Embryogenic callus induction and regeneration are useful in many aspects of plant biotechnology, especially in the functional characterization of economically important genes. However, in sugarcane, callus induction and regeneration vary across genotypes. Saccharum spontaneum is an important wild germplasm that confers disease resistance and stress tolerance to modern sugarcane cultivars, and its genome has been completely sequenced. The aim of this study was to investigate the effect of genetic variations on embryogenic callus induction and regeneration in S. spontaneum and to screen genotypes having high tissue culture susceptibility. The study was performed using nine genotypes of S. spontaneum and the following five parameters were assessed to determine the response of genotypes to embryogenic callus induction and regeneration: callus induction, embryogenic callus ratio, embryogenic callus induction, embryonic callus regeneration and regeneration capacity. All the genotypes varied significantly (P < 0.01) in all the parameters, except for embryonic callus regeneration, which was high (>80%) for all the genotypes. High broad-sense heritability (86.1–96.8%) indicated that genetic differences are the major source of genotypic variations. Callus induction was found to be strongly positively correlated with embryogenic callus induction (r = 0.890, P < 0.01) and regeneration capacity (r = 0.881, P < 0.01). Among the nine tested genotypes, VN2 was found to be the most responsive to tissue culture and could therefore be used to characterize functional genes in S. spontaneum. We also suggested an approach with potential applications in facilitating the rapid identification of sugarcane genotypes susceptible to tissue culture.

Tobacco (Nicotiana tabacum L. ‘X-73’) callus tissue cultures were used in a bioassay system for determining the effect of the following substituted 2,6-dinitroaniline herbicides on growth: trifluralin (α,α,α-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine); oryzalin (3,5-dinitro-N4,N4-dipropylsulfanilamide); benefin (N-butyl-N-ethyl-α,α,α-trifluoro-2,6-dinitro-p-toluidine); AC 92390 (N-sec-butyl-2,6-dinitro-3,4-xylidine); penoxalin [N-(1-ethylpropyl)-2,6-dinitro-3,4-xylidine]; GS-38946 (N-ethyl-N-tetrahydrofurfuryl-4-trifluoromethyl-2,6-dinitroaniline); chlornidine [N,N-di(2-chloroethyl)-4-methyl-2,6-dinitroaniline]; nitralin [4-(methylsulfonyl)2,6-dinitro-N,N-dipropylaniline]; dinitramine (N4,N4-diethyl-α,α,α-trifluoro-3,5-dinitrotoluene-2,4-diamine); isopropalin (2,6-dinitro-N,N-dipropylcumidine), and profluralin [N(cyclopropylmethyl)-α,α,α-trifluoro-2,6-dinitro-N-propyl-p-toluidine]. The molar concentration required to inhibit fresh weight gain by 50% (I50) was determined by using linear regression analysis on data from a range of five concentrations of each chemical. I50 values did not correlate with structural variations or available physical constants. Herbicides listed in order of increasing activity are AC 92390< GD-38946<profluralin = isopropalin<benefin = chlornidine = trifluralin = nitralin<oryzalin = penoxalin < dinitramine. Exogenously applied D-α-tocopherol acetate at 100 times the I50 concentrations decreased the inhibition of tissue growth by the herbicides.

Greenhouse and laboratory studies were conducted to determine the extent of cross-resistance of chlorsulfuron-resistant sugarbeet (CR1-B) to other herbicides that inhibit acetolactate synthase (ALS) and to determine the physiological basis of resistance. Cross-resistance to metsulfuron, imazaquin, and imazethapyr was not evident, while only marginal cross-resistance was observed to triasulfuron, DPX-L5300, and nicosulfuron. CR1-B was moderately resistant to chlorsulfuron and chlorimuron and was highly cross-resistant to thifensulfuron and primisulfuron. Further greenhouse studies demonstrated that CR1-B was not significantly injured by thifensulfuron and primisulfuron applied at or exceeding the field use rate. Studies with 14C-primisulfuron showed that differential absorption or metabolism of primisulfuron could not account for the observed resistance. ALS enzyme assays showed that the CR1-B ALS enzyme activity was 66, 26, and 13 times less sensitive to chlorsulfuron, thifensulfuron, and primisulfuron inhibition, respectively, compared to ALS enzyme extracted from sensitive sugarbeets. An altered ALS enzyme, which is less sensitive to sulfonylurea herbicide inhibition, appears to be the physiological basis of resistance.

Uptake, translocation, and metabolism of 14C-MBR-18337 {[N-[(4-ethylthio)-2-(trifluoromethyl)phenyl] methanesulfonamide]} in soybean [Glycine max (L.) Merr.] and johnsongrass [Sorghum halepense (L.) Pers.] plants and cell cultures were studied. Sixty-five percent of the 14C recovered from soybean (65% of applied) was on or in the treated leaf, compared to 81% of the 14C recovered from johnsongrass (43% of applied). Translocation of the 14C from the treated leaf of soybean was greater than from the treated johnsongrass leaf, and was primarily to the apical leaves of both species. MBR-18337, its sulfoxide, sulfone, and an unidentified polar component(s) were isolated from intact plants and cell cultures of both species. Soybean plants and cells contained greater proportions of the polar component(s) than johnsongrass plants and cells.

Whole plant and tissue culture experiments were conducted to determine the difference in phytotoxicity of 2,4-D and its metabolite, 2,4-DCP, to red clover. At the whole plant level, the mean concentration of 2,4-DCP (10 mM) required to cause 50% growth inhibition (I50) of shoot dry weight was 24 times greater than for 2,4-D (0.42 mM). Using callus tissue, the I50 value for 2,4-DCP (0.28 mM) was 22 times greater than for 2,4-D (0.013 mM) based on dry weights. The callus tissue was 36 and 32 times more sensitive to 2,4-DCP and 2,4-D than shoot tissue based on dry weights, respectively. These data indicate that 2,4-DCP was less phytotoxic than 2,4-D to red clover both in vitro and in vivo.

Behavior of 14C-haloxyfop-methyl {methyl 2-[4-[(3-chloro-5-(trifluoromethyl)-2-pyridinyl] oxy] phenoxy] propanoate} was examined in intact plants of soybean [Glycine max (L.) Merr. ‘Mead’], shattercane [Sorghum bicolor (L.) Moench. ♯ SORVU], and yellow foxtail [Setaria glauca (L.) Beauv. ♯ SETLU] and in cell cultures of soybean and yellow foxtail. Foliar absorption of 14C-haloxyfo-methyl by intact plants was almost complete within 48 h. Significant translocation occurred within 48 h of treatment, with soybean and shattercane plants translocating more 14C than yellow foxtail. Metabolism of 14C-haloxyfop-methyl in intact plants was similar among species. Haloxyfop-methyl was rapidly hydrolyzed to haloxyfop in treated leaves and then translocated to metabolically active areas within the plants. Significant quantities of polar products were also isolated. Soybean cells in suspension culture contained higher levels of 14C than did yellow foxtail cells. Most of the 14C in soybean cells was haloxyfop-methyl, while yellow foxtail contained mostly haloxyfop.

The metabolism of 2,4-DB [4-(2,4-dichlorophenoxy)butyric acid] in cell suspension cultures of white clover (Trifolium repens L. ‘Regal Ladino’) was investigated using (ring)- 14C 2,4-DB. The herbicide and its 14C-labeled metabolites were isolated from treated cells and medium after 48 h by partition and thin layer chromatography (TLC). The major portion of the radiolabel was in the glycoside fraction (47%) and incorporated into the cell structural material (34%). The remainder of the 14C remained either as the parent herbicide (8%) or an amino acid conjugate of 2,4-DB (10%).

Dormant line M73 wild oat caryopses were utilized to develop a system for the culture of excised embryos, to evaluate whether embryo dormancy exists, and to investigate the physiological basis for breaking dormancy. Dormant embryos cultured on N6 medium solidified with 0.25% Bacto agar displayed 70% germination in 2 d compared with approximately 20% for the other gelling agents. The non-plant-based gelling agent gellum at a concentration of 0.25% was selected for further experiments on breaking dormancy after it was determined that concentrations ≥ 0.5% decreased the rate of germination. Amending N6 medium with concentrations of 0.1 to 10 μM gibberellic acid (GA) increased the rate and extent of germination. Embryos treated with 0 to 0.01 μM GA required 6 d to attain 90% germination. Germination of dormant embryos on N6 medium without GA suggested that either true embryo dormancy did not exist in M73 or some constituent of the N6 medium promoted breaking of dormancy. Subsequent experiments indicated that the 88 mM sucrose was the constituent in the N6 medium responsible for breaking dormancy. Concentrations of sucrose from 40 to 200 mM were effective in breaking dormancy. Ten μM GA increased the rate and extent of germination of embryos cultured with 88 to 200 mM sucrose. At 88 mM, fructose, maltose, glucose, and sucrose all broke embryo dormancy. Fructose was the most active soluble sugar for breaking embryo dormancy, promoting nearly 100% germination in 4 d. As with sucrose, there was an interaction between GA and the soluble sugars in breaking dormancy. Ten μM GA with 88 mM fructose provided nearly 100% germination in 1 d. Amylose, but not amylopectin or pullulan, may substitute for soluble sugars. However, with 10 μM GA amylose, amylopectin and pullulan were equally effective in breaking dormancy. Breaking dormancy of embryos on N6 medium was independent of temperatures from 12 to 24 C in the presence of GA, but in its absence the optimum was 12 C. Application of GA to dormant caryopses significantly increased and decreased the level of glucose and sucrose, respectively, in the embryo. Gibberellic acid had a similar effect on glucose and sucrose in the endosperm tissue, except the differences were not significant at all times after treatment. The change in carbohydrate metabolism, especially in embryo tissue, may be important when considered in context with the observation that soluble sugars and GA act independently in breaking dormancy in excised M73 embryos. Breaking wild oat embryo dormancy with GA may be mainly a substitution for sugar requirement.

Field experiments established that tetraploid soybean [Glycine max (L.) Merr.] plants were relatively tolerant while diploid plants were highly susceptible to metribuzin [4-amino-6-tert-butyl-3-(methylthio)-as-triazin-5(4H)-one] applied both preemergence and postemergence. Sensitivity of diploids and tolerance of tetraploids was also found in growth chamber experiments. Autoradiographs prepared 4 days after 14C-metribuzin application showed a high level of 14C-translocation to all parts of diploid plants including meristems. In contrast, only low levels of 14C were translocated in tetraploid plants, with no 14C-movement into meristems. Tetraploid plants rapidly transformed absorbed metribuzin to nontoxic products. Diploids were incapable of inactivating absorbed metribuzin at a rate sufficient to prevent injury. Differences in absorption, translocation, and metabolism of metribuzin appeared to be the main factors in the diploid and tetraploid differential response in field and growth chamber experiments. Differences in the rate of metribuzin metabolism appeared to be the factor responsible for the differential response in diploid and tetraploid cell suspension cultures. The primary polar metabolites were conjugates of metribuzin and deaminated metribuzin [6-tert-butyl-3-(methylthio)-1,2,4-triazin-5(4H)-one] with a ratio of 8:1, respectively.

Research involved the behavior of BAS-9052 OH {2[1-(ethoxyimino)butyl]-5-[2(ethylthio)propyl]-3-hydroxy-2-cyclohexen-1-one} in soybean [Glycine max (L.) Merr.] and johnsongrass [Sorghum halepense (L.) Pers.] plants, and the fate of 14C-BAS-9052 OH in intact plants and cell cultures of both species. Microscopic examination of seedling johnsongrass plants (two- to three-leaf stage) treated with foliar applications of 0.48 μg/plant revealed disorganized apical regions and necrotic cells within the apex and leaf primordia of the shoot. Necrotic zones were also evident at the base of expanding leaves and in root apices 1 day after treatment. Following application of 14C-BAS-9052 OH, the same radioactive products were isolated from cell cultures and intact plants. Products included BAS-9052 OH and three unidentified metabolites. Greater proportions of unchanged BAS-9052 OH were extracted from the apical leaves, roots, and cell cultures of johnsongrass than of soybean. BAS-9052 OH was thermal and photo-labile, and a large portion of 14C may not have entered the plant as BAS-9052 OH. However, tolerant soybean plants and cell cultures appeared to have metabolized the herbicide more rapidly than susceptible johnsongrass plants and cell cultures. An average of 64% of the 14C remained in the treated leaf of johnsongrass compared with 48% in soybean. About one-half as much 14C was translocated to the apical leaves of susceptible johnsongrass than tolerant soybean. Therefore, differential uptake and translocation cannot account for the selectivity of BAS-9052 OH.

Bioassays using cell cultures and callus tissues of leafy spurge were devised to evaluate the potential of rhizobacteria as biocontrol agents. Rhizobacteria isolated from roots of leafy spurge seedlings were screened in suspension-cultured leafy spurge cells. Cell viability was assessed using the Evan's blue bioassay 48 h after bacterial inoculation. Among the 30 isolates tested, LS102 and LS105 consistently caused intensive cell death determined by measuring the A630 of the inoculated cell cultures. Cell death was 2.5 to 3 times higher in cultures inoculated with LS105 and LS102, respectively, than in the control. Population levels of the two isolates within cell cultures and callus tissues of leafy spurge increased during the first 48 h. Leafy spurge callus tissues were inoculated with rhizobacteria either directly or by using the Host Pathogen Interaction System (HPIS). The latter exposes calli to bacteria without any physical contact. LS102 caused cellular leakage and eventually death of the callus tissue. Callus growth was reduced by about 30 to 70% when exposed to LS102 and LS105, respectively. Results suggest that these two isolates may affect leafy spurge at the cellular level by different mechanisms. A screening method based on cell cultures and callus tissues offers a good and rapid technique for detecting deleterious rhizobacteria with potential as biocontrol agents for leafy spurge.

Satellite glial cells (SGCs) are specialized cells that form a tight sheath around neurons in sensory ganglia. In recent years, there is increasing interest in SGCs and they have been studied in both intact ganglia and in tissue culture. Here we studied phenotypic changes in SGCs in cultured trigeminal ganglia from adult mice, containing both neurons and SGCs, using phase optics, immunohistochemistry and time-lapse photography. Cultures were followed for up to 14 days. After isolation virtually every sensory neuron is ensheathed by SGCs, as in the intact ganglia. After one day in culture, SGCs begin to migrate away from their parent neurons, but in most cases the neurons still retain an intact glial cover. At later times in culture, there is a massive migration of SGCs away from the neurons and they undergo clear morphological changes, and at 7 days they become spindle-shaped. At one day in culture SGCs express the glial marker glutamine synthetase, and also the purinergic receptor P2X7. From day 2 in culture the glutamine synthetase expression is greatly diminished, whereas that of P2X7 is largely unchanged. We conclude that SGCs retain most of their characteristics for about 24 h after culturing, but undergo major phenotypic changes at later times.

N6 medium, Murashige and Skoog (MS) medium and BNBN medium were adopted in tissue cultures of Nipponbare (japonica) scutellum for performance comparison. Results indicated that, among the three kinds of culture media, the NB medium worked best in callus induction and gene transformation and was most suitable for tissue culture of Nipponbare scutellum. Based on this, the elicitor-encoding gene pemG1 from Magnaporthe grisea was introduced into the genome of Nipponbare via the Agrobacterium-mediated method. Transgenic rice plants were obtained. The integration, transcription and expression of pemG1 in rice were confirmed by polymerase chain reaction (PCR), Northern blot and Western blot, respectively. Genetic analysis showed that the transgenes were segregated normally in the progenies.

Hayachine-usuyukiso (Leontopodium hayachinense) is an alpine plant native to Mt Hayachine. This unique chrysanthemum is listed as an endangered plant by the Department of Conservation, Iwate Prefecture, and as a threatened plant by the Ministry of the Environment, Japan. We successfully cryopreserved the shoot tips from in vitro-grown L. hayachinense shoots using a vitrification protocol. Cold-hardened shoot tips were excised and pre-cultured on a solidified Murashige–Skoog medium containing 0.3 M sucrose for 1 d at 5°C. The shoot tips were then treated with loading solution for 20 min at 25°C, dehydrated in plant vitrification solution 2 for 120 min at 25°C and immersed in liquid nitrogen. The survival rate of the vitrified shoot tips was 63.3% after 30 d of regrowth. This protocol appears to be a promising technique for the cryopreservation of in vitro-grown shoots of this endangered plant.

Introduction. The worldwide production of banana and plantain, which is estimated to 106 Mt·year–1, relies on a narrow genetic basis. Thus, banana production is fragile regarding emergent diseases, and creation of new varieties of banana stands out as a real necessity for its sustainability. In banana, various improvement strategies aim to create triploid hybrids. The objective of this report is to present a simple in vitro methodology to induce stable tetraploid plants by colchicine treatment, usable as parent for obtaining triploid varieties. Materials and methods. Twenty-one diploid M. acuminata clones and three interspecific M. acuminata / M. balbisiana diploid clones were treated as proliferating culture in liquid medium with 1.25 mM of colchicine for 48 h. Plant screening has been performed by morphological identification in greenhouse, chromosome counts on root tips and flow cytometry on leave blades on vitroplants using propidium iodide staining. Results. Chromosome counts led to distinguish diploid and tetraploid plants but did not afford to detect chimeras. Flow cytometry allowed an early screening of a larger number of plants leading to detect rapidly chimerical plants. It was observed that some stable 2x / 4x cytochimeras are periclinal. Tetraploid clones were obtained with all diploid genotypes. General behaviour of tetraploids in the field was globally weaker than the corresponding diploids. Nevertheless, all the doubled-diploids flowered and crossed with diploid plants to obtain triploid progenies. Conclusion. This study has clearly shown that induction of stable doubled-diploid plants can be obtained from a wide range of genetically different bananas. These results open the way to the systematic use of doubled-diploids by banana breeding programs for the release of enhanced triploid varieties.

We studied the direct effect of intravenous anaesthetics, local anaesthetics and premedication drugs in common use for major surgery, on the spontaneous versus lectin induced proliferation of cultured normal rat peripheral blood mononuclear cells, not exposed to surgical or any other trauma prior to culture. Peripheral blood mononuclear cells were incubated in cell-culture medium in the presence or in the absence of propofol, ketamine, fentanyl, midazolam, thiopental sodium, lidocaine or etomidate. The cells proliferated either spontaneously or under stimulation with a lectin phytohaemagglutinine P for 72 h. The proliferation rate was evaluated by 2H-Thymidine incorporation. Fentanyl, thiopental sodium, lidocaine and etomidate significantly inhibited phytohaemagglutinine P induced 3H-Thymidine incorporation in cultured rat peripheral blood mononuclear cells. Counts per minute of the cultures treated with these drugs were 1667.80±745.72, 1614.1±615.00, 1688.0±615.0 and 1549±560.41, respectively, compared with the phytohaemagglutinine P stimulated positive control counts per minute, 13488±4305.6 (P<0.001 in each comparison). Propofol, ketamine and midazolam inhibited the lectin-induced cell proliferation to levels not statistically different from the baseline. Counts per minute of these cultures were 1361.90±745.73; 1108.90±751.33 and 1518.10±848.88, respectively. Compared either with the baseline 972.57±356.73 counts per minute or to the positive control culture counts per minute, 13488±4305.6 the difference was statistically significant (P<0.001) in each comparison. All the substances tested in this study proved capable of exerting direct inhibitory effect on circulating immunocompetent cells, because the latter were not subjected to any other immunosuppressive factor, be it operative trauma, blood transfusion, malnutrition, drug abuse, prior to culture. The possible theoretical and practical implications are discussed in this study.

The effects of keeping rat tympanic membranes with an artificially made pars tensa perforation in tissue culture were observed under a scanning electron microscope. After one day and onwards, spreading and thickening of the keratinizing, outer squamous epithelium (OE) was noted. In addition, ballooning of the innermost cells of the outer epithelium apposing the inner tympanic epithelium (IE) was seen. No appreciable reaction was noted in the connective tissue layer of the drum. The inner tympanic epithelium appeared to be swollen, containing spherical structures in the cytoplasm, especially close to the area of contact with the outer meatal epithelium. No complete cover of the drum defect was seen after 14 days of tissue culture. Hyperplasia and spreading of the keratinizing, outer squamous epithelium of the drum is not sufficient to achieve covering of a drum perforation and complete healing cannot take place unless supported by granulation tissue formation.