Received: 19 November, 2008. Accepted: 21 February, 2009. Original Research Paper International Journal of Plant Breeding ©2009 Global Science Books Immature Embryo Culture for Early Screening of Imidazolinone Resistance in Sunflower Gabriela Breccia 1,2 • Tatiana Vega 1,2 • Graciela Nestares 1* • María Laura Mayor 1 • Roxana Zorzoli 1,3 • Liliana Picardi 1,3 1 Cátedra de Genética, Facultad de Ciencias Agrarias, Universidad Nacional de Rosario, Campo Experimental “J. F. Villarino”, C.C. 14, S2125ZAA Zavalla, Argentina 2 Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET), Argentina 3 Consejo de Investigaciones de la Universidad Nacional de Rosario (CIUNR), Argentina Corresponding author: * gnestare@unr.edu.ar ABSTRACT The development of imidazolinone-resistant sunflower cultivars represents a great advantage in controlling weeds. This type of herbicide resistance can only be achieved with homozygosis of two resistant genes; hence breeding for this trait is time-consuming. The objectives of this study were to develop plantlets from immature sunflower embryos to evaluate imazethapyr resistance. Three methods were used, an in vitro technique and two non-sterile techniques (dish and pot assays). Three genotypes differing in imidazolinone resistance were evaluated for germination and plantlet developmental variables. Immature embryos successfully germinated in the in vitro culture and the pot germination techniques having similar efficiency. Given the simplicity of the pot assay, plantlets from pot germination were used for screening herbicide resistance at four different doses: 2.5, 5, 7.5 and 10 μM. Susceptible plants showed chlorosis and arrested growth, mainly when exposed to 7.5 and 10 μM of imazethapyr. Surviving plants were transferred to pots and placed in a greenhouse and successfully completed their lifecycle. The techniques described herein would allow selecting sunflower inbreds for imidazolinone resistance when shortening breeding cycle. _____________________________________________________________________________________________________________ Keywords: Helianthus annuus L., herbicide resistance, in vitro culture, pot germination Abbreviation: ALS, acetolactate synthase INTRODUCTION Weed competition causes substantial yield loss in sunflower (Helianthus annuus L.). Competition experiments showed that weeded conditions produced a 20 to 53% loss in yield compared to weed-free conditions (Blamey and Zollinger 1997). Genes for resistance to acetolactate synthase (ALS)- inhibiting herbicides in sunflower have been introgressed from resistant wild populations (ANN-PUR and ANN- KAN) into elite inbred lines for the purpose of developing herbicide resistant cultivars and hybrids (Al-Khatib et al. 1998; Miller and Al-Khatib 2002). Bruniard and Miller (2001) proposed a digenic model for the inheritance of imidazolinone resistance. In this model, a major gene (Imr 1 ) has a semidominant type of gene action and a second gene (Imr 2 ) acts as modifier when the major gene is present. According to these authors, complete resis- tance in sunflower can only be achieved with homozygosity of both resistant genes (Imr 1 Imr 1 Imr 2 Imr 2 ) in an inbred line or hybrid. The development of resistant elite inbred lines to further inclusion in hybrid programs involves introgression of the resistance genes into elite germplasm by backcrossing. Besides being a time-consuming process, it also implies one or more generations of self pollination and progeny testing to correctly classify and select the resistant phenotypes (Bruniard and Miller 2001). Operationally, identification of imidazolinone resistant genotypes involves applying herbi- cide to plants grown in the field or greenhouse, being a time- consuming and costly task. Initially, a laboratory technique that allows early screening resistant genotypes would be a useful tool to help reducing time and resources when breed- ing for imidazolinone resistance. Embryo culture is one of the earliest in vitro culture me- thodologies applied to practical problems and has proven of greatest value to breeders (Bridgen 1994). Its major applica- tion in sunflower plant breeding has been for overcoming incompatibility in interspecific crosses (Chandler and Beard 1983; Denat et al. 1991; Kräuter et al. 1991; Sukno et al. 1999). Immature embryo culture has also been a technique extensively used for shortening the breeding cycle in this species, allowing four to five generations per year (Plotni- kov 1983; Alissa et al. 1986; Azpiroz et al. 1987; Jambhul- kar 1995). In contrast, non-sterile germination techniques represent a simpler and cheaper alternative to in vitro culture of immature sunflower embryos (Paul and Barthou 1994; Torresán et al. 1996). Culture of immature embryos for rapid fixation of inbred lines will be of particular interest only if selection can be achieved at the same time. Given that breeding for imidazolinone resistance requires a considerable amount of time and resources, culture of immature embryos followed by selection of herbicide-resistant plantlets could be useful in saving resources and shortening the cycle of develop- ment of new varieties. The present study aimed to develop an efficient method to select imidazolinone-resistant genotypes while shortening breeding cycle. The objectives were to a) compare three different techniques for obtaining plantlets from immature sunflower embryos and b) evaluate different doses of ima- zethapyr for the selection of genotypes. MATERIALS AND METHODS Plant material Three sunflower inbred lines were used in this study: HA425B, 1058-1 and HA89B, which were described as imidazolinone resis- tant (Imr 1 Imr 1 Imr 2 Imr 2 ), intermediate (Imr 1 Imr 1 imr 2 imr 2 ) and sus- ceptible (imr 1 imr 1 imr 2 imr 2 ), respectively (Bruniard 2001; Bruniard ®