Epi-illumination Light Microscopy Research Articles

Live imaging of developmental processes in a living meristem of Davidia involucrata (Nyssaceae).

Morphogenesis in plants is usually reconstructed by scanning electron microscopy and histology of meristematic structures. These techniques are destructive and require many samples to obtain a consecutive series of states. Unfortunately, using this methodology the absolute timing of growth and complete relative initiation of organs remain obscure. To overcome this limitation, an in vivo observational method based on Epi-Illumination Light Microscopy (ELM) was developed and tested with a male inflorescence meristem (floral unit) of the handkerchief tree Davidia involucrata Baill. (Nyssaceae). We asked whether the most basal flowers of this floral unit arise in a basipetal sequence or, alternatively, are delayed in their development. The growing meristem was observed for 30 days, the longest live observation of a meristem achieved to date. The sequence of primordium initiation indicates a later initiation of the most basal flowers and not earlier or simultaneously as SEM images could suggest. D. involucrata exemplarily shows that live-ELM gives new insights into developmental processes of plants. In addition to morphogenetic questions such as the transition from vegetative to reproductive meristems or the absolute timing of ontogenetic processes, this method may also help to quantify cellular growth processes in the context of molecular physiology and developmental genetics studies.

Floral development in Scutellaria pinnatifida (Lamiaceae): the ontogenetic basis for sepal reduction.

The Scutellaria is a Labiatae genus (subfamily Scutellarioideae) with a highly specialised floral structure. The genus is characterised by a peculiar two-lobed calyx with a projecting appendage, named the scutellum. Here, we present a detailed analysis of floral development, using epi-illumination light microscopy, to clarify open questions about its floral organisation. Floral whorls appeared in an acropetal sequence, with a marked temporal overlap of petal and stamen appearance. Organ appearance in each whorl proceeded unidirectionally from the abaxial to the adaxial side. Significant developmental features included the formation of the scutellum, reduction of sepal lobes and formation of a three-lobed nectary disc. Our study revealed that both loss of organ initiation and fusion of primordia are responsible for the reduction in sepal members in Scutellaria. The nectary structure was markedly different from most other studied Lamiaceae.

Comparative floral ontogeny of single-flowered and double-flowered phenotypes of Alcea rosea (Malvaceae)

A comparative study of floral ontogeny in single- and double-flowered Alcea rosea L. was conducted using epi-illumination light microscopy. In both floral types, floral differentiation starts with the appearance of three epicalyx lobes, which subsequently subdivide to produce a 7–10-parted epicalyx. Five sepals appear then in a unidirectional or possibly spiral sequence. In single flowers, a corolla-androecium common primordium is formed and subsequently differentiated into five androecial sectors (= primary androecial primordia). Petals are developed at the base of the androecial sectors and secondary androecial primordia are initiated centrifugally in two rows on each sector. Later, tertiary androecial primordia are formed by the subdivision of secondary androecial primordia, which then differentiate into androecial units. Three types of double flowers were identified regarding androecial development. The first type of double flowers shows a more or less disorganised nature. However, 10 proliferation zones can be indentified in the proximal and distal tips of the androecial sectors. In the second and third types of double flowers, androecial development follows similar developmental pathways to that of single flowers. However, in second-type double flowers, the secondary androecial primordia differentiate into petals and the stamens then develop from the free space between the two rows of secondary androecial primordia. In third-type double flowers, after complete primordial partitioning, some primordia on the marginal parts of each androecial sector develop into petaloids or intermediate appendages. The gynoecium appears similarly in both floral types as numerous congenitally united carpel primordia. The double-flowered phenotypes of Alcea appear to fit the criteria for homoheterotopy with complete or partial replacement of stamens with petals, as well as for neoheterotopy, with the formation of stamens in a new position. Based on mutant phenotypes, it is suggested that different functions possibly contribute to the proliferation and differentiation of common primordia.

Comparative floral development in Lamioideae (Lamiaceae): Marrubium, Phlomis, and Stachys

A comparative study of floral ontogeny and development was carried out on three genera (Marrubium L., Phlomis L., Stachys L.), representing three tribes of Lamioideae (Marrubieae, Phlomideae, Stachydeae) using epi-illumination light microscopy. The sequence of organ whorl appearance in all three genera is sepals, petals plus stamens, and carpels. Sepal appearance is reversed unidirectional starting from the adaxial side in all except Phlomis, which is unidirectional. Order of petal appearance is bidirectional in Marrubium and Stachys, and simultaneous in Phlomis. Stamens appear unidirectionally starting from the adaxial side in all except in Phlomis, which has an abaxial to adaxial unidirectional sequence. Significant developmental features distinguishing the three genera from each other include (1) weakly monosymmetric, elongated calyx tube, five-lobed corolla, divergent anthers with thecae transverse to the filament, unequally bifid stigma and ovary with glandular hairs in Marrubium; (2) actinomorphic hairy calyx, four-lobed corolla and unequally bifid stigma in Phlomis; and (3) glabrous calyx, equally bifid stigma and symmetric disc nectary in Stachys. Our results indicate some potential for floral ontogenetic features in delimiting the different tribes. The hypothetical evolutionary pathway of organogenesis sequences is discussed.

Comparative inflorescence and floral ontogeny in the genus Mentha (Mentheae: Nepetoideae: Lamiaceae): variable sequences of organ appearance and random petal aestivation

The genus Mentha is a taxonomically complex genus, characterized by large morphological variations. Only a few, frequently overlapping, characters are of value in taxonomy. Comparative floral developmental studies provide an opportunity for better understanding the systematic relationships among different species. The inflorescence and floral ontogeny of three Mentha L. species (M. piperita L., M. pulegium L. and M. suaveolens Ehrh.) were investigated using epi-illumination light microscopy. All three species studied have thyrses with the same developmental pathway. The lack of higher order bracts and the monochasial branching of the higher order inflorescence apices were found as special features of inflorescence ontogeny. Sepals appear unidirectionally from the adaxial side in all except for M. pulegium which shows a modified unidirectional sequence. Variable sequences of petal and stamen appearance from unidirectional to reversed unidirectional sequence are present in all. Significant ontogenetic features include (1) appearance of the corolla as a rim before petal lobes become visible and (2) instability in petal aestivation. Morphological features including densely hairy calyx, five-lobed corolla tube, smaller adaxial stamens and hairy ovary with included style distinguish M. pulegium from the other species. On the basis of our results floral ontogenetic features could be considered important characters for delimiting or diagnosing different sections in the genus Mentha. Variable sequences of petal lobe appearance and instability in petal aestivation were found as unusual developmental characters.

Inflorescence and floral ontogeny in Crocus sativus L. (Iridaceae)

Inflorescence and floral ontogeny of the perennial, herbaceous crop Crocus sativus L. were studied using epi-illumination light microscopy. After production of leaves with helical arrangement a determinate inflorescence forms which becomes completely transformed into a single terminal flower. In some cases, bifurcation of the inflorescence meristem yields two or three floral meristems. The order of floral organs initiation is outer tepals – stamens – inner tepals – carpels. Stamens and outer tepals are produced from the lateral bifurcation of three common stamen-tepal primordia. Within each whorl, organs start developing unidirectionally from the adaxial side, except for the stamens which begin to grow from the abaxial side. Specialized features during organ development include interprimordial growth between tepals forming a perianth tube, fusion at the base of stamen filaments, and formation of an inferior ovary with unfused styles.

Floral ontogeny in Astragalus compactus (Leguminosae: Papilionoideae: Galegeae): variable occurrence of bracteoles and variable patterns of sepal initiation

Comparative studies of floral ontogeny represent a growing field that promise to provide new insights on floral evolution. Floral ontogenetic information has been used successfully in Leguminosae for re-examining phylogenetic relationships at different levels. Using epi-illumination light microscopy, we present original ontogenetic data in Astragalus compactus, which was chosen because of its unusual arrangement of inflorescence and variable occurrence of bracteoles on flowers. Based on our results, uncommon ontogeny of the inflorescence led to the arrangement of flowers in four different positions. Variation was observed not only in the presence of bracteoles, but also in the order of sepal initiation in flowers of the same inflorescence. Surprisingly, besides the widely stated unidirectional pattern, bidirectional, sequential and an atypical unreported order were observed. High degree of overlapping between whorls and formation of two types of common primordia also were found. The variable occurrence of bracteoles suggests that the species is in an intermediate state towards fully lacking of bracteoles. We propose that the variability of the sequence of sepal initiation is possibly a consequence of the function of mechanical forces generated by surrounding leaves. Relationships between mechanical force and auxin signalling are discussed.

Inflorescence and floral ontogeny inOsteospermum ecklonis(Asteraceae)

Development of the capitulum inflorescence with different types of florets in Asteraceae is an interesting issue in the field of plant evolution and development. In this study, ontogeny of the inflorescence and florets of Osteospermum ecklonis (DC.) Norl., an ornamental and evergreen subshrub, was investigated using epi-illumination light microscopy. The initiation and subsequent development of florets on the highly convex inflorescence apex occurred acropetally, except for the ray florets, which showed a lag in initiation. Organogenesis in disc florets started with unidirectional initiation of corolla lobes from the adaxial side and then proceeded by simultaneous appearance of five stamen and finally two median carpel primordia. Significant developmental features included the lack of pappus differentiation, formation of nonsyngenesious stamens, and formation of the ovule-less ovary. Ray florets showed significant differences from disc florets as reflected by the zygomorphic shape of floral apex and shift of floral merosithy from pentamery to tetramery. Also, expansion of corolla lobes to form the ligule and the formation of staminodia were observed. It is hypothesized that the actinomorphic pentamerous disc florets are most primitive among the family from which the tetramerous ray florets are derived. Accordingly, ray florets evolved from disc florets under long-term selective pressure and play a crucial role in enhancing reproductive success.

Comparison of floral ontogeny in wild-type and double-flowered phenotypes of Syringa vulgaris L. (Oleaceae)

A comparative study of floral ontogeny in normal and double-flowered phenotype of Syringa vulgaris was conducted using the epi-illumination light microscopy. In the wild phenotype, floral differentiation starts with calyx inception and the formation of four sepals in orthogonal positions (two median and two lateral). The corolla emerges as a continuous ring-like structure leading to the appearance of four petal lobes alternating with the sepals. Androecium was formed by initiation of two stamen primordia in transverse plane and finally the bicarpellate gynoecium emerges in median position. In the case of the double-flowered lilac, there are supernumerary petals in an additional whorl. In double-flowered phenotype, a ring meristem is formed acropetally after the first petal whorl initiates. Stamens and carpels initiated similarly in double-flowered as well as in wild-type plants. However, position of stamens deviates from the typical transverse situation. It seems that the appearing of an extra petal whorl did not exhibit any adverse effect on the initiation of other whorls, in terms of organ identity. Therefore, it would be suggested that the double-flowered phenotype of Syringa represents a case of neoheterotopy, with formation of an extra petal whorl, rather than a case of homoheterotopy with transformation of an ancestral androecium whorl into petals.

Inflorescence and floral ontogeny in Jasminum fruticans (Oleaceae)

The inflorescence and floral ontogeny of the oleaceous tropical bush, Jasminum fruticans L. were studied using epi-illumination light microscopy and compared with other Oleaceae. The flowers are arranged in a thyrsoid inflorescence with a determinate pattern of development. Deviations from the common pathway of floral ontogeny in Oleaceae observed in J. fruticans are characterised by pentamery in calyx and corolla, diagonal position of sepals, some deviation from acropetal and simultaneous initiation of floral organs. Sepals are helically initiated, beginning with the abaxial sepal. Formation of petal lobes from the ring meristem also is not simultaneous with the initiation of three petals before the others. Concurrently with the formation of corolla ring, two stamens are initiated sequentially. Two median carpels are the last organs to be initiated. Unusual features of organ enlargement include the quincuncial-imbricate aestivation of the corolla and formation of trichomes on the distal portion of petals and stamens.

Inflorescence and floral development in Astragalus lagopoides Lam. (Leguminosae: Papilionoideae: Galegeae)

Inflorescence and floral organogenesis and development of the bushy perennial legume Astragalus lagopoides of the section Hymenostegis were studied by means of epi-illumination light microscopy. Based on our observations, the primordia of lanceolate racemose inflorescences are born in the axils of leaves. Each inflorescence apex initiates acropetally bracts and floral apices for some time and then eventually ceases meristematic activity and forms an oblong-shaped terminal structure. The formation of such atypical terminal protrusion on the inflorescence meristem is judged to be a diagnostic feature for well-organized cessation of meristem morphogenesis. Pentamerous perfect flowers of the plant show strong zygomorphy and marked overlap in time of initiation among different organ primordia. Unexpectedly, sepal initiation is bidirectional starting from the lateral sides of the floral apex. Other significant developmental feature includes the existence of two types of common primordia, which are formed successively. From the primary common primordia there are produced antesepalous stamens and secondary common primordia. In comparison, the five secondary common primordia subdivide into a petal and an antepetalous stamen primordia. Initiation of two different types of common primordia is possibly the result of rising overlap in time of initiation of organs and demonstrates an advanced developmental style in the genus Astragalus.

Floral development in Astragalus caspicus Bieb. (Leguminosae: Papilionoideae: Galegeae)

Floral organogenesis and development of the bushy perennial legume Astragalus caspicus were studied using epi-illumination light microscopy techniques. Based on our observations, flowers are in axillary two-flowered racemes, initiate all 21 floral organs and show precocious appearance of zygomorphy. The order of floral organ initiation is unidirectional in whorls starting from the abaxial position of the flower with a high degree of overlap. Another important ontogenetic feature is the existence of two successive common primordial stages categorized as primary and secondary. The primary common primordia produce antesepalous stamens and secondary common primordia. In contrast, the five secondary common primordia subdivide into a petal and an antepetalous stamen primordia. Our findings on floral ontogeny of A. caspicus provide new evidence for the complex and variable floral initiation and development in legumes. The floral apex with strong overlapping initiation of different organs illustrates a paradox in which different capabilities must be presumed to exist simultaneously. Moreover, two extraordinary types of common primordia represent possibly an advanced evolutionary trend where time intervals between the initiations of different floral organs in Papilionoideae are shortened.

Early floral development ofHeliconia latispatha(Heliconiaceae), a key taxon for understanding the evolution of flower development in the Zingiberales

We present new comparative data on early floral development of Heliconia latispatha, an ecologically and horticulturally important tropical plant within the order Zingiberales. Modification of the six members of two androecial whorls is characteristic of Zingiberales, with a reduction in number of fertile stamen from five or six in the banana families (Musaceae, Strelitziaceae, Lowiaceae, and Heliconiaceae) to one in Costaceae and Zingiberaceae and one-half in Marantaceae and Cannaceae. The remaining five infertile stamens in these later four families (the ginger families) are petaloid, and in Costaceae and Zingiberaceae fuse together to form a novel structure, the labellum. Within this developmental sequence, Heliconiaceae share with the ginger families the possession of an antisepalous staminode, a synapomorphy that has been used to place Heliconiaceae as sister to the ginger family clade. Here, we use epi-illumination light microscopy and reconstruction of serial sections to investigate the ontogeny of the Heliconia flower with emphasis on the ontogeny of the staminode. We compare floral development in Heliconia with that previously described for other species of Zingiberales. A comparison of floral structure and development across Zingiberales is presented to better understand the evolution of the flower in this charismatic group of tropical plants.

Application of Epi-Illumination Light Microscopy for Study of Floral Ontogeny in Fruit Trees

Application of Epi-Illumination Light Microscopy for Study of Floral Ontogeny in Fruit Trees

Morphological and anatomical development in the Vitaceae. X. Comparative ontogeny and phylogenetic implications ofCissus quadrangularisL.

Although the grape family, Vitaceae, is of great economic importance, there is little understanding of the relationships between and within the genera. Recent DNA-based phylogenies of the Vitaceae have disagreed with traditional classifications by grouping some Cissus species (e.g., Cissus antarctica Vent.) in a clade with Vitis species, while placing other Cissus species (e.g., Cissus quadrangularis L.) in a monophyletic clade. This study investigates Cissus phylogeny using epi-illumination light microscopy and histological ontogenetic comparisons of vegetative and reproductive characters of Vitis rotundifolia (Forssk.) Vahl, Vitis ‘Ventura’, Vitis riparia Michx., C. antarctica, and C. quadrangularis. Ten characters limited to the Cissus species (connection of stipules, precocious axillary bud initiation, prophyll growth not obviously unequal, timing of mature shoot architecture on axillary buds, lack of spiral inflorescence branching, absence of calyptra, petals closely enveloping anthers, large gynoecial disk, ovary buried in the disk, and production of copious amounts of nectar) support the traditional phylogenies: the monophyly of Vitis and the exclusion of Cissus species from Vitis. The comparison of C. antarctica (Vitis–Cissus clade) and C. quadrangularis (monophyletic Cissus clade) supports the division of Cissus into two clades based on supernumerary buds, stipule connectivity, uncommitted primordium shape, and inflorescence branching patterns.

Morphological and anatomical development in the Vitaceae. IX. Comparative ontogeny and phylogenetic implications ofVitis rotundifoliaMichx.

The grape family, Vitaceae, is an economically important fruit family. Although the Vitaceae have been used for conservation, viticulture, and horticulture, little is known about the phylogenetic relationships of this family. Recent phylogenetic reconstructions using DNA data have disagreed with traditional classifications and other DNA-based phylogenies by grouping Vitis and Cissus species within one clade and linking Vitis rotundifolia Michx. (muscadine grape) more closely with these Cissus species. This study uses comparative developmental morphology as an independent method to investigate whether V. rotundifolia is more similar to Cissus antarctica Vent. (kangaroo vine) than other Vitis species. Results obtained using epi-illumination light microscopy and histology were compared with those from similar studies of Vitis ‘Ventura’, Vitis riparia Michx., and C. antarctica. Twelve vegetative and floral characters including axillary bud dorsiventrality, uncommitted primordium shape, calyptra, and ring-shaped gynoecial disk placed V. rotundifolia in with other Vitis species. Only two characters, stipule timing and simple tendrils, were common to C. antarctica and V. rotundifolia, suggesting that V. rotundifolia is more similar to Vitis spp. than to C. antarctica, and supporting traditional classifications contrary to ITS1 and trnL DNA phylogenies.

Morphological and anatomical development in the Vitaceae. VIII. Comparative development of three Cyphostemma (Vitaceae) species reveals important vegetative and reproductive differences among the species

Vegetative and floral development of Cyphostemma simulans (C.A. Small) Wild & Drummond, Cyphostemma juttae (Dinter & Gilg) Descoings, and Cyphostemma mappia (Lam) Galet were compared using epi-illumination light microscopy and paraffin sectioning. Cyphostemma simulans is a liana with leaf-opposed tendrils and inflorescences, which both arise from the shoot apical meristem, whereas the other two species are shrubs that lack tendrils and their main shoot terminates in an inflorescence. A shoot apical meristem of C. simulans can be overtopped by an inflorescence, making the architecture appear to develop like a sympodium, which is the architecture shared by the other two species. On the other hand, a terminal inflorescence of C. juttae or C. mappia can be placed in a leaf-opposed position by a precocious axillary branch, making the architecture appear to develop like a monopodium, which is the typical architecture of the Vitaceae. The floral development of all three species is similar in early ontogeny, except for a difference in calyx development between C. mappia and the other two species. Later developmental differences of the septum and gynoecium, in addition to flower colour and phenology, are distinct characteristics that distinguish flowers of each species. Therefore, this study is the first to demonstrate differences in the floral characteristics of Cyphostemma. In addition, it shows how each architecture type (monopodium or sympodium) can resemble the other and so provides stronger evidence about how the unique architecture of the Vitaceae has originated.

Morphological and anatomical development in the Vitaceae. VII. Floral development in Rhoicissus digitata with respect to other genera in the family

This study forms part of our series of investigations on genera in the Vitaceae and is the first developmental study for the genus Rhoicissus. Vegetative and reproductive development of shoot apices of Rhoicissus digitata (L.f.) Gilg et Brandt were examined using epi-illumination light microscopy and scanning electron microscopy. Leaf-opposed tendrils or inflorescences, typical of the shoot architecture in the Vitaceae, were present at every node. Macroscopically, the shoot appears to grow either monopodially or sympodially. At the microscopic level, however, shoot development is sympodial; the shoot apical meristem bifurcates unequally, with the larger portion forming an uncommitted primordium, which will become either an inflorescence or a tendril, and the smaller portion (in the position of the axillary bud) forming the new shoot apical meristem. Floral primordia first initiate three sepals followed by a calyx ring on which the last two sepal primordia form. The five petals are initiated in a whorl followed by the five stamens in a petal-opposed position. There is no evidence of a common petal-stamen primordium in this species. The gy noecium is initiated as a ring primordium. Subsequently, the four ovules are initiated at the base of the two septa that grow out from the inner gynoecial wall. The nectary disc forms as an outgrowth of the gynoecium base. Mature flowers have greenish petals and a red nectariferous disc. Flowers are bisexual, and seed germination is approximately 63%. Unlike previous studies in Vitis and Parthenocissus, Rhoicissus appears to have few putatively derived floral developmental characters, which would support its relatively basal position in current phylogenies for the family.Key words: Vitaceae, morphology, development, shoot architecture, flower.

Thorn and hook ontogeny in Artabotrys hexapetalus (Annonaceae)

Artabotrys hexapetalus is widely planted in the tropics and is known as "climbing ylang-ylang," an ornamental liana or woody climber. New natural sprouts, or water shoots, and those induced by the damage of Hurricane Andrew (24 August 1992) were collected and fixed in formalin/acidic acid/alcohol. Seeds from these plants were planted and grown in a greenhouse where seedling morphology was observed and young material collected and fixed. The development of lateral plagiotropic and orthotropic shoots was studied using both epi-illumination light microscopy and scanning electron microscopy. A series of buds develops in the axils of leaves on the orthotropic shoot. At the lateral margins of the axillary shelf, plagiotropic shoots form that will develop into either vegetative shoots, or thorns, or sympodial shoots that bear hooks and flowers. In between the two marginal buds, a series of median vertical buds develop that either remain dormant or grow out as renewal orthotropic shoots. Previous work that suggested that the plagiotropic shoot buds were displaced out of the median vertical series of supernumerary buds is not supported. The sympodial development of plagiotropic branches as inflorescence hooks is documented.

A new approach to the study of apical meristem development using laser scanning confocal microscopy

Epi-illumination light microscopy and scanning electron microscopy have been standard techniques for developmental studies of shoot apices. Recently, laser scanning confocal microscopy has gained popularity as a tool for biological imaging. We have adapted laser scanning confocal microscopy to study development in whole shoot apices. It was tested on angiosperm and fern apices using three fluorescent dyes; acriflavine, safranin O, and acid fuchsin, and compared with epi-illumination light microscopy and scanning electron microscopy. In all cases, acid fuchsin proved to be the best fluorochrome for examining shoot apices; having a high affinity for cell walls and nuclear material. The images produced with laser scanning confocal microscopy were sharper and clearer than images generated with epi-illumination light microscopy and scanning electron microscopy. Laser scanning confocal microscopy allows one to map patterns of cell division on the surface of an apical meristem, which is extremely difficult using other techniques such as scanning electron microscopy or epi-illumination light microscopy. Since the laser scanning light microscope records images digitally a method for digital plate production is described. Our methods can easily be applied to study the development of other plant structures on a cellular level such as root apical meristems, floral meristems, stomata, or trichomes, and reproductive organs in lower plants.Key words: confocal microscopy, apical meristem, development, fluorochrome, cytokinesis.