Volume 2 Number 2 2008

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CONTENTS AND ABSTRACTS

Ravindra B. Malabadi (India/Canada), Jaime A. Teixeira da Silva (Japan), K. Nataraja (India) Green Fluorescent Protein in the Genetic Transformation of Plants (pp 86-109)

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Invited Review: The genetic transformation of plants is a promising method for introducing and modifying various desirable traits. Several marker genes (β-glucuronidase, GUS; luciferase, LUC; or β-galactosidase, LacZ) have been successfully used in genetic transformation protocols. Relatively recently, green fluorescent protein (GPF) marker has become popular due to its many advantages in plant transformation studies. Most importantly, it does not require the addition of any interfering substances like exogenous substrates or enzymes. It also allows for the monitoring of transgenic expression from early stages of transformation through the recovery of living transgenic plants without the need to sacrifice valuable, sometimes sparse, transgenic material. However, the use of GFP is associated with some limitations, mostly related to its low levels of expression, and high toxicity when it is expressed at high levels in plants. This review aims to provide a broad overview of the use of GFP in a wide spectrum of plants.

Satvinder Kaur Mann, Prem Lal Kashyap, Gulzar Singh Sanghera, Gurpreet Singh, Sondeep Singh (India) RNA Interference: An Eco-Friendly Tool for Plant Disease Management (pp 110-126)

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Review: Efficient control of plant pathogens affecting economically important crop species represents one of the major challenges for sustainable agriculture production. Though plant breeding has been the classical means of manipulating the plant genome to develop resistant cultivar for controlling plants diseases, the advent of genetic engineering provides an entirely new approach. Currently, the area planted with crops genetically modified for resistant to disease is less compared with that of crops for tolerance to herbicide, or resistant to insects, numerous strategies are being pursued to render plants resistant to fungi, bacteria, viruses and nematodes. Recently, RNA interference (RNAi) technology has emerged to be a promising therapeutic weapon to mitigate the inherent risks like use of specific transgenes, marker genes or gene control sequences associated with development of traditional transgenics as disease-resistant transgenic plants can be produced within a regulatory framework. The advantage of RNAi as a novel gene therapy against fungal, viral and bacterial infection in plants lies in the fact that it regulates gene expression via mRNA degradation, translation repression and chromatin remodeling through small non-coding RNAs. Mechanistically, the silencing processes are guided by processing products of the dsRNA trigger, which are known as small interfering RNAs (siRNAs) and microRNAs (miRNAs). The application of tissue-specific or inducible gene silencing, with the use of appropriate promoters to silence several genes simultaneously should enhance researchers’ ability to protect crops against destructive pathogens. This review updates the current state on the use of RNAi, molecular principles underlying the biology of this phenomenon, development of RNAi technologies in relation to plants and discusses strategies and applications of this technology in plant disease management to save the green world from pathogenic intruders in eco-friendly manner.

Omid Karami (Iran) Factors Affecting Agrobacterium-mediated Transformation of Plants (pp 127-137)

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Review: Plant transformation technology has become a versatile platform for cultivar improvement as well as for studying gene function in plants. The development of an efficient method for genetic transformation is a prerequisite for the application of molecular biology to the improvement of a given crop species. Agrobacterium-mediated genetic transformation is the dominant technology used for the production of genetically modified transgenic plants. Extensive research aimed at improving the molecular machinery of Agrobacterium responsible for the generation and transport of the bacterial DNA into the host cell has resulted in the establishment of many recombinant Agrobacterium strains and technologies currently used for the successful transformation of numerous plant species. Many factors influencing Agrobacterium-mediated transformation of plants have been investigated and elucidated. These factors include bacterial strains and cell density, plant species and genotype, plant growth regulators and antibiotics, explant, explant wounding, light and temperature. Before attempting stable transformation of any new species, it is useful to optimize the factors influencing transformation efficiency, as this can reduce future costs in labor and materials. The studies of such factors hold great promise for the future of plant biotechnology and plant genetic engineering as they might help in the development of conceptually new techniques and approaches needed today to expand the host range of Agrobacterium and to control the transformation process and its outcome during the production of transgenic plants. Here, I review some of the main factors that influence Agrobacterium-mediated genetic transformation and discuss their possible roles in this process.

Pankaj Kumar Bhowmik, Saikat Kumar Basu (Canada) Current Developments, Progress, Issues and Concerns in Producing Transgenic Peas(Pisum sativum L.) (pp 138-150)

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Invited Review: Pea (Pisum sativum L.) is an important grain legume crop and has a global economic value due to its protein content both for human and animal consumption. The productivity and value of peas has been successfully enhanced by the introduction of stably inherited traits such as pest, disease and herbicide resistance, and enhanced quality of pea proteins. In this review, we present an assessment of the current developments, progress, issues and concerns in developing transgenic pea lines, briefly highlighting the global pulse industry and legume genomics. This review discusses why Agrobacterium has been successful in pea, what other alternatives have been tested, the extent to which they have yielded transgenic pea lines, and their potential agronomic utility. The GM food aspect and research related to transgenic peas as a food and feed source have also been investigated.

Masoud Tohidfar, Gholamreza Salehi Jouzani (Iran) Genetic Engineering of Crop Plants for Enhanced Resistance to Insects and Diseases in Iran (pp 151-156)

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Invited Mini-Review: Huge yield losses and deterioration of quality of cultivated plants occur due to continuous exposure of plants to pathogens and insect pests. The excessive use of chemical pesticides and fertilizers in modern agriculture result in a deterioration of soil fertility and through intracellular accumulation pesticide-resistant mutants of insects and plant pathogens have emerged worldwide. A number of plant species have been successfully transformed for resistance to insects, bacteria, viral, fungal pathogens and nematodes. These transgenic plants have been extensively field tested meeting the stringent biosafety guidelines and released for commercial cultivation since 1990. Genetically modified pest resistant crops have been attracting attention recently as alternatives to chemical pesticides in Iran. Iran is the first country in the region producing two transgenic plants (rice resistant to pests and cotton resistant to pests and disease) ready for release after the regulation processes. The first field trial on transgenic plants in Iran was conducted in 2004. GM rice and cotton are now under biosafety assessments. Furthermore, research on other transgenic plants such as rapeseed, wheat, palm, corn, alfalfa and potato for enhanced pest and disease resistance is underway. Currently, the National Biosafety Committee has submitted the draft of the National Biosafety Law (NBL) to the parliament for ratification. Ratification of the NBL will help to release these GM crops in the near future and modern biotechnology will certainly become one of the important components of agriculture in Iran.

Madappattuparambil Ravindran Beena, Rakesh Tuli, Aparna Dutta Gupta, Pulugurtha Bharadwaja Kirti (India) Transgenic Peanut (Arachis hypogaea L.) Plants Expressing cryIEC and Rice Chitinase cDNA (Chi 11) Exhibit Resistance against Insect Pest Spodoptera litura and Fungal Pathogen Phaeoisariopsis personata (pp 157-164)

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Original Research Paper: Spodoptera litura has been identified as a common insect pest of peanut, causing severe yield losses. A synthetic cry1EC gene had been specially designed and proven effective against the larvae of this polyphagous Lepidopteran insect. We have successfully introduced the synthetic cry1EC gene alone and along with a rice chitinase cDNA, Chi 11 into JL-24, a popular variety of peanut in India. The transgenic plants were raised from cotyledonary node explants through Agrobacterium tumefaciens-mediated transformation. The synergistic effect of both genes was studied in enhancing mortality of the target insect. The presence and expression of the synthetic cry gene and selectable marker (nptII) gene in regenerated plants were confirmed by PCR, Southern and Western blot analyses. Inheritance of the transgene was confirmed in the T₁ and T₂ generations. Insect bioassays on the transgenic plants carrying both genes showed enhanced resistance against the target insect larvae compared to plants harboring the cry gene alone. A fungal bioassay on plants carrying both genes showed varied resistance against the fungal pathogen Phaeoisariopsis personata,which causes late leaf spot disease, in addition to resistance against the insect pest Spodoptera. This is the first report on peanut engineered with both cry gene and chitinase against herbivores and leaf spot disease.

Jeung-Sul Han, Chang Kil Kim (Republic of Korea) Enhanced Accumulation of Metals in Bottle Gourd Plants Expressing an Arabidopsis Cation/H⁺ Exchanger (pp 165-169)

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Original Research Paper: In plants, cation sequestration from the cytosol to the vacuole is thought to be an important component for ion tolerance and its potential for use in phytoremediation. The Arabidopsis vacuolar exchanger CAX2 may be a key mediator in this machinery. Here we demonstrate that transgenic bottle gourd plants expressing CAX2 have more calcium (Ca²⁺) than wild-type. In addition, expression of CAX2 in bottle gourd showed a significant increase of the two metals tested (Mn²⁺ and Cd²⁺). Furthermore, transgenic bottle gourd seedlings showed no phenotypic alterations during the whole growth period. These results indicate that intact CAX2 has broad substrate specificity, and suggest that the modulation of this exchanger might be meaningful for the future strategies to improve plant ion tolerance.

Danial Kahrizi, Ali Hatef Salmanian (Iran) Substitution of Ala183Thr in aroA Product of E. coli (k12) and Transformation of Rapeseed (Brassica napus) with Altered Gene Confers Tolerance to Roundup (pp 170-175)

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Original Research Paper: Roundup is a non-selective broad-spectrum herbicide that inhibits the aroA gene product or 5-enolpyruvylshikimste 3-phosphate synthase (EPSPS), a key enzyme in the aromatic amino acid biosynthesis in microorganisms and plants. The manipulation of the bacterial aroA gene in order to reduce its affinity to Roundup and its transformation into plants is one of the most effective methods for production of Roundup-tolerant plants. In this research, we studied alanine183 of E. coli (k12) EPSPS enzyme. This amino acid is an important residue for EPSPS-Roundup binding. We used site-directed mutagenesis (SDM) to induce a point mutation in E. coli aroA gene in order to convert alanine183 to threonine (Ala183Thr). The manipulated aroA gene was cloned in pUC18 as a universal cloning vector and pBI121 as a plant expression vector. Molecular analysis and sequencing showed that the manipulated gene has been correctly changed and cloned in the correct orientation in both plasmids. Recombinant pBI121 containing an altered aro A gene was transferred to rapeseed (B. napus) via Agrobacterium tumefaciens-mediated transformation. Roundup tolerance was assayed in putative transgenic plants. Statistical analysis of Roundup challenging data showed that there is a significant difference between transgenic and control plants. The survival frequencies of transgenic plants in 1, 2.5, 5, 7.5 and 10 mM Roundup were 29, 22, 14, 5, and 0%, respectively whereas the non-transformed control plants were unable to survive even 1 mM Roundup. The presence and copy numbers of the construct in transgenic plants were confirmed by PCR and southern blot analysis, respectively.

Sreeramanan Subramaniam, Maziah Mahmood, Xavier Rathinam (Malaysia) Transfer of β-1,3-glucanase Gene into Banana for Tolerance to Fusarium Wilt Disease Race 1 using Agrobacterium-mediatedTransformation System (pp 176-185)

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Original Research Paper: Agrobacterium-mediated transformation of single buds in vitro grown banana cv. ‘Rastali’ (AAB) was done using the binary vector pROKla-Eg, harboring the soybean endo β-1,3-glucanase gene (Eg). Plasmid pROKla-Eg contained a neomycin phosphotransferase gene (nptII) as the selectable marker to identify the transformants. Treatment A contained kanamycin (kan) at 100 mgl^-1 and treatment B contained geneticin G-418 at 50 mgl^-1 in both MS medium supplemented 5 mgl^-1 of BAP together with 2.7 g of gelrite agar. Single buds derived from multiple bud clumps, were the target explants for transformation. An assay was performed to identify the minimum concentration required for two antibiotics (carbenicillin and cefotaxime) that was most effective against Agrobacterium strains, LBA 4404, and the effect on tissue regeneration capacity. Even though the transformation frequency based on kan selection medium (treatment A) was higher, no transformants could be confirmed based on PCR and Southern blot analyses, compared to the use of geneticin (G-418) selection medium (treatment B). These results suggested that the use of G-418 as a selection agent is preferable to kan due to the lower concentration required to allow for the small numbers of putative transgenic cells in a large population of non–transformed ones to undergo multiplication and also reduced the occurrence of chimeras. The transgenic banana plantlets were inoculated with 2 × 10⁶ spores ml^-1 conidial suspension of Fusarium oxysporum f. sp. cubense (race 1) to evaluate the degree of tolerance and to investigate the effectiveness of the bioassay system as a potential tool for early screening. An assay of protein extract from the transgenic plantlets showed a significant increase in EG enzyme activity over the untransformed plantlets. The present Agrobacterium-mediated transformation protocol reported here is suitable for the use of tiny meristem tissues to obtain fungal disease tolerant or resistant banana through genetic engineering.

Reynald Tremblay, Xiaofeng Wang, Anthony M. Jevnikar, Shengwu Ma (Canada) Expression of a Fusion Protein Consisting of Cholera Toxin B Subunit and an Anti-Diabetic Peptide (p277) from Human Heat Shock Protein in Transgenic Tobacco Plants (pp 186-191)

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Original Research Paper: A DNA construct encoding cholera toxin B subunit (CTB) and an anti-diabetic peptide from human heat shock protein 60 (hsp60) as a fusion protein was produced and transferred into low-nicotine tobacco through Agrobacterium-mediated transformation. Integration of the fusion gene into the plant genome was confirmed by PCR. The transcriptional expression of the fusion gene was demonstrated by RT-PCR. Western blot analysis further verified the synthesis and assembly of the fusion protein into pentamers in transgenic tobacco. G_M1-ELISA showed that the plant-derived fusion protein retained G_M1-ganglioside receptor binding specificity. The fusion protein accounted approximately for 0.1% of the total leaf protein. The production of transgenic plants expressing CTB-p277 offers an opportunity to test plant tissues for oral peptide antigen therapy against autoimmune diabetes by inducing oral tolerance.