A revised medium for rapid growth and bio assays with tobacco tissue cultures
TL;DR: In vivo redox biosensing resolves the spatiotemporal dynamics of compartmental responses to local ROS generation and provide a basis for understanding how compartment-specific redox dynamics may operate in retrograde signaling and stress 67 acclimation in plants.
Abstract: In experiments with tobacco tissue cultured on White's modified medium (basal meditmi hi Tnhles 1 and 2) supplemenk'd with kiticthi and hidoleacctic acid, a slrikin^' fourlo (ive-told intTease iu yield was ohtaitu-d within a three to Tour week j^rowth period on addition of an aqtteotis exlrarl of tobacco leaves (Fi^'ures 1 and 2). Subse(iueutly it was found Ihiit this jnoniotiou oi' f^rowih was due mainly though nol entirely to inorj^auic rather than organic con.stitttenls in the extract. In the isolation of Rrowth factors from plant tissues and other sources inorj '̂anic salts are fre(|uently carried along with fhe organic fraclioits. When tissue cultures are used for bioassays, therefore, il is necessary lo lake into account increases in growth which may result from nutrient elements or other known constituents of the medium which may he present in the te.st materials. To minimize interference trom rontaminaitis of this type, an altempt has heen made to de\\eh)p a nieditmi with such adequate supplies of all re(iuired tnineral nutrients and cotntnott orgattic cottslitueitls that no apprecial»le change in growth rate or yield will result from the inlroduclion of additional amounts in the range ordinarily expected to be present in tnaterials to be assayed. As a point of referetice for this work some of the culture media in mc)st common current use will he cotisidered briefly. For ease of comparis4)n Iheir mineral compositions are listed in Tables 1 and 2. White's nutrient .solution, designed originally for excised root cultures, was based on Uspeuski and Uspetiskaia's medium for algae and Trelease and Trelease's micronutrieni solution. This medium also was employed successfully in the original cttltivation of callus from the tobacco Iiybrid Nicotiana gtauca x A', tanijadorffii, atitl as further modified by White in 194̂ ^ and by others it has been used for the
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TL;DR: A large number of morphologically normal, fertile, transgenic rice plants were obtained by co-cultivation of rice tissues with Agrobacterium tumefaciens, and sequence analysis revealed that the boundaries of the T-DNA in transgenic Rice plants were essentially identical to those intransgenic dicotyledons.
Abstract: Summary
A large number of morphologically normal, fertile, transgenic rice plants were obtained by co-cultivation of rice tissues with Agrobacterium tumefaciens The efficiency of transformation was similar to that obtained by the methods used routinely for transformation of dicotyledons with the bacterium Stable integration, expression and inheritance of transgenes were demonstrated by molecular and genetic analysis of transformants in the R0, R1 and R2 generations Sequence analysis revealed that the boundaries of the T-DNA in transgenic rice plants were essentially identical to those in transgenic dicotyledons Calli induced from scutella were very good starting materials A strain of A tumefaciens that carried a so-called ‘super-binary’ vector gave especially high frequencies of transformation of various cultivars of japonica rice that included Koshihikari, which normally shows poor responses in tissue culture
3,475 citations
TL;DR: Somatic reversion of plants with white flowers to phenotypically parental violet flowers was associated with a coordinate rise in the steady-state levels of the mRNAs produced by both the endogenous and the introduced CHS genes, indicating that expression of the introduced chalcone synthase gene was not alone sufficient for suppression of endogenous CHS transcript levels.
Abstract: We attempted to overexpress chalcone synthase (CHS) in pigmented petunia petals by introducing a chimeric petunia CHS gene. Unexpectedly, the introduced gene created a block in anthocyanin biosynthesis. Forty-two percent of plants with the introduced CHS gene produced totally white flowers and/or patterned flowers with white or pale nonclonal sectors on a wild-type pigmented background; none of hundreds of transgenic control plants exhibited such phenotypes. Progeny testing of one plant demonstrated that the novel color phenotype co-segregated with the introduced CHS gene; progeny without this gene were phenotypically wild type. The somatic and germinal stability of the novel color patterns was variable. RNase protection analysis of petal RNAs isolated from white flowers showed that, although the developmental timing of mRNA expression of the endogenous CHS gene was not altered, the level of the mRNA produced by this gene was reduced 50-fold from wild-type levels. Somatic reversion of plants with white flowers to phenotypically parental violet flowers was associated with a coordinate rise in the steady-state levels of the mRNAs produced by both the endogenous and the introduced CHS genes. Thus, in the altered white flowers, the expression of both genes was coordinately suppressed, indicating that expression of the introduced CHS gene was not alone sufficient for suppression of endogenous CHS transcript levels. The mechanism responsible for the reversible co-suppression of homologous genes in trans is unclear, but the erratic and reversible nature of this phenomenon suggests the possible involvement of methylation.
2,994 citations
Cites background from "A revised medium for rapid growth a..."
...concentration of medium MS (Murashige and Skoog, 1962) sup? plemented with 0....
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TL;DR: It is reported here that H2O2 from this oxidative burst not only drives the cross-linking of cell wall structural proteins, but also functions as a local trigger of programmed death in challenged cells and as a diffusible signal for the induction in adjacent cells of genes encoding cellular protectants.
Abstract: Microbial elicitors or attempted infection with an avirulent pathogen strain causes the rapid production of reactive oxygen intermediates. We report here that H2O2 from this oxidative burst not only drives the cross-linking of cell wall structural proteins, but also functions as a local trigger of programmed death in challenged cells and as a diffusible signal for the induction in adjacent cells of genes encoding cellular protectants such as glutathione S-transferase and glutathione peroxidase. Thus, H2O2 from the oxidative burst plays a key role in the orchestration of a localized hypersensitive response during the expression of plant disease resistance.
2,740 citations
Cites background or methods from "A revised medium for rapid growth a..."
...Northern Blots Total cellular RNA was extracted and hybridized in Northern blots as described by Pepper et al. (1994). Hybridization probes were bean PAL5 and CHS7 cDNAs (Edwards et al....
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...in the dark in Murashige and Skoog (1962) medium supplemented with 0....
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TL;DR: Hundreds of Arabidopsis genes were found that outperform traditional reference genes in terms of expression stability throughout development and under a range of environmental conditions, and the developed PCR primers or hybridization probes for the novel reference genes will enable better normalization and quantification of transcript levels inArabidopsis in the future.
Abstract: Gene transcripts with invariant abundance during development and in the face of environmental stimuli are essential reference points for accurate gene expression analyses, such as RNA gel-blot analysis or quantitative reverse transcription-polymerase chain reaction (PCR). An exceptionally large set of data from Affymetrix ATH1 whole-genome GeneChip studies provided the means to identify a new generation of reference genes with very stable expression levels in the model plant species Arabidopsis (Arabidopsis thaliana). Hundreds of Arabidopsis genes were found that outperform traditional reference genes in terms of expression stability throughout development and under a range of environmental conditions. Most of these were expressed at much lower levels than traditional reference genes, making them very suitable for normalization of gene expression over a wide range of transcript levels. Specific and efficient primers were developed for 22 genes and tested on a diverse set of 20 cDNA samples. Quantitative reverse transcription-PCR confirmed superior expression stability and lower absolute expression levels for many of these genes, including genes encoding a protein phosphatase 2A subunit, a coatomer subunit, and an ubiquitin-conjugating enzyme. The developed PCR primers or hybridization probes for the novel reference genes will enable better normalization and quantification of transcript levels in Arabidopsis in the future.
2,694 citations
Cites methods from "A revised medium for rapid growth a..."
...…were also harvested from 14-d-old Arabidopsis (Col-0) wildtype plants that were grown vertically on half-strength Murashige and Skoog medium (Murashige and Skoog, 1962), supplemented with 0.5% (w/v) Suc and solidified with 0.7% agar, at 22 C under a 16-h-day (140 mmol m22 s21)/ 8-h-night…...
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TL;DR: These spruce tumors represent a promising addition to the roster of materials in which the processes of tumefaction can be profitably studied.
Abstract: tumor sectors of a single individual and the physiological comparison of these cell lines in tissue cultures. While this is theoretically possible in animal and human tumors, it is fraught with such technical difficulties that it has not yet been accomplished over more than very brief periods, whereas in these spruce tumors it can be done with relative ease. We believe that these tumors represent a promising addition to our roster of materials in which the processes of tumefaction can be profitably studied.
226 citations
TL;DR: The influence of various concentrations of salts and vitamins in the basal medium on growth of tobacco and sunflower tissue cultures was studied in three salt solutions.
Abstract: PATHOLOGICAL CELL proliferation in plants is of special interest because of the possible relationship to diseased growth in animals and man. Therefore, the crown-gall disease incited by Phytomonas tumefaciens (Smith and Town.) Bergey et al. has been studied extensively. The early literature on crowngall has been reviewed by Riker and Berge (1935) and by Levine (1936). Although much work has been done with crown-gall bacteria, little is known yet about the physiology of the host cells, because of certain technical difficulties involved in such studies. The plant tissue culture technique offers certain opportunities for studies of tissue metabolism not possible with intact plants. For example, isolated cell masses may be cultured in vitro on a medium which, except for traces of impurities, contains only known chemicals. There are no neighboring cells, tissues, and organs to influence development. The tissue cultures either grow or fail to grow because of the materials originally either absent from or present in the medium and because of the tissue metabolites. The influences of temperature, pH# sucrose concentration, certain plant extracts, and crown-gall bacterial products on growth of plant tissue cultures in vitro have been reported earlier (Hildebrandt, Riker, and Duggar, 1945, 1946). Certain of these extracts at low concentrations caused increased growth of tissue cultures, but often they inhibited growth of the cultures at higher concentrations. Since certain extracts stimulated growth of tissue cultures, this stimulation might have been due either to the introduction of more favorable quantities of certain of the constituents of the basic medium or to factors not present in it. For example, certain preliminary experiments suggested that higher concentrations of iron resulted in better growth of the tissue cultures. Therefore, the influence of various concentrations of salts and vitamins in the basal medium on growth of tobacco and sunflower tissue cultures was studied in three salt solutions. A report on part of the work herein reported has already appeared in an abstract (Hildebrandt, Riker, and Duggar, 1944). MATERIALS AND METHODS.-The materials and methods were essentially the same as those described earlier (Hildebrandt, Riker, and Duggar, 1945). 1 Received for publication March 15, 1946. This work was supported in part by the International Cancer Research Foundation and the Wisconsin Alumni Research Foundation. Published with the approval of the Director of the Wisconsin Agricultural Experiment Station. 2 The writers are indebted to Eugene Herrling for preparing figures 1 and 2. Tobacco and sunflower tissues were used. The tobacco tissue was from the hybrid Nicotiana gtauca Grah. 9 X N. langsdorfii Weinm. d and was isolated and supplied by Dr. P. R. White. The second tissue was isolated from "secondary" crown galls on sunflower (Helianthus annuus L. var. Giant Russian). Both tissues were free of crown-gall bacteria and were capable of unlimited grctwth in vitro. The composition of the basal medium (White, 1942) is repeated later for comparison with improved media. Reagent grade chemicals, except sucrose, ferric tartrate, boric acid, and potassium iodide, were used, and a record was kept of the impurities in the salts. The agar was leached by -washing in distilled water at least 3 times daily over a 3to 4-day period. All experimental cultures were incubated in 125 ml. Erlenmeyer flasks containing 50 ml. of medium. The flasks were washed previously in potassium dichromate-sulfuric acid cleaning solution and rinsed in tap and distilled water. The experimental cultures were maintained in the dark at 260 + 10C. in a special culture room. Twenty-four tissue pieces (four per flask) were used per treatment, and each experiment was repeated three times unless otherwise noted. To determine the amount of growth the tissue pieces were removed from the flasks after a 6-week incubation period and weighed individually. All data were analyzed statistically to determine significance between treatments.3 Media prepared according to the triangular method were used to study the influence of increasing and decreasing concentrations of each of the constituents of the basal medium. Ferric tartrate, ferric sulfate, and thiamine hydrochloride were sterilized by filtration; all others were autoclaved. The three salts under test were added aseptically to the culture flasks to avoid precipitation. The growth on nineteen different media of representative sunflower tissue and the concentrations of the three salts under test from one such experiment are shown in figure 1, A and B, respectively, as an example of the method. A tissue grown on the unchanged basal control medium is shown at the upper left of the triangle; tissues grown on media without one of the three salts under test are shown at the upper right of the triangle (fig. 1, A). The omission of one nutrient did not mean necessarily that those materials were lacking completely because, for example, of impurities in the washed agar, the salts, the sugar, and in the carry-over of various nutrients with the seed tissue pieces, which were grown on the basal medium.
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