Damage in needle tissues after infection with Chrysomyxa rhododendri increases cuticular conductance of Picea abies in winter

Chrysomyxa rhododendri is a rust which infects Picea abies growing near the alpine timberline. Attacked needles are normally shed, but few remain on shoots. We hypothesised that these needles increase transpiration of Picea during winter. Partly damaged, completely damaged and healthy needles of an infected tree as well as healthy needles of a resistant tree were compared in a microscopy analysis, and needle conductance of shoots was measured gravimetrically. Despite needle shedding, more than 6% of needles remaining on infected tree shoots were damaged. Partly damaged needles showed local brownish areas in the periphery and completely damaged needles necrotic parenchyma and epidermal tissues. Cuticular conductance of affected shoots was up to 25.23?±?2.75 mmol m^?2 s^?1 at moderate water potential and thus twofold higher than in the resistant tree. Needle shedding reduces negative effects of Chrysomyxa infections during summer, but remaining damaged needles impair tree water relations in winter.     

Effects of some growth regulators on in vitro flowering of Streptocarpus nobilis

Leaf discs from vegetative Streptocarpus nobilis plants were cultured in vitro in media with cytokinin (BAP or K at 0.35 mg.1^?1) and auxin (IAA, NAA or 2,4-D at 0.1 mg.1^?1). Under short days (8-h photoperiod) in medium with IAA and BAP, floral buds developed in 100% of the cultures; under long days (16-h photoperiod) only shoots were formed. In medium with IAA and K, flowering was reduced. Flowers rarely formed in medium containing NAA and K, but roots developed profusely. NAA + BAP promoted leafy shoots which rarely flowered later. The effect of 2,4-D was to inhibit flowering completely and to induce callusing and formation of teratomous structures.     

Photoconversion of riboflavin to lumichrome in plant tissues

Free flavins have been extracted from shoots of etiolated corn (Zea mays L., var. Burpee Snowcross) and from yeast cells and separated from other substances by absorption on resorcinol-formaldehyde resin and talc columns and by thin layer chromatography. Riboflavin was the only free flavin present. Extracts of etiolated shoots of oats (Avena sativa L., var. Multiline E-69 and Clinford) yielded riboflavin plus a second free flavin previously demonstrated in oats. The areas of the chromatograms expected to contain lumichrome were completely clear. After illumination of any of the three organisms with artificial light (1100 ft-c) or sunlight for 6 hours, lumichrome (7,8-dimethylalloxazine) was found. In corn shoots after irradiation by sunlight, the amount of lumichrome present was equivalent to 2.5% of the total free flavin. Lumichrome was identified by thin layer chromatography in six solvent systems (including two two-dimensional systems), by its characteristic fluorescence in acetic acid, by its absorption spectrum, and by formation of a characteristic hydrate in ammonia-containing solutions. A comparison was made with in vitro photolysis of riboflavin and the possible role of photolysis of riboflavin (either free or bound) and of lumichrome formation in photo-responses of plants is discussed. Placing the shoots in the dark for 4 hours after irradiation in sunlight for 6 hours led to no detectable loss of the lumichrome which had been formed.     

Development of tyloses in the xylem vessels of Meili grapevine and their effect on water transportation

Shoot pruning could cause short-term damages to vines. In response to damage, tyloses develop in shoot xylem vessels interfering free water and mineral transportation. In this study, the tylosis development at different nodes of the current-year and perennial shoots of sixty three-year-old grapevines (Vitis vinifera L.) after pruning was investigated. The results showed that tyloses at the nodes closest to the trunk developed rapidly; and tylosis development initiated at the time when the size of vessel-ray pit was greater than that of parenchyma cell-parenchyma cell pit. In current-year shoots, tyloses were formed in up to 87% of the vessels, and 40% of the vessels were completely blocked by tyloses. In wound-induced perennial shoots, 30% of the vessels were completely blocked by tyloses. When few vessels were blocked by tyloses, new vessels could differentiate, and water transportation system may be restored. However, when tyloses developed in a large number of vessels and the large number of the vessels were blocked, the original capability of water transport was decreased (the largest decrease was 21.1% in this study), resulting in dehiscence or shrinkage cracking in this area. The study proved that the tylosis formation in functional vessels limited the water transport efficiency.     

Estimation of dieback process caused by herbivory in an endangered root-sprouting shrub species,Paliurus ramosissimus (Lour.) Poir., using a shoot-dynamics matrix model

Mortality, a critical parameter for population dynamics, is difficult to measure in long-lived trees or clonal herbaceous species because of the extremely low frequency of deaths. A model based on shoot recruitment would be helpful to estimate the population fate of species without a sufficient number of observed deaths. Existing matrix models are applicable to the dynamics of physiologically independent shoots, but not to physiologically dependent ones. We developed a shoot-dynamics model for plants with physiologically-dependent shoots, and used the model to estimate the effects of herbivory and conservation measures on the dynamics of a long-lived, shoot-sprouting shrub species, Paliurus ramosissimus (Rhamnaceae). Two populations of the endangered shrub have been severely damaged by herbivory by sika deer. The damage was mainly to new sprouting shoots. No deaths of individual plants were observed during an 8-year survey, and we could not estimate mortality. Thus, prediction of population dynamics based on births and deaths of individuals was impossible. Because P. ramosissimus is a shoot-sprouting species, we instead estimated the decline of individuals using a shoot-dynamics model. Using this model, we estimated the time to an 80 % decrease in shoot number per individual in the two populations at 37.8 and 37.2 years. These lengths suggest an immediate need for conservation measures to prevent herbivory even though no death of any individual was observed in the field survey. The estimated recovery times from the herbivory damage were 28.7 and 29.2 years if herbivory of new shoots is completely prevented by conservation measure.     

Factors Influencing the Tissue Culture and the Agrobacterium tumefaciens-Mediated Transformation of Hybrid Aspen and Poplar Clones

Tissue culture conditions and transformation have been established for both aspen and poplar. The use of previously described culture conditions resulted in shoot tip necrosis in the shoot cultures and necrosis of stem and leaf explants. Shoot tip necrosis could be overcome by buffering the medium with 2-(N-morpholino)ethanesulfonic acid and Ca-gluconate and by growing the shoots below 25°C. Necrosis of the explants was probably due to an accumulation of ammonium in the explants and could be overcome by adapting the NO₃⁻/NH₄⁺ ratio of the media. Stem explants of established shoot cultures of the aspen hybrid Populus alba × P. tremula and of the poplar hybrid Populus trichocarpa × P. deltoides were cocultivated with Agrobacterium strains having chimeric bar and neo genes on their disarmed tDNAs. Transformed aspen shoots were obtained from 30 to 40% of the explants, while transformed poplar shoots were obtained from 10% of the explants. Extracts from the transformed trees contained high phosphinotricin acetyltransferase and neomycin phosphotransferase activities, and the trees contained one to three copies of the chimeric genes. The transformed trees were completely resistant to the commercial preparations of the herbicide phosphinotricin (glufosinate), while control trees were not.     

The effect of 2,4-dinitrophenol on the absorption and translocation of calcium by pumpkin plants

The study deals with the absorption and translocation of Ca²⁺ by án intact plant of pumpkinCucurbita pepo L. and with the effect of various concentrations of 2,4-dinitrophenol (DNP) on these processes. The absorption of Ca²⁺ was reduced by the application of this inhibitor, but not completely stopped. The translocation of Ca²⁺ into shoots was affected more expressively: it was almost completely inhibited by higher DNP concentrations. The uptake of Rb⁺ is aubstantially more influeneed than that of Ca²⁺: higher inhibitor concentrations caused its complete cessation.     

Mineral cost of carnivory in aquatic carnivorous plants

Tissue N, P, K, Ca, and Mg content was estimated in traps and photosynthetic and carnivorous shoots in five aquatic carnivorous plant species from an outdoor culture: Aldrovanda vesiculosa, Utricularia vulgaris, U. reflexa, U. intermedia, and U. stygia, for the determination of the mineral cost of carnivory. In three species with monomorphic shoots (A. vesiculosa, U. vulgaris, U. reflexa), tissue P and K content in traps was significantly higher than that in their photosynthetic shoots, whereas N content was about the same. In U. stygia and U. intermedia with dimorphic shoots, tissue N and P content was markedly the highest in photosynthetic shoots followed by traps, while it was lowest in carnivorous shoots. In all five species, trap K content was significantly (2–4 times) higher than that in photosynthetic and carnivorous shoots. In all species, the values of the mineral cost of carnivory – the proportion of mineral nutrient amount contained in traps or carnivorous shoots to that in the total plant biomass – were within 19–61% for N, 33–76% P, 51–78% K, 26–70% Ca, and 34% for Mg. A new concept of the ecological cost-benefit relationships of plant carnivory, based on the mineral benefit of prey capture and mineral costs associated with trap production, is introduced for aquatic carnivorous plants. The evolution of this plant group is considered to show the optimization of these mineral cost-benefit relationships.     

Effect of host-plant manipulation by a gall-inducing insect on abundance of herbivores on chestnut trees

We investigated plant-mediated effects of the stem gall wasp, Dryocosmus kuriphilus Yasumatsu (Hymenoptera: Cynipidae), on other herbivores on the chestnut tree Castanea crenata. In the early season, leaves emerged earlier and in greater numbers on galled shoots than on ungalled shoots. On galled shoots the leaf to shoot biomass ratio was lower and the leaves were physically different. In May and June the concentration of nitrogen in leaves was higher on galled shoots than on ungalled shoots. In July, the water content of leaves was lower on galled shoots. In May and June, the number of aphids, Myzocallis kuricola Matsumura (Homoptera: Aphidoidea), on leaves was higher on galled shoots than on ungalled shoots, but the opposite was true at the end of July. Laboratory experiments showed that aphid fecundity and body weight decrease were higher in May and June when aphids fed on leaves on a galled shoot than when they fed on those on ungalled shoots. In contrast, aphid performance in July was greater on ungalled leaves than on galled leaves. Our findings suggest that gall initiation in a chestnut tree affected aphid performance by affecting host plant quality.     

Shoot growth dynamics and the mode of competition of two rhizomatousPolygonum species in the alpine meadow of Mt. Fuji

The growth dynamics of shoot populations and the mode of competition between intra-clonal shoots of two rhizomatous species,Polygonum cuspidatum andPolygonum weyrichii, were investigated at the Houei crater (c. 2380 m a.s.l.) of Mt. Fuji. Three genetic individuals ofP. cuspidatum (large, medium and small) and five genetic individuals ofP. weyrichii were selected for this research. The growth dynamics of shoot populations were analysed using the diffusion model. InP. cuspidatum, linearG(t,x) functions (mean of absolute growth rates of shoots of sizex at timet in the diffusion model) with positive slopes were found in terms of shoot weight in three genetic individuals. The linearG(t,x) functions suggest that smaller-sized shoots were supported by means of physiological integration and not suppressed by larger-sized shoots. As in other clonal plant species, size-independentD(t,x) functions (variance of absolute growth rates of shoots of sizex at timet in the diffusion model) were found inP. cuspidatum. A similar tendency was also found in the five genetic individuals ofP. weyrichii. There was large variance of RGR in terms of shoot weight irrespective of values ofC _r(t,x), defined as (total leaf area of shoots larger thanx at timet/total leaf area of all shoots), in both thePolygonum species. This means that there was a higher probability of growth for smaller shoots, suggesting that competition between shoots was symmetric (two-sided) in bothP. cuspidatum andP. weyrichii.     

Response to Anoxia in Rice and Wheat Seedlings: Changes in the pH of Intracellular Compartments, Glucose-6-Phosphate Level, and Metabolic Rate

³¹P nuclear magnetic resonance spectroscopy was used to measure intracellular pH in living tissues. Oxygen deprivation caused fast cytoplasmic acidification from pH 7.4 to 7.0 in shoots of rice, Oryza sativa L. var arborio, a species highly resistant to anoxia. Acidification was complete after 10 minutes of anoxia. Alkalinization of both cytosplasm and vacuole followed thereafter. In the anoxia intolerant wheat shoots, Triticum aestivum L. var MEK, the same treatment caused a sharper cytoplasmic acidification, from pH 7.4 to 6.6, which occurred during a period of 2 hours. Cytoplasmic acidification continued with progress of anoxia and there was no vacuolar alkalinization comparable to the one observed in rice. In wheat oxyen, withdrawal also caused the reduction of both glucose-6-phosphate level and of metabolic rate. It also induced heavy losses of inorganic phosphate from tissues. Conversely, in rice, glucose-6-phosphate level and metabolic rate were increased and inorganic phosphate leakage from tissues was completely absent. These results are discussed in relation to the mechanisms of plant resistance to anoxia.     

Growth correlations inBryophyllum leaves and exogenous growth regulators

Growth correlations in leaves ofBryophyllum may be recognized by the development of marginal shoots varying in their particular lamina regions, the correlative inhibition increasing from the top to the base. Cytokinins extend their promoting action inB. crenatum leaves farther in the apical than in the basal direction. In the uppermost leaves ofB. daigremontianum they evoke the development of marginal shoots with flowers in the apical and of vegetative shoots in the basal region. Less suitable for this research are auxins, gibberellins, and triiodobenzoic acid which are unable to stimulate the development of marginal shoots on leaves grown out under short days if not supplemented by cytokinins. Only TIBA induces under long days formation of adventitious shoots directly from the lamina surface on leaves developed below the terminal ring fasciation exhibiting at free ends of connate leaves normal marginal shoots. Under short days a complete reduction of lateral teeth takes place inB. verticillatum both on the extremity of ring fasciation and on the uppermost leaves, the marginal shoots being formed under the subsequent long days only in the angles between the ring fasciation parts. Furthermore the correlative inhibition of the leaf causes anisophylly in marginal shoots in the same way as that of axillaries inBryophyllum.     

Identification and quantification of phenolic compounds in Hypericum perforatum L. transgenic shoots

Regeneration of transgenic shoots was achieved from Hypericum perforatum L. hairy roots on hormone-free MS/B₅ medium for a period of 4 weeks under a photoperiod of 16-h light. A control experiment was set up with root segments obtained from in vitro grown seedlings. Investigations have been made to study the production of phenolic compounds in non transgenic and transgenic shoot cultures. Six groups of phenolic compounds such as phenolic acids, flavonols, flavan-3-ols, naphtodianthrones, phloroglucinols, and xanthones were recorded in the transgenic shoots. Chlorogenic acid was found as the most representative phenolic acid in shoot extracts. With regard to the class of quercetin derivatives in transformed shoots, quercetin 6-C-glucoside usually dominated among the glycosides followed by quercitrin and hyperoside. The analysis of flavan-3-ols in transgenic shoots resulted in the identification of epicatechin and proanthocyanidin dimers. One of the main achievements in this study was considerably enhanced hypericin and pseudohypericin production in transgenic shoots. The concentration of identified naphtodianthrones was about 12-fold higher in transformed shoots compared to control. Chromatographic analysis of phloroglucinols in transgenic shoots resulted in the identification of hyperforin, while its homolog adhyperforin was detected in traces. A twofold higher content of hyperforin was observed in transgenic shoots compared to control. Although mangiferin was found as the main representative xanthone in shoot extracts, several other xanthones identified as γ-mangostin isomers, trihydroxy-1-methoxy-C-prenyl xanthone, garcinone E, and banaxathone E were de novo synthesized in transformed shoots. Therefore, H. perforatum transgenic shoots could be considered as a source for rapid and increased production of naphtodianthrones and other specific phenolic compounds.     

Improved method of in vitro regeneration in Leucaena leucocephala — a leguminous pulpwood tree species

Leucaena leucocephala is a fast growing multipurpose legume tree used for forage, leaf manure, paper and pulp. Lignin in Leucaena pulp adversely influences the quality of paper produced. Developing transgenic Leucaena with altered lignin by genetic engineering demands an optimized regeneration system. The present study deals with optimization of regeneration system for L. leucocephala cv. K636. Multiple shoot induction from the cotyledonary nodes of L. leucocephala was studied in response to cytokinins, thidiazuron (TDZ) and N⁶-benzyladenine (BA) supplemented in half strength MS (½-MS) medium and also their effect on in vitro rooting of the regenerated shoots. Multiple shoots were induced from cotyledonary nodes at varied frequencies depending on the type and concentration of cytokinin used in the medium. TDZ was found to induce more number of shoots per explant than BA, with a maximum of 7 shoots at an optimum concentration of 0.23 µM. Further increase in TDZ concentration resulted in reduced shoot length and fasciation of the shoots. Liquid pulse treatment of the explants with TDZ did not improve the shoot production further but improved the subsequent rooting of the shoots that regenerated. Regenerated shoots successfully rooted on ½-MS medium supplemented with 0.54 µM α-naphthaleneacetic acid (NAA). Rooted shoots of Leucaena were transferred to coco-peat and hardened plantlets showed ≥ 90 % establishment in the green house.Key words: Cotyledonary nodes, Multiple shoot induction, Pulse treatment, TDZ     

Analysis of the cellulose synthase genes associated with primary cell wall synthesis in Bambusa oldhamii

The synthesis of cell wall polysaccharides is highly active in rapidly growing bamboo shoots. We cloned a set of BoCesA cDNAs that encode cellulose synthase from bamboo (Bambusa oldhamii) and investigated the expression patterns of the BoCesA2, BoCesA5, BoCesA6 and BoCesA7 genes. The four BoCesA genes were differentially expressed in the different parts of growing bamboo shoots, in various organs, and in multiple shoots that were cultured in vitro. They were down-regulated by α-naphthaleneacetic acid and differentially affected by thidiazuron in the multiple shoots. In situ RT-PCR analyses demonstrated that BoCesA2, BoCesA5, BoCesA6, and BoCesA7 mRNAs were present throughout the base and the internode regions of the etiolated shoots that emerged from pseudorhizomes, and in the internode regions of the juvenile branch shoots that emerged from nodes of mature bamboo culms; however, the expression of the four genes in the lignified internode of the branch shoot was predominantly detected in the center of the vascular bundles. Our results for cDNA cloning, expression analyses, and phylogenetic analysis suggest that the 10 BoCesA genes cloned from the etiolated bamboo shoots participate in cellulose synthesis in the primary cell walls of the growing bamboo, and that at least three additional BoCesA genes involved in cellulose synthesis in the secondary walls may be present in the bamboo genome. The expressions of BoCesA genes may be under fine control in response to the various developmental stages and physiological conditions of bamboo.     

Potato leafroll virus (PLRV) and Potato virus Y (PVY) influence vegetative growth, physiological metabolism, and microtuber production of in vitro-grown shoots of potato (Solanum tuberosum L.)

Effects of Potato leafroll virus (PLRV) and Potato virus Y (PVY) on vegetative growth, physiological metabolism and microtuber production were investigated using in vitro shoot cultures. The results showed that parameters of shoot growth including bud break percentage, shoot length, and node number and length were markedly reduced in the diseased shoots. These negative effects were much more pronounced in shoots co-infected with PLRV and PVY than in those singly infected with either PLRV or PVY. The inhibitive effects on root developments measured by root number and length were observed only in shoots co-infected with PLRV and PVY. Significantly lower contents of chl-a, chl-b and total chl were found in virus infected shoots than in healthy ones. There were striking differences in contents of total soluble protein observed between healthy shoots and PLVR and PVY co-infected ones. The content of total soluble sugar was highest in shoots co-infected with PLRV and PVY, and lowest in healthy shoots. Furthermore, there were no significant differences found in the level of endogenous indole-acetic acid among healthy shoots verses virus infected shoots. However, the level of zeatin-ribosome was much higher in healthy shoots than in virus infected ones. Yet, both healthy and single PLRV infected shoots produced similar levels of gibberillic acid 3, which were much higher than those of PVY single-infected shoots and PLRV and PVY co-infected shoots. Also, there were no significant differences in the number of microtubers among healthy shoots, PLVR single or PVY single infected shoots, but shoots co-infected with PLRV and PVY produced the lowest number of microtubers. Overall, the healthy shoots produced the largest size of microtubers and the highest percentage of microtubers ≥5 mm in diameter.     

Characteristics of teratomas regenerated in vitro from octopine-type crown gall

Crown galls induced by infection of tobacco plants with Agrobacterium tumefaciens strain C58-Cl(pTiB6S3) were excised and cultured in vitro. After about one year of culture on medium-lacking phytohormones, two noncloned lines spontaneously formed shoots. Leaf explants from shoots of tumor-line T5 were capable of growing on hormone-free medium, and the resulting mixture of organized and unorganized tissue synthesized octopine. Detached leaves from T5 shoots also synthesized octopine. These results establish that shoots from this octopine-type tumor contain transformed cells and are true crown-gall teratomas.     

Interorgan photosynthate partitioning in alfalfa

The net partitioning of current photosynthate among vegetative organs of nodulated alfalfa (Medicago sativa L.) was investigated by determining radiolabel distribution from upper and lower source leaves, at different times of day, and during successively longer chase periods. Photosynthate was exported sooner and more completely by a fully expanded lower than by a fully expanded upper source leaf. The radiolabel pulse from the lower source leaf peaked in the main stem within 1.5 hours, in the crown and nodules after 3 hours, in the unexpanded leaves and apex of the main stem after 6 hours, and in the shoots growing from leaf axils on the main stem after 24 hours. The results suggest that the crown, apex, axillary shoots, and nodules both imported and mobilized photosynthate originating at a lower source leaf, while roots and shoots growing from the crown showed net accumulation. The pulse from the upper source leaf was initially rapidly exported by the main stem and imported by the root between 1.5 and 3 hours, but there was no net change of label content of these organs during the ensuing 21 hours. Rapidly growing organs had the highest concentrations, and the largest organs had the highest content of radiolabel. These results provide new information about the accumulation and circulation of photosynthate within the alfalfa plant.     

In vitro plant regeneration, secondary metabolite production and antioxidant activity of micropropagated Aloe arborescens Mill

Aloe species are highly-prized for their ornamental value and have been utilized for centuries in traditional medicine. Due to their habitat loss and exploitation for medicinal and ornamental plant trade, many species in this genus have become threatened. One of the most important globally rated medicinal species in Aloe genus is A. arborescens. The current study evaluated the roles of different aromatic cytokinin types and concentrations on direct organogenesis, in vitro bioactive secondary metabolite production and antioxidant activity of regenerated shoots of A. arborescens. There was an increase in the number of adventitious shoots produced per explant with an increase in concentration in cultures treated with meta-topolin (mT), meta-topolin riboside (mTR), meta-methoxytopolin (MemT) and benzyladenine riboside (BAR), reaching an optimum at either 5.0 or 7.5???M. Overall, the treatment with 5.0???M mT gave the largest number of transplantable shoots (regenerated shoots with length greater than 10?mm). Rooted shoots were successfully acclimatized after 8?weeks with a survival frequency above 90?% and no observable morphological abnormalities. Variable amounts of total iridoids, phenolics, flavonoids and condensed tannins were detected in regenerated shoots from all the cytokinin treatments. An increased free-radical scavenging activity with an increase in concentration was recorded in regenerated shoots from mT and mTR treatments, reaching an optimum at 7.5???M concentration. The present study shows that the choice of cytokinin type and concentration exogenously supplied during tissue culture markedly influences not only shoot proliferation but also the in vitro production of bioactive secondary metabolites.