Roots of willow (Salix viminalis L.) show marked tolerance to oxygen shortage in flooded soils and in solution culture

Responses to soil flooding and oxygen shortage were studied in field, glasshouse and controlled environment conditions. Established stools ofSalix viminalis L., were compared at five field sites in close proximity but with contrasting water table levels and flooding intensities during the preceding winter. There was no marked effect of site on shoot extension rate, time to half maximum length or final length attained. When rooted cuttings were waterlogged for 4 weeks in a glasshouse, soil redox potentials quickly decreased to below zero. Shoot extension was slowed after a delay of 20 d, while, in the upper 100 mm of soil, formation and outgrowth of unbranched adventitious roots with enhanced aerenchyma development was promoted after 7 d. At depths of 100–200 mm and 200–300 mm, extension by existing root axes was halted by soil flooding, while adventitious roots from above failed to penetrate these deeper zones. After 4 weeks waterlogging, all arrested root tips recommenced elongation when the soil was drained; their extension rates exceeding those of roots that were well-drained throughout. Growth in fresh mass was also stimulated. The additional aerenchyma found in adventitious roots in the upper 100 mm of soil may have been ethylene regulated since gas space development was inhibited by silver nitrate, an ethylene action inhibitor. The effectiveness of aerenchyma was tested by blocking the entry of atmospheric oxygen into plants with lanolin applied to lenticels of woody shoots of plants grown in solution culture. Root extension was halved, while shoot growth remained unaffected. H Lambers Section editor     

Genetic relationships between growth characters in Salix viminalis grown in Sweden

From an eight by eight factorial crossing with Salix viminalis, 40 of the 64 families obtained were selected for further analysis. Fourteen seedplants from each of these 40 families were planted in two pairs of contrasting environments: sand and clay soil, and low and high nutrient supply. The material in the soil contrast was harvested after 1, 4 and 6 years of growth. The material in the nutrient contrast was harvested each year for 3 years and analysed after the first and the third harvests. The correlation between number of shoots and weight in the clay environment changed from being negative in the first harvests to positive at the last harvest, compared with the sand environment where this correlation was positive in all years. In the nutrient contrast this correlation was positive at the last harvest in the high nutrient environment, but no correlation could be detected in the low nutrient environment. The differences in correlations between environments may be due to a different allocation of nutrients in the plants, depending on whether the plant is under stress or not. The data suggests that the genetic relationship between growth components is the same over age and environments when the plants are grown without stress.     

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.     

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.     

Plant regeneration from indica rice (Oryza sativa L.) protoplasts

Rice (Oryza sativa L.) plants of the indica cultivar IR54 were regenerated from protoplasts. Conditions were developed for isolating and purifying protoplasts from suspension cultures with protoplast yields ranging from 1·10⁶ to 15·10⁶ viable protoplasts/1 g fresh weight. Protoplast viability after purification was generally over 90%. Protoplasts were cultured in a slightly modified Kao medium in a Petri plate by placing them onto a Millipore filter positioned on top of a feeder (nurse) culture containing cells from a suspension culture of the japonica rice, Calrose 76. Plating efficiencies of protoplasts ranged from 0.5 to 3.0%; it was zero in the absence of the nurse culture. Protoplast preparations usually contained no contaminating cells, and when present, the number of cells never exceeded 0.1% of the protoplasts. After three weeks the Millipore filter with callus colonies were transferred off feeder cells and onto a Linsmaier and Skoog-type medium for an additional three weeks. Selected callus colonies that had embryo-like structures were then transferred to regeneration medium containing cytokinins, and regeneration frequencies up to 80% were obtained. Small shoots emerged and were transferred to jars for root development prior to transferring to pots of soil and growing the plants to maturity in growth chambers. Of the cytokinins evaluated, N⁶-benzylaminopurine was the most effective in promoting shoot formation; however, kinetin was also somewhat effective. Regeneration medium could be either an N₆ or Murashige and Skoog basal medium. Of 76 plants grown to maturity, 62 were fertile, and the plant heights averaged about three-fourths the height of seed-grown plants.Two other suspension cultures of IR54, one developed from the protoplast callus of the initial IR54 line, and the other developed from callus produced by mature seeds, have yielded protoplasts capable of regenerating plants when using cells of the Calrose 76 suspension as a nurse culture. In addition, protoplasts obtained from three-week-old primary callus of immature embryos of IR54 were capable of regenerating plants when using the same culture conditions.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - pcy packed cell volume - BAP N⁶-benzylaminopurine - FDA fluorescein diacetate - FW fresh weight - IAA indole-3-acetic acid Media AA Muller and Grafe (1978) - CPW Frearson et al. (1973) - Kao^* Kao (1977) - LS Linsmaier and Skoog (1965) - MS Murashige and Skoog (1962) - N₆ Chu et al. (1975) - PCM Ludwig et al. (1985)     

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     

Callus formation from cell culture protoplasts of corn (Zea mays L.)

Summary Routine procedures for the isolation of large numbers of protoplasts from an established cell culture of Zea mays and for the induction of sustained divisions leading to secondary cell cultures have been developed. The critical factors seem to be associated with neither specific enzymatic conditions for the isolation nor specific culture conditions for the protoplasts but with the quality of the culture used for protoplast isolation.     

Heat shock proteins in willow (Salix viminalis)

The heat shock response in three vegetatively propagated clones of Salix viminalis L. was studied. In the clone 78198, synthesis of a total of 58 proteins was induced or increased by heat shock. Of these proteins, 39 were found in both leaves and callus, 8 only in leaves, and 11 only in callus. The number of heat shock proteins differed between the three clones studied. The molecular weights of the heat shock proteins ranged from 18000 to over 94000. The optimal synthesis of heat shock proteins took place at 37–40°C, but several proteins could be induced at 25–30°C. The synthesis of the majority of the proteins present at a normal growth temperature (20°C) was not completely blocked by the heat shock. More than 12 h was needed for the reappearance of the normal protein synthesis pattern after heat shock.     

Callus proliferation from the protoplasts of embryogenic cells of Quercus serrata

Embryogenic culture was induced from the immature embryos of Quercus serrata using Marashige and Skoog's medium (MS) containing 0.1 M each of 2,4-d and BAP, and subcultured for seven months before isolation of protoplasts by using 1% Cellulase RS in 0.6 M mannitol solution. Efficient colony formation was obtained when protoplasts were cultured in a liquid MS medium containing 0.6 M mannitol, 3% sucrose and combination of 0.1 M or 1 M each of 2,4-d and BAP. Excluding ammonium nitrate from the MS medium resulted in the decrease of the percentage of colony formation. From colonies, both agar culture and liquid culture were sustained in the MS media without mannitol containing no plant growth regulator, or containing 0.1 M of BAP in combination with 0.1 M or 1 M of 2,4-d.Abbreviations BAP 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - MS medium after Murashige & Skoog (1962).     

Electrofusion of protoplasts from celery (Apium graveolens L.) with protoplasts from the filamentous fungus Aspergillus nidulans

A method was developed for electrofusion of higher-plant protoplasts from celery and protoplasts from the filamentous fungus Aspergillus nidulans. Initially, methods for the fusion of protoplasts from ecch species were determined individually and, subsequently, electrical parameters for fusion between the species were determined. Pronase-E treatment and the presence of calcium ions markedly increased celery protoplast stability under the electrical conditions required and increased fusion frequency with A. nidulans protoplasts. A reduction in protoplast viability was observed after electrofusion but the majority of the protoplasts remained viable over a 24-h incubation period. A small decline in protoplast respiration rate occurred during incubation but those celery protoplasts fused with A. nidulans protoplasts showed elevated respiration rates for 3 h after electrofusion.Abbreviations AC alternating current - DC direct current     

Plant regeneration from embryogenic cell suspension-derived protoplasts of rubber

Embryogenic cell suspensions of rubber derived from immature inflorescences and inner integuments of immature fruits released 3.1 ± 0.2 × 10⁷ protoplasts g^-1 f. wt. (mean ± s.e.m, n = 10) and 3.2 ± 0.2 × 10⁷ protoplasts g^-1 f. wt., with mean viabilities of 83 ± 2% and 77 ± 8%, respectively. Sustained mitotic division was observed only when protoplasts were cultured in KPR liquid medium on nitrocellulose membranes overlying the same semi-solid medium containing Lolium multiflorum nurse cells. Protoplast-derived cell colonies were produced within 2 months of culture. Protoplast-derived cell colonies proliferated, upon subculture to MS-based regeneration medium, with 40% of the protoplast-derived calli developing somatic embryos. The latter germinated into plants on the same medium after 3 months of culture. This revised version was published online in June 2006 with corrections to the Cover Date.     

Salix martiana,a regularly hermaphrodite willow

Sex distribution in a neotropical willow,Salix martiana Leybold, has long escaped the attention of botanists. Its catkins are regularly composed of female, hermaphrodite and male flowers. The present paper discusses some aspects of its possible morphological and/or evolutionary significance.     

Cadmium and copper uptake and translocation in five willow (Salix L.) species

The efficacy for phytoremediation of five willow species was tested by experimental copper and cadmium uptake in a greenhouse hydroponic system. Five treatments included two concentrations (5 and 25 microM for each metal) and a control. Metal concentrations in solution as well as solution uptake were monitored. Metal resistance was assessed through effects on the dry weight of roots and shoots. The willow species tested were generally resistant of increased Cu and Cd content. Metal accumulation was found in all plant organs of all species. Growth and transpiration were not decreased by 5 microM of copper and 25 microM of cadmium in the solution for most species. 25 microM copper caused injury and reduced the dry weight for all species after 21 d. Salix nigra was highly resistant of both Cu and Cd and accumulated more metals than other species. Future field study should be conducted to confirm the findings and feasibility of the phytoremediation technology using those species.     

Callus proliferation from leaf protoplasts using phenylurea type cytokinin, 4-PU,inBetula platyphylla VAR.Japonica

Summary Protoplasts were isolated from leaves ofBetula platyphylla var.japonica using a 0.6M mannitol solution containing 1% Cellulase Onozuka R-10 and 1% Driselase. The cell division and colony formation were largely enhanced using Murashige and Skoog (1962) liquid medium at half strength (1/2 MS), containing 0.6M mannitol, 0.09M sucrose, and factorial combinations of 0.1–30 μM N-(2-chloro-4-pyridyl)-N′-phenylurea (4-pu) and 0.1–10 μM 1-naphthaleneacetic acid (NAA) or 0.1–30 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The optimal protoplast density was 5–7 × 10⁴/ml. Continuous callus proliferation from protoplasts was achieved by transferring colonies to fresh 1/2 MS agar medium containing 1 μM NAA and 1 μM 4-pu with no mannitol. It appeared that supplementation of the medium with phenylurea type cytokinin, 4-pu gave the successful callus proliferation from the protoplasts ofB. platyphylla.     

Genetics of breeding characters with possible effects on biomass production in Salix viminalis (L.)

 Genetic variation in number of sylleptic shoots, wood density, dry matter content, flower production, budbreak, growth cessation, insect susceptibility and rust susceptibility and the relationships of these characters with other growth characters were studied. Forty of sixty-four families in an 8×8 factorial crossing with Salix viminalis were used. Fourteen plants from each family were planted in the framework of a nutrient experiment with optimal and suboptimal nutrient availability and in a sandy soil. The analyses showed high additive genetic variances for all characters studied. Characters measured in both nutrient environments did not show any genotype×environment interaction. Both in the nutrient and in the sand environment insect susceptibility was negatively correlated to different growth characters. This was also the case for rust susceptibility in the sand environment. The highest negative correlation was found between number of days to budburst and weight, indicating the importance of early budburst for production. Ideas on important characteristics for an Salix ideotype are discussed. Received: 2 March 1998 / Accepted: 13 August 1998     

Ethylene production in the leaves of basket willow (Salix viminalis L.) during the growing season

The production of ethylene in the leaves ofSalix viminalis L. increased gradually from May till October. The emanation from the leaves, the main constituent of which was ethylene, inhibited the extension growth and the formation of the dry matter of rye seedlings more or less equally during the whole growing season. This indicates that the effect of ethylene on the growth is not, to any great extent, dependent on its concentration.     

Embryogenic callus formation from maize protoplasts

Maize (Zea mays L.) protoplasts have been obtained which divide rapidly and produce a callus that differentiates to form somatic embryos. The somatic embryos can be induced to form roots and small leaf-like structures. The genotype was the hybrid A188xBlack Mexican Sweet. Protoplasts were prepared from an embryogenic suspension culture derived from a Type II callus which had been selected from Type I callus produced by immature zygotic embryos. The basal medium for the suspension culture was N6 (C.C. Chu et al., 1975, Scientia Sinica 18, 659–668). The 2,4-dichlorophenoxyacetic acid concentration of the suspension culture was critical for subsequent protoplast growth and was optimal at 4.0 mg.l. Protoplasts had to be cultured in a low-osmoticum medium (0.3 M mannitol) for subsequent cell divisions to occur. The protoplasts have been transformed transiently with the gene chloramphenicol acetyltransferase (CAT) containing the 35S promoter obtained from cauliflower mosaic virus (CaMV-35S).Abbreviations FDA fluorescein diacetate - ABA abscisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Primary callus as source of totipotent barley (Hordeum vulgare L.) protoplasts

Summary Primary callus of barley (Hordeum vulgare L.) derived from scutella (cv. Dissa) and anthers (cv. Igri) was used for protoplast isolation and plant regeneration. The protoplasts were embedded in agarose and cultured with nurse cells. The plating efficiency varied from 0.1% to 0.7%. Shoots regenerated from the developing callus. Plantlets were transferred to soil and cultivated in the greenhouse three to five months after protoplast isolation. All plants were normal in morphology, and most of them flowered and set seeds.     

Plant regeneration fromBrassicanigra (L.) Koch stem protoplasts

Summary Protoplasts ofBrassica nigra (L.) Koch were isolated from stem peels of bolting racemes and cultured in 1.5 ml of VN1 liquid medium. The protoplasts in the liquid medium were plated on top of half strength MS medium supplemented with 400 mg/liter glutamine, 15 mg/liter glutathione, 50 mg/literl-serine, 0.25 mg/liter 6-benzylaminopurine, 0.5 mg/liter 2,4-dichlorophenoxyacetic acid, 1.5% sucrose, and 5% mannitol, pH, 5.7, solidified with 0.3% agarose. Ten percent of calli obtained from the protoplasts developed into plantlets within 4 wk after transfer onto 2N regeneration medium which contains MS salts plus 200 mg/liter casein hydrolysate, 0.625 mg/liter 6-benzylaminopurine, 0.625 mg/liter kinetin, 0.625 mg/liter 6-(γ,γ-dimethylallylamino)-purine, 0.625 mg/liter zeatin, 0.5 mg/liter 1-naphthaleneacetic acid, 1.5% sucrose, and 0.4% agarose. THis is the first report of plant regeneration fromB. nigra protoplasts.     

Studies on the movement of indole auxins in willow (Salix viminalis L.)

Summary Auxin activity was detected in honeydew obtained from the aphid Tuberolachnus salignus (Gmelin) feeding on willow (Salix viminalis). Active uptake of ¹⁴C-indolyl-3-acetic acid (IAA) into the sieve tubes was demonstrated by irrigating the cambial surface of willow bark with ¹⁴C-IAA solution and assaying aphid stylet exudate. When, however, ¹⁴C-IAA was applied to the peridermal tissues of the bark or to a mature leaf most of the radioactivity (collected in honeydew or stylet exudate) co-chromatographed with indolyl-3-acetyl-aspartic acid (IAAsp). The presence of IAAsp in honeydew was not affected by extraction procedure or by aphid metabolism. Honeydew obtained from willow treated with ¹⁴C-tryptophan contained only ¹⁴C-tryptophan. When ¹⁴C-IAA was applied in agar to the cut end of willow segments the radioactivity was found to move in a basipetally polar manner. The direction of movement of radioactivity in the sieve tubes, however, was found to be influenced by the proximity of the roots. Nevertheless, there was evidence that endogenous auxin in the sieve tubes does move in a predominantly basipetal direction.