T REPRO N DU LA C The International Journal of Plant Reproductive Biology 7(2) pp.128-134, 2015 P T I F V O E

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DOI 10.14787/ijprb.2015 7.2.128-134 T

Reproductive biology of Feronia limonia (L.) Swingle syn. Limonia acidissima (Rutaceae)

Seema Chauhan

Academy of Life Sciences, 8/13 I Kaushalpur Bye Pass Road, Agra-282005, India

*e-mail: [email protected]

Received : 13.08.2013; Revised: 23.01.2015; Accepted & Published on line: 01.05.2015

ABSTRACT

Feronia limonia (L.) Swingle syn. Limonia acidissima L. (Rutaceae) commonly known as wood-apple or elephant apple is a deciduous slow growing tree. Trees flower in April-June. The flowers arranged in terminal cymes bear yellow petals, 8-10 pink stamens with dark red anthers and pentacarpellary gynoecium. A nectariferous disk develops at the base of the ovary which is covered by large number of long tapering trichomes. Flowers are cross-pollinated in nature by honey bees (Apis cerana indica) showing facultative xenogamous breeding system. Fruits are berry, round, hard, woody; the rind is scurfy thick, with numerous small, white seeds. Seeds ripen in early October through March.

Keywords: Elephant apple, breeding system, nectariferous disk

INTRODUCTION family-Aurantioideae of the family Rutaceae. It is commonly known as kaith, kath or wood-apple or Studies on reproductive biology, an important elephant apple. The fruits are rich in vitamin C and used interdisciplinary area are essential for developing as a liver and cardiac tonic and when unripe, it is used as effective strategies for both in situ and ex situ an astringent means of halting diarrhoea and dysentery conservation of the species (Moza & Bhatnagar 2007). and effective treatment for cough, sore throat and Owing to the increasing and immediate concern for diseases of the gums. Feronia limonia a medicinally augmenting food supply, knowledge on reproductive important species is threatened due to over exploitation, biology has been mostly utilized for herbaceous crops. increasing urbanization and industrialization. Moreover, Trees have been neglected and they have not received the it is less frequently cultivated due to higher seedling attention they deserve. This is largely due to several mortality and the out breeding nature results in poor difficulties in conducting researches e.g. their large size, regeneration, genetic variability in natural population prolonged juvenility, long life cycles, in frequent and inferior germplasm (Aslam et al. 2010). Rotting of flowering and inaccessible flowers, selection of plus fruit pulp and seeds caused by several saprophytic fungi trees, breeding, progeny testing and selection of elites (Aspergillus, Penicillium and Rhizopus) results in highly (Tandon et al. 2005). Feronia limonia (L.) Swingle syn. reduced seed germination. In order to conserve this Limonia acidissima L., native of Indomalaya ecozone to threatened medicinally important tree species, the Bangladesh, India and Sri Lanka is a monotypic genus in reproductive biology has been undertaken. the sub tribe- Balsamocitrinae; tribe- Citreae; sub- 2015 Reproductive Biology of Feronia limonia- Chauhan 129

MATERIAL & METHODS sucrose following the method of Hauser & Morrison (1964); and by Fluorochromatic reaction (FCR) test as Present investigation was undertaken on five given by Heslop-Harrison & Heslop-Harrison (1970). marked trees growing at following sites in Agra city (stretching across 26° 44' N to 27° 25' N and 77° 26' E to Number of ovules/flower- Number of ovules/flower 78° 32' E) of Uttar Pradesh (India): 1. Raja Balwant was recorded by the method after Stelly et al. (1984). Singh College; 2. Mental Hospital; 3. Moti Lal Nehru Floral buds fixed in formalin-acetic-alcohol (F.A.A.) Park; 4. Cantonment Park and 5. Ram Bagh during the were hydrated and stained in Mayer’s hemalum (using years 2012 and 2013. Sass’s modification of 20 g instead of 50 g of alum per liter), for 1-2 days. Destained with 2.0% acetic acid for Phenological data on leaf fall, leaf renewal, initiation of 1-2 days and rinsed with tap water for 2-24 hours. flowering, peak flowering period, last stages of Dehydrated, infiltrated with xylene and cleared with flowering and time of fruiting was recorded. Flowering methyl salicylate in the series 2:1, 1:2 (xylene: methyl phenology (time of anthesis, anther dehiscence, stigma salicylate) for 15+ minutes each. The number of ovules receptivity) from five marked branches on different from the cleared ovaries were counted using plants was collected. Number of flowers/inflorescence, stereomicroscope. size of floral parts, fruit-set percentage, and number of seeds/fruit was recorded. Breeding system : Flowering phenology—The number of flowers on five 1. Open pollination-Floral buds on 20 inflorescences marked branches each on ten marked plants were before anthesis were tagged and left for open counted periodically throughout the flowering period. pollination. The beginning, peak and end of flowering, as well as the 2. Hand pollinations experiments- The stigmas at the relative flowering intensity (average number of time of maximum receptivity in the emasculated and flowers/inflorescence x average number of bagged flowers were pollinated with the pollen inflorescences/individual) was registered following the collected from freshly dehisced anthers from the procedure of Dafni (1992). Number of simultaneously flowers of the same tree or from flowers of different open flowers/inflorescence/branch/tree/day was trees between 0900-1100 h. The number of fruits counted. Time of opening of flowers, anther dehiscence, formed in each hand pollination experiment was and stigma receptivity was recorded. Time of opening of recorded. new flowers i.e. when the petals separated to expose the androecium and gynoecium was recorded in the marked Pollination biology—Observations on the floral floral buds. Stigma receptivity was determined by hand- visitors and their behaviour were made following the pollinating 100 stigmas at different stages of procedure given by Faegri & van der Pijl (1979) and development and in vivo pollen germination on stigmatic Dafni (1992). Insect visitors on the flowers were surface was recorded by aniline blue fluorescence observed as potential pollinators. Their behaviour on the method after Martin (1959). Formation of fruits was flower, their flying pattern across inflorescences, time of observed periodically in tagged floral buds on marked visit and kind of resource collected were recorded. plants. The longevity of flowers was observed by the Captured individuals were pinned and identified with method after Gill et al. (1998). These changes were the help of Dr. Girish Maheshwari, School of recorded every day throughout the flowering period. Entomology, St. John’s College, Agra. Pollen load of Pollen production/anther/flower—Mature anthers their body parts was counted under stereomicroscope were crushed in lactophenol-glycerine with aniline blue. (Zeiss). A known dilution was placed on the grid and 10 replicate Nectar—Volume of nectar from individual flowers (25 counts were made using a hemocytometer (Barrett 1985). from each marked plant) was measured using 20 µl Pollen viability—Pollen viability was checked by 0.2% microcapillary tubes. Nectar volume was computed with TTC (2, 3, 5-triphenyltetrazolium chloride) solution in the following equation after Cruden & Hermann (1983). 130 The International Journal of Plant Reproductive Biology 7(2) pp.128-134, 2015 July, 7 (2)

Scanning electron microscopy (SEM)— For SEM unilocular, with parietal placentation. A nectary disc studies, fresh floral parts (calyx, corolla, anthers and arises as a receptacular outgrowth below the ovary pistils) and pollen grains were fixed in 3% independently from stamens (Fig. 2e). Caris et al. glutaraldehyde. These were dehydrated through aqueous (2006) have also observed a nectar disc below ovary in

acetone series, dried with CO2 in a HCP-2 Hitachi Cneorum tricoccon (Rutaceae) and found the presence critical point dryer. The samples were coated with gold of two kinds of unicellular hairs on the outer surface of (20nm) in a SCD 020 sputter coating Unit (Polorn ovary. The long hairs were present all over the surface, Equipment Ltd., Welford, England) and observed in a while short club-shaped once were restricted near slits in Philips EM 50, SEM at All India Institute of Medical the ovary and the swollen emergence at the base of the Science, New Delhi. style. Caris et al. (2006) observed that the regions at the base of the style stained slightly darker indicating OBSERVATIONS & DISCUSSION secretory activity. Style is erect with capitate and wet type of stigma bearing several hairy papillae (Fig.2 d). Feronia limonia is a deciduous, slow-growing, erect tree of 10±2 (n=5) meter height with a few upward Nectar-The nectariferous disc develops below the ovary. reaching branches bending outward near the summit Several unicellular, long tapering trichomes arise from where they are subdivided into slender branch lets the base of this disc (Figs. 2e, f). The nectariferous disc drooping at the tips (Fig. 1a). Bark is ridged, fissured and and trichomes start secreting nectar in the morning hours scaly; on some of the zigzag twigs, there are 2-5 cm long between 0630-0800 h (115± 25.7 µL/flower). As the day sharp spines. Leaves are alternate and pinnately progresses, nectar secretion gradually increases and compound; 7.5-12.5 cm long with 5-7 leaflets. Each between 1030-1130 h it reaches to the maximum leaflet is 30±5 mm long and 15±5 mm broad, dark-green, (214.72±35.12 µL/flower). Later the quantity decreases leathery, notched at the apex, dotted with mild lemon- gradually and by the evening (1700-1800 h) it is scented oil glands (Figs.1b, c). minimum (58±12.3 µL/flower). Ren & Tang (2012) observed that the nectaries at the base of the ovary in Ruta Phenology- The leaves shed in October-November; graveolens (Rutaceae) produce large quantity of nectar flush of new leaves appear in February-March. and nectar secretion continues after nectar removal. Flowering is initiated in February and continues up to May and fruiting takes place in May and June (Fig. 1b). Flowering phenology—Flowers open between 0600- The mature fruits remain attached even after the 0800 h; Anthers dehisce between 0700-0900 h by a commencement of flowering in the next season. Kumar longitudinal slit (Figs. 2a, d-arrows). Stigma in open & Kalavathy (2013) have recorded phenological cycle in flowers becomes receptive between 0900-1100 h. Feronia limonia trees growing in north Gujarat, India. Pollen biology—The pollen grains are tri- (Fig. 1b), All the phenological events recorded by them are 15-20 tetra- and penta-colporate (Fig.2c). The exine is days ahead to those recorded at Agra. reticulate and steriate (Figs. 2b & c). Grant et al. (2000) Floral biology—The flowers are dull-red or greenish, have described six pollen types in the sub-family- up to 1.25 cm wide, borne in small, loose, terminal Aurantioideae of Rutaceae. According to them the cymes (Fig. 1c). The flowers are regular, bisexual, pollen grains of Feronia limonia are of Citrus media type pentamerous, hypogynous. Calyx consists of five free and the pollen of this type are both 4 or 4/5 colporate and green sepals with valvate aestivation (Fig. 1d). Corolla the pollen grains of Feronia limonia are tetra- and penta- consists of 4-5 free yellow petals with imbricate colporate, circular in polar out line or circular or slightly aestivation. Androecium comprises 8-10 stamens with elliptical. in equatorial out line. According to Perveen & their small filaments united into a disc (Figs. 1c, d). Qaiser (2005) pollen grains of 7 species of the family Anthers are yellow when young, but turn pink (Fig. 1c) Rutaceae in Pakistan are symmetrical, isopolar, 3-5 and finally become blood red (Fig. 1d). Gynoceium is colporate, Tectum striate- reticulate or reticulate- penta-carpellary, syncarpous. Ovary is superior, refulate. Each anther produces 3157±156 (n=50) pollen 2015 Reproductive Biology of Feronia limonia- Chauhan 131

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Fig. 1— a-h. Feronia limonia. a. Tree, b. Tree with fruits c. Flowering twig, d. Inflorescence (p: pistil; st: stamen); e. Hover fly (arrow), f. Honey bee on the open flower (arrow). 132 The International Journal of Plant Reproductive Biology 7(2) pp.128-134, 2015 July, 7 (2)

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c

Fig. 2 a-f. Scanning electron microphotographs of floral parts. a. Part of flower showing stamens (st), disc (di), trichomes (t) and a petal (p), b. Single anther dehisced through a longitudinal slit (arrow), c. pollen grains (po) on the stigmatic surface, d. dehisced anther (st), papillate (pa) stigma (stg.), e. disc with several trichomes (ti), f. magnified view of trichomes (ti) and petals. (Bars a, b and e: 200 µm; c: 10 µm; d and f: 100 µm). 2015 Reproductive Biology of Feronia limonia- Chauhan 133

grains and per flower there are 25266±1714 (n=50). ovule ratio suggested facultative geitonogamy in Aegle Pollen viability as tested by 0.2% TTC and FCR test was marmelos (Rutaceae). 93±5% and 81.5% respectively. Fruits-Fruits are berry, round to oval, 7±4 cm in Pollination biology-Large quantity of pollen and nectar diameter, with a hard, woody, grayish-white, scurfy rind attracts honey bees (Apis cerana indica) (Fig.1f), about 6 mm thick, pulp light brown, mealy, odorous, halictid bees (Lasioglossum spp.), wasps (Vespa spp.), resinous, astringent, acidic, with numerous small, white yellow butter flies (Eurema spp.) and hoverfly or seeds scattered in the fruit pulp (Fig.1 c, f, g) and the marmalade (Episyrphus spp.) (Fig. 1e). They forage fruits ripen in early October and remain attached on the mainly around the centre of the flower and collect nectar. trees even after the plants are in full bloom in the next The main pollinators are Apis cerana indica and flowering season. Mature fruits drop down and crack Lasioglossum spp. Honey bees and halictid bees in large and the pulp along with seeds quickly rot due to the numbers forage the flowers between 1000-1330 h. infestation of several saprophytic fungi e.g. Aspergillus, They come in contact with the dehisced anthers and Penicillium and Rhizopus (Fig. 1 h). collect pollen which is transferred on the hairy stigmatic In the light of foregoing observations it is concluded surface of other flowers. On the other hand, butter flies that this tree species shows facultative xenogamous (Eurema spp.), wasps (Vespa spp.), and hoverfly breeding system. For the conservation of this (Episyrphus spp.) also visit flowers in the morning medicinally important tree, the seeds of mature fruits hours (0800-1100 h) but are only nectar robbers. collected from the trees should either be grown in vitro or Number of ovules/flower- There are 79±18 in the nursery and the seedlings thus raised should be ovules/flower and pollen-ovule ratio is 319.8 indicating transferred into its natural habitat. facultative xenogamous breeding system in this tree Acknowledgements- Sincere thanks are due to the (Cruden 1977). Department of Science & Technology, New Delhi for financial assistance. Thanks are due to the Incharge, Breeding behavior—There was 78.9±25% fruit set in Electron microscopy facility, ITRC, Lucknow and All open pollinated flowers. The bagged flowers failed to India Institute of Medical Sciences, New Delhi for SEM produce fruits, indicating self-incompatible nature of the facility. plant. In the emasculated bagged flowers there was 18.4±8% and 81.6±34% fruit set by geitonogamy and LITERATURE CITED xenogamy respectively. The fruit set percentage in a solitary tree growing for last 25 years at Agra campus of Aslam S, Zahan M, Akter S, Banu TA & Habib A 2010. Raja Balwant Singh College was significantly poor. A Mass propagation of Feronia limonia L. through tissue young plant developed near the older one from the self culture. Bangladesh J. Sci. Indus. Res. 45(1) 75-78. sown seed during last 5 years and is flowering now. Interestingly, the fruit set percentage on the older tree Bala R & Kaul Veenu 2010. Floral traits in relation to increased significantly (Chauhan SVS Personal breeding system in Murraya koenigii (L.) Spreng. Inter. J. Plant Repro. Biol. 2(1) 79-84. communication), indicating facultative xenogamous nature of the tree. This is further supported by low Barrett SCH 1985. Floral trimorphism and pollen-ovule ratio of the tree (Cruden 1977). Bala & monomorphisim in island population of Eicchornia Kaul (2010) have observed mixed mating system in paniculata (Pontederiaceae). Bot. J. Linnean Soc. 25(1) Murraya koengii (Rutaceae). According to them, higher 41-60. pollen-ovule ratio, male-biased sex-allocation ratios and results of pollination experiments support mixed mating Caris P, Smets E, Custer K De & Craene LP Donse De system in Murraya koengii. Sharma et al. (2011) on the 2006. Floral ontogeny of Cneorum tricoccon L. basis of their hand-pollination experiments and pollen- (Rutaceae). Pl. Syst. Evol. 257 223-232. 134 The International Journal of Plant Reproductive Biology 7(2) pp.128-134, 2015 July, 7 (2)

Cruden RW 1977. Pollen-ovule ratios, a conservative species in north Gujrat region (NGR), Gujrat, India. indicator of breeding systems in flowering plants. Inter. J. Environ. 2(1) 60-69. Evolution 31 32-46. Martin FW 1959. Staining and observing pollen tubes in Cruden RW & Hermann SM 1983. Studying nectar? the style by means of fluorescence. Stain Tech. 34 125- Some observations on the art. In: Bentley B & Elias T 128. (eds.) The biology of nectaries. Columbia University Press, New York. Pp. 223-241. Moza MK & Bhatnagar AK 2007. Plant reproductive biology studies crucial for conservation. Curr. Sci. Dafni A 1992. Pollination ecology: a practical 92(9) 1207. approach. New York. Oxford University Press, New York, Pp.250. Perveen A & Qaiser M 2005. Pollen flora of Pakistan- XLV- Rutaceae. Pak. J. Bot. 37(3) 495-501. Faegri K & Pijl van Der L 1979. The Principles of Pollination Ecology. Pergamon Press, London. Ren Ming-Xun & Tang Jing-Yu 2012. Up and down: stamen movements in Ruta graveolens (Rutaceae) Gill GE, Ryan JR, Fowler T & Mort SA 1998. Pollination enhance both outcrossing and delayed selfing Ann. Bot. Biology of Symphonia globulifers (Cluiaceae) in Central DOI:10.109/aob/mcs181 French Guiana. Biotropica 30 139-144. Sharma Lalita, Rana Anita & Chauhan Seema 2011. Grant M, Blackmore S & Morton C 2000. Pollen Reproductive biology of Aegle marmelos (L. Correa morphology of the subfamily Aurantioideae (Rutaceae). (Rutaceae) Inter. J. Plant Repro. Biol. 3(2) 151-154. Grana 39(1) 8-20. Stelly DM, Peloquin SJ, Palmer RG & Crane CF 1984. Hauser EJP & Morrison JH 1964. The cytochemical Mayer’s hemalum methyl salicyate; a stain clearing reduction of nitro blue teetrazolium as an index of pollen technique for observation within whole ovules. Stain viability. Am. J. Bot. 51(1) 748-752. Tech. 59 155-161. Heslop-Harrison J & Heslop-Harrison Y 1970. Tandon R, Gupta P, Sunnichan VG, Shivanna KR & Evaluation of pollen viability by enzymaticallyinduced Mohan Ram HY 2005. Reproductive biology of some fluorescence: intercellular hydrolysis of fluorescence Indian trees of economic importance. In: Chaturvedi SN diacetate. Stain Tech. 45 115-120. & Singh KP (eds.) Plant Reproductive and Molecular Kumar R & Kalavathy S 2013. Human threat on Biology. Aaavishkar Publishers, Distributors, Jaipur. Pp. phenological cycle of selected dry deciduous tree 10-29.