Difference between revisions of "Team:SCAU-China/Protocol"

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   </div>
 
   </div>
 
    
 
    
−
    <div class="container animated">
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    <div class="container">
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<div class="col-sm-12">
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<div class="row col">
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<div style="margin-bottom:30px">
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<div class="h1_font_size">Protocol</div>
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<div align="center">
+
−
<h1>Protocol</h1>
+
<div class="h2_font_size">Vector construction protocol</div>
−
</div>
+
−
</div>
+
<div class="h2_font_size">Transformation of rice</div>
−
<div style="margin-bottom:30px">
+
<div class="h3_font_size">1.Brief on the procedure:</div>
−
<div class="row">
+
<div class="p_font_size">Callus induction→ Subculture of callus → Co-culture of callus with Agrobacterium → Selection of resistant callus→ Differentiation of resistant callus → Rooting the seedling → Hardening the seedling →Transplanting the seedling.</div>
−
<div class="col-sm-1"></div>
+
<div class="h3_font_size">2.Detailed steps</div>
−
<div class="col-sm-10">
+
<div class="h4_font_size">2.1 Callus induction</div>
−
<h2>Vector construction protocol</h2>
+
<div class="p_font_size"><strong>2.1.1 Medium Preparation</strong></div>
−
+
<table class="table">
−
</div>
+
<thead>
−
</div>
+
<th>Component</th>
−
</div>
+
<th>Dosage</th>
−
<div style="margin-bottom:30px">
+
</thead>
−
<div class="row">
+
<tbody>
−
<div class="col-sm-1"></div>
+
<tr>
−
<div class="col-sm-10">
+
<td>10×N6 macroelement solution</td>
−
<h2>Transformation of rice</h2>
+
<td>50 mL</td>
−
<h3>1.Brief on the procedure:</h3>
+
</tr>
−
<p>Callus induction→ Subculture of callus → Co-culture of callus with Agrobacterium → Selection of resistant callus→ Differentiation of resistant callus → Rooting the seedling → Hardening the seedling →Transplanting the seedling.</p>
+
<tr>
−
<h3>2.Detailed steps</h3>
+
<td>1000×B5 microelement solution</td>
−
<h4>2.1 Callus induction</h4>
+
<td>0.5 mL</td>
−
<p><strong>2.1.1 Medium Preparation</strong></p>
+
</tr>
−
<p><table class="table">
+
<tr>
−
<thead>
+
<td>100×B5 vitamin solution</td>
−
<th>Component</th>
+
<td>5 mL</td>
−
<th>Dosage</th>
+
</tr>
−
</thead>
+
<tr>
−
<tbody>
+
<td>100×Ferric salt solution</td>
−
<tr>
+
<td>5 mL</td>
−
<td>10×N6macroelement solution</td>
+
</tr>
−
<td>50 mL</td>
+
<tr>
−
</tr>
+
<td>0.5 mg/mL 2,4-D</td>
−
<tr>
+
<td>3 mL</td>
−
<td>1000×B5microelement solution</td>
+
</tr>
−
<td>0.5 mL</td>
+
<tr>
−
</tr>
+
<td>Casein enzymatic hydrolysate</td>
−
<tr>
+
<td>150 mg</td>
−
<td>100×B5vitamin solution</td>
+
</tr>
−
<td>5 mL</td>
+
<tr>
−
</tr>
+
<td>L-Proline</td>
−
<tr>
+
<td>250 mg</td>
−
<td>100×Ferric salt solution</td>
+
</tr>
−
<td>5 mL</td>
+
<tr>
−
</tr>
+
<td>L-Glutamine</td>
−
<tr>
+
<td>250 mg</td>
−
<td>0.5 mg/mL 2,4-D</td>
+
</tr>
−
<td>3 mL</td>
+
<tr>
−
</tr>
+
<td>Sucrose</td>
−
<tr>
+
<td>15 g</td>
−
<td>Casein enzymatic hydrolysate</td>
+
</tr>
−
<td>150 mg</td>
+
<tr>
−
</tr>
+
<td colspan="2">Add distilled water until the medium volume reach 500 mL</td>
−
<tr>
+
</tr>
−
<td>L-Proline</td>
+
<tr>
−
<td>250 mg</td>
+
<td colspan="2">Blend with magnetic stirrer</td>
−
</tr>
+
</tr>
−
<tr>
+
<tr>
−
<td>L-Glutamine</td>
+
<td colspan="2">Adjust medium pH to 5.8 with 1 mol/L NaOH</td>
−
<td>250 mg</td>
+
</tr>
−
</tr>
+
<tr>
−
<tr>
+
<td>Phytagel</td>
−
<td>Sucrose</td>
+
<td>1.5 g</td>
−
<td>15 g</td>
+
</tr>
−
</tr>
+
</tbody>
−
<tr>
+
</table>
−
<td colspan="2">Add distilled water until the medium volume reach 500 mL</td>
+
<div class="p_font_size"><strong>2.1.2 Experimental procedures</strong></div>
 +
<div class="p_font_size">(1)Put 400 rice seeds into a 50 mL Erlenmeyer flask.</div>
 +
<div class="p_font_size">(2)Prepare 75% ethanol solution.</div>
 +
<div class="p_font_size">(3)Pour some 75% ethanol solution into the Erlenmeyer flask which contain the seeds. Keep shaking the Erlenmeyer flask in 1 minute at most.</div>
 +
<div class="p_font_size">(4)Decant the 75% ethanol from the Erlenmeyer flask.</div>
 +
<div class="p_font_size">(5)Add about 35 mL distilled water to the Erlenmeyer flask immediately and keep shaking for 40 seconds. Decant the water. Repeat this step for 5 times.</div>
 +
<div class="p_font_size">(6)Prepare 1.5% sodium hypochlorite solution.</div>
 +
<div class="p_font_size">(7)Pour some 1.5% sodium hypochlorite solution into the Erlenmeyer flask and seal the Erlenmeyer flask with flask sealing film. Shake the Erlenmeyer flask in a shaker for 30 minutes.</div>
 +
<div class="p_font_size">(8)Decant the 1.5% sodium hypochlorite solution.</div>
 +
<div class="p_font_size">(9)Add about 35 mL distilled water to the Erlenmeyer flask immediately and keep shaking for 40 seconds. Decant the water, Repeat this step for 5 times.</div>
 +
<div class="p_font_size">(10)Repeat step 6 to 9.</div>
 +
<div class="p_font_size">(11)Pick out seeds by a sterile tweezer and put seeds on a sterile filter paper.</div>
 +
<div class="p_font_size">(12)Place seeds in the laminar flow cabinet and dried by blowing for about 2 hours.</div>
 +
<div class="p_font_size">(13)Put sterile seeds on the surface of the callus initiation medium and seal plates with parafilm.</div>
 +
<div class="p_font_size">(14)Incubate seeds in the dark for about 14 days.</div>
 +
<div class="h4_font_size">2.2 Subculture of callus</div>
 +
<div class="p_font_size"><strong>2.2.1 Medium preparation</strong></div>
 +
<table class="table">
 +
<thead>
 +
<th>Component</th>
 +
<th>Dosage</th>
 +
</thead>
 +
<tbody>
 +
<tr>
 +
<td>10×N6macroelement solution</td>
 +
<td>50 mL</td>
 +
</tr>
 +
<tr>
 +
<td>1000×B5microelement solution</td>
 +
<td>0.5 mL</td>
 +
</tr>
 +
<tr>
 +
<td>100×B5vitamin solution</td>
 +
<td>5 mL</td>
 +
</tr>
 +
<tr>
 +
<td>100×Ferric salt solution</td>
 +
<td>5 mL</td>
 +
</tr>
 +
<tr>
 +
<td>0.5 mg/mL 2,4-D</td>
 +
<td>3 mL</td>
 +
</tr>
 +
<tr>
 +
<td>Casein enzymatic hydrolysate</td>
 +
<td>150 mg</td>
 +
</tr>
 +
<tr>
 +
<td>L-Proline</td>
 +
<td>250 mg</td>
 +
</tr>
 +
<tr>
 +
<td>L-Glutamine</td>
 +
<td>250 mg</td>
 +
</tr>
 +
<tr>
 +
<td>Sucrose</td>
 +
<td>15 g</td>
 +
</tr>
 +
<tr>
 +
<td colspan="2">Add distilled water until the medium volume reach 500 mL</td>
 +
</tr>
 +
<tr>
 +
<td colspan="2">Blend with magnetic stirrer</td>
 +
</tr>
 +
<tr>
 +
<td colspan="2">Adjust medium pH to 5.8 with 1 mol/L NaOH</td>
 +
</tr>
 +
<tr>
 +
<td>Phytagel</td>
 +
<td>1.5 g（3 g/L）</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<div class="p_font_size"><strong>2.2.2 Experimental procedures</strong></div>
 +
<div class="p_font_size">(1)Place the callus initiation medium in the laminar flow cabinet.</div>
 +
<div class="p_font_size">(2)Use a sterile tweezer to divide callus from seeds and put callus on the subculture medium. Seal plates with parafilm.</div>
 +
<div class="p_font_size">(3)Incubate callus in the dark for 5 days.</div>
 +
<div class="h4_font_size">2.3 Co-culture of callus with Agrobacterium.</div>
 +
<div class="p_font_size"><strong>2.3.1 Medium preparation</strong></div>
 +
<div class="p_font_size">(1)</div>
 +
<table class="table">
 +
<thead>
 +
<th>Component</th>
 +
<th>Dosage</th>
 +
</thead>
 +
<tbody>
 +
<tr>
 +
<td>10×MSmacroelement solution</td>
 +
<td>25 mL</td>
 +
</tr>
 +
<tr>
 +
<td>1000×B5microelement solution</td>
 +
<td>0.25 mL</td>
 +
</tr>
 +
<tr>
 +
<td>100×B5 vitamin solution</td>
 +
<td>2.5 mL</td>
 +
</tr>
 +
<tr>
 +
<td>100×Ferric salt solution</td>
 +
<td>2.5 mL</td>
 +
</tr>
 +
<tr>
 +
<td>0.5 mg/mL 2,4-D</td>
 +
<td>1.5 mL</td>
 +
</tr>
 +
<tr>
 +
<td>Casein enzymatic hydrolysate</td>
 +
<td>125 g</td>
 +
</tr>
 +
<tr>
 +
<td>Inositol</td>
 +
<td>0.5 g</td>
 +
</tr>
 +
<tr>
 +
<td>Sucrose</td>
 +
<td>7.5 g</td>
 +
</tr>
 +
<tr>
 +
<td>Glucose</td>
 +
<td>7.5 g</td>
 +
</tr>
 +
<tr>
 +
<td colspan="2">Add distilled water until medium volume reach 250 mL</td>
 +
</tr>
 +
<tr>
 +
<td colspan="2">Blend with magnetic stirrer</td>
 +
</tr>
 +
<tr>
 +
<td colspan="2">Adjust medium pH to 5.2 with 1 mol/L NaOH</td>
 +
</tr>
 +
<tr>
 +
<td>1000×Acetosyringone<br>(Add before using the medium)</td>
 +
<td>0.25 mL</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<div class="p_font_size">(2)</div>
 +
<table class="table">
 +
<thead>
 +
<th>Component</th>
 +
<th>Dosage</th>
 +
</thead>
 +
<tbody>
 +
<tr>
 +
<td>10×MSmacroelement solution</td>
 +
<td>25 mL</td>
 +
</tr>
 +
<tr>
 +
<td>1000×B5microelement solution</td>
 +
<td>0.25 mL</td>
 +
</tr>
 +
<tr>
 +
<td>100×B5 vitamin solution</td>
 +
<td>2.5 mL</td>
 +
</tr>
 +
<tr>
 +
<td>100×Ferric salt solution</td>
 +
<td>2.5 mL</td>
 +
</tr>
 +
<tr>
 +
<td>0.5 mg/mL 2,4-D</td>
 +
<td>1.5 mL</td>
 +
</tr>
 +
<tr>
 +
<td>Casein enzymatic hydrolysate</td>
 +
<td>125 g</td>
 +
</tr>
 +
<tr>
 +
<td>Inositol</td>
 +
<td>0.5 g</td>
 +
</tr>
 +
<tr>
 +
<td>Sucrose</td>
 +
<td>7.5 g</td>
 +
</tr>
 +
<tr>
 +
<td>Glucose</td>
 +
<td>7.5 g</td>
 +
</tr>
 +
<tr>
 +
<td colspan="2">Add distilled water until medium volume reach 250 mL</td>
 +
</tr>
 +
<tr>
 +
<td colspan="2">Blend with magnetic stirrer</td>
 +
</tr>
 +
<tr>
 +
<td colspan="2">Adjust medium pH to 5.3 with 1 mol/L NaOH</td>
 +
</tr>
 +
<tr>
 +
<td>Agar</td>
 +
<td>3.5 g</td>
 +
</tr>
 +
<tr>
 +
<td>1000×Acetosyringone<br>(Add before dividingthe medium into plates)</td>
 +
<td>0.25 mL</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<div class="p_font_size"><strong>2.3.2 Experimental procedures</strong></div>
 +
<div class="p_font_size">(1)Place the subculture medium into the laminar flow cabinet.</div>
 +
<div class="p_font_size">(2)Pick out the callus and put them on a sterile filter paper. Dry the surface of callus by blowing.</div>
 +
<div class="p_font_size">(3)Add 0.25 mL Acetosyringone to co-culture liquid medium.</div>
 +
<div class="p_font_size">(4)Pour about 35 mL co-culture liquid medium into a 50 mL Erlenmeyer flask.</div>
 +
<div class="p_font_size">(5)Use a sterile stainless steel spoon to collect Agrobacterium transforment strain and suspend in the co-culture liquid medium. The co-culture liquid medium contain Agrobacterium also called Agrobacterium suspension.</div>
 +
<div class="p_font_size">(6)Adjust the OD600 of Agrobacterium suspension to 0.1.</div>
 +
<div class="p_font_size">(7)Seal the Erlenmeyer flask with flask sealing film. Shake the Erlenmeyer flask in a shaker in 27 ℃ for 30 minutes.</div>
 +
<div class="p_font_size">(8)After 30 minutes, take the Erlenmeyer flask out and put it in the laminar flow cabinet. Pick callus into the Agrobacterium suspension and gentle shake the Erlenmeyer flask for 20 minutes. Ensure that each callus contact with the Agrobacterium suspension directly.</div>
 +
<div class="p_font_size">(9)Decant the Agrobacterium suspension. Pick out callus and put them on a sterile filter paper.<br>Dry the surface of callus by blowing.</div>
 +
<div class="p_font_size">(10)At the same time, put a sterile filter paper on the surface of the co-culture medium. Dry the surface of the co-culture medium by blowing.</div>
 +
<div class="p_font_size">(11)When the surface of callus become dry, put them on the surface of the filter paper above the co-culture medium.</div>
 +
<div class="p_font_size">(12)Seal plates with parafilm. Incubate in the dark at 25 ℃ for 3 days.</div>
 +
<div class="h4_font_size">2.4 First time selection of resistant callus</div>
 +
<div class="p_font_size"><strong>2.4.1 Medium preparation</strong></div>
 +
<table class="table">
 +
<thead>
 +
<th>Component</th>
 +
<th>Dosage</th>
 +
</thead>
 +
<tbody>
 +
<tr>
 +
<td>10×N6macroelement solution</td>
 +
<td>50 mL</td>
 +
</tr>
 +
<tr>
 +
<td>1000×B5 microelement solution</td>
 +
<td>0.5 mL</td>
 +
</tr>
 +
<tr>
 +
<td>100×B5 vitamin solution</td>
 +
<td>5 mL</td>
 +
</tr>
 +
<tr>
 +
<td>100×Ferric salt solution</td>
 +
<td>5 mL</td>
 +
</tr>
 +
<tr>
 +
<td>0.5 mg/mL 2,4-D</td>
 +
<td>3 mL</td>
 +
</tr>
 +
<tr>
 +
<td>Casein enzymatic hydrolysate</td>
 +
<td>150 mg</td>
 +
</tr>
 +
<tr>
 +
<td>L-Proline</td>
 +
<td>250 mg</td>
 +
</tr>
 +
<tr>
 +
<td>L-Glutamine</td>
 +
<td>250 mg</td>
 +
</tr>
 +
<tr>
 +
<td>Sucrose</td>
 +
<td>15 g</td>
 +
</tr>
 +
<tr>
 +
<td colspan="2">Add distilled water until the medium volume reach 500 mL</td>
 +
</tr>
 +
<tr>
 +
<td colspan="2">Blend with magnetic stirrer</td>
 +
</tr>
 +
<tr>
 +
<td colspan="2">Adjust medium pH to 5.8 with 1 mol/L NaOH</td>
 +
</tr>
 +
<tr>
 +
<td>Phytagel</td>
 +
<td>3 g</td>
 +
</tr>
 +
<tr>
 +
<td>1000×Hygromycin B<br>(Add before dividing the medium into plates)</td>
 +
<td>0.5 mL</td>
 +
</tr>
 +
<tr>
 +
<td>1000×Cefazolin sodium<br>(Add before dividing the medium into plates)</td>
 +
<td>0.5 mL</td>
 +
</tr>
 +
<tr>
 +
<td>1000×Carbenicillin sodium<br>(Add before dividing the medium into plates)</td>
 +
<td>0.5 mL</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<div class="p_font_size"><strong>2.4.2 Experimental procedures</strong></div>
 +
<div class="p_font_size">(1)Place the co-culture medium in the laminar flow cabinet.</div>
 +
<div class="p_font_size">(2)Add 1 mL 1000×Cefazolin sodium and 1 mL 1000×Carbenicillin sodium to 1 L sterile distilled water. This solution is Agrobacterium eluent.</div>
 +
<div class="p_font_size">(3)Pick out callus and put them in a 50 mL Erlenmeyer flask.</div>
 +
<div class="p_font_size">(4)Add about 35 mL Agrobacterium eluent to the Erlenmeyer flask. Shake the Erlenmeyer flask for one minute. Decant the Agrobacterium eluent.</div>
 +
<div class="p_font_size">(5)Repeat step 4 for about 20 times. Ensure that most of the Agrobacterium on callus are eluted.</div>
 +
<div class="p_font_size">(6)Pick out callus and put them on a sterile filter paper. Dry the surface of callus by blowing.</div>
 +
<div class="p_font_size">(7)Put callus on the selecting medium.</div>
 +
<div class="p_font_size">(8)Seal plates with parafilm.</div>
 +
<div class="p_font_size">(9)Culture in the dark for 30 days.</div>
 +
<div class="h4_font_size">2.5 Second time selection of resistant callus</div>
 +
<div class="p_font_size"><strong>2.5.1 Medium preparation</strong></div>
 +
<table class="table">
 +
<thead>
 +
<th>Component</th>
 +
<th>Dosage</th>
 +
</thead>
 +
<tbody>
 +
<tr>
 +
<td>10×N6 macroelement solution</td>
 +
<td>50 mL</td>
 +
</tr>
 +
<tr>
 +
<td>1000×B5 microelement solution</td>
 +
<td>0.5 mL</td>
 +
</tr>
 +
<tr>
 +
<td>100×B5 vitamin solution</td>
 +
<td>5 mL</td>
 +
</tr>
 +
<tr>
 +
<td>100×Ferric salt solution</td>
 +
<td>5 mL</td>
 +
</tr>
 +
<tr>
 +
<td>0.5 mg/mL 2,4-D</td>
 +
<td>3 mL</td>
 +
</tr>
 +
<tr>
 +
<td>Casein enzymatic hydrolysate</td>
 +
<td>150 mg</td>
 +
</tr>
 +
<tr>
 +
<td>L-Proline</td>
 +
<td>250 mg</td>
 +
</tr>
 +
<tr>
 +
<td>L-Glutamine</td>
 +
<td>250 mg</td>
 +
</tr>
 +
<tr>
 +
<td>Sucrose</td>
 +
<td>15 g</td>
 +
</tr>
 +
<tr>
 +
<td colspan="2">Add distilled water until the medium volume reach 500 mL</td>
 +
</tr>
 +
<tr>
 +
<td colspan="2">Blend with magnetic stirrer</td>
 +
</tr>
 +
<tr>
 +
<td colspan="2">Adjust medium pH to 5.8 with 1 mol/L NaOH</td>
 +
</tr>
 +
<tr>
 +
<td>Phytagel</td>
 +
<td>3 g</td>
 +
</tr>
 +
<tr>
 +
<td>1000×Hygromycin B<br>(Add before dividing the medium into plates)</td>
 +
<td>0.5 mL</td>
 +
</tr>
 +
<tr>
 +
<td>1000×Cefazolin sodium<br>(Add before dividing the medium into plates)</td>
 +
<td>0.5 mL</td>
 +
</tr>
 +
<tr>
 +
<td>1000×Carbenicillin sodium<br>(Add before dividing the medium into plates)</td>
 +
<td>0.5 mL</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<div class="p_font_size"><strong>2.5.2 Experimental procedures</strong></div>
 +
<div class="p_font_size">(1)Place the first time selecting medium in the laminar flow cabinet.</div>
 +
<div class="p_font_size">(2)Pick out callus and put them on the second time selecting medium.</div>
 +
<div class="p_font_size">(3)Seal plates with parafilm.</div>
 +
<div class="p_font_size">(4)Culture in the dark for 30 days.</div>
 +
<div class="h4_font_size">2.6 Differentiation of resistant callus</div>
 +
<div class="p_font_size"><strong>2.6.1 Medium preparation</strong></div>
 +
<table class="table">
 +
<thead>
 +
<th>Component</th>
 +
<th>Dosage</th>
 +
</thead>
 +
<tbody>
 +
<tr>
 +
<td>10×N6 macroelement solution</td>
 +
<td>50 mL</td>
 +
</tr>
 +
<tr>
 +
<td>1000×MS microelement solution</td>
 +
<td>0.5 mL</td>
 +
</tr>
 +
<tr>
 +
<td>100×B5 vitamin solution</td>
 +
<td>5 mL</td>
 +
</tr>
 +
<tr>
 +
<td>100×Ferric salt solution</td>
 +
<td>5 mL</td>
 +
</tr>
 +
<tr>
 +
<td>0.5 mg/mL 6-B,A</td>
 +
<td>3 mL</td>
 +
</tr>
 +
<tr>
 +
<td>0.1 mg/mL NAA</td>
 +
<td>5 mL</td>
 +
</tr>
 +
<tr>
 +
<td>Sorbitil</td>
 +
<td>9.1 g</td>
 +
</tr>
 +
<tr>
 +
<td>Sucrose</td>
 +
<td>10 g</td>
 +
</tr>
 +
<tr>
 +
<td colspan="2">Add distilled water until the medium volume reach 500 mL</td>
 +
</tr>
 +
<tr>
 +
<td colspan="2">Blend with magnetic stirrer</td>
 +
</tr>
 +
<tr>
 +
<td colspan="2">Adjust medium pH to 5.8 with 1 mol/L NaOH</td>
 +
</tr>
 +
<tr>
 +
<td>Phytagel</td>
 +
<td>2 g</td>
 +
</tr>
 +
<tr>
 +
<td>1000×Hygromycin B<br>(Add before dividing the medium into plates)</td>
 +
<td>0.5 mL</td>
 +
</tr>
 +
<tr>
 +
<td>1000×Cefazolin sodium<br>(Add before dividing the medium into plates)</td>
 +
<td>0.5 mL</td>
 +
</tr>
 +
<tr>
 +
<td>1000×Carbenicillin sodium<br>(Add before dividing the medium into plates)</td>
 +
<td>0.5 mL</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<div class="p_font_size"><strong>2.5.2 Experimental procedures</strong></div>
 +
<div class="p_font_size">(1)Place the first time selecting medium in the laminar flow cabinet.</div>
 +
<div class="p_font_size">(2)Pick out callus and put them on the second time selecting medium.</div>
 +
<div class="p_font_size">(3)Seal plates with parafilm.</div>
 +
<div class="p_font_size">(4)Culture in the dark for 30 days.</div>
 +
<div class="h4_font_size">2.6 Differentiation of resistant callus</div>
 +
<div class="p_font_size"><strong>2.6.1 Medium preparation</strong></div>
 +
<table class="table">
 +
<thead>
 +
<th>Component</th>
 +
<th>Dosage</th>
 +
</thead>
 +
<tbody>
 +
<tr>
 +
<td>10×N6 macroelement solution</td>
 +
<td>50 mL</td>
 +
</tr>
 +
<tr>
 +
<td>1000×MS microelement solution</td>
 +
<td>0.5 mL</td>
 +
</tr>
 +
<tr>
 +
<td>100×B5 vitamin solution</td>
 +
<td>5 mL</td>
 +
</tr>
 +
<tr>
 +
<td>100×Ferric salt solution</td>
 +
<td>5 mL</td>
 +
</tr>
 +
<tr>
 +
<td>0.5 mg/mL 6-B,A</td>
 +
<td>3 mL</td>
 +
</tr>
 +
<tr>
 +
<td>0.1 mg/mL NAA</td>
 +
<td>5 mL</td>
 +
</tr>
 +
<tr>
 +
<td>Sorbitil</td>
 +
<td>9.1 g</td>
 +
</tr>
 +
<tr>
 +
<td>Sucrose</td>
 +
<td>10 g</td>
 +
</tr>
 +
<tr>
 +
<td colspan="2">Add distilled water until the medium volume reach 500 mL</td>
 +
</tr>
 +
<tr>
 +
<td colspan="2">Blend with magnetic stirrer</td>
 +
</tr>
 +
<tr>
 +
<td colspan="2">Adjust medium pH to 5.8 with 1 mol/L NaOH</td>
 +
</tr>
 +
<tr>
 +
<td>Phytagel</td>
 +
<td>2 g</td>
 +
</tr>
 +
<tr>
 +
<td>1000×Hygromycin B<br>(Add before dividing the medium into plates)</td>
 +
<td>0.5 mL</td>
 +
</tr>
 +
<tr>
 +
<td>1000×Cefazolin sodium<br>(Add before dividing the medium into plates)</td>
 +
<td>0.5 mL</td>
 +
</tr>
 +
<tr>
 +
<td>1000×Carbenicillin sodium<br>(Add before dividing the medium into plates)</td>
 +
<td>0.5 mL</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<div class="p_font_size"><strong>2.6.2 Experimental procedures</strong></div>
 +
<div class="p_font_size">(1)Place the second time selecting medium in the laminar flow cabinet.</div>
 +
<div class="p_font_size">(2)Pick out those yellow callus that generate after two times of selection. Put them on the differentiation medium.</div>
 +
<div class="p_font_size">(3)Seal plates with parafilm.</div>
 +
<div class="p_font_size">(4)Culture under the condition of 14 hours light and 10 hours dark a days, for about 25 days.</div>
 +
<div class="p_font_size">(5)After 25 days, green spot will appear on the surface of the resistant callus.</div>
 +
<div class="p_font_size">(6)Place the differentiation medium in the laminar flow cabinet.</div>
 +
<div class="p_font_size">(7)Choose callus which has green spot. Transfer them to a new differentiation medium.</div>
 +
<div class="p_font_size">(8)Seal cuture bottles.</div>
 +
<div class="p_font_size">(9)Culture under the condition of 14 hours light and 10 hours dark a days, for about 30 days.</div>
 +
<div class="h4_font_size">2.7 Rooting the seedling</div>
 +
<div class="p_font_size"><strong>2.7.1 Medium preparation</strong></div>
 +
<table class="table">
 +
<thead>
 +
<th>Component</th>
 +
<th>Dosage</th>
 +
</thead>
 +
<tbody>
 +
<tr>
 +
<td>10×N6 macroelement solution</td>
 +
<td>50 mL</td>
 +
</tr>
 +
<tr>
 +
<td>1000×MS microelement solution</td>
 +
<td>0.5 mL</td>
 +
</tr>
 +
<tr>
 +
<td>100×N6vitamin solution</td>
 +
<td>5 mL</td>
 +
</tr>
 +
<tr>
 +
<td>100×Ferric salt solution</td>
 +
<td>5 mL</td>
 +
</tr>
 +
<tr>
 +
<td>0.1 mg/mL NAA</td>
 +
<td>5 mL</td>
 +
</tr>
 +
<tr>
 +
<td>Sucrose</td>
 +
<td>10 g</td>
 +
</tr>
 +
<tr>
 +
<td colspan="2">Add distilled water until the medium volume reach 500 mL</td>
 +
</tr>
 +
<tr>
 +
<td colspan="2">Blend with magnetic stirrer</td>
 +
</tr>
 +
<tr>
 +
<td colspan="2">Adjust medium pH to 5.8 with 1 mol/L NaOH</td>
 +
</tr>
 +
<tr>
 +
<td>Phytagel</td>
 +
<td>2 g</td>
 +
</tr>
 +
<tr>
 +
<td>1000×Hygromycin B<br>(Add before dividing the medium into plates)</td>
 +
<td>0.5 mL</td>
 +
</tr>
 +
<tr>
 +
<td>1000×Cefazolin sodium<br>(Add before dividing the medium into plates)</td>
 +
<td>0.5 mL</td>
 +
</tr>
 +
<tr>
 +
<td>1000×Carbenicillin sodium<br>(Add before dividing the medium into plates)</td>
 +
<td>0.5 mL</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<div class="p_font_size"><strong>2.7.2 Experimental procedures</strong></div>
 +
<div class="p_font_size">(1)Place the differentiation medium in the laminar flow cabinet.</div>
 +
<div class="p_font_size">(2)Carefully pick the seedling out with a sterile tweezer. Transfer it to rooting medium.</div>
 +
<div class="p_font_size">(3)Seal culture bottles.</div>
 +
<div class="p_font_size">(4)Culture under the condition of 14 hours light and 10 hours dark a days, for about 20 days.</div>
 +
<div class="h4_font_size">2.8 Hardening the seedling</div>
 +
<div class="p_font_size"><strong>2.8.1 Experimental procedures</strong></div>
 +
<div class="p_font_size">(1)Choose seedling where are 8 cm high and grown strong roots.</div>
 +
<div class="p_font_size">(2)Open the lid of culture bottle. Add appropriate amount of distilled water.</div>
 +
<div class="p_font_size">(3)Culture in the greenhouse for 7 days.</div>
 +
<div class="h4_font_size">2.9 Transplanting the seedling</div>
 +
<div class="p_font_size"><strong>2.9.1 Experimental procedures</strong></div>
 +
<div class="p_font_size">(1)After hardening the seedling, take the seeding out and remove the medium on its roots with running water.</div>
 +
<div class="p_font_size">(2)Transplant the seedling to soil. Make sure that the seedling isn’t submergence or insolated.</div>
  
−
</tr>
+
<div class="h2_font_size">Extraction and detection</div>
−
<tr>
+
<div class="h3_font_size">1. The method of extracting the astaxanthin in rice endosperm</div>
−
<td colspan="2">Blend withmagnetic stirrer</td>
+
<div class="p_font_size">1. Take 0.1 g rice seeds into mortar. After grinding it into powder on the ice, add 2 mL of methanol in mortar, and continue to grind for 5 minutes.</div>
−
</tr>
+
<div class="p_font_size">2. Transfer the powder to a 2 mL centrifuge tube, spin at 10000 rpm for 10 min in the dark and low temperature condition to extract </div>
−
<tr>
+
<div class="p_font_size">3. Centrifuge at 8000rpm at 4 ℃for 5 minutes and collect supernatant.</div>
−
<td colspan="2">Adjust medium pH to 5.8 with 1 mol/L NaOH</td>
+
<div class="p_font_size">4. Extract the sediment with methanol repeatedly, until the precipitation become white.</div>
−
</tr>
+
<div class="p_font_size">5. Centrifuge again(8000rpm, 4 ℃, 5 min)and collect supernatant .</div>
−
<tr>
+
<div class="p_font_size">6. Merge supernatant, concentrate supernatant into powder in the dark and low temperature conditions</div>
−
<td>Phytagel</td>
+
<div class="p_font_size">7. Add 600 µL methanol into dry astaxanthin extraction.</div>
−
<td>1.5 g</td>
+
<div class="h3_font_size">2. HPLC(High Performance Liquid Chromatography)determination of astaxanthin</div>
−
</tr>
+
<div class="p_font_size">After enrichment, put the samples into a 2 mL brown bottle through 0.22µm syringe-driven filter, then detect the samples by HPLC. The mobile phase is methanol: water = 95:5. The flow rate is 1 mL/min. Column temperature is room temperature. The detection wavelength is 480 nm.</div>
−
</tbody>
+
<div class="h3_font_size">3. Standard curve for the determination of astaxanthin</div>
−
</table></p>
+
<div class="p_font_size">Dissolve accurately 10 mg of standard sample of astaxanthin in 1mL methylene chloride, and dilute with methanol to 100mL in a brown volumetric flask and get the 0.1 mg/mL solution. Dilute the above-mentioned solution with methanol into 2µg/mL, 4µg/mL, 6 µg/mL, 8 µg/mL, 10 µg/mL. Take 20µL sample to detect separately according to the conditions that mentioned above. With peak area as the ordinate, the concentration of the standard sample for horizontal, apply linear regression analysis, and get the regression equation.</div>
−
<p><strong>2.1.2 Experimental procedures</strong></p>
+
<div class="h3_font_size">4.Conclusion</div>
−
<p>(1)Put 400 rice seeds into a 50 mL Erlenmeyer flask.</p>
+
<div class="p_font_size">The method of detecting the astaxanthin content in transgenic rice endosperm: grind transgenic rice seeds into powder on the ice, extract the sediment with methanol repeatedly, until the precipitation become white, concentrate the supernatant into powder, dissolved in methanol, use the high performance liquid chromatography (HPLC) to measure the content of astaxanthin.</div>
−
<p>(2)Prepare 75% ethanol solution.</p>
+
<div class="h3_font_size">References</div>
−
<p>(3)Pour some 75% ethanol solution into the Erlenmeyer flask which contain the seeds. Keep shaking the Erlenmeyer flask in 1 minute at most.</p>
+
<div class="p_font_size">【1】Y. J. Zhong, J. C. Huang, J. Liu, Y. Li, Y. Jiang, Z. F. Xu, G. Sandmann, F. Chen, Functional characterization of various algal carotenoid ketolases reveals that ketolating zeaxanthin efficiently is essential for high production of astaxanthin in transgenic Arabidopsis. Journal of Experimental Botany. 62, 10, 3659–3669 (2011)</div>
−
<p>(4)Decant the 75% ethanol from the Erlenmeyer flask.</p>
+
<div class="p_font_size">【2】J. C. Huang, Y. J. Zhong, G. Sandmann, J. Liu, F. Chen, Cloning and selection of carotenoid ketolase genes for the engineering of high-yield astaxanthin in plants. Planta. 236, 691–699 (2012)</div>
−
<p>(5)Add about 35 mL distilled water to the Erlenmeyer flask immediately and keep shaking for 40 seconds. Decant the water. Repeat this step for 5 times.</p>
+
<div class="p_font_size">【3】J. C. Huang, Y. J. Zhong, J. Liu, G. Sandmann, F. Chen, Metabolic engineering of tomato for high-yield production of astaxanthin. Metabolic Engineering. 17, 59–67(2013) </div>
−
<p>(6)Prepare 1.5% sodium hypochlorite solution.</p>
+
<div class="p_font_size">【4】J. Breitenbach, C. Bai, S. M. Rivera, R. Canela, T. Capell, Paul Christou, C. f. Zhu, G. Sandmann, A novel carotenoid, 4-keto-a-carotene, as an unexpected by-product during genetic engineering of caroteno genesis in rice callus. Phytochemistry. 98, 85–91(2014) </div>
−
<p>(7)Pour some 1.5% sodium hypochlorite solution into the Erlenmeyer flask and seal the Erlenmeyer flask with flask sealing film. Shake the Erlenmeyer flask in a shaker for 30 minutes.</p>
+
−
<p>(8)Decant the 1.5% sodium hypochlorite solution.</p>
+
−
<p>(9)Add about 35 mL distilled water to the Erlenmeyer flask immediately and keep shaking for 40 seconds. Decant the water, Repeat this step for 5 times.</p>
+
−
<p>(10)Repeat step 6 to 9.</p>
+
−
<p>(11)Pick out seeds by a sterile tweezer and put seeds on a sterile filter paper.</p>
+
−
<p>(12)Place seeds in the laminar flow cabinet and dried by blowing for about 2 hours.</p>
+
−
<p>(13)Put sterile seeds on the surface of the callus initiation medium and seal plates with parafilm.</p>
+
−
<p>(14)Incubate seeds in the dark for about 14 days.</p>
+
−
+
−
<h4>2.2 Subculture of callus</h4>
+
−
<p><strong>2.2.1 Medium preparation</strong></p>
+
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+
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+
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+
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</div>
+
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</div>
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<div style="margin-bottom:30px">
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<div class="row">
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<div class="col-sm-1"></div>
+
−
<div class="col-sm-10">
+
−
<h2>Extraction and detection</h2>
+
−
<p>As our project is to use the rice as a bioreactor and produce astaxanthin,and we also hope that aSTARice can serve as a kind of food in human’s daily lives,so we are very concerned about the safety of aSTARice.</p>
+
−
<p>We believe that risks are mainly embodied in the following two aspects: one is that the encoding products of marker genes might be toxic and allergenic potentially for human or livestock. Antibiotic gene might be transferred into gut microbes’ genome of humans or animals , increasing the resistance of the microbes of the antibiotic, thus result in reducing the effectiveness of antibiotics in clinical treatment;the other one is that marker genes could be spread through pollens or other methods into other organisms,which is called genetic drift. Genetic drift can transfer antibiotic genes to weeds or other plants,which is threatening the ecological environment.</p>
+
−
<p>In order to reduce the effects of antibiotic marker gene, we use a technique called selectable marker-free technique in the project, so the final aSTARice is an marker-free transgenic plants(MFTPs).We design a Cre-loxP based side-direct recombinant system to knock out hygromycin phosphotransferase gene,minimizing the effect of the selectable maker gene.</p>
+
−
<p>In order to reduce the risk of genetic drift as well as the threat to the ecological environment, we add isolation area to surround the experimental fields.By isolating the transgenic rice,we reduce the possibility of genetic drift duel to the transfer of the pollen,sufficiently guaranteeing the safety of the project.Whats more,we are also analyzing  coding products by HPLC and trying to make sure that there is no harmful products in our rice.</p>
+
−
<p>Rice was chose as the chassis though it isn’t included in the white line,but we checked-in with the official website in the beginning.Because we are the first team who choose rice as chassis,we have to asses the safety for rice.</p>
+
−
<p>Rice is the self-pollinated plant so that the gemotype in the individuals is pure and homogeneous.The probabilities that two flowers take place cross-pollinated are just 1%，and the distance of the spread of rice pollen are hardly 1meter.</p>
+
−
<p>The phenomena of pollen escape are the main ways which lead to the flow of exogenous gene of transgene plants.Recently,the reaches of transgene plants verified that the exogenous gene of transgene plants will flow to the same species or weeds and even the conventional species.As igemers,we took safety into consideration carefully so that we saw plentiful papers ,and that we found a conclusion that under the close distance less than 1% adjacent non-genetically modified plants take place gene flow.If we increase the distance to 5~10 meters,the probabilities decrease to 0.001%~0.0001%,which will not happen in theory.when planting rice,we follow seriously the safe regulations of transgene plantsSo we have faith that it is impossible for gene flow come up under this plant distance.</p>
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Revision as of 11:27, 14 October 2016

SCAU

Protocol
Vector construction protocol
Transformation of rice
1.Brief on the procedure:
Callus induction→ Subculture of callus → Co-culture of callus with Agrobacterium → Selection of resistant callus→ Differentiation of resistant callus → Rooting the seedling → Hardening the seedling →Transplanting the seedling.
2.Detailed steps
2.1 Callus induction
2.1.1 Medium Preparation
Component Dosage
10×N6 macroelement solution 50 mL
1000×B5 microelement solution 0.5 mL
100×B5 vitamin solution 5 mL
100×Ferric salt solution 5 mL
0.5 mg/mL 2,4-D 3 mL
Casein enzymatic hydrolysate 150 mg
L-Proline 250 mg
L-Glutamine 250 mg
Sucrose 15 g
Add distilled water until the medium volume reach 500 mL
Blend with magnetic stirrer
Adjust medium pH to 5.8 with 1 mol/L NaOH
Phytagel 1.5 g
2.1.2 Experimental procedures
(1)Put 400 rice seeds into a 50 mL Erlenmeyer flask.
(2)Prepare 75% ethanol solution.
(3)Pour some 75% ethanol solution into the Erlenmeyer flask which contain the seeds. Keep shaking the Erlenmeyer flask in 1 minute at most.
(4)Decant the 75% ethanol from the Erlenmeyer flask.
(5)Add about 35 mL distilled water to the Erlenmeyer flask immediately and keep shaking for 40 seconds. Decant the water. Repeat this step for 5 times.
(6)Prepare 1.5% sodium hypochlorite solution.
(7)Pour some 1.5% sodium hypochlorite solution into the Erlenmeyer flask and seal the Erlenmeyer flask with flask sealing film. Shake the Erlenmeyer flask in a shaker for 30 minutes.
(8)Decant the 1.5% sodium hypochlorite solution.
(9)Add about 35 mL distilled water to the Erlenmeyer flask immediately and keep shaking for 40 seconds. Decant the water, Repeat this step for 5 times.
(10)Repeat step 6 to 9.
(11)Pick out seeds by a sterile tweezer and put seeds on a sterile filter paper.
(12)Place seeds in the laminar flow cabinet and dried by blowing for about 2 hours.
(13)Put sterile seeds on the surface of the callus initiation medium and seal plates with parafilm.
(14)Incubate seeds in the dark for about 14 days.
2.2 Subculture of callus
2.2.1 Medium preparation
Component Dosage
10×N6macroelement solution 50 mL
1000×B5microelement solution 0.5 mL
100×B5vitamin solution 5 mL
100×Ferric salt solution 5 mL
0.5 mg/mL 2,4-D 3 mL
Casein enzymatic hydrolysate 150 mg
L-Proline 250 mg
L-Glutamine 250 mg
Sucrose 15 g
Add distilled water until the medium volume reach 500 mL
Blend with magnetic stirrer
Adjust medium pH to 5.8 with 1 mol/L NaOH
Phytagel 1.5 g（3 g/L）
2.2.2 Experimental procedures
(1)Place the callus initiation medium in the laminar flow cabinet.
(2)Use a sterile tweezer to divide callus from seeds and put callus on the subculture medium. Seal plates with parafilm.
(3)Incubate callus in the dark for 5 days.
2.3 Co-culture of callus with Agrobacterium.
2.3.1 Medium preparation
(1)
Component Dosage
10×MSmacroelement solution 25 mL
1000×B5microelement solution 0.25 mL
100×B5 vitamin solution 2.5 mL
100×Ferric salt solution 2.5 mL
0.5 mg/mL 2,4-D 1.5 mL
Casein enzymatic hydrolysate 125 g
Inositol 0.5 g
Sucrose 7.5 g
Glucose 7.5 g
Add distilled water until medium volume reach 250 mL
Blend with magnetic stirrer
Adjust medium pH to 5.2 with 1 mol/L NaOH
1000×Acetosyringone
(Add before using the medium)		 0.25 mL
(2)
Component Dosage
10×MSmacroelement solution 25 mL
1000×B5microelement solution 0.25 mL
100×B5 vitamin solution 2.5 mL
100×Ferric salt solution 2.5 mL
0.5 mg/mL 2,4-D 1.5 mL
Casein enzymatic hydrolysate 125 g
Inositol 0.5 g
Sucrose 7.5 g
Glucose 7.5 g
Add distilled water until medium volume reach 250 mL
Blend with magnetic stirrer
Adjust medium pH to 5.3 with 1 mol/L NaOH
Agar 3.5 g
1000×Acetosyringone
(Add before dividingthe medium into plates)		 0.25 mL
2.3.2 Experimental procedures
(1)Place the subculture medium into the laminar flow cabinet.
(2)Pick out the callus and put them on a sterile filter paper. Dry the surface of callus by blowing.
(3)Add 0.25 mL Acetosyringone to co-culture liquid medium.
(4)Pour about 35 mL co-culture liquid medium into a 50 mL Erlenmeyer flask.
(5)Use a sterile stainless steel spoon to collect Agrobacterium transforment strain and suspend in the co-culture liquid medium. The co-culture liquid medium contain Agrobacterium also called Agrobacterium suspension.
(6)Adjust the OD600 of Agrobacterium suspension to 0.1.
(7)Seal the Erlenmeyer flask with flask sealing film. Shake the Erlenmeyer flask in a shaker in 27 ℃ for 30 minutes.
(8)After 30 minutes, take the Erlenmeyer flask out and put it in the laminar flow cabinet. Pick callus into the Agrobacterium suspension and gentle shake the Erlenmeyer flask for 20 minutes. Ensure that each callus contact with the Agrobacterium suspension directly.
(9)Decant the Agrobacterium suspension. Pick out callus and put them on a sterile filter paper.
Dry the surface of callus by blowing.
(10)At the same time, put a sterile filter paper on the surface of the co-culture medium. Dry the surface of the co-culture medium by blowing.
(11)When the surface of callus become dry, put them on the surface of the filter paper above the co-culture medium.
(12)Seal plates with parafilm. Incubate in the dark at 25 ℃ for 3 days.
2.4 First time selection of resistant callus
2.4.1 Medium preparation
Component Dosage
10×N6macroelement solution 50 mL
1000×B5 microelement solution 0.5 mL
100×B5 vitamin solution 5 mL
100×Ferric salt solution 5 mL
0.5 mg/mL 2,4-D 3 mL
Casein enzymatic hydrolysate 150 mg
L-Proline 250 mg
L-Glutamine 250 mg
Sucrose 15 g
Add distilled water until the medium volume reach 500 mL
Blend with magnetic stirrer
Adjust medium pH to 5.8 with 1 mol/L NaOH
Phytagel 3 g
1000×Hygromycin B
(Add before dividing the medium into plates)		 0.5 mL
1000×Cefazolin sodium
(Add before dividing the medium into plates)		 0.5 mL
1000×Carbenicillin sodium
(Add before dividing the medium into plates)		 0.5 mL
2.4.2 Experimental procedures
(1)Place the co-culture medium in the laminar flow cabinet.
(2)Add 1 mL 1000×Cefazolin sodium and 1 mL 1000×Carbenicillin sodium to 1 L sterile distilled water. This solution is Agrobacterium eluent.
(3)Pick out callus and put them in a 50 mL Erlenmeyer flask.
(4)Add about 35 mL Agrobacterium eluent to the Erlenmeyer flask. Shake the Erlenmeyer flask for one minute. Decant the Agrobacterium eluent.
(5)Repeat step 4 for about 20 times. Ensure that most of the Agrobacterium on callus are eluted.
(6)Pick out callus and put them on a sterile filter paper. Dry the surface of callus by blowing.
(7)Put callus on the selecting medium.
(8)Seal plates with parafilm.
(9)Culture in the dark for 30 days.
2.5 Second time selection of resistant callus
2.5.1 Medium preparation
Component Dosage
10×N6 macroelement solution 50 mL
1000×B5 microelement solution 0.5 mL
100×B5 vitamin solution 5 mL
100×Ferric salt solution 5 mL
0.5 mg/mL 2,4-D 3 mL
Casein enzymatic hydrolysate 150 mg
L-Proline 250 mg
L-Glutamine 250 mg
Sucrose 15 g
Add distilled water until the medium volume reach 500 mL
Blend with magnetic stirrer
Adjust medium pH to 5.8 with 1 mol/L NaOH
Phytagel 3 g
1000×Hygromycin B
(Add before dividing the medium into plates)		 0.5 mL
1000×Cefazolin sodium
(Add before dividing the medium into plates)		 0.5 mL
1000×Carbenicillin sodium
(Add before dividing the medium into plates)		 0.5 mL
2.5.2 Experimental procedures
(1)Place the first time selecting medium in the laminar flow cabinet.
(2)Pick out callus and put them on the second time selecting medium.
(3)Seal plates with parafilm.
(4)Culture in the dark for 30 days.
2.6 Differentiation of resistant callus
2.6.1 Medium preparation
Component Dosage
10×N6 macroelement solution 50 mL
1000×MS microelement solution 0.5 mL
100×B5 vitamin solution 5 mL
100×Ferric salt solution 5 mL
0.5 mg/mL 6-B,A 3 mL
0.1 mg/mL NAA 5 mL
Sorbitil 9.1 g
Sucrose 10 g
Add distilled water until the medium volume reach 500 mL
Blend with magnetic stirrer
Adjust medium pH to 5.8 with 1 mol/L NaOH
Phytagel 2 g
1000×Hygromycin B
(Add before dividing the medium into plates)		 0.5 mL
1000×Cefazolin sodium
(Add before dividing the medium into plates)		 0.5 mL
1000×Carbenicillin sodium
(Add before dividing the medium into plates)		 0.5 mL
2.5.2 Experimental procedures
(1)Place the first time selecting medium in the laminar flow cabinet.
(2)Pick out callus and put them on the second time selecting medium.
(3)Seal plates with parafilm.
(4)Culture in the dark for 30 days.
2.6 Differentiation of resistant callus
2.6.1 Medium preparation
Component Dosage
10×N6 macroelement solution 50 mL
1000×MS microelement solution 0.5 mL
100×B5 vitamin solution 5 mL
100×Ferric salt solution 5 mL
0.5 mg/mL 6-B,A 3 mL
0.1 mg/mL NAA 5 mL
Sorbitil 9.1 g
Sucrose 10 g
Add distilled water until the medium volume reach 500 mL
Blend with magnetic stirrer
Adjust medium pH to 5.8 with 1 mol/L NaOH
Phytagel 2 g
1000×Hygromycin B
(Add before dividing the medium into plates)		 0.5 mL
1000×Cefazolin sodium
(Add before dividing the medium into plates)		 0.5 mL
1000×Carbenicillin sodium
(Add before dividing the medium into plates)		 0.5 mL
2.6.2 Experimental procedures
(1)Place the second time selecting medium in the laminar flow cabinet.
(2)Pick out those yellow callus that generate after two times of selection. Put them on the differentiation medium.
(3)Seal plates with parafilm.
(4)Culture under the condition of 14 hours light and 10 hours dark a days, for about 25 days.
(5)After 25 days, green spot will appear on the surface of the resistant callus.
(6)Place the differentiation medium in the laminar flow cabinet.
(7)Choose callus which has green spot. Transfer them to a new differentiation medium.
(8)Seal cuture bottles.
(9)Culture under the condition of 14 hours light and 10 hours dark a days, for about 30 days.
2.7 Rooting the seedling
2.7.1 Medium preparation
Component Dosage
10×N6 macroelement solution 50 mL
1000×MS microelement solution 0.5 mL
100×N6vitamin solution 5 mL
100×Ferric salt solution 5 mL
0.1 mg/mL NAA 5 mL
Sucrose 10 g
Add distilled water until the medium volume reach 500 mL
Blend with magnetic stirrer
Adjust medium pH to 5.8 with 1 mol/L NaOH
Phytagel 2 g
1000×Hygromycin B
(Add before dividing the medium into plates)		 0.5 mL
1000×Cefazolin sodium
(Add before dividing the medium into plates)		 0.5 mL
1000×Carbenicillin sodium
(Add before dividing the medium into plates)		 0.5 mL
2.7.2 Experimental procedures
(1)Place the differentiation medium in the laminar flow cabinet.
(2)Carefully pick the seedling out with a sterile tweezer. Transfer it to rooting medium.
(3)Seal culture bottles.
(4)Culture under the condition of 14 hours light and 10 hours dark a days, for about 20 days.
2.8 Hardening the seedling
2.8.1 Experimental procedures
(1)Choose seedling where are 8 cm high and grown strong roots.
(2)Open the lid of culture bottle. Add appropriate amount of distilled water.
(3)Culture in the greenhouse for 7 days.
2.9 Transplanting the seedling
2.9.1 Experimental procedures
(1)After hardening the seedling, take the seeding out and remove the medium on its roots with running water.
(2)Transplant the seedling to soil. Make sure that the seedling isn’t submergence or insolated.
Extraction and detection
1. The method of extracting the astaxanthin in rice endosperm
1. Take 0.1 g rice seeds into mortar. After grinding it into powder on the ice, add 2 mL of methanol in mortar, and continue to grind for 5 minutes.
2. Transfer the powder to a 2 mL centrifuge tube, spin at 10000 rpm for 10 min in the dark and low temperature condition to extract
3. Centrifuge at 8000rpm at 4 ℃for 5 minutes and collect supernatant.
4. Extract the sediment with methanol repeatedly, until the precipitation become white.
5. Centrifuge again(8000rpm, 4 ℃, 5 min)and collect supernatant .
6. Merge supernatant, concentrate supernatant into powder in the dark and low temperature conditions
7. Add 600 µL methanol into dry astaxanthin extraction.
2. HPLC(High Performance Liquid Chromatography)determination of astaxanthin
After enrichment, put the samples into a 2 mL brown bottle through 0.22µm syringe-driven filter, then detect the samples by HPLC. The mobile phase is methanol: water = 95:5. The flow rate is 1 mL/min. Column temperature is room temperature. The detection wavelength is 480 nm.
3. Standard curve for the determination of astaxanthin
Dissolve accurately 10 mg of standard sample of astaxanthin in 1mL methylene chloride, and dilute with methanol to 100mL in a brown volumetric flask and get the 0.1 mg/mL solution. Dilute the above-mentioned solution with methanol into 2µg/mL, 4µg/mL, 6 µg/mL, 8 µg/mL, 10 µg/mL. Take 20µL sample to detect separately according to the conditions that mentioned above. With peak area as the ordinate, the concentration of the standard sample for horizontal, apply linear regression analysis, and get the regression equation.
4.Conclusion
The method of detecting the astaxanthin content in transgenic rice endosperm: grind transgenic rice seeds into powder on the ice, extract the sediment with methanol repeatedly, until the precipitation become white, concentrate the supernatant into powder, dissolved in methanol, use the high performance liquid chromatography (HPLC) to measure the content of astaxanthin.
References
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【2】J. C. Huang, Y. J. Zhong, G. Sandmann, J. Liu, F. Chen, Cloning and selection of carotenoid ketolase genes for the engineering of high-yield astaxanthin in plants. Planta. 236, 691–699 (2012)
【3】J. C. Huang, Y. J. Zhong, J. Liu, G. Sandmann, F. Chen, Metabolic engineering of tomato for high-yield production of astaxanthin. Metabolic Engineering. 17, 59–67(2013)
【4】J. Breitenbach, C. Bai, S. M. Rivera, R. Canela, T. Capell, Paul Christou, C. f. Zhu, G. Sandmann, A novel carotenoid, 4-keto-a-carotene, as an unexpected by-product during genetic engineering of caroteno genesis in rice callus. Phytochemistry. 98, 85–91(2014)