Studies on the relationship between ploidy level,morphology, the concentration of some phytohormones and the nicotine concentration of haploid and doubled haploid tobacco (Nicotiana tabacum L.) and NICA plants

As compared to doubled haploid plants of the same origin, haploid tobacco plants are characterized by narrow leaves and in these leaves the endogenous concentration of gibberellins was considerably higher than in doubled haploids. This higher GA activity is almost entirely due to elevated levels of polar gibberellins. The same leaf shape as in haploids could be induced by GA₃ sprays to doubled haploids. A similar leaf shape was also observed on tissue culture derived so called NICA plants displaying the morphology of tobacco plants as described by Dudits et al. (1987) from whom the plant material was obtained as a gift. Here, in the leaves of a special strain with narrow lamina again a much higher gibberellin activity was detected than in the leaves of plants of the original tobacco strain. Histochemical determination of the relative DNA content indicated that leaves of NICA were chimaeras containing 1C cells besides cells with higher C values. Obviously, haploidy is somehow related to the endogenous gibberellin activity in tobacco plant material with consequences on the morphological appearance of 1n plants. Comparing some haploid and doubled haploid strains in tissue culture and pot and field experiments in several years apparently the genotype of the plant material is more significant for nicotine concentration than the ploidy level.Abbreviations DW dry weight - FW fresh weight - LSI leaf shape index     

Hormonal and histological studies related to in vitro banana bud formation

Shoot apices of Musa subgroup AAA `Grande Naine' were used for in vitro culture establishment. The endogenous hormone levels and their effects on bud formation were evaluated during a 75-day period. Cytokinins, IAA and ABA were separated by HPLC and quantified by means of ELISA. Enzymatic degradation of IAA was determined by the colorimetric method. Explants were maintained on establishment medium for 60 days. The endogenous cytokinins were higher in the basal portion of the explant. Subculture to proliferation medium (65 to 75 days) resulted in a substantial increase of cytokinins in the basal portion and in a decline in the apical portion. 2iP was the predominant cytokinin in the tissue. The endogenous level of IAA and the IAA/cytokinin ratio decreased after the 65th day of culture. The level of ABA was reduced from the time of inoculation up to the 75th day of culture. Histological analysis indicated that buds formed at the leaf base at the 65th day of culture. This revised version was published online in August 2006 with corrections to the Cover Date.     

Molecular cloning,characterization, and expression of a cDNA encoding an endochitinase gene from the mycoparasite Stachybotrys elegans

Stachybotrys elegans is a mycoparasite of the soilborne plant pathogenic fungus Rhizoctonia solani. The mycoparasitic activity of S. elegans is correlated with the production of cell wall degrading enzymes such as chitinases. This report details the cloning by RACE-PCR and characterization of a full-length cDNA clone, sechi44, that appears to encode an extracellular endochitinase. An analysis of the sechi44 sequence indicates that this gene contains a 1269-bp ORF and encodes a 423-aa polypeptide. The SECHI44 protein has a calculated molecular weight of 44.1kDa and pI of 5.53. Since the SECHI44 protein also appears to encode a signal peptide, an extracellular location for the corresponding protein is predicted. Comparison of SECHI44 sequence with known sequences of fungal endochitinases revealed that SECHI44 is grouped with endochitinases from other mycoparasites. Real-time quantitative RT-PCR analysis showed an elevated level of expression of sechi44 (21-fold) in chitin-rich (induced) as compared to no-carbon (non-induced) culture conditions. In dual culture, the temporal expression of sechi44 increased after 2 days of contact with R. solani, reaching a 10-fold increase after 9 days, followed by a decrease to basic expression level at 12 days. Interestingly, inhibition of sechi44 expression was observed when S. elegans hyphae were in close proximity with R. solani hyphae.     

The effects of glutamine of the maintenance of embryogenic cultures of Cryptomeria japonica

Summary Embryogenic tissues of sugi (Cryptomeria japonica) were induced on a modified Campbell and Durzan (CD) medium containing 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 600 mg l⁻¹ glutamine, and subcultured in the medium of the same composition for over 1 yr. This resulted in a mixed culture of embryogenic and non-embryogenic cells. When embryogenic cells were isolated and cultured independently, their capacity to form embryogenic aggregates was lost. Thus, the non-embryogenic cells present within a mixed culture system were essential to the formation of embryogenic aggregates. When embryogenic tissues were isolated and cultured independently on a high glutamine-containing (2400 mg l⁻¹) medium, dry weights and endogenous levels of glutamine increased, and the tissue could generate a large number of embryogenic aggregates. Amino acid analysis of embryogenic and non-embryogenic cells from the maintenance culture indicated a higher level of glutamine was present in the latter. The high endogenous level of glutamine in the non-embryogenic portion of mixed cell masses may be the supplier of glutamine for maintaining the embryogenic property of the tissues.     

Auxin levels,antiauxin(s) and androgenic plantlet formation in isolated pollen cultures ofNicotiana tabacum

Summary Anthers ofNicotiana tabacum var. Badischer Burley contain endogenous auxins, one of these was identified as indoleacetic acid. At the developmental stage shortly after the first pollen mitosis the anthers contain equivalents of 0.1 mg IAA per kg fresh weight. This endogenous auxin level is maintained during the eight-day preculture of the anthers prior to isolated pollen culture. However, in anthers of short-day plants, which are characterized by a high proportion of embryogenic pollen at the end of preculture (Heberle-Bors andReinert 1979), an increase of the auxin level till the fourth day of culture is detectable.Preculture of anthers in the presence of an inhibitor of auxin synthesis (7-azaindole) and an antiauxin (-(o-chlorophenoxy)-isobutyric acid) results in enhanced plantlet yield by pollen cultures. The significance of these observations for androgenesis is discussed.     

Levels and immunolocalization of endogenous cytokinins in thidiazuron-induced shoot organogenesis in carnation

We evaluated the capacity of the plant growth regulator thidiazuron (TDZ), a substituted phenylurea with high cytokinin-like activity, to promote organogenesis in petals and leaves of several carnation cultivars (Dianthus spp.), combined with 1-naphthaleneacetic acid (NAA). The involvement of the endogenous auxin indole-3-acetic acid (IAA) and purine-type cytokinins was also studied. Shoot differentiation was found to depend on the explant, cultivar and balance of growth regulators. TDZ alone (0.5 and 5.0 micromol/L) as well as synergistically with NAA (0.5 and 5.0 micromol/L) promoted shoot organogenesis in petals, and was more active than N6-benzyladenine. In petals of the White Sim cultivar, TDZ induced cell proliferation in a concentration-dependent manner and, on day 7 of culture, the proportion of meristematic regions in those petals allowed the prediction of shoot regeneration capacity after 30 days of culture. Immunolocalization of CK ribosides, N6-(delta2-isopentenyl)adenosine, zeatin riboside (ZR) and dihydrozeatin riboside (DHZR), in organogenic petals showed them to be highly concentrated in the tips of bud primordia and in the regions with proliferation capacity. All of them may play a role in cell proliferation, and possibly in differentiation, during the organogenic process. After seven days of culture of White Sim petals, NAA may account for the changes found in the levels of IAA and DHZR, whereas TDZ may be responsible for the remarkable increases in N6-(delta2-isopentenyl)adenine (iP) and ZR. ZR is induced by low TDZ concentrations (0.0-0.005 micromol/L), whereas iP, that correlates with massive cell proliferation and the onset of shoot differentiation, is associated with high TDZ levels (0.5 micromol/L). In addition to the changes observed in quantification and in situ localization of endogenous phytohormones during TDZ-induced shoot organogenesis, we propose that TDZ also promotes growth directly, through its own biological activity. To our knowledge, this study is the first to evaluate the effect of TDZ on endogenous phytohormones in an organogenic process.     

甘蔗幼叶脱分化及愈伤组织形成过程中内源细胞分裂素和ABA的变化

甘蔗幼叶片,叶鞘和幼茎在含有2.4-D的培养条件下脱分化的情况有明显差异。它们除了在形态学上有区别外,这三种材料原有的细胞分裂素和ABA的水平明显不同。在幼叶片脱分化及愈伤组织形成过程中,内源细胞分裂素和ABA的水平也发生了明显变化。据此我们认为在甘蔗组织培养中2.4-D可能通过调节内源激素的水平及其相互作用,引起培养物中某些生理生化过程发生改变,从而进行脱分化和愈伤组织形成。     

Abscisic acid and stress treatment are essential for the acquisition of embryogenic competence by carrot somatic cells

Studies of carrot embryogenesis have suggested that abscisic acid (ABA) is involved in somatic embryogenesis. A relationship between endogenous ABA and the induction of somatic embryogenesis was demonstrated using stress-induced system of somatic embryos. The embryonic-specific genes C-ABI3 and embryogenic cell proteins (ECPs) were expressed during stress treatment prior to the formation of somatic embryos. The stress-induction system for embryogenesis was clearly distinguished by two phases: the acquisition of embryogenic competence and the formation of a somatic embryo. Somatic embryo formation was inhibited by the application of fluridone (especially at 10⁻⁴ M), a potent inhibitor of ABA biosynthesis, during stress treatment. The inhibitory effect of fluridone was nullified by the simultaneous application of fluridone and ABA. The level of endogenous ABA increased transiently during stress. However, somatic embryogenesis was not significantly induced by the application of only ABA to the endogenous level, in the absence of stress. These results suggest that the induction of somatic embryogenesis, in particular the acquisition of embryogenic competence, is caused not only by the presence of ABA but also by physiological responses that are directly controlled by stresses.     

Stringently and developmentally regulated levels of a cytoplasmic double-stranded RNA and its high-efficiency transmission via egg and pollen in rice

A very restricted amount of high-molecular-weight double-stranded RNA (dsRNA) has been found in healthy japonica rice plants. We discriminated dsRNA-carrying rice plants from noncarriers. The endogenous dsRNA was localized in the cytoplasm (about 100 copies per cell) and was transmissible to progeny plants by mating. In crosses between carriers and noncarriers, the RNA was transmitted efficiently to F1 plants via both egg and pollen. The rice dsRNA was maintained at an almost constant level by host plant cells from generation to generation. The high-efficiency transmission of the endogenous dsRNA to progeny plants appears to depend on the autonomously controlled replication of the dsRNA localized in cytoplasmic vesicles. However, an increase in copy number (about 10-fold) of the dsRNA was observed during the suspension culture of host cells. The number of copies of dsRNA returned to the original low value in regenerated plants, suggesting that the copy number is stringently and developmentally regulated in rice cells.     

植物激素对体细胞胚胎发生的诱导与调节

以作者自己的工作为背景,结合国内外近几年的有关报道,综述了几种外源和内源激素对植物体细胞胚胎发生的诱导与调节作用。外源生长素和细胞分裂素是诱导离体培养细胞分化与增殖所必需的,2,4-D是诱导胚性愈伤组织的重要激素。在体细胞胚胎发生中内源激素含量和代谢的平衡起着关键的作用,而且外源和内源激素对诱导体细胞胚胎发生起相互调节作用。ABA在提高体细胞胚胎发生频率和质量上具有重要作用,同时,外源与内源ABA对体细胞胚胎发生起相互促进作用。本文还较为深入地讨论了这些激素诱导体细胞胚胎发生的可能作用机制。 Abstract:The paper summarizes the induced and regulatory effects of a few exogenous and endogenous hormones in plant somatic embryogenesis by our studies and related international reports.The exogenous auxin and cytokinin are necessary to induced differentiation and proliferation of cells of culture in vitro.2,4-D is an important hormone of induced embryogenic calluses.The contents and the metabolic balances of endogenous hormones have key effects for somatic embryogenesis.In addition,the exogenous and endogenous hormones have mutual regulatory effects for somatic embryogenesis.ABA has an important effect to improving the frequency and quality of somatic embryogenesis.Meanwhile,the exogenous and endogenous ABA have mutual promoted effects for somatic embryogenesis.The paper discusses possible mechanism of hormones-induced somatic embryogenesis in a deep-going way.     

Elicitor induction of furanocoumarin biosynthetic pathway in cell cultures ofRuta graveolens

Treatment with an autoclaved culture homogenate of the yeastRhodotorula rubra induces rapid accumulation of acridone epoxides, furoquinolines and furanocoumarins in cell cultures ofRuta graveolens (L). The increased accumulation is preceeded by an induction of enzymes of the biosynthetic pathways. In the case of furanocoumarins induction was shown for phenylalanine ammonia-lyase (PAL), 4-coumarate: CoA ligase (4-CL) and S-adenosyl-l-methionine: xanthotoxol O-methyltransferase (XOMT). For PAL and 4-CL time courses of induced activity showed an early maximum, 8–12 h after treatment, whereas XOMT was found to reach its maximum later, about 36–42 h after treatment. The elicitor dose-response curve showed saturation at an elicitor concentration of 1%. At any time during the whole culturing period cells responded to elicitiation but the maximum enzyme activities induced were lower at the late stages. Experiments with different suspension culture strains, a shoot teratoma culture and hydroponically grown sterile photomixotrophic plants were performed to assess the influence of differentiation on constitutive activities of these enzymes and their inducibility by elicitation. Constitutive furanocoumarin accumulation was positively correlated with the level of differentiation. Although induction of PAL, 4-CL and XOMT activity always accompanied induced furanocoumarin accumulation no absolute correlation existed between induced enzyme activities and the induced product level or relative product increase.Abbreviations 4-CL 4-coumarate:CoA ligase - COMT S-adenosyl-l-methionine:caffeic acid 3-O-methyltransferase - PAL phenylalanine:ammonia-lyase - XOMT S-adenosyl-l-methionine:xanthotoxol O-methyltransferase     

Efficient secretion of human endostatin in the yeast, Pichia pastoris

Endostatin is a 20 kDa COOH-terminal fragment of collagen XVIII that inhibits angiogenesis and tumor growth. The cDNA coding for human endostatin in human fetal liver has been cloned into the secreting expression organism Pichia pastoris, and the high level expression of human endostatin has been achieved (about 200 mg of endostatin in 1 l of culture). The recombinant human endostatin was purified to homogeneity by heparin-affinity column, and showed antiproliferative effect on rat brain micro-vascular endothelia cells.     

Aerobic,phenol-induced TCE degradation in completely mixed,continuous-culture reactors

BothPseudomonas putida F1 and a mixed culture were used to study TCE degradation in continuous culture under aerobic, non-methanotrophic conditions. TCE mass balance studies were performed with continuous culture reactors to determine the total percent removed in the reactors, and to quantify the percent removed by air stripping and biodegradation. Adsorption of TCE to biomass was assumed to be negligible. This research demonstrated the feasibility of treating TCE-contaminated water under aerobic, non-methanotrophic conditions with a mixed-culture, continuous-flow system.Initially glucose and acetate were fed as primary substrates. Pnenol, which has been shown to induce TCE-degrading enzymes, was fed at a much lower concentration (20mg/L). Little degradation of TCE was observed when acetate and glucose were the primary substrates. After omitting glucose and acetate from the feed and increasing the phenol concentration to 50mg/L, TCE biotransformation was observed at a significant level (46%). When the phenol concentration in the feed was increased to 420mg/L, 85% of the incoming TCE was estimated to have been biodegraded. Under the same conditions, phenol utilization by the mixed culture was greater than that ofP. putida F1, and TCE degradation by the mixed culture (85%) exceeded that ofP. putida F1 (55%). The estimated percent-of-TCE biodegraded by the mixed culture was consistently greater than 80% when phenol was fed at 420mg/L. Biodegradation of TCE was also observed in mixed-culture, batch experiments.     

Effects of endogenous pyrogen and prostaglandin E2 on hypothalamic neurons in rat brain slices

We investigated the effects of endogenous pyrogen and prostaglandin E2 (PGE2) on the preoptic and anterior hypothalamic (POAH) neurons using brain slice preparations from the rat. Partially purified endogenous pyrogen did not change the activities of most of the neurons in the POAH region when applied locally through a micropipette attached to the recording electrode in proximity to the neurons. This indicates that partially purified endogenous pyrogen does not act directly on the neuronal activity in the POAH region. The partially purified endogenous pyrogen, applied into a culture chamber containing a brain slice, facilitated the activities in 24% of the total neurons tested, regardless of the thermal specificity of the neurons. Moreover, PGE2 added to the culture chamber facilitated 48% of the warm-responsive, 33% of the cold-responsive, and 29% of the thermally insensitive neurons. The direction of change in neuronal activity induced by partially purified endogenous pyrogen appears to be almost the same as that induced by PGE2 when these substances were applied by perfusion to the same neuron in the culture chamber. These results suggest that partially purified pyrogen applied to the perfusate of the culture chamber stimulates some constituents of brain tissue to synthesize and release prostaglandin, which in turn affects the neuronal activity of the POAH region.     

The polyamine spermine induces cystocarp development in the seaweed <Emphasis Type="Italic">Grateloupia</Emphasis> (Rhodophyta)

Polyamines such as putrescine, spermidine and spermine are ubiquitous aliphatic amines involved in reproductive events in plants and algae, and first become evident through changes in endogenous levels during reproductive development. To examine whether the differences observed in polyamines, during carposporogenesis, in the red alga Grateloupia, followed a specific pattern as is seen in other organisms, infertile axes (i.e. not showing cystocarps) were excised from the same holdfast of female fertilized individuals (i.e. showing cystocarps in other axes), and cultivated until the cystocarps became visible. Changes in the endogenous levels of free putrescine, spermidine and spermine were monitored over the 8 days of culture. The activity of enzymes related to polyamine metabolism, such as l-ornithine decarboxylase (ODC), diamine oxidase and polyamine oxidase, was measured at the beginning and end of the experimental period. Up to 50% of the infertile axes became fertile and produced cystocarps at a density of 1.91 ± 0.1 cystocarps mm⁻² after 8 days. The endogenous content of spermine increased markedly over the first 5 days of culture, then decreased to the initial level by day 8. Spermidine followed a similar pattern to spermine, whereas putrescine remained at high levels, until day 5 when it decreased abruptly. The activity of ODC was less on day 8 than on day 0, whereas the activities of diamine oxidase and polyamine oxidase increased. In parallel experiments with explants from infertile axes, exogenously added spermine (10⁻⁶ M) increased the number of cystocarps, and reversed the effect of cyclohexylamine (CHA), which is known to inhibit polyamine synthesis in Grateloupia. Serial sectioning and microscopic observation of specimens from explants cultivated in 10⁻⁶ M spermine indicated that cystocarp development was induced. The results suggest that, during transition from infertile to fertile spermine is accumulated, thus favouring the development of cystocarps, given the presumed role of spermine as an inducing agent.     

Levels of endogenous polyamines and NaCl-inhibited growth of rice seedlings

The role of endogenous polyamines in the control of NaCl-inhibited growth of rice seedlings was investigated. Putrescine, spermidine and spermine were all present in shoots and roots of rice seedlings. NaCl treatment did not affect spermine levels in shoots and roots. Spermidine levels in shoots and roots were increased with increasing concentrations of applied NaCl. NaCl at a concentration of 50 mM, which caused only slight growth inhibition, drastically lowered the level of putrescine in shoots and roots. Addition of precursors of putrescine biosynthesis (L-arginine and L-ornithine) resulted in an increase in putrescine levels in NaCl-treated shoots and roots, but did not allow recovery of the growth inhibition of rice seedlings induced by NaCl. Pretreatment of rice seeds with putrescine caused an increase in putrescine level in shoots, but could not alleviate the inhibition effect of NaCl on seedling growth. The current results suggest that endogenous polyamines may not play a significant role in the control of NaCl-inhibited growth of rice seedlings.Abbreviations PUT putrescine - SPD spermidine - SPM spermine     

Changes in endogenous polyamines during the formation of somatic embryos from isogenic lines of Dactylis glomerata L. with different regenerative capacities

The endogenous levels of polyamines (PAs) in leaf-base explants isolated from plants of two isogenic lines of Dactylis glomerata L., differing in their competence for somatic embryogenesis, were compared. Leaf-bases isolated from plants with a high level of competence for somatic embryogenesis (HEC) contained four times the level of polyamines compared to those isolated from plants with a low level of competence for somatic embryogenesis (LEC). When the levels of individual polyamines in the HEC and LEC lines were compared, leaf-bases from plants of the HEC line had much lower PUT/SPD ratios than those from the LEC line. When changes in the levels of PAs were monitored during the first 28 d of culture, on a medium which promotes initiation of somatic embryogenesis, leaf-base cultures from plants of the HEC line showed a 50% increase in the levels of PAs during the first 7 d of culture, after which time levels began to decline. By day 21, levels had dropped below those found in freshly isolated leaf bases. While PUT and SPM levels increased by about 30%, the greatest increase was shown by SPD, which increased by more than 100% during the first 7 d of culture, before declining. In contrast much smaller changes in PA levels were found when leaf-bases from plants of the LEC line were cultured.