Volume 3 Special Issue 1 2009

Guest Editors

Madhugiri Nageswara-Rao, Jaya R. Soneji

University of Florida, IFAS, Citrus Research & Education Center, USA

www.crec.ifas.ufl.edu/index.htm

CONTENTS AND ABSTRACTS

Herb Aldwinckle (USA), Mickael Malnoy (Italy) Plant Regeneration and Transformation in the Rosaceae (pp 1-39)

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Invited Review: The plant family Rosaceae consists of over 100 genera and 3,000 species that include many important fruit, nut, ornamental, and lumber crops. Members of this family provide high-value nutritional foods and contribute desirable aesthetic and industrial products. A complete annotated genome sequence is expected momentarily for apple, and in the near future for peach and strawberry. These sequences will accelerate the next wave of research exploring and comparing the form and function of the many genomes that define the striking differences in morphology and physiology among the Rosaceae, e.g., a herbaceous plant vs. a tree, or a fleshy rather than a dehiscent fruit. Among the many dividends of this research will be the development of superior products for consumers, a better understanding of the genetic elements that contribute to horticultural traits of interest, and an enhanced vision of Rosaceae evolution. It will also answer some of the fundamental questions of plant biology, particularly around the regulation of plant architecture, which may be best answered by species within this family. This review serves as a synopsis of the genetic engineering resources available to study the function and production of new Rosaceae varieties of benefit to the consumer.

Sameer Joshi (The Netherlands), José Miguel Soriano (The Netherlands/Spain), Jan Schaart (The Netherlands), Giovanni Broggini, Iris Szankowski (Switzerland), Evert Jacobsen, Frans Krens, Henk J. Schouten (The Netherlands) Approaches for Development of Cisgenic Apples (pp 40-46)

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Invited Mini-Review: Introgression of genetic traits from wild apple germplasm (Malus spp.) into commercial apple cultivars is a painstakingly slow process. For e.g. introgression of the Vf gene from Malus floribunda 821 for resistance to apple scab, caused by the fungus Venturia inaequalis, took more than 80 years due to genetic drag and the long juvenile period of apple. In order to speedup the classical breeding, molecular techniques can be applied to enrich existing commercial apple varieties with functional alleles from sexually compatible plants, preventing genetic drag and keeping the genetic makeup of the commercial cultivar. This concept is named “cisgenesis”. This paper describes several approaches and considerations for development of cisgenic apples and stacking of genes. Also we provide an overview of isolated alleles from apple available for cisgenesis at the moment and in the near future.

Jaya R. Soneji, Madhugiri Nageswara Rao (USA) Genetic Engineering of Pineapple (pp 47-56)

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Invited Review: Pineapple is an important crop for tropical countries. It is consumed as fresh fruit as well as processed or canned, dehydrated and juice products. Even though it is grown in more than 82 countries around the world, there is a remarkable lack of commercial varieties. ‘Smooth Cayenne’ is the only cultivar which dominates the trade and pineapple industry. Conventional breeding has yielded very poor results making genetic engineering particularly suitable for genetic improvement of pineapple. Tissue culture regeneration has been widely reported in pineapple making genetic engineering more amenable. Genetic engineering also offers the means for manipulating horticulturally important traits without altering the cultivar phenotype. This review provides an overview of the genetic transformation efforts carried out in pineapple.

Bijaya Ketan Sarangi, Shweta Kalve, Ram Awatar Pandey, Tapan Chakrabarti (India) Transgenic Plants for Phytoremediation of Arsenic and Chromium to Enhance Tolerance and Hyperaccumulation (pp 57-86)

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Review: Phytoremediation of metals and other environmental pollutants is gaining importance as a cost-effective method for pollution mitigation and envisages sustainable development. This paper envisages prospects of phytoremediation for mitigation of heavy metal pollutants from the environment, with particular reference to arsenic (As) and chromium (Cr). Genetically engineered tailor-made plants have much potential for selective uptake, accumulation and sequestration of heavy metals. Recent developments in this area and state-of-the-art technology foresee genetically engineered plants with an ability to prevent accumulation of As in aerial parts of experimental plant systems, which could be extrapolated to edible plants such as rice, wheat and others. Similarly, hypereaccumulation in plant biomass is another important approach for removal of these toxic metals from the land and water ecosystems and mitigation of As and Cr pollution. The mechanisms of As hyperaccumulation by the hyperaccumulator plants has opened up scope for genetic engineering other prospective plant species to enhance hyperaccumulation of toxic metals in their aerial biomass. This review enumerates the mechanisms of hyperaccumulation in the plant systems, the potential genes that could be engineered to develop tailor made genetically engineered plants aimed for phytoremediation of As and Cr and other metals in general.

Jinfu Lü (China), Feifei Qin (China/Japan), Chengrong Wang, Ran Wang, Gang Ma, Yan Liu (China), Huilian Xu (Japan) Agrobacterium tumefaciens-mediated Transformation of Broccoli (Brassica oleracea L. var. italica) Hypocotyls with BO-ACO2 Gene (pp 87-91)

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Original Research Paper: Ethylene metabolism is associated with postharvest senescence in fruits and vegetables. It is important to elucidate the mechanisms of ethylene production, especially at the molecular level. Therefore, vectors carrying the sense or antisense BO-ACO2 gene and a neomycin phosphotransferase II(NPT-II) gene have been constructed according to Homology-Dependent Gene Silencing (HDGS). In addition, in the present research, a genetic transformation system of broccoli (Brassica oleracea L. var. italica) by Agrobacterium tumefaciens was established. Factors that were known to influence genetic transformation were evaluated to optimize the transformation system. Hypocotyls from 10-day-old seedlings were used for transformation. The transformation protocol was divided into 3 periods including pre-cultivation, co-cultivation and keeping-cultivation, and the most appropriate time for each period was 2 days, 72 hours and 20 days, respectively. The critical determinant was 0.2 (OD₆₀₀) for Agrobacterium density and 5 min for infection. The kanamycin (Kan) selection pressures for bud differentiation and root induction were 30 and 15 mg L^-1, respectively. Carbenicillin disodium salt (Carb) at 300 mg L^-1 was used to suppress bacteria reproduction. In conclusion, using the optimized protocol, seven positive transgenic plants were successfully obtained and the BO-ACO2 and NPT-II genes detected by PCR were mobilized into broccoli. The transgenic plants produced from this work will be important in elucidating the effects of the introduced BO-ACO2 gene on ethylene biosynthesis and post-harvest senescence in transgenic broccoli.

Syeda Zinia Rashid (Malaysia/Japan), Masaharu Kyo (Japan) Ectopic Expression of WOX5 Dramatically Alters Root-tip Morphology in Transgenic Tobacco (pp 92-96)

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Original Research Paper: This paper examines the effects of ectopic expression of WUSCHEL (WUS)-RELATED HOMEOBOX5 (WOX5) on the morphology of transgenic tobacco plantlets and seedlings under the control of a chemical-inducible expression system. In the presence of the expression inducer, β-estradiol, the root-tip region developed a swelling of callus tissue from which adventitious shoots emerged. Except at the root tip itself, no abnormality was evident in any other parts of treated plantlets such as in the leaf, stem or other parts of the root. Transgenic seedlings also generated ectopic shoots from similar root swellings after treatment with the inducer. Expression analyses reveal that WOX5 may induce transdifferentiation of root-tip cells that possess competence for the formation of an apical shoot meristem. It is suggested that WOX5 may execute a common functional regulation in transgenic tobacco as previously reported in the case of WUS, during the process of adventitious shoot formation, and from the root-tip region.

Yogendra Kalenahalli Narasimhamurthy, Paramanahally Hanumanthegowda Ramanjini Gowda, Sandesh Hakkare Swamidatta, Gunnaiah Raghavendra, Asha Velayudhan Nair, Ningaraju Toresuragondanahalli Malatheshaiah, Deepak Nanjappa (India) Production and Characterization of Hepatitis B Recombinant Vaccine in Tobacco (Nicotiana tabacum cv. ‘Kanchun’) (pp 97-101)

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Original Research Paper: Immunization with Hepatitis B vaccine is the most effective means of preventing Hepatitis B virus infection and its consequences. Plants are a potential source of Hepatitis B surface Antigen (HBsAg) that is not dependent upon process technology to ensure protein folding and particle assembly. A plant-based HBsAg expression system makes possible the testing of an oral immunization strategy by simply feeding plant samples. The primary means of transformationis Agrobacterium-mediated gene transfer which has provided a reliable means of creating transformants in a wide variety of species and also can express important pharmaceutical products. Leaf explants of tobacco were transformed with the Hepatitis B surface antigen gene along with nptII as an antibiotic selection marker gene. The presence of the HBsAg gene in putative transformants was confirmed by the presence of a 900-bp band in PCR analysis. The crude protein obtained from the transformed tobacco plants was tested by SDS-PAGE for the presence of a 24 kDa protein. Western-blot and ELISA confirmed the antigen specificity and immunogenic nature of the Hepatitis B surface antigen S-protein. T₁ generation seeds obtained from transgenic tobacco plants were tested for inheritance analysis by germination in the presence of 100 ppm kanamycin. These showed a 3: 1 segregation ratio indicating Mendelian inheritance. Transgenic plants hold promise as low-cost vaccine production systems and this study emphasizes the integration and stability of recombinant protein expressed in tobacco plants.

Shahin Sharif Ali (Ireland/India), Padma Yealla, Bidyut Kumar Sarmah (India) Bar as a Potential Selectable Marker Gene to Obtain Putative Transformants in Indian Chickpea (Cicer arietinum L.) Cultivars (pp 102-106)

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Original Research Paper: A reproducible and efficient transformation method was demonstrated for two Indian desi chickpea (Cicer arietinum L.) cultivars using bar as a selectable marker gene. In order to select the transformants in tissue culture medium, 2.5 mg/l of phosphinothricin was optimized to use as a selective agent in regeneration and selection medium. Cotyledonary explants containing half embryonic axes were infected and co-cultivated with Agrobacterium strain harboring binary plasmid (pBK16.2) containing a chimeric cre (Cyclization recombination) gene of bacteriophage P1 and a bar gene as selectable marker gene. The frequencies of putative transformed plants were 1.23% in cv. ‘ICCV 89314’ and 1.12% in cv. ‘Vijay’. Explant to plant duration was 61-71 days. Putative transformed plants/shoots that were selected on regeneration and selection medium were confirmed by PCR analysis for the presence of transgenes. A “leaf painting assay” using 0.6 mg/ml of phosphinothricin confirmed expression of the bar gene in the putative transformed plants. This protocol generated a slightly higher frequency of putative transformed plants in our laboratory when compared to generation of transformed chickpea plants using nptII as the selection marker gene.

Mitra Mohammadi Bazargani, Masoud Tohidfar, Behzad Ghareyazie, Gholamreza Salehi Jouzani, Badraldin Ebrahim Sayed-Tabatabaei, Reihaneh Golabchian (Iran) Transformation of Iranian Cotton Varieties Using Shoot Apex (pp 107-112)

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Original Research Paper: The optimization of gene transfer into Iranian cotton commercial varieties by shoot apices is presented. Shoot apices of two Iranian varieties, ‘Sahel’ and ‘Varamin’ was used as explants for Agrobacterium-mediated transformation. Three Agrobacterium strains, harboring the plasmid vector pBI121 containing the β-glucuronidase (gus) gene, were used under the control of the cauliflower mosaic virus (CaMV) 35S promoter. The neomycin phosphotransferase (nptII) gene was used as the selectable marker. Inoculated shoot apices were placed onto cotton co-cultivation medium. Shoot regeneration was achieved within 3-4 weeks on MS basal medium supplemented with modified B₅ vitamins. Transformed shoot apices were selected on selective medium containing 50 mg l^-1 kanamycin and 200 mg l^-1 cefotaxime. Putative transgenic shoot apices were subsequently regenerated on half-strength agar-solidified MS basal medium supplemented with 0.1 mg l^-1 indole-3-butyric acid (IBA) and modified B₅ vitamins. The presence of gus and nptII genes in the transgenic plants was verified by histochemical GUS assay and PCR analysis, respectively. The transformation frequency of ‘Sahel’ and ‘Varamin’ using LBA4404 strain was 3.7 and 5.5%, respectively. The c² test of T₁ transgenic cotton plants in greenhouse conditions indicated that the inheritance of the gus gene followed a Mendelian ratio for a single gene (3:1). Our investigations on T₁ lines confirmed the stability of the gus gene and its expression.

A. Manoj Kumar, S. Sundaresha, Rohini Sreevathsa (India) Resistance to Alternaria Leaf Spot Disease in Transgenic Safflower (Carthamus tictorius L.) Harboring a Rice Chitinase Gene (pp 113-118)

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Original Research Paper: Safflower (Carthamus tinctorius L.) is an important oilseed crop of semi-arid regions. Yield losses due to fungal diseases are enormous in the cultivation of this crop. Overexpression of pathogenesis-related (PR) proteins leads to increased resistance to pathogenic fungi in several crops. The PR protein chitinase hydrolyses a major cell wall component, chitin of pathogenic fungi and acts as a plant defense barrier. We report in this paper, overexpression of a rice chitinase in transgenic safflower cv. ‘A-1’ and its resistance towards Alternaria carthami. PCR was used to confirm stable integration of the chitinase gene in transgenic safflower plants. When screened for resistance against A. carthami, these plants showed not only a reduction in the number of spots but also a delay in the onset of disease. Overall the method resulted in a transformation efficiency of 7.72% on analysis of T₁ plants. The results demonstrate the potential of a PR protein from a heterologous source in developing fungal disease-resistant safflower.

Jaime A. Teixeira da Silva, Michio Tanaka (Japan) Optimization of Particle Bombardment Conditions for Hybrid Cymbidium (pp 119-122)

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Original Research Paper: The conditions to maximize 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 have been optimized using two plasmids, pBI121 and pWI-GUS. Although callus tended to be much more sensitive to particle bombardment than PLBs, thus affecting regeneration, it was possible to obtain conditions for high level transient and stable GUS expression. The conditions for callus and PLBs were: microcarrier (gold for both explant types), number of shots (2 for both explant types), rupture disk pressure (1100 and 1350 psi), target distance (6 cm for both explant types), explant pre-culture (3, 6 or 9 days for both explant types) period, respectively.