Screening Multiple Soybean Cultivars (MG 00 to MG VIII) for Somatic Embryogenesis Following Agrobacterium-Mediated Transformation of Immature Cotyledons

improvements on the efficiency of the embryogenic culture system and reduced culture time have been reA total of 15 soybean [Glycine max (L.) Merr.] cultivars representported (Samoylov et al., 1998a, 1998b; Santarém et al., ing maturity groups 00 to VIII were assessed for their embryogenic and transformation potentials. All cultivars were identified as embryo1998; Santos et al., 1997), this system remains highly genic under hygromycin selection following Agrobacterium-mediated genotype-dependent. Moreover, this system is not very transformation of immature cotyledons. Histochemical -glucuronidesirable because of low transformation efficiency, chidase (GUS) assays of induced hygromycin-resistant somatic embryos merism, and complex patterns of introduced DNA in (SEs) showed that 13 out of 15 cultivars were amenable for transformatransgenic plants (Hadi et al., 1996; Trick and Finer, tion. Wide variations among different genotypes were observed for 1998). their embryogenic capacity under hygromycin selection. Three cultiGiven that there is no efficient transformation system vars, Cisne, Council, and Kunitz, were highly embryogenic yielding for a wide range of soybean cultivars, several studies more than 50% responding explants and 1.5 to 2.4 hygromycin-resishave evaluated the potential of various soybean genotant SEs per responding explant. The transformation potential of multitypes for somatic embryogenesis (Parrott et al., 1989; ple soybean cultivars was highly correlated with the embryogenic potential of immature cotyledons under hygromycin selection. It was possible Bailey et al., 1993; Meurer et al., 2001; Tomlin et al., to distinguish between highly and poorly embryogenic genotypes by 2002). However, these studies have primarily focused on visually observing the phenotype of cultured immature cotyledons. the embryogenic response of soybean genotypes under For highly embryogenic cultivars, the induction of somatic embryos nonselective conditions. Meurer et al. (1998) have sugmainly originated from actively dedifferentiating and browning/negested this strategy may be misleading because infected crotic tissues along the margins of the abaxial side of cultured cotytarget tissues may differentiate differently in the presledons. ence of Agrobacterium and selective agents. For example, Hartweck et al. (1988) and later Santarém et al. (1997) have reported that when the adaxial side of imS the first transgenic soybean plants were demature cotyledons is oriented upward on a nonselective veloped via Agrobacterium-mediated transformamedium, high induction of somatic embryogenesis is tion of cotyledonary nodes (Hinchee et al., 1988), imobserved. Whereas, Ko et al. (2003) have reported that provements have been made by means of hypervirulent frequencies of somatic embryogenesis and transformaA. tumefaciens strains, addition of thiol compounds to tion are higher when the abaxial side of explants is the cocultivation medium, or sonication of explants oriented upward on the selective medium. (Hood et al., 1993; Torisky et al., 1997; Trick and Finer, In this report, soybean cultivars representing a wide 1997; Olhoft et al., 2001). However, successful transforrange of maturity groups (MG 00 to MG VIII) have mation has been limited to a few soybean cultivars in been screened for their embryogenic and transformalimited maturity groups (Hinchee et al., 1988; Di et al., tion potentials using the protocol described by Ko et 1996; Trick and Finer, 1998; Yan et al., 2000; Donaldson al. (2003). To our knowledge, this is the first report on and Simmonds, 2000; Olhoft and Somers, 2001; Olhoft screening multiple soybean cultivars of various genetic et al., 2003). backgrounds and maturities for somatic embryogenesis An alternative target tissue–transformation protocol under hygromycin selection. We also report on observed that is widely used for soybean transformation is that of phenotypic differences of immature cotyledonary exproliferative embryogenic suspension cultures–particle plants that are associated with genotypic variability for bombardment (Finer and McMullen, 1991; Sato et al., embryogenic response. 1993; Parrott et al., 1994). These cultures consist of dense globular-stage clumps of embryogenic tissues deMATERIALS AND METHODS rived from secondary globular embryos, initially formed on immature cotyledons, and maintained by routine subPlant Material culture in a liquid medium. However, this regeneration Fifteen soybean cultivars were selected to represent the system relies on the lengthy process of liquid propagarange of pedigree diversity in the USA as defined by Gizlice tion before and/or following transformation. Although et al. (1996). These cultivars belonged to different maturity groups, based on photoperiod requirements and time to flowT.-S. Ko and S.S. Korban, Dep. of Natural Resources and Environmenering, with lower maturity groups representing more northerntal Sciences, Univ. of Illinois, Urbana, IL 61801; R.L. Nelson, USDAadapted germplasm and higher maturity groups representing ARS, Soybean/Maize Germplasm, Pathology, and Genetics Research more southern-adapted germplasm. The cultivars were GlaUnit, Dep. of Crop Sciences, Univ. of Illinois, Urbana, IL 61801. Recier (MG 00), Council (MG 0), MN0301 (MG 0), MN1301 ceived 14 Oct. 2003. *Corresponding author ([email protected]). (MG I), Savoy (MG II), Olympus (MG II), NE3399 (MG III), Published in Crop Sci. 44:1825–1831 (2004).  Crop Science Society of America Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; GUS, -Glucuronidase; HPT, Hygromycin phosphotransferase; SEs, somatic embryos. 677 S. Segoe Rd., Madison, WI 53711 USA