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Methodology for starch gel electrophoresis and protocols for isozymes of 32 plant genera

R.M.L. Ballve^I,II; H.P. Medina Filho^II; R. Bordignon^II; M.M.A. Lima^II

^IFundação IAC, Av. Barão de Itapura 1481, Sala A, 13020-902 Campinas, SP, Brasil
^IIInstituto Agronômico de Campinas, Seção de Genética, Caixa Postal 28, 13001-970 Campinas, SP, Brasil. Send correspondence to H.P.M.F°.

ABSTRACT

Starch gel electrophoresis is a well recognized technique for providing 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 technique need to be made. Usually this is a time consuming process because of several factors involved in electrophoresis per se, in the extraction of enzymes and staining reactions and also due to complex interactions among them. During many years of isozyme work with the equipment of Tanksley (Report of the Tomato Cooperative 29: 37-38, 1979), which permits the analysis of up to seven enzymes and 48 samples per gel, a number of factors and interactions were tested and a general methodology 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 Borate (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 run at 30 mA up to 300 V; staining with 25 ml for each slice; and fixation 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, Solanum, Persea, Ricinus, Saccharum, Bixa, Boehmeria, Bromelia, Ananas, Paracoffea, Psylanthus, Microcitrus, Fortunella, Citropsis, Feroniella, Swinglea, Tnphasia, Murraya and Clausena. More detailed investigations of methodology were carried out in Citrus, Poncirus, Severinia, Carica, Coffea, Zea, Coix, Stevia, Allium, Panicum and Euterpe. This methodology has been succesfully applied in the early identification of hybrids and nucellar clones of citrus and its relatives.

Keywords: starch gel; electrophoresis; isozymes; plant.

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