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Introduction
Flowering is an attractive system for the study of development in plants.
The complex process of flowering comes about through a major change in the life
history of the plants: a switch from indeterminate growth to development of
reproductive structures (Poethig, 1990). The process
of flowering typify all aspects of plant organogenesis, i.e., differentiate
cell division, cellular differentiation and alterations in gene expression (Mizukami
and Ma, 1992). It also provides an excellent experimental system for the
study of environmental and internal control of development. Flowers, consisting
of genetically identical sets of organs, and the possibility of environmental
manipulations of the process, offer a greater degree of flexibility in the experiments
(Meyer, 1966). The process is also of great practical
importance to the mankind, since agriculture is based on the control of flowering
and the resultant fruits and seeds (Drews and Goldberg,
1989).
Over the past two decades, flower development has attracted widespread attention
as an excellent model system for studying organogenesis in plants at molecular
level (Smyth et al., 1990). Several genes have
been identified through genetic analysis that control floral organ formation
(Wellmer et al., 2006). The sequence of events
during floral morphogenesis has been described in detail for many species including
Arabidopsis thaliana (Muller, 1961; Hill
and Lord, 1989; Smyth, 2005) Brassica napus
(Polowick and Sawhney, 1986), Lycopersicon esculentum
(Sekhar and Sawhney, 1987) and Nicotiana tabacum
(Hicks and Sussex, 1970). The genetic basis for the
control of flowering is not well established in all systems. However, the discovery
of modern gene identification, isolation and cloning techniques have resulted
in a renewed interest in the study of flower development. Scanning electron
microscopy (SEM) provides a 3-D image as well as improved resolution at higher
magnifications which allows a detailed analysis of developmental patterns in
early primordial development (Veit, 2006).
Material and Methods
SEM is used extensively to study ontogeny of floral organs (Polowick
and Sawhney, 1986). The samples were fixed in freshly prepared formalin-acetic
acid-ethanol system for 2 hours in order to preserve the structural features
of tissues for SEM preparation,. The fixative contained 3.7% (v/v) formaldehyde,
50% (v/v) ethyl alcohol and 5% (v/v) acetic acid. Fixation was followed by dehydration
with ethanol concentrations/grading of 30%, 45%, 70%, 90% and 100% v/v. The
samples were allowed to stay in each grade of ethanol for at least 4 minutes.
Dehydration was further continued in a graded series of acetone, using the same
dilutions and time as used for ethanol. Dehydrated samples were stored in 100%
acetone if required.
The dehydrated samples were dried in a Polaron E-2000 critical point drying
apparatus, using CO2 as the exchange medium. The dried samples were
mounted onto stubs, using a double-sided adhesive tape. Edges of the tape were
painted with silver solution. The silver streak was brought directly in contact
with the sample at a point for efficient electrical conductivity. The stubs
were then placed in a gold-palladium sputter coater (Polaron E-5000). Gold plating
was done up to a thickness of 20 nm at 40 mA for 1 minute using argon gas. Observations
were made using a JSM-840 scanning electron microscope with a fitted camera.
Acceleration voltage used during the observations was 10 KV.
Results and Discussion
Floral morphology
Flowers in N. rustica are arranged in a loose panicle (compound raceme),
which grows in an indeterminate fashion (Fig. 1). Lower flowers
may be solitary in leaf axils. Individual flowers (Fig. 2)
are generally large and showy. Five sepals, fused into a tube constitute the
calyx which is ¾ to 1.0 inches in length. The calyx tube extends to about three
quarters the length of the corolla tube and possesses 5 distinct lobes. Five
petals, which are interior and alternate to the sepals, are connately fused
along most of their length. The corolla limb is slaver-shaped with distinct
lobes. The corolla tube is funnel-shaped, more or less evenly expanding and
yellow in colour. Five stamens are adnately fused to the bases of the petals,
with the anthers inserted and situated either below, level with or above the
stigma. The carpels have a two-celled superior ovary possessing a long slender
style and a blunt bi-lobed stigma. Generally, in angiosperms, the ovule is located
within the pistil, which consists of one or several fused carpels (Favaro
et al., 2003). Inflorescence of N. rustica is shown Fig.
3 (not discussed a,b and c parts).
Floral Organogenesis
Review of literature shows that details about floral differentiation for
the amphiploid species N. tabacum are available (Mandel
et al., 1992, Koltunow et al., 1990).
However, detailed floral organogenesis maps for N. rustica have not been
reported, though floral homeotic mutants have been reported for this species.
SEM analysis of the initiation of flowers in N. rustica showed that
the earliest sign of a shift from the vegetative growth to flower development
was marked by a change in the surface of the apical bud from a slightly flat
structure to a dome shape structure. Floral buds arose sequentially on the inflorescence
apex in a phyllotactic spiral pattern (Fig. 1). A key event
during early flower development is the specification of different types of floral
organs (Wellmer et al., 2006). In N. rustica,
during the development of individual flowers on the inflorescence, the bracts
became progressively distinct from the flower primordia. Further morphological
differentiation of the flower primordium occurred in a typical acropetal sequence
of sepals, petals, stamen and carpels in a quick succession. Organ primordia
in the first three whorls occupied alternate positions with respect to the organ
primordia in adjacent whorls. Sepals and petal primordia showed a connate fusion
soon after initiation, while stamens became adnately fused to the petals during
development. Veit (2006) had reported that the organ
primordia were initiated by the peripheral zone of the meristem, while the central
zone remained undifferentiated to allow organogenesis to continue indefinitely.
However, in case of N. rustica floral development, there is a switch
to determinate growth of flower formation; therefore, the peripheral as well
as central zones were observed to be involved in organ development. Major distinguishable
features of floral organogenesis which appeared sequentially in N. rustica
are described below and are shown in Fig. 4-A to 4-H:
| i) |
A single sepal primordium appeared in the abaxial position
of the floral meristem early in flower differentiation. The largest sepal
primordium in Figs. 4-B and 4-C corresponds
to the first sepal. |
| ii) |
Inception of another four sepal primordia followed, one of them prominent
in the adaxial position (Figs. 4-B, 4-C)
preceding the others. |
| iii) |
Initiation of five-petal primordia alternating with the sepal primordia
was the next stage in the flower organogenesis. As shown in Figs.
4-C and 4-D, the petal primordia arose more or less
simultaneously. |
| iv) |
It was followed by five stamen primordia which were seen adnately fused
to the petal primordia (Fig. 4-E). The stamen and petal
primordia initially developed separately but became fused in later stages. |
| v) |
Initiation of two carpel primordia occurred in the form of an inward invagination,
with two crescent-shaped structures at the edges (Fig. 4-F).
The two carpels later fused to form a single ovary, followed by the development
of a single style which began as a tube (Fig. 4-G and
4-H). |
| vi) |
Differentiation of anthers took place from the stamen primordia which
grew in length and later gave rise to an elongated stalk. In relatively
mature buds, only the stalks remained fused to the petals while the anthers
stayed loose and separate (Figs. 4-G and 4-H). |
| vii) |
The styles became capped with a bi-lobed stigma which marked the end of
organ formation in a developing flower, though all organs continued their
growth till the opening of flowers and their maturity (Fig.
4-H). |
| viii) |
During stage 7 of the flower development the sepals fully enclosed a developing
bud, petals grew out of the sepals just before the flower opened. |
Flower development is a continuous process, nevertheless, it has been arbitrarily
divided into stages, characterised by landmark events for comparison between
species. A comparison of different developmental stages in flower morphogenesis
of N. rustica, N. tabacum and A. Thaliana is shown in Table
1. It is clear from the table that differentiation of N. rustica
flowers was generally similar to the flower development in N. tabacum.
On the other hand, the flower development in N. rustica was different
from flower development in A. thaliana, mainly in relation to the number
of flower organs in each whorl and their rate of growth in relation to each
other. The relative similarity of flower organ differentiation in N. rustica
and N. tabacum could be attributed to the similarity of floral morphology
and architecture between the flowers of the two species which are taxonomically
related among themselves but are different from A. thaliana. Minor differences
between the flower development of N. rustica and N. tabacum were
ultimately reflected in the floral morphology of the two species, such as the
comparatively slower growth of petal, sepal and anthers in N. rustica
flowers resulted in relatively shorter and compact flowers rather than the long
flowers in N. tabacum.
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Figure 1: Wild type inflorescence of
N. rustica.
Figure 2: Wild type flower of N. rustica.
Figure 3: Wild type flower organs of N. rustica; (a) Sepals,
(b) Petals, (c) Stamens, (d) Carpels.
Figure 4: (A to H): Scanning electron micrographs
of different stages in the development of wild type flowers in N. rustica.
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Table 1: Developmental stages in floral
organogenesis in N. rustica, N. tabacum and A. thaliana.
The stages correspond for N. rustica and N. tabacum, however
species-specific differences in N. rustica flower development are
listed. Correspondence with A. thaliana is only approximate. |
Finer differences, such as, the early connate fusion of W1 and W2 organs in
the N. rustica which occurred later in N. tabacum, formed the
basis for differences in floral morphology between the two related species.
The early connate fusion of W1 and W2 organs was reflected in the relatively
more ‘entire’ margins of the calyx and corolla in N. rustica as compared
to that in N. tabacum. Comparatively slower, petal, sepal and anther
filament growth in N. rustica flowers resulted in relatively shorter,
more compact flowers, rather than the longer flowers in N. tabacum. Styles
were initiated much later in flower development in N. rustica as compared
to N. tabacum flowers. This delay in initiation is evident in the difference
in relative lengths of styles in the two species.
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