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May 21, 2001 - Agricultural Research Organization, the Volcani Center, PO Box 6, Bet Dagan 50250, Israel; 3Current address: UMR 406 INRA/UAPV, Ecologie ...
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Are flowers morphologically adapted to take advantage of electrostatic forces in pollination? Blackwell Science Ltd

Yiftach Vaknin1,3, Samuel Gan-mor2, Avital Bechar2, Beni Ronen2 and Dan Eisikowitch1 1

Department of Plant Sciences, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel Aviv 69978, Israel; 2Institute of Agricultural Engineering,

Agricultural Research Organization, the Volcani Center, PO Box 6, Bet Dagan 50250, Israel; 3Current address: UMR 406 INRA/UAPV, Ecologie des invertebres, Site Agroparc, 84914 Avignon cedex 9, France

Summary Author for correspondence: Yiftach Vaknin Tel: +4 32 72 26 39 Fax: +4 32 72 26 02 Email: [email protected] Received: 21 May 2001 Accepted: 23 July 2001

• The relationship between floral morphology and electrostatic pollination was studied here. To test the effects of floral morphology on pollen deposition on the stigma and other floral parts by means of electrostatic forces, metal replicas of almond (Amygdalus communis) flowers were constructed and then dusted with electrostatically charged and uncharged almond pollen. The pollen was applied to the flowers with a specially designed electrostatic powder-coating device. • Pollen deposition on the flower was found to be higher when the pollen was electrostatically charged than when it was not. Most of the charged pollen grains were deposited on the corolla extremities and on the stigma, whereas uncharged pollen grains were evenly distributed on the entire flower. • Stigma exsertion was the most important morphological feature of the flower promoting pollen deposition on the stigma when electrostatic charge was used. Large flowers with corollas showed higher electrodeposition on the corolla than smaller, narrower ones. • These results collectively imply that morphological features of a plant might be adaptations to take advantage of electrostatic forces. This provided us with a very important tool for future research on floral morphology and pollination biology. Key words: Artificial flowers, electrodeposition, electrostatic pollination, floral morphology, stigma exsertion. © New Phytologist (2001) 152: 301–306

Introduction The contribution of electrostatic forces to pollination in nature has been discussed over the last 20 yr (Buchmann & Hurley, 1978; Eisikowitch, 1981; Corbet et al., 1982; Erickson & Buchmann, 1983; Schroeder, 1995; Endress, 1997; Vaknin et al., 2000). However, confirmation that these processes actually occur in nature was obtained only recently (Schwartz, 1991; Gan-Mor et al., 1995). The mechanism of electrostatic pollination has been suggested by several researchers (Hardin, 1976; Corbet et al., 1982; Erickson & Buchmann, 1983). It was based on the process of inductive charging: when a charged body is brought into the vicinity of a grounded electrode, a quantity of charge of the opposite sign is forced to flow up from the earth, onto the electrode. This maintains the electrode at ground potential in

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the presence of the charged body (Law, 1975). In the case of electrostatic pollination, the same process should occur. When an electrically charged insect approaches a flower, a charge of opposite polarity flows into the plant’s stem and flowers, as a result of the electric field between the insect and the flower. This electric field strengthens as the separation diminishes. The distribution of the electric field around the plant and the flower varies according to their geometry. The electric fields should be greatest near sharp points such as petal edges and exserted anthers or stigmas (Dai & Law, 1995). The temporary forces of attraction created between the airborne insect and the flower can initiate the detachment of some pollen grains from the flower and help in their deposition on the insect’s body. The same forces can initiate pollen detachment from the body of the insect and help in its deposition on the flower. These processes also depend upon physical variables

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such as the magnitude of the charge and its location, dielectric properties of the intervening media, and the morphology of the flower (Dai & Law, 1995). The process also depends upon environmental variables such as rh, air-ion concentration and mobility, and variations in the Earth’s ambient electric field (Law et al., 1996). The geometries involved in electrostatic deposition (electrodeposition) processes of pollen grains are extremely complex. Dai & Law (1995) and Bechar et al. (1999) both independently developed mathematical models to describe the system of a charged body, either insect or pollen cloud, approaching a flower with an exserted stigma. They both constructed a 3-D finite-element model for the analysis of the transient electric field produced by a charged body as it approaches and enters a flower. The models showed that the electric field near a flower was dependent on the geometry of the flower: as the flower’s opening angle increased, or as the style length increased, the electric field near the stigma rose and that near the petals decreased. The morphology and size of animal-pollinated flowers are considered to be the result of multiple selective requirements such as display and adaptation for pollinators (Faegri & van der Pijl, 1979; Young & Stanton, 1990; Armbruster, 1996; Galen, 1996; Kobayashi et al., 1997), and also from constraints such as the need to protect the pollen and the nectar from outside disturbances such as wind, rain and radiation (Armbruster, 1996), and the need for cross-pollination (Lloyd & Yates, 1982; Thomson & Stratton, 1985; Proctor et al., 1996). Despite the increasing knowledge of floral morphology, most of our knowledge of electrostatic pollination has remained speculative and theoretical ( Vaknin et al., 2000). Moreover, morphology of an entomophilous flower is much more complex than that presented in the mathematical models. These models were limited to very simple floral structures, and did not address floral size and relationships between floral size and stigma exsertion. In the present article we aim to determine the effects of floral morphology on electrostatic deposition of pollen on stigmas and petals by using metal flowers that are scaled to the actual size of almond (Amygdalus communis L.) flowers.

Fig. 1 (a) Five areas on the artificial flower on which pollen counts were taken, four on the corolla and one on the stigma. (b) Measured morphological parameters of the artificial flower: corolla width (CW), style length (SL), stigma exsertion (SE).

Materials and Methods In order to test the effects of floral morphology on electrostatically assisted pollen deposition, we constructed metal replicas of insect-pollinated almond flowers. Each ‘flower’ consisted of a style made out of a 24-mm-long, 1.5 mm-diameter cylindrical steel rod and a five-petal corolla made out of a 0.15mm-thick copper sheet (Fig. 1). Style length was easily modified from 0 to 24 mm, and the flexible petals at the base of the flower could be bent to modify the corolla angle between the upright style and the petals. We selected five equal areas on the flower for our pollen counts: four on one of the petals (areas 1–4 in Fig. 1a) and one on the tip of the style (i.e. the ‘stigma’, in Fig. 1a). Each of

Fig. 2 A schematic of a system for electrostatic pollination of artificial flowers.

these areas was stained with a black dye to provide a dark background for accurate pollen counts (Fig. 1a). We constructed a system for applying electrostatically charged almond pollen to the artificial flower (Fig. 2): a vertically mounted 80 × 80-cm metal grid with 10-cm spacing was connected to ground and a model flower was attached to its centre. Then, we positioned a specially designed electrostatic powder-coating device with its outlet facing the flower from a distance of 2 m (Bechar et al., 1999). We used almond

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Fig. 3 Style length and corolla angle of artificial flowers in experiments on electrostatic pollination. Corolla angle and style length are in brackets.

pollen of cv. ‘Fritz’, which was kept at –20°C before use, and was blown into the gun head by an air stream during the experiment. A high-voltage (50 kV ) generator created a corona discharge that ionized the air and charged the pollen by ion bombardment in the region close to the gun outlet. The pollen was then transported to the grounded flower by a combination of aerodynamic and electrostatic forces. This pollen cloud simulated an approaching insect carrying pollen grains on its body (Bechar et al., 1999). We applied 2 g of pollen for 15 s each time and immediately started pollen counts on the five designated areas on the flower. We photographed each of the five designated areas with a video camera (WV-CL320, Panasonic, Inc., Japan) mounted on a stereoscope that provided 100× magnification. The pictures were recorded by a personal computer (PC) with U-lead (USA) PC video software. We counted the pollen grains directly on the monitor display, and calculated pollen density as the number of pollen grains per mm2. In order to assess the efficiency of pollen deposition on the flower we applied uncharged and charged pollen grains to a fully opened flower model with a corolla angle of 90° and a 12-mm-long style ( N = 5). Our hypothesis was that without the electrostatic charge, pollen grains would be more evenly distributed and less affected by floral morphology. The morphological properties of the metal flowers were varied as follows (Fig. 3): (a) Five lengths of the style, 0, 3, 6, 12 and 24 mm, with corolla angle of 90°. (b) Five lengths of the style, 0, 3, 6, 12 and 24 mm, with corolla angle of 60°. (c) Three corolla angles of 0, 30 and 180° with a style length of 12 mm. Each floral morphology was electrostatically dusted five times. We used digital calipers having an accuracy of ± 0.1 mm to take several measurements of each of the treated flowers (Fig. 1b): corolla diameter, style length and stigma exsertion. Positive values of stigma exsertion meant that the style projected above the petal extremities, whereas negative values meant that it was entirely below the petal extremities. We arbitrarily decided to use corolla width (diameter) as an assessment of floral size.

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Analysis Statistical analyses were performed using SPSS 7.5.1 (SPSS Inc., Chicago, IL, USA, 1996–89) software. All counts of pollen grains on the flowers were square-root transformed before analysis to adjust for normality. Comparisons of pollen deposition on the stigma between charged and uncharged applications of pollen were done by means of one-tailed t-test. ANOVA and Fisher’s LSD tests were used to compare pollen depositions among the five designated areas of the flower, for each treatment separately. ANOVA and Fisher’s LSD tests were used to compare among pollen depositions on flowers of 12 mm style length, having various corolla angles and therefore differing corolla widths and stigma exsertions. We first compared pollen densities on the stigmas of all the floral morphologies. We then compared pollen depositions on all the floral areas including the stigma, for each floral morphology separately. All means are given with their standard errors.

Results Pollen deposition on the stigma under electrostatic pollination was significantly greater by 33%, than under uncharged pollination (19.3 ± 0.32 and 14.5 ± 2.15 pollen grains, respectively, one-tailed t-test, t8 = 2.097, P = 0.035, N = 5). We compared the pollen density distributions under charged and uncharged pollination, and found that under charged application the pollen density on the corolla was highest on the corolla extremities (34.4 ± 2.51 grains on area 1; 27.8 ± 2.48 grains on area 2) and was diminished significantly towards the base of the style (22.6 ± 1.91 grains on area 3; 21.7 ± 1.30 grains on area 4 at the base of the style) (ANOVA, F(4,20) = 10.399, P < 0.0001). By contrast, under uncharged pollination, the pollen density did not vary significantly among the floral parts (15.6 ± 1.17 pollen grains on area 1; 15.4 ± 1.13 pollen grains on area 2; 15.6 ± 2.03 pollen grains on area 3 and; 14.1 ± 1.75 pollen grains on area 4) (ANOVA, F(4,20) = 0.179, P = 0.9466). When we compared pollen electrodeposition on the stigma and corolla of flowers with differing corolla angles but with the same style length (12 mm), we found the 90° corolla angle

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Fig. 4 Effects of various corolla angles (0 – 180°) but the same style length (12 mm) on pollen electrodeposition on artificial flowers. Squares, area 1; diamonds, area 2; downward triangles, area 3; upward triangles, area 4; circles, stigma. Values are means ± SE. N = 5.

had the maximum amount of pollen deposited on the stigma and the effect was more pronounced in the more distal areas of the petal (Fig. 4). The metal flowers with reflexed petals (180°) had less deposition, comparable to those with petals bent outward (60°). The 90° flowers were also the largest in terms of corolla diameter, and this may account for their increased attraction of pollen on distal areas. It appears that, as the corolla angle was reduced from 60° to 30° and 0°, the consequent reductions in both floral size and stigma exsertion caused decreased pollen deposition on the stigma and corolla. It also appears that the same series of changes lead to the pollen densities being the same on all floral parts, including the stigma (Fisher’s LSD, P > 0.05) (Fig. 4). We studied the effects of stigma exsertion in flowers of two morphologies, one with flat flowers (90° corolla angle and 49.3 mm corolla width) and the other with saucer-shaped flower (60° corolla angle and 43.9 mm corolla width). In flat flowers pollen deposition increased with increasing stigma exsertion (Fig 5a). In the saucer-shaped flower, the style was generally less exserted, and pollen deposition on all floral areas remained relatively low and unchanged as the stigma exsertion increased (Fig. 5b).

Discussion Our study highlights the importance of electrostatic pollination and the manner in which floral morphology determines the distribution of pollen grains attracted to the flower. By charging the pollen grains we clearly increased pollen deposition on the entire flower as compared with uncharged pollen.

Most of the charged pollen grains were deposited on the corolla extremities and on the stigma, whereas uncharged pollen grains were evenly deposited on the entire flower, with no preference for any area on the flower. The results of this study confirmed some of the predictions of the mathematical models by Dai & Law (1995) and Bechar et al. (1999), particularly the increase of pollen deposition on the stigma with increase of the flower’s opening angle and of stigma exsertion. They also brought to our attention the importance of the complexity of floral morphology in electrostatic pollination, especially stigma exsertion – corolla diameter relationships. We showed that stigma exsertion above the petal extremities was very important in its effect on electrostatic pollination: the greater the stigma exsertion, the more pollen was deposited on the stigma. Moreover, in a fully opened flower, the more the stigma was exserted while corolla diameter remained unchanged, the more pollen was deposited on the entire flower and ‘wasted’ to the corolla (Fig. 5a). We also showed that corolla diameter played a crucial role in electrostatic pollination: fully opened flowers (90° corolla angle) received far more pollen grains than flowers with a smaller corolla diameter (180° corolla angle), but with the same stigma exsertion (12 mm) (Fig. 4). Moreover, when the petals were partially folded (60° corolla angle), stigma exsertion had no significant effect on pollen deposition on the entire flower (Fig. 5b). We suggest that both the stigma and the corolla form an electric ‘target’ that potentially attracts more charged pollen grains. Increasing the dimensions of either one will result in an increase of pollen deposition on the entire flower.

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Fig. 5 Effects of stigma exsertion on efficiency of pollen electrodeposition on artificial flowers. (a) Corolla angle 90° and diameter of 49.3 mm; (b) corolla angle 60° and diameter of 43.9 mm. Squares, area 1; diamonds, area 2; downward triangles, area 3; upward triangles, area 4; circles, stigma. Values are means ± SE. N = 5.

Kobayashi et al. (1997) suggested that corolla diameter could directly enhance pollen transfer efficiency through several possible mechanisms: a larger flower is more conspicuous and might attract more pollinators; a wider corolla would enhance pollen transfer efficiency if pollen were deposited on body parts that pollinators cannot groom efficiently; a larger corolla helps to deposit pollen on body parts that contact recipient stigmas more often than others; and larger corollas could reduce wastage of pollen grains (if more are deposited, fewer are dropped). We suggest that a larger corolla will also enhance pollen deposition when sufficient electrostatic forces are involved, either in natural pollination by insects or in supplementary pollination in agriculture (Vaknin et al., 2001). Although anther exsertion was not tested in the present study, we assume that the electrostatic forces work in a similar way with the anthers. The greater their exsertion, the more pollen the approaching insect removes. In conclusion, despite the fact that we know very little of the mechanisms by which natural selection drives the evolution of floral shape and size (Galen, 1996; Conner, 1997), our study contributes some information on very important morphological features of electrostatically assisted plant pollination (i.e. stigma exsertion and corolla diameter). This knowledge could be a very important tool in future research on the effects of floral morphology on pollination biology, in both natural and agricultural systems.

Acknowledgements This study was prepared as part of the requirements of Yiftach Vaknin’s PhD in the Department of Plant Sciences, Tel-Aviv University.

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Schroeder CA. 1995. Electrostatic effects with avocado pollen. California Avocado Society Yearbook 79: 201–204. Schwartz Y. 1991. Pollen harvesting by electrostatic and aerodynamic techniques. MSc thesis (In Hebrew). The Technion – Israel Institute of Technology, Israel: The Faculty of Agricultural Engineering. Thomson JD, Stratton DA. 1985. Floral morphology and cross-pollination in Erythronium grandiflorum (Liliaceae). American Journal of Botany 72: 433–437. Vaknin Y, Gan-Mor S, Bechar A, Ronen B, Eisikowitch D. 2001. Improving pollination of almond (Amygdalus communis L. Rosaceae) using electrostatic techniques. Journal of Horticultural Science and Biotechnology. 76: 208–212. Vaknin Y, Gan-Mor S, Bechar A, Ronen B, Eisikowitch D. 2000. The role of electrostatic forces in pollination. Plant Systematics and Evolution 222: 133–142. Young HJ, Stanton ML. 1990. Influences of floral variation on pollen removal and seed production in wild radish. Ecology 71: 536 –547.

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