METHODOLOGY FOR STARCH-GEL ELECTROPHORESIS AND PROTOCOLS FOR ISOZYMESOF 32 PLANT GENERA

Starch gel electrophoresis is a well recognized technique for providin g genetic markers in plant species. The technique is easy, inexpensive and allows simultaneous analysis of a great number of individuals and several isozyme systems. However for application of this methodology to a new species, several tests and a general adaptation of the techni que need to he made. Usually this is a time consuming process because of several factors involved in electrophoresis per se, in the extracti on of enzymes and staining reactions and also due to complex interacti ons among them. During many years of isozyme work with the equipment o f Tanksley (Report of the Tomato Cooperative 29: 37-38, 1979), which p ermits the analysis of up to seven enzymes and 48 samples per gel, a n umber of factors and interactions were tested and a general methodolog y that considerably reduces the number of tests necessary to adapt the technique to a new species was developed. Standard conditions are: 12 % SIGMA starch; gel buffer TRIS (0.018 M)-Citrate (0.036 M); gel pH (7 .9-8.3), electrode buffer Berate (0.3 M) pH 8.3; 25 minutes pulse and 60 minutes run with a current of 25 mA and up to 150 V; 180 minutes ru n at 30 mA up to 300 V; staining with 25 ml for each slice, and fixati on with 50% glycerol. The variables remaining for further testing are the choice of tissue, grinding buffers and enzyme activity. Thirty-two genera were studied including, Gluillielma, Leucaena, Lycopersicon, S olanum, Persea, Ricinus, Saccharum, Bixa, Boehmeria, Bromelia, Ananas, Paracoffea, Psylanthus, Microcitrus, Fortunella, Citropsis, Feroniell a, Swinglea, Triphasia, Murraya and Clausena. More detailed investigat ions of methodology were carried out in Citrus, Poncirus, Severinia, C arica, Coffea, Zea, Coix, Stevia, Allium, Panicum and Euterpe. This me thodology has been succesfully applied in the early identification of hybrids and nucellar clones of citrus and its relatives.