Aloka Kumari and A. K. Pandey
Plant Systematics Research Centre
University Department of Botany
T.M.Bhagalpur University;Bhagalpur-812007, India.
aloka_kumari04@yahoo.com
Abstract
Safflower is basically self-pollinating crop. However, cross pollination increases the number of seed setting. If anther dehiscence occurs before style elongates, the stigma pushes through mass of pollen, becomes coated with pollen and gets self fertilized. If anther dehiscence occurs after style elongates, stigma passes through the anther tube without becoming pollen coated, then cross pollination is needed. Absence of pollinators results in self-pollination. Anthesis of disc florets takes place between 06:30 h to 08:00 h. The number of pollen grains produced in safflower per anther/per flower is comparatively low (236+74 grains/anther or 906+372 grains/flower). In safflower highest seed set (%) was obtained with stigma pollinated 24h after anthesis. Stigma remains receptive up to 32-56 hours after anthesis. There is about 85.99% seed setting in the open pollinated heads followed by 38.15% (in muslin cloth) and 35.54% (in butter paper) of the flowers set seeds under bagged condition. Polyacrylamide gel electrophoresis shows that, 3 Protein bands (RP 0.21, 0.30 and 0.42) are present in both pollen and stigma. The percentage similarity is just 50%; indicating 50% chances for compatibility. Key Words: Safflower, Carthamus tinctorius, Self incompatibility, PAGE.
Introduction
Safflower (Carthamus tinctorius L.) an important oil seed crop belonging to the tribe Cardueae of the family Asteraceae. The seeds yield creamish, edible, semidrying oil which is used for culinary purposes, for anointing the body, as an illuminant, for soap manufacture and in paints. Seeds are also used for feeding the cage birds. The press cake is used for livestock feed and as manure (Anonymous, 1976; Li and Mundel, 1996). The crop is predominantly cultivated in Spain across North Africa and West Asia to India, many being indigenous to the Mediterranean region. Presently, safflower is cultivated in USA, Mexico, Ethiopia, Australia, Argentina, Russia, China, India, Egypt, Italy, Germany and Spain. Safflower producing States in India are: Karnataka, Madhya Pradesh, Maharashtra, Andhra Pradesh, Orissa and free radical catalyst. Tetra Methylene Diamine (TEMED) was added to control polymerization. Riboflavine or Ammonium persulphate was used as a catalyst, which was freshly prepared in order to have better results. The porosity of gel was determined by the relative concentrations of acrylamide monomer and bis acrylamide (Davies, 1964). 200 mg pollen and pistil sample was crushed with 2 ml of 1 M phosphate buffer (pH- 6.2). After 24 hours it was centrifuged for the collection of supernatant. The collected supernatant was used for qualitative determination of protein. However, stepwise working protocol for qualitative determination of proteins is as follows: (i) Run was carried out with a standard electrophoretic system and the gels were removed from the tubes with the help of 21 gauge needle by pushing water between gel and the glass tubes with the help of 21 gauge needle by pushing water between gel and the glass tube wall. (ii) After this, these gels were treated separately for protein studies. (iii) For studying proteins, gels were stained in 1.25% (w/V) coummasie blue in 7% glacial acetic acid for 25 |
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Bihar. India is largest producer of safflower in the world. Attempts are being made to improve the crop by conventional breeding techniques as well as by using biotechnological methods. Safflower is predominantly a self-pollinating crop, with the genetic potential of over 90% for selfing, although environmental conditions may result in outcrossing exceeding 50% (Li & Mundel 1996). Heterogeneity builds up quickly in safflower populations. To enhance their genetic homogeneity, plants selected as parents for genetic studies and breeding purpose are selfed by covering the flower for one or two successive generation with cloth or paper bags. To ensure planned crossing, flowers are emasculated by removing the anther tubes, along with the upper portion of the corolla and petal lobes, in the late bud stage (Knowles 1980). The next day, when the styles have elongated, the emasculated florets are fertilized with pollen from preselected flowers or head. However, a massemasculation technique (Deshmuk & Ranga Rao 1989), takes less time than emasculation of individual florets and allows more efficient production of crossed seeds.
Materials and methods.
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At 50% flowering stage, flowering branches of different plants with young unopened heads were bagged. When seed setting was completed and heads started drying, seeds were counted from both bagged and opened heads. Initially seeds from three different districts of Bihar viz. two area of Bhagalpur districts (Mohanpur diara, Bikramshila) and one area of Khagaria district (Parbatta), were cultivated following completely randomized design (CRD) with four replicates for each (Figure 1). However as no marked difference was noticed in their characteristics, data for seed setting from different plots was pooled. The data presented is an average of 90 heads for both bagged and open inflorescences..
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The Polyacrilamide Gel Electrophoresis technique involves the co-polymerization of acrylamide (CH2(NHCOCH=CH2)) with a suitable quantity of cross linking compound N-N methylene Bisacrylamide (CH2(NHCOCH=CH2)) with a suitable setting varied. It was 38.15% in muslin cloth, 35.54% in butter paper and 31.58% in news paper respectively (Table1).
Table 1: Self incompatibility test in different experimental conditions |
minutes. (iv) Gels were destained with 7% glacial acetic acid for atleast 24 h till visualization of sharp bands.
Results and Discussions
Safflower is basically self-pollinating crop. However, cross pollination increases the number of seed setting. If anther dehiscence occurs before style elongates, the stigma pushes through mass of pollen, becomes coated with pollen and gets self fertilized. If anther dehiscence occurs after style elongates, stigma passes through the anther tube without becoming pollen coated, then cross pollination is needed. Absence of pollinators results in self-pollination. Anthesis of disc florets takes place between 06:30 h to 08:00 h. The number of pollen grains produced in safflower per anther/per flower is comparatively low (236+74 grains/anther or 906+372 grains/flower). Observations were also made on the morphology of pollen and stigma. Open pollinated and bagged heads showed During present study bagging experiments reveal that there was about 85.99% seed
Table2. (a) R.P. Value of protein in Pollen grains and pistil of safflower. (b) R.P Value of Pollen grains of safflower and pistil showing presence and absence of protein bands. Flower in safflower are borne on a capitulum which consists of single types of flower. The disc florets are hermaphrodite where as ray florets are absent. Pollen grains are spherical, and yellow. They are tricolpate, triporate, with irregularly circular pores and short germinal furrow which is broad in the center.The echinulate exine has spines with circular base and sharp pointed vertical projections. Upper surface of stigma shows numerous papillae covered with uniformely thickened cuticle overlaid with a layer of pallicle. The stigma is of dry type. A floret opens and liberates early in the morning, the style emerges about mid day and the stigma lobes separate and curl backwards in the evening. Unlike other Cardueae members, the stigma lobes in safflower rarely curl sufficiently to touch the pollen in their own style (Weiss, 1983), a circumstance which also helps to explain failure of this plant to set seeds readily under bagged conditions. Earlier, Knowles (1958) studied the influence of insects in seed setting in this plant where plants were allowed to flower in cages from which bees were either introduced or excluded. Plants visited by bees had 45 seeds per head while those without bees set only 16 seeds per head. Formation of seeds under |
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In the present study Polyacrylamide (PAGE) was employed for separation of protein in safflower. For analysis of proteins, pollen and pistil were selected. During electrophoretic separations of the proteins, five bands were observed in the pistil of safflower followed by pollen of safflower with four bands. Based on the present study it is concluded that protein is an important parameter for separating species. Three Protein bands were approximately matched to each other but one of them two extra bands present in stigma of safflower while, one band of pollen of safflower. Therefore from these observations, I conclude that protein bands show that the plant is self-compatible and in this plant protein doesn’t work as barrier. R.P.Value and zymograms of Carthamus tinctorius L. showed in table 2. Proteins 1 & 5 (RP 0.13 PP 0.46) were noted to be specific for stigma while Protein 6 (RP 0.6) for pollens. 3 Protein bands (RP 0.21, 0.30 and 0.42) was present in both pollen and stigma. The percentage similarity is just 50%; indicating 50% change for crossability / compatibility.
bagged conditions shows that there is some selfing in this crop. Seed setting under muslin bags has been reported to take place only if the weather is fair, particularly if the sky is not cloudy and there is low humidity in the air (Chavan, 1961). It shows that temperature and humidity influence compatibility behaviour of safflower. Safflower is homomorphic and of sporophytic type. Our observations have shown that in incompatible pollination, pollen germination was usually inhibited on the stigma surface. If pollen grains germinate some how their tubes twist over the surface of the papillae and do not penetrate the basal portion. Detailed studies on this aspect is in progress and will appear is in progress and will appear elsewhere. References
1 Anonymous 1976 Medicinal Plants ; Vol II pp 354-355 (Madras: Orient Longman Ltd.)
2 Chavan VM 1961 Niger and Safflower. Indian Central Oilseeds Committee. Examiner Press, Bombay.
3 Deshmuk A K & V Ranga Rao 1989 A new and efficient method to achieve mass hybridization in safflower without emasculation: A re-appraisal of currently followed emasculation techniques; In Proceedings of Second International Safflower Conference, Hyderabad, India, 9-13 Jan. 1989; pp 157-161 eds V.Ranga Rao and M.Ramachandran (Hyderabad: Indian Society of Oilseeds Research, Directorate of Oilseeds Research)
4 Knowles P F 1958 Safflower; Ad. Agron. 10 289- 323
5 Knowles P F 1980 Safflower: In Hybridization of Crop plants; pp 535-547 eds. W.R.Fehr and H.H.Hadley (Wisconsin, U.S.A: Am. Soc. Argon., Madison)
6 Li D & H H. Mundel 1996 Safflower (Carthamus tinctorius L.): Promoting the conservation and use of underutilized and neglected crops 7. (Rome, Italy: Institute of Plant Genetics and Crop Plant Research, Gatersleben / International Plant Genetic Resources Institute)
7 Weiss EA 1983 Oil crops. Longman, London.
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