T REPRO N DU LA C The International Journal of Reproductive Biology 10(1) Jan., 2018, pp.38-43 P T I F V O E

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S H Reproductive biology of spectabilis Roth. T Somnath Kar* and B. K. Datta Plant and Biodiversity Laboratory, Department of Botany, Tripura University Suryamaninagar -799022, Tripura, India *e-mail: [email protected] Received: 10.05.2017; Revised: 03.06.2017; Accepted and Published online: 01.09.2017 ABSTRACT The present communication deals with flowering phenology, floral morphology, and pollen biology of Crotalaria spectabilis Roth. Flowering commences in October and lasts up to end of March. Initiation of floral bud takes 8±1 days from the vegetative shoots and blooms in 11±2 days. Fruit formation takes place in November and continues till April. Flowers open between 14:00 to 16:30 h, and are protandorus, and the anthers dehisce before anthesis begins. Pollen grains remain viable for 3 days. The flowers of Crotalaria spectabilis, exhibit two types of stamens, short filaments with long narrow anthers and long filaments with small rounded anthers.The flowers are visited by butterflies, beetles and bees throughout the day. Key words : Crotalaria spectabilis, reproductive biology, dimorphic anthers, floral visitors.

Crotalaria () commonly known as rattle pod is a Floral visitors—Visiting pollinators and their activity were large genus with more than 700pecies (Le Roux et al. 2013), observed and recorded at all the four sites. During peak mainly distributed around the tropical and subtropical region flowering period, pollinators were observed at day as well as at of the world. In India, the genus Crotalaria is represented by night for one month continuously over three flowering 81species, out of which 27 species are endemic;15 species are seasons. Insects were trapped, collected and identified by the listed in the Red Data Book of Indian (Nayar and Sastry entomologists, at Ecology and Biodiversity Lab, Department 1987, Subramaniam and Pandey 2013). Crotalaria spectabilis of Zoology , Tripura University. is a robust shrub commonly known as Showy Rattlebox, pipuli Pollen morphology—For pollen morphology, acetolysis jhunjhun in Bengali, ghungri or sanni or dhandhania in Hindi, methodology was followed (Erdtman 1952). The size of the kaadusenabu in kanada, jhunjhania in Tripuri and dingala in pollen grains was measured by mounting the pollen in Marathi. The entire plant is of great medicinal importance glycerine jelly (Wodehouse 1935) using standard ocular (Jain and De Filipps 1991, Parrotta 2001). In spite of its micrometer. excessive use for medicinal purposes, this species is gradually Pollen Production—For detailed study of pollen production decreasing due to habitat fragmentation and urbanization. So mature anthers were crushed in lactophenol-glycerine with there is an urgent need for its conservation and it can be aniline blue and counts were made using a hemocytometer achieved by understanding its reproductive biology. (Barrett 1985) MATERIAL AND METHODS Pollen viability—Pollen viability was assessed by fluoro chromatic reaction test (FCR) test (Heslop-Harrison and Study sites—Present investigation was under taken at four Heslop-Harrison 1970). sites (Table 1). In vitro Pollen germination—Pollen germination is the first critical morphogenetic event in the pollen towards fulfilling its Table 1–Sites of study and their geographical location ultimate function of discharge of male gametes in the embryo Sites Geographical position sac. It is therefore important to understand the physiology and College Tilla 23°49´46.76"N and 91°18’2.74"E biochemistry of pollen germination. The stigma provides a Tripura University campus 23°45´51.55"N and 91°15’33.17"E suitable site for pollen germination, however studies on in vivo are not easily feasible because of the complications involving Malaynagar 23°45´13.48´´ N and 91º18´43.80´´ E in pistillate tissue (Biswas et al. 2013). It is possible to Jirania 23º48´48.18´´ N and 91º25´50.27´´ E germinate pollen grains of a number of taxa using rather a Floral Biology— Five healthy plants growing in their natural simple nutrient medium and to achieve a reasonable length of habitat were selected from each site. Phenological events (bud tube growth. In vitro pollen germination was carried out in break, flowering, fruiting, shedding of leaves, fresh leaf different concentration of sucrose (2%, 5%, 10%, 15%, 20%, emergence, etc.) were recorded over a period of three 25%, 30%, 35% and 40%) alone and in combination with flowering seasons (2014-2015, 2015-2016 and 2016-2017). 100 ppm boric acid and calcium carbonate respectively. Floral morphology, floral biology was studied by various Reproductive Success—According to Shivanna and Johri methods given by Kearns and Inouye (1993) were studied with (1985), rather than the total pollen per flower or per plant ratio, the help of hand lens and dissection microscopy. the pollen: ovule ratio is more accurate measures of 2018 Reproductive biology of Crotalaria spectabilis Roth. 39

reproductive success. The pollen ovule ratio was measured when maximum and minimum temperatures ranged between following the method of Cruden (1977). 14,.4 -17.2oC and 7.2 – 12.7oC respectively. The peak flowering, continued for 35 - 45 days when maximum and RESULTS AND DISCUSSION minimum temperatures ranged between 12.2-24.7oC and 11.8 Floral phenology—Commencement of floral bud in –19.9oC respectively. Thereafter, decline started till cessation, Crotalaria spectabilis Roth. occurred in by the second week of when maximum and minimum temperatures fluctuated October or first week of November, when maximum and between 20.6- 28.3oC and 14.5 – 22.3oC respectively. Thus the minimum temperatures ranged between 25.5 - 31.7oC and 15.8 plant remained in blooming stage from second week of –19.8oC, respectively. Flowering remained moderate, October to last week of March over wide range of temperature quantified in terms of numbers per m2, for about 45 to 55 days, showing in Table 2.

Table 2–Duration of different parameters of Crotalaria spectabilis plant in relation to ambient temperature.

Sl. No. Plant parameters Time intervals Duration Ambient temperature ( ºC ) ( Days) Ranges Averages Max Min Max Min 1 Initiation of floral bud Second week of October – first week of 20 – 32 25.5 - 31.7 15.8 – 19.8 28.9 17.7 November 2 Moderate flowering Second week of November to first week 45 – 55 14.4 -17.2 7.2 –12. 7 15.3 9.6 of January 3 Peak flowering Second week January to last week of 35 - 45 12.2-24.7 11.8 – 19. 9 19.2 16.4 February 4 End of flowering First week of March to first week of 25-35 20.6- 28.3 14.5 – 22.3 26.5 19.3 April

Floral bud Initiation takes 7 to 9 days to develop from Bracts are lanceolates to ovate in shape, leafy green in colour vegetative shoots and floral buds take 9 to 12 days to bloom and persistent in nature. Flowers are zygomorphic, bisexual and converted into mature fruits flowers take 15 to 20 days. and hypogynous. Pedicels are larger than calyx. Calyx Flowers started opening in the afternoon between gamosepalous, green in colour, 1.5 cm in length, glabrous and 14:00–16:30h. Crotalaria spectabilis possesses dimorphic persistent, campanulate in shape, upper teeth large, triangular anthers and all of them are fertile. However, the long anther shaped, apex acute and 0.5 mm broad at the base. The lower 3 dehisces in bud condition between 11:00-12:00 h and the teeth smaller and less deeply cut, linear to lanceolate in shape, small anther dehisces after 48 hrs of the dehiscence of the long acute apex. The flowers have a typical papilionaceous corolla. anther. The adaptive advantage of this mechanism may result The petals are yellow in colour ringed with purple , 5 in in pollination success which was supported by the work done number, size of - banner is 2.4cm x 2.7 cm , keel is 1.8cm x by Sarala et al. (1999). The length of the filament of dimorphic 1.2 cm and wing is 1.7 cm x 0.7 cm. stamens are 10 (5 + 5) in stamens varied in different day flowering. In bud condition, number, monadelphous. Stamens are two types. Five stamens the length of the filament of the stamens having small rounded have short filaments with long narrow anthers and other 5 anthers the short anthers were shorter than the stamens having stamens have long filaments with small rounded anthers and long narrow anthers, whereas in 2nd day of flowering, the both types of stamens are alternate with one another. Long filaments of stamens bearing smaller anthers become 3 times anthers are basifixed and short anthers are dorsifixed. longer than the stamens having long narrow anthers. Filaments of the stamens having long anther is 1.5 cm in According to Escaravage et al. (2001), differential upward length and anthers are 0.3 cm in length where as Stamen movement of filaments might represent a strategy to increase having short anther have 1.8 cm long filament with 0.1 cm the chances of both pollen donation and pollen reception. long anther. Pollens are trizonocolporate and prolate. Style 1 Stamen dimorphism and its possible consequences may play a in number, long, slender and 1.0cm – 1.1cm in length. Stigma - direct effect on the breeding system of C.spectabilis and which 1, capitate, 0.05 – 0.1 cm in length. Fruits are cylindrical pod, may ensure its reproductive success. Longevity of the flowers 3.0 – 3.8 cm in length and 0.8- 1.1 cm in breadth, inflated that are more than 3 days and ranged between 74 to 78 h (mean ± darken at maturity. Seeds are 0.5 cm x 0.4 cm in size, kidney SD = 76.25 ± 2.25). shaped, small, and brown to black in colour at maturity. Floral morphology—The inflorescence of C. spectabilis is a Number of seeds/ pod is 20 – 28 in number and each seed is terminal raceme. Peduncle is stout and 16 - 35 cm length. 0.023 ± 0.0017g in weight. 40 The International Journal of Plant Reproductive Biology 10(1) Jan. 2018, pp.38-43

Table 3–Pollen morphological information of Crotalaria spectabilis Pollen viability—According to Dafni and Firmage Roth (2000),pollen viability is considered to be an important parameter of pollen quality. Pollen viability refers to the Pollen attributes Observations ability of complete the post pollination events on a Pollen type Tricolporate, zonoaperturate , prolate compatible, receptive stigma and effect fertilization. Pollen size and viability are good markers of the course of Polar axis Longer anther (26.757-)27.331 ± 0.627 microsporogenesis (Bhowmik and Datta 2012). Nyine and (-28.179) µm Shorter anther (27.594-)28.914 ± 0.837 Pillay (2007) emphasized that pollen grain viability (-29.871) µm assessment through the staining method seem to express the Equatorial diameter Longer anther (27.051-) 27.93 ± 0.837 (- germination potential, but not its occurrence. A number of 29.122) µm authors have discussed the terms used to describe the viability Shorter anther (28.483-)29.455 ± 0.765 of pollen grains and their ability to germinate and fertilize (-30.189) µm ovules, and have recommended different terms such as pollen P/E ratio Longer anther (0.968) 0.977 ± 0.009 (- sterility, stainability, viability, germinability, stigmatic 0.989) µm Shorter anther (0.949-) 0.982 ± 0.021 (- germinability, and fertilization ability, pollen quality (Dafni 0.999) µm and Firmage 2000, Klein 2000). Exine size Longer anther (1.334-) 1.847 ± 0.333 (- Results show that pollen grains of C. spectabilis, have 2.17) µm high viability which may ensures high chance of successful Shorter anther (1.253-) 1.687 ± 0.313 (- fertilization. In FDA test, viability of the pollens of longer 2.138) µm anther is more than shorter anther pollen, shown in the figure Pollen kit Present 1. Pollen viability percentage in long and short anther pollen in The number of pollen 29697.6 ± 3023.35 (Long Anther) and FDA test it was 85.94 and 81.59 in long and short anther pollen / anther 5654 ± 224.45 (Short anther) respectively. Floral visitors—During field observations a total of 12 floral Pollen germination—According to Pfahler et al. 1997, in visitors were recorded for Crotalaria spectabilis Roth. Their vitro pollen germination and pollen tube growth are very name, family name and visiting are given in table no 3. useful for explaining the lack of fertility. Pollen germination Pollination activity started from 7 am and remains up to 5 pm and the growth of pollen tubes are important and necessary for but pollination done by Thrips took place in day as well in proper fertilization and seed formation in flowering plants. night. Among the 12 pollinators of Crotalaria spectabilis In present study , pollen grains of long anther showed the Roth. Lampides boeticus L and Bombus sp. were the most highest germination rate ( 78.59 %) in 20 % sucrose solution frequent. Activity of Lampides boeticus L. were noticed where as in 5% sucrose solution it showed highest pollen tube between 9 am to 3 pm. Whereas Xylocopa sp remain active development ( 283.25 ± 14.23µm) .In case of short anther, between 9 am to 12 pm and again from 2 pm to 4 pm. Bombus pollen grains showed the highest germination rate (58.52% sp and three Apis sp were frequent throughout the day. )and pollen tube development (181.62 ± 12.96 µm) in 20% Pollinator activity were noticed during different day sucrose solution. flowering. Pollen from long anthers in 100 ppm boric acid and 10 % sucrose solution showed the highest pollen germination rate Table 4 – Showing floral visitors of Crotalaria spectabilis Roth. (75.23 ± 6.02) and pollen tube development (385.61 ± Sl. Name of flower visitor Family of the Visiting time 14.52µm). Pollens of short anther shows highest pollen No. visitors germination rate and pollen tube length (53.06 ± 4.1) and 1 Apis dorsata Apidae Day pollen tube development (308.92 ± 13.72 µm) respectively in 2 Apis indica Apidae Day 100 ppm boric acid and 5 % sucrose solution. 3 Apis mellifera Apidae Day 85.94 4 Aulacophora nigripennis Chrysomelidae Day 81.59 5 Bombus sp Apidae Day 6 Lampides boeticus Lyeaenidae Day 7 Megacopta sp Plastuspididae Day 8 Musca sp Muscidae Day 9 Telamonia dimidiate Saticidae Day 10 Thomisus sp Thomibicidae Day 11 Thrips Thrpidae Day and night Long anther pollen Small anther pollen 12 Xylocopa sp Violacea Day Fig 1. Percentage of pollen viability of long and short anthers of Crotalaria spectabilis Roth . 2018 Reproductive biology of Crotalaria spectabilis Roth. 41

Fig. 2A. , B. Calyx and Corolla, C. Androecium and gynoecium, D. Ovary section, E. Fruit, F. Dimorphic condition in bud condition, G. Dimorphic condition in 1st day of flowering, H. Dimorphic condition in 2nd day of flowering, I. Pollen grain, J. Pollen viability in FDA test, K.Pollen germination in 20 % sucrose solution, L. and M. Pollinators of Crotalaria spectabilis Roth. 42 The International Journal of Plant Reproductive Biology 10(1) Jan. 2018, pp.38-43

This is attributed to the fact that sucrose is necessary for Normally C. spectabilis indicates a potential to produce proper pollen nutrition, osmotic control and in combination 16327 seeds on an average which is the absolute/ maximum with boric acid promoted pollen germination (Sidhu and reproductive potential (Rm), whereas, the ecological/realized Malik 1986). Boron combines with sugar and form a reproductive potential (Re) under natural conditions was, sugar-borate complex which is translocated with greater only 49.32% (8052 seeds per plant on Avg.) of its absolute/ facility than sugar molecules without boric acid (Gauch maximum reproductive potential. This may be due to the and Duggar 1953). Boron may enhance the sucrose uptake presence of some strong ecological constraint (s). Factors and also stimulate germinating ability which has been responsible for low ecological reproductive potential remain confirmed by the study of other co-workers (Pal et al. to be investigated. 1989, Gupta et al. 1989, Mandal et al. 1982, Bhattacharya Acknowledgement—The authors are thankful to the Head, et al. 1997, Mohi-ud-din et al. 2007 and Biswas et al. Department of Botany, Tripura University for providing 2008). necessary facilities. First author is thankful to UGC, New

It is also observed that the addition of CaCO3 with Delhi for providing financial assistance in form of JRF, sucrose solution results in the increase of pollen germination UGC BSR FELLOWSHIP [F.TU/FIN(233) / (P)/12-05]. The percentage as well as pollen tube development. A tip-focused authors are thankful to Ecology and Biodiversity Lab, calcium gradient is required for pollen tube growth and is Department of Zoology, Tripura University for identification involved in regulation of cytoskeleton and vesicle dynamics in of floral visitors. pollen. Calcium-dependent kinases regulate ion channels in pollen (Stein horst and Kudla 2013) The optimum result REFERENCES was obtained in 10% sucrose solution supplemented with 100 Barrett SCH 1985. Floral trimorphism and monomorphisim ppm CaCO3 solution. Pollen grains of long anther which in island population of Eicchornia paniculata (Bot. J. showed highest germination rate i.e. 75.23 ± 6.02 and pollen Linnean Soc. 25(1) 41-60. tube development 301.88 ± 13.45 µm. Whereas, pollen grains of short anther showed highest germination rate Bhattacharya A, Mondal S and Mandal S 1997. In vitro pollen i.e. 71.55 ± 9.76 and pollen tube development 302.04± germination of Delonix regia (Boj.) Raf. Sci. Cult. 63 16.04 µm. (5/6) 143-144. This is attributed to the fact that sucrose is necessary for Bhowmik S and Datta BK 2012. In vitro pollen proper pollen nutrition, osmotic control and in combination germination in Eichhornia Crassipes (Mart.) Solms : with CaCO3 promoted pollen germination. An insight into its preferred mode of clonal

Table 5–Floral Attribute of Crotalaria spectabilis Roth. reproduction. Notulae Scientia Biologicae. 4(2) 65-71. Floral Attribute Values

Number of Pollen per anther 29697.6 ± 3023.35 (Long Anther) Biswas K, Mondal S and Mandal S 2008. Studies on in vitro and 5654 ± 224.45 (Short anther) pollen germination of Solanum surattense Burm. f. and Number of Pollen per flower 176758 ±15258.11 Solanum nigrum L. Sci. Cult. 74(3-4) 149 -152. Number of ovule per flower 22 ± 2 Biswas K, Mondal S. and Mandal S 2013. Floral Biology and Pollen / Ovule Ratio 7894.32 : 1 pollination of Solanum sisymbrifolium Lamk. Reproductive success—According to Shivanna and Johri International Journal of Current Research. 5(6) 1429- (1985), rather than the total pollen per flower or per plant 1433. ratio, the pollen: ovule ratio is more accurate measures of Cruden RW 1977. Pollen-ovule ratios: a conservative reproductive success. The high P/O ratio i.e. 7894.32: 1 along indicator of breeding systems in flowering plants. with high pollen production i.e. 176758 ± 15258.11 in Evolution. 31 32-46. Crotalaria spectabilis attributes to its high seed set and also ratified with the result reported by Cruden (1977), indicates Dafni A and Firmage D 2000. Pollen viability and that the species to be xenogemous. Present study shows longevity practical, ecological and evolutionary that the number of inflorescences per plant ranged from 30 to implications. Plant Systematics and Evolution. 222 76 (Mean 51.3 ± 10.54) and number of flowers per 113-132. inflorescence ranged from 9 to 19(Mean 14.6 ± 2.63). Erdtman, G. 1952. Pollen morphology and plant Fruit set per inflorescence ranged from 5 to 9 (Mean 7.2± 0.97) taxonomy—Angiosperms. Almqvist and Wiksell, while seeds per fruit were 20 to 28 (Mean 21.8 ± 2.49) on an Stockholm, Sweden. Pp. 539. average. 2018 Reproductive biology of Crotalaria spectabilis Roth. 43

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