Spatial patterns of benthic diatoms,carbohydrates and mud on a tidal flat in the Ems-Dollard estuary

The chlorophyll a content and two operational fractions of carbohydrate (water extractable and EDTA extractable) were measured every three months during one year along transects on a tidal flat in the Ems-Dollard estuary (The Netherlands). Chlorophyll a was used as an indicator of microphytobenthos biomass, which was composed predominantly of epipelic diatoms. Both carbohydrate fractions correlated significantly with chlorophyll a. EDTA extractable carbohydrates were more resistant towards degradation than the water extractable fraction. During most of the year, concentrations of chlorophyll a and carbohydrates were low, but in June, high concentrations of up to 90 g chlorophyll a/g sediment were found in a narrow zone running parallel to the channel. Maximum concentrations of water extractable carbohydrates and EDTA extractable carbohydrates ranged between 800–1200 and 600–800 g/g sediment, respectively. The mud content was high (± 90%) at the margin of the tidal flat. This was not limited to the growth season of the diatoms, but was observed throughout the year. This indicated that the high mud content at the mudflat margin was mainly caused by hydrodynamic factors, and not by biostabilization. In June, exceptionally high diatom densities were found in sediment with a high mud content. There was only minor evidence that biostabilization by epipelic diatoms lead to a further increase in the mud content of the sediment.     

Rhodospirillum sodomense,sp. nov., a Dead Sea rhodospirillum species

A new species of halophilic anoxygenic purple bacteria of the genus Rhodospirillum is described. The new organism, isolated from water/sediment of the Dead Sea, was vibrio-shaped and an obligate halophile. Growth was best at 12% NaCl, with only weak growth occurring at 6% or 21% NaCl. Growth occurred at Mg²⁺ concentrations up to 1 M but optimal growth was obtained at 0.05–0.1 M Mg²⁺. Bromide was well tolerated as an alternative anion to chloride. The new organism is an obligate phototroph, growing photoheterotrophically in media containing yeast extract and acetate or a few other organic compounds. Growth of the Dead Sea Rhodospirillum species under optimal culture conditions was slow (minimum t_d 20 h). Cells contained bacteriochlorophyll a and carotenoids of the spirilloxanthin series and mass cultures were pink in color. Absorption spectra revealed the presence of a B875 (light-harvesting I) but no B800/B850 (light-harvesting II) photopigment complex. The new organism shares a number of properties with the previously described halophilic phototrophic bacterium Rhodospirillum salinarum and was shown to be related to this phototroph by 16S rRNA sequencing. However, because of its salinity requirements, photosynthetic properties, and isolation from the Dead Sea, the new phototroph is proposed as a new species of the genus Rhodospirillum, R. sodomense.     

Tolerance of peanut to excess boron

The tolerance ofArachis hypogaea cv. Shulamit to high concentrations of B in nutrient solution, [B]o, was determined under greenhouse conditions that promoted the production of vegetative dry matter. Plants grew in large containers in which a root zone of nutrient solution was separated from a pod zone of soil. Grain yield was reduced at a calculated [B]o-threshold of 0.29 mM, which was associated with a concentration of B in the vegetative shoots that was approximately four times larger than the control. Symptoms of B toxicity occurred on leaves as young as the third unfolded leaf from the shoot apex before the [B]o-threshold. Excess B caused a relatively larger decrease in pod number than in vegetative shoot weight, which was high in all treatments (78 g d.wt/plant) and it did not decrease single grain weight. It was suggested that the tolerance of grain development to excess B was a consequence of the high ratio of vegetative matter to pod number.     

Mycelial growth characteristics in a split-plate culture of four strains of the genus <Emphasis Type="Italic">Suillus</Emphasis>

A split-plate method with two media in different concentrations in each compartment was applied for the mycelial growth of four strains of Suillus luteus, S. grevillei, S. granulatus, and S. bovinus. As the glucose concentration in the A-side (the side containing higher concentrations of glucose) increased, the mycelial growth in both A- and B-sides (the side containing lower concentrations of glucose) increased. The mycelial density in both sides and B/A ratio (the ratio of the mycelial growth in the B-side to that in the A-side) also increased, and the colony morphology changed. In both A- and B-sides, the colony area reached maximum at 10g/l glucose in the A-side in most cases and at 33.3g/l in several cases. The results indicated nutrients are translocated from mycelia in the A-side to those in the B-side. High concentration of phosphate or fructose + glucose in the A-side induced better mycelial growth in the B-side. Addition of high concentrations of phosphate to one side enhanced mycelial growth in the other side. Low-temperature incubation promoted the growth in the B-side. Our split-plate culture method will be useful for qualitative study of translocation in ectomycorrhizal fungi.     

Improvement of Kefir yeast by mutation with N-methyl-N- nitrosoguanidine

A commercial culture of Kefir grew well at 30°C and 37°C with NaCl up to 4% (w/v) but at 42°C and 4% (w/v) NaCl it grew very slowly. These conditions were selected for the mutation with N-methyl-N-nitrosoguanidine in order to improve cell yield at high temperatures and salt concentration. The mutated cells had higher biomass and growth rate compared with the original culture. The improvement of Kefir at high temperatures and salt concentrations offers advantages for uses in single cell protein production and alcohol production from whey containing high salt.     

Sediment toxicity in the Kattegat and Skagerrak

Sediments were sampled from 62 sites in the Kattegat and Skagerrak, which are located between the Baltic and the North Sea in the Western Atlantic, during autumn 1989 and spring 1990. From each site 5 to 6 samples were taken wit ha box-corer. After mixing to composite samples on board, transport and storage (at 4 °C for 2 to 4 weeks), the samples were tested for toxicity to Daphnia magna and Nitocra spinipes. Immobility in Daphnia after exposure to 16 percent sediment (wet wt) in reconstituted standardized water (ISO, 1982) ranged from 0 to 88 percent after 24 h and from 3 to 95 percent after 48 h. For Nitocra the toxicity, determined as the 96-h LC50 (% wet wt) at 7 salinity, ranged from > > 32 percent (nontoxic) to 1.8 percent (most toxic). All exposures were made in duplicates and the effects obtained in the duplicates with the same sediment were correlated to each other. However, sediment toxicity to Daphnia and Nitocra was not. The test with Nitocra, which was made at several concentrations of sediment, was considered to give the most reliable picture of sediment toxicity in the Kattegat and Skagerrak. This ambient toxicity assessment identified three areas with toxic sediment, (1) the Göta älv estuary (outside the city of Göteborg) and its surroundings, (2) the Bay of Laholm in southern Kattegat, which is an area with periodic oxygen depletion and where repeated mussel kills have occurred during the last decade, and (3) an area in the open Skagerrak northwest of Skagen (the tip of the Jutland peninsula). Sediments, which had been stored at 4 °C, were tested again after 6 to 13 mos with the Nitocra test. Stored sediment toxicity was poorly correlated with fresh sediment toxicity. The average detoxification during storage was 5 times, but the range was 3 orders of magnitude, from 17 times more toxic to 73 times less toxic. The reasons for the observed areal and storage differences in sediment toxicity are so far not understood.     

Degradation of phenol and phenolic compounds by a defined denitrifying bacterial culture

Phenol, a major pollutant in several industrial waste waters is often used as a model compound for studies on biodegradation. This study investigated the anoxic degradation of phenol and other phenolic compounds by a defined mixed culture of Alcaligenes faecalis and Enterobacter species. The culture was capable of degrading high concentrations of phenol (up to 600 mg/l) under anoxic conditions in a simple minimal mineral medium at an initial cell mass of 8 mg/l. However, the lag phase in growth and phenol removal increased with increase in phenol concentration. Dissolved CO₂ was an absolute requirement for phenol degradation. In addition to nitrate, nitrite and oxygen could be used as electron acceptors. The kinetic constants, maximum specific growth rate _max; inhibition constant, K _i and saturation constant, K _s were determined to be 0.206 h^–1, 113 and 15 mg phenol/l respectively. p-Hydroxybenzoic acid was identified as an intermediate during phenol degradation. Apart from phenol, the culture utilized few other monocyclic aromatic compounds as growth substrates. The defined culture has remained stable with consistent phenol-degrading ability for more than 3 years and thus shows promise for its application in anoxic treatment of industrial waste waters containing phenolic compounds.     

Maintaining turbidity and current flow in laboratory aquarium studies, a case study using Sabellaria spinulosa

Many aquatic organisms rely on the suspension of particulate matter for food or for building materials, yet these conditions are difficult to replicate in laboratory mesocosms. Consequently, husbandry and experimental conditions may often be sub-optimal. The Vortex Resuspension Tank (VoRT) is a simple and reliable system for the resuspension of food or sediments using an enclosed airlift. The particle rain from the lift is mixed in the tank by two water inputs that provide directional current flow across the study organism(s). The vortex mixing creates a turbulent lateral water flow that allows the distribution of particulate matter outwards from the sediment outflow. By calibrating a VoRT it is possible to control sedimentation rate by manipulating water and air flow rates. As an example application, three VoRTs were maintained under different sediment loadings to assess the sediment fraction utilisation and tube growth rates of the tube-building polychaete worm Sabellaria spinulosa. S. spinulosa consistently utilised a lower mean particle size than that of the background sediment when provided with well sorted medium sands. Under sediment starved conditions, there was net erosion of colonies whereas under intermediate and high sediment rates there was consistent cumulative growth throughout a 15 d experiment. This highlights the importance of suspended sediment for S. spinulosa and also the suitability of the VoRT system for maintaining organisms with suspended matter requirements.     

Relationship of cellular phosphorus in the cyanobacterium Nodularia to phosphorus availability in the Peel-Harvey Estuarine System

A laboratory-determined critical concentration of phosphorus in the cyanobacterium Nodularia spumigena was used to assess the phosphorus sufficiency of field populations in the Peel-Harvey Estuarine System, Western Australia. Nutrient addition bioassays indicated that phosphorus was the limiting nutrient. Since river flow, and thus phosphorus input, to the estuary ceases 1–2 months before the onset of Nodularia blooms, the phosphorus contained in these blooms was derived from sediment release and nutrient recycling. A progressive increase in the mean cellular phosphorus concentration of Nodularia from 1981 to 1983 indicated that sediments were releasing a greater amount of phophorus each year. Although evidence suggests that the biomass of recent Nodularia blooms is now limited by physical factors, the additional phosphorus contained in each bloom will increase the supply of phosphorus to other organisms. Cellular phosphorus concentrations in Nodularia appeared to be correlated with river phosphorus loadings in the Peel Inlet but not in the Harvey Estuary.     

The importance of sampling immature leaves for the diagnosis of boron deficiency in oilseed rape (Brassica napus cv. Eureka)

Plant analysis can diagnose boron (B) deficiency when the standards used have been properly developed by establishing that a close relationship exists between B concentration in a plant part and its physiological function. The purpose of the present study was to demonstrate the importance of choosing the growing immature leaves for B deficiency diagnosis and for establishing critical B concentrations for the diagnosis of B deficiency in oilseed rape (Brassica napus). In Experiment 1, the plants were subject to seven levels of B supply using programmed nutrient addition, for the estimation of critical B concentrations in plant parts for shoot growth. In Experiment 2, the plants were treated with two levels of B supply in solution: 10 (+B) and 0 (-B) M B, for the estimation of functional B requirements for leaf elongation. The results showed that critical B concentrations varied amongst the plant parts sampled and decreased with leaf age. As B taken up by roots is largely phloem-immobile, B concentrations in mature leaves are physiologically irrelevant to plant B status at the time of sampling, giving rise to a significant over- or underestimation of the B requirement for plant growth. By contrast, a growing, immature leaf, in this case the youngest open leaf (YOL), was the most reliable plant part for B deficiency diagnosis. Critical B concentrations developed from both methods were comparable-i.e. 10–14 mg B kg^–1 dry matter in the YOL at vegetative growth stages up to stem elongation.     

Fine sediment effects on feeding and growth in the invertebrate grazers Potamopyrgus antipodarum (Gastropoda,Hydrobiidae) and Deleatidium sp. (Ephemeroptera,Leptophlebiidae)

The influence of fine sediment (<63 m diameter) upon the assimilation rates of the snail Potamopyrgus antipodarum, and the mayfly Deleatidium sp. were determined by allowing individuals to feed upon ¹⁴C radiolabelled periphyton which had been contaminated with varying quantities of sediment (sediment:food ratios of: 0:1, 1:1, 5:1, 10:1, 50:1, 100:1 [dry weight]). For both grazers, the assimilation rate falls in direct proportion to the sediment fraction in the (sediment+food) matrix. In a second experiment the growth of P. antipodarum was monitored over 3 months when fed organic matter that had been contaminated with fine sediment at ratios of 0:1, 1:1, 5:1, 10:1, 50:1, 100:1, 500:1. In contrast to the monotonic relation between sediment and short-term assimilation, growth rates (mm shell height d^–1) were highest at intermediate levels of sediment contamination (5:1 and 10:1 by dry weight) and lowest in the treatment with no sediment added. Growth rates were significantly lower, and mortality high, at sediment:food contamination ratios above 50:1. The reasons for the contrast between the results from the short-term and the long-term experiments are unknown at present, but the fact that snail growth was greatest at intermediate levels of sediment contamination might indicate that they derive trace nutrients from ingested sediment.     

Two-dimensional gel electrophoresis analysis of the abundance of virulent exoproteins of group A streptococcus caused by environmental changes

Group A streptococci regulate the expression of virulence factors in response to environmental change. In order to investigate this mechanism, the growth of group A streptococci and the abundance of virulent exoprotein production in culture supernatant were analyzed by two-dimensional gel electrophoresis (2-D electrophoresis) under several culture conditions. Judging from alterations in their growth, group A streptococci were affected by various environmental stresses. Under high O₂ and low CO₂ concentrations, streptococcal pyrogenic exotoxin B (SpeB) and streptococcal pyrogenic exotoxin F (SpeF) significantly decreased, and the streptococcal inhibitor of complement (Sic) increased . At 30 °C, increases in endo--N-acetylglucosaminidase (EndoS) and -amylase were also detected, while at 41 °C EndoS became undetectable and SpeB and SpeF decreased. Sic, SpeF and mitogenic factor 3 (Mf3) decreased when cells were cultured in higher NaCl concentrations, and EndoS disappeared following culture of the cells in high glucose concentration. An increase in acid phosphatase and a decrease in several other proteins were detected when the cells were cultivated in high iron concentrations. These results suggest that group A streptococci have a versatile adaptation system that responds to several environmental stresses by altering the level of exoprotein production.Abbreviations SpeB Streptococcal pyrogenic exotoxin B - SpeF Streptococcal pyrogenic exotoxin F - Sic Streptococcal inhibitor of complement - EndoS Endo--N-acetylglucosaminidase     

The influence of NaCl on the degradation rate of dichloromethane byHyphomicrobium sp.

The degradation of dichloromethane by the pure strainHyphomicrobium GJ21 and by an enrichment culture, isolated from a continuously operating biological trickling filter system, as well as the corresponding growth rates of these organisms were investigated in several batch experiments. By fitting the experimental data to generally accepted theoretical expressions for microbial growth, the maximum growth rates were determined. The effect of NaCl was investigated at salt concentrations varying from 0 to 1000 mM. Furthermore the dichloromethane degradation was investigated separately in experiments in which a high initial biomass concentration was applied. The results show that microbial growth is strongly inhibited by increased NaCl concentrations (50% reduction of _max at 200–250 mM NaCl), while a certain degree of adaptation has taken place within an operational system eliminating dichloromethane. A critical NaCl concentration for growth of 600 mM was found for the microbial culture isolated from an operational trickling filter, while a value of 375 mM was found for the pure cultureHyphomicrobium GJ21. The substrate degradation appears to be much less susceptible to inhibition by NaCl. Even at 800 mM NaCl relatively high substrate degradation rates are still observed, although this process is again dependent on the NaCl concentration. Here the substrate elimination is due to the maintenance requirements of the microorganisms. The inhibition of the dichloromethane elimination was also investigated in a laboratory scale trickling filter. The results of these experiments confirmed those obtained in the batch experiments. At NaCl concentrations exceeding 600 mM a considerable elimination of dichloromethane was still observed for during several months of operation. These observations indicate that the inhibition of microbial growth offers a significant control parameter against excessive biomass growth in biological trickling filters for waste gas treatment.     

Effect of CO2 enrichment and nitrogen availability on resource acquisition and resource allocation in a grass,Bromus mollis

Summary The effects of CO₂ enrichment on the growth, biomass partitioning, photosynthetic rates, and leaf nitrogen concentration of a grass, Bromus mollis (C₃), were investigated at a favorable and a low level of nitrogen availability. Despite increases in root: shoot ratios, leaf nitrogen concentrations were decreased under CO₂ enrichment at both nitrogen levels. For the low-nitrogen treatment, this resulted in lower photosynthetic rates measured at 650 l/l for the CO₂-enriched plants, compared to photosynthetic rates measured at 350 l/l for the non-enriched plants. At higher nitrogen availability, photosynthetic rates of plants grown and measured at 650 l/l were greater than photosynthetic rates of the non-enriched plants measured at 350 l/l. Water use efficiency, however, was increased in enriched plants at both nitrogen levels. CO₂ enrichment stimulated vegetative growth at both high and low nitrogen during most of the vegetative growth phase but, at the end of the experiment, total biomass of the high and low CO₂ treatments did not differ for plants grown at low nitrogen availability. While not statistically significant, CO₂ tended to stimulate seed production at high nitrogen and to decrease it at low nitrogen.     

Effects of environmental conditions on cell growth and poly-β-hydroxybutyrate accumulation in Alcaligenes eutrophus

Influences of the control of glucose and oxygen concentrations on cell growth and poly--hydroxybutyrate (PHB) accumulation in Alcaligenes eutrophus were studied. Glucose affects both biosynthesis and glycolysis directly and the other pathways indirectly. PHB accumulation could also be stimulated under oxygen limitation conditions, but the final PHB content within the cells was less than in the case of nitrogen limitation. When the culture was shifted from the PHB accumulation state to balanced growth conditions, PHB degradation occurred in the cells. The cell growth was inhibited by high PHB content within the cells.     

Diffusion of Fe(II) from an iron propagation cage and its effect on tissue iron and pigments of macroalgae on the cage

Iron propagation cages were settled on sand and/or rock beds in coastal areas of Hokkaido. The cage was oxidized by dissolved oxygen and the released Fe(II) diffused into the seawater around the cage. Fe(II) concentrations in the range of 10–50 nM were detected within a 20-m distance around the cage. For comparison, in the Japan Sea, the total iron concentration is less than 2 nM.Laminaria japonica was grown in an indoor semi-continuous culture system. The critical Fe level for maintaining maximum growth, and the subsistence Fe level for survival were measured. The concentrations obtained were 14–21 and 8 g Fe g^–1 tissue, respectively. Iron found inL. japonica growing on rocks and/or rock beds in the Japan Sea was close to the subsistence level. However, the Fe level inL. japonica on the cage in the Japan Sea was considerably higher. The concentrations of chlorophyll-a and fucoxanthin collected from the cage were significantly higher for sporophytes, demonstrating that iron is a very important element for the growth of seaweeds.     

Auxin autonomy in cultured tobacco teratoma tissues transformed by an auxin-mutant strain ofAgrobacterium tumefaciens

We have studied the mechanism of auxin autonomy in tobacco (Nicotiana tabacum L.) crowngall tissues transformed by the auxin-mutant (tms ^–) A66 strain of Agrobacterium tumefaciens. Normally, tms ^– tobacco tumor tissues require the formation of shoots to exhibit auxin-independent growth in culture. We have isolated from tms ^– tobacco cells several stable variants that are fully hormone-independent and grow rapidly as friable, unorganized tissues, thus mimicking the growth and morphology of tms ⁺ tobacco cells that produce high levels of auxin. However, none of the variants contained the high levels of auxin found in tms ⁺ tumor cells. The variants could be divided into two classes with respect to their response to applied auxin. The first class was highly sensitive to applied auxin: low concentrations (1 M) of -naphthaleneacetic acid (NAA) severely inhibited growth and markedly stimulated the accumulation of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC). The second class of variants showed a low sensitivity to applied auxin: growth was promoted by concentrations of NAA up to 10 M, and growth inhibition and high ACC levels were observed only at high NAA concentrations (100 M). Unorganized variants with low auxin sensitivity were also isolated from a variant line with high auxin sensitivity. The isolation of tumor cells that exhibited the growth phenotype of tms ⁺ cells while retaining the low auxin content and low auxin sensitivity of tms ^– cells indicates that full hormone autonomy, characteristic of wild-type crown-gall tumors, can be achieved by a mechanism that is independent of changes in the auxin physiology of the cells.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - IAA indole-3-acetic acid - MACC N-malonyl ACC - NAA naphthaleneacetic acid - tms tumor morphology shooty, the auxin biosynthesis locus of Agrobacterium Ti plasmids The authors thank Dr. Andrew Binns (University of Pennsylvania, Philadelphia, USA) for providing cell lines TA6-5 and TA66C3-78, and Mr. James Dacey for preparation of the composite photograph used in Fig. 1. Support for this work by the National Science Foundation (DMB84-17087) and the U.S. Department of Agriculture (86-CRCR-1-2150) is gratefully acknowledged.     

Relationship of aluminium and calcium to net CO2 exchange among diverse Scots pine provenances under pollution stress in Poland

Light-saturated net photosynthesis (A_sat), dark respiration (RD), and foliar nutrient content of eight European Scots pine (Pinus sylvestris L.) provenances were measured at experimental sites in western Poland. Two-year-old seedlings were planted in 1984 at two sites with similar soils in areas of contrasting air pollution. One site was near a point source of SO₂ and other pollutants, and another 12 km to the southeast in an area free of acute air pollution was treated as a control. The eight provenances were from a large north-tosouth latitudinal range (60 to 43° N). At the heavily polluted site Scots pine trees exhibited lower growth rates and crown dieback and deformation. Soil pH, Ca and Mg were at least 10 times lower, and Al 10 times higher at the polluted than the control site. In 1991, concentrations of Al, P, Ca, S, Mn, Fe, and Zn in oneyear old Scots pine foliage were higher and Mg lower at the polluted than control site. At both sites foliar Mg levels were within the range considered deficient (0.6 mg g^-1), and at the polluted site, Al concentrations were very high (670 to 880 g g^-1). In all provenances, RD of one-year-old needles was higher (by 22% on average) and A_sat was lower (by 37% on average) at the polluted than the control site. The ratio of A_sat: RD was half as great in all provenances at the polluted (4 to 6) than control site (8 to 11). Provenances of southern origin had greater increases in RD and water-use efficiency at the polluted site than other provenances. Within the polluted site alone, or across both sites, A_sat in Scots pine was negatively correlated to the Al: Ca ratio (p<0.001, r=–0.93). Across sites RD increased with needle N and Al (multiple regression, p<0.001). The data suggest that at the polluted site there is excessive soil Al and deficient Mg availability, low needle Mg and high Al concentrations and high Al: Ca ratios, and that these have resulted in reduced photosynthetic capacity and increased respiration.     

Actinidia deliciosa in vitro II. Growth and exogenous carbohydrates utilization by explants

Changes in sugar composition (sucrose, glucose and fructose) of medium, callus, stem and leaves of in vitro proliferating explants of Actinidia deliciosa C.F. Liang, Hayward were analyzed together with explant growth at 0, 15, 30, 45 and 60 days of culturing. Autoclaving hydrolyzes a small part of the initial sucrose of the medium into glucose and fructose. In presence of Actinidia explants the initial sucrose decreased to 32% after 15 days of culturing, to 4% after 30 days and to 0.08% at the end of the culture period (60 days). Sucrose increase in the explants did not parallel with its decrease in the medium. Sucrose presence in the explants was evident only during the last month of culturing. After 15 days of culturing a large increase of glucose and fructose was found in the medium but it did not equal the hydrolyzed sucrose. The level of these two monosaccharides remained stable in the medium until the 30th day, then significantly decreased in the second month of culture; neither were completely exhausted at the end of the culture. In the whole explant the highest amount of glucose and fructose was reached after 30 days of culturing.The balance of the three sugars in the medium-explant system, as % distribution of carbon atoms, showed a utilization throughout the whole culture period.Qualitative analyses performed on medium, callus and leaves at 0, 15, and 30 days of culturing revealed the presence of glucose and fructose only and no significant amounts of other hexoses or pentoses. Starch accumulation in the leaves was also observed throughout the culturing.Paper No. 724     

A revised scheme for mass propagation of Easter Lily

Lilium longiflorum Thunb., commonly known as Easter Lily is widely propagated by vegetative means for its high ornamental value as a pot plant. Following in vitro technique, mass propagation has been achieved through direct production of bulblets from the explant as well as regeneration from callus. The chromosome analysis of the progeny derived from callus even from long term culture, did not reveal any marked variability in chromosome morphology. The stable nature of callus maintained in modified MS medium in long term culture has been confirmed. Along with rapid growth, the regenerating capacity of calli has been maintained for 3 years of culture in the above medium. Following shake culture, large number of bulblets could be obtained from such differentiated calli within 3–4 weeks. The shake culture technique of calli is ideally suited for securing stable regenerants on a mass scale in this species.Abbreviations MS Murashige & Skoog's medium - NAA -napthaleneacetic acid - IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzyladenine