Scanning Electron Microscopy Volume 1986 Number 1 Part I Article 30 3-8-1986 Plant Ultrastructure in the Scanning Electron Microscope Susan H. Barnes British Museum Stephen Blackmore British Museum Follow this and additional works at: https://digitalcommons.usu.edu/electron Part of the Biology Commons Recommended Citation Barnes, Susan H. and Blackmore, Stephen (1986) "Plant Ultrastructure in the Scanning Electron Microscope," Scanning Electron Microscopy: Vol. 1986 : No. 1 , Article 30. Available at: https://digitalcommons.usu.edu/electron/vol1986/iss1/30 This Article is brought to you for free and open access by the Western Dairy Center at DigitalCommons@USU. It has been accepted for inclusion in Scanning Electron Microscopy by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. SCANNING ELECTRON MICROSCOPY /1986/1 (Pages 281-289) 0586-5581/86$1 . 00+05 SEM Inc., AMF O'Hare (Chicago), IL 60666 - 0507 USA PLANTULTRASTRUCTURE IN THE SCANNINGELECTRON MICROSCOPE Susan H. Barnes * and Stephen Blackmore British Museum (Na tura l History), Cromwell Road, London SW? SBD, England (Received for publication December 30, 1985, and in revised form March 08, 1986) Abstract Introduction Preparative techniqu e s which have been us ed The combination of high resolution scanning to study internal details of plant cells in the ele ctron microscopy and new preparatory scanning elec tron microscope are reviewed . A techniques have provided fine structural number of me thods hav e previously been described information concerning the in t ernal features of which involve selective extraction of materials cells . He re we review the development of th ese from freeze-fractured s urfaces and can be techniques from a botanical perspective and referred to as freeze-fracture and cytoplasmic present new findings in the field of pollen maceration. One of these t echniques which ontogeny . involves an extended period of cytoplasmic The tough, secondary walls of some plant maceration with dilute osmium tetroxide has been ce l ls (e.g. wood, diatoms, pollen grains) lend applie d to th e study of Cichorium intybus themselves to observation when cytoplasmic (chicory) pollen ontogeny. The results obtained, contents are absent or have been removed. incl udin g changes in th e numbers of mitochondria However, if images of delicate membranous and the form of endoplasmic reticulum during th e components are needed mor e elaborate methods of co ur se of dev el opment demonstrate th e value of preparatlon must be employed. An early advance the approach in showing the three dimensional involved th e snapping of critical point dried arrangement of organelles and wall layers. The material to reveal the internal surfaces of cells findings emphasise that pollen grains with (Troughton and Donaldson, 1972; Kessel and Shih, similar mature morphologies may differ in th e 1974). details of their ontogeny. Epoxy r es ins have bee n used to give support and rigidity to cells of soft tissues. Wodzicki and Humphre ys (1973) introduced a method in which leaves were embedded in resin and fractured. In a study of developing peristome teeth of th e moss Funaria Jarvis (1975) dissolved the resin from blocks which had been sec tion ed for transmission electron microscopy. Chambers and Hamilton (1973) dissolved incompletely polymerised resin from cut surfaces of wheat nodes to reveal the internal details of transfer cell walls. Frozen specimens have ideal characteristics for fracturing. Low temperature scanning elec tr on microscopy has been used to examine frozen hydrated plant cells . Where necessary, moisture may be sublimed partially, to etch fractured surfaces or totally, to achieve permanent freeze-dried specimens (e.g. Read and Beckett, 1985). The absence of artefacts caused ~ words: Cichorium intybus, Compositae: by chemical treatm ent and critical point drying Lactuceae, echinolophate, exine, freeze-fracture is a significant advantage of this approach (see and cytoplasmic maceration, ontogeny, plant reviews by Echlin and Moreton, 1976 and Robards, ultrastructure, pollen, primexine, tapetum. 1984). To obtain a high er level of d e tail of *Address for correspondence: organelles and membrane systems more complex British Museum (Natural History), Cromwell Road, procedures involving chemical fixation are London SW? 5BD, England. needed. Haggis (1970) pioneered this approach Telephon e : 01 - 589 6323 ex t 348 and many developments followed. Lim (1971) 281 S.H. Barnes and S.Blackmore devised a method in which specimens too small to in aldehyde fixed root tips by maceration in handle were allowed to self-fracture when frozen dilute aldehyde followed by dilute osmium in 75% ethanol. Sybers and Ashraf (1973) tetroxide. As an alternative to fracturing, fractured specimens after dehydration to absolute Inoue and Osatake (1984) freeze - polished thin ethanol. This t echniq ue was adapted by Humphreys specimens to expose intracellular components et al. (1974) for a variety of plant and animal before maceration. This development has yet to be specimens. In their study of Lilium and Solanum eval uat ed with plant material but may well prove Whellan et al. (1974) freeze-fractured fixed, to be very valuable. rehydrated anthers in Freon 22. Although their Preparative techniques which involve micrographs show what may be disruption of some ex t rac tion of th e cytoplasmic matrix af t er cytoplasmic components, th e chromosomes appear freeze -fracturin g can be described und er th e well preserved. The work of Yamada e t al. general heading of "freeze-fracture and (1983a,b) and Osumi e t al. (1984) on th e fine cytoplasmic maceration." The ex tended maceration structure of isolated etioplas t prolamellar method (Barnes and Blackmore, 1984a) has recently bodies provides a recent demonstration of the been us ed to study pollen development in results obtainable by fracturing dehydrated Compositae (Blackmore and Barnes, 1985; Barnes material. and Blackmore, 1986) and Liliaceae (Dickinson and The method of Haggis et al. (1976) involved Sheldon, 1986). Her e we demonstrate th e cryoprotection and freeze-fracturing before applicabili t y of this approach by presenting new fixation. The use of a cryoprotectant enabled findings on pollen ontogeny in Cichorium fracturing to be carried out in an aqueous intyb us L. (Compositae : Lactuceae). medium. When the fragments were thaw ed in Many workers have used the transmiss ion fixative some mobile cytoplasmic components were electron microscope to st udy pollen wall ext racted from th e fractured surfaces, which was morphogenesis since the initial research of necessary to provide observable thr ee dimensional Heslop-Harrison (1962, 1963). Pollen grains have structure. This method was later named the diverse and elabora t e walls and th e processes by "freeze-fracture, thaw, fix" techniqu e (Haggis which th ey are deposited have now been documented and Phipps-Todd, 1977; Haggis and Bond, 1979). in a wide range of plants (see reviews by Heslop­ Maruyama (1983) studied chromosomes of Vicia faba Harrison, 1968; Knox, 1984; Blackmore and Crane, using a method involving thaw-fixat~after 1986). However, several fundamental questions freeze-fracturing of fresh unfixed root tips. including th e mechanism by which patterning is Excellent images of organelles and imparted to the developing wall have yet to be membranous systems of animal cells were obtained answered (Dickinson and Sheldon, 1986). The by Tanaka (1981) and Tanaka and Nagura (1981) study of sporogenesis in the scanning elec tron using their O-D-O method which introduced osmium microscope may provide a more comple t e tetroxide fixation in hypotonic buffer prior to understanding of th e processes involved by freeze-fracturing in dimethyl sulphoxide. revealing the spatial relationships between Extrac tion of mobile components was achieved by organelles and developing wall layers. treatment with a very dilute solution of osmium The echinolophate pollen of I:_ intybus has tetroxide in the same hypotonic buffer. When a characteris t ic pat t ern of spiny ridges. The applied to our studies of botanical materials we highly derived nature of this pattern and the found that this technique resulted in incomplete stratification of the wall are of taxonomic maceration of th e cytoplasmic matrix. However, significance (Tomb, 1975; Blackmore, 1981, 1982, successful results were achieved with onion root 1984). Our programme of research aims to tips by Nagura e t al. (1983). By greatly contrast the development of pollen morphology in extending the macerating period, from two days to closely related species. two weeks, we obtained improved extraction of the stroma in chloroplast membrane systems (Barnes Materials and Methods and Blackmore, 1984a). This adaptation of Tanaka's original method has proved s ucc essful Cichorium intyb us was cultivated at Chelsea when applied to a range of botanical specimens Physic Garden, London. Following th e procedure (Barnes and Blackmore, 1984b; Blackmore and described in our earlier work (Barnes and Barnes, 1986). Cytoskeletal components in Blackmore, 1984a,b ) truncat ed anthers at various mesophyll cells were revealed by another stages of maturity were fixed in 1% osmium adaptation of Tanaka's t echniq ue. Fresh, unfixed tetroxide in M/15 phosphate buffer pH 7.4 for 2- l eaves were freeze-fractured in liquid nitrogen 16 hours at room temperature. The fixed anthers without the use of a cryoprotectant, macerat ed were washed in buffer and treated with 15%, 30% for two days and then fixed in osmium tetroxide and 50% aqueous dime thyl sulphoxide (DMSO) for 30 (Blackmore et al., 1984; Barnes et al., 1985; minutes in each solution. They were then frozen Giordano et al., 1985). Other variations have on a liquid nitrogen cooled metal block and proved useful when applied to animal tissu es . freeze-fractured with a razor blade and hammer. Tanaka and Mitsushima ( 1984 ) studied mat erial Fragments were thawed in 50% DMSO, rinsed in that had been perf us ed with an aldehyde prior to buffer and transf erred to buffered 0 .