ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/1: 173–178, 2005 USING ENZYME POLYMORPHISM TO IDENTIFY THE GAMETIC ORIGIN OF FLAX REGENERANTS ZUZANA BARTOŠOVÁ, BOHUŠ OBERT, TOMÁŠ TAKÁC ˇ , ANDREJ KORMUT ˇ ÁK, AND ANNA PRET ˇ OVÁ * Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Akademická 2, P.O. Box 39/A, 950 07 Nitra, Slovak Republic Received December 6, 2004: revision accepted April 29, 2005 Anther culture is currently the most successful method for production of doubled haploid lines in flax. Recently, ovary culture was also described as a good source of doubled haploids. In this contribution we investigated the incorporation of enzyme polymorphism of acid phosphatase and peroxidase as molecular markers for the gametic origin of flax plants derived from anther and ovary cultures. Key words: Anther culture, ovary culture, isozymes, haploids, flax. INTRODUCTION Flax is a very important economic fiber and oilseed plant. The use of anther and ovary cultures has advan- tages for flax breeding programs. Both are considered a tool for production of haploid plant material and homozygous lines supporting breeding, and more re- cently also for molecular marker studies. Anther culture is currently the most successful method for production of doubled haploid lines in flax (Fried et al., 1995; Bergmann and Fried, 1996; Chen et al., 1998; Pretova and Obert, 2000; Obert et al., 2004a,b), but its efficiency is still very low. Flax gy- nogenesis was first reported by Bartošová et al. (2003). The conditions under which the flax donor plants are cultivated seem to be one of the limiting factors for good androgenic response (Bartošová and Pret ’ ová, 2004). Both androgenesis and gynogenesis can be useful for breeding purposes when the gametic origin of the re- generated plants is confirmed. In both cases, regener- ation can easily take place not only from the microspore or the egg cell in the ovule, but also from the surround- ing diploid somatic tissue (anther wall and/or ovary tissue). For this reason, identification of the gametic origin of the regenerants is very important. For dis- crimination of doubled haploids originated from hybrid flax lines via anther cultures, morphological markers are the main ones used so far (Tejklova, personal communication). Chen et al. (2001) employed several markers (RAPD, ISSR) of dominant phenotype and codominant markers (PCR/RFLP) for identification of presumed flax doubled haploids of androgenic origin. We have chosen the polymorphism of peroxidase (EC 1.11.1.7) and acid phosphatase (EC 3.1.3.2) for our studies. Peroxidases in plants catalyze a large variety of reactions (Siegel, 1993). The involvement of peroxi- dases in stress-related physiological processes (Low and Merida, 1996) as well as in plant-pathogen inter- actions have been demonstrated (Montalbini et al., 1995; Wojtaszek, 1997). The isozyme patterns of per- oxidases are intensively studied for cultivar identifica- tion in plant breeding, seed marketing and other fields of agriculture (Samec et al., 1998). Acid phosphatase is one of the critical lytic enzymes in plants, and has been reported as a marker for differentiation processes in plant development (Coppens and de Wite, 1990; Feirer and Simon, 1991). Acid phosphatase is present in tis- sues characterized by high metabolic activity, and in- dicates the need for high nutrient uptake (Raghavan, 1977). Both enzymes used in our investigations have been studied in flax during callus formation and root and shoot regeneration (McDougal et al., 1992, 1993). The objectives of our studies were (1) to identify the gametic origin of flax regenerants, and (2) to use the enzyme polymorphism of acid phosphatase and PL ISSN 0001-5296 © Polish Academy of Sciences, Cracow 2005 *e-mail: anna.pretova@savba.sk Abbreviations: RAPD – random amplified polymorphic DNA; ISSR – inter-simple sequence repeat amplification; PCR/RFLP – PCR primers produce a DNA fragment, which after digestion with an enzyme yields special RFLP (restriction fragment length poly- morphism) patterns.