Callus production from willow (Salix viminalis L.) protoplasts

Protoplasts were isolated from cell suspensions of Salix viminalis (basket willow) clone 78-0-90 and S. schwerinii clone 77-0-77, using cellulysin and macerase in modified Woody Plant medium. For clone 78-0-90, 6.3 · 10⁶ ± 1.9 · 10⁶ protoplasts were obtained per gram fresh weight. Cell divisions started two days after protoplast isolation and gave rise to callus which has been maintained in culture for up to four years. Protoplast yield from the clone 77-0-77 was lower (less than 10⁶ protoplasts per gram cells), cell division was infrequent and no callus was obtained. Protoplasts were also isolated from the leaves of willow shoot cultures using cellulysin and pectolyase, but these did not show cell divisions.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - MS medium Murashige & Skoog (1962) medium - WP medium Woody Plant medium (Lloyd & McCown 1981)     

Distribution of assimilated carbon in plants and rhizosphere soil of basket willow (Salix viminalis L.)

Willow is often used in bio-energy plantations for its potential to function as a renewable energy source, but knowledge about its effect on soil carbon dynamics is limited. Therefore, we investigated the temporal variation in carbon dynamics in willow, focusing on below-ground allocation and sequestration to soil carbon pools. Basket willow plants (Salix viminalis L.) in their second year of growth were grown in pots in a greenhouse. At five times during the plants growth, namely 0, 1, 2, 3 and 4 months after breaking winter dormancy, a subset of the plants were continuously labelled with ¹⁴CO₂ in an ESPAS growth chamber for 28 days. After the labelling, the plants were harvested and separated into leaves, first and second year stems and roots. The soil was analysed for total C and ¹⁴C content as well as soil microbial biomass. Immediately after breaking dormancy, carbon stored in the first year stems was relocated to developing roots and leaves. Almost half the newly assimilated C was used for leaf development the first month of growth, dropping to below 15% in the older plants. Within the second month of growth, secondary growth of the stem became the largest carbon sink in the system, and remained so for the older age classes. Between 31 and 41% of the recovered ¹⁴C was allocated to below-ground pools. While the fraction of assimilated ¹⁴C in roots and root+soil respiration did not vary with plant age, the amount allocated to soil and soil microbial biomass increased in the older plants, indicating an increasing rhizodeposition. The total amount of soil microbial biomass was 30% larger in the oldest age class than in an unplanted control soil. The results demonstrate a close linkage between photosynthesis and below-ground carbon dynamics. Up to 13% of the microbial biomass consisted of carbon assimilated by the willows within the past 4 weeks, up to 11% of the recovered ¹⁴C was found as soil organic matter.     

Periodicity of response to abscisic acid in lateral buds of willow (Salix viminalis L.)

Aseptically cultured lateral buds of Salix viminalis L. collected from field-grown trees exhibited a clear periodicity in their ability to respond to exogenous abscisic acid (ABA). Buds were kept unopened by ABA only when the plants were dormant or entering dormancy. Short days alone did not induce bud dormancy in potted plants but ABA treatment following exposure to an 8-h photoperiod prevented bud opening although ABA treatment of buds from long-day plants did not. Naturally dormant buds taken from shoots of field-grown trees and cultured in the presence of ABA opened following a chilling treatment. In no cases were the induction and breaking of dormancy and response to ABA correlated with endogenous ABA levels in the buds.Abbreviations ABA abscisic acid - GA₃ gibberellic acid - HPLC high-performance liquid chromatography - LD long day - MeABA methyl ABA - PAR photosynthetically active radiation - SD short day     

Inoculation of willows (Salix spp.) with ectomycorrhizal fungi on mined boreal peatland

Inoculation with ectomycorrhizal fungi was explored as a means to improve productivity of experimental short-rotation plantations of the willowsSalix viminalis andSalix dasyclados for biomass production on surface-mined peatlands in northern Finland. Both willow species formed ectomycorrhizas withAmanita spp.,Cortinarius purpurascens, Entoloma nidorosum, otherEntoloma spp.,Hebeloma crustuliniforme, H. pusillum, Laccaria bicolor, andPaxillus involutus in greenhouse experiments.Field trials on a mined peatland site revealed (after one growing season) statistically significant growth stimulation after inoculation due to mycorrhiza formation in both willow species: plants inoculated withEntoloma were sometimes twice as large as control plants. However, such effects were observed only in plots receiving normal phosphate fertilization as opposed to low phosphate application, and were not consistent from season to season. With the inoculum of other species (Cortinarius, Hebeloma andPaxillus) some evidence of growth enhancement was found in the field, but these results were sometimes attributable to non-symbiotic effects of inoculation.     

Enhanced ethylene production by primary roots of Zea mays L. in response to sub-ambient partial pressures of oxygen

Ethylene production by primary roots of 72–h-old intact seedlings of Zea mays L. cv. LG11 was studied under ambient and sub-ambient oxygen partial pressures (pO₂) using a gas flow-through system linked to a photoacoustic laser detector. Despite precautions to minimize physical perturbation to seedlings while setting-up, ethylene production in air was faster during the first 6h than later, in association with a small temporary swelling of the roots. When roots were switched from air (20–8kPa O₂) to 3 or 5kPa O₂ after 6h, ethylene production increased within 2—3 h. When, the roots were returned to air 16 h later, ethylene production decreased within 2—3 h. The presence of 10kPa CO₂ did not interfere with the effect of 3kPa O₂. Transferring roots from air to 12–5kPa did not change ethylene production, while a reduction to 1 kPa O₂ induced a small increase. The extra ethylene formed in 3 and 5 kPa O₂ was associated with plagiotropism, swelling, root hair production, and after 72 h, increased amounts of intercellular space (aerenchyma) in the root cortex. Root extension was also slowed down, but the pattern of response to oxygen shortage did not always match that of ethylene production. On return to air, subsequent growth patterns became normal within a few hours. In the complete absence of oxygen, no ethylene production was detected, even when anaerobic roots were returned to air after 16 h.     

Growth responses of selected clones of birch (Betula pendula roth.,B. pubescens Ehrh.) and willow (Salix caprea L.,S. cinerea L.) to nitrogen in solution culture

Summary Growth of selected clones of birch and willow obtained from nitrogen deficient soils was compared with that of unselected controls in pot experiments using three levels of nitrogen. Unselected controls of both genera continued shoot growth, albeit very slowly, with a very low level of nitrogen (5 ppm), while selected clones of birch grew significantly more than the controls. Selected clones of willow, on the other hand, ceased shoot growth after 10 weeks with this low nitrogen treatment. Nevertheless, they remained healthy, their leaves containing similar concentrations of nitrogen to those of the plants which continued stem growth throughout the experiment. Furthermore, they had very high root: shoot ratios compared with those of control willows and both selected and unselected birch. The two genera may have developed different mechanisms for tolerating low nitrogen, birch producing a small, relatively efficient root system; willow a larger but less efficient one. Both appear equally effective in ensuring survival on low-nitrogen sites in the field since all the selected clones were obtained from such sites and have survived well in field trials on similar sites. Both birch and willow responded toincreased nitrogen availability with increasing shoot growth and a relative decline in root growth. However, whereas in willow the unselected plants responded significantly more than selected clones, a similar but less markeddifference was found in birch. It appears that in both genera, as in herbaceous plants originating from nitrogen deficient sites, selected clones are less able to respond to increasing nitrogen supply than control plants from more fertile habitats. Attempts to correlate the response of the selected clones to nitrogen in this experiment with that to added nitrogen fertilizer in field trials has been unsuccessful. Further work is required to determine the importance of the many interacting factors which influence the response of young trees to nitrogen under the unusual field conditions associated with restored mineral workings.     

Components of resistance of leaf rust (Melamspora laricii epitea Kleb./Melampsora ribesii-viminalis Kleb.) in Salix viminalis L.

Selection on partial resistance components, namely latent period, development rate of uredia and number and size of uredia, has been suggested as a means to achieve durable resistance. Three experiments were carried out in growth chambers to assess the impact of environmental and genetic factors on these components in the Salix-Melampsora host-pathogen system. They confirmed the environmental ability of the Melampsora-Salicaceae relationship and provided no definite answer on the possibility of attaining durable resistance through selection on partial resistance components. On the one hand, there is a large amount of heritable variation among clones for most components; on the other hand, all components were extremely sensitive to environmental conditions and isolates, making the development of standard selection methods difficult.     

Constitutive expression of the SAP1 gene from willow (Salix discolor) causes early flowering in Arabidopsis thaliana

SAP1-1 and SAP1-2 were isolated from the male reproductive buds of willow (Salix discolor, clone S365). SAP1-1 differs from SAP1-2 based on a few nucleotide substitutions, but the sizes of their full-length cDNAs are identical. The deduced amino acid sequences of SAP1-1 and SAP1-2 were 98% similar and contain the same C-terminal amino acid motif “GYGA” like that of PTAP1-2 from Populus trichocarpa. The expression patterns of SAP1 in various parts of the male reproductive buds of S. discolor implicate this gene in the formation of the inflorescence meristems, bracts, and floral meristems. To characterize the functions of SAP1, we assessed Arabidopsis thaliana transformed with 35S∷SAP1-1. A total of 52 transgenic T₁ lines were obtained, and a 3:1 segregation ratio was obtained in the T₂ generation of each line. In the T₃ generation, five homozygous transgenic lines were obtained, which were used for further analysis. Screening of transgenic lines was greatly facilitated by the detection of GFP expression starting with germinating seeds. Phenotypes of the homozygous transgenic lines included early flowering, conversion of inflorescence branches to solitary flowers, formation of terminal flowers, and formation of flowers with greater number of petals, stamens, and pistils. Northern analysis showed similar expression levels in all five lines. This study provides the first functional analysis of an APETALA1 (AP1)/SQUAMOSA (SQUA) homolog from a dioecious species and suggests that SAP1 is a homolog of the AP1/SQUA gene.     

Shoot growth dynamics and photosynthetic response to increased nitrogen availability in the alpine willow Salix glauca

Plants subjected to increases in the supply of resource(s) limiting growth may allocate more of those resources to existing leaves, increasing photosynthetic capacity, and/or to production of more leaves, increasing whole-plant photosynthesis. The responses of three populations of the alpine willow, Salix glauca, growing along an alpine topographic sequence representing a gradient in soil moisture and organic matter, and thus potential N supply, to N amendments, were measured over two growing seasons, to elucidate patterns of leaf versus shoot photosynthetic responses. Leaf-(foliar N, photosynthesis rates, photosynthetic N-use efficiency) and shoot-(leaf area per shoot, number of leaves per shoot, stem weight, N resorption efficiency) level measurements were made to examine the spatial and temporal variation in these potential responses to increased N availability. The predominant response of the willows to N fertilization was at the shoot-level, by production of greater leaf area per shoot. Greater leaf area occurred due to production of larger leaves in both years of the experiment and to production of more leaves during the second year of fertilization treatment. Significant leaflevel photosynthetic response occurred only during the first year of treatment, and only in the dry meadow population. Variation in photosynthesis rates was related more to variation in stomatal conductance than to foliar N concentration. Stomatal conductance in turn was significantly related to N fertilization. Differences among the populations in photosynthesis, foliar N, leaf production, and responses to N fertilization indicate N availability may be lowest in the dry meadow population, and highest in the ridge population. This result is contrary to the hypothesis that a gradient of plant available N corresponds with a snowpack/topographic gradient.     

Formation and function of secondary aerenchyma in hypocotyl, roots and nodules of soybean (Glycine max) under flooded conditions

Flooding is a major problem in many areas of the world and soybean is susceptible to the stress. Understanding the morphological mechanisms of flooding tolerance is important for developing flood-tolerant genotypes. We investigated secondary aerenchyma formation and function in soybean (Glycine max) seedlings grown under flooded conditions. Secondary aerenchyma, a white and spongy tissue, was formed in the hypocotyl, tap root, adventitious roots and root nodules after 3 weeks of flooding. Under irrigated conditions aerenchyma development was either absent or rare and phellem was formed in the hypocotyl, tap root, adventitious roots and root nodules. Secondary meristem partially appeared at the outer parts of the interfascicular cambium and girdled the stele, and then cells differentiated to construct secondary aerenchyma in the flooded hypocotyl. These morphological changes proceeded for 4 days after the initiation of the flooding. After 14 days of treatment, porosity exceeded 30% in flooded hypocotyl with well-developed secondary aerenchyma, while it was below 10% in hypocotyl of irrigated plants that had no aerenchyma. When Vaseline was applied to the hypocotyl of plants from a flooded treatment to prevent the entry of atmospheric oxygen into secondary aerenchyma, plant growth, especially that of roots, was sharply inhibited. Thus secondary aerenchyma might be an adaptive response to flooding.     

The role of active oxygen in iron tolerance of rice (Oryza sauva L.)

Summary Iron tolerance of rice (Oryza sativa L.) was investigated using an oxygen depleted hydroculture system. Treatment with high concentrations of Fe²⁺ induced yellowing and bronzing symptoms as well as iron coatings at the root surface. Root and shoot growth were inhibited by increasing iron concentration in the medium. All symptoms were more pronounced in an iron sensitive cultivar (IR 64) compared to an iron tolerant one (IR 9764-45-2). Superoxide dismutase and peroxidase activity of root extracts of IR 97 were about twice that of IR 64 in untreated control plants. No significant increase of peroxidase activity was detected with increasing iron concentration in the medium. Catalase activity of IR 64 was slightly higher than that of IR 97, independent of iron concentration.Abbreviations SOD Superoxide dismutase (EC 1.15.1.1) - POD peroxidase (EC 1.11.1.7) - EDTA ethylenediamintetraacetic acid - fwt fresh weight - Hepes (N-[2-hydroxyethyl]piperazine-N-[2-ethanesulfonic acid]) - BSA bovine serum albumin - IR 97 IR 9764-45-2 an iron tolerant rice cultivar - IR 64 iron sensitive rice cultivar - PM plasma membrane     

The aerenchymatous phellem of Lythrum salicaria (L.): a pathway for gas transport and its role in flood tolerance

While the importance of cortical aerenchyma in flood tolerance is well established, this pathway for gaseous exchange is often destroyed during secondary growth. For woody species, therefore, an additional pathway must develop for oxygen to reach submerged tissues. In this paper we examine the potential for the aerenchymatous phellem (cork) of Lythrum salicaria L. to provide a pathway for gas transport from shoots to roots and assess its importance in flood tolerance. Plants in which the continuity of the aerenchymatous phellem between shoots and roots was broken showed a significant reduction in oxygen levels in roots, but no difference in carbon dioxide levels compared with controls that retained an intact phellem. These plants also had a greater total shoot height and shoot dry weight, and an increase in shoot/root dry mass ratios compared with controls. Total dry weight was not significantly affected by this treatment. This study is the first to show that the aerenchymatous phellem can provide a pathway for gaseous exchange between roots and shoots and can influence plant morphology and patterns of resource allocation. This suggests that this tissue may play a significant role in the flood tolerance of a woody plant.     

Susceptibility of willow clones (Salix spp.) to herbivory by Phyllodecta vulgatissima (L.) and Galerucella lineola (Fab.) (Coleoptera, Chrysomelidae)

Levels of damage by mixed natural infestations of the leaf-feeding chrysomelid beetles, Phyllodecta vulgatissima (L.) (the blue willow beetle) and Galerucella lineola (Fab.) (the brown willow beetle), were determined in replicated field plots of 24 Salix clones at Long Ashton (Bristol, UK) during 1993–94. Over the same period, the host plant preferences of both chrysomelids were investigated in a standard multiple-choice laboratory procedure, where beetles were enclosed in Petri dishes with leaf discs cut from young pot-grown trees propagated from shoot cuttings taken from 20 of the 24 willow clones represented in the field study. The laboratory experiments indicated that P. vulgatissima and G. lineola had similar host plant preferences in the range of willows examined (r >0.85). In both field and laboratory, the least preferred Salix clones and hybrids were those of 5. eriocephala, followed by S. purpurea, S. burjatica, S. dasyclados and S. triandra. Clones of S. eriocephala and S. purpurea were frequently rejected altogether in laboratory tests. Most preferred were clones of S. viminalis and several hybrids of S. viminalis, S. aurita, S. caprea and S. cinerea. These results substantiate the reports that P. vulgatissima and G. lineola are deterred from feeding on willows which have relatively high concentrations of phenolic (salicylate) glucosides in the leaves. The least preferred willows, particularly S. eriocephala, S. purpurea and S. burjatica, could be of great potential value in plant breeding for resistance to these willow beetle pests.     

Physiological and biochemical adaptations of Cynodon dactylon (L.) Pers. from the Salt Range (Pakistan) to salinity stress

Naturally adapted salt tolerant populations provide a valuable material for exploring the adaptive components of salt tolerance. Under this aspect, two populations of Cynodon dactylon (L.) Pers. were subjected to salt stress in hydroponics. One was collected from a heavily salt-affected soil in the vicinity of a natural salt lake, Uchhali Lake, in the Salt Range of the Punjab province of Pakistan, and the other from a normal non-saline habitat from the Faisalabad region. The NaCl treatments in Hoagland's nutrient solution were: Control (no salt), 50, 100, 150 and 200 mM of NaCl. After 8 weeks of growth in hydroponics produced biomass, ion relations, and photosynthetic capacity were measured in the differently adapted ecotypes. In the ecotype of C. dactylon from the Salt Range, shoot dry weight was only slightly affected by varying levels of salt. However, in contrast, its root weight was markedly increased. On the other hand, the ecotype from Faisalabad (non-saline habitat) showed a marked decrease in shoot and root dry weights under saline regimes. The ecotype from the Salt Range accumulated relatively less amount of Na⁺ in the shoot than did that from Faisalabad, particularly at higher salt levels. Shoot or root K⁺ and Ca²⁺ contents varied inconsistently in both ecotypes under salt stress. All the photosynthetic parameters, leaf water potential and osmotic potential, and chlorophyll content in both ecotypes were adversely affected by salt stress, but all these physiological attributes except turgor potential and soluble sugars were less affected at high salinities in the salt tolerant ecotype from Salt Range. This ecotype accumulated significantly higher organic osmotica (total free amino acids, proline, total soluble proteins, and total soluble sugars) under saline conditions than its intolerant counterpart. Overall, the salt tolerant ecotype of C. dactylon from the Salt Range showed high salt tolerance due to its restricted uptake of Na⁺ accompanied by an increased uptake of K⁺ and Ca²⁺ in the roots as well as shoot due to its higher photosynthetic capacity and accumulation of organic osmotica such as free amino acids and proline under saline conditions.     

Root aeration in rice (Oryza sativa): evaluation of oxygen, carbon dioxide, and ethylene as possible regulators of root acclimatizations

Adventitious roots of rice (Oryza sativa) acclimatize to root-zone O(2) deficiency by increasing porosity, and induction of a barrier to radial O(2) loss (ROL) in basal zones, to enhance longitudinal O(2) diffusion towards the root tip. Changes in root-zone gas composition that might induce these acclimatizations, namely low O(2), elevated ethylene, ethylene-low O(2) interactions, and high CO(2), were evaluated in hydroponic experiments. Neither low O(2) (0 or 0.028 mol m(-3) O(2)), ethylene (0.2 or 2.0 microl l(-1)), or combinations of these treatments, induced the barrier to ROL. This lack of induction of the barrier to ROL was despite a positive response of aerenchyma formation to low O(2) and elevated ethylene. Carbon dioxide at 10 kPa had no effect on root porosity, the barrier to ROL, or on growth. Our findings that ethylene does not induce the barrier to ROL in roots of rice, even though it can enhance aerenchyma formation, shows that these two acclimatizations for improved root aeration are differentially regulated.     

Ventilation and respiration in roots of one-year-old seedlings of grey mangrove Avicennia marina (Forsk.) Vierh.

Avicennia marina (Forsk.) Vierh. was grown from seed for 12 months in artificially tidal tanks providing a range of duration and depth of inundation. Plant growth characteristics were measured at harvest. Root aerenchyma development was estimated by pycnometry, root respiration rates by manometry, and the oxygen supply capacity of the above-ground portions of the plant was determined using oxygen electrode chambers. The mass per plant at harvest was influenced by the extent of inundation during growth with maximal growth at intermediate-length (1.5 to 6.5 h per tide) inundation periods. Those plants that had been submerged the longest (8.5 h per tide) had the least root tissue. The oxygen conductance of the stem base plus any pneumatophores showed a maximum in plants grown under intermediate inundation. Oxygen demand and internal gas space per unit dry weight of root were independent of extent of inundation. During high tide the plants grown at inundation periods of more than about 3–5 hours per tide were likely to become anaerobic. This may constitute a physiological limit for this species at the bottom of the tidal range.     

Effects of N6-benzyladenine on shoots of five willow clones (Salix spp.) cultured in vitro

Growth and differentiation in shoot cultures of five willow clones on media of different BA concentrations were compared. The tendency of axillary shoots to develop on shoot cultures depended on the genotype, the type of shoot and the number of previous subcultures. The optimum concentration for shoot multiplication was either 5×10^-7 M or 10^-6 M. On BA concentrations of 10^-5 M or higher, browning and death of shoots occurred. Depending on the genotype, shoot elongation was best on media containing 0–5×10^-7 M BA. Rooting ability was also genotype dependent. Prolonged culture in vitro improved the rooting ability of the two most reluctant clones. BA concentrations of 5×10^-7 M or higher inhibited rooting almost completely, but this was not a permanent effect. All clones could be rooted on medium containing 10^-6 M NAA. Shoots were transferred to greenhouse conditions and rooted with varying degrees of success depending on shoot size and genotype.     

Factors toxic to beech (Fagus sylvatica L.) seedlings in acid soils

The effects of highly and moderately acid soils on total biomass, biomass partitioning, fine root characteristics and nutritional status of beech seedlings (Fagus sylvatica L.) were studied in a growth chamber experiment. In Haplic Arenosols seedlings grew slowly but equally well without damage symptoms in a highly acid and a moderately acid soil horizon. The moderately acid A_h+B_w-horizon of a Eutric Cambisol was favourable to seedling growth. The fine root development was reduced in the highly acid A+B_w-horizon of a Dystric Cambisol and in the A_h+E-horizon of a Haplic Podzol, the latter of which also caused increased mortality. Seedling growth in the B₂-horizon of the Haplic Podzol was vigorous, in spite of a higher level of extractable Al and lower base saturation as compared with the A_h+E-horizon. These results are interpreted in relation to soil acidity, soil Al and nutritional status of the seedlings. We conclude that neither Al-toxicity nor nutrient deficiency cause the damage symptoms observed in the A_h+E-horizon of a Haplic Podzol and the fine root reduction in the A+B_w-horizon of a Dystric Cambisol. The damage symptoms of the PZh_A treatment seems to be more the result of H-toxicity or H-related factors other than nutrient shortage or Al-toxicity. Other pH-related toxic factors are discussed.     

The influence of soil moisture and flooding on formation of VA-endo- and ectomycorrhizae in Populus and Salix

Native mixtures of extomycorrhizal fungi were found to infect Populus and Salix roots primarily in very moist but well drained soils in both the field and in controlled experiments (0 to –0.2 MPa), whereas native mixtures of VA-endomycorrhizal fungi infected roots over a much wider range of soil moisture (flooded to –3.4 MPa). Although a moisture gradient experiment showed endomycorrhizal formation was greater in moist soil than in very dry or flooded soils, this pattern was reversed in field transects along drainage gradients. Infection by VA-endomycorrhizal fungi in the field was the lowest where infection by ectomycorrhizal fungi was high, which suggests possible antagonism among the fungal symbionts. The narrow moisture range for ectomycorrhizal formation, and antagonism among endo- and ectomycorrhizal fungi, apparently combine to produce the mycorrhizal distributions found in nature.     

Patterns of clonal diversity in three species of sub-arctic willow (Salix lanata, Salix lapponum and Salix herbacea)

Sub-arctic willow scrub is an endangered habitat in Britain, and typically occurs on steep crags inaccessible to grazing animals. These willows can reproduce both sexually and asexually, although the relative importance of each is unknown. Knowledge of reproductive mode is important for the design of grazing management and restoration programmes. Accordingly, clonality was assessed in the largest stand of sub-arctic willow scrub in the UK, focusing on Salix lanata and S. lapponum. Little evidence of clonal growth was detected; most individuals possessed distinct multi-locus genotypes. Thus despite the capacity for vegetative reproduction, and seedlings being rarely observed, sexual reproduction is the predominant means of perpetuation and dispersal at this site. We also examined clonal growth in a common willow species (Salix herbacea) that occupies a different habitat type (exposed mountain tops and ridges). Multiple individuals shared identical genotypes up to 7 m apart, suggesting an important role for clonal growth in local patch formation in this species.