Morphological and cytological characteristics of somatic hybrids of Nicotiana tabacum L. (+) N. megalosiphon Heurk. et Müll

Summary Somatic hybrid plants of Nicotiana tabacum (bar) (+) Nicotiana megalosiphon (npt II) were recovered after polyethylene glycol (PEG) mediated fusion. Hybrid calluses were selected on the basis of their dual resistance to bialaphos and kanamycin or UV inactivation of the donor species (Nicotiana megalosiphon) protoplasts. The hybrid nature of the individual clones obtained was confirmed by AFLP analysis. An array of plants were recovered including self-fertile hybrid plants with N. tabacum or N. megalosiphon phenotype, self-sterile plants with N. tabacum habit, leaf and intermediate flower morphology, self-sterile plants with N. megalosiphon habit, abnormal leaves and intermediate flowers, and self sterile plants of N. megalosiphon type with abnormal characters. Viable pollen was observed in hybrid plants from the third group. The hybrids possessed a nuclear DNA content near that of the diploid tobacco or N. megalosiphon, and also near that of the tetraploid genome size of N. megalosiphon. The results provide evidence for nonpreferential loss of one of the parental genomes and spontaneous asymmetrization of hybrid plants. The present study shows that by means of somatic hybridization a great genetic diversity in the hybrid clones can be achieved.     

Effect of gamma-radiation on the growth of primary and “long-term” tissue cultures (Nicotiana tabacum L.)

Growth of primary (cv. Wisconsin 38) and long-term (cv. Virginia Bright Italia) tissue cultures obtained fromNicotiana tabacum L. pith was affected by exposure to a single dose of gamma-radiation ranging from 1 to 150 krad. The inhibitory effect was proportional to the applied dose. Changes in the degree of inhibition during the subculture interval were ascertained by quantitative evaluation of the growth response. Primary cultures proved to be several times more radiosensitive than long-term cultures, the capacity to recover from radiation damage of the cultures participating in the resulting manifestation of the effect. The possible reasons for these differences are discussed.     

Effect of Rhizobium–Azospirillum coinoculation on nitrogen fixation and yield of two contrasting Phaseolus vulgaris L. genotypes cultivated across different environments in Cuba

Azospirillum spp. have shown potential to enhance nodulation and plant growth of legumes when coinoculated with Rhizobium. The effect of Azospirillum on the Rhizobium-legume symbiosis is, however, dependent on the host genotype used. Previous greenhouse experiments identified two genotypes of common bean (Phaseolus vulgaris L.), BAT477 and DOR364, contrasting in nodulation response to Azospirillum when coinoculated with Rhizobium. Genetic analysis revealed a genetic basis (Quantitative Trait Loci) on the bean genome related to the differential responsiveness to Azospirillum between the two bean genotypes. In this study, on-station and on-farm field experiments in different regions in Cuba were conducted to evaluate the agronomic relevance of the differences in response to Azospirillum–Rhizobium coinoculation between the two genotypes BAT477 and DOR364. It was observed that Azospirillum–Rhizobium coinoculation as compared to single Rhizobium inoculation increased the amount of fixed nitrogen and the yield of DOR364 across all sites. For BAT477, on the contrary, a negative effect of Azospirillum–Rhizobium coinoculation on yield and nitrogen fixation was observed on most of the sites as compared to single Rhizobium inoculation. The modified stability regression equations resulting from this study may contribute to predict how a combination of genotype and inoculum will perform at a certain environmental setting. This study highlights the importance of genotype × inocula interactions in agricultural outputs and establishes a link between greenhouse phenotype, genetic background and performance in the field.     

Temporal and Hormonal Control of β-1,3-Glucanase in Phaseolus vulgaris L

The endo-beta-1, 3-glucanase (beta-1, 3-glucan 3-glucanhydrolase, EC 3.2.1.6) extracted from Phaseolus vulgaris L. cv. Red Kidney had a pH optimum of 5 and a temperature optimum of 50 C. Excision of plant tissue resulted in an increase in beta-1, 3-glucanase activity after a 6-hour lag period. The increase could be prevented by indole-3-acetic acid, gibberellic acid, and cytokinins. Ethylene (half-maximal concentration = 0.1 microliter/liter) promoted the synthesis of beta-1, 3-glucanase, and 10% CO(2) overcame some of the ethylene effect. Cycloheximide prevented the induction of beta-1, 3-glucanase, but actinomycin D and chromomycin A(3) had only a partial effect.The amount of callose in sieve tube cells correlated with levels of beta-1, 3-glucanase, suggesting that this enzyme played a role in the degradation of beta-1, 3-glucans.     

Effects of Long-Term Exposure to Low-Power 915 MHz Unmodulated Radiation on Phaseolus vulgaris L.

The morphophysiological response of Phaseolus vulgaris L. to low-power electromagnetic radiation was investigated in order to assess the potential harmful effects of long-term continuous exposure. The plants were grown in two separate electromagnetic field (EMF) shielded rooms, in a controlled, greenhouse-like environment. One batch was continuously irradiated during the growth period (from sowing to maturity) and the other one was used as a reference. An unmodulated signal at 915 MHz (the central frequency between the uplink and downlink of the GSM900 mobile communications band) was used, with a maximum power density of 10 mW/m² measured near the plants. The plants were analyzed using ultraviolet–visible, statistical, morphometric, and electron microscopy methods. Significant differences were observed regarding the height of the plants, number of inflorescences, and chlorophyll and carotenoid content, all closely connected with the ultrastructural changes observed in the leaves. The irradiated batch grew higher (19% increase in plant height, 20% increase in stem and leaves' dry mass), with 18% fewer inflorescences, and extremely long roots (34% increase in dry mass). The ultrastructure of the irradiated leaves showed irregular cells and a higher content of plastoglobules in the chloroplasts. All results indicate that the irradiated plants suffered significant morphological modifications during their long-term exposure to the specific EM radiation. Bioelectromagnetics. © 2020 Bioelectromagnetics Society     

Effect of chitosan on tobacco mosaic virus（TMV） accumulation,hydrolase activity,and morphological abnormalities of the viral particles in leaves of N. tabacum L. cv. Samsun

The effect of chitosan on the development of infection caused by Tobacco mosaic virus（TMV） in leaves of Nicotiana tabacum L. cv. Samsun has been studied. It was shown that the infectivity and viral coat protein content in leaves inoculated with a mixture of TMV（2 μg/mL） and chitosan（1 mg/mL） were lower in the early period of infection（3 days after inoculation）, by 63% and 66% respectively, than in leaves inoculated with TMV only. Treatment of leaves with chitosan 24 h before inoculation with TMV also caused the antiviral effects, but these were less apparent than when the virus and polysaccharide were applied simultaneously. The inhibitory effects of the agent decreased as the infection progressed. Inoculation of leaves with TMV together with chitosan considerably enhanced the activity of hydrolases（proteases, RNases） in the leaves, in comparison with leaves inoculated with TMV alone. Electron microscope assays of phosphotungstic acid（PTA）-stained suspensions from infected tobacco leaves showed that, in addition to the normal TMV particles（18 nm in diameter, 300 nm long）, these suspensions contained abnormal（swollen, “thin” and “short”） virions. The highest number of abnormal virions was found in suspensions from leaves inoculated with a mixture of TMV and chitosan. Immuno-electron microscopy showed that “thin” virus particles, in contrast to the particles of normal diameter, lost the ability to bind to specific antiserum. It seems that the chitosan-induced activation of hydrolases stimulates the intracellular degradation of TMV particles and hence hydrolase activation may be considered to be one of the polysaccharide-mediated cellular defense mechanisms that limit virus accumulation in cells.     

Immuno-localization of Calmodulin in Unfertilized and Fertilized Embryo Sacs in Nicotiana tabacum var.macrophylla

Light microscopic immunohistochemical techniques with horse radish peroxidase(HRP)-conjugated second antibody and protein A-gold immunoelectron microscopic techniques were used to study the distribution of calmodulin (CaM) in unfertilized and fertilized embryo sacs in Nicotiana tabacum var. mocrophylla. Before fertilization, CaM was richer in the egg apparatus cells and antipodal cells than in the central cell. During the course from pollination to fertilization, the persistent synergid contained more CaM than the degenerated synergid. Meanwhile, two distinct bands rich in CaM were observed between the egg apparatus and the central cell, and gradually fused with each other appearing arc shape. When the two polar nuclei had fused, this CaM-rich band began to disappear. After fertilization, CaM level was still high in the zygote and the persistent synergid but low in the endosperm cells. Although there was no evidence about the polar distribution of CaM in the zygote, distinguishable difference, however, existed between the apical cell and the basal cell of a proembryo, being higher in the former than in the latter. The function of CaM during double fertilization and early embryogenesis as well as the temopral relationship between the CaM-rich band and the actin corona reported by other investigators are discussed.     

The effect of γ-irradiation on a polygenically inherited character in Nicotiana tabacum L. in relation to selection after treatment of aged seeds

• 1.1. γ-Ray sensitivity of 2-, 3- and 5-year old seeds of Nicotiana tabacum (var. Amarelinho Tietê) was studied. In the R₁, it was confirmed that germination rate, survival rate and seedling height decrease with age of seeds and with radiation dose, and that LD₅₀ is also dependent on age of seeds.
• 2.2. Polygenic inheritance of corolla length in the R₂ and R₃ raised from the irradiated aged seeds was studied. The results show that the variabilities in the R₂ and R₃ differed depending on age and radiation dose. Older seeds had higher variances due to irradiation and higher estimated “heritabilities” than younger seeds in the R₂ as well as R₃. The realized heritabilities did not show such clear-cut results. Among the realized heritabilities there was considerable interaction between age of seeds and radiation dosage, with the highest value being for low dose (15800 R) of the oldest seeds. These results suggest that selection for polygenic characters following irradiation of older seeds might be more effective than using younger seeds, not only in the R₂ but also in the R₃ and perhaps also in the later generations.

     

Studies on synthetic hybrids of british species of Senecio: I Senecio viscosus L. × S. vulgaris L.

Summary

Four aquatic hyphomycetes and one terrestrial fungus were examined for their responses to the phenoxy herbicides (±)-MCPP and 2,4-D as both single and binary preparations with respect to hyphal extension, sporulation and respiration. Hyphal extension of all species was unaffected at concentrations less than 100 mg l^?1. At higher concentrations there was a variable inhibitory response to the herbicides but no clear pattern was observed between the five fungi. The binary herbicide mixture had a weak synergistic effect on inhibition of growth rate. For the four aquatic hyphomycetes sporulation was reduced at several herbicide concentrations, but there was no consistent reduction over the experimental period. Flagellospora curvula and Clavariopsis aquatica showed increased sporulation at 100 and 1000 mg l^?1 for only some herbicide combinations. The respiration rates of the fungi varied with species and herbicide concentration and ranged between stimulation at 100 mg l^?1 to inhibition at 4000 mg l^?1. The results indicate that the five fungi are not likely to be severely effected by the phenoxy herbicides at concentrations normally occurring in the field. The possible effects of these herbicides on nutrient cycling are briefly discussed.     

Apoptotic cell death induces temperature-sensitive lethality in hybrid seedlings and calli derived from the cross of Nicotiana suaveolens×N. tabacum

Hybrid lethality expressed in the interspecific hybrid of Nicotiana suaveolens Lehm. ×N. tabacum L. cv. Hicks-2 is one of the mechanisms for reproductive isolation and it is temperature-sensitive. Apoptotic changes were detected in the cells of hybrid seedlings and calli expressing lethality at 28 °C but not under high-temperature conditions (36 °C), when the lethality is suppressed. Condensation of chromatin, fragmentation of nuclei and cytoplasmic reduction are the cytological changes associated with apoptosis leading to hybrid lethality. Fragmentation of nuclei was correlated with the lethal symptoms in both hybrid seedlings and calli, as confirmed by fluorimetry of the nuclear DNA using laser scanning cytometry. Agarose gel analysis of DNA extracted from hybrid seedlings and calli showing lethal symptoms revealed a specific ladder pattern suggesting nucleosomal fragmentation which is one of the biochemical changes of apoptosis. In-situ detection using terminal deoxyribonucleotidyl transferase-mediated dUTP-fluorescein nick end labeling (TUNEL) showed that this process occurred in distinct stages on each organ of hybrid seedlings and centripetally in hybrid calli. From these results, we confirmed that cell death inducing hybrid lethality was indeed apoptosis. Received: 23 December 1999 / Accepted: 5 April 2000     

Genomic factors responsible for abnormal morphology of pollen tubes in the interspecific cross Nicotiana tabacum ×N. rustica

Nicotiana tabacum shows unilateral pollen-pistil incongruity with N. rustica. If N. tabacum is pollinated with N. rustica, growth of the pollen tube is arrested in the middle of the style, and abundant callose deposition, tube swelling and tube winding are observed. An attempt was made to clarify the genomic factors responsible for this pollen-pistil incongruity. N. tabacum was pollinated with N. paniculata or N. undulata, progenitors of amphidiploid N. rustica. When pollinated with N. undulata, growth of the pollen tube was arrested in the middle of the style and showed abnormal morphology similar to that with N. rustica, but when pollinated with N. paniculata the pollen tube reached near the base of the style and was almost normal in appearance. These observations suggest that the factors responsible for the pollen tube abnormality of N. rustica are derived from the N. undulata genome.We also used N. sylvestris, N. glutinosa and N. otophora as pistilar parents and N. rustica or its progenitors as pollen parents to examine the genomic factors of the pistilate parents. The pollen tube features of these three species in the pistils of N. sylvestris were similar to those in the pistil of N. tabacum. Received: 25 October 1999 / Revision accepted: 2 February 2000     

Is a decreased water potential after withholding oxygen to roots the cause of the decline of leaf-elongation rates in Zea mays L. and Phaseolus vulgaris L.?

Leaf-elongation rates of Zea mays L. and Phaseolus vulgaris L. were measured in plants grown for 4 d in nutrient solution bubbled with N₂ and in soil-grown waterlogged Phaseolus plants. Leaf water potential in both species was lower 3–4h after replacing aeration by N₂-bubbling. In Zea, the water potential after 24 h or more was the same in control plants and plants with N₂ treatment. In Phaseolus, the water potential of inundated plants and plants with N₂ treatment was always lower than those of control plants. The leaf-elongation rate of both species was always lower in plants treated with N₂, especially during light periods. In Zea, the elongation rate was lowest in the first 24 h, whilst in Phaseolus it was lowest on the last (fourth) day of treatment. There was no difference between N₂ treatment and inundation experiments. It is concluded that during the first hours of treatment the leaf-elongation rate was reduced as a consequence of the lower water potential. Thereafter, however, elongation rates were lower than could be expected on the basis of the plant's water relations.Abbreviations LER leaf elongation rate - PEG-200 polyethylene-glycol 200 - RWC relative water content     

Genetic analysis of single-locus and epistatic QTLs for seed traits in an adapted × nuña RIL population of common bean (Phaseolus vulgaris L.)

Key message

The QTLs analyses here reported demonstrate the significant role of both individual additive and epistatic effects in the genetic control of seed quality traits in the Andean common bean.

Abstract

Common bean shows considerable variability in seed size and coat color, which are important agronomic traits determining farmer and consumer acceptability. Therefore, strategies must be devised to improve the genetic base of cultivated germplasm with new alleles that would contribute positively to breeding programs. For that purpose, a population of 185 recombinant inbred lines derived from an Andean intra-gene pool cross, involving an adapted common bean (PMB0225 parent) and an exotic nuña bean (PHA1037 parent), was evaluated under six different—short and long-day—environmental conditions for seed dimension, weight, color, and brightness traits, as well as the number of seed per pod. A multi-environment Quantitative Trait Loci (QTL) analysis was carried out and 59 QTLs were mapped on all linkage groups, 18 of which had only individual additive effects, while 27 showed only epistatic effects and 14 had both individual additive and epistatic effects. Multivariate models that included significant QTL explained from 8 to 68  % and 2 to 15 % of the additive and epistatic effects, respectively. Most of these QTLs were consistent over environment, though interactions between QTLs and environments were also detected. Despite this, QTLs with differential effect on long-day and short-day environments were not found. QTLs identified were positioned in cluster, suggesting that either pleiotropic QTLs control several traits or tightly linked QTLs for different traits map together in the same genomic regions. Overall, our results show that digenic epistatic interactions clearly play an important role in the genetic control of seed quality traits in the Andean common bean.     

Spontaneous and induced reversion rates in a double heterozygous mutant of Nicotiana tabacum var. xanthi n.c. —Dose-response relationship

The ability of the double heterozygous mutant a₁⁺/a₁a₂⁺/a₂(greenish-yellow) to revert phenotypically towards green was used to quantify the dose-response relationship at 0, 8, 16, 32, 64, 128 and 256 R of gamma-rays from a ⁶⁰Co-source. All the studies undertaken up to now on the genetic nature of the events responsible for the phenotypic reversion suggest that the changes take place mostly at locus a₁⁺−a₁.The present experiment was split into two parts: a low-dose (0–64 R) and a higher-dose (64–256 R) analysis, in which the responses were quadratic and linear, respectively.The spontaneous reversion rate was estimated to be 10⁻⁵.     

Effects of drought stress on the evergreen Quercus ilex L., the deciduous Q. robur L. and their hybrid Q. × turneri Willd.

Five-year-old trees of deciduous Quercus robur L., evergreen Q. ilex L., and their semideciduous hybrid, Q. × turneri Willd. (var. pseudoturneri), growing in pots, were subjected to drought stress by withholding water for 18–22 days, until leaf water potentials decreased below ?2 MPa. Gas-exchange rates, oxygen evolution, and modulated chlorophyll (Chl) fluorescence measurements revealed that by strong stomata closure and declining photosynthetic capacity down to approximately 50%, all three taxa responded with strongly reduced photosynthesis rates. In Q. robur, photochemical quenching of the drought-stressed plants was much lower than in nonstressed controls. Dissection of the occurring events in the photosynthetic electron transport chain by fast Chl fluorescence induction analysis with the JIP-test were discussed.