Plant Cell Reports (1989) 7:701-703 Plant Cell Reports © Springer-Verlag 1989 Analysis of lipid composition and morphological characteristics in soybean regenerantsl D.F. Hildebrand, T. R. Adams, M.L. Dahmer, E. G. Williams, and G.B. Collins Received August 1, 1988/Revised version received January 24, 1989 - Communicated by G. C. Phillips ABSTRACT A study was conducted to examine the extent of somaclonal variation of soybean plants, Glycine max (L.) Merrill cv. 'McCall', regenerated via somatic embryogenesis from cultured immature cotyledons using two different protocols. The sexual progeny of regenerants were compared with normal, seed-derived populations for morphological characteristics and fatty acid composition of seeds. First generation progeny of regenerants showed greater phenotypic variation than the control population, but this variation was not observed in the second generation. No stable somaclonal variants for fatty acid composition of the seed oil or morphological characteristics were observed, indicating that this somatic embryogenesis system should be adaptable for transformation with minimal generation of unwanted variation. ABBREVIATIONS N•, l-naphtaleneacetic acid; 2,4-D, 2,4- dichlorophenoxyacetic acid; IBA, indolebutyric acid. INTRODUCTION Several reports have described the regeneration of whole soybean plants in which plants did not arise from preexisting meristems (Lazzeri et al, 1985; Barwale et al, 1986; Ranch et al, 1985). Establishment of de novo regeneration in soybean tissue cultures opens the possibility of recovering culture-induced genetic variability or somaclonal variation in regenerated plants (Evans and Sharp, 1983; Larkin et al, 1984; Baertlein and McDaniel, 1987; Grabosch et al, 1987). Such variation may affect traits that could be useful in plant breeding. In soybean breeding, additional variation for fatty acid composition of the seed oil is desirable because natural variation appears to be limited. Mutation breeding and recurrent selection have had some I j / Contribution from the Department of Agronomy, University of Kentucky, Lexington, KY 40546. The research, was supported by the American Soybean Association and the Lubrizol Genetics Company, published with the approval of the director of the Kentucky Agricultural Experiment Station as Journal Article Number 88-3-264. Offprintrequ~ to.'D.F. Hildebrand success in developing commercial soybean lines with desired oil compositions (e.g. a 50-70% decrease in linolenic acid), but selection of soybean lines with further changes in seed oii composition is still desirable (Burton et al, 1983; Hammond and Fehr, 1984; Wilcox and Cavins, ]985). The purpose of this study was to evaluate the nature of variability, particularly for fatty acid composition of seed lipids, in plants regenerated from tissue culture by two protocols. MATERIALS AND METHODS Plant materials Plants of Glycine max (L.) Merrill cv. 'McCall' were grown in the greenhouse with natural illumination supplemented by high pressure sodium lamps to a 14 h photoperiod with 22/28 C night/day temperatures. Three populations were compared: (N) Normal, seed derived control plants. (J) Sexual progeny of nine plants regenerated after ca. 5 months in culture using the protocol of Lazzeri et ai.(1985) with 15 mg/l NAA. First and second generation progeny are designated JR 1 and JR 2. (T) Sexual progeny of a singl6 plant regenerated by a protocol developed for transformation as follows: An immature zygotic embryo (3.5 mm long) was excised from a surface sterilized pod. The embryo axis was removed and the isolated cotyledons were plated on modified MS medium (Lazzeri et al, 1985) containing 10 mg/L 2,4-D in a 2 cm x 10 cm plastic petri dish. After 10 days the cotyledons were inoculated with an overnight culture ofAgrobacterium tumefaciens washed and resuspended in MS medium without hormones. This Agrobacterium contained pAN/Kan-] (kindly supplied by Don Merlow, Agrigenetics Advanced Research Co., Madison, WI) which is a disarmed octopine-type Ti plasmid with neomycin phosphotransferase as the selectable marker (Facciotti et al, 1985; Umbeck et al,1987). After 2 days exposure to Agrobacterium, the cotyledons were replated on the same medium with the addition of 400 #g/ml Mefoxin (sodium cefoxitin) and 50 #g/ml Kanamycin. After 5 days, a well developed somatic embryo (>2 mm long) was transferred to MS BKZNmedium (Lazzeri eta], 1985). This embryo was transferred after