Theor Appl Genet (1990) 80:409-416 ~(~ N .... ' ICS 9 Springer-Verlag 1990 The expression and perpetuation of inherent somatic variation in regenerants from embryogenic cultures of Pennisetum glaucum (L.) R. Br. (pearl millet) E M. Morrish 1, W.W. Hanna 2 and I.K. Vasil 1,, 1 Laboratory of Plant Cell and Molecular Biology, Vegetable Crops Department, University of Florida, Gainesville, FL 32611, USA 2 USDA/ARS, Coastal Plain Experiment Station, Tifton, GA, 31793, USA Received November 3, 1989; Accepted April 11, 1990 Communicated by P. L. Pfahler Summary. Genetic analysis was conducted on the quali- tative and quantitative traits of sexual progeny derived from embryogenic cultures of two inbred lines of Pen- nisetum glaucurn (L.) R. Br. (pearl millet). These lines included a genetically stable inbred of Tift 23BE and a genetic marker line, derived from Tift 23BE, which bore qualitative genetic markers for a dominant purple plant trait (P) and two recessive traits, early flowering (%) and yellow stripe (ys). Tissue culture regenerant populations (R0) and progeny populations (RI) produced from these plants by selfing showed no qualitative genetic variation when derived from the genetically stable inbred Tift 23BE. In contrast, stably inherited qualitative variation for a number of genetic markers was observed in R0, R1, and R 2 progeny of the genetic marker line. In a popula- tion of 1,9l I plants regenerated over a 12-month period, 0.02% of the population lost or showed reduced expres- sion of the purple plant trait and 92% of plants were chlorophyll deficient. Plants showing reduction or loss of anthocyanin synthesis also flowered later. None of the purple plants showed any significant variation in flower- ing time. The incidence of chlorophyll deficiency in- creased with time in culture, 51% of the progeny regener- ated after 1 month were chlorophyll deficient, while 100% of the plants regnerated after 12 months were chlorophyll deficient. Qualitative variation was also ob- served in control populations of the genetic marker line where 1 plant in a total of 1,010 lacked purple pigmenta- tion and a total of 6% showed chlorophyll variation in the first generation (So). The presence of qualitative vari- ation in controls suggests that the inherent variation present in the original explant was expressed and perpet- uated in vitro. Quantitative variation was observed for a number of traits in the first sexual cycle (R1) of the marker line but did not occur in a subsequent generation, suggesting that this variation was epigenetic. Key words: Gramineae - Genetic analysis - Penniseturn glaucurn - Somatic embryogenesis - Tissue culture Introduction Experience gained from studies on cultured cells and regenerated plants has demonstrated that the in vitro cul- ture of plant cells can result in the production of epige- netic, physiological, cytological, or genetic variation (Meins 1983; Orton 1984; D'Amato 1986; Gould 1986). This variation is considered a potential source of novel breeding lines (Larkin 1985). Such widespread manifes- tation of somatic variation, however, creates a major problem if tissue culture is to be used in conjunction with genetic engineering for plant improvement, since the suc- cess of this procedure depends on the maintenance of genetic fidelity of the introduced gene or genes. It is, therefore, imperative to establish culture and regenera- tion procedures that reduce or eliminate variation. Such a system may be established through developing a clearer understanding of the potential sources of somatic varia- tion in cultured cells and the factors that control its expression in regenerated plants. With this knowledge we may be able to minimize the production of somatic vari- ation where genetic fidelity is required or directionally maximize its expression in cases where somatic variation in vitro is desirable as, e.g., in the selection of useful and stable mutants. Available evidence indicates that the pathway of plant regeneration, genetic architecture of the donor plant material, genotype, and the duration of culture are among the most significant factors affecting both the nature and number of variants produced in tissue culture (e.g., Edallo et al. 1981; Fukui 1983; Oono 1985; McCoy