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International Journal of Agricultural Science and Research (IJASR) ISSN 2250-0057 Vol. 3, Issue 4, Oct 2013, 39-42 © TJPRC Pvt. Ltd.

TYPIFICATION OF STIGMA IN PSOPHOCARPUS TETRAGONOLOBUS (L.) DC. EAPEN P. KOSHY1, BLESSYMOLE K. ALEX2 & PHILIP JOHN3 1

Department of Tissue Engineering, Jacob School of Biotechnology and Bioengineering,

Sam Higginbottom Institute of Agriculture, Technology and Sciences, Allahabad, Uttar Pradesh, India 2

Department of Molecular and Cellular Engineering, JSBB, SHIATS, Allahabad, Uttar Pradesh, India 3

Department of Science, Walthamstow Science Academy, Walthamstow, London

ABSTRACT The reproductive biology of flowering plants is important for determining barriers to seed and fruit set, for conservation, and for understanding pollination and breeding systems that regulate the genetic structure of populations.Psophocarpus tetragonolobus (L.) DC., commonly known as ‘winged bean’ is an underutilized leguminous crop, which has drawn worldwide attention of the scientists mainly due to its food value. Its seeds are highly proteinaceous in nature and can wipe away malnutrition from under developed countries. But there are several pit falls in developing this as a prominent food crop. An in depth knowledge of the reproductive organs of the plant is very much essential for overcoming these problems.The study of factors that control the reproductive process in higher plants necessitates a thorough knowledge of the pollen and gynoecium. While considerable information exists on the pollen and the ovary, not much is known about the stigma. In this context, the stigma morphology of Psophocarpus tetragonolobus L. Dc. was analyzed using both stereo and scanning electron microscopy. The study revealed that the stigma of winged bean is of the wet type with finger like papillae on the rim and thus coming under Group III according to the general classification.

KEYWORDS: Papillae, Psophocarpus tetragonolobus, Scanning Electron Microscopy, Wet Stigma, Winged Bean INTRODUCTION Psophocarpus tetragonolobus (L.) DC. (Winged bean), a leguminous plant of high economic importance and food value, is often neglected due to lack of documented information of the plant. Several characters of wild plants still exists in most of the winged bean accessions, viz.viny and indeterminate habit of growth, photosensitivity, pod shattering (when dry on the vine), presence of toxic substances in the raw dry beans and in the rind of the tubers, uneven germination rate, low yield, and above all, tremendous diversity of form in all characters. To overcome these problems of this crop, conventional plant breeding techniques and tissue culture techniques can play a significant role. A thorough knowledge of the floral biology of the plant is necessary for successful plant breeding techniques. The pistil is the most vital organ of a flower since it contains the egg deep seated within its ovary and protects the embryo that develops from it after fertilization. Basically it has three main parts, the upper stigma of varied nature and morphology, the middle style, which is long and flexible and the basal ovary that contains the egg. These parts, though morphologically different, serve the purpose of receiving the pollen grain selectively and aiding its growth and allowing the male gametes to make a successful fertilization. As the stigma is the recipient of the pollen grains, the initial interaction takes place between these two structures. Stigma morphology is variable and has been conveniently classified by Heslop – Harrison and Shivanna (1977) covering around 900 genera belonging to 250 families. The classification is mainly based on the stigma morphology and the amount of secretion at the receptive period. Thus angiosperm stigma either falls into wet or dry type depending on the presence or


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Eapen P. Koshy, Blessymole K. Alex & Philip John

absence of secretion on the stigma. Again the presence of low or medium sized papillae that are either unicellular or multicellular in uniseriate or multiseriate arrangement or the complete absence of papillae makes an angiosperm stigma subject to further categorization.Based on the amount of secretion present during the receptive period and the nature of the stigmatic papillae, the two basic types have been further classified (Heslop – Harrison et al., 1975; Heslop– Harrison and Shivanna, 1977)as follows; Dry stigma (without apparent fluid secretion) Group I

Plumose with receptive cells dispersed on multiseriate branches (Gramineae). Discontinuities along the receptive surface.

Group II

Receptive cells concentrated in distinct ridges, zones or heads.

Surface non-papillate

Surface distinctly papillate o

Papillae unicellular

o

Papillae multicellular o

Papillae uniseriate

o

Papillae multiseriate

Wet Stigma (Surface Secretions Present During Receptive Period) Group III Group IV

Receptive surface with low to medium papillae; secretion fluid flooding interstices. Receptive surface non-papillate; cells often necrotic at maturity, usually with more surface fluid than group III.

The study of its developmental anatomy (Konar and Linskens, 1966) showed that the stigma could be separated into two zones, an upper zone consisting of the epidermis and a lower secretory zone consisting of 1-3 layers of cells. Below this, the lower zone of parenchymatous ground tissue is present. Though a few studies on mature stigma, style and stigma interaction of plants do exist, no detailed studies have been made on its developmental aspects. The present investigation is designed with an objective to understand the reproductive biology of Psophocarpus tetragonolobus (L.) DC.through the analysis of ultrafine structures of stigma as shown by stereo and scanning electron microscopy.

MATERIALS AND METHODS Morphological characters of the stigma were studied using stereomicroscope as well as Scanning Electron Microscope (SEM). Tissue Processing for Stereomicroscopy Fresh stigma was observed under a trinocular stereomicroscope and photographed. Scanning Electron Microscopic Studies SEM preparations were made according to Falk (1980). The steps used in the processing for SEM are as follows. Fresh stigma was fixed with 3% gluteraldehyde in 0.1M phosphate buffer at pH.7 for 6 hours. The fixed material was


Typification of Stigma in Psophocarpus tetragonolobus (L.) DC.

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washed four times thoroughly in cold phosphate buffer for 15 minutes each at 40C.The fixed materials were dehydrated through acetone series of 25%, 50%, 75% and 100% for 10 minutes each and stored in 70% acetone. Finally the tissues were kept in 100% acetone, for 30 minutes and in isoamyl acetate for 5 minutes at room temperature.The dehydrated stigmas were critical point dried in a critical point drier (H.C.P-2 Hitachi).The dried specimens were mounted on stubs using double sided adhesive tape and coated with gold in a sputter coater (Model E-101-Hitachi). The coated specimens were observed under SEM (S-2400, Hitachi).

RESULTS AND DISCUSSIONS The stigma is round at the tip and is densely pubescent at the margin (Figure 1-2). The surface of the stigma has a free flowing exudate, which is secreted, by glandular cells. The stigmatic surface shows globular, unicellular papillae cells with finger like structures, arising from the rim of the stigmatic head, which are quite evident from the SEM observations (Figure 3-4).

Figure 1: Stereomicroscopic View of Wet Stigma Surface Showing the Globular Head and the Papillae on the Rim of Stigma

Figure 2: Enlarged View of the Wet Stigma

Figure 3: SEM View of the Stigmatic Surface (X30)

Figure 4: SEM View of Stigma Showing the Finger Like Projections on the Surface of the Stigma (X 1000)


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Eapen P. Koshy, Blessymole K. Alex & Philip John

The stigma surface of P. tetragonolobus is of the wet papillate type. At the time of anthesis, the stigma secretions become watery. According to Hesslop- Harrison and Shivanna (1977), this stigma comes under Group III of the wet type. Group III wet type stigma has been reported in many plants (Crestiet al., 1982; John, 1989). The watery nature of the stigmatic exudate makes the papillae visible. The papillae cells are considered to be modified epidermal cells taking active part in secretion and are responsible for the secretion on the stigma surface (Considine and Knox, 1979; Owens and Horsefiled, 1982). In wet stigma, receptivity often coincides with accumulation of stigmatic secretion (Heslop – Harrison and Shivanna, 1977). Studies on the stigma surface secretions of the wet stigma of Petunia (Konar and Linskens, 1966; Herrero and Dickinson, 1979) and Acacia (Kernick and Knox, 1989) have shown that the major exudates are not produced until anthesis.

CONCLUSIONS SEM studies reveal that the stigma is round at the tip and is densely pubescent at the margin. The stigmatic surface shows globular, unicellular papillae cells with finger like structures, arising from the rim of the stigmatic head. The surface is with a free flowing exudate and thus the stigma of Psophocarpus tetragonolobus comes under Group III.

REFERENCES 1.

Considine, J.A. and Knox, R.B.(1979). Development and histochemistry of the pistil of grape, Vitisvinifera. Annals of Botany. 43:11 – 22.

2.

Cresti, M., Ciampolini, F., Van Went, J.L. and Wilms, H.J.(1982). Ultrastructure and histochemistry of Citrus limon L. stigma. Planta 156: 1 – 9.

3.

Falk, R.H.(1980). Scanning Electron Microscopy – SEM. Inc., Amfo’Hare (Chicago) IL, USA.

4.

Herrero, M. and Dickinson, H. G.(1979). Pollen-pistil incompatibility in Petunia hybrida. Changes in the pistil following compatible and incompatible intraspecific crosses. Journal of Cell Science. 36:1-18.

5.

Heslop-Harrison, J., Heslop-Harrison, Y. and Barber, J. (1975). The stigma in incompatability responses. Proceedings of the Royal Society of London. Series B. 188:287-297.

6.

Heslop-Harrison, Y. and Shivanna, K. R.(1977). The receptive surface of the angiosperm stigma.Annals of Botany.41:1233-1258.

7.

John, P.(1989). Development, ultrastructure, cytochemical and experimental studies of the pistil of Catharanthusroseus L. G. Don (Apocynaceae) and WithaniasomniferaDunal (Solanaceae). (Unpublished doctoral thesis).M.S University, Baroda, Gujarat, India.

8.

Kernick, J. and Knox, R.B.(1989). Pollen-pistil interactions in leguminosae (Mimosoideae) cf. The components of the sexual reproduction process and the pathway to in vivo fertilization in higher plants. Owens, S.J.Botany Journal of Scotland.46(2):287-310.

9.

Konar, R. N. and Linskens, H. F.(1966). The morphology and anatomy of the stigma of Petunia hybrida.Planta71:356-371.

10. Owens, S.J. and Horsefield, N.J., 1982. A light and electron-microscopic study of stigma in Aneilemaand Commelina species (Commelinaceae).Protoplasma. 42: 26 – 36.


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