High Temperatures during Bloom Can Inhibit Pollen Germination and Tube Growth, and Adversely Affect Fruit Set in the Prunus domestica Cultvars Improved French and Muir Beauty M.T. DeCeault and V.S. Polito Department of Plant Sciences University of California Davis, California USA Keywords: Prunus domestica, pollination, pollen germination, pollen tube growth, fruit set, temperature Abstract Prune production areas in California experienced unusually high temperatures during three of the past four bloom seasons (2004, 2005, 2007). This has been accompanied by marked reductions in fruit set for many growers. We have been investigating the effects of temperature on pollen germination and pollen tube growth. Pollen from Improved French and Muir Beauty was incubated on agarsolidified medium on a temperature-gradient bar. Pollen germination and pollen tube growth were determined from these samples. Interpolations of parabolic regressions indicate optimum temperatures for pollen germination are 22.6 C for Improved French and 22.2 C for Muir Beauty, and temperature optima for pollen tube growth are 24.0 C for Improved French and 24.3 C for Muir Beauty. In vivo analyses of pollen tube growth for Improved French were conducted under ambient and elevated temperature conditions for two years, one characterized by relatively cool temperatures during bloom and one with warmer conditions. Flower samples were collected at intervals after hand pollination and pollen tube growth analyzed from aniline blue stained samples examined under a fluorescence microscope. Results indicated that supraoptimal temperatures inhibit pollen tube growth at the upper style. In addition, results of our experiments indicate mid-day microsprinkler irrigation may moderate temperatures during bloom sufficiently to enhance fruit set under high temperature conditions. INTRODUCTION Literature spanning many years suggests temperature plays an important role in determining the success of nearly all reproductive processes. Previous work on other Prunus crops examining temperature effects on reproductive events shows that high temperatures may adversely effect pollen germination and tube growth as well as ovule development and longevity (Cerovic et al., 2000; Jefferies et al., 1982; Moreno et al., 1992; Szabo and Schauz, 2000; Thompson and Liu, 1973; Weinbaum et al., 1984). While temperature has been studied in regard to its effects on prune (Prunus domestica L.) reproductive biology, all previous work was conducted using cultivars not grown in California. These data show that the effects of temperature on reproductive processes vary among cultivars. We have conducted an investigation looking at different temperature parameters on pollen germination, tube growth and ovule longevity in prune flowers of two cultivars grown in California. Prune production areas in California have experienced unusually high temperatures during three of the past four bloom seasons (2004, 2005, 2007). This has been accompanied by reduced set for many growers. With the prospects of global climate change raising the likelihood of more frequent occurrences of high temperature episodes, we initiated a project to determine temperature parameters that affect pollen germination, pollen tube growth and fruit set in prune flowers. Our first objective was to model temperature responses for pollen germination and pollen tube growth for Improved French and Muir Beauty based on in vitro analyses and to use these regressions to Proc. 9 th IS on Plum & Prune Genetics, Breeding & Pomology Ed.: F. Sottile Acta Hort. 874, ISHS 2010 163
interpolate temperature optima and to elucidate supraoptimal responses. A second objective was to conduct in vivo experiments at ambient and modified temperature conditions. Finally, we initiated a preliminary experiment to determine the efficacy of using irrigation to moderate high temperatures during bloom. MATERIALS AND METHODS Analysis of pollen germination and pollen tube growth in vitro was done in 2006 and 2007. Freshly collected pollen was incubated on an agar-solidified medium consisting of 1.0 mm Calcium Chloride, 1.0 mm Boric Acid, 10% Sucrose and 1.2% Agar in petri plates maintained on a temperature gradient bar. The temperature gradient apparatus consisted of a meter-long aluminum bar with channels cut into both ends. Ethylene glycol (automobile antifreeze) solutions were pumped through the channels. The solution at one end was cooled by a chilling unit and the solution at the other end of the bar was heated with a submersible heater. When the bar reached equilibrium, petri plates with media were brought to temperature equilibrium on the bar and inoculated with pollen. Pollen tube growth was stopped by fixation after three hours and the plates were evaluated for pollen germination percentages and pollen tube growth. Pollen tubes were measured from digital photomicrographs using image analysis software. The data from each replication was fitted to a parabolic regression (second order polynomial) with optima found at the vertex (-b/2a) and summarized using SAS Version 9.1 Software. For the in vivo experiments an Improved French tree was enclosed and covered with polyethylene sheeting allowing for the accumulation of heat. In 2006 a small greenhouse heater was used to raise the temperature in the tree enclosure. In 2007 solar radiation alone was used. For the irrigation experiment conducted in 2007, we used two micro-sprinklers on each side of the tree irrigated for up to 5 midday hours with 40 L/h of water. Temperature was monitored with data loggers placed in appropriate locations approximately 2.5 m high in the enclosed and irrigated space as well as an adjacent ambient tree that served as a control. Flowers were hand pollinated as they emerged over several days. Pistils were collected at two-day intervals following pollination. The pistil samples were fixed in ethanol-acetic acid solution. Style portions were softened and squashed to reveal pollen tubes. The squashed styles were stained with alkaline aniline blue, a fluorescent stain for callose, a pollen tube cell wall component. Stained samples were observed in a fluorescence microscope, and pollen tube growth in several portions of the style was determined for pollen tube kinetic and dynamic studies. Ovary portions were dissected to remove the ovules, however ovule fluorescence was conspicuously absent from the primary ovules of Improved French prune and we were unsuccessful in determining ovule viability. RESULTS AND DISCUSSION In Vitro Pollen Germination and Tube Growth Responses to Temperature Pollen germination regressions for Improved French and Muir Beauty pollen are shown in Figure 1. Pollen tube lengths for both cultivars are shown in Figure 2. Temperature optima for germination and tube growth (Table 1) range from 22.2 to 24.3ºC in Improved French prune and fall off sharply at extremely hot temperatures. In Vivo Pollen Tube Growth for Improved French Three treatments for studies of in vivo pollen tube growth were used in 2006, a relatively cool year: Ambient temperature and two elevated temperatures from polyethylene-enclosed trees. Pollen tubes first reached the style base six days after anthesis (DAA) in all treatments (Fig. 5). At 8 DAA the number of pollen tubes at the style base continued to increase in flowers from the ambient temperature treatment, but a decline was seen in the high temperature treatments. These trends continued through 10 DAA by which time no pollen tubes were visible in the two high temperature treatments. The lack of visible 164
pollen tubes in the high temperature treatments at 10 DAA likely indicates senescence of tubes that had reached that point at 6 DAA. Three treatments were also used in 2007, a relatively warm year: Ambient temperature, elevated temperature within a polyethylene enclosure and microsprinkler irrigated; Figures 6 and 7 show temperature conditions for these treatments over the course of our experiments. The microsprinkler irrigation treatment was effective at reducing maximum temperature during high temperature events (Fig. 8). Pollen tube growth to the base of the style was completely inhibited in the high temperature treatment (Fig. 9) and a greater number of pollen tubes grew in the styles of flowers under the microsprinkler treatment for the early portion of the bloom period (Fig. 10). CONCLUSIONS Our results show that the optimum temperatures for pollen germination and pollen tube growth for Improved French and Muir Beauty cultivars of Prunus domestica are approximately 22 to 24ºC. Above this optimum, pollen tube growth decreased rapidly. At high daily maximum temperatures, pollen tubes began to senesce in the style. The effects of high temperatures on pollen tube growth can preclude fruit set. Preliminary experiments with microsprinkler irrigation during high temperature events indicated that this may be useful as a management tool to moderate high temperatures during bloom. Further research is underway to refine and expand these results. ACKNOWLEDGEMENTS We thank the California Dried Plum Board for financial assistance and Carolyn DeBuse, Ted DeJong and Katy Pinney for their assistance on this project. Literature Cited Cerovic, R., Ruzic, D. and Micic, N. 2000. Viability of plum ovules at different temperatures. Ann. Appl. Biol. 137:53 59. Jefferies, C.J., Brain, P., Stott, K.G. and Belcher, A.R. 1982. Experimental systems and a mathematical model for studying temperature effects on pollen-tube growth and fertilization in plum. Plant, Cell and Environ. 5:231 236. Moreno, Y.M., Miller-Azarenko, A.N. and Potts, W. 1992. Genotype, temperature, and fall-applied ethephon affect plum flower development and ovule longevity. J. Amer. Soc. Hort. Sci. 117(1):14 21. Szabo, Z. and Schauz, R. 2000. Floral biology of plum. Int. J. Hort. Sci. 6:11 27. Thompson, M.M. and Liu, L.J. 1973. Temperature, fruit set, and embryo sac development in Italian prune. J. Amer. Soc. Hort. Sci. 98:193 197. Weinbaum, S.A., Parfitt, D.E. and Polito, V.S. 1984. Differential cold sensitivity of pollen germination in two Prunus species. Euphytica 33:419 426. Tables Table 1. Optimum temperatures for pollen germination and pollen tube growth determined from the regression curves shown in Figures 1 and 2. Cultivar Optimum ( C) Pollen germination Improved French 22.6 Pollen germination Muir Beauty 22.2 Pollen tube growth Improved French 24.0 Pollen tube growth Muir Beauty 24.3 165
Figures Fig. 1. Regression curve for Improved French and Muir Beauty prune pollen germination vs. temperature. R=0.84 for Improved French and 0.83 for Muir Beauty. Fig. 2. Regression curve for Improved French and Muir Beauty prune pollen tube length vs. temperature. R=0.41 for Improved French and 0.38 for Muir Beauty. 166
Figs. 3 and 4. Mean and maximum daily temperatures for the three treatments in 2007. 50 40 Ambient Medium - High High % of Flowers 30 20 10 0 0 2 4 6 8 10 Days After Anthesis Fig. 5. Pollen tubes at the base of the style in flowers from ambient, medium-high and high temperature treatments. Figs. 6 and 7. Mean and maximum daily temperatures for the three treatments in 2007. 167
Fig. 8. Hours recorded above temperature thresholds for 2007 field treatments. 40 30 Ambient Irrigation High % of Flowers 20 10 0 0 2 4 6 8 10 Days After Anthesis Fig. 9. Pollen tube growth in the style for ambient, irrigation treatment and high temperature treatment. Data show percentage of flowers with at least one pollen tube present at the base of the style. 1.4 1.2 Ambient Irrigation High Number of Pollen Tubes 1.0 0.8 0.6 0.4 0.2 0.0 0 2 4 6 8 10 Days After Anthesis Fig. 10. Number of pollen tubes present at the base of the style for ambient, irrigation treatment and high temperature treatment. 168