title: State of the art of large-scale genetic purity testing of hybrid vegetable seeds using isoelectric focusing at PetoSluis.
abstract: During the past ten years we have been engaged in developing and applying isoelectric focusing techniques to test the genetic quality of vegetable seeds. We started with isoelectric focusing using carrier ampholytes (IEF-CA), and continued research with isoelectric focusing in immobilized pH gradients (IEF-IPG) and two-dimensional electrophoresis (IPG-DALT). In addition, we have developed equipment and procedures for large-scale seed and seedling homogenization, sample preparation and semi-automatic gel staining. Moreover, we have optimized the sample application and gel running setup for large-scale analysis. We have developed hybrid purity (inbred) testing methods for all important vegetables, e.g., melon, cole crops, tomato, pepper, watermelon, squash, cucumber, radish etc. using either IEF-CA or IEF-IPG of seed or seedling proteins, followed by specific or general protein staining. To indicate the efficiency of the equipment and procedures developed we present results of two of our hybrid purity test methods, namely for brassica using polymorphism for phosphoglucomutase (PGM) from dry seeds, and for tomato using polymorphism for alcohol dehydrogenase (ADH) from imbibed seeds. We show that one person can routinely analyze 1536 individual seeds per day at a cost of about US +0.11 per seed for chemicals, materials, and electrophoresis equipment.
authors: PetoSluis, Research and Development Department, Enkhuizen, The Netherlands.
authors: H C
authors: HC
authors: Burg
authors: B M
authors: BM
authors: van den Berg
mesh_terms: D000426:Alcohol Dehydrogenase / Q000235:genetics; D002021:Buffers; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D006824:Hybridization, Genetic; D007525:Isoelectric Focusing / Q000379:methods*; D010733:Phosphoglucomutase / Q000235:genetics; D011110:Polymorphism, Genetic; D012639:Seeds / Q000201:enzymology; D014675:Vegetables / Q000201:enzymology / Q000235:genetics*
chemical_list: D002021:Buffers; D000426:Alcohol Dehydrogenase; D010733:Phosphoglucomutase