add 9 ml of 8% mannitol to a petri dish . using a spatula , put 7 day old moss from 2 - 3 ppnh4 plates of 5- in the petri dish containing mannitol . add 3 ml of 2% driselase ( sigma d9515 - 25 g ) to the petri dish . incubate the petri dish at room temperature with gentle shaking for 1 hour . filter the protoplast suspension through a 100 m mesh ( bd falcon 352350 ) . re - suspend the protoplasts very gently with 500 l of 8% mannitol . add 9.5 ml 8% mannitol in the culture tube repeat the filtration and re - suspension steps ( 1.6 , 1.7 and 1.8 ) two more times . take 10 l of the protoplast solution and count the protoplasts in a hemocytometer . multiply the number of protoplast in a 16 square area by 10,000 to obtain the number of protoplasts per ml ( see figure 1 ) . re - suspend protoplasts in mmg solution at the concentration of 1.6 million protoplasts / ml . incubate the protoplast suspension at room temperature for 20 minutes . packaging limited , uk ) ; allow cellophane to hydrate on the plate surface for at least 5 min . with a spatula , re - suspend protoplasts in melted 2 ml of prm - t ( before adding it to the protoplasts this solution can be kept liquid at 42c ) . plate 1 ml of re - suspend protoplasts per prm - b plate covered by cellophane . 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 . growth chamber is set for a 16 hr . the dna used in this example was a supercoiled 7.8 kb plasmid carrying a hygromycin resistant cassette . the pictures were taken two weeks after the plants were moved to the selection plates . 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 previously . 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 45c water bath for 3 minutes . the mixture was incubated in a room temperature water bath immediately after heat shock for 17 min . 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 ) . note the 16 squares required for counting protoplasts ( see 1.11 ) ; the image shows a count of 24 protoplasts ( 240,000 cells/ ml ) . a : 15g ; b : 30g ; c : 60g ; d : 120g . add 9 ml of 8% mannitol to a petri dish . using a spatula , put 7 day old moss from 2 - 3 ppnh4 plates of 5- in the petri dish containing mannitol . add 3 ml of 2% driselase ( sigma d9515 - 25 g ) to the petri dish . incubate the petri dish at room temperature with gentle shaking for 1 hour . filter the protoplast suspension through a 100 m mesh ( bd falcon 352350 ) . re - suspend the protoplasts very gently with 500 l of 8% mannitol . add 9.5 ml 8% mannitol in the culture tube repeat the filtration and re - suspension steps ( 1.6 , 1.7 and 1.8 ) two more times . take 10 l of the protoplast solution and count the protoplasts in a hemocytometer . multiply the number of protoplast in a 16 square area by 10,000 to obtain the number of protoplasts per ml ( see figure 1 ) . re - suspend protoplasts in mmg solution at the concentration of 1.6 million protoplasts / ml . incubate the protoplast suspension at room temperature for 20 minutes . packaging limited , uk ) ; allow cellophane to hydrate on the plate surface for at least 5 min . with a spatula , remove any air bubbles trapped between the cellophane and the plate . dilute the mixture with 3 ml of w5 solution . re - suspend protoplasts in melted 2 ml of prm - t ( before adding it to the protoplasts this solution can be kept liquid at 42c ) . plate 1 ml of re - suspend protoplasts per prm - b plate covered by cellophane . wrap the plates with micropore ( 3 m , health care ) . keep the plates at 25c in a growth chamber . 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 . the dna used in this example was a supercoiled 7.8 kb plasmid carrying a hygromycin resistant cassette . the pictures were taken two weeks after the plants were moved to the selection plates . 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 previously . 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 45c water bath for 3 minutes . the mixture was incubated in a room temperature water bath immediately after heat shock for 17 min . 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 ) . note the 16 squares required for counting protoplasts ( see 1.11 ) ; the image shows a count of 24 protoplasts ( 240,000 cells/ ml ) . protoplasts were transformed with the indicated amount of supercoiled plasmid . a : 15g ; b : 30g ; c : 60g ; d : 120g . the method presented here enables a straightforward and consistent dna transformation into physcomitrella patens protoplasts . this method was originally developed for arabidopsis , but here we demonstrated that it performs well with protoplasts of physcomitrella patens , a simpler and different plant . possible modifications for optimization include protoplast concentration in mmg , incubation time in mmg and peg / ca solution . we choose to use 60 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 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 experiments . in addition the absence of top agar allows for easy plant isolation for immunostaining and rt - pcr .
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 .