T REPRO N DU LA C The International Journal of Reproductive Biology 9(1) Jan., 2017, pp.18-24 P T I F V O E

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S H Standardization of pollen germination medium for eriocarpum – an T endemic, endangered tree of Central and Western Himalayas Kadambini Das*, Rajesh Tandon and A. K. Bhatnagar Department of Botany, University of Delhi, Delhi 110 007, India *e-mail : [email protected] Received: 12.09.2015; Revised: 25.06.2016; Accepted: 25.08.2016; Published online: 01.09.2016 ABSTRACT Skewed gender ratio of unisexual in a population could be detrimental to optimal fruit/seed-set and sustenance of a species. In instances of non-availability of sufficient pollen donors or quality pollen donors, storage of pollen for supplemental pollination has proved to be a reliable strategy. However, testing the germinability and viability of pollen grains under in vitro conditions at the time of collection or after storage is a pre-requisite. Optimization of pollen germination media, especially in case of threatened and endemic plant species, may help in their breeding and conservation to ensure seed formation in years of low yield and poor recruitment. Here, we present the details of a standardized pollen germination medium for Pittosporum eriocarpum, an endangered tree species, which does not respond to Brewbaker and Kwack’s (BK) medium, usually employed to ascertain pollen germinability. The optimized BK medium, improves pollen germination and the pollen tube growth responses up to 75% and 111.76 µm long, respectively; in control the response was only 28% and 60.08 µm long. Storage of pollen of the species at low temperature (4°C) helped in maintaining the viability up to a significant period of time (six months). Keywords : RET trees, pollen germination, pollen tube growth, germination medium. Abbreviations : relative humidity (RH), room temperature (RT), International Union for Conservation of Nature (IUCN), World Conservation Monitoring Centre (WCMC). Pittosporum eriocarpum is an endemic and endangered under near-optimum germination conditions. BK medium [WCMC, 1998 (IUCN 2015.2, B1+2c ver 2.3)] tree species of (Brewbaker and Kwack 1963) is the most frequently used and Central and Western Himalaya. It has an uneven patchy has been found to be suitable for over 86 plant species. distribution in the mixed forests of Uttarakhand (Mussoorie, However, species do not always respond Dehradun, and Nainital) and Himachal Pradesh (Kullu). The satisfactorily to BK medium, and thus adjustments in the populations are sub-dioecious and comprise of male, female concentration of the medium components is required and sexually labile individuals. These sexually labile sometimes (Brewbaker and Kwack 1964). Therefore, pollen individuals behave as male (set a negligible amount of ‘male germination media recommended so far range from simple fruits’ due to leaky dioecy) in one year and female in sugar/boric acid media (Stanley and Linskens 1964) to subsequent year or vice versa (personal observation). The complex ones containing polyethylene glycol (PEG) staminate and pistillate flowers are differentiated on separate formulated by Zhang and Croes (1982), Shivanna et al. (1997) trees, and flowering occurs from the first week of April to the and Tandon et al. (1999), and the more complex media using third week of May. The natural populations of the species are polyamines (Vikas et al. 2012) and amino acids (Read et al. dwindling at an alarming rate, mainly due to fragmentation, 1993, Jayaprakash and Sarla 2001, Tupý and Ríhová 1984). pollarding, habitat loss and overexploitation. This renders The present paper provides the details of formulation of poor chances of mating between the conspecifics leading to an optimized pollen germination medium for P. eriocarpum reduced natural recruitment. and storability of its pollen at low temperature conditions. In vitro pollen germination studies not only help in MATERIAL AND METHODS understanding different aspects of pollen biology but are also crucial for assisted breeding programmes, in which regular Collections of sample–Samples were collected from male monitoring of germinability of pollen samples, including trees located at the study site at Nainital (N 29º22’37.6" E those that are stored, is required (Heslop-Harrison 1987, 70º28’11.2"). Collections were done separately for storage as Shivanna and Rangawamy 1992). Manual pollination using well as testing viability and germinability of pollen grains at pollen from a distant plant donor (i.e. between individuals of different intervals of time period after anther dehiscence. For different populations) often enhances fruit-set and results in testing viability, flowers (n=25) were tagged on the day of greater amount of seed-set than when the pollen is sourced anthesis (DOA). Pollen were collected each day at 08.00 h in from the same population (personal observation). Thus, the morning from the day of anthesis (DOA) up to 4 days after before attempting any breeding experiment, testing the anthesis (DAA) i.e. on DOA, 1 DAA, 2 DAA, 3 DAA, 4 DAA germinability of pollen from different sources is necessary. and viability test was conducted. Anther dehiscence occurs Methods for testing pollen grain viability usually employ two days prior to anthesis. However, observations have been either the fluorochromatic reaction test or germinability test collected from the DOA, as it is the time when pollen grains 2017 Standardization of pollen germination medium for Pittosporum eriocarpum...... 19

are actually available to the pollinators. For storage, fresh drop cultures for pollen germination were raised in various polliniferous material (n=300 anthers) was collected at the concentrations of sucrose (10, 15, 20 and 25%). For each peak time of flowering from the mature floral buds, two days combination, pollen grains were pooled from five freshly before anthesis (DBA) as anther dehiscence occurs two days dehisced anthers and uniformly spread onto a clean glass slide; prior to anthesis. three replicates were used. The slide was maintained for 30 Morphology of pollen grains–Freshly collected pollen min in an improvised humidity chamber, IHC, (RHÃ95%) for grains were stained with safranine (1%), mounted with pre-hydration of pollen grains. A drop of sucrose solution was glycerine and observed under Zeiss Primo Star photo added to the pollen grains and kept in IHC for 30 min at RT microscope (Göttingen, Germany) for morphological studies. (25±2°C). Pollen grains were considered germinated when the For studying the pollen surface, anthers were fixed in freshly tubes measured twice the diameter of the grain (Tuinstra and prepared Karnowsky’s fluid for 12 h at 4°C (Karnowsky Wedel 2000). The germination response of pollen was 1965), buffered with 0.1 M sodium phosphate buffer at pH 7.2. expressed as percentage. After the standardization of the After fixation, samples were washed once with buffer and osmoticum, the percent of pollen germination was tested in dehydrated in a graded acetone series (10 to 100%) at room different modified BK media. First, we tested the BK medium temperature, RT, (25±2°C) for 20 min each. Using liquid CO2, with a slight modification by the addition of polyethylene the samples were critical point dried by Polaron Critical Point glycol, PEG 400 (10%). The other modifications were: boric

Drier (Serial No. 12–09–R–711/A) at 31.5°C with 1100 p.s.i. acid (H3BO3: 100, 200, 300 and 400 mg/L), calcium nitrate

Dehydrated samples were mounted on aluminium stubs and (CaNO3: 300, 400 and 500 mg/L), magnesium sulphate coated with gold particles (100A° thickness) under vacuum on (MgSO4: 200, 300 and 400 mg/L), and potassium nitrate sputter coating unit (JEOL JFC–1600 Auto Fine Coater, (KNO3: 100, 200, 300 and 400 mg/L) supplemented with Tokyo, Japan) for 19 sec (Hill and West 1982). The coated sucrose (20%) and PEG (10%). The length of the pollen tubes material was examined and photographed with JEOL (n=10) was measured using a calibrated ocular micrometer. JSM–6610LV (Tokyo, Japan) Scanning Electron Microscope The concentrations which gave maximum response were at an accelerating voltage of 3-5 KV. chosen for formulating the optimized BK medium. Estimation of viability–Viability of the pollen grains was Storage of pollen grains–Pollen grains were completely tested using fluorochromatic reaction test (Shivanna and released from the anthers by teasing them with the help of fine Rangaswamy 1992). Fresh pollen grains were suspended in a pair of forceps and a needle, collected in an aluminium foil sheet. drop of fluorescein diacetate (2 mg/mL in 20% sucrose Multiple sets of pollen grains (each for one time use) were placed solution) and placed in dark under humidity chamber in aluminium foil cups and kept over dry silica in a desiccator. (Relative Humidity, RH Ã90%) for 10 min at RT (25±2°C). After 24 h pollen grains from each cup were transferred to Pollen grains with bright yellow-green fluorescence were eppendorfs and sealed with parafilm (modified from Chanon considered viable, under an epifluorescence microscope and Jourdon 1998). For long term storage, the tubes were kept in (Zeiss Axio Scope A.1, Göttingen, Germany). a desiccator and were maintained at 4°C in the refrigerator. For Numbers of cells in freshly released pollen–Fresh pollen assessing the germination response of the stored pollen sample, grains were pooled from anthers on the DOA. A stock solution the samples were first kept at room temperature for 20 min and of ethidium bromide, EtBr (0.1%) was prepared and diluted then prehydrated for 30 min in an IHC. ten times to make the working solution (Shivanna and All statistical analyses were carried out using SPSS Rangaswamy 1992). A drop of EtBr solution was placed on version 22.0 (IBM®, SPSS®, AmosTM). The per cent data was glass slides with pollen grains, mounting in the same solution arcsine transformed before performing the one-way ANOVA; and observed under the epifluorescence microscope (Zeiss difference among the means was analyzed through post-hoc Axio Scope A.1, Göttingen, Germany) with UV-2A type filter Tukey’s test (pÂ0.05). having EX 330-380 exciter filter, DM 400 beam filter and BA RESULTS 420 barrier filter. Fertility of pollen grains–Pollen grains collected from the The pollen grains of P. eriocarpum are released as monads staminate flowers on the DOA were stained with 0.1% (Fig. 1A) and at 2-celled stage (Fig. 1D). Pollen grains are acetocarmine, mounted with glycerine. Preparations were isopolar to spheroid (Figs. 1A, 1C), smooth, small (22.3±2.39 studied at 40x using Zeiss Primo Star photo microscope µm), and trizonocolporate (Fig. 1C). On the DOA, nearly 17% (Göttingen, Germany) equipped with Power Shot G 10 camera of the pollen grains in a flower are sterile. The sterile pollen (Göttingen, Germany). grains were shriveled (Fig. 1B), lacked cytoplasmic contents. In vitro pollen germination and pollen tube growth–To The viability of the fresh pollen is 64. 46±7.40% and it declines standardize the osmoticum of the germination medium, sitting to 30.93±4.52% 4 DAA (Fig. 1D; 2). Pollen grains stored at 20 The International Journal of Plant Reproductive Biology 9(1) Jan 2017, pp.18-24

Fig. 1–Pollen biology. A. Isopolar to spheroidal pollen monads stained with safranine. B. Dark red (fertile) pollen grains stained with acetocarmine (indicated with an arrow) and light pink (sterile) pollen grains. C. Scanning electron micrograph of a tri-colporate, spherical, pollen grains with smooth exine. D. Fluorescence micrograph of pollen grain stained with FDA. Only the bright green fluorescent (arrow) pollen grains are the viable ones. Inset shows pollen grains at 2-celled stage as visualized after staining with Ethidium bromide.

Fig. 2–Viability and fertility of pollen grains. Per cent (mean ± SE) viable Fig. 3–Comparative effects of different treatments (control and pollen grains (dotted bars) at different time intervals after floral anthesis modified Brewbaker and Kwack's medium) on per cent pollen (DOA: Day of anthesis; DAA: Day after anthesis) and per cent (mean ± germination (mean ± SE) and tube growth (mean ± SE). Control: SE) fertility at the time of anthesis (DOA). Columns followed by same Modified B&K Medium + 10% PEG; O B&K: Optimized B&K Medium. letters are not significantly different as analyzed through one-way ANOVA using SPSS 22.0. 2017 Standardization of pollen germination medium for Pittosporum eriocarpum...... 21

Table 1–Details of in vitro pollen germination and tube growth in various media tested. The pollen grains were prehydrated for 30 min, and an incubation period of 90 min was used in all the treatments. Experiment Outcome Germination (%) Pollen tube length (µm) Experiment 1: Concentrations of sucrose (a) 10% 8.15±1.77a 23.84±0.56a (b) 15% 20.64±5.04ab 36.78±1.51b (c) 20% 24.59±6.09b 61.27±0.54c (d) 25% 15.05±5.39ab 41.99±0.42b Experiment 2: Modified BK medium (+ 10% PEG) 28.70±2.52a 60.08±6.14a

Experiment 3: H3BO3 (mg/L) + BK with 20% sucrose (a) 100 50.44±2.05b 94.4±4.57ab (b) 200 52.98±2.40b 109.36±3.74b (c) 300 69.26±5.26c 136.64±4.56d (d) 400 53.62±4.51b 103.44±2.71c

Experiment 4: CaNO3 (mg/L) + BK with 20% sucrose (a) 300 41.61±1.16b 140.8±4.28c (b) 400 50.55±1.11c 131.72±1.96c (c) 500 48.97±1.91b 95.04±1.72b

Experiment 5: MgSO4 (mg/L) + BK with 20% sucrose (a) 200 57.71±1.19a 69.36±3.25ab (b) 300 48.55±0.71a 83.76±4.21b (c) 400 38.94±0.89a 97.92±2.99ab

Experiment 6: KNO3 (mg/L) + BK with 20% sucrose (a) 100 49.59±1.20a 81.2±5.87b (b) 200 55.09±4.61a 72.72±3.93ab (c) 300 57.27±1.00a 143.12±4.55c (d) 400 39.72±1.12a 71.68±3.13ab Experiment 7: Optimized BK medium (20% sucrose + b b 10% PEG + 300 mg/L H3BO3 + 400 mg/L CaNO3 + 74.71±2.43 111.76±4.47

200 mg/L MgSO4 + 300 mg/L KNO3)

Response in different sucrose concentrations was compared separately and letters with different alphabets are significantly different as analyzed through one-way ANOVA. For rest of the experimental procedure, modified BK medium was treated as control. Per cent values were first arcsine transformed. Response of pollen grains, in terms of germination and tube growth, obtained in the different concentration of a particular mineral salt was compared to control after subjecting them to one-way ANOVA using SPSS version 22.0. Values denoted with different alphabets were significantly different. low temperature (4°C) for six months exhibited 60% viability, 57.27±1.00%) and the tube length (131.72±1.96, 69.36±3.25, i.e. a 10% decline from the time of storage. 143.12±4.55µm), respectively. Thus, the final optimized Initially no germination response was achieved after 30 pollen germination medium for P. eriocarpum (Experiment 7) min of incubation in BK medium supplemented with various was comprised of sucrose (20%), boric acid (300 mg/L), concentrations of sucrose at RT (25±2°C). However, the calcium nitrate (400 mg/L), magnesium sulphate (200 mg/L), pollen grains responded to germination after the incubation potassium nitrate (300 mg/L) and PEG (10%). This medium period was enhanced to 90 min. With this composition, could significantly enhance the germination response up to sucrose at 20% gave the best response of germination 74.71±2.43% and tube length up to 111.76±4.47 µm (Fig. 3). (24.59±6.09%) and tube growth (61.27±0.54 µm long) Storage of pollen grains at low temperature conditions (4°C) (Experiment 1, Table 1). Further modification of BK medium, maintained a significant amount of viability (60.47±4.17%) by the addition of PEG could marginally augment the even after six months from the time of anther dehiscence. germination response (Experiment 2, Table 1). This medium DISCUSSION was considered the Modified BK medium for improvisation in the remaining experiments (Experiments 3-7). Pittosporum eriocarpum is a subdioecious tree species An attempt was made to test various concentrations of and sets fruits only through xenogamy. The fresh pollen of the mineral salts. BK medium supplemented with boric acid (300 species exhibits 70% fertility and tends to lose viability mg/L) was able to enhance the germination response up to rapidly (~4 days). As the species is an endangered one, three folds (69.26±5.26%) and showed highest pollen tube assisted pollinations may prove useful in the event of length (136.64±4.56 µm). The other components CaNO3 (400 pollination failure in the natural populations. Viability of mg/L), MgSO4 (200 mg/L), KNO3 (300 mg/L) also enhanced pollen grains refers to their ability to complete post pollination the germination percentage (50.55±1.11, 57.71±1.19, events on compatible, receptive stigma, while fertility is the 22 The International Journal of Plant Reproductive Biology 9(1) Jan 2017, pp.18-24

ability to produce two functional male gametes. Assisted Brewbaker and Kwack 1963, Steer and Picton 1984, Nobiling pollination may also be pursued with stored pollen grains and Reiss 1987), as it plays a regulatory role in tip-oriented when there is paucity of sufficient pollen donors in the growth, elongation and guidance of pollen tube (Pierson et al. population. Our attempts to store the pollen showed that it is 1996, Holdaway-Clarke et al. 1997). In the present study, a possible to store the pollen at low temperature up to six months significant increase in the germination rate of P. eriocarpum without affecting the viability. As the pollen do not appear to pollen in the Ca supplemented medium indicated its positive be recalcitrant, it would be possible to store the pollen of the influence. The pollen grain of P. eriocarpum appears to lack species at ultra-low temperature (-150 to -196°C). Pollen sufficient endogenous level of Ca to continue with the tube storage at ultra-low temperature has been successful in many growth, as its exogenous application improved the response. plant species (Barnabás and Kovács 1997, Tandon et al. 2007). Also, it has been shown that Ca is important for the synthesis of Storage of pollen also requires their regular assessment for cell wall components (Roy et al. 1999) and its intra- and extra- germinability. Although 2-celled pollen systems are amenable cellular streaming (Holdaway-Clarke and Hepler 2003). No for in vitro germination in BK medium (Brewbaker and significant increase was observed in the germination rate in Kwack 1963), the pollen grains of P. eriocarpum failed to media supplemented with potassium (K) and magnesium respond well in vitro. Thus it became essential to optimize a (Mg), indicating the absence of any significant response even pollen germination medium for the species. at higher concentrations. However, these minerals (K and Mg) The initial attempts indicated that a higher concentration significantly increased the tube growth at 300 mg/L and 200 of sucrose is essential for the pollen of P. eriocarpum to mg/L, respectively. K and Mg are known to play an assistive germinate in vitro. The response was conducive with 20% role in maximizing the association of Ca to the cell wall sucrose in the BK medium. However, at higher concentrations (Brewbaker and Kwack 1963), which imparts structural of sucrose an inhibitory response was observed which could rigidity, permeability and ion selectivity to the wall. be due to an increased rate of respiration and membrane Our study demonstrates that the P. eriocarpum pollen permeability (Bair and Loomis 1941, Zhang and Croes 1982). might be deficient in both B and Ca, thus requiring a high PEG was added to the germination medium, as it is known to concentration of them for germination and tube growth. Under provide a good osmoticum (Read et al. 1993). It is not in vivo conditions the same nutrients are perhaps supplied by metabolized by the pollen grains (Subbaiah 1984) and reduces the stigmatic exudate for attaining successful fertilization and pollen rupture by regulating the permeability of plasma seed-set. High requirement of Ca and B is in accordance with membranes (Shivanna and Sawhney 1995). the natural habitat of the species as it flourishes well in high Pollen grains are usually deficient in boron (B) and Ca-rich soil. Therefore, the optimized media found most essentially require it for tube wall formation (Dickinson 1978, suitable for P. eriocarpum consisted of sucrose (20%), H3BO3

Obermeyer and Blatt 1996, Stanley and Loewus 1964). It (300 mg/L), KNO3 (300 mg/L), CaNO3 (400 mg/L), MgSO4 stimulates translocation and metabolism of sucrose by (200 mg/L) and PEG (10%). forming an ionizable sugar-borate complex (Gauch and Acknowledgements—Present research work was supported Dugger 1953, Sisler et al. 1956). Montaner et al. (2003) has by “All India Coordinated Research Project on Reproductive argued that B may be critical more for the tube growth than for Biology of Four RET Tree Species of Central and Western germination of pollen. Thus, B is an essential component for Himalaya” sanctioned by the Ministry of Environment, most of the pollen germination media. Its exogenous Forests & Climate Change, Government of India, New Delhi, application, as observed in many economically important India (Sanction No. 22/2/2010-RE). trees, significantly influences both pollen germination and REFERENCES tube growth (Lewis 1980, Nyomora et al. 2000, Wang et al. 2003, Wojcik and Wojcik 2003). In P. eriocarpum, lower Bair RA and Loomis WE 1941. The germination of maize concentrations of B resulted in low germination and tube pollen. Sci. 94 168-169. growth. At a higher concentration (400 mg/L), B inhibited Barnabás B and Kovács G 1997. Storage of pollen. In: both germination and tube growth. According to Brewbaker Shivanna KR and Sawhney VK (eds.) Pollen and Majumder (1961), most species require B at a Biotechnology for Crop Production and Improvement. concentration of 100 ppm. However, in the present work the Cambridge University Press, UK Pp 293-314. amount of B required was significantly higher (300 mg/L). In both in vitro and in vivo conditions, optimal pollen Brewbaker JL and Kwack BH 1963. The essential role of germination and continued tube growth requires a high level of calcium ion in pollen germination and pollen tube calcium (Ca) (300-5000 ppm) (Todd and Bretherick 1942, growth. Am. J. Bot. 50(9) 859-865. 2017 Standardization of pollen germination medium for Pittosporum eriocarpum...... 23

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