Volume 1 Number 2 2007

CONTENTS AND ABSTRACTS

Miguel Angel Gómez Lim (Mexico) Transgenic Plants in Therapeutically Valuable Protein Production (pp 256-266)

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Invited Review: Over the last 15 years a growing number of research groups worldwide have focused on plants as biofactories for the production of heterologous proteins. The reason is that plants provide a number of advantages over conventional recombinant systems including low cost, increased safety and scalable production, among others. The skepticism that received this technology when first envisaged has turned into a cautious optimism. A wide variety of proteins can be produced in plants and they are almost indistinguishable from their native counterparts. Even though there are still several issues that need refining such as boosting expression and ensuring correct post-translational processing and protein stability in plant tissues, molecular farming can potentially provide unlimited quantities of recombinant proteins for use as diagnostic and therapeutic tools. The low cost of plant-based vaccines make them ideal for large-scale programs in poor countries. Vaccinating the nearly 33 million children that each year remain unvaccinated for vaccine-preventable diseases would have profound effects on leveling the health inequities all over the world (Thanavala et al. 2006). It is hoped that the issue of IP does not represent an insurmountable obstacle to this end.

Sean M. Bulley (New Zealand), Mickaël Malnoy (USA/Italy), Ross G. Atkinson (New Zealand), Herb S. Aldwinckle (USA) Transformed Apples: Traits of Significance to Growers and Consumers (pp 267-279)

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Invited Review: The first report of transformed apple plants in 1989 raised expectations for new apple cultivars that would be better tasting, healthier and easier to grow. Although, many different traits have been now been introduced successfully into apple, no transformed cultivars have yet made it to commercial production. Most early reports on transformed apple described ‘proof of concept’ experiments involving the development of regeneration protocols, and the choice of appropriate promoters and selectable markers. More recently the focus has moved onto functional testing of traits of potential commercial interest. These traits can be grouped into two categories: horticultural production traits and fruit-focussed traits. Production traits of interest include bacterial, fungal and pest resistance, dwarfing, propagation, stress resistance, precocity, storage life and self fertility. Examples of fruit-focussed traits include novel health properties, flavour, reduced browning, colour, and reduced allergenicity. This review will consider reports of characters introduced into apple that are useful to growers and consumers, and looks toward future trends, targets and challenges.

Miguel A. Quesada, Carmen Martín-Pizarro, Juan A. García-Gago, Sara Posé, Nieves Santiago, Rafael Sesmero, Fernando Pliego-Alfaro, José A. Mercado (Spain) Transgenic Strawberry: Current Status and Future Perspectives (pp 280-288)

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Invited Mini-Review: Strawberry (Fragaria x ananassa Duch.) world production has increased notably in the last 25 years, mainly due to the development of new cultivars adapted to a wide range of environmental conditions. However, the octoploid nature of this species, its high heterozygosity and the lack of natural resistance to several diseases, hamper the improvement of strawberry by conventional breeding. The high ability of strawberry tissues to regenerate in vitro has allowed the application of transgenic technologies to a high number of commercial cultivars, and genetic engineering appears now as a powerful tool to overcome some of the problems not resolved by conventional breeding. Most transformation studies in strawberry have been devoted to improve fruit quality and pest resistance, two of the major challenges in strawberry production. It is remarkable that positive results have been achieved in the manipulation of both traits and, for example, fruits with an extended postharvest shelf life or plants with enhanced tolerances to several fungal pathogens have been obtained. Strawberry is propagated vegetatively by runners instead of seeds. In spite of this handicap, the technology to eliminate marker genes has been tested successfully in this species. The application of these protocols opens the possibility to develop new transgenic plants free of unnecessary genes, which would facilitate the commercialization of transgenic strawberry. However, as it happens with other fruits, the negative public acceptance of genetically modified food may constrain the future development of transgenic strawberry. In this review, we outline the major trends in genetic transformation of strawberry, focusing on those advances that could be useful in its genetic improvement.

Ravindra B. Malabadi (Portugal/India), K. Nataraja (India) Genetic Transformation of Conifers: Applications in and Impacts on Commercial Forestry (pp 289-313)

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Invited Review: This review highlights methods of gene transfer technology in trees, particularly conifers, and its possible applications to commercial forestry. Two methodologies have mainly been used on conifers: biolistic and Agrobacterium-mediated genetic transformation. DNA transfer using the latter method makes the use of physical processes only to achieve transformation. Since there is no dependence on living organisms, i.e. bacteria, the limitations inherent in organisms such as Agrobacterium tumefaciens do not apply. Information of mechanisms for T-DNA transfer to plant cells by A. tumefaciens is provided, focusing on the role played by the different components of the virulence system. There are many differences between both methods of gene transfer technology, and these are highlighted in conifer transgenic systems. The first transgenic trees produced by biolistics following the cloning of mature Indian pines, Pinus roxburghii, and success in the Agrobacterium-mediated transformation of Himalayan blue pine (P. wallichiana) are some of the major breakthroughs in forest biotechnology that might help in solving current problems of tree breeding. There are many problems in traditional breeding programs such as a slow production due to long maturation times and the slow growth rate of trees; however, biotechnological approaches have the potential to provide significant improvement in tree growth and quality. Despite the large number of marker genes that exist for plants, only a few have been used for most research and improvement studies in conifers. As the production of transgenic plants is labor-intensive, expensive and difficult for most recalcitrant conifers, practical issues govern the choice of selectable marker genes that are used. This review will also focus some of the important applications of this technology, including the deployment of transgenic plants under field conditions, and its impacts on the environment.

Jean-François Trontin (France), Christian Walter (New Zealand), Krystyna Klimaszewska, Yill-Sung Park (Canada), Marie-Anne Lelu-Walter (France) Recent progress in genetic transformation of four Pinus spp. (pp 314-329)

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Invited Review: Advances in conifer tissue culture and genetic transformation offer new opportunities in the field of genetic engineering. Genetic transformation is an important tool for breeders because it allows them to introduce valuable genes that might otherwise be difficult to integrate into elite genotypes. Combined with mass vegetative propagation, such as somatic embryogenesis, regeneration of transgenic conifers could result in accelerated tree improvement. Genetic transformation requires the development of an efficient gene delivery system. Extensive work has been done in Pinus spp. to improve transformation methods, either by DNA-coated particle bombardment or by co-culture with Agrobacterium tumefaciens. This review describes the most recent progress made in genetic transformation in the genus Pinus, with emphasis on four important forest and plantation species grown in Europe (P. pinaster), New Zealand, Australia and South America (P. radiata), and North America (P. taeda and P. strobus). The biosafety issues associated with potential deployment of transgenic pine varieties in commercial forestry are highlighted.

P.A.C. Sparrow, J.A. Irwin, C.M. Goldsack, L. Østergaard (UK) Brassica Transformation: Commercial Application and Powerful Research Tool (pp 330-339)

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Invited Review: Transformation systems are now available for all six of the main economic Brassica species; with a wealth of introduced traits reported (reviewed in Cardoza and Stewart 2004; Christey et al. in press). Agrobacterium-mediated transformation remains the method of choice for Brassica, yet despite the significant progress in enhancing efficiencies some genotypes remain recalcitrant to transformation. Recent advances in our understanding of the genetics behind transformation have enabled researchers to identify more readily transformable genotypes for use in routine high-throughput systems. These developments open up exciting new avenues to use model Brassica genotypes as powerful research tools for understanding gene function in complex genomes. In this paper advances in Brassica transformation methodologies and applications are reviewed.

Dragan Vinterhalter (Serbia), Tatjana Sretenović-Rajičić (Germany/The Netherlands), Branka Vinterhalter, Slavica Ninković (Serbia) Genetic Transformation of Brassica oleracea Vegetables (pp 340-355)

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Invited Review: Brassica oleracea L. with its numerous subvarieties is one of the most important vegetable species in the world. Constant improvement of various agronomical traits is a permanent task of Brassica breeders for which methods of genetic engineering have been adopted recently. However transformation in Brassica vegetables is not yet a routine. Development of successful transformation protocols based on common transformation procedures has been presented in many reports. Transformation success depends on many factors including genotype, explant type, gene introduction technique and the construct itself. In this review we present recent data on transformation of Brassica vegetables using both Agrobacterium tumefaciens and A. rhizogenes strains, as well as some other alternative approaches.

Yingying Cao, Yi Tu, Marcel Labandera, Shamila Abeynayake, Stephen Panter, Aidyn Mouradov, German Spangenberg (Australia) Designer Pasture Plants: from Single Cells to the Field (pp 356-363)

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Invited Mini-Review: Pasture occupies more land area than any other crop and it is of tremendous value as livestock feed. Ryegrasses (Lolium spp.) and white clover (T. repens) are among the most important forage plants in the world. The demand for high quality forages continues to grow. Elite pasture plants need to demonstrate tolerance to both biotic and abiotic stresses, including drought-stress, low temperatures, diseases and insect pests, without compromising their forage quality and productivity. Generation of these advanced forages is beyond the scope and speed of conventional plant breeding. Functional genomics has greatly increased our understanding of mechanisms that determine the genetic, molecular and biochemical basis of economically important traits in forage plants and allow plants to develop and adapt to a dynamic environment. In the post-genomics era, we need to convert this information into practical benefits for farmers and the agricultural sector. This has required multi-disciplinary approaches that exploit advances in molecular genetics, functional genomics and computational biology as well as close collaboration with plant breeders. This review discusses recent progress in finding the molecular and biochemical bases of quality traits in white clover and ryegrass which will enable the development of transgenic ‘designer’ cultivars with improved forage quality, yield and stress tolerance.

Malgorzata Czernicka, Maria Klein (Poland) New Rhododendron Cultivars through Genetic Engineering (pp 364-367)

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Mini-Review: The aim of our study was to review the results of genetic engineering of rhododendrons which have been published in the scientific literature so far. Genetic engineering has great potential to improve rhododendrons, but current protocols are complicated and time-consuming. Assessing each study revealed more and more factors that have a significant impact on the efficiency of genetic transformation. Much work still needs to be done in order to optimize the process, but gene transfer experiments that have been carried out thus far have already proved to be successful. Rhododendrons with a more efficient root system, allowing for higher iron uptake under low iron stress soil conditions have been obtained.

Marina Laura, Giafar Safaverdi, Andrea Allavena (Italy) Osteospermum ecklonis Marker-Free Transgenic Plants: Analysis of Insertion by AL-PCR (pp 368-375)

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Original Research Paper: The generation of “marker-free” plants is a response to public concern and technical reasons (preclusion to use the same gene during further steps of transformation and gene silencing consequent to marker gene pyramiding by conventional breeding). A further step towards breeding of an advanced generation of transgenic plants is to avoid the insertion of DNA not essential for the correct expression or silencing of the useful transgene(s). To accomplish these purposes, transformation vectors derived from pGreen II 0000 were prepared. The original T-DNA of 814 bp was completely replaced with a new T-DNA sequence of 44 bp containing exclusively multiple cloning sites; rolC and rolABC were then cloned into the Hind III/Eco RI and Eco RI sites, respectively. Following Agrobacterium-mediated transformation of the ornamental species Osteospermum ecklonis (DC.) Norl. (Cape daisy) and screening of regenerated shoots by PCR amplifying the rolA and rolC gene fragments, three plant clones that had at least one complete copy of the rolABC genes, and which expressed the transcript and showed the expected phenotype (high number of branches, profuse flowering, compact plant habit) were identified. Adaptor ligation PCR was performed on these plants and several products were identified at both sides of the insert. Sequencing of amplification products revealed a complicated scenario far from the classical type of integration reported for Agrobacterium. Integration cases identified were classified as: correct integration, mis-recognition of left border (LB) and right border (RB) or re-arrangements of the T-strand. None of the three marker-free plant clones expressing rol ABC had integrated a single copy of the genes. In addition, backbone sequences were found in all plants. These data indicate that these materials need further steps of conventional breeding to select progenies that had integrated the transgene(s) by inserting the minimum amount of DNA required for its correct expression. Strategies to improve the correct recognition of LB as T-strand terminations are finally discussed based on current knowledge.

Ravindra B. Malabadi (Portugal/India), K. Nataraja (India) Production of Transgenic Plants via Agrobacterium tumefaciens-Mediated Genetic Transformation in Pinus wallichiana (Himalayan Blue Pine) (pp 376-383)

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Original Research Paper: We established for the first time an Agrobacterium-mediated genetic transformation system for the Himalayan blue pine. Embryogenic tissue derived from vegetative shoot apices of mature trees of Pinus wallichiana were inoculated with A. tumefaciens strain EHA105 fused with a binary vector pBI121. The plasmid pBI121, containing the neomycin phosphotransferase II (nptII) gene providing kanamycin resistance as a selectable marker and the β-glucuronidase (uidA) reporter gene, was used in the transformation studies. GUS activity was used to monitor transient expression of the uidA gene and to further test lines selected on kanamycin-containing medium. The integration of the transgene (nptII) was confirmed by PCR followed by southern and northern blot analyses. These results demonstrated that a stable and enhanced transformation system could be established in P. wallichiana. This provides an opportunity to transfer economically important genes into Himalayan blue pines.

N. Nirupa, M.N.V. Prasad, S.K. Jami, P.B. Kirti (India) Optimization of Agrobacterium-Mediated Overexpression of Osmotin-Ferritin Genes in Brassica juncea (pp 384-392)

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Original Research Paper: The binary vector p35SGUSINT mobilized into Agrobacterium strain GV2260 was used for transformation of cotyledonary petiole explants of Brassica juncea (L.) Czern cv. ‘Pusa Jaikisan’ and transient GUS expression was used as the basis for identifying the most appropriate conditions for transformation. Genetic transformation is influenced by a number of factors which affect the efficiency. Some of the crucial factors like explant age, preculture period, and bacterial density and use of non-ethylene agent like silver nitrate were evaluated to optimize a protocol for cotyledonary petiole explants. The efficiency of the method optimized with the GUS construct has also been applied for introducing the osmotin-ferritin construct into B. juncea. The transfer of the foreign GUS gene into B. juncea was demonstrated through GUS assays, PCR and Southern blot analyses. The osmotin-ferritin transformants were confirmed in the T₀ and T₁ generations for the integration and expression of the osmotin and ferritin transgenes through PCR, Southern and Western blots.