Irradiation (X-ray; 5-15 Gy) of protoplasts treated with plasmid-DNA and PEG yielded higher transformation rates in comparison to non-irradiated protoplasts transformed by the same method. This could be demonstrated for four plant species. The irradiation doses used did not affect the total number of colonies regenerated without selection pressure, but resulted in 3-6-fold enhancement of hygromycin- or kanamycin-resistant colonies. Plant regeneration frequencies of transformed colonies derived from irradiated and non-irradiated protoplasts were similar in tobacco as well as in Petunia. Higher integration rates of foreign DNA as a consequence of an increased recombination machinery in irradiated cells may be responsible for the enhancement of the number of stably transformed colonies.

An efficient procedure has been developed for inducing direct somatic embryogenesis, organogenesis, and regeneration of plants from tissue cultures of peanut (Arachis hypogaea L.). Thin transverse sections of the cotyledons and juvenile leaves were cultured on Murashige and Skoog medium supplemented with N6-benzylaminopurine (BAP) or a substituted phenylurea, thidiazuron (TDZ). Somatic embryos or shoot buds differentiated from cut surfaces of the cotyledons and midrib region of the leaves. The application of BAP induced differentiation of shoot buds whereas the treatment with TDZ resulted in the production of somatic embryos. Somatic embryos developed into plants after subculturing on a basal meduim. Agar-solidified medium was found to be superior to the liquid medium for the development of embryos and shoot buds. The procedure of TDZ-induced somatic embryogenesis and plant regeneration was successfully applied to three genotypes of peanut. A distinct feature of this study is the induction of the morphogenic competence in cultures of seedling expiants of peanut that so far have remained recalcitrant to somatic embryogenesis in vitro. Key words: peanut, Arachis hypogaea, shoot regeneration, somatic embryogenesis, thidiazuron, plant regeneration.

In planta differentiation of somatic embryos was induced in seedlings of peanut (Arachis hypogaea L.) obtained from mature seeds germinated on a medium supplemented with thidiazuron (TDZ: N-phenyl-N(1)- (1,2,3 thiadiazol-yl)urea). At optimum levels of TDZ (10 μM), all germinating seeds produced embryogenic seedlings, and somatic embryos developed in the apical region and on the surface of cotyledons and hypocotyls. These somatic embryos matured, germinated, and formed shoots which eventually developed into whole plants. Thidiazuron-induced direct embryogenesis from morphologically intact seedlings may provide an excellent experimental system for investigating somatic embryogenesis and the morphoregulatory role of TDZ.

Somatic embryogenesis was induced in hypocotyl explants of geranium (Pelargonium x hortorum) cultured on media supplemented with various concentrations of N-phenyl-N'-1,2,3-thiadiazol-5-ylurea (thidiazuron). In less than 2 weeks, somatic embryos were observed in treatments containing levels of thidiazuron (TDZ) ranging from 0.2 to 1.0 micromolar. The use of N'-benzylaminopurine in combination with indole-3-acetic acid also evoked embryogenesis, but the efficiency of somatic embryo production was significantly lower than that obtained with TDZ. Hypocotyl culture for only 2 days on TDZ-supplemented medium before transfer to a basal medium was sufficient for inducing somatic embryogenesis. This distinction between the induction and expression of embryogenesis may provide an experimental system for studying the developmental biology of somatic embryogenesis. Substitution of the auxin-cytokinin requirement for the induction of somatic embryogenesis by TDZ suggests the possibility of a novel mode of its action by modulation of endogenous growth regulators.TDZ3, a substituted phenylurea (N-phenyl-N'-1,2,3-thiadiazol-5-ylurea; Dropp), is primarily used as a cotton defoliant (1) and has been shown in various cytokinin bioassays to exhibit strong cytokinin-like activity similar to that of N6-substituted adenine derivatives (12). In many cytokinin-dependent callus cultures, the application of TDZ evoked a comparable or higher degree of growth response than adenine-based cytokinins (5, 20). The morphogenetic responses in which TDZ has been found to mimic cytokinin-like activity include release of lateral buds from dormancy (21) and shoot formation in cultures of a wide variety of plant species (7,9 cytokinins, or it may induce the synthesis and (or) accumulation of endogenous cytokinins (5, 11). The latter notion is based on the effects of the high ability of TDZ in inducing cytokinin-dependent shoot regeneration and modulation of endogenous levels of cytokinins. In this communication, we demonstrate the high efficiency of TDZ in stimulating somatic embryogenesis in geranium hypocotyl cultures, a response usually mediated by an appropriate combination of an auxin and a cytokinin. Our results suggest for the first time that auxin(s) may also be involved in the induction and/or expression of TDZ-induced morphogenic differentiation. MATERIALS AND METHODSSeeds of the diploid zonal geranium (Pelargonium x hortorum Bailey) cv Scarlet Orbit Improved were surface sterilized by dipping in 95% ethanol for 30 s and then immersing in a 1.2% solution of sodium hypochlorite containing two drops of Tween 20 per 200 mL. Seeds were agitated in this solution for 20 min and then rinsed three times in sterile distilled water. Ten seeds were aseptically cultured per 100 x 15 mm Petri dish on 30 mL of MS (13)

The identification of molecular markers for economically significant traits should greatly improve the speed and efficiency of all peanut (Arachis hypogaea L.) breeding programs. Development and phenotypic evaluation of recombinant inbred line (RIL) populations of peanut, along with molecular genotyping, will be essential for association of markers with traits. The primary objectives of this research were to develop 16 structured RIL populations that can be used by the peanut research community, and to begin high-resolution phenotyping of these populations. Crosses were made using a 2 by 8 (common by unique) factorial nested association mapping design. Parents were selected to attempt to maximize genetic diversity while meeting practical breeding objectives. First, two modern runner cultivars (Tifrunner and Florida-07) were selected as common parents because runner cultivars account for about 80% of the production in the U.S. Second, the eight unique parents were selected to supply diversity across market classes and botanical varieties and are donors of favorable alleles for enhancing drought tolerance and resistance to most important disease of peanut in the U.S. The eight unique parents are N08082olJCT (a Bailey derived high oleic breeding line), C76-16, NC 3033, SPT 06-06, SSD 6 (PI 576638), OLin, New Mexico Valencia A, and Florunner. The 16 populations were advanced using summer and winter nurseries. Input from multiple disciplines has resulted in a prioritized list of populations and traits that should be examined, and seed increase has begun to provide the community with material for extensive phenotyping. In-depth phenotyping and genotyping of these populations should result in markers that can be deployed by breeding programs for the development of improved cultivars.