Hormone-free medium will support callus production and subsequent shoot regeneration from whole plant leaf explants in some sugarbeet (Beta vulgaris L.) populations.

Sugarbeet plants representing 14 of 16 germplasm sources (4 to 5 plants per source) produced callus from leaf disks on a hormone-free Murashige and Skoog based medium. Overall, 49.2% of explants from partially expanded leaves of whole plants initiated callus (53 of 74 plants tested), in an average time of 96.7 days. The time to callus was considerably longer than the 4-6 weeks observed when 1 mg/L N(6)-benzyladenine has been used in the medium. Shoots were regenerated on the hormone-free medium without subculture from callus of eight individual genotypes, representing 3 of the 14 populations that produced callus. Shoots produced by 'Gartons White Knight' and 'L53' appeared to be of somatic embryo origin. Rhizogenic calli were also produced by the same three populations that regenerated shoots. Significant differences among populations were found for frequency of root formation from leaf disks and time to callus. Variation among plants within a population was significant for four of the five traits examined. The results indicate the ease of hormone autonomization in sugarbeet, and should be of value in designing regeneration media for a wider range of beet germplasm.

Morphogenetic effects of 2,4-dichlorophenoxyacetic acid on pinto bean (Phaseolus vulgaris L.) leaf explants in vitro.

Roots, callus and/or globular structures were produced on primary leaf and distal cotyledon explants of pinto bean (Phaseolus vulgaris L. cv. UI 114) cultured on semisolid MS medium with a wide range of 2,4-D concentrations (0.01 to 80 mg/L) with either 0 or 1.0 mg/L kinetin. Explants rooted at lower 2,4-D concentrations than at those favoring globule formation on callus, although roots, callus and globules often developed from the same explant. Isolated opaque green globular structures developed when callus initiated on media with 3 or more mg/L 2,4-D was subcultured in liquid MS + 30 mg/L 2,4-D. These structures multiplied with a fresh weight doubling time of 8-9 days in MS + 30 mg/L 2,4-D. Although this multiplicative behavior and opaque color were reminiscent of embryoids reported for other species, no cotyledons or roots were seen.

Ectodermal control of the avascular zone of the peripheral mesoderm in the chick embryo.

Prospective skin ectoderm is underlaid by a relatively thick (100 +/- 20 micrometer) avascular zone of mesoderm in most regions of the early embryo. To determine whether or not the ectoderm exercises a role in the establishment and maintenance of the avascular zone, trypsin-isolated pieces of backskin ectoderm from chick or quail embryos were implanted as a sheet into a slit cut deep into the capillary bed of the wing bud of host chick embryos of stages 19-23. In sham operations, slits were cut at various anteroposterior levels, and the wing was allowed to heal. At intervals of 3-48 hr after these operations, embryos were injected with India ink, fixed, and cleared. Implants formed flattened vesicles, usually in continuity with host ectoderm, but sometimes completely internalized. Periderm cells from each side of the vesicle faced each other, and the cells of the cuboidal layer faced an avascular mesodermal layer at least 100 micrometer thick at all points. The implantation of prospective skin ectoderm resulted in the formation of an avascular zone in normally vascularized mesoderm of the wing bud. In contrast, the vascular bed of the limb bud abutted directly on implants of Millipore filters or of Silastic silicone (Dow Corning). Likewise, the capillary bed came in direct contact with implants of retinal pigment epithelium, an ectodermal derivative normally in close contact with the vascular choroid coat of the eye. These results, taken in conjunction with earlier experiments that show the necessity of the apical ectodermal ridge for the formation of the marginal veins of the limb bud, suggest that epithelial-mesenchymal interactions are involved in important aspects of vasculogenesis in early embryos.

Nicotine Biosynthetic Enzyme Activities in Nicotiana tabacum L. Genotypes with Different Alkaloid Levels.

Young plants of five Nicotiana tabacum L. genotypes were examined for activity of nicotine biosynthetic enzymes. Genotypes near isogenic except at two loci each with two alleles controlling nicotine level were used in a comparison of the four homozygous allelic combinations producing high, high intermediate, low intermediate, and low nicotine levels in a "Burley 21" background. Putrescine N-methyltransferase (EC 2.1.1.53) and quinolinic acid phosphoribosyltransferase (EC 2.4.2.19) activities in root tissue of these four genotypes were proportional to leaf nicotine level, whereas N-methylputrescine oxidase activity in root tissue differed in proportion and ranking. Quinolinic acid phosphoribosyltransferase activities in leaf tissue were lower than in roots, but no differences were found among the four genotypes. The homozygous recessive alleles at either locus affect levels of all three enzyme activities examined in roots. Each locus seems to be involved in regulation of nicotine metabolism, but whether directly as a regulatory locus or indirectly through the metabolic product of a structural locus is not known.No difference was observed between enzymic oxidation of putrescine and N-methylputrescine by leaf and root extracts of Burley 21 (a high nicotine, low nornicotine genotype) and a high nornicotine cultivar, "Robinson Medium Broadleaf." Putrescine was utilized as a substrate to a greater extent than N-methylputrescine by leaf extracts compared with root extracts of both cultivars. It was concluded that genetic differences in levels of nicotine and nornicotine were not due to differences in enzymic oxidation of these two precursors during alkaloid biosynthesis.