MATERIALS AND METHODSPreparation of Plant TissuesStigmas were harvested from both autosterile(D07) and autofertile (K25) lines of Vicia fabain the days leading to anthesis. Samples wererapidly frozen in liquid nitrogen and attachedto the cold stage (kept at -20oC) with carbonsticky pads.Scanning Electron MicroscopyPlant specimens were examined in a Carl ZeissEVO LS 15 SEM with variable pressure capabil-ity at 20 kV and imaged using a variable pres-sure secondary electron (VPSE) detector.In addition, the SEM was equipped with aCoolstage from Deben UK Ltd, Suffolk, and theimages presented in this note were obtainedwith the stigmas cooled to approximately -20°C. In order to reduce charging artifacts, alow pressure of about 30 Pa of air was used.This very low pressure is sufficient to compen-sate for specimen charging and to provide agas phase scintillation signal for the VPSEdetector.The Coolstage, shown in Figure 3, is a Peltierdevice that is able to reduce the temperatureof the specimen. Extended periods of SEMimaging are then possible as hydration of thespecimen is maintained. This approach offersan alternative to the full environments SEM ifdynamic processes involving water are notrequired.BIOGRAPHYTimothy Baldwin has aPhD in botany from theUniversity of Reading.After a post-doctoralposition at the JohnInnes Centre, Norwich,he became a lecturer inthe Botany Department at Universiti SainsMalaysia. In 1999 he returned to the UK andwas appointed a post-doctoral researchassociate in the Department of Biochemistryat the University of Cambridge. Since 2001,Tim has been senior lecturer in plant sci-ences at the University of Wolverhampton.His research interests include the role of theplant cell wall in plant growth and develop-ment, pollination biology, orchid conserva-tion and plants used in traditional Chinesemedicine.ABSTRACTThe cell walls of the stigma and style areimportant zones for cell-cell recognition,nutrition, guidance and protection of thepollen tube along the transmitting tract.The SEM data presented here was a compo-nent of a larger study the main objective ofwhich was to investigate pistil developmentand pollination in the crop species Vicia fabaL. (the faba bean). The data demonstratethat there is a developmentally regulateddifference in the structural integrity of thestigmatic surface in autofertile (K25) andautosterile (D07) lines of the faba bean. Inthe autofertile lines the stigmatic surfaceruptures two days prior to flower opening(anthesis), whereas in the autosterile plantsthe stigma remains intact, until anthesis.The VPSEM technique used in the currentstudy shows this difference to great effectand as such is an invaluable tool for use inplant cell biology.KEYWORDSscanning electron microscopy, variable pres-sure, cryo electron microscopy, plant sci-ences, pollinationAUTHOR DETAILSDr Timothy C. Baldwin, Senior Lecturer -Plant Sciences, School of Applied Sciences, University of Wolverhampton, Wolverhampton WV1 1SB, UKTel: +44 (0) 1902 322142Email: email@example.comMicroscopy and Analysis25(7):21-22 (EU), 2011VPSEM INPLANTSCIENCESINTRODUCTIONGrain legumes are extremely important inworld agriculture yet information on the struc-ture, composition, and functioning of theirsolid stigma and open style is limited. Globalfood production is underpinned by plantbreeding programmes centred on importantcrops including the faba bean (Vicia fabaL.)(Figure 1), soybean, pea, and the commonbean. Understanding the mechanism of polli-nation in such species enhances the effective-ness of these programmes.The faba bean is particularly suitable forstudy because it produces numerous largeflowers with straight styles that are easy to dis-sect and is partially dependent upon bees forpollination.For the faba bean there are both autofertileand autosterile lines. Autofertile lines do notrequire a pollinating insect but do not havevery large yields. Autosterile lines do need apollinating insect and have relatively high cropyields.The aim for plant scientists is to compareautosterile and autofertile lines so that crossesbetween the two lines can be developed. Theoverall aim is increased food production.In this article the development of the fababean stigmatic papillae was observed in thedays leading up to anthesis (flower opening)using low-temperature variable pressure scan-ning electron microscopy (VPSEM).Variable Pressure Scanning Electron Micro-scopy of Vicia fabaStigmatic PapillaeWen Chen, Fred Stoddard and Timothy C. BaldwinSchool of Applied Sciences, University of Wolverhampton, UK Figure 1: Vicia faba. From Prof. Dr Otto Wilhelm Thomé Flora von Deutschland,Österreich und der Schweiz 1885, Gera, Germany. www.biolib.deMICROSCOPY AND ANALYSISNOVEMBER 201121Figure 2: Photomicrograph showing the general anatomy of the faba beanpistil. Key: (b) -stigma, (c) -apical region, (d) -middle region, (e) -basal region, ov -ovary, sh -stylar hairs, stg -stigma, sty -style.Scale bar = 500 ?m.
RESULTS AND DISCUSSIONFigure 2 shows the general anatomy of thefaba bean pistil and the positions of thestigma, style, stylar hairs and ovary.Figure 4 shows the structure of faba beanstigmas from autofertile K25 (a-e) andautosterile D07 (f-j) lines at d-4 (a, f), d-3 (b, g),d-2 (c, h), d-1 (d, i) and anthesis (e, j). The stig-matic cuticle started to become ruptured(arrowed) at 2 days pre-anthesis in K25 whilstremaining intact until anthesis in D07.Figure 5 shows close ups of faba bean stig-matic papillae.These data show the differences in stigmaticmorphology in autofertile and autosterilelines of faba bean in the days prior to floweropening and pollination which clearly indicatewhy each line is autofertile or autosterilerespectively. They also demonstrate the utilityof VPSEM for similar studies in other plantsspecies where changes in surface morphologycorrelate with biological function.REFERENCES1.Chen, W., Stoddard, F. L. and Baldwin, T. C. Int. J. Plant Sci.167(5): 919-932, 2006.2.Chen W. and Baldwin T. C. An improved method for thefixation, embedding and immunofluorescence labelling ofresin-embedded plant tissue. Plant Molecular BiologyReporter, 25:25-37, 2007.ACKNOWLEDGEMENTSWe are very grateful to Professor W. Link, Uni-versity of Göttingen, for supplying the seeds ofthe inbred lines; to Mr Robert Hooton, Univer-sity of Wolverhampton, who assisted with thecultivation of the faba beans and to Ms Bar-bara Hodson for help and advice with the SEM.W. C. was financially supported by a Universityof Wolverhampton PhD studentship, grantnumber RS 328, 'A molecular and structuralinvestigation of autofertility and autosterilityin Vicia faba(faba bean)'. The project wasassociated with European Commission grantQLK5-CT-2002-02307, 'Faba bean breeding forsustainable agriculture in Europe' (acronymEU-Faba).©2011 John Wiley & Sons, LtdFigure 3 (above): The cold stage used on the VP SEM. Courtesy of Carl Zeiss NTS.MICROSCOPY AND ANALYSISNOVEMBER 222011Figure 4 (right):Structure of stigma from K25 (a-e) and D07 (f-j) at d-4 (a, f), d-3 (b, g),d-2 (c, h), d-1 (d, i) and anthesis (e, j). The stigmatic cuticle started tobecome ruptured (arrowed) at 2 days pre-anthesis in K25 whilst remain-ing intact until anthesis in D07. Scale bars = 40 ?m.Figure 5:Close up of faba bean stigmatic papillae (a) and (b).Scale bars = 20 ?m.