Apple Pollen Tube Growth Rates Are Regulated by Parentage and Environment

A greater understanding of apple (Malus *domestica) pollen tube growth rates can improve crop load management in commercial orchards. Specifically, applications of caustic bloom thinning chemicals need to occur when enough, but not too many, flowers have been fertilized to achieve crop load densities that balance yields with marketable fruit sizes. In this study, the pollen tube growth rates of five crabapple (Malus sp. cultivars were measured in the styles of three maternal cultivars at 12, 18, 24, and 30 8C after 24 hours in a growth chamber. Pollen tube growth rates were greatest for 'Selkirk' and 'Thunderchild' at 12 8C, and greatest for 'Indian Summer' 'Selkirk' and 'Thunderchild' at 24 8C. Pollen tube growth increased with increasing temperatures until 24 8C. There were minimal pollen tube growth rate increases between 24 and 30 8C. Overall, 'Snowdrift' had the slowest pollen tube growth rate of the five evaluated crabapple genotypes. At 24 and 30 8C, 'Indian Summer' and 'Thunderchild' pollen tubes reached the base of the style most frequently, and 'Snowdrift' pollen tubes the least frequently. Pollen tube growth rate was also influenced by the maternal cultivar, with Golden Delicious having relatively faster pollen tube growth than Fuji at 24 and 30 8C. Interactions among paternal and maternal genotypes as well as temperature after pollination reveal complex biological and environmental relationships that can be used to develop more precise crop load management strategies for apple orchards. Apple pollination occurs when pollen is transferred, often by an insect vector, from the anthers of one blossom to the stigma of another. After pollen grains are deposited, they are rehydrated by stigmatic secretions, and pollen tube growth begins (Dennis, 2000; Jackson, 2003) Genetically compatible pollen tubes grow through the stigma and style and toward the ovaries. At the end of the style, the pollen tube enters the ovary through the micropyle, where two sperm cells are released into the ovule. Fertilization occurs when one nucleus fuses with the egg cell, and the second nucleus fuses with the embryo sac, initiating seed formation (Dennis, 2000) Investigations into apple pollen biology have been underway for over a century (Adams, 1916) and have recently been reviewed by Ram irez and Davenport (2013) In commercial apple orchards, cross pollination between two distinct genotypes results in greater fertilization rates and thus a greater fruit set, even in orchards containing self fertile cultivars (Dennis, 2003) Various Malus species, colloquially referred to as crabapples, are preferred pollinizers in commercial orchards due to their long bloom periods, disease resistance, and abundant pollen production (Fitzgerald, 2005) In particular, the crabapple Malus baccata 'Manchurian' has been widely planted in commercial orchards as a pollinizer. However, it has fallen into disfavor in recent years due to its high susceptibility to canker diseases, which could limit apple exports into Asian markets (Hansen, 2014) Other popular crabapple pollinizers include 'Snowdrift' 'Thunderchild' 'Indian Summer' 'Mt. Blanc' 'Wickson Crab' and 'Chestnut Crab' The pollen tube growth rate of 'Snowdrift' has been used to develop a temperature driven pollen tube growth model that can predict the time between pollination and fertilization (Peck et al. 2016; Yoder et al. 2013) Themodel is now being used by commercial apple growers to precisely calculate when to apply bloom thinning chemicals (Lehnert, 2014) The majority of bloom thinning chemicals are caustic and prevent pollination or fertilization by destroying flower parts and inhibiting pollen germination and pollen tube growth. Liquid lime sulfur (calcium polysulfide) is the most studied and commercially used bloom thinning chemical (McArtney et al. 2006) Although Snowdrift pollen tube growth can differ by as much as 3 fold among maternal cultivars, published reports on the pollen tube growth rates of other crabapple species and cultivars are limited (Embree and Foster, 1999; McArtney et al. 2006; Stott, 1972; Williams, 1965) Successful application of blossom thinning chemicals is highly dependent on understanding the rate of pollen tube growth among different pollen donor genotypes (Jackson, 2003) Analyzing the pollen Received for publication 13 May 2016. Accepted for publication 29 Aug. 2016. This project was funded by Virginia Agricultural Experiment Station and Virginia Tech's Department of Horticulture. We thank Abby Kowalski, Ashley Thompson, Taylor Mackintosh, and David Carbaugh for their assistance in the field and lab, Mizuho Nita for guidance with statistics, and Roger Harris and Tony Wolf for review of the manuscript. Corresponding author.  gmp32@cornell.edu. 548  tube growth rates of additional pollen donors could contribute to the selection of improved pollinizers for apple orchards. The objectives of this study were to determine how pollen tube growth rates were affected by paternal (pollen source) and maternal cultivars, temperatures after pollination, and the interaction among these factors. We did this by comparing the pollen tube growth rates of five crabapple cultivars on three maternal culinary apple cultivars at four temperatures.  Experiments were conducted at Virginia Tech's Alson H. Smith Jr. Agricultural Research and Extension Center (AREC) in Winchester, VA. In 2013 and 2014, pollen was collected from mature 'Evereste' 'Indian Summer' 'Selkirk' 'Snowdrift' and 'Thunderchild' trees located at the AREC. Only healthy blossoms in the late pink flower stage (when petals still covered the sexual organs and before dehiscence of anthers) were used (Chapman and Catlin, 1976) Pollen was collected by removing anthers from the blossoms using a fine toothed comb, allowing them to air dry, and then pressing the anthers through a 200 mesh sieve (74 mm between wires) Pollen was stored in microcentrifuge tubes at 12 C until use. Pollen collected in 2013 and 2014 was combined for each crabapple cultivar. Pollen viability was scored in two samples for each crabapple cultivar after 24 h at 21 C on an agarose (10 g L) sucrose (100 g L) and boric acid (10 mg L) medium by calculating the percent germination (Williams and Maier, 1977) Mature 'Autumn Rose Fuji''M.9' 'Golden Delicious' 'M.27' and 'Cripps Pink''M.9' trees grown in 19 L root bags (Lacebark, Stillwater, OK) were the maternal cultivars selected for this study. Whole trees (including the bagged root system) were removed from the orchard in Jan. 2015, placed in 19 L buckets, and kept in cold storage to accumulate sufficient winter chill hours to complete endodormancy. 'Cripps Pink' and 'Fuji' trees were kept at 4.4 C for 400 h, and 'Golden Delicious' trees were kept at 4.4 C for 700 h; after the chilling hours had been met, trees were moved into a greenhouse (mean temperature 24 C) to force bloom. Maternal flowers used in the experiment were selected during the late pink flower stage. During each temperature trial, pollen from the five paternal cultivars was hand pollinated onto 35 flowers from each maternal cultivar; seven unique pollinated flowers for each paternal cultivar treatment. An additional seven emasculated, nonpollinated flowers were left on the tree to serve as nonpollinated controls. All side blooms and extra flower clusters were removed from the tree to prevent cross contamination. Flowers were tagged to identify the pollen source, and a sterilized, size 6 artist's brush was used to apply pollen directly to the stigma. Pollen was applied in a uniform way until pollen grains were visible on the stigma. Immediately after pollination, trees were placed into a temperature controlled growth chamber (Intellus Control System; Percival Scientific, Perry, IA) for 24 h under a 12 h photoperiod. For each maternal cultivar, the experiment was repeated at four ambient air temperatures: 12, 18, 24, and 30 C for the duration of the experiment. After 24 h in the growth chamber, flowers were removed from the tree in the order they were pollinated and placed into glass vials filled with a 5% aqueous sodium sulfite (Amresco, Solon, OH) solution. Storage vials containing the flowers were placed in boiling water for 20 min to soften the tissue. The pistillate portions were then excised from each flower and rinsed with distilled water. The styles were individually detached from the ovary using forceps and scalpel. The styles were stained with a water soluble fluorescent solution containing 0.01% aniline blue dye (MP Biomedicals, Solon, OH) in 0.067 M K2HPO4 for pollen tube visualization, then pressed between two microscope slides (Embree and Foster, 1999) Slides were viewed at 10* and 40* with a light microscope (Eclipse Ci; Nikon, Table 1. Percent crabapple pollen germination, Spring 2015 first bloom and petal fall dates, and flower color of crabapple trees used for paternal cultivar pollen collection. Cultivars were all planted in 1999 on M.9 rootstocks. Paternal cultivar Pollen germination 2015 First bloom 2015 Petal fall Flower color Evereste 65 22 Apr. 4 May White Indian Summer 75 17 Apr. 28 Apr. Pink Selkirk 85 15 Apr. 22 Apr. Pink Snowdrift 65 22 Apr. 4 May White Thunderchild 90 17 Apr. 24 Apr. Pink Fig. 1. 'Thunderchild' pollen tube growth in a 'Fuji' apple 24 h after pollination at a constant temperature of 24 C, visualized with water soluble fluorescent solution containing 0.01% aniline blue dye in 0.067 M K2HPO4 and magnified 10* using a light microscope and a fluorescent light source.  Japan) and fluorescent light source (Intensilight C HGFI; Nikon) Histological examination for each individual style was conducted as described in Yoder et al. (2009) and Yoder et al. (2013) and included pollen germination/tube growth on the stigmatic surface (0% t