Journal of Integrative Plant Biology 2008, 50 (3): 375–383 High-throughput Procedure for Single Pollen Grain Collection and Polymerase Chain Reaction in Plants Ping-Hua Chen 1,2† , Yong-Bao Pan 1† * and Ru-Kai Chen 2 ( 1 USDA-ARS, Southern Regional Research Center, Sugarcane Research Laboratory, Houma, LA 70360, USA; 2 Key Laboratory of Eco-physiology & Genetic Improvement for Sugarcane, Ministry of Agriculture, Fujian Agriculture and Forestry University, Fujian Normal University, Fuzhou 350002, China) Abstract Single pollen grain polymerase chain reaction (PCR) has succeeded in several species, however only limited numbers of pollen grains were involved due to difficulties in pollen isolation and lysis. This has limited its application in genetic analysis and mapping studies in plants. A high-throughput (HT) procedure for collecting and detecting genetic variation in a large number of individual pollen grains by PCR is reported. The HT procedure involved the collection of individual pollen grains by a pair of special forceps and the lysis of pollen grains in a heated alkali/detergent solution followed by neutralization with a tris-ethylenediamine tetraacetic acid (TE) buffer. These resulting template solutions yielded PCR reactions involving the 5S ribosomal RNA intergenic spacers, randomly amplified polymorphic DNA, and simple sequence repeats markers. Using this procedure, one person with experience could collect and process up to 288 single pollen grain PCR reactions per day. The method worked well on sugarcane, corn, Miscanthus spp., snap bean, sorghum, and tomato. The ability to collect and conduct PCR on individual pollen grains on a large scale offers a new approach to genetic analyses and mapping studies in an easily controllable environment with a considerable cost reduction. The method will also significantly benefit studies in species that are difficult subjects for classical genetic research. Key words: DNA marker; high-throughput; polymerase chain reaction; single pollen grain. Chen PH, Pan YB, Chen RK (2008). High-throughput procedure for single pollen grain collection and polymerase chain reaction in plants. J. Integr. Plant Biol. 50(3), 375–383. Available online at www.jipb.net Polymerase chain reaction (PCR) has succeeded on single cells and single pollen grains for studying genetic variation (Li et al. 1990; Zhang et al. 1992; Petersen et al. 1996; Matsunaga et al. 1999; Aziz and Sauve 2003; Parducci et al. 2005), gene expression (Shoemaker et al. 2005), and genotyping (Li and Yeung 2002; Aziz et al. 2005). However, only limited numbers of pollen grains were involved in these studies (Matsunaga et al. Received 11 Apr. 2007 Accepted 5 Aug. 2007 Supported in part by Grower/processor Check-off Funds administrated by the American Sugar Cane League of the USA., Inc., Thibodaux, Louisiana, USA (to Y.-B. Pan) and the Chinese 948 Project (2003-Q06) (to P.-H. Chen). † These authors contributed equally to this work. ∗ Author for correspondence. Tel: +1 985 853 3165; Fax: +1 985 868 8369; E-mail: <ypan@srrc.ars.usda.gov>. C  2008 Institute of Botany, the Chinese Academy of Sciences doi: 10.1111/j.1744-7909.2007.00624.x 1999; Aziz and Sauve 2003; Parducci et al. 2005) except for one report involving 60 pollen grains (Aziz et al. 2005). Single cells, such as human sperm, could be isolated with the aid of flow cytometry (Li et al. 1990; Zhang et al. 1992), a micromanipulator (Shoemaker et al. 2005), or a capillary tube (Li and Yeung 2002), while single pollen grains were collected with laser-mediated manipulation (Matsunaga et al. 1999), micromanipulator (Aziz and Sauve 2003; Haliyo and Regnier 2003; Aziz et al. 2005), or specifically modified glass micropipettes (Parducci et al. 2005). A micromanipulator was primarily used for picking up single pollen grains; however, the efficiency was low and the number of pollen grains being picked up never exceeded 60 in a single study (Aziz et al. 2005). Laser-mediated manipulation was highly effective in isolating single pollen grains, but the instrument was expensive. The thick and rigid walls (Santos and Mariath 1999; Parre and Geitmann 2005) and the sizes (0.005 to 0.25 mm in diameter) of mature pollen grains (Pinar and Inceoglu 1999; Perveen and Qaiser 2001; Sarkissian and Harder 2001) facilitate the picking up of individual pollen grains under a dissecting microscope by