1. Mohammad Ghaffarzadeh, Carla Taylor, and Michael Lauer Pioneer Hi-Bred International, Inc. Field Measurement of Maize Pollen Abstract The quantity and purity of hybrid seed produced by a seed-bearing genotype of maize (Zea mays L) can be limited by pollen amount and temporal distribution during flowering. The seed industry and researchers have attempted to measure and establish spatial and temporal distribution of maize pollen using various instruments. Labor, consistency and accuracy of collected data, environmental constraints, and costs significantly impact utility of existing methodologies. The objectives of this work were to design a reliable measuring tool and processing technique to quantify maize pollen from seed production fields. The development of an automated pollen trap increased the capability to collect pollen grains shed on a daily basis in a large number of fields. Various pollen collection techniques were compared with automated pollen traps. Several modifications were made to improve automated pollen trap operation and optimize retention media and quantification methods. The quantity of pollen in samples collected with the liquid automated pollen trap compared favorably to pollen collected on dry media traps. A liquid media was identified that slightly reduced pollen grain size but did not allow water uptake by the pollen, thus preventing rupture. The liquid media allowed high throughput counting. Rainfall during pollen collection had limited effects on sample integrity. Background Pollen shed in the field has been estimated using sticky traps (Fonseca et al., 2002) or liquid filled containers (Fig. 1). The pollen sticky traps were constructed on a black surface covered with double-sided tape (Fonseca et al., 2002). Sticky traps or liquid traps were placed at corn ear height, removed daily and replaced with fresh traps during flowering (Fig. 1). Total pollen shed by a tassel can be estimated using air-permeable bags. References Fonseca A.E., M.E. Westgate, R.T. Doyle. 2002. Application of fluorescence microscopy and image analysis for quantifying dynamics of maize pollen shed. Crop Sci. 42:2201-2206. Additional Information To order an automated pollen trap or to submit your suggestions and comments please visit: http://www.pollentrap.com/index.htm Or contact: Joel Peterson Systems Embedded Software 3880 N. Main St., Suite C East Peoria, IL 61611 Phone: (309) 699-7800 To learn more about the coulter counter principles visit: http://scooter.cyto.purdue.edu/pucl_cd/flow/vol2/6/coulter. To learn more about Zeiss Axiovert 200M inverted microscope imaging visit: http://www.zeiss.de/4125681F004CA025/Contents- Frame/C7B4C762D6F8A09A85256B4B0075CE56 Pollen grain count Sticky traps: Images were obtained to estimate the number of pollen grains captured on each slide. The number of pollen grains per unit area was averaged across 10 images per slide (Fig. 2). Objectives Design an automated pollen trap with the capability to capture samples of pollen and airborne particles at preset sample times and intervals to determine temporal and spatial pollen load within a field. Figure 3. Coulter Counter Multisizer II can be used to estimate the number of pollen grains collected in liquid traps. Software provides the total number of particles within a particular range (corn pollen range 55  m-110  m). Liquid traps: Isotonic solution was used to preserve integrity of captured pollen. Number of pollen grains in each sample was estimated using a particle size analyzer. Developments 2001 – 2002: attributes of automated pollen traps were identified and the first prototype was assembled (Fig. 4). Figure 4. First prototype automated pollen trap was designed for wide range of capabilities including dual tray to hold sticky slides or containers of isotonic solution. This model had only ON and OFF and two timing setups. 2003: Model A (Fig. 5) was produced in large quantities and used at various sites and research plots. This model was equipped with a programmable electronic controller including several settings. Sample tray rotation options included 2, 4, 8, 12, and 24 hr interval settings. Delay settings allowed traps to be deployed before pollen shed began and programmed to start at a set date and time. Several evaluation protocols and quality control procedures were established. Shelf life, durability under field conditions, and water resistance evaluations were conducted. Results from evaluations and improvements were incorporated into the next advanced model. Figure 5. Model A automated pollen trap and sampling tray to hold isotonic solution containers. Field tests for placement and comparison between sticky traps and liquid media traps cells were conducted. Opening of approximately 25 cm 2 at ear height within the row were used to collect pollen. Figure 6. Position of the trap and collection opening within the corn row at silk height. Future modification and improvements may be incorporated such as a rain sensor and shutter to protect the samples during intense rain events. Evaluation and Process Improvement Two common sampling techniques: pollen sticky dry trap and liquid pollen trap were evaluated. Field issues and estimation process issues were identified. B Figure 1. A) Traps were placed at ear height within a row during flowering or B) whole tassel was placed inside a breathable bag to capture pollen. A Total pollen production per tassel can be estimated. After flowering pollen grains were separated and counted. Issues: throughput, sample integrity, need for specific microscope with fluorescing capability. Sample collection, processing and storage required extreme care. Trap warping caused inconsistent depth of field limiting imaging ability and slowing throughput. Both type of traps were removed daily and replaced with fresh traps during flowering. The automated pollen trap was designed to improve sampling capability and increase throughput. 2004 – 2005: Model B (Fig. 7) with new capabilities and functionalities was designed and tested. This model was designed for mass production with UV resistant material and water proof components. Rotation options of the sample collection tray were increased and ranged from 1 to 48 hr with both liquid and slide capturing options (Fig. 8). At the end of the sampling interval the tray rotates to a covered position to protect the samples in case of delay in trap removal. Figure 7. Model “B” with digital display, easy access control mode and both liquid traps and sticky trap trays. With minor modification this automated pollen trap can be used to collect pollen for a range of wind- pollinated crops. Various airborne particles or disease spores may also be collected with these traps. Figure 8. Clear glass background (with use of modified slide frame) allows high throughput digital microscope imaging and assure sample integrity. Available slide frame types & sizes tested. Sticky trap Liquid trap Figure 2. (L) Example of digital images of fluorescing pollen taken from pollen traps collected at varying pollen shed densities (source: Fonseca et al., 2002). Numbers in the left top corner indicate the number of pollen grains in each image. (R) Corn pollen grains imaged by digital microscopy using an epifluoresence Zeiss Axiovert 200M inverted microscope (source: Hanselman and Ghaffarzadeh, unpublished) LR