Single-cell cloning of a crown gall from protoplasts regenerated in hormone-free medium. Establishment of pure transformed cell-lines of Catharanthus roseus

Protoplasts enzymatically isolated from cell line of Catharanthus roseus G. Don crown gall, were cultured at high density (10⁵ P ml^-1) in modified B5 liquid medium (Gamborg et al. 1976). In the absence of growth regulators C. roseus protoplasts were able to regenerate a cell-wall, divide and, subsequently, yield very numerous clones in the absence of growth regulators. After two weeks, the cultures were greatly diluted in order to obtain clones of single-cell origin. Most of the clones individually transferred onto solid medium can proliferate indefinitely, without growth regulators. Among analyzed clones, 90% were nopaline positive. Their ajmalicine and serpentine content was compared with that of the parental crown gall line, and was found to be low. The CR10 protoplasts were very easy to grow, they were an interesting model for the development of pure tumorous lines. Moreover, we found that the tumorous protoplasts were useful for cell fusion experiments or for the delicate culture of tree protoplasts.Abbreviations B5 Gamborg et al. (1976) medium - 2,4-d 2,4-dichlorophenoxyacetic acid - Kin Kinetin - NAA naphthalene acetic acid - BA N⁶ (benzyl) adenine     

Agrobacterium radiobacter strains K84, K1026 and K84 Agr‐ produce an antibiotic‐like substance,active in vitro against A. tumefaciens and phytopathogenic Erwinia and Pseudomonas spp.

Agrobacterium radiobacter strains K84, K1026 and K84 Agr^‐ produced in vitro an antibiotic‐like substance (ALS 84), different from agrocin 84 and observed in mannitol‐glutamate medium. Twenty five out of 39 A. tumefaciens strains of biovars 1, 2 and 3 were sensitive to ALS 84 regardless of their sensitivity to agrocin 84. Sensitivity in A. tumefaciens strain C58 was not encoded by the Ti‐plasmid. Most isolates tested of Erwinia carotovora subsp. carotovora E. carotovora subsp. atroseptica, Pseudomonas corrugata P. cichorii and unidentified isolates from galls were also sensitive to this substance. ALS 84 was not affected by the proteases studied, nor by treatment at 62°C for 30 min and had a bacteriostatic effect. The production of ALS 84 might play a role in the complex mechanism of biological control of crown gall, especially in strains resistant to agrocin 84 and sensitive to ALS 84, and by the creation of an ecological niche favourable to A. radiobacter strains K84, K1026 or K84 Agr^‐.     

A tooth crown morphology framework for interpreting the diversity of primate dentitions

Variation in tooth crown morphology plays a crucial role in species diagnoses, phylogenetic inference, and the reconstruction of the evolutionary history of the primate clade. While a growing number of studies have identified developmental mechanisms linked to tooth size and cusp patterning in mammalian crown morphology, it is unclear (1) to what degree these are applicable across primates and (2) which additional developmental mechanisms should be recognized as playing important roles in odontogenesis. From detailed observations of lower molar enamel–dentine junction morphology from taxa representing the major primate clades, we outline multiple phylogenetic and developmental components responsible for crown patterning, and formulate a tooth crown morphology framework for the holistic interpretation of primate crown morphology. We suggest that adopting this framework is crucial for the characterization of tooth morphology in studies of dental development, discrete trait analysis, and systematics.     

药用植物红芽大戟的个体生态学研究

报道了式芽大戟生长地的气候与植被状况,土壤条件以及它的生长动态。还研究了红芽大戟的越冬与繁殖。结果表明：红芽大戟是一种主要生长和分布在热带及南亚热带的药用植物。在广东它的生长动态呈双峰曲线,生长地土壤为砖红壤和红壤,砂茂越冬和根颈繁殖是引种驯化和生产上值得应用的方法。     

Silent T-DNA genes in plant lines transformed by Agrobacterium tumefaciens are activated by grafting and by 5-azacytidine treatment

Summary A shooty tumor induced by a shooter mutant of an octopine strain of Agrobacterium tumefaciens was cloned. One clone obtained (TS038) behaved aberrantly in that it grew as a shooty tumor tissue on phytohormone free medium, but did not contain octopine synthase activity. In line TS038 the genes for octopine synthase and for the enzymes involved in agropine and mannopine synthesis were present, but were not transcribed. However, the above genes became active in TS038 tumor shoots after grafting as well as after treatment with the hypomethylating agent 5-azacytidine. After an unusually long incubation period in the growth cabinet shoot cultures appeared to have developed small shoots from the top of the leaves. This unusual form of differentiation was found to be accompanied by the induction of octopine synthase activity.     

Plant tumor reversal associated with the loss of foreign DNA

Summary Transformation of plant tissues into crown gall tumors has been associated with the transfer of a portion of a tumor-inducing plasmid (Ti-plasmid) into plant DNA. Various laboratories have regenerated normal-appearing plants from a number of crown gall tumors. This study investigates the fate of the foreign DNA in a series of tissues derived from various parts of a plant regenerated from the tumor BT-37 by Braun and his coworkers. It was found that all the foreign DNA sequences were lost from tissues that had lost all their tumorous traits; whereas the plasmid DNA sequences were still present in tissues that appeared normal but still exhibited tumorous traits when returned to tissue culture media. From these studies it would appear that the presence of the Ti-plasmid sequences in the plant DNA is required for the maintenance of the transformed state. Presented in the Symposium on Gene Transfer, Differentiation and Neoplasia in Plant and Animal Cells at the 30th Annual Meeting of the Tissue Culture Association, Seattle, Washington, June 10–14, 1979. This symposium was supported in part by Grant CA 26748 from the National Cancer Institute, DHEW, and Grant RD-67 from the American Cancer Society.     

Temperature-sensitive loss of tumor characteristics in a tobacco crown gall strain

Summary Three independently isolated tobacco crown gall strains incited byAgrobacterium tumefaciens C58 required phytohormone (auxin and cytokinin) supplements in the basal medium to grow, at 37°C. Six other tobacco crown gall strains incited, respectively, byA. tumefaciens IIBV7, B6, CGIC, A6NC, 27 and AT4 expressed, at 37°C, the tumor characteristic of ability to grow in vitro on medium lacking phytohormones. Nopaline was not detectable in C58 tumors cultured at 37°C, but octopine was produced by B6 tumor tissues incibated at the elevated temperature. C58 tumor strains kept at 37°C for 1 week or more lost the ability to express tumor characteristics at 27°C such as tissue morphology, growth on basal medium lacking phytohormones and nopaline production. Heat-treated C58 tissues also differed from the original tumor strain in regeneration ability and phytohormone requirements of explants; i.e. explants from regenerated, heart-treated C58 tumors required both auxin and cytokinin for growth in vitro.     

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered perfusion can aid research into the mechanisms of retinal pathologies. Intravital video microscopy of fluorescent tracers can be used to measure vascular diameters and bloodstream velocities of the retinal vasculature, specifically the arterioles branching from the central retinal artery and of the venules leading into the central retinal vein. Blood flow rates can be calculated from the diameters and velocities, with the summation of arteriolar flow, and separately venular flow, providing values of total retinal blood flow. This paper and associated video describe the methods for applying this technique to mice, which includes 1) the preparation of the eye for intravital microscopy of the anesthetized animal, 2) the intravenous infusion of fluorescent microspheres to measure bloodstream velocity, 3) the intravenous infusion of a high molecular weight fluorescent dextran, to aid the microscopic visualization of the retinal microvasculature, 4) the use of a digital microscope camera to obtain videos of the perfused retina, and 5) the use of image processing software to analyze the video. The same techniques can be used for measuring retinal blood flow rates in rats.