Name: Efficient Polyethylene Glycol (PEG) Mediated Transformation of the Moss Physcomitrella patens (Video) | JoVE Journal
Uploaded: 2011-04-19T13:00:00.000+00:00
Duration: 4 min 54 s
Description: Learn an efficient PEG-mediated transformation method for Physcomitrella patens protoplasts in plant genetic engineering. This streamlined protocol enables reliable gene expression studies and facilitates transient RNAi experiments.

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5 minutes

L.V. is supported by NSF (IOS 1002837)

1. Making the Protoplasts
<ol>
<li>Add 9 mL of 8% mannitol to a Petri dish.</li>
<li>Using a spatula, put 7 day old moss from 2-3 PPNH4 plates of 5- in the Petri dish containing mannitol.</li>
<li>Add 3 mL of 2% Driselase (Sigma D9515-25G) to the Petri dish.</li>
<li>Incubate the Petri dish at room temperature with gentle shaking for 1 hour.</li>
<li>Filter the protoplast suspension through a 100 μm mesh (BD Falcon 352350).</li>
<li>Spin the filtered suspension at 250 g for 5 minutes. Remove the supernatant.</li>
<li>Re-suspend the protoplasts very gently with 500 μL of 8% mannitol.</li>
<li>Add 9.5 mL 8% mannitol in the culture tube. Make sure protoplasts are fully suspended.</li>
<li>Repeat the filtration and re-suspension steps (1.6, 1.7 and 1.8) two more times.</li>
<li>Take 10 μL of the protoplast solution and count the protoplasts in a hemocytometer.</li>
<li>Multiply the number of protoplast in a 16 square area by 10,000 to obtain the number of protoplasts per mL (See Figure 1).</li>
</ol>
2. Transformation
<ol>
<li>Spin the protoplast suspension at 250 g for 5 minutes. Remove the supernatant.</li>
<li>Re-suspend protoplasts in MMg solution at the concentration of 1.6 million protoplasts / mL.</li>
<li>Incubate the protoplast suspension at room temperature for 20 minutes.</li>
<li>Add 600 μL of protoplast suspension into a culture tube containing 60 μg DNA. Swirl the tube gently.</li>
<li>Add 700 μL of PEG/Ca solution into the protoplast / DNA mixture. Swirl the tube gently until whole mixture is homogeneous.</li>
<li>Incubate the mixture at room temperature for 30 minutes.</li>
<li>During this waiting period, cover PRM-B plates with cellophane (80 mm diameter circles, A.A. Packaging Limited, UK); allow cellophane to hydrate on the plate surface for at least 5 min.</li>
<li>With a spatula, remove any air bubbles trapped between the cellophane and the plate.</li>
<li>Dilute the mixture with 3 mL of W5 solution.</li>
<li>Spin the mixture at 250 g for 5 minutes. Remove the supernatant.</li>
<li>Re-suspend protoplasts in melted 2 mL of PRM-T (before adding it to the protoplasts this solution can be kept liquid at 42°C). Plate 1 mL of re-suspend protoplasts per PRM-B plate covered by cellophane. Wrap the plates with Micropore (3M, Health Care). Keep the plates at 25°C in a growth chamber.</li>
<li>Alternatively, protoplasts can be re-suspended in 1 mL of liquid plating medium and plated 0.5 mL on a PRM-B plate covered with cellophane. This allows faster regeneration, but the number of regenerating plants is lower.</li>
<li>Growth chamber is set for a 16 hr. light and 8 hr. dark cycle.</li>
<li>Move the cellophane onto to a fresh selection plate 4 days after transformation.</li>
</ol>
3. Representative Results:
An example of transformation is shown in Figure 2. The DNA used in this example was a supercoiled 7.8 kb plasmid carrying a hygromycin resistant cassette. The amount of protoplasts plated was reduced to 50% for photography. The pictures were taken two weeks after the plants were moved to the selection plates. The transformation efficiency is not affected by the DNA concentration up to 60 μg. Higher concentrations of DNA are detrimental to the efficiency of transformation. The data showed that this method delivers consistent result over a wide range of DNA amounts. This method can also generate stable transformants effectively as shown in Table 1; we can get about 4 stable transformants per microgram of linearized DNA, which is much higher than what was reported previously14.
We also tested the effect of heat shock on transformation efficiency by transforming a supercoiled plasmid carrying a gene for a fluorescent protein as a marker. The heat shock was carried out 10 minutes after room temperature incubation (within Step 2.6) by incubating the mixture in a 45°C water bath for 3 minutes. The mixture was incubated in a room temperature water bath immediately after heat shock for 17 min. Results were recorded 22 hours after transformation. The ratios of transformants obtained with and without heat shock were very similar (~25%), but we observed an adverse effect of heat shock on the viability of the protoplasts (data not shown).
<img src="/files/ftp_upload/2560/2560fig1.jpg" alt="Microscope image of particulate grid for particle size analysis, 100 μm scale, grid calibration method." data-altupdatedat="1788235205000" data-alt="Figure 1"> 
Figure 1. View of protoplasts in hemocytometer. The section needed for protoplast counting is indicated by a red square. Note the 16 squares required for counting protoplasts (see 1.11); the image shows a count of 24 protoplasts (240,000 cells/ mL).
<img src="/files/ftp_upload/2560/2560fig2.jpg" alt="Bacterial colony growth on agar plates; comparative study; experiment results; microbiology analysis." data-altupdatedat="1788235205000" data-alt="Figure 2"> 
Figure 2. Pictures of 18 day old transient transformants. Protoplasts were transformed with the indicated amount of supercoiled plasmid. A: 15μg; B: 30μg; C: 60μg; D: 120μg.
<table border="1">
<tbody>
 <tr>
 <td>Amount of DNA (μg)</td>
 <td>Efficiency (tranformants/μg DNA)</td>
 </tr>
 <tr>
 <td>15, supercoiled plasmid</td>
 <td>120±24</td>
 </tr>
 <tr>
 <td>30, supercoiled plasmid</td>
 <td>145±54</td>
 </tr>
 <tr>
 <td>60, supercoiled plasmid</td>
 <td>121±60</td>
 </tr>
 <tr>
 <td>120, supercoiled plasmid</td>
 <td>71±38</td>
 </tr>
 <tr>
 <td>60, linearized plasmid</td>
 <td>4±1</td>
 </tr>
 </tbody>
</table>
Table 1. Transformation efficiency at various DNA amounts.

<ol><li>Abel, S., Theologis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transient+transformation+of+Arabidopsis+leaf+protoplasts%3A+a+versatile+experimental+system+to+study+gene+expression%5BTitle%5D)+AND+%22Plant+J%22%5BJournal%5D)">Transient transformation of Arabidopsis leaf protoplasts: a versatile experimental system to study gene expression.</a> Plant J. 5, 421-427 (1994).</li>
<li>Wood, A. J., Oliver, M. J., Cove, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bryophytes+as+model+systems%5BTitle%5D)+AND+%22Bryologist%22%5BJournal%5D)">Bryophytes as model systems.</a> Bryologist. 103, 128-133 (2000).</li>
<li>Schaefer, D. G., Zrÿd, J. -P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+gene+targeting+in+the+moss+Physcomitrella+patens%5BTitle%5D)+AND+%22Plant+J%22%5BJournal%5D)">Efficient gene targeting in the moss Physcomitrella patens.</a> Plant J. 11, 1195-1206 (1997).</li>
<li>Schaefer, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gene+targeting+in+Physcomitrella+patens%5BTitle%5D)+AND+%22Curr.+Opin.+Plant+Biol%22%5BJournal%5D)">Gene targeting in Physcomitrella patens.</a> Curr. Opin. Plant Biol. 4, 143-150 (2001).</li>
<li>Kamisugi, Y., Cuming, A. C., Cove, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Parameters+determining+the+efficiency+of+gene+targeting+in+the+moss+Physcomitrella+patens%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">Parameters determining the efficiency of gene targeting in the moss Physcomitrella patens.</a> Nucleic Acids Res. 33, e173-e173 (2005).</li>
<li>Cove, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+moss+Physcomitrella+patens%5BTitle%5D)+AND+%22Annu.+Rev.+Genet%22%5BJournal%5D)">The moss Physcomitrella patens.</a> Annu. Rev. Genet. 39, 339-358 (2005).</li>
<li>Kamisugi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+mechanism+of+gene+targeting+in+Physcomitrella+patens%3A+homologous+recombination%2C+concatenation+and+multiple+integration%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">The mechanism of gene targeting in Physcomitrella patens: homologous recombination, concatenation and multiple integration.</a> Nucleic Acids Res. 34, 6205-6215 (2006).</li>
<li>Vidali, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+formin-mediated+actin-filament+elongation+is+essential+for+polarized+plant+cell+growth%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">Rapid formin-mediated actin-filament elongation is essential for polarized plant cell growth.</a> Proc. Natl. Acad. Sci. U.S.A. , (2009).</li>
<li>Sawahel, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transfer+of+foreign+DNA+into+Physcomitrella+protonemal+tissue+by+using+the+gene+gun%5BTitle%5D)+AND+%22Plant+Mol.+Biol%22%5BJournal%5D)">Transfer of foreign DNA into Physcomitrella protonemal tissue by using the gene gun.</a> Plant Mol. Biol. 10, 315-316 (1992).</li>
<li>Schaefer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stable+transformation+of+the+moss+Physcomitrella+patens%5BTitle%5D)+AND+%22Mol.+Gen.+Genet%22%5BJournal%5D)">Stable transformation of the moss Physcomitrella patens.</a> Mol. Gen. Genet. 226, 418-424 (1991).</li>
<li>Cho, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Particle+bombardment+mediated+transformation+and+GFP+expression+in+the+moss+Physcomitrella+patens%5BTitle%5D)+AND+%22Mol.+Cells%22%5BJournal%5D)">Particle bombardment mediated transformation and GFP expression in the moss Physcomitrella patens.</a> Mol. Cells. 9, 14-19 (1999).</li>
<li>Ashton, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+bryophyte+Physcomitrella+patens+replicates+extrachromosomal+transgenic+elements%5BTitle%5D)+AND+%22New+Phytol%22%5BJournal%5D)">The bryophyte Physcomitrella patens replicates extrachromosomal transgenic elements.</a> New Phytol. 146, 391-402 (2000).</li>
<li>Hohe, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+improved+and+highly+standardized+transformation+procedure+allows+efficient+production+of+single+and+multiple+targeted+gene-knockouts+in+a+moss%2C+Physcomitrella+patens%5BTitle%5D)+AND+%22Curr.+Genet%22%5BJournal%5D)">An improved and highly standardized transformation procedure allows efficient production of single and multiple targeted gene-knockouts in a moss, Physcomitrella patens.</a> Curr. Genet. 44, 339-347 (2004).</li>
<li>Vidali, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Profilin+is+essential+for+tip+growth+in+the+moss+Physcomitrella+patens%5BTitle%5D)+AND+%22Plant+Cell%22%5BJournal%5D)">Profilin is essential for tip growth in the moss Physcomitrella patens.</a> Plant Cell. 19, 3705-3722 (2007).</li>
</ol>

No conflicts of interest declared.

The method presented here enables a straightforward and consistent DNA transformation into Physcomitrella patens protoplasts. This method was originally developed for Arabidopsis1, but here we demonstrated that it performs well with protoplasts of Physcomitrella patens, a simpler and different plant. Therefore, this method can be applied to other plant species with minor modifications. Possible modifications for optimization include protoplast concentration in MMg, incubation time in MMg and PEG/Ca solution. We choose to use 60 &mu;g DNA in our routine experiments because the number of transformants increases linearly with the DNA concentration until this point (Table 1). We could get approximately 7000 transient transformants or 200 stable transformants on a single 60 &mu;g DNA transformation, which allows for further screening and analysis of a large population of plants.
The transformed protoplasts can be either spread with liquid plating medium or PRM-T. Although the regeneration efficiency is lower when liquid plating medium is used, we choose this method when the protoplasts were transformed with supercoiled plasmids. As showed in Table 1, transformations done with supercoiled plasmid DNA generate many more transformants and thus, we can still obtain large numbers of plants with liquid plating medium. Furthermore, spreading protoplasts with liquid plating medium allows faster regeneration, which is important for experiments involving transient phenotypes, such as RNAi knock down experiments9,15. In addition the absence of top agar allows for easy plant isolation for immunostaining and RT-PCR9,15.

<table><tbody><tr><td>PPNH&lt;sub&gt;4&lt;/sub&gt;</td><td></td><td></td><td>1.84 mM KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&lt;br/&gt; 3.4 mM Ca(NO&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;&lt;br/&gt; 1 mM MgSO&lt;sub&gt;4&lt;/sub&gt;&lt;br/&gt; 2.72 mM Diammonium tartrate&lt;br/&gt; 54 &amp;#x03BC;M FeSO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;O&lt;br/&gt; 9.93 &amp;#x03BC;M H&lt;sub&gt;3&lt;/sub&gt;BO&lt;sub&gt;3&lt;/sub&gt;&lt;br/&gt; 1.97 &amp;#x03BC;M MnCl&lt;sub&gt;2&lt;/sub&gt;.4H&lt;sub&gt;2&lt;/sub&gt;O&lt;br/&gt; 0.23 &amp;#x03BC;M CoCl&lt;sub&gt;2&lt;/sub&gt;.6H&lt;sub&gt;2&lt;/sub&gt;O&lt;br/&gt; 0.19 &amp;#x03BC;M ZnSO&lt;sub&gt;4&lt;/sub&gt;. 7H&lt;sub&gt;2&lt;/sub&gt;O&lt;br/&gt; 0.22 &amp;#x03BC;M CuSO&lt;sub&gt;4&lt;/sub&gt;. 5H&lt;sub&gt;2&lt;/sub&gt;O&lt;br/&gt; 0.10 &amp;#x03BC;M Na&lt;sub&gt;2&lt;/sub&gt;MoO&lt;sub&gt;4&lt;/sub&gt;.2H&lt;sub&gt;2&lt;/sub&gt;O&lt;br/&gt; 0.168 &amp;#x03BC;M KI&lt;br/&gt; Add 0.7% agar for solid medium.</td></tr><tr><td>PRM-B</td><td></td><td></td><td>PPNH&lt;sub&gt;4&lt;/sub&gt; with 6% mannitol and 0.8% agar. Add 10 mL 1M CaCl&lt;sub&gt;2&lt;/sub&gt; to 1 L before pouring plates. </td></tr><tr><td>PRM-T</td><td></td><td></td><td>PPNH&lt;sub&gt;4&lt;/sub&gt; with 6% mannitol and 0.6% agar. Add 0.5 mL 1M CaCl&lt;sub&gt;2&lt;/sub&gt; to 50 mL before use.</td></tr><tr><td>Liquid Plating Medium</td><td></td><td></td><td>PPNH&lt;sub&gt;4&lt;/sub&gt; with 8.5% mannitol without agar. Add 0.5 mL 1M CaCl&lt;sub&gt;2&lt;/sub&gt; to 50 mL before use.</td></tr><tr><td>2% Driselase</td><td></td><td></td><td>Add 4 g of Driselase (Sigma D9515-25G) to 200 mL of 8% mannitol. Stir gently for 30 min. at room temperature, incubate 30 min. at 4&amp;deg;C, and stir gently for 5 min. at room temperature. Spin 2,500 g for 10 min, discard pellet. Filter with 0.22 &amp;#x03BC;m, and store frozen as 10 mL aliquots. Thaw in a room temperature water bath before use.</td></tr><tr><td>MMg</td><td></td><td></td><td>0.4 M mannitol&lt;br/&gt; 15 mM MgCl&lt;sub&gt;2&lt;/sub&gt;&lt;br/&gt; 4 mM MES (pH 5.7)</td></tr><tr><td>PEG/Ca</td><td></td><td></td><td>4 g PEG4000&lt;br/&gt; 3 mL H&lt;sub&gt;2&lt;/sub&gt;O&lt;br/&gt; 2.5 mL 0.8 M mannitol&lt;br/&gt; 1 mL 1M CaCl&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>W5</td><td></td><td></td><td>154 mM NaCl&lt;br/&gt; 125 mM CaCl&lt;sub&gt;2&lt;/sub&gt;&lt;br/&gt; 5 mM KCl&lt;br/&gt; 2 mM MES (pH 5.7) </td></tr></tbody></table>

efficient polyethylene glycol (peg) mediated transformation of the moss physcomitrella patens

Efficient Polyethylene Glycol (PEG) Mediated Transformation of the Moss Physcomitrella patens

A simple and efficient method to transform Physcomitrella pantens protoplasts is described. This method is adapted from protocols for Physocmitrella protonemal protoplast and Arabidopsis mesophyll protoplast transformation1. Due to its capacity to undergo efficient mitotic homologous recombination, Physcomitrella patens has emerged as an important model system in recent years2. This capacity allows high frequencies of gene targeting3-9, which is not seen in other model plants such as Arabidopsis. To take full advantage of this system, we need an effective and easy method to deliver DNA into moss cells. The most common ways to transform this moss are particle bombardment10 and PEG-mediated DNA uptake11. Although particle bombardment can produce a high transformation efficiency12, gene guns are not readily available to many laboratories and the protocol is difficult to standardize. On the other hand, PEG mediated transformation does not require specialized equipments, and can be performed in any laboratory with a sterile hood. Here, we show a simple and highly efficient method for transformation of moss protoplasts. This method can generate more than 120 transient transformants per microgram of DNA, which is an improvement from the most efficient protocol previously reported13. Because of its simplicity, efficiency, and reproducibility, this method can be applied to projects requiring large number of transformants as well as for routine transformation.

A simple and efficient method to transform Physcomitrella pantens protoplasts is described. This method is adapted from protocols for Physocmitrella protonemal protoplast and Arabidopsis mesophyll protoplast transformation1.

A simple and efficient method to transform Physcomitrella pantens protoplasts is described. This method is adapted from protocols for Physocmitrella ...

The overall goal of this procedure is to transform SCO Metre Protoplasts. This is accomplished by first digesting the plants with Drizly. Next, the protoplasts are washed with mannitol to remove the drizly and are then incubated with DNA and peg.Finally, the transformed protoplasts are spread onto plates and incubated. Ultimately, results can be obtained. That show transformed protoplasts expressing fluorescently labeled proteins through fluorescence microscopy Demonstrating the procedure will be Y lu, a postdoc from my laboratory.The main advantage of this technique is that it is effective and consistently generates protoplasts that express the protein of interest To isolate moss Protoplasts begin by adding nine milliliters of 8%mannitol into a Petri dish using a spatula Place five to seven day old moss from two to three PP NH four plates into the mannitol. Please see the accompanying written protocol for this and additional reagent recipes. Next, add three milliliters of 2%drizly and incubate at room temperature with gentle shaking for one hour.Filter the mixture through 100 micron mesh to obtain a green homogeneous suspension. Then spin the filtrate at 250 G for five minutes. Discard the supernatant very gently.Resuspend the protoplasts in 500 microliters of 8%mannitol. Then add an additional 9.5 milliliters of 8%mannitol. Make sure that the protoplasts are fully suspended.Perform the wash steps two more times by spinning the solution again at 250 G for five minutes. Discarding the supernatant and resus suspending the protoplasts. First in 500 microliters.Then 9.5 milliliters of 8%mannitol using a hemo cytometer. Count the number of protoplasts in 10 microliters of the suspension to transform DNA into the isolated protoplasts. First, spin the protoplasts mixture at 250 G for five minutes.Remove the supernatant resuspend the protoplasts at 1.6 million per milliliter in MMG solution. Incubate the suspension at room temperature for 20 minutes. Next, add 600 microliters of the Protoplasts solution into a culture tube containing 60 micrograms of DNA.Shake.Gently add 700 microliters of PEG calcium solution and shake. Well incubate the sample at room temperature for 30 minutes during this waiting period. Cover PRMB plates with cellophane.Then dilute the suspension with three milliliters of W five solution spin at 250 G for five minutes. To remove the PEG resuspend, the protoplasts in two milliliters of PRMT plate one milliliter per PRMB plate covered with cellophane. Keep the plates at 25 degrees Celsius in a growth chamber.Finally, four days after transformation, move the cellophane onto fresh selection plates. This figure shows an example of 18 day old plants regenerated from protoplasts, transformed with 15 micrograms of super coiled plasma, DNA, carrying a hygromycin resistance cassette. Here, 30 micrograms of the same super coiled plasmid, DNA, were transformed into protoplasts.60 micrograms of plaid DNA were transformed in this sample. And finally, these plants were regenerated from protoplasts that were transformed with 120 micrograms of the same super coiled plasma DNA carrying the hygromycin resistance cassette. The transformation efficiency is not affected by DNA concentrations of up to 60 micrograms.However, higher concentrations of DNA are detrimental to this procedure. The data indicate that this method delivers consistent results over a wide range of DNA amounts Once semester. This technique can be done in three hours if it is performed properly.Following this procedure, additional methods like transient RNAi and gene knockouts can be performed in order to answer additional questions about gene function.

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Learn an efficient PEG-mediated transformation method for Physcomitrella patens protoplasts in plant genetic engineering. This streamlined protocol enables reliable gene expression studies and facilitates transient RNAi experiments.

Efficient Polyethylene Glycol (PEG) Mediated Transformation of the Moss Physcomitrella patens

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