T REPRO N DU LA C The International Journal of Plant Reproductive Biology 9(2) Jul., 2017, pp.109-114 P T I F V O E

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S H Studies on pollen biology and stigma receptivity of Garcinia imberti Bourd. T (Clusiaceae)- a critically endangered tree of Western Ghats, Kerala Rajkumar K.1, Keshavanarayan P.1, Shubharani R.1, 2 and Sivaram V.1, 2* Laboratory of Apiculture and Biodiversity Conservation, 1Department of Botany, Bangalore University, Bangalore-560056, India. V Sivaram Research Foundation, No. 132, 2nd Main, R.R. Nagar, Bangalore -560056, India. *e-mail: [email protected] Received: 14.03.2017; Revised : 01.05.2017; Accepted & Published online : 01.06.2017 ABSTRACT Garcinia imberti Bourd. (Clusiaceae) is a critically endangered tree endemic to Agasthyamalai hills of Western Ghats, Kerala, India. In the present investigation, the pollen production, stigma receptivity, pollen viability, in-vitro pollen germination and pollen tube growth was studied during 2012 and 2014. The male flowers produced 7094 pollen grains per anther and 1, 41,890per flower.The pollen grains were round in shape with an average diameter of 20-36 μm. The stigma became receptive only after the opening of flowers. The pollen viability was 89.19±2.11% as tested by2% TTC. The in vitro pollen germination was 72.73±9.20% with 67.13±21.33% long pollen tubes in 20% sucrose solution. Higher pollen-ovule ratio (70945:1) and fruit and seed-seet only by cross pollination in self-incompatible Garcinica imberti indicated obligate xenogamy. Keywords :Garcinia imberti, Clusiaceae, stigma receptivity, pollen production, viability, pollen germination, pollen tube growth.

The genus Garcinia consists of over 400 species and is is one of basic factor among several environmental conditions one of the largest genus in Clusiaceae also known as which influences pollen germination and longevity of stored Guttiferae. As per IUCN Red list and World Conservation pollen (Sharafi 2010). In vitro pollen germination is one of the Monitoring Centre (1998), Garcinia it is a critically most convenient and reliable methods used to test the viability endangered tree species distributed across the tropical regions of fresh or stored pollen. The media used for in vitro of Asia, Africa and Polynesia (Whitemore 1973). There are germination of pollen of different species ranges from simple about 30 evergreen polygamous tree species of Garcinia sucrose/boric acid media (Stanley and Linskens 1967) to distributed in India, which include G. combogia, G. complex media containing polyethylene glycol (Shivanna et xanthochymus, G. cowa, G. imberti and G. indica. Many of al. 1997, Tandon et al.1999) and various amino acids (Read et these species of Garcinia are commercially important and al.1993). Brewbakers and Kwack (1963) medium containing used as timber, medicine, resin oil, pigment, fodder and edible important ingredients like, sucrose, boron and calcium has fruits (Cox 1976). Garcinia imberti is a dioecious evergreen, been widely used and found suitable for more than 86 plant critically endangered tree species endemic to Southern species. However, some flowering plant species do not always Western Ghats, Kerala, India and only a limited number of respond satisfactorily to Brewbakers and Kwack medium, and trees were available in Agasthyamalai hills. sometimes alternative media are required (Brewbaker and Studies on pollen biology and stigma receptivity are Kwack 1963). essential for pollen-pistil interaction and in developing In the light of facts enumerated above, present effective strategies for both in situ and ex situ conservation of investigation has been undertaken to study the pollen biology the species (Moza and Bhatnagar 2007). Researches on and stigma receptivity, in the endangered tree species of various aspects of reproductive biology have been mostly Garcinia imberti. conducted in herbaceous crops and trees have been neglected. MATERIALS AND METHODS This is largely due to various difficulties including their large size, prolonged juvenility, long life cycles, inaccessible Study period and study site - The present study was flowers, breeding, progeny testing and selection of elites conducted on twenty dioecious populations of Garcinia (Tandon et al. 2005). imberti during the two consecutive flowering seasons (2012 Studies on pollen biology are crucial for assisting and 2013).The natural distribution zone and population of breeding programmes, in which regular monitoring of pollen Garcinia imberti is located in undisturbed tropical rainforest viability and longivity are essential (Heslop-Harrison 1987, between N 8˚41.204 Latitude; E 77˚11.275 Longitude of Shivanna and Rangaswamy 1992). Transfer of sufficient Agasthyamalai ranges which covers an area of 2000 sq.km2. quanity of viable pollen on receptive surface of stigma are The identification was confirmed by matching either with type essential for successful pollen-pistil interaction and seed-set specimens or the Herbarium available (BSI, MH, and TBGRI) (Dekers and Porreye, 1984, Stosser et al.1996). Temperature (Herbarium specimen numbers: 11402, 11412 and 162084). 110 The International Journal of Plant Reproductive Biology 9(2) July 2017, pp.109-114

Pollen morphology, pollen production and pollen 20% and 25%) were also used to evaluate the pollen ovule ratio - The pollen morphology was studied by germination and pollen tube growth. Pollen grains from fresh acetolysis method (Erdtman, 1969). The anthers were open flowers were collected at the time of anther dehiscence collected and crushed using glass rods and freshly prepared were dusted on a drop of Brewbaker's medium placed on clean acetolysis mixture in the ratio 9:1 from acetic anhydride and cavity slides. Germination chambers were observed under concentrated sulphuric acid was added. The mixture was light microscope and pollen germination was determined by placed in water bath at 80-90°C for 5 minutes, stirred, centrifuged and supernatant decanted. A drop of glycerine was the formula reported below: added and the samples (pollen grains) were then transferred Pollen germination (%) = No. of pollen grains germinated into storage vials from where they were mounted on slide to Total no. of pollen grains study the pollen morphology. Pollen production per anther and RESULTS flower, procedures after Dafni (1992), Shivanna and Rangaswamy (1992) and Kearns and Inouye (1993) were Stigma receptivity- The stigma receptivity was observed used. The mature anthers were crushed and transferred to in twenty flowers for three days by hydrogen peroxidase test. calibrated tubes containing 0.5 ml 70% ethanol. The pollen The stigmas at floral bud stage were non-receptive, however, grains were counted by haemocytometer and the total pollen the stigma became receptive in fresh open flowers. The grains within the 25 square counting area was determined with viscous exudates gradually increased on the stigmatic surfaces five replicates. These replicates were prepared from twenty and the entire stigmatic surface in the fresh open flowers was anthers from five different flowers. The pollen ovule (P/O) covered with exudates indicating their receptivity. The ratio was calculated using the following Cruden's (1977) receptivity of the stigma gradually decreased with age and formula: within one or two days as the stigmatic lobes became brown Pollen count No. of anthers per flower a n d completely dry, indicating complete loss of receptivity P/O ratio = × per anther No. of ovules per flower (Fig. 1 A-E). Pollen morphology and pollen viability-- The pollen Stigma receptivity— The duration of stigma receptivity grains were acetolysed after the procedure of Erdman (1969). was determined for three days after anthesis by testing stigmas The pollen grains were round in shape, spiny and light brown of 10 undamaged fresh flowers at different stage by placing in colour. The average diameter of pollen grains was 20-36 μm them in a 3% solution of hydrogen peroxidase according to (Fig.2E).. The pollen viability was assessed by different standard method (Galden and Plowright 1987, Wendel and staining solutions is presented Table 1 (Figs. 2 A-B, 3). Weeden 1989, Kearns and Inouye 1993, Dafni et al. 2005). Maximum pollen viability (89.16±2.11%) was recorded in The presence of bubbles on the stigma surface indicates that 0.2% TTC (Tryphenyl Tetrazolium Chloride) and lowest the stigma is producing the enzyme peroxidase and number of (84.35±4.12% ) in 1% acetocarmine stain. receptive stigmas was counted in each individual flower with In vitro pollen germination and pollen tube growth - the help of hand lens (Folding magnifier 10x). The percentage of pollen germination and pollen tube growth Pollen viability— The pollen viability was determined was assessed in Brewbaker and Kwack' media containing 5%, by 0.2.0% TTC (2, 3, 5-Triphenyl Tetrazolium Chloride) in 10%, 15%, 20%, 25%, sucrose was 60.83%, 64.98%, 79.10%, sucrose solution after Hauser and Morrison (1964). The fresh 81.44% and 77.30% with average length of pollen tube growth anthers were collected from ten flowers and mixed randomly. was 40.04 µm, 59.04 µm, 60.00 µm, 94.40 µm and 82.20 µm Pollen from fresh dehisced anther were sprinkled in 1-2 drops respectively (Table 2). The maximum percentage of pollen of TTC solution on a clean micro slide and a cover glass was germination (82.44%) and pollen tube growth (94.40 µm) was placed over it. Similarly the pollen grains from fresh dehisced observed with the 20% sucrose concentration and the mean anthers were placed in a drop of 1.0% Acetocarmine (1 g average of pollen germination and pollen tube growth was aceocarmine dissolved in boiling 45% glacial acetic acid and a (72.73±9.20) and (67.13±21.33) respectively. The pollen pinch of ferric acetate after cooling) and covered by a cover germination (60.83%) and the pollen tube growth (40.04 µm) slip. These were observed after incubating for 1 h in dark at were lowest in 5% sucrose concentration. room temperature (Dafni 1992). Pollen production and pollen ovule ratio- The average In vitro pollen germination-– In vitro pollen pollen grains produced per anther was 7094.5 and pollen germination was observed by hanging drop technique using grains per flower were 1, 41,890. The ovary was globose and Brewbaker and Kwack (1963) medium containing sucrose, superior with two ovules and each in one locule and the pollen

Ca(NO3)2.H2O, MgSO4.7H2O, borate (H3BO3) and KNO3. The ovule ratio (P/O) was estimated as 70945:1 according to different concentrations of sucrose solution (5%, 10%, 15%, Cruden's method (1977). 2017 Studies on pollen biology and stigma receptivity of Garcinia imberti Bourd...... 111

Nv

A B C Nv A B

Nv D E C D E — 20-36 µm

Fig.2. Pollen viability, pollen germination, pollen tube growth and Fig.1. Stigma receptivity: A) Non-receptivite at bud stage, B) Stigma pollen morphology: A) Pollen viability in TTC, B)Pollen viability in receptive on the day after anthesis, C & D. Receptive stigma showing Acetocarmine stains (Nv- Non viable, V-Viable pollen grains), C & D. high level bubbles, E) Dried stigma Pollen germination and pollen tube growth, E) Pollen showing thick

Table 1- Pollen viability in Garcinia imberti Sl. No No. of total No. of viable No. of non-viable Pollen viability (%) pollen grains pollen grains pollen grains

A B A B A B A B

1 20 30 18 24 2 6 90.00 80.00

2 39 36 36 32 3 4 92.30 88.88

3 36 42 32 36 4 6 88.88 85.71

4 41 39 36 34 5 3 87.80 87.17

5 38 40 33 32 5 8 86.84 80.00

Mean 89.16 84.35

Standard deviation 2.11 4.12

Standard error 0.94 1.84

***A. 0.2% Tetrazolium Chloride (TTC) test), B. Acetocarmine test (1.0%)

Table 2- Pollen germination and tube growth in Garcinia imberti

Sl. No Concentrations of Mean no. of Mean no. of Pollen Pollen tube growth sucrose solutions total pollen germinated pollen germination (%) length (µm)

1 5 % 87.20 53.20 60.83 40.04

2 10 % 89.20 57.80 64.98 59.04

3 15 % 97.80 77.20 79.10 60.00

4 20 % 99.40 72.60 81.44 94.40

5 25 % 99.00 76.40 77.30 82.20

Mean 67.44 72.73 67.13

Standard deviation 11.15 9.20 21.33

Standard error 2.23 4.11 9.54 112 The International Journal of Plant Reproductive Biology 9(2) July 2017, pp.109-114

pollen production, morphological homogeneity and pollen Acetocarmine (1.0%) viability are favourable for fertilization. The average number TTC (0.2%) of ovules per flower was two and the pollen ovule ratio (P/O) was calculated as 70945:1. The high pollen-ovule ratios in the flowering plants reflect the cross pollination (Jurgens et al. 2002). Higher P/O ratio recorded in self-incompatible Garcinia imberti is indicative of obligate xenogamy (Cruden). Rajkumar et al. (2015) observed fruit-set only after manual transfer of pollen from male flowers to the stigma of female flowers in this tree species confirming obligate xenogamy. The rate of stigma receptivity indicates the successful fertilization in sexual reproduction. Therefore, indentifying the optimum conditions for stigma receptivity is an important aspects of any evolution of reproductive behavior (Shivanna and Tandon 2014, Espinoza et al. 2002). In Garcinia imberti, Fig.3. Pollen viability in TTC and Acetocarmine test no receptive (no vigorous bubbles) was observed before opening the flower and more vigorous bubbles was observed Pollen germination (%) after opening of flower on the stigmatic surface. The stigma Pollen tube length (µm) receptivity of flower varies from few hours to few days (Dafni 1992). These results indicate that the receptive of stigmas was concomitant with the secretion of the exudates. The stigmatic exudates has the important functions such as the attachment of pollen at the stigmatic surface, rehydration of pollen for germination and pollen tube entry into the style (Richards 1997). The pollen viability is generally considered to indicate the ability of pollen grains to perform its function of the delivering the gametes to the embryo sac following compatible pollination (Shivanna et al.1991). Assessment of pollen viability is a pre-requisite and also important in studies on pollen storage, reproductive biology and hybridization Fig.4. Pollen germination and pollen tube growth in Brewbaker & (Heslop-Harrison et al. 1984). According to the findings of the Kwack Medium. harvested pollen grains of many Garcinia species have high viability as assessed by their stain ability like 99.4% of DISCUSSION Garcinia corymbosa, 92.5% of Garcinia forbesii, and 85% of The pollen morphological studies provide useful Garcinia cf. forbesii (Ha et al.1988). In the present information on the taxonomy of plant species and play an observation, the average pollen viability of Garcinia imberti important role in the lifecycle of flowering plants. Pollen is was similar in both experiments as 89.19±2.11% in TTC and classified on the basis of their shape, size, symmetry, polarity, 84.35±4.12% in Acetocarmine test. This characteristic is very aperture and exine sculpturing (Perveen 1993). The present significant in this genus and these findings were supported by observation revealed that the pollen grains of Garcinia imberti Takagaki et al. (1995), Han et al. (1996) and Wang (1997). were round with average diameter of 20-36 μm. Lopes and Viable pollen grains were not found in Garcinia dulcis, while Machado (1998) reported that the pollen grains of Clusia in male flowers of Garcinia cowa, Garcinia speciosa and nemorosa (Clusiaceae) were powdery, suboblate and Garcinia schomburgkiana there were 93-100% viable pollen tricolporate. Nnamani and Nwosu (2012) recorded grains (Te-chato 2007). tetracolpolate type of pollen with pistillate sculpturing In the present study, the maximum pollen germination ornamentation in Garcinia kola. The pollen grains of Garcinia was observed as 81.44% in 20% of sucrose solution and the hombroniana were identified as suboblate, granulum in average pollen germination was 72.73±9.20%. Likewise, high aperturate and circular in shape (Ibrahim et al. 2012). The percentage of pollen germination (95%) and presence of large pollen production tests showed that the male flowers produced number of pollen grains with elongated pollen tube were sufficient amount pollen grains with the average pollen observed in Coccinia grandis, a dioecious climber indicated production per anther was 7094.5 and per flower was 1, the potential of the pollen grains effecting fertilization (Shaina 41,890. Gercekcioglu et al. (1999) has reported that the high and Beevy 2015). Kumari et al. (2009) have reported that 2017 Studies on pollen biology and stigma receptivity of Garcinia imberti Bourd...... 113

percentage of pollen germination increases with increase in Espinoza F, Pessino SC, Quarin CL and Valle EM 2002. concentration of sucrose in Trichosanthes dioica Effects of pollination timing on the rate of apomictic (Cucurbitaceae). The sucrose is the best carbohydrate source reproduction revealed by RAPD markers in Paspalum for pollen germination and tube growth for most of the plants notatum. Ann. Bot. 89 165-170. investigated (Hrabetova and Tupy 1964). The pollen Galen C and Plowwright RC 1987. Testing the accurancy of germination and pollen tube elongation are two distinct using Peroxidase activity to indicate stigma receptivity, processes differing in their sensitivity to different Can. J. Bot. 65 107-111. concentrations of the medium. The present study showed the maximum pollen tube growth (94.40) in 20% sucrose Gercekcioglu R, Gunes M and Ozkan Y 1999. A study on concentrations and the average pollen tube growth was determination of pollen quality and pollen production of 67.13±21.33%. In flowering plants, the pollen tube delivers some fruit cultivars grown in Tokat ecological conditions. sperm cells to the embryo sac. The pollen tube growth Bahce. 28(1-2) 57-64 proceeds through tip extension and affected by many factors, Ha CO, Sands VE, Soepadmo E and Jong K 1988. including temperature, medium osmolarity and the Reproductive patterns of selected understory trees in availability of calcium, zinc and boron (Sawidis and Reiss Malaysian rain forest: the apomictic species. Bot. J. Linn. 1995). Soci. 97 317-332. Acknowledgement —We extend our thanks to the Ministry of Han XB, Li RQ, Wang JB and Miao C 1996. Effect of heat Environment and Forests, Government of India, New Delhi stress on pollen development and pollen viability of for extending financial assistance under the All India Pepper, Acta.Hort. Sinica. 23(4) 359-64. coordinated research project (AICRP) on “Reproductive biology of RET Tree species” (FNo.22/10/2010-RE) to Hauser EJP & Morrison JH 1964. The cytochemical reduction undertake the present investigation. The authors are thankful of nitro blue tetrazolium as an index of pollen viability. to the Department of Botany, Bangalore University, Am. J. Bot. 51(7) 748-752. Bangalore - 560056 for providing facilities and Kerala Forest Heslop-Harrison J 1987. Pollen germination and pollen-tube Department for providing permission to undertake research in growth. Int. Rev. Cytol. 107 1-78. Agasthyamalai forest range. Heslop-Harrison J, Heslop-Harrison Y and Shivanna KR REFERENCES 1984. The evaluation of pollen quality, and a further appraisal of the fluorochromatic (FCR) test Brewbaker JL and Kwack BH 1963. The essential role of procedure.Theor. Appl. Gene. 67 367. calcium ion in pollen germination and pollen tube growth. Am. J. Bot. 50 859-865. Hrabetova E and Tupy J 1964. The growth effect of some sugars and their metabolism in pollen tubes. In: Linskens Cox JEK 1976. Garcinia mangostana-mangostana. The HF(ed) Pollen Physiology and Fertilization. North- propagation of tropical fruit trees. Horticultural Review, Holland Publ, Amsterdam, the Netherlands. Pp. 95-101. No.4 Common wealth Bureau of Horticulture and plantation crops. East Mallin, Kent. Ibrahim IF, Balasundram SK, Abdullah NAP, Alias MS and Mardan M 2012. Morphological characterization of Cruden RW 1977. Pollen-Ovule Ratios: A Conservative pollen collected by Apis dorsata from a tropical Indicator of Breeding Systems in Flowering Plants. Evol. rainforest. Inter. J. Bot. 8(3) 96-106. 31(1) 32-46. Jurgens A, Witt T and Gottsberger G 2002. Pollen grain Dafni A 1992. Pollination ecology: a practical approach. numbers, ovule numbers and pollen-ovule ratios in Oxford University Press, New York, New York, USA. Pp. Caryophylloideae: correlation with breeding system, 250. pollination, life form, style number, and sexual system. Sexual Plant Repro. 14(5) 279-289. Dafni A, Keavan PG and Husband BC 2005. Practical pollination biology. Enviroquest, Cambridge. Kearns CA and Inouye DW 1993. Techniques for Pollination Biologists. University Press of Colorado. Niwot, Dekers T and Porreye W 1984. Influence of the temperature on Colorado, USA. pollen germination of different cultivars of apple and pear Kumari A, Rashmi K, Rajeev R and Arun KP 2009. In-vitro trials in vitro. Acta Hort.149 123-130. pollen germination, pollen tube growth and pollen Erdtman G 1969. Handbook of Palynology. Hafner Publishing viability in Trichosanthes diodica Roxb. Inter.J. Plant Press, New York. Repro. Bio. 1(2) 147-151. 114 The International Journal of Plant Reproductive Biology 9(2) July 2017, pp.109-114

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