Science, Technology and Development  Volume 33 Issue 3, 2014

Research Article

Elaboration of the Developmental Stages of Flower Organogenesis in Nicotiana rustica L.
Mukhtar Alam
Department of Agriculture, University of Swabi, Swabi, Khyber Pakhtunkhwa, Pakistan.

Habib Ahmad
Department of Genetics, Hazara University, Mansehra, Khyber Pakhtunkhwa, Pakistan.

Ontogeny of the flower organogenesis of Nicotiana rustica was carried out with the help of scanning electron microscopy. Eight landmarks were observed in the developmental stages of flower development in N. rustica. The recorded landmarks in the flower development of N. rustica were compared with that of Nicotiana tobacum and Arabidopsis thaliana.
    How to Cite:
Mukhtar Alam and Habib Ahmad , 2014. Elaboration of the Developmental Stages of Flower Organogenesis in Nicotiana rustica L.. Science, Technology and Development, 33: 110-114
DOI: 10.3923/std.2014.110.114

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.

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.

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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