or bio-hydrogen has already been demonstrated. Genetic modifications that enhance physiological properties of algal strains and optimization of algal production systems should further improve the potential of this auspicious technology in the future.

Sung Hyun Cho (Korea/USA), Ralph S. Quatrano (USA), Jeong Sheop Shin (Korea) Transgenesis of Physcomitrella patens (pp 99-103)

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Invited Mini-Review: The moss Physcomitrella patens has a simple life cycle, relatively few cell types during gametophytic growth, similar responses to plant growth regulators and environmental factors as seed plants, high regeneration capacity after homogenization, and dominant haploid generation, and as such, is an excellent experimental system. In addition to being able to use most molecular and biochemical methodologies, one can apply efficient gene targeting techniques to study gene function. For transformation, polyethyleneglycol-mediated protoplast transformation and particle bombardment-mediated biolistic delivery method are both efficient methods to introduce genes into gametophytic tissue. Finally, the genome of P. patens genome has been sequenced and assembled.

Stefanie Goedeke, Goetz Hensel, Eszter Kapusi, Manfred Gahrtz, Jochen Kumlehn (Germany) Transgenic Barley in Fundamental Research and Biotechnology (pp 104-117)

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Invited Review: Barley represents both a useful experimental model for a number of small-grain cereals as well as an agronomically important crop for feed and food production. In recent years, a vast amount of different barley genetic resources has been generated and collected worldwide. Together with these resources, the development of reliable transformation technologies has stimulated a variety of approaches to functional gene analysis and genetically engineered breeding lines. The technical details of the gene transfer process are central to the establishment of powerful transformation technology. In contemporary methods, agrobacteria are employed as naturally evolved and artificially optimized vehicles to integrate recombinant DNA into the barley genome. In addition, essentially three types of regenerable targets for stable Agrobacterium-based DNA-transfer are particularly useful in barley, each having specific advantages. While the use of immature zygotic embryos results in the highest efficiency of transgenic plant formation, androgenetic pollen cultures permit a more rapid production of true-breeding transgenic lines, and isolated ovules allow for the generation of transgenic plants without use of a selectable marker. Interestingly, the latter two systems are exclusively available in barley to date. In this paper, a current overview on barley transformation technologies is presented, including information on vector systems, gene transfer methods and targets, transferred coding and regulatory DNA-sequences as well as methods to generate barley without unnecessary recombinant DNA integrated in its genome. Moreover, transformation-based approaches to genetically improve barley and functionally characterise DNA-sequences associated with various aspects of crop performance are comprehensively surveyed. Further on, it is demonstrated that barley grains constitute a promising production platform for molecular farming.

Toshihiko Komari, Yuji Ishida, Yukoh Hiei (Japan) Transgenic Rice (pp 118-128)

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Invited Review: Rice is a remarkable plant and has been the staple food for nearly half of the world population as well as an excellent model plant in molecular biology and genomics. Genetic transformation has been an indispensable technique both in rice improvement and in basic studies ever since it became possible about two decades ago. Electroporation or polyethylene glycol-mediated transformation of protoplasts was employed in the beginning and followed by particle bombardment. Efficient methods employing Agrobacterium tumefaciens were then developed in the mid 1990s. Many of the gene transfers to rice have been mediated by A. tumefaciens for the last 10 years. Targets in improvement of rice crops include agronomic traits such as high yield, resistance to disease and insects, and tolerance to drought, low and high temperature, high salinity, and herbicides, modification of nutritional and quality factors such as vitamins, minerals, protein, lipid and starch, and capability of producing specific proteins and other metabolites. The scope of rice transformation experiments in basic studies is also complex, covering analysis of gene functions, assays of promoters, complementation of mutations, and tests of tissue culture protocols. Rice has especially been valuable in the examination of genes from other cereals and in the development of genomic resources like large-scale libraries of transformants with T-DNA insertions. Transformation technology has been continuously optimized to support diverse applications so that wider genotypes can be transformed at a higher efficiency. In this review, key developments in the studies of transgenic rice are reviewed with an emphasis on recent achievements.

Gábor Kocsy, Livia Simon-Sarkadi, G?bor Galiba (Hungary), Jacoba A. de Ronde (South Africa) Transformation of Soybean and Use of Transgenic Lines in Basic and Applied Research (pp 129-144)

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Invited Review: Research on transgenic soybean ensures the generation of new data for basic and applied research. Successful transformation of soybean was achieved by both particle bombardment and Agrobacterium-based methods. The introduction of transgenes is a powerful tool for increasing resistance to biotic and abiotic stresses. Thus, resistance to viral and fungal infection, nematodes and insects, tolerance to herbicides as well as drought and high temperature can be increased using transformation. In the case of the industrial use of soybean oil, the alteration of fatty acid composition widened the range of potential applications. Yield quality was also improved by changing the amino acid composition in order to fulfil the requirement necessary for soybean to be used as food or feed. The function-, organ- or developmental stage-specific expression changes of several genes were studied in transgenic soybean. Suppressed or increased expression of genes allowed the determination of the possible regulatory or functional role of their products. Up to now the desired traits have been manipulated in soybean mainly by modification of the expression of structural genes. However, in the case of abiotic stress tolerance, determined by several genes, even more success could be achieved in the future if the expression of whole regulons might be changed by the genetic manipulation of the corresponding transcription factors.

Mutasim M. Khalafalla (Sudan), Hany A. El-Shemy (Egypt) Transgenic Azuki Bean Approaches (pp 145-149)

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Invited Mini-Review: The first transgenic plants were reported in 1983. Since then, many recombinant proteins have been expressed in several important agronomic species of plants including tobacco, corn, tomato, potato, banana, alfalfa and canola. The choice of plant system was initially driven by convenience and ability to develop high frequency, routine and reproducible regeneration and genetic transformation systems. For this reason, azuki bean [Vigna angularis (Willd.) Ohwi & Ohashi] recently emerged as important transgenic grain legume crop. Azuki bean genetic transformation has taken rapid strides since the first transgenic azuki bean plant was produced 10 years ago. During the last 5 years, tremendous progress has been made to develop a high frequency, routine, and reproducible genetic transformation protocol for azuki bean through Agrobacterium-mediated transformation technology. This technology has been applied to produce azuki bean plants that withstand several a biotic stresses, as well as to gain tolerance against various pests and diseases. In addition, quality improving and increased nutritional value traits have also been introduced into azuki bean. Most of these gains were not possible through conventional breeding technologies. Moreover, using genetic transformation technology, azuki bean could be emerged as an important leguminous model plant providing the framework within which the molecular mechanisms that underlie the grain legume-specific character can be clarified.This review is an attempt to summarize the progress in transgenic azuki bean technology, with particular emphasis on agronomic and nutritional traits.

Xianlong Zhang, Shuangxia Jin (China) Transgenic Cotton: An Overview (pp 150-162)

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Invited Review: The first transgenic cotton having insect or herbicide resistance was released to the field in 1996 in the United States. Since then the rapid increase in transgenic cotton acreage within 10 years attests to the overall success of agricultural biotechnology. This review article provides an overview of genetically modified cotton and its application in agricultural production. We first critically review cotton tissue culture as the basic work of biotechnology. Then, three main transformation methods, namely, Agrobacterium-meditated, particle bombardment and pollen tube-pathway are described in this paper. The performance of transgenic cotton plants engineered for insect, disease, herbicide resistance and fibre improvement is reviewed from a perspective of the benefits and limitations. Finally, recent progress in plastid engineering research of cotton is briefly mentioned. Cotton genetic engineering shows great potential to enhance breeding programs by introducing novel traits that have eluded more traditional plant improvement methods and therefore will likely play an increasingly important role in the genetic improvement of cotton in the future.

Murugesan Dhandapani, Doo Hwan Kim, Seung-Beom Hong (Korea) Transgenic Plant Development in Catharanthus roseus: Limiting Factors and Scope (pp 163-168)

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Invited Mini-Review: This review deals with the factors limiting for the plant regeneration and genetic transformation of Catharanthus roseus (L.) G. Don. Despite its importance in producing pharmaceutically valuable terpenoid indole alkaloids, the lack of a reliable C. roseus regeneration system at a high frequency is currently a bottleneck step for the transgenic plant development. The efficiency of Agrobacterium-mediated transformation is dependent on the type of explant, explant age, pre-culture and co-culture period, vir genes and antioxidants supplementation and Agrobacterium strains. In addition, the disadvantages of negative selection markers, the utility of GFP as a visual selection marker, and the advantages of positive selection markers such as phosphomannose isomerase, tryptophan decarboxylase and feedback-resistant anthranilate synthase are discussed along with selection agents to obtain high frequency genetic transformation. To optimize the factors that are discussed in this review may successfully lead to transgenic C. roseus for the metabolic engineering of terpenoid indole alkaloids.

Gregorio Godoy-Hernández, María de Lourdes Miranda-Ham (Mexico) Marigold Biotechnology: Tissue Culture and Genetic Transformation (pp 169-174)

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Invited Mini-Review: Members of the Tagetes genus include important floricultural (cut-flower) and ornamental (pot and garden) crops, as well as plants of medicinal and ethno-pharmacological interest. Despite the use of many of these plants in the extraction of important secondary metabolites and essential oils, the greatest biotechnological emphasis has been on their in vitro tissue culture and micropropagation. Few studies have been conducted on genetic transformation, with those primarily focused on increasing yield of compounds in plants. However, the application of genetic transformation methodology requires the development of efficient techniques, not only for the transfer of foreign genes into plant cells, but also for the regeneration of whole, fertile plants from the transformed cells. Thus, the development of suitable methods for regeneration is one of the main prerequisites for genetic improvement by biotechnologic means. The purpose of our review is to describe the approaches, via organogenesis or embryogenesis, that have been applied to regenerate whole marigold (Tagetes erecta L.) plants and the current status of targeting genes, whether via Agrobacterium tumefaciens or biobalistics. The advances, applications and limitations of marigold biotechnology are discussed.

Tianchi Wang, Kui Lin-Wang (New Zealand) High Throughput Transformation of Actinidia: A Platform for Kiwifruit Functional Genomics and Molecular Breeding (pp 175-184)

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Invited Review: Plant transformation is an important tool for charactering the function of genes that will assist in the breeding of novel cultivars. Establishing a transformation platform, which can produce transformants in a highly efficient, reliable and reproducible way, is a prerequisite to using this tool for both genetic breeding programmes and functional genomic analysis. Agrobacterium-mediated transformation is still the most effective mechanism for transforming genes into the fruit species Actinidia (kiwifruit). Great progress has been made in Actinidia transformation since the first transgenic kiwifruit plants were reported 17 years ago. This progress includes expression from the fruit-related actinidin promoter, development of high transformation efficiency, the choice of Agrobacterium strain, various genes of interest, different species and field trials of transgenic plants. To date, transformation systems have been developed for three Actinidia species for varying purposes. The emerging scientific revolution, sparked by genomics-based technologies, has produced enormous amounts of gene sequence information. An Actinidia database of over 130,000 expressed sequence tags (ESTs) has been developed to exploit the genetic potential of kiwifruit. The platform of high throughput transformation of Actinidia has been set up for characterizing kiwifruit genes from the EST database in an efficient way. A range of factors, which are associated with Agrobacterium-mediated transformation systems, will be discussed. An overview will be provided on the use of transgenic technology and the new emerging concepts of transformation techniques.

Leena Tripathi, Jaindra Nath Tripathi (Uganda), Irie Vroh-Bi (Nigeria) Bananas and Plantains (Musa spp.): Transgenics and Biotechnology (pp 185-201)

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Invited Review: Bananas and plantains (Musa spp.) are the 4^th world’s most important food crop after rice, wheat and maize in terms of gross value of production. They are major staple food and source of income for millions of people in tropical and sub-tropical regions. Most of the bananas grown worldwide are produced by small-scale farmers for home consumption or for sale in local and regional markets. Many pests and diseases have significantly affected Musa cultivation. Biotechnology and transgenic technology, together with conventional methods can assist in overcoming these problems in developing new banana cultivars. Some successes in genetic engineering of Musa have been achieved, enabling the transfer of foreign genes into the plant cells. The transgenic approach shows potential for the genetic improvement of bananas using a wide set of transgenes currently available which may confer resistance to pests and diseases. The use of appropriate constructs may allow the production of pest and disease resistant plants in a significantly shorter period of time than using conventional breeding, especially if several traits can be introduced at the same time. Tissue culture techniques like somatic embryogenesis, micropropagation and embryo culture are used for germplasm exchange, conservation and generation of hybrid materials. Biotechnology also provides great prospects for Musa improvement through genomics-based approaches for gene discovery, candidate gene validation, development of molecular markers and their utilization to assist classical breeding programs. This article discusses the application of transgenic technology and biotechnology for sustainable production of bananas and plantains.

Wenwu Guo, Dingli Li, Yanxin Duan (China) Citrus Transgenics: Current Status and Prospects (pp 202-209

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Invited Mini-Review: Citrus is one important fruit crop in China and worldwide. Genetic transformation with more recently cloned target genes is an effective alternative for citrus improvement. In the past years, transformation systems using several explant sources such as protoplasts, embryogenic calluses, epicotyl segments and mature tissues have been well established and optimised. Selectable marker genes i.e. GUS and GFP are extensively used in citrus. Protoplast and embryogenic callus as explants are convenient and available at any time for transformation of seedless cultivars. Epicotyl segments as explants from seed germination are only available for seedy cultivars. Agrobacterium-mediated transformation is the most extensively used one with a few reports on PEG or electroporation-mediated protoplast transformation. With the optimized regeneration systems, genetic transformation with target genes such as CTV coat protein, green fluorescent protein (GFP, an in vivo visual marker), LFY, AP1, CiTF (to shorten juvenility), peptide D and Xa21 (for potential citrus bacterial canker resistance), Barnase (to induce seedless fruit) and abiotic stress related genes, was conducted and numerous transgenic plants were regenerated from many citrus cultivars. Transgenic lines containing the GFP gene are also being used as a visual marker in citrus somatic fusion for several purposes. The prospects of transformation for citrus improvement are discussed.

Nobuhito Mitani (Japan) Transgenic Trifoliate Orange (Poncirus trifoliata L. Raf.) (pp 210-214)

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Invited Mini-Review: Trifoliate orange (Poncirus trifoliata L. Raf.) is the only recognized species of Poncirus, which is closely related to Citrus. In Japan, trifoliate orange has been used as a rootstock for the cultivation of most citrus cultivars because many citrus cultivars are compatible with this species, which is cold-tolerant and resistant to the tristeza virus. However, some cultivars require a more diverse range of potential rootstocks. Genetic transformation of citrus genotypes by various methods has been reported, including Agrobacterium-mediated transformation, polyethylene glycol (PEG)-mediated transformation, and particle bombardment. Agrobacterium-mediated transformation using epicotyl segments has been most widely used for genetic transformation of trifoliate orange in recent years. In addition, transgenic hybrids have been obtained, including Carrizo citrange (C. sinensis ´ P. trifoliata) and Swingle citrumelo (C. paradisi ´ P. trifoliata). Introduction of foreign genes into trifoliate orange and its hybrids has resulted in diverse plant characteristics, and the suitability of these transgenic plants as rootstocks must be examined. This manuscript reviews the genetic transformation of trifoliate orange, and discusses the transformation procedures and the characteristics of the transgenic plants.

Yoshiko Koshita (Japan) Transgenic Japanese Persimmon (Diospyros kaki L.) (pp 215-218)

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Invited Mini-Review: Introduction of foreign genes into the genome of Japanese persimmon (Diospyros kaki L.) is mainly performed using a disarmed strain of Agrobacterium tumefaciens that carryies a binary vector. Plant regeneration systems from callus derived from leaf disc or hypocotyl segments prepared from seed had been adapted to Agrobacterium-mediated transformation. Several foreign genes have been introduced into the genome of Japanese persimmon by Agrobacterium-mediated gene transfer, and the characteristics of the transformants revealed that agriculturally important characteristics, such as insect resistance, salt tolerance, dwarfness, and disease resistance, had been successfully vested to Japanese persimmon. On the other hand, not only a disarmed strain of A. tumefaciens but also the wild-type of Agrobacterium rhizogenes has been used in the natural genetic transformation of Japanese persimmon. The transformants showed different growth from non-transformants, such as dwarfness, short internode and decreased rooting ability. These studies showed that genetic transformation of the Japanese persimmon is one of the most effective ways to improve the characteristics of this species. The procedure of genetic transformation and the characteristics of transgenic plants are discussed in this review.

Margit Laimer (Austria) Transgenic Grapevines (pp 219-227)

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Invited Review: Grapevine (Vitis vinifera) is one of the most valuable horticultural crops in the world. Traditionally the geographic distribution was in areas with a Mediterranean climate, but in the last decades the production area expanded to temperate areas in the Northern and Southern hemispheres. A long-term objective of grapevine breeding is to increase cultivar resistance to plant pathogens resulting in the reduced use of labour and fungicides with benefits for winegrowers, consumers and the environment. To enhance the potential of existing cultivars as well as to develop new cultivars resistant to biotic and abiotic stress factors, to overcome limiting climatic conditions, to improve traits of economic value like colour, reduced browning, improved yield, by taking advantage of the increasing knowledge available in grapevine genetics, genetic transformation is a key technology. In this review the technical approaches for creating transgenic grapevines through transformation, selection and regeneration will be discussed, beginning with the approach for virus resistance breeding, since it provided the first transgenic plants with agronomically interesting traits. The current stage of transgenic grapes with constructs conferring resistance to fungi and bacteria will be highlighted. Finally, an outlook on thoughts about new construct design in the view of discussions about safety aspects raised in public perception and hindering acceptance will be presented.

Reda E. A. Moghaieb (Egypt), Hirofumi Saneoka (Japan), Sawsan Samy Youssef, Ahmed M. EL-Sharkawy (Egypt), Kounosuke Fujita (Japan) Improvement of Salt Tolerance in Tomato Plant (Lycopersicon esculentum) by Transformation with Ectoine Biosynthetic Genes (pp 228-232)

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Original Research Paper: Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) biosynthetic genes (ect ABC) derived from the halophilic bacterium Halomonas elongata were introduced into tomato (Lycopersicon esculentum) using an Agrobacterium-mediated gene-delivery system. Stable integration of the ectoine genes into the regenerated plant genomes was confirmed by PCR and Southern blot analyses. Expression of these genes was detected in the transgenic tomato plants by Northern blot analysis. The transgenic plants exhibited the normal growth characteristics of the non-transgenic plants. The concentration of ectoine increased with increasing salinity, and the increase was higher in the roots than in the leaves. The present data indicates that the turgor values of the ectoine transgenic tomato lines increased with increasing salt concentration. The data suggests that the accumulation of ectoine in transgenic tomato plants contributed to the maintenance of osmotic potential of the cells.

Nedeljka Rosic (Australia) Agrobacterium rhizogenes-Mediated Transformation of Medicinal Plants from the Family Rhamnaceae (pp 233-236)

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Invited Mini-Review: A number of plants can be successfully transformed by Agrobacterium rhizogenes through the transfer of T-DNA from agrobacteria to the plant genome. Transgenic tissue ? hairy roots ? is produced as a result of the transformation process. This organized, genetically stable, hormone-independent transformed tissue is capable of accomplishing complex metabolic pathways, including biosynthesis and accumulation of various secondary metabolites. Somaclonal variation is often observed among the hairy root cultures. The highly-productive hairy root lines, containing a large amount of important metabolites can be selected and grown in vitro on hormone-free media for a long period of time, whilst preserving their biosynthetic capacities. Consequently, during the last decade, hairy root cultures have been recognized as an excellent system for in vitro generation of a large biomass of transgenic tissue that could be utilized for the extraction of desired metabolites or even in the development of new compounds through novel metabolic pathways. Species from the family Rhamnaceae are well known for their capacity to synthesize the aromatic carbohydrates, anthraquinones (AQs). These metabolites with laxative action are traditionally extracted from the bark of Frangulae cortex. Applying a genetic engineering approach, the hairy root cultures of Rhamnus fallax open a convenient alternative for the production of increased amount of medically important metabolites such as AQs while protecting natural recourses and environment.

Janna Ong Abdullah, Wilson Thau Lym Yong (Malaysia) Melastomataceae: Inherent Economical Values Substantiating Potential Transgenic Studies in the Family (pp 237-243)

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Invited Mini-Review: Melastomataceae is one of the largest families of flowering plants. It is comprised of approximately 4,500 species in less than 200 genera, distributed in tropical and sub-tropical regions around the world. Limited fossil records have resulted in different hypothetical viewpoints on the biogeographical history of this family. Despite uncertainties in the monophyly of this family, the most obvious synapomorphy is the acrodromous leaf venation. Members of this family consist of diverse vegetative forms: from a few centimeters tall plant to woody creepers, to shrubs and even to several meters tall tree. Even though it has vast members, widely distributed worldwide, this family is one of the least studied or exploited. For those members fortunate enough to gain the attention of scientists worldwide, the outcomes have shown that members of this family have diverse valuable properties: ornamental, medicinal, herbal, phytoremediative, hinting that there might be others with new values that have yet to be explored in this huge family. Despite a great deal of research has been carried out to improve plant traits via genetic engineering in the plant kingdom, this technology has barely scratched the surface of Melastomataceae. A lack of critical information and detailed studies on the molecular aspects of this family might have hindered the progress in this aspect. This minireview focuses on the limited transgenic work that has only recently been explored in this family, with suggestions for future research, and also reviews the biochemical studies that have been conducted extensively on members of this Melastomataceae family throughout the decades.

Xiangming Xie, Mingjia Yang, Xiaoqing He (China) Somatic Embryogenesis and Genetic Engineering of Acacia Species (pp 244-249)

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Invited Mini-Review: Acacia species are widely dispersed in tropical and sub-tropical regions of the world and many of these species are important for fuelwood, timber, shelterbelts, soil improvement and landscaping and garden ornamentals. A. mangium, A. crassicarpa, A. auriculiformis, and A. hybrid (A. mangium × A. hybrid ) have become a preferred fibre source for the pulp and paper industry because of their rapid growth, high pulp yield, high fibre quality and their ability to thrive in degraded soils. These tree species have been expandingly planted for reforestation, reclamation of wasteland, and industrial material production in Southeast Asia, especially in Indonesia, and in China. Thus the increasingly expanding plantation of these species requires clonal propagation of elite clones and efficient techniques for in vitro regeneration. However, the recalcitrance of regeneration, long generation time of trees, and the prolonged period needed for evaluation of mature traits are strong limitations for classical breeding programs in Acacia. The development of methods for in vitro regeneration including micropropagation, organogenesis and embryogenesis and genetic engineering has provided a new alternative for producing Acacia elite trees or modified genotypes. This review focuses on somatic embryogenesis in Acacia, and briefly presents research advances in genetic engineering in Acacia.

Setsuko Komatsu (Japan) Artificially Controlling Morphogenesis by Altering Plant Function Based on the Elucidation of Molecular Mechanism for Brassinosteroids and Gibberellins Signal Transduction (pp 250-255)

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Invited Mini-Review: Brassinosteroids (BRs) and gibberellins (GAs) are essential plant growth-promoting natural products that are required for normal plant elongation and during development. The underlying molecular mechanisms for signal transduction involving these phytohormones will be elucidated using the methods of molecular genetics and protein chemistry, and information from the rice genome. Altering plant function will help the next generation of rice plants with the ideal grass type having high-yield and improved grain quality, which will greatly contribute to and enhance agricultural productivity. In this review, we discuss the molecular mechanism of BR- and GA-regulated genes based on the phonotype of our constructed transgenic rice.