Two sites for adenine-nucleotide regulation of ATP-sensitive potassium channels in mouse pancreatic β-cells and HIT cells

Summary ATP-inhibited potassium channels (K(ATP)) were studied in excised, inside-out patches from cultured adult mouse pancreatic -cells and HIT cells. In the absence of ATP, ADP opened K(ATP) channels at concentrations as low as 10 m and as high as 500 m, with maximal activation between 10 and 100 m ADP in mouse -cell membrane patches. At concentrations greater than 500 m, ADP inhibited K(ATP) channels while 10 mm virtually abolished channel activity. HIT cell channels had a similar biphasic response to ADP except that more than 1 mm ADP was required for inhibition. The channel opening effect of ADP required magnesium while channel inhibition did not. Using creatine/creatine phosphate solutions with creatine phosphokinase to fix ATP and ADP concentrations, we found substantially different K(ATP)-channel activity with solutions having the same ATP/ADP ratio but different absolute total nucleotide levels. To account for ATP-ADP competition, we propose a new model of channel-nucleotide interactions with two kinds of ADP binding sites regulating the channel. One site specifically binds MgADP and increases channel opening. The other, the previously described ATP site, binds either ATP or ADP and decreases channel opening. This model very closely fits the ADP concentration-response curve and, when incorporated into a model of -cell membrane potential, increasing ADP in the 10 and 100 m range is predicted to compete very effectively with millimolar levels of ATP to hyperpolarize -cells.The results suggest that (i) K(ATP)-channel activity is not well predicted by the ATP/ADP ratio, and (ii) ADP is a plausible regulator of K(ATP) channels even if its free cytoplasmic concentration is in the 10–100 m range as suggested by biochemical studies.We would like to thank Mr. Louis Stamps for expert technical assistance and Dr. Wil Fujimoto and Ms. Jeanette Teague for generously providing HIT cells obtained from Dr. Robert Santerre at Eli Lilly. We would also like to thank Dr. Michel Vivaudou for providing the program ALEX. Support was provided by the NIH and the Department of Veterans Affairs.     

Five new species of coccidia (Apicomplexa: Eimeriidae) from Madagascan chameleons (Sauria: Chamaeleonidae)

Coprological examination of 19 Madagascan chameleons of the genera Furcifer and Brookesia revealed the presence of five new coccidian species. Isospora brygooi n. sp. from Furcifer pardalis has spherical to subspherical oöcysts with a slightly pitted wall, 20.7 (17–24.5) × 19.3 (16–23) m and broadly ellipsoidal sporocysts, 12.2 (11.5–13) × 8.1 (8–8.5) m, with Stieda and substieda bodies. Oöcysts of Eimeria glawi n. sp. from Furcifer pardalis are cylindrical to ellipsoidal, 27.7 (26–29.5) × 18.4 (17–19) m, with ellipsoidal sporocysts, 7.3 (6.5–8) × 5.2 (5–5.5) m. E. vencesi n. sp. described from F. pardalis has spherical to subspherical oöcysts, 14.3 (13–15.5) × 13.0 (12–13) m, with small granules, one to three globular polar granules and ellipsoidal sporocysts, 7.3 (6.5–8) × 5.2 (5–5.5) m. E. worthi n. sp., described from Furcifer oustaleti has spherical oöcysts, 17.9 (17.5–19.0) × 15.0 (14.5–16.0) m without a polar granule and ellipsoidal to cylindroidal sporocysts, 8.2 (7.0–9.5) × 5.8 (5.0–6.5) m. Oöcysts of E. brookesiae n. sp. from Brookesia decaryi are cylindrical, 25.6 (23–27) × 15.0 (13–16) m with ellipsoidal sporocysts, 10.1 (9–11) × 6.9 (6–7) m. Endogenous development of E. vencesi is confined to the intestine, while that of E. glawi occurs in the gall-bladder.     

Grazing by a dominant rotifer Conochilus unicornis Rousselet in a mountain lake: in situ measurements with synthetic microspheres

Grazing rates of zooplankton were analysed in the summer of 1999 in Yellow Belly Lake, an oligotrophic system in the Sawtooth Mountains of Idaho (U.S.A.). The colonial rotifer Conochilus unicornis was a dominant species in the epilimnion, with densities reaching 20 colonies l^–1 (ca. 400 ind. l^–1). Clearance rates were measured with an in situ Haney Grazing chamber and synthetic microspheres 5, 9 and 23m in diameter. At epilimnetic temperatures of around 14 °C, mean clearance rates for 5m particles ranged from 30 to 65 l ind.^–1 h ^–1. Clearance rates were 2–9 times higher on the 5m spheres than on the 9 m spheres, and C. unicornis almost never fed on the 23 m spheres. Grazing rates did not change over the diel cycle. Clearance rates declined more than 10-fold as temperatures declined from 14 °C in the epilimnion to 7 °C in the metalimnion. In the epilimnion, grazing by C. unicornis was more important than grazing by crustaceans in the community, at least on particles 9m. The results show the importance of grazing by rotifers in lakes, and the significance of spatial variations that influence grazing rates.     

Effect of unionized ammonia,viscosity and protozoan contamination on reproduction and enzyme activity of the rotifer Brachionus rotundiformis

We determined the effect of environmental stressors on the physiological condition of Brachionus rotundiformis. For two morphologically distinct B. rotundiformis strains: Hawaii (average lorica length = 222 m) and Langkawi strains (average lorica length 180 m), neonates hatched from resting eggs were exposed to different levels of unionized ammonia (0.7–9.8 mg l^–1), viscosity (relative viscosity against natural seawater = 1–1.17) and Euplotes sp. (protozoan) contamination (1–40 cells ml^–1). Increasing stress decreased fecundity and lifespan of both rotifer strains. Glucosidase and phospholipase activities were correlated with reproductive responses of both the strains exposed to unionized ammonia. When culture water viscosity was changed, the activity of esterase and phospholipase was correlated with reproductive responses of the Hawaiian strain, and glucosidase activity was correlated with those of Langkawi strain.With the protozoan contamination, esterase and glucosidase activities were correlated only with reproductive responses of the Hawaiian strain, while activity of all three enzymes was correlated to those of the Langkawi strain. Glucosidase activity proved to be a reliable indicator of stress for cultured B. rotundiformis.     

Five new species of Eimeria (Apicomplexa: Eimeriidae) from lizards

Five new species of Eimeria are described from lizards. Eimeria baltrocki n. sp. was found in the berber skink, Eumeces schneideri, from Egypt. The oöcysts are cylindroidal, averaging 38 × 18.3 m, with a single thick oöcyst wall. Most oöcysts possess a single polar granule; a micropyle and oöcyst residuum are absent. The sporocysts are ellipsoidal and average 11.5 × 8.1 m, each with a large, globular sporocyst residuum; the Stieda body is absent. Eimeria anolidis n. sp. is described form the common anole, Anolis carolinensis, from Florida, USA. The oöcysts are cylindroidal and average 31 × 15.8 m with a thick, single-layered oöcyst wall. Two polar granules are usually present; a micropyle and oöcyst residuum absent. The sporocysts are ellipsoidal and average 9.4 × 7.5 m with a globular sporocyst residuum; the Stieda body is absent. Eimeria guyanensis n. sp is recorded in the ameiva, Ameiva ameiva, from Guyana, South America. The oöcysts are spherical to subspherical, average 19.0 × 17.8 m and possess a thick, single-layered oöcyst wall. Numerous polar granules are present (n > 5); a micropyle and oöcyst residuum are absent. The sporocysts are spherical to subspherical, average 7.5 × 7.8 m and possess a compact globular sporocyst residuum; the Stieda body is absent. Eimeria phelsumae n. sp. was recovered from the giant day gecko, Phelsuma madagascariensis grandis, from Madagascar, which harboured a simultaneous infection of E. brygooi. The oöcysts measured 32 × 15 m and are cylindroidal without polar granules, a micropyle or oöcyst residuum, or a Steida body. The sporocysts are ellipsoidal and average 9.8 × 7 m, with a loosely clumped, granular sporocyst residuum; the Steida body is absent. Eimeria leiocephali n. sp. was discovered in the faeces of the ornate ground iguana, Leiocephalus barahonensis, from Haiti. The oöcysts are spherical to subspherical, 21 × 19 m, and contain a number of polar granules (n > 5); a micropyle and oöcyst residuum are absent. The sporocysts are spherical, 8 m in diameter and lack a sporocyst residuum. Eimeria turcicus and E. lineri were found in faeces of Hemidactylus turcicus turcicus from the host's country of origin, Turkey.     

Isospora lutrae n. sp. (Apicomplexa: Eimeriidae), a new coccidium from the European otter Lutra lutra (L.) (Carnivora: Mustelidae) from Spain

Parasitological examination of European otter originating from Extremadura, Spain revealed the presence of a new isosporan species. Oöcysts of Isospora lutrae n. sp. are spherical to subspherical, 31.2 (27.5–32) × 29.6 (28–31) m and have a smooth wall c. 1 m thick. Sporocysts are ellipsoidal, 18.2 (17–19) × 14.4 (14–16) m and lack Stieda and substieda bodies. A spherical sporocyst residuum is present, consisting of granules scattered among the sporozoites. Sporozoites are spindle-shaped, 12.4 × 2.5 m and have anterior and posterior refractile bodies. Based on its unique morphologic structure and host, I. lutrae is considered to be new.     

Sarcocystis inghami n. sp. (Sporozoa: Sarcocystidae) from the skeletal muscles of the Virginia opossum Didelphis virginiana in Michigan

This report describes the newly identified Sarcocystis inghami n. sp. from the skeletal muscles of opossums (Mammalia: Didelphidae) that were collected from south central Michigan (42° 43-42° 79N, 84° 18-84°mathtype="display">6'W), USA. The new species is distinguished from all species described from North and South American opossums by the distinctive morphology of the villar protrusions on the cyst wall. Sarcocysts of S. inghami are microscopic, up to 700 m long and 110 m wide. The sarcocyst wall is up to 7 m thick, with long, stalked protrusions which average 5.5 × 1.2 m. These are constricted at the base, expanded laterally, rounded off distally and occasionally bifid. The villar protrusions have numerous microtubules without electron–dense bodies that extend from the tips into the granular layer. Bradyzoites are 10.7 × 4.3 (8––12 × 4––5) m. This is the second species of Sarcocystis sarcocyst described from the Virginia opossum in North America.     

Wardium canarisi n. sp. (Cestoda: Hymenolepididae) parasite de Arenaria melanocephala (Aves: Charadrii) d'Alaska

The authors describe and illustrate Wardium canarisi n. sp. (Cestoda: Hymenolepididae), an intestinal parasite of Arenaria melanocephala (Aves: Charadrii) from Alaska, characterised by a strobila 20–40 mm long, 10 aploparaksoid hooks 19–21 m long, a short cylindrical cirrus (40 m) covered with very minute spines (0.2 m), and a short (8–18 m) and extremely narrow (1 m) copulatory vagina. These characters have no equivalent, even approximate, among the 27 species of Wardium parasitic in the Charadrii which are reviewed. The genus Debrosia Spassky, 1987 appears to be justified.     

Fecundity of the Grass Shrimp,Pandalus kessleri (Decapoda: Pandalidae), near the Southern Kuril Islands

We determined the absolute (AIF) and relative (RIF) individual fecundity in a commercially important object, the grass shrimp, Pandalus kessleri (Decapoda: Pandalidae) near the Southern Kuril Islands. In the shrimps from Izmeny Bay (Kunashir Island) and the coastal area of the Southern Kuril Islands, the AIF equaled 192–918 (averaging 505) and 162–784 (459), respectively. In larger females, the AIF increases, reaches the maximum value, and then decreases again in the largest females. In June the AIF is smaller than in September, and in larger prawns, this difference is more pronounced. The mean large (D) and small (d) diameters of eggs equaled 1972 (1665–2400) and 1538 (1347–1900) m, respectively; the D/d ratio equaled 1.28. The mean wet weight of an egg (w) was 3.43 mg (1.986–4.9) mg. From September 1 to November 30 D and d increased from 1894 to 2088 m and from 1490 to 1567 m, respectively. The factors affecting the fecundity of grass shrimp in Izmeny Bay are discussed.     

Sobolevicanthus transvaalensis n.sp. (Cestoda: Hymenolepididae) from the Cape Teal (Anas capensis Gmelin, 1789)

Summary Sobolevicanthus transvaalensis n.sp. is described from the Cape Teal, Anas capensis Gmelin, 1789, collected in the Republic of South Africa. The new species possesses 8 skrjabinoid hooks 78–88 m long (mean 85 m) and a short claviform cirrus-sac 79–143 m long and resembles S. javanensis (Davis, 1945) and S. terraereginae (Johnston, 1913). It can be distinguished from S. javanensis by its shorter cirrus-sac and smaller cirrus diameter, and by differences in the morphology of the accessory sac and vagina and in their position relative to the cirrus-sac. It can be separated from S. terraereginae on the basis of cirrus length and diameter. The basal diameter of the cirrus in S. terraereginae is three times that in S. transvaalensis. ac]19830414     

In vitro organogenesis of elephant apple (Feronia limonia)

Elephant apple (Feronia limonia L.). was micropropagated on MS medium containing 4.4 M benzyladenine and 4.6 M kinetin using cotyledon explants taken from in vitro-grown seedlings. Adventitious buds formed on the cotyledon developed into shoots that were rooted in half-strength MS medium containing 0.57 M indoleacetic acid and 0.49 M indolebutyric acid. Plants were successfully established in soil.Abbreviations BA 6-benzyladenine - IAA 3-indoleacetic acid - IBA 3-indolebutyric acid - MS Murashige & Skoog     

Micropropagation of flowering dogwood (Cornus florida) from seedlings

A method for regenerating whole plants from nodal (axillary bud) cultures of seedlings was developed for flowering dogwood (Cornus florida L.). The seed source significantly influenced the rate of proliferation, although cultures initiated from each of the seven mother trees produced some shoots. Woody plant medium (WPM) was superior to either Murashige and Skoog or Schenk and Hildebrandt basal medium. 6-Benzyladenine (BA) at 2.2 or 4.4 m stimulated the generation of significantly more useable shoots (1 cm) compared to all other concentrations (0.5–22.5 m tested. Thidiazuron (TDZ) at 0.6 and 1.1 m supported proliferation, but strongly inhibited shoot elongation. TDZ initiated cultures transferred to medium containing 4.4 m BA produced usable shoots after five additional subcultures. Shoots generated adventitious roots when exposed to either a 12-h pulse of relatively high concentrations (246–1230 m or continuous lower concentrations (0.5–49.0 m of indolebutyric acid (IBA) for longer periods. Microshoots produced the significantly greatest number of roots when subjected to 4.9 m IBA in WPM over a 4-week period. Whole plants were acclimatized to the laboratory conditions and subsequently to the greenhouse. The methodology described here should be useful in a breeding program by supplying multiple copies of unique, recombinant genotypes.     

The permeability to water of the eggs of certain marine teleosts

Summary Measurements have been made of the rate of turnover of tritiated water in the eggs of the plaice,Pleuronectes platessa and of the permeability of the vitelline membrane. The rate constant of water exchange in body cavity eggs is 1.0 h^–1, corresponding to a permeability of 0.086 ³/²/sec. When shed into sea water the rate constant of exchange declines to 0.2 h^–1 after 1 hour and to 0.02 h^–1 after one day corresponding to a permeability of 0.0017 [³/²/sec. As the embryo develops the rate of turnover increases again. The buoyancy of the egg is dependent on its low salt content which counteracts the weight of the protein. During development the water loss by osmosis reduces the egg almost to neutral buoyancy.     

Biological differences in reproductive strategy between the mosquito sibling species Anopheles gambiae sensu stricto and An. quadriannulatus

Females of the afrotropical mosquito species Anopheles gambiae Giles sensu stricto and An. quadriannulatus (Theobald) (Diptera: Culicidae) were studied for the effect of blood meal size and the frequency of blood feeding on reproductive development during the first gonotrophic cycle. To standardize the blood meals, meals were administered by enema in some experiments. The effects of insemination, mosquito size, and metabolic reserves at emergence on egg development were also investigated. Maximum insemination was reached after seven days, varying from 62% in An. quadriannulatus to 95% in An. gambiae and was significantly different (P<0.05) between the two species. Insemination had no effect on feeding success. Females of An. quadriannulatus were significantly larger than An. gambiae females (mean wing size 2.90 ±0.01 mm versus 2.82 ±0.01 mm), but the protein, glycogen, and lipid content of newly emerged females of the two species were not significantly different. Without a blood meal, larger females of both species were significantly more likely to develop oocytes up to Christopher's stage II. With one blood meal, 27% of An. gambiae became pre-gravid and 73% matured eggs. In contrast, all An. quadriannulatus females remained in the pre-gravid stage following ingestion of one blood meal. Vitellogenesis was significantly reduced in smaller-sized pre-gravid An. quadriannulatus compared to larger individuals. When given the opportunity to feed up to three times on three successive days, all females of An. gambiae matured eggs but only 85% of An. quadriannulatus did so. When 1 l of human blood was administered by enema, none of the females of either species developed eggs. With a single enema of 1.5 l of human blood, only An. gambiae developed eggs. A similar result was observed with 1 l and 1.5 l enemas of bovine blood although some An. gambiae also developed eggs with 1 l of blood. Anopheles quadriannulatus developed eggs only when given two 1 l enemas on successive days. However, the percentage of females developing eggs was significantly lower than that of An. gambiae. The implications of these differences in reproductive strategy are discussed in the light of behavioural traits in the field.     

Development of an L6 myoblast in vitro model of moniliformin toxicosis

L6 myoblasts were used as an in vitro model to investigate the role of moniliformin and its interaction with monensin in turkey knockdown syndrome and sudden death syndromes in poultry. Cell viability and microscopic and ultrastructural alterations noted in L6 myoblasts cultured in the presence of moniliformin (0.0–0.3 g/l) were compared to those observed in parallel cultures also containing one of the following compounds: selenium (0–0.004 ng/l), thiamine (0–0.3 g/l), or pyruvate (0–0.46 g/l). Marked dilation of the RER, membranous whorls, glycogen deposition, membrane-bound cytoplasmic inclusions and necrosis were observed in myoblasts exposed to 0.03/2-0.30 g moniliformin/l medium. Supplementation of medium with thiamine and pyruvate, or selenium, provided significant protection to cells exposed to 0.0–0.3 g/l or 0.0–0.15 g moniliformin/l, respectively. Dose-dependent differences in protein and ATP production were not detected. Myoblasts grown in medium containing 0–0.15 g moniliformin/l and 7.5–50.0 M A23187, beauvericin or monensin had degrees of cytotoxicity similar to parallel cultures receiving only an ionophore. L6 myoblasts were a useful model of moniliformin toxicosis. The findings of this study suggest cytotoxicity due to moniliformin in L6 myoblasts may be due in part to oxidative damage and altered pyruvate metabolism, and that moniliformin does not predispose myoblasts to ionophore toxicosis. This study supports the results of in vivo investigations in poultry that moniliformin and monensin do not act synergistically to induce knockdown or monensin toxicosis.     

Uptake of a monoclonal antibody against CEA (Tumak 431/31) in a human colon tumor (Co-112) xenografted in the nude mouse

Summary A monoclonal antibody (Tumak) against carcinoembryonic antigen (CEA) was injected into nude mice bearing a human colon carcinoma (Co-112). The tumor uptake was found to be dependent on the size of the tumors: relative uptake (percentage of the injected dose/gram tumor (% i. d./g) decreased for tumors with weights up to 1 g, although the absolute uptake (% i. d./tumor) still increased over the same weight range. In the constant region (1 g) mean relative tumor uptake was 4% i. d./g.The same tumor size dependence was found for the relative Tumak uptake in the other mouse organs studied (e.g., blood, liver, spleen and muscle). Consequently tumor/organ ratios were found to be independent of tumor size.Tumor uptake was also studied for various doses of Tumak (0.07–120 g) in tumors of 1 g. Evidence was found for a threshold dose of 0.1 g under which no serious tumor uptake appeared. From 1 to 120 g no further dependence of Tumak distribution on applied dose was found: the relative uptake of all organs remained the same but the absolute uptake increased with dose.     

A new species of Crepidostomum (Allocreadiidae: Digenea) from northeastern Finland,with comments on its possible origin

Crepidostomum wikgreni n. sp. is described from the gall-bladder and intestine of the whitefish Coregonus acronius in Lake Yli-Kitka in NE Finland. It is morphologically similar to Crepidostomum farionis, with which it occurs sympatrically and sometimes concurrently; but it differs in that the eggs are much larger, i.e. 96±6.5 m mean-length, as opposed to 71±4.7 m mean-length for C. farionis in the same host and locality.It is suggested that the new species has arisen from C. farionis after deglaciation and since c. 8,400 BP, at which time the waters of the Lake Kitka System were isolated from those in the rest of Finland and flowed eastwards into the White Sea Basin. The isolation of the White Sea Basin appears to have been maintained by watersheds running north-south close to the Fenno-Soviet border and east-west through central Karelia. It is further suggested that C. farionis currently occurring in Lake Yli-Kitka is a recent re-introduction brought about by the translocation of fish-stocks.     

Serum release of hepatic calcium-binding protein regucalcin by liver injury with galactosamine administration in rats

The incorporation and 9 desaturation of exogenous [¹⁴C]stearic acid were studied in HTC 7288c cells in suspension. We examined the uptake of the acid over a wide range of concentrations (0–160 M) after incubating the cells for 6 h in a chemically-defined medium. Under this experimental condition, the uptake of the labeled acid was more extensive than that obtained from static cultures or from monolayer of isolated hepatocytes of rats. At an external concentration of 160 Mca. 52 nmoles of acid per mg of cellular protein was taken up. The production of oleic acid from [¹⁴C]stearate (9 desaturation) correlated well with the uptake curve between 0–80 M concentration. For higher stearate concentrations, the biosynthesis of oleic acid declined substantially and a plateau of 22 nmoles/mg cellular protein was reached. The incorporation and desaturation of an initial exogeneous concentration of [¹⁴C]stearic acid (80 M) was also studied from 0–6 h. The results obtained demonstrated that the uptake of the substrate into cellular lipids was fast and non saturable. Quantitative gas-liquid chromatography of total cellular lipids under the different experimental conditions demonstrated a negative correlation between the decrease in the palmitic and palmitoleic acids and the increase in the intracellular levels of stearic and oleic acids. These analytical modifications took place with no changes in the saturated/monoenoic fatty acid ratio. This work also demonstrated a significant contribution of the stearoyl-CoA desaturase system to the high levels of oleic acid present in this kind of hepatoma cells.Abbreviations FAME Fatty Acid Methyl esters - GLC gas-liquid chromatography - HEPES N-2-hydroxyethyl-piperazine-N-2-ethanosulfonic acid - HTC Hepatoma Tissue Culture - IMEM-Zo Improved Minimal Essential Medium-zinc optional     

Daily egg production ofTemora longicornis (Copepoda,Calanoida) during winter and early spring in the Marsdiep (Southern North Sea)

Mature females of the copepodTemora longicornis from the Marsdiep tidal inlet were incubated at ambient water temperature during the winter of 1989/1990 to estimate their egg production during 24 hours. Throughout winter this production remained at a level of 0 to 10 eggs per female. In March a steep rise to 60 to 80 eggs coincided with an equally steep rise in chlorophyll-a from 4 to 20 g.l^–1. Excess food given during incubation tended to increase the observed production rate only in October at relatively high temperature. Additive effects of rising food level and temperature on daily egg production were found in spring. In March and April 1988 and 1990 the observed maximum daily egg production per female increased from about 20 at 5°C and 5 g.l^–1 chlorophyll to 70 at almost 10°C and 20 g.l^–1. The egg production in winter gives a prebloom potential to maintain a stock of premature stages ready to utilize the algal spring bloom. Early spawning before April can also contribute to the development of the abundance peak of adults in May–June. Due to growth rates increasing with temperature in the months March and April, most of the adults at their peak density may have hatched from eggs before the period of maximum egg production rate at the end of April. The exceptionally high production rate observed at the end of March may be related to a developingPhaeocystis bloom.     

Micropropagation of the nickel hyperaccumulator,Hybanthus floribundus (Family Violaceae)

A protocol for micropropagation of the nickel hyperaccumulator Hybanthus floribundus (Lindley) F. Muell. (Shrub Violet) is described in this paper. Healthy callus was first produced from stem and leaf explants on a medium containing half strength Murashige and Skoog medium with 5 M N ⁶-benzylaminopurine (BA) and 0.5 M -naphthaleneacetic acid (NAA). Numerous shoots (>20 shoots per callus) were also successfully grown from callus on this medium. The exposure time of shoots to auxin was critical for successful in vitro rooting. Best rooting efficiency was obtained by transferring shoots to auxin medium (100 M indole-3-butyric acid) for 24 h and then to a medium without growth regulators (about 75% of treated shoots produced healthy roots). Importantly, cloned shoots retained their ability to hyperaccumulate nickel.