POLLEN: DEVELOPMENT AND PHYSIOLOGY
CONTENTS POLLEN GRAIN COUNTING USING A CELL COUNTER A TOOLKIT FOR TEASING APART THE EARLY STAGES OF POLLEN–STIGMA INTERACTIONS IN ARABIDOPSIS THALIANA DECAPITATION CROSSES TO TEST POLLEN FERTILITY MUTATIONS FOR DEFECTS IN STIGMA-STYLE PENETRATION ISOLATION OF THE PISTIL-STIMULATED POLLEN TUBE SECRETOME RESTRICTED POLLINATION FOR TRACING INDIVIDUAL POLLEN TUBES IN A PISTIL SEMI-IN VIVO ASSAY FOR POLLEN TUBE ATTRACTION ISOLATION AND CLONING OF SUPPRESSOR MUTANTS WITH IMPROVED POLLEN FERTILITY WORK?OW TO CHARACTERIZE MUTANTS WITH REPRODUCTIVE DEFECTS INTERNALLY CONTROLLED METHODS TO QUANTIFY POLLEN TUBE GROWTH AND PENETRATION DEFECTS IN ARABIDOPSIS THALIANA ARABIDOPSIS THALIANA POLLEN TUBE CULTURE FOR MULTI-OMICS STUDIES LARGE-SCALE ANALYSIS OF POLLEN VIABILITY AND OXIDATIVE LEVEL USING H2DCFDA-STAINING COUPLED WITH FLOW CYTOMETRY OBTAINING MUTANT POLLEN FOR PHENOTYPIC ANALYSIS AND POLLEN TUBE DUAL STAINING GALVANOTROPIC CHAMBER FOR CONTROLLED REORIENTATION OF POLLEN TUBE GROWTH AND SIMULTANEOUS CONFOCAL IMAGING OF INTRACELLULAR DYNAMICS ANALYZING INTRACELLULAR GRADIENTS IN POLLEN TUBES SILICONE CHAMBERS FOR POLLEN TUBE IMAGING IN MICROSTRUCTURED IN VITRO ENVIRONMENTS ANALYZING THE IMPACT OF PROTEIN OVEREXPRESSION ON CA2+ DYNAMICS AND DEVELOPMENT IN TOBACCO POLLEN TUBES IMAGING AND ANALYSIS OF THE CONTENT OF CALLOSE, PECTIN, AND CELLULOSE IN THE CELL WALL OF ARABIDOPSIS POLLEN TUBES GROWN IN VITRO CHARACTERIZATION OF GROWTH BEHAVIOR AND THE RESULTING FORCES APPLIED BY POLLEN TUBES IN A 3D MATRIX FLOW CHAMBER ASSAY TO IMAGE THE RESPONSE OF FRET-BASED NANOSENSORS IN POLLEN TUBES TO CHANGES IN MEDIUM COMPOSITION QUANTI?CATION OF MECHANICAL FORCES AND PHYSIOLOGICAL PROCESSES INVOLVED IN POLLEN TUBE GROWTH USING MICRO?UIDICS AND MICROROBOTICS MEASURING EXOCYTOSIS RATE IN ARABIDOPSIS POLLEN TUBES USING CORRECTED FLUORESCENCE RECOVERY AFTER PHOTOCONVERSION (CFRAPC) TECHNIQUE TESTING POLLEN TUBE PROTEINS FOR IN VIVO BINDING TO PHOSPHATIDIC ACID BY N-BUTANOL TREATMENT AND CONFOCAL MICROSCOPY CHAPTER POLLEN GRAIN COUNTING USING A CELL COUNTER ABSTRACT THE NUMBER OF POLLEN GRAINS IS A CRITICAL PART OF THE REPRODUCTIVE STRATEGIES IN PLANTS AND VARIES GREATLY BETWEEN AND WITHIN SPECIES IN AGRICULTURE, POLLEN VIABILITY IS IMPORTANT FOR CROP BREEDING IT IS A LABORIOUS WORK TO COUNT POLLEN TUBES USING A COUNTING CHAMBER UNDER A MICROSCOPE HERE, WE PRESENT A METHOD OF COUNTING THE NUMBER OF POLLEN GRAINS USING A CELL COUNTER IN THIS METHOD, THE COUNTING STEP IS SHORTENED TO 3 MIN PER ?OWER, WHICH, IN OUR SETTING, IS MORE THAN ?VE TIMES FASTER THAN THE COUNTING CHAMBER METHOD THIS TECHNIQUE IS APPLICABLE TO SPECIES WITH A LOWER AND HIGHER NUMBER OF POLLEN GRAINS, AS IT CAN COUNT PARTICLES IN A WIDE RANGE, FROM 0 TO 20,000 PARTICLES, IN ONE MEASUREMENT THE CELL COUNTER ALSO ESTIMATES THE SIZE OF THE PARTICLES TOGETHER WITH THE NUMBER BECAUSE ABORTED POLLEN SHOWS ABNORMAL MEMBRANE CHARACTERISTICS AND/OR A DISTORTED OR SMALLER SHAPE, A CELL COUNTER CAN QUANTIFY THE NUMBER OF NORMAL AND ABORTED POLLEN SEPARATELY WE EXPLAIN HOW TO COUNT THE NUMBER OF POLLEN GRAINS AND MEASURE POLLEN SIZE IN ARABIDOPSIS THALIANA, ARABIDOPSIS KAMCHATICA, AND WHEAT (TRITICUM AESTIVUM) KEY WORDS POLLEN NUMBER, POLLEN SIZE, POLLEN VIABILITY, CELL COUNTER (CASY CELL COUNTER), ARABIDOPSIS THALIANA, ARABIDOPSIS KAMCHATICA, WHEAT (TRITICUM AESTIVUM) INTRODUCTION POLLEN GRAIN NUMBER HAS LONG BEEN STUDIED FROM AGRICULTURAL AND EVOLUTIONARY VIEWPOINTS [1–3] FOR EXAMPLE, THE NUMBER OF POLLEN GRAINS WAS SHOWN TO HAVE DECREASED DURING RICE DOMESTICATION [4] THE NUMBER OF VIABLE POLLEN GRAINS IS CRITICAL FOR BREEDING BECAUSE DECREASED YIELD DUE TO POLLEN ABORTION CAN BE INDUCED BY DIVERSE ABIOTIC STRESSES, SUCH AS LOW/HIGH TEMPERATURE, SALT STRESS, AND OSMOTIC SHOCK [5, 6] TO CONTROL POLLEN MASS IS A MAJOR CHAL- LENGE TO INCREASE THE EF?CIENCY OF HYBRID BREEDING IN WHEAT [7] IN AN EVOLUTIONARY CONTEXT, SHIFTS FROM OUTCROSSING TO SEL?NG ARE ONE OF THE MOST FREQUENT EVOLUTIONARY TRANSITIONS IN ANGIOSPERMS [8] SEL?NG PLANTS TEND TO SHOW A SET OF TRAITS TERMED SEL?NG SYNDROME, SUCH AS SMALLER ?OWER SIZE, REDUCED SCENT AND NECTAR, AND A REDUCED POLLEN GRAIN NUMBER (OR LOW POLLEN–OVULE RATIO) THE ,