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Volume 5 Number 1 2011
CONTENTS AND ABSTRACTS
Gulzar S. Sanghera, Prem L. Kashyap, Gurpreet Singh (India), Jaime A. Teixeira da Silva (Japan) Transgenics: Fast Track to Plant Stress Amelioration (pp 1-26)
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Review: Crop researchers are under increasing pressure to breed designer crops that are able to survive a plethora of biotic and abiotic threats while enhancing their nutritional or other inherent value. Conventional crop breeding is no longer able to meet the challenges of the 21st century. Genetic transformation is a realistic and viable means of modifying traits of economic significance in crops that ultimately provide a solution to solve the global problems of hunger and malnutrition. Genetically modified crops can now overcome biotic (pathogens and insect pests) and abiotic stresses (herbicides, drought, salinity, salt, etc.) while maintaining the same productivity. This review focuses on the significant achievements of genetic transformation in crops built to be tolerant to different biotic and abiotic stresses.
N. Saikishore, K.B.R.S. Visarada, S.V. Rao, N. Seetharama (India) Progress and Prospects for Agrobacterium-Mediated Genetic Transformation in Sorghum in Comparison to Other Cereals (pp 27-34)
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Review: Transgenic technology in sorghum, Sorghum bicolor L. (Moench), especially the Agrobacterium-mediated method, has picked up momentum in the recent past. High throughput Agrobacterium-mediated transformation in sorghum is prerequisite for use as a tool in functional genomics through T-DNA insertional mutagenesis and for development of marketable transgenic crop products. Despite equivalent efficiency in transient expression with other cereals, recovery of transgenic plants in sorghum is quite low, which can be attributed to a loss of regeneration potential over subsequent prolonged subculture and treatment with agents for decontamination of Agrobacterium. It can be overcome by right-choice of starting material with high regeneration and effective in vitro culture methods. Immature inflorescences are resourceful for high regeneration in sorghum, but recovery of plants subsequent to Agrobacterium-based methodology is very low. In vitro methods based on seed as the starting material in sorghum stand inefficient. Alternatively, immature embryos are frequently used explants for genetic transformation and these protocols need further improvement for increasing the efficiency of transformation in sorghum. Thus, the critical factors in Agrobacterium-mediated genetic transformation in sorghum include i) moderation of Agrobacterium-infection parameters ii) decontamination procedures with least phytotoxic effects iii) efficient plant regeneration and iv) specialized vectors for high efficiency transformation. The current review presents the achievements in Agrobacterium–based genetic transformation of sorghum and possible recommendations in the light of developments in other cereals.
Xia Dong, Barbara Meisel, Annette Block, Johanna Graßmann, Viviane Radl, Nicole Weinert (Germany), Remo Meincke, Gabriele Berg (Austria), Gerhard Wenzel, Michael Schloter, Volker Mohler (Germany) Expression Analysis of Zeaxanthin Epoxidase of Genetically Engineered Zeaxanthin-rich Potatoes in Comparison to Conventional Cultivars under Field Conditions (pp 35-42)
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Original Research Paper: Two genetically engineered (GE) zeaxanthin-rich potato (Solanum tuberosum L.) clones, derived from potato cultivar ‘Baltica’ were evaluated under open-field conditions with respect to agronomic performance, stability and tuber-specific expression of the inserted zeaxanthin epoxidase (zep) gene. Data collected from two field sites totalling four environments in Germany demonstrated that general morphology and tuber yield of GE potato clones were not impaired by the metabolic changes in tuber tissue. Quantitative real-time PCR analysis of zep gene expression in leaves, roots and tubers collected at three different developmental stages from the two GE potato clones and the conventional counterpart clone ‘Baltica’ showed that the transgene maintained its ability to induce the accumulation of zeaxanthin in tubers, while no significant zep expression changes were found in leaves and roots. The results clearly demonstrated that the tuber-specific promoter led to a strict tissue-specific expression of the inserted gene in the two GE potato clones in each of the four environments. Additionally, HPLC measurement showed that the tubers from two GE clones contained 19.5 to 58.7 µg/g dw of zeaxanthin, while the zeaxanthin content in the tubers of ‘Baltica’ was under detection level. HPLC results together with qRT-PCR results confirmed the inverse relationship between zep expression level and the accumulation of zeaxanthin in GE tubers. Furthermore, zep expression analysis of four other conventional cultivars showed that gene expression differed in a similar or even greater range among the four conventional cultivars investigated than the variation between GE clones and ‘Baltica’.
Prashantkumar Shrishail Hanjagi, Sashidhar Vudayagiri Radhakrishnan, Sushma Muragendra Awaji, Rohini Sreevathsa (India) Improvement of Salt Tolerance in Putatively Transgenic Rice Plants Overexpressing AVP1,a Vacuolar H+-Pyrophosphatase (pp 43-49)
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Original Research Paper: Alleviating salt stress in plants is an important aspect of crop improvement. Salt stress affects plant growth and development in many different ways. To maintain growth and productivity plants must adapt to stress conditions and exercise specific tolerance mechanisms. In the present study, AVP1, a vacuolar H+-PPase gene from Arabidopsis thaliana was overexpressed in rice (var-Vikas) by Agrobacterium mediated in planta transformation technique. To screen putative T1 plants for salt tolerance, stringent salt screening test was followed and root and shoot growth of T1 putative transformants was used as a selection criterion. Some of the transgenics showed significantly higher root and shoot growth compared to wild type. PCR analysis confirmed the integration of the transgene in the rice genomic DNA. Physiological studies such as chlorophyll (Chl) estimation, membrane integrity, cell viability tests were also conducted to assess their levels of tolerance at T1 generation. Some of the T1 transformants showed lower percent reduction in Chl content, higher cell viability after NaCl treatment compared to wild type (WT). These results clearly demonstrate that transgenic rice plants overexpressing AVP1, a vacuolar H+ proton pump have better salt-tolerance.
Manoj Kumar A, Sundaresha S, Rohini Sreevathsa (India) Transgenic Sunflower (Helianthus annuus L.) with Enhanced Resistance to a Fungal Pathogen Alternaria helianthi (pp 50-56)
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Original Research Paper: Sunflower (Helianthus annuus L.) is mainly grown for its oil all over the world. Like any other crop it also suffers major yield losses due to viral, bacterial and fungal diseases. Overexpression of PR proteins leads to increased resistance to pathogens in several crops. The PR-2 protein, β-1,3-glucanase contributes to plant defenses against fungal infection. We report in this paper, overexpression of a tobacco β-1,3-glucanase gene in transgenic sunflower and its resistance towards Alternaria helianthi. Molecular analysis by Southern dot blots, PCR analysis confirmed stable integration of the β-1,3-glucanase gene in sunflower transgenics. When these transgenic plants screened for resistance against A. helianthi, the transgenics showed not only reduction in the number of spots but also delay in the onset of Alternaria blight. The results demonstrate the potentiality of a PR protein from a heterologous source in developing resistant sunflower to Alternaria blight.
Farzaneh Kordbacheh, Hamideh Ofoghi, Ali Akbarzadeh, Mahyat Jafari, Ali-Hatef Salmanian (Iran) High Level Expression and Chloroplast Targeting of Human Calcitonin (hCT) in Transgenic Tobacco Plants (pp 57-61)
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ABSTRACT
Original Research Paper: Human calcitonin (hCT) is a 32-amino acid hormone which plays important roles in the regulation of calcium metabolism, treatment of osteoporosis, inhibition of osteoclastic activity and Paget bone disease. Recombinant hCT has been previously produced at low concentrations in plant-based systems with biological activity. To investigate if hCT could be effectively produced and accumulate at high concentrations in a plant system, we established transgenic tobacco plant lines expressing an optimized synthetic gene construct. Several strategies were applied to increase production levels including a strong over expression promoter, modified codon usage and organelle targeting. An hCT expression construct with tobacco codon preference was synthesized and attached to a chloroplastic signal sequence from the Rubisco small subunit gene of Brassica napus L. The construct was created by designing specific primers, using Polymerase Chain Reaction (PCR) and overlapping extension methods. Agrobacterium-mediated transformation was used to establish transgenic tobacco plants. The transgenic plants were analyzed by molecular methods and hCT concentration was determined by quantitative EASIA. hCT was expressed in the cytoplasm and accumulated in the chloroplast with a 26% efficiency. This is the first report describing expression and chloroplast targeting of hCT in transgenic plants.
Peyman Norouzi, Morad Jafari, Mohammad Ali Malboobi, Behzad Ghareyazie, Abazar Rajabi (Iran) Inheritance of Transgene and Resistance to a Lepidoptran Pest, Spodoptera littoralis, in Transgenic Sugar Beet Plants Harboring a Synthetic cry1Ab Gene (pp 62-66)
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Original Research Paper: Two transgenic sugar beet T0 lines, 7233-3 and 7233-8, carrying cry1Ab gene were selfed and crossed with a single cross cytoplasmic male sterile line. One hundred and ninety F1 plants derived from the transgenic lines were screened using polymerase chain reaction analysis. Statistical test confirmed the 1: 1 Mendelian ratio for the transgene in F1 progenies indicating a single locus insertion into the nuclear genome. Stable expression of the transgene under the maize-phosphoenol pyruvate carboxylase promoter in F1 progenies was confirmed by western blot analysis. The approximate quantity of protein expressed in the progenies was estimated to be about 0.1% of total leaf soluble protein. Insect bioassay in some progenies of the transgenic plants revealed an enhanced resistance to the prodenia pest (Spodotera littoralis).
Nasrin Moshtaghi, Abdolreza Bagheri, Ahmad Sharifi (Iran) A Comparison of Two Selectable Marker Gene Systems Used in the Transformation of Chickpea (Cicer arietinum L.) (pp 67-71)
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Original Research Paper: Different selectable marker genes are used in the genetic transformation of plants, but the efficiency of these genes in the selection of transgenic plants is different. In this study, we have used two different selectable marker gene systems in an established chickpea transformation protocol, and have compared the effectiveness of each selection system based on transformation efficiency. Using the herbicide resistance bar gene, together with a low concentration of the selective agent Phosphinothricin, results in greater transformation efficiency than using the antibiotic resistance nptII gene with a high concentration of the selective agent Kanamycin. In addition, modification of the rooting media and the use of a grafting method further improved the transformation efficiency of both selection systems. The transformation efficiency using the nptII and bar genes as selectable markers was 0.37% and 4.3%, respectively .
Ningaraju Thoresuragondanahalli Malatheshaiah, Krishnaraj Pu, Krishnamurthy Kempagangaiah, Sandesh Hakkare Swamidatta, Yogendra Kalenahalli Narasimhamurthy, Kuruvinashetti Mahaling Shrishailappa (India) Cloning, Expression and Development of Transgenic Tobacco using ChiA gene from Native Isolate of Serratia marcescens 141 (pp 72-77)
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Original Research Paper: Serratia marcescens is one of the most effective Gram-negative bacteria for degradation of chitin 1,4-β–glucosamine, which is the major structural component of the exoskeleton of insects and crustaceans and also occurs in the cell wall of several fungi. Chitinases are of biotechnological importance because nature uses these enzymes as a plant biocontrol agent against fungal elicitors. Chitinases hydrolyse the β-1-4 linkage in chitin to soluble oligosaccharides, mainly chitobiose, which is hydrolyzed to GlcNAc by chitinase. Different isolates of S. marcescens obtained from soils of Western Ghats were screened for their chitinolytic activity from total DNA which was isolated from the most efficient strain. A 1691-bp amplicons was obtained after PCR amplification with ChiA-specific primers. The amplicon was cloned into pTZ57R/T vector. When sequenced, it showed 99% homology with reported sequences both at nucleotide and translated amino acid levels. The ChiA gene was expressed in Escherichia coli BL21 by subcloning into prokaryotic expression vector pET28a (+) and in tobacco by subcloning into plant transformation vector pHS100.
Jaime A. Teixeira da Silva, Michio Tanaka (Japan) Optimization of Particle Bombardment Conditions for Hybrid Cymbidium: Part II (pp 78-82)
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Research Note: Four factors (delayed selection, osmoticum pre-treatment of plant material, choice of plasmid, age and developmental stage of plant material) affecting β-glucuronidase (GUS) transient and stable expression in protocorm-like bodies (PLBs) and embryogenic callus of hybrid Cymbidium Twilight Moon ‘Day Light’ through particle bombardment were optimized, leading to as much as 100% GUS expression in 24-month old plantlets and up to T3 generation PLBs following bombardment with pWI-GUS and selection on 50-100 mg/l kanamycin sulphate. The ideal conditions for callus and PLB transformation were: delayed selection (1 week after bombardment), osmoticum pre-treatment of plant material (0.3 M raffinose was better than an equimolar amount of mannitol and sorbitol, i.e. 0.4 M each), choice of plasmid (pWI-GUS showed 6-fold more GUS expression than pBI121), and age and developmental stage of plant material (1-week-old PLBs). The bar selector gene could also be inserted using pMSP38. These, as well as our previous results, pave the way for repeatable and systematic transformation of Cymbidium hybrids using other genes of interest inserted into plasmid cassettes.
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