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Boron (B) is essential for growth and development, with the B requirement differing depending on the particular organs and tissues, but the molecular mechanisms underlying the preferential distribution of B to different tissues are poorly understood. We investigated the role of a rice gene (OsBOR1) encoding a B efflux transporter in the distribution of B to different tissues under different B supplies. OsBOR1 was highly expressed in the nodes at all growth stages. The OsBOR1 protein shows polar localization at the distal side of bundle sheath cells in nodes and xylem parenchyma cells of elongating leaf sheath, but in the mature leaf sheath and blade at the proximal side of bundle sheath cells. Furthermore, the expression of OsBOR1 was not affected by external B fluctuations, but the OsBOR1 protein was gradually degraded in response to high B. Knockout of this gene altered B distribution, decreasing the distribution of B to new leaves and panicles but increasing B distribution to old leaves. These results indicate that OsBOR1 expressed in nodes and leaf sheath is involved in the preferential distribution of B to different tissues in rice. Furthermore, the OsBOR1 undergoes degradation in response to high B for fine regulation of B distribution to different tissues.

Pits are regions in the cell walls of plant tracheary elements that lack secondary walls. Each pit consists of a space within the secondary wall called a pit chamber, and a modified primary wall called the pit membrane. The pit membrane facilitates transport of solutions between vessel cells and restricts embolisms during drought. Here we analyzed the role of an angiosperm-specific TPX2-like microtubule protein MAP20 in pit formation using Brachypodium distachyon as a model system. Live cell imaging was used to analyze the interaction of MAP20 with microtubules and the impact of MAP20 on microtubule dynamics. MAP20-specific antibody was used to study expression and localization of MAP20 in different cell types during vascular bundle development. We used an artificial microRNAs (amiRNA) knockdown approach to determine the function of MAP20. MAP20 is expressed during the late stages of vascular bundle development and localizes around forming pits and under secondary cell wall thickenings in metaxylem cells. MAP20 suppresses microtubule depolymerization; however, unlike the animal TPX2 counterpart, MAP20 does not cooperate with the γ-tubulin ring complex in microtubule nucleation. Knockdown of MAP20 causes bigger pits, thinner pit membranes, perturbed vasculature development, lower reproductive potential and higher drought susceptibility. We conclude that MAP20 may contribute to drought adaptation by modulating pit size and pit membrane thickness in metaxylem.

Leaf function is intimately related to the size, shape, abundance and position of cells and chloroplasts. Anatomy has long been assessed and quantified in two dimensions with 3D structure inferred from 2D micrographs. Serial block face scanning electron microscopy (SBF-SEM) was used to reconstruct 95 cells and 1173 chloroplasts from three wheat and nine chickpea leaves (three samples each from three chickpea genotypes). Wheat chloroplast volume was underestimated by 61% in mesophyll cells and 45% in bundle sheath cells from 2D micrographs, whereas chickpea mesophyll chloroplast volume was underestimated by 60% using simple geometrical models. Models of chickpea spongy and palisade cells both under- and overestimated surface area and volume by varying degrees. These models did not adequately capture irregular shapes such as flattening of chloroplasts or lobed spongy mesophyll cells. It is concluded that simple geometrical models to estimate chloroplast and cell 3D volume and surface area from 2D micrographs are inadequate, and that SBF-SEM has strong potential to contribute to improved understanding of leaf form and function.

Leaves with high photosynthetic capacity require high transpiration capacity. Consequently, hydraulic conductance, stomatal conductance, and assimilation capacities should be positively correlated. These traits make independent demands on anatomical space, particularly due to the propensity for veins to have bundle sheath extensions that exclude stomata from the local epidermis. We measured density and area occupation of bundle sheath extensions, density and size of stomata and subsidiary cells, and venation density for a sample of extant angiosperms and fossil and living nonangiosperm tracheophytes. For most nonangiosperms, even modest increases in vein density and stomatal conductance would require substantial reconfigurations of anatomy. One characteristic of the angiosperm syndrome (e.g. small cell sizes, etc.) is hierarchical vein networks that allow expression of bundle sheath extensions in some, but not all veins, contrasting with all-or-nothing alternatives available with the single-order vein networks in most nonangiosperms. Bundle sheath modulation is associated with higher vein densities in three independent groups with hierarchical venation: angiosperms, Gnetum (gymnosperm) and Dipteris (fern). Anatomical and developmental constraints likely contribute to the stability in leaf characteristics - and ecophysiology - seen through time in different lineages and contribute to the uniqueness of angiosperms in achieving the highest vein densities, stomatal densities, and physiological rates.

The primary thickening growth of Moso (Phyllostachys edulis) underground shoots largely determines the culm circumference. However, its developmental mechanisms remain largely unknown. Using an integrated anatomy, mathematics and genomics approach, we systematically studied cellular and molecular mechanisms underlying the growth of Moso underground shoots. We discovered that the growth displayed a spiral pattern and pith played an important role in promoting the primary thickening process of Moso underground shoots and driving the evolution of culms with different sizes among different bamboo species. Different with model plants, the shoot apical meristem (SAM) of Moso is composed of six layers of cells. Comparative transcriptome analysis identified a large number of genes related to the vascular tissue formation that were significantly upregulated in a thick wall variant with narrow pith cavity, mildly spiral growth, and flat and enlarged SAM, including those related to plant hormones and those involved in cell wall development. These results provide a systematic perspective on the primary thickening growth of Moso underground shoots, and support a plausible mechanism resulting in the narrow pith cavity, weak spiral growth but increased vascular bundle of the thick wall Moso.

To understand the molecular mechanisms of rice aerial organ development, we identified a mutant gene that caused a significant decrease in the width of aerial organs, termed ABNORMAL VASCULAR BUNDLES (AVB). Histological analysis showed that the slender aerial organs were caused by cell number reduction. In avb, the number of vascular bundles in aerial organs was reduced, whereas the area of the vascular bundles was increased. Ploidy analysis and the in situ expression patterns of histone H4 confirmed that cell proliferation was impaired during lateral primordia development, whereas procambium cells showed a greater ability to undergo cell division in avb. RNA sequencing (RNA-seq) showed that the development process was affected in avb. Map-based cloning and genetic complementation demonstrated that AVB encodes a land plant conserved protein with unknown functions. Our research shows that AVB is involved in the maintenance of the normal cell division pattern in lateral primordia development and that the AVB gene is required for procambium establishment following auxin signaling.

In C4 photosynthesis CO2 assimilation and reduction are typically coordinated across mesophyll (M) and bundle sheath (BS) cells, respectively. This system consequently requires sufficient light to reach BS to generate enough ATP to allow ribulose-1,5-bisphosphate (RuBP) regeneration in BS. Leaf anatomy influences BS light penetration and therefore constrains C4 cycle functionality. Using an absorption scattering model (coded in Excel, and freely downloadable) we simulate light penetration profiles and rates of ATP production in BS across the C3 , C3 -C4 and C4 anatomical continua. We present a trade-off for light absorption between BS pigment concentration and space allocation. C3 BS anatomy limits light absorption and benefits little from high pigment concentrations. Unpigmented BS extensions increase BS light penetration. C4 and C3 -C4 anatomies have the potential to generate sufficient ATP in the BS, whereas typical C3 anatomy does not, except some C3 taxa closely related to C4 groups. Insufficient volume of BS, relative to M, will hamper a C4 cycle via insufficient BS light absorption. Thus, BS ATP production and RuBP regeneration, coupled with increased BS investments, allow greater operational plasticity. We propose that larger BS in C3 lineages may be co-opted for C3 -C4 and C4 biochemistry requirements.

474 I. 474 II. 475 III. 475 IV. 477 V. 477 VI. 477 VII. 479 VIII. 481 482 References 482 SUMMARY: A significant proportion of terrestrial biomass is constituted of xylem cells that make up woody plant tissue. Xylem is required for water transport, and is present in the vascular tissue with a second conductive tissue, phloem, required primarily for nutrient transport. Both xylem and phloem are derived from cell divisions in vascular meristems known as the cambium and procambium. One major component that influences several aspects of plant vascular development, including cell division in the vascular meristem, vascular organization and differentiation of vascular cell types, is a signalling module characterized by a peptide ligand called TRACHEARY ELEMENT DIFFERENTIATION INHIBITORY FACTOR (TDIF) and its cognate receptor, PHLOEM INTERCALATED WITH XYLEM (PXY). In this review, we explore the literature that describes signalling components, phytohormones and transcription factors that interact with these two central factors, to control the varying outputs required in vascular tissues for normal organization and elaboration of plant vascular tissue.

Plants rapidly release photoassimilated carbon (C) to the soil via direct root exudation and associated mycorrhizal fungi, with both pathways promoting plant nutrient availability. This study aimed to explore these pathways from the root's vascular bundle to soil microbial communities. Using nanoscale secondary ion mass spectrometry (NanoSIMS) imaging and (13) C-phospho- and neutral lipid fatty acids, we traced in-situ flows of recently photoassimilated C of (13) CO2 -exposed wheat (Triticum aestivum) through arbuscular mycorrhiza (AM) into root- and hyphae-associated soil microbial communities. Intraradical hyphae of AM fungi were significantly (13) C-enriched compared to other root-cortex areas after 8 h of labelling. Immature fine root areas close to the root tip, where AM features were absent, showed signs of passive C loss and co-location of photoassimilates with nitrogen taken up from the soil solution. A significant and exclusively fresh proportion of (13) C-photosynthates was delivered through the AM pathway and was utilised by different microbial groups compared to C directly released by roots. Our results indicate that a major release of recent photosynthates into soil leave plant roots via AM intraradical hyphae already upstream of passive root exudations. AM fungi may act as a rapid hub for translocating fresh plant C to soil microbes.

Bundle sheath extensions (BSEs) are key features of leaf structure whose distribution differs among species and ecosystems. The genetic control of BSE development is unknown, so BSE physiological function has not yet been studied through mutant analysis. We screened a population of ethyl methanesulfonate (EMS)-induced mutants in the genetic background of the tomato (Solanum lycopersicum) model Micro-Tom and found a mutant lacking BSEs. The leaf phenotype of the mutant strongly resembled the tomato mutant obscuravenosa (obv). We confirmed that obv lacks BSEs and that it is not allelic to our induced mutant, which we named obv-2. Leaves lacking BSEs had lower leaf hydraulic conductance and operated with lower stomatal conductance and correspondingly lower assimilation rates than wild-type leaves. This lower level of function occurred despite similarities in vein density, midvein vessel diameter and number, stomatal density, and leaf area between wild-type and mutant leaves, the implication being that the lack of BSEs hindered water dispersal within mutant leaves. Our results comparing near-isogenic lines within a single species confirm the hypothesised role of BSEs in leaf hydraulic function. They further pave the way for a genetic model-based analysis of a common leaf structure with deep ecological consequences.

Magnesium (Mg) is among the most abundant mineral elements in plants, yet the knowledge of which genes control its accumulation in specific tissues and organelles lags behind that of many other mineral elements. Only in recent years has identification of important molecular play ers begun to take shape. In this issue of New Phytologist, Conn et al. (pp. 583–594) shed additional light on two Mg transporters that play important roles in accumulation of Mg in leaf cell vacuoles. Using subcellular-level ion measurements on leaves, gene expression measurements after single-cell sampling, a genetic approach, and clever use of calcium (Ca) and Mg supply to plants or detached leaves, Conn et al. have demonstrated that vacuoles of mesophyll rather than epidermal or bundle sheath cell types of Arabidopsis leaves are the main sites of Mg accumulation. They have also shown the effects of mutations in MRS2-1 and MRS2-5 genes on this accumulation and a role for these

C4 photosynthesis is an adaptation that evolved to alleviate the detrimental effects of photorespiration as a result of the gradual decline in atmospheric carbon dioxide levels. In most C4 plants, two cell types, bundle sheath and mesophyll, cooperate in carbon fixation, and, in so doing, are able to alleviate photorespiratory losses. Although much of the biochemistry is well characterized, little is known about the genetic mechanisms underlying the cell-type specificity driving C4 . However, several studies have shown that regulation acts at multiple levels, including transcriptional, post-transcriptional, post-translational and epigenetic. One example of such a regulatory mechanism is the cell-specific accumulation of major photorespiratory transcripts/proteins in bundle sheath cells, where ribulose-1,5-bisphosphate carboxylase/oxygenase is localized. Although many of the genes are expressed in the bundle sheath, some are expressed in both cell types, implicating post-transcriptional control mechanisms. Recently, ultra-high-throughput sequencing techniques and sophisticated mass spectrometry instrumentation have provided new opportunities to further our understanding of C4 regulation. Computational pipelines are being developed to accommodate the mass of data associated with these techniques. Finally, we discuss a readily transformable C4 grass--Setaria viridis--that has great potential to serve as a model for the genetic dissection of C4 photosynthesis in the grasses.

Summary Secondary growth from vascular cambia results in radial, woody growth of stems. The innovation of secondary vascular development during plant evolution allowed the production of novel plant forms ranging from massive forest trees to flexible, woody lianas. We present examples of the extensive phylogenetic variation in secondary vascular growth and discuss current knowledge of genes that regulate the development of vascular cambia and woody tissues. From these foundations, we propose strategies for genomics-based research in the evolution of development, which is a next logical step in the study of secondary growth.

In Arabidopsis, the compartmentation of sugars into vacuoles is known to be facilitated by sugar transporters. However, vacuolar sugar transporters have not been studied in detail in other plant species. To characterize the rice (Oryza sativa) tonoplast monosaccharide transporters, OsTMT1 and OsTMT2, we analysed their subcellular localization using green fluorescent protein (GFP) and expression patterns using reverse-transcription polymerase chain reaction (RT-PCR), performed histochemical beta-glucuronidase (GUS) assay and in situ hybridization analysis, and assessed sugar transport ability using isolated vacuoles. Expression of OsTMT-GFP fusion protein in rice and Arabidopsis revealed that the OsTMTs localize at the tonoplast. Analyses of OsTMT promoter-GUS transgenic rice indicated that OsTMT1 and OsTMT2 are highly expressed in bundle sheath cells, and in vascular parenchyma and companion cells in leaves, respectively. Both genes were found to be preferentially expressed in the vascular tissues of roots, the palea/lemma of spikelets, and in the main vascular tissues and nucellar projections on the dorsal side of the seed coats. Glucose uptake studies using vacuoles isolated from transgenic mutant Arabidopsis (tmt1-2-3) expressing OsTMT1 demonstrated that OsTMTs are capable of transporting glucose into vacuoles. Based on expression analysis and functional characterization, our present findings suggest that the OsTMTs play a role in vacuolar glucose storage in rice.

We investigated changes in mineral nutrient uptake and cellular expression levels for metal transporter genes in the cadmium (Cd)/zinc (Zn) hyperaccumulator, Thlaspi caerulescens during whole plant and leaf ontogenesis under different long-term treatments with Zn and Cd.- Quantitative mRNA in situ hybridization (QISH) revealed that transporter gene expression changes not only dependent on metal nutrition/toxicity, but even more so during plant and leaf development. The main mRNA abundances found were: ZNT1, mature leaves of young plants; ZNT5, young leaves of young plants; MTP1 (= ZTP1 = ZAT), young leaves of both young and mature plants.- Surprisingly different cellular expression patterns were found for ZNT1 and ZNT5, both belonging to the ZIP family of transition metal transporters: ZNT1, photosynthetic mesophyll and bundle sheath cells; ZNT5, nonphotosynthetic epidermal metal storage cells and bundle sheath cells. Thus, ZNT1 may function in micronutrient nutrition while ZNT5 may be involved in metal storage associated with hyperaccumulation.- Cadmium inhibited the uptake of Zn, iron (Fe) and manganese (Mn), probably by competing for transporters or by interfering with the regulation of transporter gene expression. Cadmium-induced changes in cellular expression for ZNT1, ZNT5 and MTP1 could also be part of plant acclimatization to Cd toxicity. Defence against Cd toxicity involved enhanced uptake of magnesium (Mg), calcium (Ca) and sulphur (S).

Summary • Brassica juncea is a potential candidate plant for phytoremediation of a number of heavy metals, but little is known about the phytotoxicity of chromium (Cr) for this plant in Cr(III)- and Cr(VI)-contaminated soils. • Chromium distribution and phytotoxicity at the whole plant and cellular levels were studied using chemical, light microscopy, scanning electron microscopy and transmission electron microscopy analyses. • Bioavailability of Cr in soils was low, but the uptake significantly increased at phytotoxic levels. Chromium from Cr(VI)-contaminated soils was more phytotoxic than from Cr(III)-contaminated soils. Chromium causes growth retardation, reduces the number of palisade and spongy parenchyma cells in leaves, results in clotted depositions in the vascular bundles of stems and roots, and increases the number of vacuoles and electron dense materials along the walls of xylem and phloem vessels. • Our results suggest that B. juncea is not a good candidate for phytoremediation of soils with lower Cr. However, it is able to accumulate significant amounts of Cr in both shoots and roots at higher soil-Cr concentrations despite severe phytotoxic symptoms.

The development of fruits of blackcurrant (Ribes nigrum) cv. Ben Alder from flower to maturity was studied non-invasively by nuclear magnetic resonance (NMR) microscopy, using attached and detached fruits, and the images were compared with those from low temperature scanning electron microscopy (LTSEM) and conventional resin histology. The NMR images derived from 2-D and 3-D datasets showed the previously unreported growth of arillar tissues to the extent that they almost completely occlude the locular cavity, but LTSEM and resin histology revealed that no fusion occurs between the arillar tissues and the gelatinous sheath surrounding each seed, or between the arillar tissues and the endocarp. The discontinuities between these tissues cause magnetic inhomogenities which result in these structures being clearly resolved by gradient echo imaging sequences. During seed maturation the endosperm changed from high (bright) to low (dark) signal intensity as lipid reserves formed and solidified, whereas the gelatinous sheath had high signal intensity throughout maturation. The high lipid concentration in the seed was manifested by chemical shift effects in the images and the increasing viscosity of the endosperm was reflected in the decrease in spin–lattice (T1) relaxation times. The funiculi, throughout development of seeds, appeared in NMR images with low signal intensity and 3-D surface-rendered reconstructions illustrated the complexity of the spatial array of seeds and funiculi arising from parietal placentas within the loculus. All other vascular tissues in the pericarp and placentas were resolved as a small bright core surrounded by a dark region, within a matrix of moderate signal intensity. Conventional microscopical studies then showed that the bright core discernible by NMR imaging encompassed an entire vascular bundle whereas the darker surrounding region represented small parenchyma cells with pronounced intercellular gas spaces. Other regions of the pericarp which included extremely large parenchyma cells, however, had few intercellular spaces and consequently gave rise to brighter regions of the image.

After cooling the roots and shoot apex of barley, there is a reduction in sucrose export from, and an increased total accumulation of carbohydrate and induction of fructan synthesis in, source leaves. We investigated carbohydrate accumulation in individual leaf cells. Using the microsampling technique and microfluorometric enzymatic assay, the amounts of various carbohydrates were measured in mesophyll, bundle sheath and epidermal cells. Epidermal cells take no part in carbohydrate partitioning and have very low concentrations of sugars even when leaf export is reduced. Even without cooling, however, there are remarkable changes in sucrose concentration (up to 200 mM) in mesophyll and bundle sheath cells during the photoperiod. The activity of acid invertase in samples from individual cells was very low, c. 3 μmol sucrose (g f. wt)−1 h−1. Interrelationships between these cell types and the control of fructan metabolism are discussed.

summary The dominant gene N-1 in cultivated oats gives rise to a phenotype characterized by a thin papery lemma from which the grain readily threshes free, a multiflorous habit, and rachillas that are elongated in comparison to those of husked oats. This study was undertaken in order to identify the stages at which morphological and biochemical development of naked and husked oats begin to diverge, and to look for correlated differences in gene products (mRNAs and proteins). Spikelet morphology in naked and husked oats was shown to diverge early in panicle development: the first visible difference was premature cessation of growth of the third and subsequent florets in husked oats. Lignin deposition in the lemma occurred later and followed a similar time-course in both genotypes, though in naked oats it was restricted to the vascular bundles. Mature lemmas of the naked genotype closely resembled glumes in appearance and in lignification pattern. Genotypic differences in extractable polypeptide complements were apparent only late in development, coinciding with the onset of lignification, and were not associated with comparable differences in translatable mRNA profiles. The normal (n-1) allele at the naked locus is likely to encode a transcription factor with properties similar to those of previously-characterized homeotic gene products in plants.

summary We report on the structure of N2-fixing nodules formed on the stem of Discolobium pulchellum Benth., an aquatic legume in the subfamily Pupilionoideae, tribe Aeschynomeneae, from the Hooded areas in the ‘Pantanal Matogrossense’ region of Brazil. The stern (and root) nodules were obligately aquatic, requiring permanent submergence in water or flooded soil, and receive oxygen via profuse aerenchyma covering The lower stem. Of the 69 isolates of rhizobia isolated from stem and root nodules, 70% were fast-growing acid producers and 38% were slow growers. The rhizobia were not photosynthetic. Nodules were connected to the stem, and the vascular system from the stem branched throughout the nodule, penetrating the infected, tissue within finger-like ingrowths of cortex. In both stem and root nodules, infected tissue was aeschynomenoid or desmodioid, that is, without uninfected (interstitial) cells. The infected cells in stem nodules were vacuolate, with visible infection threads. The inner cortex was rich in amyloplasts and contained the components of an oxygen diffusion barrier (a boundary cell layer without intercellular spaces and glycoprotein occlusions of intercellular spaces in other cell layers). The mid-cortex, external to the boundary layer, consisted of loosely-packed cells and these were continuous with stem aerenchyma. The outer part of the nodules was made up of phellogen-derived cells forming a periderm, or ‘corky’ layer of cells. The periderm formed large lenticels above cortical vascular bundles. These lenticels also connected with the stem aerenchyma. Root nodules differed only in that infected cells were not vacuolate, bacteroids were larger and contained more poly-β-hydroxybutyrate (PHB) and there was less aerenchyma/lenticellular tissue. Stem and root nodule structure is discussed in terms of adaptations to O2 constraints in an aquatic environment.

SUMMARY The fluorescent tracers sulphorhodamine G (SR) and pyrene trisulphonate (PTS) were used to explore the functions of cells and tissues within the pine needle, following their progress after feeding through the transpiration stream. The distributions of tracer in the needles were stabilized for fluorescence microscopy by rapid freezing and freeze-substitution, and anhydrous embedding and sectioning. After short pulse + chase times (up to 2 h), SR and PTS accumulated at higher-than-xylem concentrations in the transfusion tracheids on the flanks of the vascular strand, but did not pass out through the endodermis. The accumulations occurred locally where the transpiration water was separated from the tracers and passed into the symplast of the transfusion parenchyma and endodermis. After a 24 h water chase, SR had entered the symplast through the transfusion parenchyma, and spread through the endodermis and palisade. It is argued that this is evidence of active H+-ATPase systems lowering the external pH of the transfusion parenchyma, and characterizes these cells as scavenging cells similar to those found in the bundle sheath systems of legume leaves. The transport of SR through the endodermis and palisade is the first clear evidence that this tracer can also function as a symplastic tracer. The hypothesis that the transfusion parenchyma acts to scavenge solutes from the transpiration stream was tested by loading the stream with [14C]aspartate and examining the subsequent distribution of 14C by dry autoradiography. After a pulse + chase of (0.75 + 3) h, 14C was found concentrated in the transfusion parenchyma, and at even higher levels in the Strasburger cells. It is proposed that major functions of the transfusion tissue of gymnosperms are (a) the concentrating of solutes from the transpiration stream and (b) the retrieval from the stream of selected solutes that are returned to the phloem through the Strasburger cells, or forwarded through the endodermis to the palisade.

Nuclear magnetic resonance (NMR) micro-images of mature red raspberry (Rubus idaeus L.) fruits and their separated components have been produced under a variety of experimental conditions with the objective of understanding factors which determine image quality in different types of tissue. By varying the delay time between image accumulation sequences it has been shown that water molecules in drupelet surface layers in contact with other drupelets, and in the vascular bundles and traces in the receptacle and drupelets have shorter relaxation times than those in other tissues of the fruit. By performing separate T1 - > and T2 -weighted imaging sequences it was shown that the short relaxation times in the vascular tissue was primarily a T1 effect. This is probably caused by paramagnetic ions because electron paramagnetic resonance (EPR) spectroscopy showed significant amounts of the manganese(n) ion in a spectrum of an aqueous extract of receptacle tissue. The cause of the short relaxation times in the drupelet surface layers is less clear, but we suggest that epicuticular waxes or the suberized cuticular layer of the epidermis might be responsible. The appearance of entire seeds, including the mature embryos, as featureless black regions in the images has been shown to be due to the lower concentration of mobile protons in these structures compared with that in the surrounding mesocarp, and not to differences in relaxation properties of the protons. Consequently, internal detail of the seeds could only be obtained by separating them from the fruit.

SUMMARY Intermediate veins of mature barley leaves were exposed by partial enzymic removal of mesophyll cells. Lucifer Yellow was iontophoresed into a variety of cell types and its symplastic transfer followed by fluorescence microscopy. Dye transfer from mesophyll cells into the parenchymatous bundle sheath (PBS) and from PBS to mestome sheath (MS) occurred readily. Dye transfer around and along both PBS and MS was rapid. Following injection into PBS or MS dye could be seen moving within sieve tubes, thus providing a demonstration of potential mesophyll-to-sieve tube transfer.

summary Red raspberry (Rubus idaeus) fruits were subjected to nuclear magnetic resonance (NMR) micro-imaging to study the changes in spatial distribution of mobile protons (1H) during a 3-day ripening period while the fruit was still attached to the receptacle. All imaging measurements were made using the protons from a single absorption peak at 4.6 ppm, which corresponds to water, and data were collected by pulsed gradient spin-echo and gradient-echo methods with voxel dimensions of 70 × 70 × 500 μm. Individual drupelets were clearly visible by this non-invasive histological method in distal transverse ‘slices’ of a newly harvested red ripe fruit viewed by spin-echo; highest mobile proton densities were found at the lines of contact between drupelets. The mesocarp tissues of each drupelet showed intermediate grey tones with some evidence of radial striations. In more proximal ‘slices’ the surface of the receptacle and its central vascular cylinder had the highest mobile proton densities. The seeds produced black images in all ‘slices’, indicative of low mobile proton densities or long relaxation times. After 3 days the proximal ring of drupelets separated from the receptacle, the spaces between drupelets enlarged slightly and the boundaries between drupelets were less pronounced. Vascular bundles in the receptacle lost their whitish radiating pattern in proximal ‘slices’ almost completely, indicating that mobile protons were less abundant after abscission of drupelets. The same ‘slices’ viewed by gradient echo were less discriminating for localization of Seeds and pronounced tangential wave patterns, often overlaying radial striations, presented more complex images than those seen by spin echo.

summary This light and electron microscope study of the iridescent blue leaves of the fern Teratophyllum rotundifoliatum, reveals several novel anatomical and cytological features. In this extreme shade species [0.4—-(3)–4 W m−2] of lowland tropical rainforest in peninsular Malaya, the greater part of the lamina between the simple collateral vascular bundles, consists only of epidermal cells. From 4–(6.9)–12 large chloroplasts 9.4–(17.9)–27 μm in diameter are located adjacent to the inner walls of the lens-shaped upper epidermal cells. Those in the tessellated lower epidermal cells are smaller [5.0–(7.6)–19.5 μm], much more numerous [21–(53)–83] and regularly distributed around the periphery. They are identical in size and ultrastructure to the chloroplasts in the guard cells of the stomata. Whereas the upper epidermal chloroplasts and those in the stelar tissues contain very little starch, numerous starch grains occupy up to Sl-9% of the volume of the lower epidermal chloroplasts. As in other extreme-shade plants the epidermal chloroplasts in T. rotundifoliatum contain numerous large grana, with no preferred orientation, and integranal lamellae completely filling the stroma. The numbers of thylakoids per granum in the lower epidermal cells [9–(29.3)–84] are significantly smaller than those in the upper [22–(86)–28O]. The latter appear to be the largest yet recorded in land plants and this probably accounts for the remarkably low chlorophyll a: b ratio (1.8) in T. rotundifoliatum. Record amounts of chlorophyll per unit leaf fresh weight and per unit leaf area in T. rotundifoliatum reflect the high proportion of leaf cells containing chloroplasts. The unusual features of leaf anatomy and cytology in T. rotundifoliatum appear to be associated with maximizing the efficiency of absorption of very low diffuse radiation.

summary Xylem, as a mixture of elements of large and small diameter, presents a paradox when viewed as a conductor of sap flow, since the quantity carried is determined by only the largest diameter elements. The paradox may be s resolved, and an important function for the smallest elements revealed, by consideration of flow in leaky tubes. It is shown how the balance between axial flow and leakage becomes critical at a certain diameter, called the critical diameter (D0). Below D0, no axial flow can occur. When a stack of connected tracheary elements of different diameters is assembled into a xylem wedge, as in the major veins of dicotyledon leaves, this wedge will act as a functional unit. Elements with D > D0 have axial flow, but those where DD0 have only radial flow. The mixture of elements then acts to concentrate solutes of the sap in the smallest elements at the apex of the wedge, left behind as the water leaves. The balance between radial and axial flow is also self-adjusting to changes in the longitudinal gradient of pressure. Consideration is given to the functions of these graded stacks in the metaxylem of leaves, of stem vascular bundles, within secondary wood, and in roots. It is shown how some xylem fibres may be expected to mediate exchanges of water and solutes, and may act as regulators of these exchanges in space and time. Some experimental demonstrations are presented of the predictions of this hypothesis.

summary In primary leaves of Vigna sinensis Endl. seedlings growing in the presence of colchicine, some giant polyploid cells at the phloem pole of the vascular bundles differentiate into an extraordinary cell type acquiring most of the prominent features of the sieve elements. These aberrant sieve elements (a-SEs) show some exceptional features not found in normal sieve-tube elements. A heavy secondary thickening is deposited on their walls, which in surface view exhibits a reticulate or ‘pitted’ appearance. The thickenings reach the same depth into the cytoplasm and show a diffuse orientation of cellulose microfibrils. Amorphous callose is deposited around some fine cytoplasmic strands penetrating the secondary wall up to the primary one along the longitudinal and particularly the transverse walls. In the latter walls sieve pores are not formed. At first, the a-SEs undergo a partial protoplasmic degradation like normal sieve-tube elements but finally autolyse completely. During this process, the major portion of the unmasked primary and secondary wall is dissolved. The observations show that (a) a vascular bundle initiation and development proceeds in the affected primary leaves, in the absence of cell divisions.‘Vascular bundles' are formed consisting of a single row of cells, in which a-SE differentiation dominates. (b) The ‘sieve element differentiation’ is initiated without differential divisions. (c) Although the microtubules arc absent, the cells determined to become a-SEs activate another morphogenetic mechanism to control a more or less patterned deposition of a secondary wall layer.

SUMMARY Mature tulip plants were treated with 14CO2 in June after flower senescence, and the fate of 14C-labelled products followed at irregular intervals by fractionation, chromatography and autoradiography until the senescence of the above-ground parts and, later, following replanting, when reserves were mobilized for the growth of new shoots, new daughter bulbs and roots. Following feeding with 14CO2, sucrose was identified as the main mobile sugar which was transported down the vascular bundles of the shoot to the daughter bulbs where it accounted for 74% of the total plant radioactivity after 7 days. In the daughter bulb conversion occurred progressively with time to starch and non-starch ethanol-insoluble material. After replanting in mid-September, both shoots and roots showed high radioactivity (10% and 14% respectively of total activity, 60 days after replanting). Vascular tissues were strongly labelled indicating their role in the redistribution of carbohydrates which were mainly sucrose and fructose polymers in the daughter bulbs and sucrose and glucose in the growing organs.

SUMMARY Ornithogalum ovules are bitegmic: the inner is initiated first, but both attain maturity at the same time. Both integuments are derived from the epidermal layer of the nucellus and are covered by a cuticle. Inner integument cells define the micropyle and those which line it are secretory. An outer integument is present only on the distal (non-funicular) side of the ovule. Its proximal portion apparently is fused congenitally with the funiculus. A funiculus attaches the ovule (and seed) to placental tissue. Proliferation of cells at the base of the funiculus contributes to development of the obturator. Funicular curvature causes the ovules to assume an anatropous orientation. A procambial strand branches from a ventral vein of the carpels and differentiates acropetally in the funiculus. At ovule maturity, vascular tissue terminates in the chalaza a few cells removed from the hypostase. Terminal cells of the vascular bundle consist only of tracheary elements with annular thickenings.

Summary Helianthus internode segments were fitted with apical donor blocks containing [14C]IAA and [3H]sucrose, and plain agar basal receiver blocks. After transport, donors, tissue and receivers were oxidized in an ‘Oxymat’ and 14C and 3H counted separately. Ageing the segments before transport, or pretreatment with TIBA, greatly reduced the amount of 14C in the receivers, but had no effect on the 3H count. Profiles of 14C were determined after transport of [14 C]IAA through similar segments ringed to various depths, or with the pith interrupted. Interruption of the vascular bundles, but not of the pith, caused accumulation of I4C above the cut in segments transporting IAA basipetally; no accumulation was caused by ringing segments transporting IAA acropetally. It is concluded that active polar transport of IAA takes place in or near the phloem, but by a different mechanism from the one involved in sucrose transport through the phloem of intact plants.

SUMMARY The effect of Endogone infection on the stem anatomy and on the reproduction in tomato, maize, strawberry and petunia was examined. In tomato, petunia and maize plants, the infection increased the amount of vascular tissue. The lignification of the xylem was greater in mycorrhizal tomato and petunia plants and more vascular bundles were produced in the maize plants. Mycorrhizal infection stimulated flower production in petunia, the formation of fruit in strawberry and the development of pollen in maize.

Summary From reviews of the anatomy and cell contents of grasses it would appear that crystals of calcium compounds do not occur. However, 13 scattered reports of such crystals in grasses have been discovered in the literature. As a result of observations made by the author, calcium oxalate crystals have been found in bundle sheath cells and adjacent mesophyll in leaves of Melinis minutiflora (Panicoideae).

Summary Thin sections of the oldest uncrushed sieve elements in two vascular bundles have been examined by electron microscopy. They are shown to contain bundles of about 50 P-protein tubules which are no different in appearance from tubules in immature sieve elements. Tubules in these old sieve elements have remained interlinked with ‘side-arms’ throughout the time that the sieve elements were translocating. The accumulation of callose on each sieve element pore, which signifies senescence, may incidentally protect the tubules from changes in pressure whilst they are fixed. The generally held conclusion that P-protein tubules change into narrow striated filaments and disperse from their parietal positions as the sieve element reaches maturity is re-examined. In the development of the sieve element the central body of the P-protein crystal is shown to be present very early, at a stage when the sieve element and the companion cell are otherwise still difficult to distinguish.

SUMMARY The development of raphide-forming cells and the differentiation of calcium oxalate needles were investigated in young banana stems using fluorescence microscopy. By employing highly sensitive procedures for the detection of carbohydrates, proteins and RNA, six functional stages in raphide-cell ontogeny were defined: (1) the appression of cytoplasm to the cell wall in young idioblasts; (2) the synthesis of a polysaccharide matrix with early lacunae; (3) the formation in the lacunae of chambers, the envelope of which contains RNA and protein; (4) the synthesis of glycoproteins and polysaccharides in the chamber envelopes; (5) initiation of the formation of closely packed raphide bundles and the beginning of the crystallization of calcium oxalate and (6) new crystals are formed in a polysaccharide lattice produced in the mucilaginous bundle sheath. The results presented are compared with those of other investigations on the pattern of idioblast development and calcium oxalate deposition in higher plants.

Summary The variation in over 20 leaf characters including morphological, anatomical and cellular features have been examined in nine Triticum genotypes at three levels of ploidy. The characters included transectional leaf area, leaf width, distance between vascular bundles, volume occupied by each cell type, cell size and number, and chloroplast size and number. Analysis of these characters showed that the major components of the variation were related to the ploidy level and the genotype of the species and cultivars examined. The measured characters most strongly influenced by ploidy level were cell size, chloroplast number and leaf thickness. Genetic variation accounted for the variation in all other measured characters. The interaction of genetic variation with the major effects attributable to ploidy level creates a characteristic anatomical ‘fingerprint’ for each genotype. The observation that photosynthetic capacity is negatively correlated with the ratio of mesophyll cell size to nuclear genome size, and with no other character, is discussed.

Entry of water and subsequent germination of hard seeds of Acacia kempeana occurs after the strophiole lifts and cracks. The ‘opening’ of a strophiole is induced by heat and is effected through the splitting of thin-walled cells lying beneath short palisade cells within the strophiole. Estimated pressure increases of greater than 25 % within the vascular bundle, which passes directly under the thin-walled cells, might influence strophiolar lifting. The vascular bundle between the hilum and the adjacent strophiole is virtually devoid of xylern vessels, thereby preventing water entry through the hilum. However, following the strophiole, the vascular bundle obtains a maximum number of about 35 xylern vessels and is itself enclosed in this region by a noticeable group of over 150 thin-walled cells. Many of these thin-walled cells appear crushed along the route of the vascular bundle through the mesophyll layer to the end opposite the hilum. At the non-hilum end, the vascular bundle almost touches the internal side of the seed coat and thus entry of water would be facilitated through this zone to the internal endosperm and embryo.

The petals of Caltha palustris are unusual in that they contain chromoplasts belonging to two different ultrastructural classes. Other forms of plastid are also present and one of these provides the petal with a reflective starch layer. These different types of plastid and several distinctive features of petal morphology all point to a type of petal which is particularly well-adapted to encourage pollination of the flower by insects. chromoplast. It is common for plastids in adjacent cell layers of the same organ to differ from each other in structure and for these differences first to become apparent during very early stages of organ development. The developing chloroplasts in adjacent layers within the primary leaf of Phaseolus vulgaris L. and those within the green root of Azolla pinnata R. Br. both show such cell layer-related differences (Whatley, 1979; Whatley & Gunning, 1981). However, these layer-related variations in plastid ultrastructure are mainly minor and quantitative, as illustrated by small differences in plastid size and in the amounts of starch and thylakoid material present. More fundamental qualitative differences in plastid ultrastructure are typical of only a few specialized types of cell like the bundle sheath cells of some C4 plants and the sieve elements of seed plants (Whatley, 1983a). The petals of Caltha are unusual in that just such major qualitative differences in ultrastructure are shown by the plastids (including the two classes of chromoplast) in each of the three uppermost cell layers. In addition, a fourth type of plastid - a true chloroplast - is present in cells adjacent to the veins.

SUMMARY Illuminated leaf discs of Amaranthus paniculatus formed alanine rapidly after infiltration with aspartate. a-oxoglutarate and glutamate accelerated aspartate-dependent alanine production. Alanine was also formed by A. paniculatus leaf discs infiltrated with oxalacetate; this production was stimulated only by glutamate. Our results confirm that alanine can be formed rapidly from aspartate in C4 plants. Aspartate-dependent alanine production was stimulated greatly by light. The stimulatory effect of light could almost be replaced by ATP in presence of sodium ions. These findings are discussed in relation to a possible light-dependent active transport of amino acids into the bundle sheath, mediated by Na+-dependent ATPase activity.

Summary Vessel endings in various parts of the alfalfa (Medicago sativa L.) shoot were mapped by an India ink movement method. Stems were excised at various locations between the fifth and sixth nodes below the apex and placed in vials of India ink. Vessel endings were determined by examining the distance to which ink penetrated in the transpiration stream; the particles of India ink are too large to pass from vessel to vessel through pit membranes. The highest frequencies of vessel endings were found (1) in the petiolules and blades of the leaflets, (2) in the pulvinus of the petiole where the leaf traces emerge from the stem and anastomose while traversing the abscission zone, and (3) in the stem nodes where leaf traces arise and vascular bundles anastomose. The frequency of vessel endings was low and uniformly distributed throughout both internodes and petioles. Vessels were longest in the internodes and shortest in the leaflets. The regions where vessel endings were grouped coincided with those determined in other studies to be particularly susceptible to vascular blocking by macromolecules such as those produced by vascular wilt pathogens.

SUMMARY In Vicia faba, phloem fibres are shown not to take part in the active polar transport of auxin which takes place exclusively through the vascular bundles of young internode segments. Though transport of auxin through parenchyma/fibre strips does not seem to be active, it is slightly polarized in favour of the basipetal direction.

Summary Cavitation has been studied by acoustic detection in preserved and fresh vascular bundles and in whole leaves of Plantago and other plants. ‘Clicks’, indicating cavitation vibrations, were produced as vascular bundles lost water in a manner similar to that of Ricinus leaves (Milburn, 1973a), but the process was more rapid and freely suspended bundles jerked as they cavitated, like spore elator systems. Clicks were counted in a semi-quantitative study of both cavitation and the reverse process, the restoration of water-filled xylem conduits. Restoration was greatly enhanced by compression or vacuum infiltration treatments in comparison with simple soaking: apparently gas entrainment is the major obstacle preventing refilling. Cavitation within wilting leaves was much less extensive than when isolated bundles are dried, indicating that cavitation is generally incomplete under natural conditions. Xylem sap tensions at which cavitation is incipient were estimated approximately by balancing cavitation against the external humidity. Sap tensions at incipient cavitation could be measured more accurately using a pressure bomb, however. These tensions were surprisingly low, covering a range of tensions (5-15 bar) expected to occur under field conditions. Cavitation was detected in Plantago under natural field conditions and in other plants also, especially Tussilago, Further implications are discussed.

SUMMARY If a very young leaf blade of Coleus blumei Benth. is excised, the differentiation of primary xylem in the traces belonging to the treated leaf virtually ceases and all the cells in that half of the internode below the leaf are reduced in size. If the excised blade is replaced by auxin paste, differentiation of xylem proceeds almost normally and cell size is restored. Autoradiographs of sections of internodes treated apically with radioactive auxin paste indicate that after translocation auxin is heavily concentrated in the vascular bundles.

IN a set of six serial sections primarily designed to exhibit the structure of a cone of Sphenophyllum Dawsoni occur a number of foliar organs having such thick-walled cells that these organs form a conspicuous feature of the slides. Further study has borne out the early opinion that these foliar organs present certain features of novelty, and may clear up some points regarding the lepidodendroid cone, so an account of them has seemed worth putting on record. These slides were prepared by Mr James Lomax, who has furnished the following data: "Loc. Upper Foot Mine, Shore, Littleborough, Lancashire." The state of preservation of this material is for the most part admirable, even certain thin-walled cells of the vascular area being preserved in some of the sections. The light brown colour of the cell walls appears to indicate rapid penetration of the "petrifying" liquid. The general features are brought out in P1. III, figs. i and 2, which show the exceedingly simple shape of these objects. They are almost flat, although some sections indicate a slight concavo-convex shape, i.e. the scales were very slightly trough-shaped. No projecting midrib or lateral grooves have been found, but the organs were thickest at the middle, tapering gradually right and left to a very thin margin. Measurements of the thickness of nineteen scales were made at the region of the single central vascular bundle. The thinnest scale measured o045 mm. and the thickest o go mm., with an average of o-67 mm. Sections extending from midregion to edge of a scale were scarce, but seven half-scales were measured, the minimum width being 9-I8 mm., the maximum 205 mm., and average I3-07 mm., all being calculated for width of a whole scale. The length of a slightly slanting longitudinal section (broken off at each end) was 32 mm. The similarity of the various transverse sections indicates that the scales were lanceolate, and the dimensions clearly show that they were not acicular. The plan of the scale as seen in transverse section (P1. III, fig. 2) shows a single central bundle, and at a short distance right and left

The morphological nature of the staminate flower of Cucurbitaceae was a subject of longstanding discussion, and unfortunately its exact nature has not yet been clearly understood. Stamens in the Cucurbitaceae have been described usually as two and a half, or three, or five (see references). The difficulty of comprehension of the monothecal type of the anther unusual in flowering plants involved arguments for considering the double thecal stamens (Clarke, I879) as complete and the odd one with one theca as a half-stamenone theca in the latter case being considered not developed. In order to determine the real significance of this interesting structure, I have undertaken a thorough morphological study of the staminate flowers of Lagenaria leucantha (Duch.) Rusby (L. vulgaris Ser.) along with other species. The plants were raised at the glasshouse of the Cambridge University Botanic Garden from seeds procured from India and other sources. Seeds were sown in late May or early June, and flowering started from the beginning of September. Serial microtome sections of the male flower buds of different ages were cut at I0-I 5 /t thickness. The materials were fixed and killed in Belling's modification of Navashin's fixative (Lee, I937). Light green in equal parts of xylol and clove oil or fast green dissolved in clove oil and safranin dissolved in 50 % alcohol were found suitable combination-stains for distinguishing lignified from cellulose tissues. Freehand sections of mature flower buds or flowers were employed whenever necessary. For determining the courses of vascular strands from the pedicel to the floral members thick longisections of flower buds or semi-open flowers were cut by razor, boiled in pure lactic acid for about half an hour and mounted in lactic acid in grooved slides and sealed in gold-size. Illustrations were drawn under microprojection in a dark chamber. In following the courses of vascular bundles from the flower stalk to their entrance to different floral members we start with I2-I4 vascular bundles in two rings, one set of bundles being opposite the ridges of the pedicel and the other set opposite the furrows. But before entering the receptacle (fused calyx and corolla) ten bundles, five in the outer and five in the inner circle, are noticed. At the base of the receptacle and below the threelobed glandular pistillodium, these inner and outer vascular bundles develop branches and form more or less a ring of vascular tissue from the inner edges of which smaller and weaker branches develop and pass centrally to the pistillodium (Fig. i). The vascular bundles of the pedicel meet at the base of the receptacle and form a sort of circular ring (siphonostele). From the outside of this ring fifteen traces in five groups (i.e. three traces in a group) pass to the sepals. A group of three traces passes to each sepal, the middle trace forming the sepal main and the two side ones constituting the sepal laterals. At

Summary By sequential visual and photographic recording, the courses of the initiation of bending and the subsequent rates of curvature of the petiole and stem have been followed when a growth regulator is applied to (a) the leaf surface above the midrib of one primary leaf, (b) a partially excised short tongue of the midrib, and (c) the cotyledonary node at the junction of a cotyledon. In a comparison of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) it was found that though the times taken to initiate bending are greatly dependent on the site of application, there are no significant differences between the two compounds. On the other hand, for indole-3-acetic acid (IAA), the initiation times are shorter and the equi-effective doses are much smaller. Once bending has been initiated, for 2,4-D and 2,4,5-T the degree of curvature increases progressively, whereas for IAA the initial positive rate eventually becomes negative. Using 2,4-D and 2,4,5-T, labelled with carbon-14 in the carboxyl group, autoradiographs demonstrated that entry into the vascular strands via the tongue of midrib take place in about 5 minutes, while for applications to the cotyledonary node radioactivity is present in the tip of the primary leaf within the same time. On the basis of these and other observations, it is concluded that under the controlled conditions selected, the initiation time for the bending of the stem following an application of either 2,4-D or 2,4,5-T to the leaf surface I cm from the tip can be broken down into (a) 79 minutes for enough of the auxin to pass from the leaf surface to the underlying vascular bundles, (b) 61 minutes to reach the first internode, and (c) 24 minutes to induce curvature. For IAA the corresponding figures for (b) and (c) are 31 and 17 minutes. The velocity of downward transport in the phloem is estimated at 10–12 cm/hour for 2,4-D and 2,4,5-T and 20–24 cm/hour for IAA. From differences in the initiation times for applications of 2,4-D to the tip and base of the leaf the velocity was estimated to be 14–25 cm/hour. Simultaneously with observations on the course of bending, the amounts of [14C]2,4-D and [14C]2,4,5-T. present in the tissues were followed. When bending is initiated the petiole and first internode contain 0.0016 and 0.008 μg respectively. The distribution of 2,4-D applied to one of the primary leaves was examined for periods up to 27 hours. Penetration into the leaf and subsequent export from the leaf are greatest when the application is made to the tongue of midrib, less for an application to the lower leaf surface and least for the upper surface. The pattern of accumulation is dependent on the amount exported. In general, accumulation is highest in the first internode and upper hypocotyl. At the end of 27 hours no 2,4-D is yet present in the opposite leaf.

The use of lactophenol in conjunction with cotton blue staining, first described by Klebahn (I9g0), for demonstrating the presence of fungal hyphae in infected plant material is now a well-known technique, and there must be many mycologists who have noticed that under the clearing action of lactophenol plant tissues become almost transparent except for lignified xylem elements. It was this observation which led the writer, in 1939, to develop a rapid clearing technique, using pure lactic acid (B.P.) instead of lactophenol, for the morphological study of a wide range of botanical material. A method of clearing by means of lactic acid, which was similar to this in some respects, was described by Simpson (1929) and has since become widely used in America, though it does not appear to be well known in England. The technique described here was designed primarily for examining the internal vascular structure of the flower and has subsequently been found most useful for examining the arrangement of the sporangia within the sori of ferns; it has also been used to demonstrate the vascular bundles within the fertile spikes of members of the Ophioglossaceae and to show the arrangement of the sporangia within the cones of species of Selaginella. The principal advantages of the technique lie in its rapidity, in the fact that no staining is required, that it may be applied equally to fresh material, spirit material and dried herbarium specimens, and that preparations may be moflnted permanently in the clearing fluid. For studying the vascular structure of large or medium-sized flowers, longitudinal and transverse hand-cut slices of tissue up to 2 mm. in thickness may be used, while small flowers can often be examined whole, and for studying the. contents of a fern sorus it is sufficient to use a portion of the frond without any preliminary dissection. Satisfactory results have been obtained with dried herbarium material, both of ferns and of angiospermous flowers, after soaking out by means of the modified Juel technique described by Tillson & Bamford (I938). Before proceeding 'with the clearing process, it is necessary first to decolorize the tissues. Thus, if fresh material is used, all chlorophyll must be removed. Spirit material, though free from chlorophyll, is often coloured brown, and herbarium material usually contains a similar brown coloration. This can usually be removed by treatment with alkaline hydrogen peroxide (':2o vols.'), though the process may take 24 hr. or more. The clearing process consists of heating the material for a few minutes in pure lactic acid (B.P.) kept at ioo0 C. in a boiling water-bath. The preparation can then be examined in a watch-glass under a binocular microscope, and any dissection which may be required can then be carried out. Examination at this stage will also reveal any air bubbles which there may be within the tissues, and which must be removed under a vacuum pump before the preparation is mounted permanently.