the normal development of the male gametophyte of some plant species can be redirected into 
 embryogenic development , resulting in the formation of embryos able to differentiate into 
 haploid plants ( reynolds , 1997 ) .
pollen 
 embryogenesis is of importance in the breeding of certain crop species as it simplifies the 
 fixation of meiotic recombination events via the doubling of the chromosome complement of 
 haploid regenerants .
barley is particularly amenable to pollen embryogenesis , a feature 
 which has helped achieve the crop s status as the leading model temperate cereal 
 ( maraschin et al .
, 2005a ) . 
 defining reliable structural markers for pollen embryogenesis , which has proved to be 
 difficult
to date ( pechan and smykal , 2001 ) , 
 requires the in vivo microscopy of cultured immature pollen ( krens et al . , 1998 ) .
previous 
 efforts to track the fate of individual pollen ( bolik 
 and koop , 1991 ; kumlehn and lrz , 
 1999 ; indrianto et al . , 
 2001 ; maraschin et al .
, 
 2005b 
 , 2008 ) suffered from the inadequate 
 delivery of signalling molecules produced by co - cultivated embryogenic pollen , a process 
 which is inevitably compromised when the target pollen grains have to be immobilized in a 
 gel matrix .
signalling has been shown to be essential for the definition of cell identity 
 and proliferation in plant cell cultures ( kumlehn and 
 lrz , 1999 ; kumlehn et 
 al . , 1999 ) .
in addition , these experiments have had to rely on the 
 acquisition of a series of images , in which the time - interval between their capture has been 
 of the order of several hours ; such a long separation can be expected to fail to record a 
 number of important cellular events .
a method whereby immature barley pollen , arranged in a monolayer 
 within a liquid medium , develops normally and its growth can be followed over a period of 
 several weeks is demonstrated here .
the automated capture of z - stacks of images allowed for 
 an unprecedented level of temporal resolution to be achieved .
the method should prove 
 valuable for elucidating the ultrastructural features and the molecular machinery important 
 for the initiation of pollen embryogenesis and other cellular processes occurring in 
 developing pollen .
( 2005 ) was applied to raise barley ( hordeum 
 vulgare l. ) cv .
 igri ( saatzucht ackermann , irlbach , germany ) 
 plants as a source of immature pollen amenable to pollen embryogenesis .
construction of a live - cell 
 chamber was based on a two - well chambered coverglass ( thermo scientific - cat .
155380 ) 
 in combination with an 0.4 m hydrophilic , diaphanous ptfe membrane of a 12 mm 
 millicell cell culture insert ( millipore - cat .
picm01250 ) ( hereafter referred to as a 
  ptfe membrane ) and a plastic mask fabricated from a 
 countess cell counting chamber slide ( invitrogen , cat .
1e ) adjusted in size to fit 
 precisely within the well of the chambered coverglass ( fig . 

. a square hole of size approximately 77 mm was cut in its centre using 
 a heated scalpel blade ( fig .
one of the ptfe 
 membranes was then placed over the square hole and , using a pair of heated forceps , the 
 edge of the membrane was fused to the plastic mask ( fig . 

( b ) a 12 mm millicell cell culture insert with its 0.4 m 
 hydrophilic ptfe membrane . ( c ) a ptfe membrane in the process of being detached by 
 forceps .
200 000ml 
 immature barley pollen was concentrated 10-fold by removing 900 l of medium from a 
 1ml volume using a 30 m mesh - coated 1ml pipette tip ( fig .
approximately 100 l concentrated cell suspension was pipetted 
 onto the lid of a plastic petri dish ( fig .
2c ) , which 
 ensured that the droplet took on a convex shape . the droplet was left undisturbed for 
 35min to allow dead cells and detritus to precipitate while the viable cells 
 accumulate at the surface of the droplet .
then , a ptfe membrane was brought into contact 
 with the droplet for 23 s , allowing a thin layer of medium containing viable cells 
 to adhere .
the membrane was blotted gently with a filter paper to remove as much of the 
 culture medium as possible , and was then placed face down in the well of the chambered 
 coverglass ( fig .
2d ) , avoiding the formation of air 
 bubbles between the membrane and the cover slip .
this created a monolayer of immature 
 pollen cells in direct contact with the cover slip ( fig . 
 2d , 2j ) .
50 l of starvation medium ( smb1 ) ( coronado et al . , 2005 ) 
 semi - solidified by the addition of 6 mg ml agarose ( type i , low eeo ; 
 sigma - aldrich , steinheim , germany ) ( fig .
after a 
 second plastic mask was placed above the agarose layer ( fig . 

2f , 2 g ) , 1ml of embryogenic pollen culture 
 was pipetted into the chamber to ensure a sufficient supply of signalling molecules for 
 the test pollen ( fig .
2i ) . the combination of agarose 
 and the second mask ensured that the optical focal plane was free of contamination from 
 the feeder cells ( fig .
in order to avoid 
 interference from condensation on the inner surface of the chamber lid , a smooth edged 
 1515 mm gap was cut into the lid of the chamber and covered with a sterile cover 
 slip , which was replaced every day ( fig .
the set - up ensured 
 that the test pollen remained immersed in liquid culture medium , thereby providing 
 suitable culture conditions .
after 2 d , the starvation medium was withdrawn using a 30 
 m mesh - coated 1ml pipette tip ( fig .
1h ) , and 
 replaced by 1ml kbp nutrient medium ( coronado 
 et al . ,
this step had to be done with care to avoid 
 mechanical disturbance of the test pollen .
schematic illustrations of the home - made live - cell chamber cover slip used for 
 time - lapse recording of pollen embryogenesis .
( c ) a 
 droplet of highly concentrated pollen suspension on the hydrophobic lid of a plastic 
 petri dish .
( d ) a monolayer of pollen adhering to a ptfe membrane positioned , along 
 with a plastic mask , within a chamber coverglass . ( e ) covering the ptfe membrane with 
 smb1 medium semi - solidified with agarose .
( i ) the addition of 1ml pollen suspension and the covering of 
 the hole with a cover slip ; the live - cell chamber slide is now ready for monitoring . 

( j ) the pollen monolayer is sandwiched between the bottom slide of the live - cell 
 chamber and a ptfe membrane .
ip , immature pollen . in a series of preliminary trials , in which non - immobilized 
 cultured pollen were used as a control
, it was established that neither 5min nor 3min 
 interval recordings had any detrimental effect on pollen vitality and development . 
 reducing the time interval to only 2min , however , caused the cultures to die between 
 35 d after the start of the recordings .
therefore the 3min time - interval was 
 selected as the standard to perform live - cell imaging of pollen embryogenesis .
z - stacks of 
 nine images with a total scanning time of 40 s were acquired every 3min .
pollen 
 development was monitored for a period of up to 28 d using a hene 633 nm laser line .
the 
 optical interval of the z - stacks was gradually increased from 2 m to 4 m 
 over the course of the monitoring period to take account of the expansion of the test 
 pollen . in vivo microscopy
was performed using a zeiss lsm 510 meta ( carl 
 zeiss , jena , germany ) equipped with a 20 objective ( na 0.75 ) .
the microscope was 
 installed in a dark room kept at a constant 25 c to avoid focus drift . from every 
 individual z - stack
finally , 5069 images were used to 
 compile a time - lapse movie depicting the course of pollen development ( see supplementary 
 video s1 at jxb online ) .
( 2005 ) was applied to raise barley ( hordeum 
 vulgare l. ) cv .
 igri ( saatzucht ackermann , irlbach , germany ) 
 plants as a source of immature pollen amenable to pollen embryogenesis .
construction of a live - cell 
 chamber was based on a two - well chambered coverglass ( thermo scientific - cat .
155380 ) 
 in combination with an 0.4 m hydrophilic , diaphanous ptfe membrane of a 12 mm 
 millicell cell culture insert ( millipore - cat .
picm01250 ) ( hereafter referred to as a 
  ptfe membrane ) and a plastic mask fabricated from a 
 countess cell counting chamber slide ( invitrogen , cat .
1e ) adjusted in size to fit 
 precisely within the well of the chambered coverglass ( fig . 

1 g ) . a square hole of size approximately 77 mm was cut in its centre using 
 a heated scalpel blade ( fig .
one of the ptfe 
 membranes was then placed over the square hole and , using a pair of heated forceps , the 
 edge of the membrane was fused to the plastic mask ( fig . 

( b ) a 12 mm millicell cell culture insert with its 0.4 m 
 hydrophilic ptfe membrane . ( c ) a ptfe membrane in the process of being detached by 
 forceps .
200 000ml 
 immature barley pollen was concentrated 10-fold by removing 900 l of medium from a 
 1ml volume using a 30 m mesh - coated 1ml pipette tip ( fig .
approximately 100 l concentrated cell suspension was pipetted 
 onto the lid of a plastic petri dish ( fig .
2c ) , which 
 ensured that the droplet took on a convex shape . the droplet was left undisturbed for 
 35min to allow dead cells and detritus to precipitate while the viable cells 
 accumulate at the surface of the droplet .
then , a ptfe membrane was brought into contact 
 with the droplet for 23 s , allowing a thin layer of medium containing viable cells 
 to adhere .
the membrane was blotted gently with a filter paper to remove as much of the 
 culture medium as possible , and was then placed face down in the well of the chambered 
 coverglass ( fig .
2d ) , avoiding the formation of air 
 bubbles between the membrane and the cover slip .
this created a monolayer of immature 
 pollen cells in direct contact with the cover slip ( fig . 
 2d , 2j ) .
50 l of starvation medium ( smb1 ) ( coronado et al . , 2005 ) 
 semi - solidified by the addition of 6 mg ml agarose ( type i , low eeo ; 
 sigma - aldrich , steinheim , germany ) ( fig .
after a 
 second plastic mask was placed above the agarose layer ( fig . 
 2f , 2 g ) , 1ml of embryogenic pollen culture 
 was pipetted into the chamber to ensure a sufficient supply of signalling molecules for 
 the test pollen ( fig .
2i ) . the combination of agarose 
 and the second mask ensured that the optical focal plane was free of contamination from 
 the feeder cells ( fig .
in order to avoid 
 interference from condensation on the inner surface of the chamber lid , a smooth edged 
 1515 mm gap was cut into the lid of the chamber and covered with a sterile cover 
 slip , which was replaced every day ( fig .
the set - up ensured 
 that the test pollen remained immersed in liquid culture medium , thereby providing 
 suitable culture conditions .
after 2 d , the starvation medium was withdrawn using a 30 
 m mesh - coated 1ml pipette tip ( fig .
1h ) , and 
 replaced by 1ml kbp nutrient medium ( coronado 
 et al . ,
this step had to be done with care to avoid 
 mechanical disturbance of the test pollen .
schematic illustrations of the home - made live - cell chamber cover slip used for 
 time - lapse recording of pollen embryogenesis .
( c ) a 
 droplet of highly concentrated pollen suspension on the hydrophobic lid of a plastic 
 petri dish .
( d ) a monolayer of pollen adhering to a ptfe membrane positioned , along 
 with a plastic mask , within a chamber coverglass .
( e ) covering the ptfe membrane with 
 smb1 medium semi - solidified with agarose .
( i ) the addition of 1ml pollen suspension and the covering of 
 the hole with a cover slip ; the live - cell chamber slide is now ready for monitoring . 

( j ) the pollen monolayer is sandwiched between the bottom slide of the live - cell 
 chamber and a ptfe membrane .
ip , immature pollen . in a series of preliminary trials , in which non - immobilized 
 cultured pollen were used as a control
, it was established that neither 5min nor 3min 
 interval recordings had any detrimental effect on pollen vitality and development . 
 reducing the time interval to only 2min , however , caused the cultures to die between 
 35 d after the start of the recordings
. therefore the 3min time - interval was 
 selected as the standard to perform live - cell imaging of pollen embryogenesis .
z - stacks of 
 nine images with a total scanning time of 40 s were acquired every 3min .
pollen 
 development was monitored for a period of up to 28 d using a hene 633 nm laser line .
the 
 optical interval of the z - stacks was gradually increased from 2 m to 4 m 
 over the course of the monitoring period to take account of the expansion of the test 
 pollen . in vivo microscopy
was performed using a zeiss lsm 510 meta ( carl 
 zeiss , jena , germany ) equipped with a 20 objective ( na 0.75 ) .
the microscope was 
 installed in a dark room kept at a constant 25 c to avoid focus drift . from every 
 individual z - stack
finally , 5069 images were used to 
 compile a time - lapse movie depicting the course of pollen development ( see supplementary 
 video s1 at jxb online ) .
the movie shown here is representative of a total of eight individual trials lasting 
 between 14 d and 28 d that were performed to prove the usefulness of the system ( see 
 supplementary video s1 at jxb online ) .
every trial comprised 
 between 4 and 15 observed pollen and showed similar patterns of pollen development and 
 similar pollen types were identified . without the possibility of agitating the culture 
 medium , the rate of development
was significantly delayed to the extent that it took as long 
 as 28 d for embryogenic microcalli to emerge from the exine . in
conventionally grown 
 embryogenic pollen , exine breakage typically occurs in the third week of cultivation . 

nevertheless , the cultures appeared to retain a high level of viability which was not 
 compromised by the recordings .
tracking of four pre - mitotic pollen over the course of 28 d 
 resulted in a set of 91 242 images .
the first mitotic division occurred in pollen # 1 after 2 
 d ( fig .
the nuclear division took place at the 
 cell periphery opposite the pollen aperture and produced two equal - sized nuclei . over the 
 course of the following days , a synchronized series of mitotic divisions occurred , 
 generating a multicellular structure ( fig . 

despite this mitotic activity , the dimensions of this pollen remained 
 constant over the first 14 d ( fig .
h ) , but 
 expanded considerably thereafter . in pollen # 2 , the first pollen mitosis occurred after 3 d 
 ( fig .
3b ) , following this the large vacuole remained 
 for approximately 10 d ( fig .
3c , 3d ) . thereafter , the pair of nuclei migrated towards the centre , so that 
 the cytoplasm assumed a star - like pattern .
no further mitoses were observed , and this pollen retained a constant size 
 over the entire period of observation .
starting at day 15 , detectable starch granules began 
 to accumulate within the cells ( fig .
pollen # 3 did not develop at all and collapsed on day 
 seven ( fig .
e ) before the first pollen mitosis took 
 place at the cell periphery on day seven , resulting in the generation of a pair of 
 similar - sized nuclei ( fig .
several further 
 synchronized series of mitoses subsequently converted this pollen grain into an expanding 
 multicellular structure ( fig .
none of the 
 four test pollen described above had its exine ruptured by day 28 , although rupture was 
 observed in some pollen grains of other trials that were run under the same conditions . for 
 comparison , conventionally cultured embryogenic pollen ( non - immobilized , incubated in 35 mm 
 petri dishes )
the results revealed that there are no significant differences between 
 non - immobilized and immobilized pollen with respect to viability , developmental pattern , and 
 ratio of identified cell types ( dead pollen , starch accumulated pollen , multicellular 
 structures ) .
representative example of in vivo microscopy of cultured immature 
 pollen over a 28 d period .
( a  i ) individual pollen # 1 and # 4 underwent pollen 
 embryogenesis and generated a multicellular structure .
( e , f ) after just one mitotic 
 division , development was arrested in pollen # 2 , and starch granules began to 
 accumulate .
( f  i ) pollen # 3 did not show any sign of development and died after 7 
 d. n , nucleus . bar=10 m . the entire series of images from which those shown in fig .
3 have been selected is available online in the form 
 of a time - lapse video ( see supplementary video s1 at jxb online ) .
the experimental set - up described here provided appropriate growing conditions while 
 simultaneously permitting high - quality imaging of cellular events .
it also allowed for the 
 culture medium to be completely exchanged , which was not possible in previous attempts to 
 perform time - lapse imaging where the targets were immobilized within a solidified medium 
 ( kumlehn and lrz , 1999 ; indrianto et al .
, 2001 ; maraschin et al . , 2005b 
 ) and were thus subject to sub - optimal conditions since they had limited access to 
 the signalling molecules produced by freely growing feeder cells .
the presence of the ptfe 
 membrane ensured that the test pollen were trapped within the liquid medium , while at the 
 same time allowing for the free flow of nutrients and signalling molecules . since the test 
 pollen were in close contact to the cover slip of the chamber coverglass , it was possible to 
 use high numerical aperture objectives to acquire high quality images .
it has been shown previously that well - spaced cells undergoing pollen 
 embryogenesis can be successfully observed by light microscopy , since spacing avoids the 
 problems of cell overgrowth and optical interference from non - target cells ( kumlehn and lrz , 1999 ; indrianto et al . ,
this requirement was satisfied by 
 separating the monolayer of observed pollen from a highly concentrated ( 200 000 pollen 
 ml ) feeder population , using a thin layer of solidified medium 
 sandwiched between two permeable ptfe membranes .
previous observations of the in 
 vitro development of wheat ( indrianto 
 et al . , 2001 ) and barley ( bolik and koop , 1991 ; kumlehn and lrz , 1999 ; 
 maraschin et al . , 
 2005a , b , 2008 ) embryogenic pollen invariably used time - intervals of one to several hours . 

although this level of temporal resolution may be sufficient to identify major events , such 
 long time - intervals are not well suited for the documentation of the full detail of events 
 associated with pollen embryogenesis .
the temporal resolution was increased here to 3min and 
 our set - up has proved to be able to maintain the test pollen in a viable state for at least 
 28 d. cultured immature pollen characteristically develops at a variable 
 rate , as illustrated by the differential performance of the four individual pollen shown in 
 fig .
3 . three contrasting fates , as also outlined by 
 maraschin et al . 

( 2005a , b ) , were observed : some cells underwent proliferation , and 
 subsequently formed embryogenic microcalli ; some were arrested in development after initial 
 rounds of mitosis had occurred ; and some failed to develop at all .
( 2005a , b ) reported that , prior to the first 
 mitosis of pollen - embryogenesis - competent pollen of wheat and barley , the nucleus migrates 
 to a central position , leading to the cytoplasm taking on a star - like appearance within the 
 highly vacuolated pollen .
this feature has been considered to be a predictive marker for the 
 initiation of pollen embryogenesis . by contrast , in both pollen # 1 and # 4 ( as well as in 
 many other pollen not shown here ) , the first mitosis took place while the nucleus was still 
 at the cell periphery . a star - like pattern did occur after the first pollen mitosis in 
 pollen # 2 ( fig .
3e , 3f ) , which went on to accumulate starch rather than switch to embryogenic 
 development . in our view , therefore , the formation of the star - like cytoplasm is not a 
 reliable morphological marker for embryogenic pollen development .
the goal of the 28 d study was a test - case to establish the technical 
 limits of our set - up . in a typical experiment ,
such long running times are not necessary for 
 detailed scientific observations focusing on the initiation of pollen embryogenesis but 
 could be useful to study cell proliferation and embryo formation .
we suggest that the improvement in efficiency and temporal resolution 
 achieved by the cell culture method described here will facilitate a more comprehensive 
 analysis of the key events in pollen embryogenesis , leading potentially to the 
 identification of molecular triggers .
in particular , its application to transgenic material 
 expressing fluorophore - labelled cellular structures such as nuclei , chromatin or components 
 of the cytoskeleton will generate a wealth of detailed information concerning the process of 
 pollen embryogenesis .
supplementary data can be found at jxb online . supplementary video s1 . a time - lapse movie ( 5069 images , 7 pictures per second ) depicting 
 the course of pollen development over 28 d.
 pollen embryogenesis provides exciting opportunities in the areas of breeding and 
 biotechnology as well as representing a convenient model for studying the process of plant 
 cell proliferation in general and embryogenesis in particular . 
 a cell culture system was 
 devised in which immature barley pollen could be cultured as a monolayer trapped between 
 the bottom glass - cover slip of a live - cell chamber and a diaphanous ptfe membrane within a 
 liquid medium over a period of up to 28 d , allowing the process of embryogenesis to be 
 tracked in individual pollen . 
 z - stacks of images were automatically captured every 3min , 
 starting from the unicellular pollen stage up to the development of multicellular , 
 embryogenic structures . 
 the method should prove useful for the elucidation of 
 ultrastructural features and molecular processes associated with pollen embryogenesis . 
Introduction
Materials and methods
Plant material and the induction of pollen embryogenesis
Construction and use of a live-cell chamber cover slip
Results
Discussion
Supplementary data