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<document id="7F4A1208785FF03FB2B96957BCB15B5F" ID-CLB-Dataset="298096" ID-DOI="10.1515/bot-2018-0028" ID-GBIF-Dataset="c4450f0c-1dae-40b4-b98b-29f058a8503e" ID-ISSN="1437-4323" ID-Zenodo-Dep="11360085" IM.bibliography_approvedBy="carolina" IM.illustrations_approvedBy="carolina" IM.materialsCitations_approvedBy="felipe" IM.metadata_approvedBy="felipe" IM.tables_approvedBy="carolina" IM.taxonomicNames_approvedBy="carolina" IM.treatments_approvedBy="carolina" checkinTime="1716847565839" checkinUser="felipe" docAuthor="Ramessur, Anusha Devi, Bothwell, John H., Maggs, Christine A., Gan, Sook Yee &amp; Phang, Siew Moi" docDate="2018" docId="038F146EFFCEA12F507596C46B70DF8F" docLanguage="en" docName="BotMar.61.5.499-510.pdf" docOrigin="Botanica Marina (Warsaw, Poland) 61 (5)" docSource="http://dx.doi.org/10.1515/bot-2018-0028" docStyle="DocumentStyle:9102D1D0F376D6FBA22A1B5DFA71D3A3.2:BotMar.2015-.journal_article.closed" docStyleId="9102D1D0F376D6FBA22A1B5DFA71D3A3" docStyleName="BotMar.2015-.journal_article.closed" docStyleVersion="2" docTitle="Agrobacterium LBA" docType="treatment" docVersion="4" lastPageNumber="503" masterDocId="FFB66C16FFCDA12A534D904D6923D946" masterDocTitle="Agrobacterium-mediated gene delivery and transient expression in the red macroalga Chondrus crispus" masterLastPageNumber="510" masterPageNumber="499" pageNumber="502" updateTime="1717626902964" updateUser="ExternalLinkService">
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<mods:title id="1B13BEFC9DBAA64FABAC727AE1996DC0">Agrobacterium-mediated gene delivery and transient expression in the red macroalga Chondrus crispus</mods:title>
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<mods:namePart id="0E5EE2F91365848220BC718342559378">Ramessur, Anusha Devi</mods:namePart>
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<mods:namePart id="779CF61D5D5C34630BD86613BF8196A0">Bothwell, John H.</mods:namePart>
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<mods:namePart id="D4FDB946AD492CA6B1E86DA639605ED2">Maggs, Christine A.</mods:namePart>
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<mods:namePart id="00A3A49EF8A598A41A1CB9FB8CA11E2A">Gan, Sook Yee</mods:namePart>
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<mods:namePart id="A7EF2E20CA0B57AE6FD67DB0DF61B3B8">Phang, Siew Moi</mods:namePart>
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<mods:title id="2FCB7917C7960D61A191D1E9F3202EF2">Botanica Marina</mods:title>
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<mods:date id="AC3D35FC4F0C5D31D1FF211857B3CC5C">2018</mods:date>
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<mods:title id="85F5EEA46E3DC2F4BCD114EEFBBEF427">Warsaw, Poland</mods:title>
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<mods:number id="506290FC03E64194EBC96001F8D5CA25">2018-09-12</mods:number>
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<mods:number id="5F433F3B8227998C870841CF7B7501F0">61</mods:number>
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<treatment id="038F146EFFCEA12F507596C46B70DF8F" ID-DOI="http://doi.org/10.5281/zenodo.11494493" ID-GBIF-Taxon="232143092" ID-Zenodo-Dep="11494493" LSID="urn:lsid:plazi:treatment:038F146EFFCEA12F507596C46B70DF8F" httpUri="http://treatment.plazi.org/id/038F146EFFCEA12F507596C46B70DF8F" lastPageId="5" lastPageNumber="503" pageId="3" pageNumber="502">
<subSubSection id="C33CF6F3FFCEA129507596C46D89DF89" pageId="3" pageNumber="502" type="nomenclature">
<paragraph id="8B99A578FFCEA129507596C46D89DF89" blockId="3.[824,1412,1673,1744]" pageId="3" pageNumber="502">
<heading id="D0D11214FFCEA129507596C46D89DF89" fontSize="12" level="2" pageId="3" pageNumber="502" reason="7">
<emphasis id="B952796AFFCEA129507596C46D89DF89" bold="true" pageId="3" pageNumber="502">
<taxonomicName id="4C26DEFBFFCEA129507596C46D64DFE1" ID-CoL="QZ9" authority="LBA" authorityName="LBA" box="[824,1095,1673,1703]" class="Alphaproteobacteria" family="Rhizobiaceae" genus="Agrobacterium" kingdom="Bacteria" order="Rhizobiales" pageId="3" pageNumber="502" phylum="Proteobacteria" rank="genus">
<emphasis id="B952796AFFCEA129507596C46D26DFE1" bold="true" box="[824,1029,1673,1703]" italics="true" pageId="3" pageNumber="502">Agrobacterium</emphasis>
LBA
</taxonomicName>
4404 cells successfully attached to
<taxonomicName id="4C26DEFBFFCEA129509096FC6D41DF96" authority="Stackhouse, 1797" box="[989,1122,1713,1744]" class="Florideophyceae" family="Gigartinaceae" genus="Chondrus" kingdom="Plantae" order="Gigartinales" pageId="3" pageNumber="502" phylum="Rhodophyta" rank="genus">
<emphasis id="B952796AFFCEA129509096FC6D41DF96" bold="true" box="[989,1122,1713,1744]" italics="true" pageId="3" pageNumber="502">Chondrus</emphasis>
</taxonomicName>
cells
</emphasis>
</heading>
</paragraph>
</subSubSection>
<subSubSection id="C33CF6F3FFCEA12F507596BB6B70DF8F" lastPageId="5" lastPageNumber="504" pageId="3" pageNumber="502" type="description">
<paragraph id="8B99A578FFCEA129507596BB6C33DEDF" blockId="3.[824,1452,1782,1945]" pageId="3" pageNumber="502">
Under the
<collectionCode id="ED373DBDFFCEA12950FE96BB6AC4DE48" box="[947,999,1782,1806]" pageId="3" pageNumber="502">SEM</collectionCode>
, short, rod-shaped bacterial cells about 0.8 × 2 µm were seen attached in large numbers (an estimated average of one bacterial cell per µm
<superScript id="7C530830FFCEA129564297776C35DE0E" attach="left" box="[1295,1302,1850,1864]" fontSize="6" pageId="3" pageNumber="502">2</superScript>
) to the
<taxonomicName id="4C26DEFBFFCEA129562097776A44DE30" class="Florideophyceae" family="Gigartinaceae" genus="Chondrus" kingdom="Plantae" order="Gigartinales" pageId="3" pageNumber="502" phylum="Rhodophyta" rank="genus">
<emphasis id="B952796AFFCEA129562097776A44DE30" bold="true" italics="true" pageId="3" pageNumber="502">Chondrus</emphasis>
</taxonomicName>
thallus surface at the wound sites. Whole colonies were observed in a fibrillar mesh (
<figureCitation id="131DB9FDFFCEA12957E497CC6C27DEDF" box="[1193,1284,1921,1945]" captionStart="Figure 2" captionStartId="4.[136,193,514,534]" captionTargetBox="[136,1270,174,483]" captionTargetId="figure-315@4.[136,1270,174,483]" captionTargetPageId="4" captionText="Figure 2: Intimate contact and adhesion of bacteria to wound sites of Chondrus thalli. Thalli co-cultivated for 48 h in induction medium were fixed and viewed under a scanning electron microscope: (A) control thallus incubated without bacteria showing a clean bacterium-free surface; (B) close up view of rod-shaped bacteria attached to thallus surface at wound site; (C) bacterial film at wound sites on thallus surface." figureDoi="http://doi.org/10.5281/zenodo.11360093" httpUri="https://zenodo.org/record/11360093/files/figure.png" pageId="3" pageNumber="502">Figure 2</figureCitation>
).
</paragraph>
<caption id="DF59F5F0FFC9A12E53C5924F6B7ADB28" ID-DOI="http://doi.org/10.5281/zenodo.11360093" ID-Zenodo-Dep="11360093" httpUri="https://zenodo.org/record/11360093/files/figure.png" pageId="4" pageNumber="503" startId="4.[136,193,514,534]" targetBox="[136,1270,174,483]" targetPageId="4" targetType="figure">
<paragraph id="8B99A578FFC9A12E53C5924F6ABEDB50" blockId="4.[136,1414,513,622]" box="[136,925,513,534]" pageId="4" pageNumber="503">
<emphasis id="B952796AFFC9A12E53C5924F6ABEDB50" bold="true" box="[136,925,513,534]" pageId="4" pageNumber="503">
Figure 2: Intimate contact and adhesion of bacteria to wound sites of
<taxonomicName id="4C26DEFBFFC9A12E5044924C6A41DB53" box="[777,866,513,533]" class="Florideophyceae" family="Gigartinaceae" genus="Chondrus" kingdom="Plantae" order="Gigartinales" pageId="4" pageNumber="503" phylum="Rhodophyta" rank="genus">
<emphasis id="B952796AFFC9A12E5044924C6A41DB53" bold="true" box="[777,866,513,533]" italics="true" pageId="4" pageNumber="503">Chondrus</emphasis>
</taxonomicName>
thalli.
</emphasis>
</paragraph>
<paragraph id="8B99A578FFC9A12E53C592526B7ADB28" blockId="4.[136,1414,513,622]" pageId="4" pageNumber="503">Thalli co-cultivated for 48 h in induction medium were fixed and viewed under a scanning electron microscope: (A) control thallus incubated without bacteria showing a clean bacterium-free surface; (B) close up view of rod-shaped bacteria attached to thallus surface at wound site; (C) bacterial film at wound sites on thallus surface.</paragraph>
</caption>
<paragraph id="8B99A578FFC9A12E53C592FF6802DBBE" blockId="4.[136,762,690,760]" pageId="4" pageNumber="503">
<emphasis id="B952796AFFC9A12E53C592FF6802DBBE" bold="true" pageId="4" pageNumber="503">
Protocol for eliminating
<taxonomicName id="4C26DEFBFFC9A12E529592FF6B86DB96" box="[472,677,690,720]" class="Alphaproteobacteria" family="Rhizobiaceae" genus="Agrobacterium" kingdom="Bacteria" order="Rhizobiales" pageId="4" pageNumber="503" phylum="Proteobacteria" rank="genus">
<emphasis id="B952796AFFC9A12E529592FF6B86DB96" bold="true" box="[472,677,690,720]" italics="true" pageId="4" pageNumber="503">Agrobacterium</emphasis>
</taxonomicName>
using cefotaxime
</emphasis>
</paragraph>
<paragraph id="8B99A578FFC9A12E53C5935768E1DAFB" blockId="4.[136,764,794,957]" pageId="4" pageNumber="503">
The results of testing cefotaxime concentrations of 500 and
<quantity id="4CDE089DFFC9A12E53F393706835DA13" box="[190,278,829,853]" metricMagnitude="-4" metricUnit="kg" metricValue="8.0" pageId="4" pageNumber="503" unit="mg" value="800.0">800 mg</quantity>
<superScript id="7C530830FFC9A12E526F93706815DA0C" attach="left" box="[290,310,828,853]" fontSize="6" pageId="4" pageNumber="503">l−1</superScript>
confirmed that this cefotaxime-supplemented wash protocol ensured that residual
<taxonomicName id="4C26DEFBFFC9A12E51CB93136999DADC" class="Alphaproteobacteria" family="Rhizobiaceae" genus="Agrobacterium" kingdom="Bacteria" order="Rhizobiales" pageId="4" pageNumber="503" phylum="Proteobacteria" rank="genus">
<emphasis id="B952796AFFC9A12E51CB93136999DADC" bold="true" italics="true" pageId="4" pageNumber="503">Agrobacterium</emphasis>
</taxonomicName>
and other culturable bacteria were eliminated on the thalli within 6 days (
<tableCitation id="C6A490C3FFC9A12E522B93E86897DAFB" box="[358,436,933,957]" captionStart="Table 1" captionStartId="4.[136,186,1325,1345]" captionTargetPageId="4" captionText="Table 1: Confirmation of the suitability of 500 and 800 mg l−1 cefotaxime in eliminating bacteria." httpUri="http://table.plazi.org/id/DF59F5F0FFC9A12E53C5956068EBDC18" pageId="4" pageNumber="503" tableUuid="DF59F5F0FFC9A12E53C5956068EBDC18">Table 1</tableCitation>
).
</paragraph>
<paragraph id="8B99A578FFC9A12E53C594456B49DD08" blockId="4.[136,655,1032,1102]" pageId="4" pageNumber="503">
<heading id="D0D11214FFC9A12E53C594456B49DD08" fontSize="12" level="2" pageId="4" pageNumber="503" reason="7">
<emphasis id="B952796AFFC9A12E53C594456B49DD08" bold="true" pageId="4" pageNumber="503">
<taxonomicName id="4C26DEFBFFC9A12E53C594456876DD60" ID-CoL="QZ9" box="[136,341,1032,1062]" class="Alphaproteobacteria" family="Rhizobiaceae" genus="Agrobacterium" kingdom="Bacteria" order="Rhizobiales" pageId="4" pageNumber="503" phylum="Proteobacteria" rank="genus">
<emphasis id="B952796AFFC9A12E53C594456876DD60" bold="true" box="[136,341,1032,1062]" italics="true" pageId="4" pageNumber="503">Agrobacterium</emphasis>
</taxonomicName>
successfully delivered pCAMBIA 1301 into
<taxonomicName id="4C26DEFBFFC9A12E52D894626B39DD08" box="[405,538,1071,1102]" class="Florideophyceae" family="Gigartinaceae" genus="Chondrus" kingdom="Plantae" order="Gigartinales" pageId="4" pageNumber="503" phylum="Rhodophyta" rank="genus">
<emphasis id="B952796AFFC9A12E52D894626B39DD08" bold="true" box="[405,538,1071,1102]" italics="true" pageId="4" pageNumber="503">Chondrus</emphasis>
</taxonomicName>
thalli
</emphasis>
</heading>
</paragraph>
<paragraph id="8B99A578FFC9A12E53C594386C20DD01" blockId="4.[136,764,1141,1235]" lastBlockId="4.[794,1421,688,1095]" pageId="4" pageNumber="503">
The methodology for transformation developed using pCAMBIA 1301 followed by the established wash protocol included controls to detect any false positives occurring from indigenous activity of beta-glucoronidase in
<taxonomicName id="4C26DEFBFFC9A12E560392FD6A6ADBAA" class="Florideophyceae" family="Gigartinaceae" genus="Chondrus" kingdom="Plantae" order="Gigartinales" pageId="4" pageNumber="503" phylum="Rhodophyta" rank="genus">
<emphasis id="B952796AFFC9A12E560392FD6A6ADBAA" bold="true" italics="true" pageId="4" pageNumber="503">Chondrus</emphasis>
</taxonomicName>
(unwounded and untransformed thalli,
<figureCitation id="131DB9FDFFC9A12E565992986CA2DBAB" box="[1300,1409,725,749]" captionStart="Figure 3" captionStartId="5.[166,223,476,496]" captionTargetBox="[166,1451,174,445]" captionTargetId="figure-608@5.[166,1451,174,445]" captionTargetPageId="5" captionText="Figure 3: Histochemical staining and low-power microscopic analysis of Chondrus thallus segments for transient expression of GUS 7 days after co-cultivation. (A) Pin-pricked thalli co-cultivated without bacteria, (B) pin-pricked thalli co-cultivated with untransformed LBA4404 bacteria, (C) unwounded and untransformed thalli, (D) pin-pricked thalli co-cultivated with LBA4404/pRI 910 (no GUS gene), (E) thalli transformed with LBA4404 (pCAMBIA 1301), scale bar: 1 cm." figureDoi="http://doi.org/10.5281/zenodo.11360095" httpUri="https://zenodo.org/record/11360095/files/figure.png" pageId="4" pageNumber="503">Figure 3C</figureCitation>
), indigenous activity from LBA4404 bacterial cells (pin-pricked thalli co-cultivated with untransformed
<taxonomicName id="4C26DEFBFFC9A12E561F93546AE6DA13" authority="LBA" authorityName="LBA" class="Alphaproteobacteria" family="Rhizobiaceae" genus="Agrobacterium" kingdom="Bacteria" order="Rhizobiales" pageId="4" pageNumber="503" phylum="Proteobacteria" rank="genus">
<emphasis id="B952796AFFC9A12E561F93546AA4DA12" bold="true" italics="true" pageId="4" pageNumber="503">Agrobacterium</emphasis>
LBA
</taxonomicName>
4404 bacteria,
<figureCitation id="131DB9FDFFC9A12E573C93706DC2DA13" box="[1137,1249,829,853]" captionStart="Figure 3" captionStartId="5.[166,223,476,496]" captionTargetBox="[166,1451,174,445]" captionTargetId="figure-608@5.[166,1451,174,445]" captionTargetPageId="5" captionText="Figure 3: Histochemical staining and low-power microscopic analysis of Chondrus thallus segments for transient expression of GUS 7 days after co-cultivation. (A) Pin-pricked thalli co-cultivated without bacteria, (B) pin-pricked thalli co-cultivated with untransformed LBA4404 bacteria, (C) unwounded and untransformed thalli, (D) pin-pricked thalli co-cultivated with LBA4404/pRI 910 (no GUS gene), (E) thalli transformed with LBA4404 (pCAMBIA 1301), scale bar: 1 cm." figureDoi="http://doi.org/10.5281/zenodo.11360095" httpUri="https://zenodo.org/record/11360095/files/figure.png" pageId="4" pageNumber="503">Figure 3B</figureCitation>
), physiological response of algal cells to wounding (pin-pricked thalli cultivated without bacteria,
<figureCitation id="131DB9FDFFC9A12E571493CF6DE5DADC" box="[1113,1222,898,922]" captionStart="Figure 3" captionStartId="5.[166,223,476,496]" captionTargetBox="[166,1451,174,445]" captionTargetId="figure-608@5.[166,1451,174,445]" captionTargetPageId="5" captionText="Figure 3: Histochemical staining and low-power microscopic analysis of Chondrus thallus segments for transient expression of GUS 7 days after co-cultivation. (A) Pin-pricked thalli co-cultivated without bacteria, (B) pin-pricked thalli co-cultivated with untransformed LBA4404 bacteria, (C) unwounded and untransformed thalli, (D) pin-pricked thalli co-cultivated with LBA4404/pRI 910 (no GUS gene), (E) thalli transformed with LBA4404 (pCAMBIA 1301), scale bar: 1 cm." figureDoi="http://doi.org/10.5281/zenodo.11360095" httpUri="https://zenodo.org/record/11360095/files/figure.png" pageId="4" pageNumber="503">Figure 3A</figureCitation>
) and vector components (pin-pricked thalli co-cultivated with
<taxonomicName id="4C26DEFBFFC9A12E565A93EE6AA7DA99" authority="LBA" authorityName="LBA" class="Alphaproteobacteria" family="Rhizobiaceae" genus="Agrobacterium" kingdom="Bacteria" order="Rhizobiales" pageId="4" pageNumber="503" phylum="Proteobacteria" rank="genus">
<emphasis id="B952796AFFC9A12E565A93EE6A6FDA99" bold="true" italics="true" pageId="4" pageNumber="503">Agrobacterium</emphasis>
LBA
</taxonomicName>
4404 bearing a construct where the
<emphasis id="B952796AFFC9A12E5641938B6C19DAA6" bold="true" box="[1292,1338,966,992]" italics="true" pageId="4" pageNumber="503">
<collectionCode id="ED373DBDFFC9A12E5641938B6C19DAA6" box="[1292,1338,966,992]" pageId="4" pageNumber="503">GUS</collectionCode>
</emphasis>
gene is absent; pRI 910 binary vector,
<figureCitation id="131DB9FDFFC9A12E573C93A76DFCDD44" box="[1137,1247,1002,1026]" captionStart="Figure 3" captionStartId="5.[166,223,476,496]" captionTargetBox="[166,1451,174,445]" captionTargetId="figure-608@5.[166,1451,174,445]" captionTargetPageId="5" captionText="Figure 3: Histochemical staining and low-power microscopic analysis of Chondrus thallus segments for transient expression of GUS 7 days after co-cultivation. (A) Pin-pricked thalli co-cultivated without bacteria, (B) pin-pricked thalli co-cultivated with untransformed LBA4404 bacteria, (C) unwounded and untransformed thalli, (D) pin-pricked thalli co-cultivated with LBA4404/pRI 910 (no GUS gene), (E) thalli transformed with LBA4404 (pCAMBIA 1301), scale bar: 1 cm." figureDoi="http://doi.org/10.5281/zenodo.11360095" httpUri="https://zenodo.org/record/11360095/files/figure.png" pageId="4" pageNumber="503">Figure 3D</figureCitation>
). None of these controls were stained. However, thalli transformed with pCAMBIA 1301 were stained blue (
<figureCitation id="131DB9FDFFC9A12E57C194626DD5DD01" box="[1164,1270,1071,1095]" captionStart="Figure 3" captionStartId="5.[166,223,476,496]" captionTargetBox="[166,1451,174,445]" captionTargetId="figure-608@5.[166,1451,174,445]" captionTargetPageId="5" captionText="Figure 3: Histochemical staining and low-power microscopic analysis of Chondrus thallus segments for transient expression of GUS 7 days after co-cultivation. (A) Pin-pricked thalli co-cultivated without bacteria, (B) pin-pricked thalli co-cultivated with untransformed LBA4404 bacteria, (C) unwounded and untransformed thalli, (D) pin-pricked thalli co-cultivated with LBA4404/pRI 910 (no GUS gene), (E) thalli transformed with LBA4404 (pCAMBIA 1301), scale bar: 1 cm." figureDoi="http://doi.org/10.5281/zenodo.11360095" httpUri="https://zenodo.org/record/11360095/files/figure.png" pageId="4" pageNumber="503">Figure 3E</figureCitation>
).
</paragraph>
<paragraph id="8B99A578FFC9A12E505794E96DD7DDAC" blockId="4.[794,1406,1188,1258]" pageId="4" pageNumber="503">
<emphasis id="B952796AFFC9A12E505794E96DD7DDAC" bold="true" pageId="4" pageNumber="503">Manipulating transformation conditions can enhance transformation efficiency</emphasis>
</paragraph>
<paragraph id="8B99A578FFC9A12E5057955C6C4ADFC5" blockId="4.[794,1422,1297,1667]" pageId="4" pageNumber="503">
As shown in
<figureCitation id="131DB9FDFFC9A12E50EA955C6D21DC6F" box="[935,1026,1297,1321]" captionStart="Figure 4" captionStartId="5.[167,224,975,995]" captionTargetBox="[167,792,654,944]" captionTargetId="figure-449@5.[306,788,676,873]" captionTargetPageId="5" captionText="Figure 4: Effect of cultivation medium, wounding method and acetosyringone on Agrobacterium transformation efficiency. The transformation efficiencies of Chondrus thalli segments co-cultivated with pCAMBIA 1301-transformed Agrobacterium in seawater and Induction Medium (IM) were compared. Using seawater as co-cultivation medium, the wound-related treatments, namely biolistics wounding, wounding using pin-pricks and no wounding, were compared. The effect of acetosyringone (100 µM) was also studied. Data are expressed as means ± SE (n = 4). The Mann-Whitney non-parametric test was conducted to compare between two groups." figureDoi="http://doi.org/10.5281/zenodo.11360097" httpUri="https://zenodo.org/record/11360097/files/figure.png" pageId="4" pageNumber="503">Figure 4</figureCitation>
, among the pin-pricked thalli, a significant increase in transformation efficiency (higher percentage of thallus segments stained blue) was observed when seawater was used in preference to Induction Medium (Mann-Whitney test, p = 0.029) in the presence of acetosyringone. However, no transformation was detected in the unwounded thallus segments or those wounded by bombardment when co-cultivation was conducted using seawater. In addition, without the presence of acetosyringone, transformation occurred at a significantly lower efficiency of less than 10% (Mann-Whitney, p = 0.004).
</paragraph>
<caption id="DF59F5F0FFC9A12E53C5956068EBDC18" ID-Table-UUID="DF59F5F0FFC9A12E53C5956068EBDC18" httpUri="http://table.plazi.org/id/DF59F5F0FFC9A12E53C5956068EBDC18" pageId="4" pageNumber="503" startId="4.[136,186,1325,1345]" targetBox="[136,764,1417,1690]" targetIsTable="true" targetPageId="4" targetType="table">
<paragraph id="8B99A578FFC9A12E53C5956068EBDC18" blockId="4.[136,694,1324,1374]" pageId="4" pageNumber="503">
<emphasis id="B952796AFFC9A12E53C595606B91DC7F" bold="true" box="[136,690,1324,1345]" pageId="4" pageNumber="503">
Table 1: Confirmation of the suitability of 500 and 800 mg
<superScript id="7C530830FFC9A12E51E995606B91DC7F" attach="left" box="[676,690,1324,1345]" fontSize="5" pageId="4" pageNumber="503">l−</superScript>
</emphasis>
1 cefotaxime in eliminating bacteria.
</paragraph>
</caption>
<paragraph id="8B99A578FFC9A12E52E395C46BD8DFDC" pageId="4" pageNumber="503">
<table id="F92657D8FFC95ED553C595C46BDFDFDC" box="[136,764,1417,1690]" gridcols="4" gridrows="8" pageId="4" pageNumber="503">
<tr id="3516A73AFFC95ED553C595C46BDFDCDB" box="[136,764,1417,1437]" gridrow="0" pageId="4" pageNumber="503" rowspan-0="1" rowspan-1="1">
<th id="76C7CE46FFC95ED552E695C46BDFDCDB" box="[427,764,1417,1437]" colspan="2" colspanRight="1" gridcol="2" gridrow="0" pageId="4" pageNumber="503">
<emphasis id="B952796AFFC9A12E52E395C46BDFDCDB" bold="true" box="[430,764,1417,1437]" pageId="4" pageNumber="503">Mean number of colonies (per plate)</emphasis>
</th>
</tr>
<tr id="3516A73AFFC95ED553C595F86BDFDC8C" box="[136,764,1461,1482]" gridrow="1" pageId="4" pageNumber="503" rowspan-1="1">
<th id="76C7CE46FFC95ED553C595F86988DC8C" box="[136,171,1461,1482]" gridcol="0" gridrow="1" pageId="4" pageNumber="503">
<emphasis id="B952796AFFC9A12E53C595FB6988DC8C" bold="true" box="[136,171,1462,1482]" pageId="4" pageNumber="503">Day</emphasis>
</th>
<td id="76C7CE46FFC95ED552E695F86BDFDC8C" box="[427,764,1461,1482]" colspan="2" colspanRight="1" gridcol="2" gridrow="1" pageId="4" pageNumber="503">
<emphasis id="B952796AFFC9A12E52E695FB6BC0DC8C" bold="true" box="[427,739,1462,1482]" pageId="4" pageNumber="503">Concentration of cefotaxime (mg l</emphasis>
<emphasis id="B952796AFFC9A12E51A895FB6BD6DC84" bold="true" box="[741,757,1461,1474]" pageId="4" pageNumber="503">− 1</emphasis>
<emphasis id="B952796AFFC9A12E51B895FB6BDFDC8C" bold="true" box="[757,764,1462,1482]" pageId="4" pageNumber="503">)</emphasis>
</td>
</tr>
<tr id="3516A73AFFC95ED553C595AE6BDFDCB1" box="[136,764,1507,1527]" gridrow="2" pageId="4" pageNumber="503" rowspan-0="1">
<td id="76C7CE46FFC95ED5525A95AE6877DCB1" box="[279,340,1507,1527]" gridcol="1" gridrow="2" pageId="4" pageNumber="503">
<emphasis id="B952796AFFC9A12E520595AE6877DCB1" bold="true" box="[328,340,1507,1527]" pageId="4" pageNumber="503">0</emphasis>
</td>
<td id="76C7CE46FFC95ED552E695AE6B0BDCB1" box="[427,552,1507,1527]" gridcol="2" gridrow="2" pageId="4" pageNumber="503">
<emphasis id="B952796AFFC9A12E514E95AE6B04DCB1" bold="true" box="[515,551,1507,1527]" pageId="4" pageNumber="503">500</emphasis>
</td>
<td id="76C7CE46FFC95ED551DE95AE6BDFDCB1" box="[659,764,1507,1527]" gridcol="3" gridrow="2" pageId="4" pageNumber="503">
<emphasis id="B952796AFFC9A12E519A95AE6BD8DCB1" bold="true" box="[727,763,1507,1527]" pageId="4" pageNumber="503">800</emphasis>
</td>
</tr>
<tr id="3516A73AFFC95ED553C5965C6BDFDF63" box="[136,764,1553,1573]" gridrow="3" pageId="4" pageNumber="503">
<th id="76C7CE46FFC95ED553C5965C6988DF63" box="[136,171,1553,1573]" gridcol="0" gridrow="3" pageId="4" pageNumber="503">
<emphasis id="B952796AFFC9A12E53C5965C69B7DF63" bold="true" box="[136,148,1553,1573]" pageId="4" pageNumber="503">0</emphasis>
</th>
<td id="76C7CE46FFC95ED5525A965C6877DF63" box="[279,340,1553,1573]" gridcol="1" gridrow="3" pageId="4" pageNumber="503">
&gt;
<emphasis id="B952796AFFC9A12E526E965C6877DF63" bold="true" box="[291,340,1553,1573]" pageId="4" pageNumber="503">1000</emphasis>
</td>
<td id="76C7CE46FFC95ED552E6965C6B0BDF63" box="[427,552,1553,1573]" gridcol="2" gridrow="3" pageId="4" pageNumber="503">
&gt;
<emphasis id="B952796AFFC9A12E52BA965C6B0BDF63" bold="true" box="[503,552,1553,1573]" pageId="4" pageNumber="503">1000</emphasis>
</td>
<td id="76C7CE46FFC95ED551DE965C6BDFDF63" box="[659,764,1553,1573]" gridcol="3" gridrow="3" pageId="4" pageNumber="503">
&gt;
<emphasis id="B952796AFFC9A12E5186965C6BDFDF63" bold="true" box="[715,764,1553,1573]" pageId="4" pageNumber="503">1000</emphasis>
</td>
</tr>
<tr id="3516A73AFFC95ED553C596636BDFDF04" box="[136,764,1582,1602]" gridrow="4" pageId="4" pageNumber="503">
<th id="76C7CE46FFC95ED553C596636988DF04" box="[136,171,1582,1602]" gridcol="0" gridrow="4" pageId="4" pageNumber="503">
<emphasis id="B952796AFFC9A12E53C5966369B7DF04" bold="true" box="[136,148,1582,1602]" pageId="4" pageNumber="503">2</emphasis>
</th>
<td id="76C7CE46FFC95ED5525A96636877DF04" box="[279,340,1582,1602]" gridcol="1" gridrow="4" pageId="4" pageNumber="503">
&gt;
<emphasis id="B952796AFFC9A12E526296636870DF04" bold="true" box="[303,339,1582,1602]" pageId="4" pageNumber="503">300</emphasis>
</td>
<td id="76C7CE46FFC95ED552E696636B0BDF04" box="[427,552,1582,1602]" gridcol="2" gridrow="4" pageId="4" pageNumber="503">
<emphasis id="B952796AFFC9A12E528D96636B0BDF04" bold="true" box="[448,552,1582,1602]" pageId="4" pageNumber="503">0.75 ± 0.95</emphasis>
</td>
<td id="76C7CE46FFC95ED551DE96636BDFDF04" box="[659,764,1582,1602]" gridcol="3" gridrow="4" pageId="4" pageNumber="503">
<emphasis id="B952796AFFC9A12E51DE96636BDFDF04" bold="true" box="[659,764,1582,1602]" pageId="4" pageNumber="503">1.25 ± 0.95</emphasis>
</td>
</tr>
<tr id="3516A73AFFC95ED553C596066BDFDF26" box="[136,764,1611,1632]" gridrow="5" pageId="4" pageNumber="503">
<th id="76C7CE46FFC95ED553C596066988DF26" box="[136,171,1611,1632]" gridcol="0" gridrow="5" pageId="4" pageNumber="503">
<emphasis id="B952796AFFC9A12E53C5960669B7DF19" bold="true" box="[136,148,1611,1631]" pageId="4" pageNumber="503">4</emphasis>
</th>
<td id="76C7CE46FFC95ED5525A96066877DF26" box="[279,340,1611,1632]" gridcol="1" gridrow="5" pageId="4" pageNumber="503">
&gt;
<emphasis id="B952796AFFC9A12E526296066870DF19" bold="true" box="[303,339,1611,1631]" pageId="4" pageNumber="503">300</emphasis>
</td>
<td id="76C7CE46FFC95ED552E696066B0BDF26" box="[427,552,1611,1632]" gridcol="2" gridrow="5" pageId="4" pageNumber="503">
<emphasis id="B952796AFFC9A12E528D96066B0BDF19" bold="true" box="[448,552,1611,1632]" pageId="4" pageNumber="503">0.25 ± 1.60</emphasis>
</td>
<td id="76C7CE46FFC95ED551DE96066BDFDF26" box="[659,764,1611,1632]" gridcol="3" gridrow="5" pageId="4" pageNumber="503">
<emphasis id="B952796AFFC9A12E51DE96066BDFDF19" bold="true" box="[659,764,1611,1632]" pageId="4" pageNumber="503">2.00 ± 0.50</emphasis>
</td>
</tr>
<tr id="3516A73AFFC95ED553C596246BDFDF3B" box="[136,764,1641,1661]" gridrow="6" pageId="4" pageNumber="503">
<th id="76C7CE46FFC95ED553C596246988DF3B" box="[136,171,1641,1661]" gridcol="0" gridrow="6" pageId="4" pageNumber="503">
<emphasis id="B952796AFFC9A12E53C5962469B7DF3B" bold="true" box="[136,148,1641,1661]" pageId="4" pageNumber="503">6</emphasis>
</th>
<td id="76C7CE46FFC95ED5525A96246877DF3B" box="[279,340,1641,1661]" gridcol="1" gridrow="6" pageId="4" pageNumber="503">
&gt;
<emphasis id="B952796AFFC9A12E526296246870DF3B" bold="true" box="[303,339,1641,1661]" pageId="4" pageNumber="503">300</emphasis>
</td>
<td id="76C7CE46FFC95ED552E696246B0BDF3B" box="[427,552,1641,1661]" gridcol="2" gridrow="6" pageId="4" pageNumber="503">
<emphasis id="B952796AFFC9A12E515696246B04DF3B" bold="true" box="[539,551,1641,1661]" pageId="4" pageNumber="503">0</emphasis>
</td>
<td id="76C7CE46FFC95ED551DE96246BDFDF3B" box="[659,764,1641,1661]" gridcol="3" gridrow="6" pageId="4" pageNumber="503">
<emphasis id="B952796AFFC9A12E51D296246BDFDF3B" bold="true" box="[671,764,1641,1661]" pageId="4" pageNumber="503">1.00 ± 1.2</emphasis>
</td>
</tr>
<tr id="3516A73AFFC95ED553C596CB6BDFDFDC" box="[136,764,1670,1690]" gridrow="7" pageId="4" pageNumber="503">
<th id="76C7CE46FFC95ED553C596CB6988DFDC" box="[136,171,1670,1690]" gridcol="0" gridrow="7" pageId="4" pageNumber="503">
<emphasis id="B952796AFFC9A12E53C596CB69B7DFDC" bold="true" box="[136,148,1670,1690]" pageId="4" pageNumber="503">8</emphasis>
</th>
<td id="76C7CE46FFC95ED5525A96CB6877DFDC" box="[279,340,1670,1690]" gridcol="1" gridrow="7" pageId="4" pageNumber="503">&gt;300</td>
<td id="76C7CE46FFC95ED552E696CB6B0BDFDC" box="[427,552,1670,1690]" gridcol="2" gridrow="7" pageId="4" pageNumber="503">0</td>
<td id="76C7CE46FFC95ED551DE96CB6BDFDFDC" box="[659,764,1670,1690]" gridcol="3" gridrow="7" pageId="4" pageNumber="503">0</td>
</tr>
</table>
</paragraph>
<paragraph id="8B99A578FFC9A12E53C596FB6B49DED1" blockId="4.[136,764,1718,1943]" pageId="4" pageNumber="503">
<tableNote id="76C0A4F6FFC9A12E53C596FB6B49DED1" pageId="4" pageNumber="503" targetBox="[136,764,1417,1690]" targetPageId="4">
<emphasis id="B952796AFFC9A12E53C596FB6983DE1B" bold="true" pageId="4" pageNumber="503">
<taxonomicName id="4C26DEFBFFC9A12E53C596FB682CDF8C" box="[136,271,1718,1738]" class="Alphaproteobacteria" family="Rhizobiaceae" genus="Agrobacterium" kingdom="Bacteria" order="Rhizobiales" pageId="4" pageNumber="503" phylum="Proteobacteria" rank="genus">
<emphasis id="B952796AFFC9A12E53C596FB682CDF8C" bold="true" box="[136,271,1718,1738]" italics="true" pageId="4" pageNumber="503">Agrobacterium</emphasis>
</taxonomicName>
co-cultured with thalli was eliminated by consecutive washes and incubated in fresh antibiotic. Every 2 days, the thallus segments were rinsed for 4 min with sterile seawater six times before incubation for 48 h in fresh seawater with replenished cefotaxime. A thallus segment was streaked on a LBA plate which was incubated at 28
</emphasis>
°
<emphasis id="B952796AFFC9A12E53E5970568E4DE3C" bold="true" pageId="4" pageNumber="503">
C for up to 4 days to check for the persistence of
<taxonomicName id="4C26DEFBFFC9A12E511297046BC5DE1B" box="[607,742,1865,1885]" class="Alphaproteobacteria" family="Rhizobiaceae" genus="Agrobacterium" kingdom="Bacteria" order="Rhizobiales" pageId="4" pageNumber="503" phylum="Proteobacteria" rank="genus">
<emphasis id="B952796AFFC9A12E511297046BC5DE1B" bold="true" box="[607,742,1865,1885]" italics="true" pageId="4" pageNumber="503">Agrobacterium</emphasis>
</taxonomicName>
. This was conducted in triplicates (n
</emphasis>
= 3). Colonies appearing from periodic streaks of thalli on LBA plates were counted.
</tableNote>
</paragraph>
<paragraph id="8B99A578FFC9A12E505796836C12DE52" blockId="4.[794,1355,1742,1813]" pageId="4" pageNumber="503">
<emphasis id="B952796AFFC9A12E505796836C12DE52" bold="true" pageId="4" pageNumber="503">
Successful expression obtained with the
<taxonomicName id="4C26DEFBFFC9A12E505796BB6ABCDE53" box="[794,927,1782,1813]" class="Florideophyceae" family="Gigartinaceae" genus="Chondrus" kingdom="Plantae" order="Gigartinales" pageId="4" pageNumber="503" phylum="Rhodophyta" rank="genus">
<emphasis id="B952796AFFC9A12E505796BB6ABCDE53" bold="true" box="[794,927,1782,1813]" italics="true" pageId="4" pageNumber="503">Chondrus</emphasis>
</taxonomicName>
-specific expression cassette
</emphasis>
</paragraph>
<paragraph id="8B99A578FFC9A12F505797766B70DF8F" blockId="4.[794,1421,1850,1945]" lastBlockId="5.[166,794,1366,1737]" lastPageId="5" lastPageNumber="504" pageId="4" pageNumber="503">
Using the
<taxonomicName id="4C26DEFBFFC9A12E50C297776D0CDE15" box="[911,1071,1850,1875]" class="Alphaproteobacteria" family="Rhizobiaceae" genus="Agrobacterium" kingdom="Bacteria" order="Rhizobiales" pageId="4" pageNumber="503" phylum="Proteobacteria" rank="genus">
<emphasis id="B952796AFFC9A12E50C297776D0CDE15" bold="true" box="[911,1071,1850,1875]" italics="true" pageId="4" pageNumber="503">Agrobacterium</emphasis>
</taxonomicName>
transformation protocol developed using pCAMBIA 1301, the constructed pRI 910 (Ac-
<collectionCode id="ED373DBDFFC9A12E505797CC6A69DEDF" box="[794,842,1921,1945]" pageId="4" pageNumber="503">GUS</collectionCode>
) was delivered into thallus cells using seawater as the co-cultivation medium and with the same controls as for pCAMBIA 1301. Blue colouration was apparent at the sites of tear, cut or wounding on
<taxonomicName id="4C26DEFBFFC8A12F514195D76B8FDCF5" box="[524,684,1434,1459]" class="Alphaproteobacteria" family="Rhizobiaceae" genus="Agrobacterium" kingdom="Bacteria" order="Rhizobiales" pageId="5" pageNumber="504" phylum="Proteobacteria" rank="genus">
<emphasis id="B952796AFFC8A12F514195D76B8FDCF5" bold="true" box="[524,684,1434,1459]" italics="true" pageId="5" pageNumber="504">Agrobacterium</emphasis>
</taxonomicName>
-mediated transformed thalli when compared to controls (
<figureCitation id="131DB9FDFFC8A12F51FC95F36A2EDC90" box="[689,781,1470,1494]" captionStart="Figure 5" captionStartId="6.[136,193,1369,1389]" captionTargetBox="[146,1043,198,1329]" captionTargetId="figure-212@6.[136,1043,174,1339]" captionTargetPageId="6" captionText="Figure 5: GUS expression in Chondrus thallus segments transformed with LBA 4404/pRI 910 (Ac-GUS). (A) Surface view of control thallus not co-cultivated with Agrobacterium, (B) surface view of transformed thallus showing GUS expression at wound sites, (C) section through untransformed thallus, (D) thallus section within wounded area showing blue medullary cells at wound spots, (E) cortical cells from untransformed control thallus, (F) GUS-expressing cortical cells in transformed thallus, (G) medullary filaments from untransformed control thallus, (H) medullary filaments from transformed thallus." figureDoi="http://doi.org/10.5281/zenodo.11360099" httpUri="https://zenodo.org/record/11360099/files/figure.png" pageId="5" pageNumber="504">Figure 5</figureCitation>
). Transverse sections through the tissue revealed blue localization of the
<collectionCode id="ED373DBDFFC8A12F5235964E6884DF5D" box="[376,423,1539,1563]" pageId="5" pageNumber="504">GUS</collectionCode>
precipitate within the peripheral cortical cells and central medullary filaments. In general, a darker staining intensity was observed with pRI 910 (Ac-
<collectionCode id="ED373DBDFFC8A12F53EB962669F9DFC5" box="[166,218,1643,1667]" pageId="5" pageNumber="504">GUS</collectionCode>
). Furthermore, a higher transformation efficiency of 85% was obtained with this
<taxonomicName id="4C26DEFBFFC8A12F52A596C16B6DDFE0" box="[488,590,1676,1702]" class="Florideophyceae" family="Gigartinaceae" genus="Chondrus" kingdom="Plantae" order="Gigartinales" pageId="5" pageNumber="504" phylum="Rhodophyta" rank="genus">
<emphasis id="B952796AFFC8A12F52A596C16B6DDFE0" bold="true" box="[488,590,1676,1702]" italics="true" pageId="5" pageNumber="504">Chondrus</emphasis>
</taxonomicName>
-specific construct as opposed to 52% with pCAMBIA 1301.
</paragraph>
</subSubSection>
</treatment>
</document>
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