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      Synergistic Action of D-Glucose and Acetosyringone on Agrobacterium Strains for Efficient Dunaliella Transformation

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          Abstract

          An effective transformation protocol for Dunaliella, a β-carotene producer, was developed using the synergistic mechanism of D-glucose and Acetosyringone on three different Agrobacterium strains (EHA105, GV3101 and LBA4404). In the present study, we investigated the pre-induction of Agrobacterium strains harboring pMDC45 binary vector in TAP media at varying concentrations of D-glucose (5 mM, 10 mM, and 15mM) and 100 μM of Acetosyringone for co-cultivation. Induction of Agrobacterium strains with 10 mM D-glucose and 100 μM Acetosyringone showed higher rates of efficiency compared to other treatments. The presence of GFP and HPT transgenes as a measure of transformation efficiency from the transgenic lines were determined using fluorescent microscopy, PCR, and southern blot analyzes. Highest transformation rate was obtained with the Agrobacterium strain LBA4404 (181 ± 3.78 cfu per 10 6 cells) followed by GV3101 (128 ± 5.29 cfu per 10 6 cells) and EHA105 (61 ± 5.03 cfu per 10 6 cells). However, the Agrobacterium strain GV3101 exhibited more efficient single copy transgene (HPT) transfer into the genome of D. salina than LBA4404. Therefore, future studies dealing with genetic modifications in D. salina can utilize GV3101 as an optimal Agrobacterium strain for gene transfer.

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          A synthetic gene coding for the green fluorescent protein (GFP) is a versatile reporter in Chlamydomonas reinhardtii.

          The use of Chlamydomonas reinhardtii as a model system has been hindered by difficulties encountered in expressing foreign genes. We have synthesised a gene encoding green fluorescent protein (GFP) adapted to the codon usage of C. reinhardtii (cgfp). After verifying the gene was functional in Escherichia coli, the cgfp was fused in frame to the phleomycin resistance gene ble from Streptoalloteichus hindustanus and expressed in C. reinhardtii under control of the rbcS2 promoter and intron sequences. The GFP-fluorescence was seen only in the nucleus demonstrating the nuclear accumulation of the Ble-GFP fusion protein. The cgfp was also fused to the chlamyopsin gene, cop, and expressed in C. reinhardtii under control of the cop promoter. The eyespot became fluorescent indicating that the opsin-GFP fusion protein was correctly directed into the eyespot along with the endogenous unmodified opsin. We conclude that cgfp provides a useful tool to visualize protein synthesis and localisation in vivo in C. reinhardtii and possibly in related green algal species.
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            Quantitative real-time PCR assay for determining transgene copy number in transformed plants.

            The development of transgenic events can be limited by many factors. These include expression levels, insert stability and inheritance, and the identification of simple insertion events. All of the factors can be related to the copy number of the transgene. Traditionally, copy number has been determined by laborious blotting techniques. We have developed an alternative approach that utilizes the fluorogenic 5' nuclease (TaqMan) assay to quantitatively determine transgene copy level in plants. Using this assay, hundreds of samples can be analyzed per day in contrast to the low throughput encountered with traditional methods. To develop the TaqMan copy number assay, we chose to utilize our highly efficient Agrobacterium-mediated transformation system of maize. This transformation procedure generates predominantly low copy number insertion events, which simplified assay development. We have also successful applied this assay to other crops and transformation systems.
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              Estimating the copy number of transgenes in transformed rice by real-time quantitative PCR.

              In transgenic plants, transgene copy number can greatly influence the expression level and genetic stability of the target gene, making estimation of transgene copy number an important area of genetically modified (GM) crop research. Transgene copy numbers are currently estimated by Southern analysis, which is laborious and time-consuming, requires relatively large amounts of plant materials and may involve hazardous radioisotopes. We report here the development of a sensitive, high-throughput real-time (RT)-PCR technique for estimating transgene copy number in GM rice. This system uses TaqMan quantitative RT-PCR and comparison to a novel rice endogenous reference gene coding for sucrose phosphate synthase (SPS) to determine the copy numbers of the exogenous beta-glucuronidase (GUS) and hygromycin phosphotransferase (HPT) genes in transgenic rice. The copy numbers of the GUS and HPT in primary rice transformants (T0) were calculated by comparing quantitative PCR results of the GUS and HPT genes with those of the internal standard, SPS. With optimized PCR conditions, we achieved significantly accurate estimates of one, two, three and four transgene copies in the T0 transformants. Furthermore, our copy number estimations of both the GUS reporter gene and the HPT selective marker gene showed that rearrangements of the T-DNA occurred more frequently than is generally believed in transgenic rice.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                28 June 2016
                2016
                : 11
                : 6
                : e0158322
                Affiliations
                [001]School of Bio-Sciences and Technology, VIT University, Vellore – 632 014, Tamil Nadu, India
                Instituto de Biología Molecular y Celular de Plantas (IBMCP), SPAIN
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: KMG. Performed the experiments: RS. Analyzed the data: RS KMG. Contributed reagents/materials/analysis tools: RS. Wrote the paper: RS KMG.

                Author information
                http://orcid.org/0000-0003-1576-5324
                Article
                PONE-D-16-03543
                10.1371/journal.pone.0158322
                4924854
                27351975
                068b476b-3b9b-46b2-9907-a3925c41c91b
                © 2016 Srinivasan, Gothandam

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 22 February 2016
                : 14 June 2016
                Page count
                Figures: 7, Tables: 2, Pages: 13
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100001412, Council of Scientific and Industrial Research;
                Award ID: 38(1327)/12/EMR-II
                Award Recipient : Gothandam KM
                This work was financially supported by Council for Scientific and Industrial Research (Grant No. 38(1327)/12/EMR-II dated 03.04.2012), India. Funders have no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript.
                Categories
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                Biology and Life Sciences
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                Molecular Biology Techniques
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