Effect of decapitation on absorption,translocation, and phytotoxicity of imazamethabenz in wild oat (Avena fatua L.)

The release of apical dominance by the physical destruction in situ of the apical meristem and associated leaf primordia (decapitation) promoted the growth of tillers in non-herbicide-treated wild oat plants, as indicated by increased tiller lengths and fresh weights. At 96 h after [¹⁴C] herbicide treatment following decapitation, the absorption of [¹⁴C]imazamethabenz and total translocation of radioactivity were respectively increased by 28% and 49%. By 96 h after [¹⁴C]imazamethabenz application, the radioactivity detected in the roots of decapitated plants was 45% higher than that in the roots of nondecapitated plants while the radioactivity in tillers of decapitated plants was 2.6-fold that in tillers of intact plants. Decapitation together with foliar spraying of imazamethabenz at 200 g ha^–1 further reduced tiller fresh weight, greatly decreased the total tiller number, and thereafter significantly increased overall phytotoxicity by 32% as measured by total shoot fresh weight. The results of this study support the hypothesis that main shoot apical dominance limits translocation of applied imazamethabenz to lateral shoots, rendering tillers less susceptible to growth inhibition by the herbicide.     

Gibberellin-photoaffinity labeling of wild oat (Avena fatua L.) aleurone protoplasts

Aleurone protoplasts of wild oat (Avena fatua L.), and subcellular fractions isolated from them, were photoaffinity labeled using the synthetic gibberellin (GA) derivative GA₄-17-yl-1-(1-thia)propan-3-ol-4-azido-5-[¹²⁵I]iodosalicylate. Labeled polypeptides were identified by electrophoresis under denaturing conditions followed by autoradiography. GA-photoaffinity labeling of both intact protoplasts and isolated subcellular fractions led to the covalent attachment of the reagent to many polypeptides. A 50 kD polypeptide in the soluble fraction of homogenates of aleurone protoplasts GA-photoaffinity labeled in vivo showed specific binding. The biologically active GA₁, GA₄ and GA₄-17-yl-1(1-thia)propan-3-ol-4-azidosalicylate completed for binding whereas the biologically inactive GA₈ and GA₃₄ did not. The GA-photoaffinity labeling characteristics of this polypeptide suggested that it might interact specifically with biologically active GAs in vivo. Attempts to detect specific GA-binding in in vitro GA-photoaffinity labeling experiments met with only limited success perhaps indicating the labile nature of specific binding observed in vivo. The potential of GA-photoaffinity labeling for identifying GA-binding proteins in aleurone and other GA-responsive tissues is discussed.Abbreviations azido IAA = 5-azido-7-[³H]indole-3-acetic acid - azido NPA = 5-azido-[3,6-³H]1-N-napthylpthalamic acid - BTP = 1,3-bis(Tris(hydroxymethyl)methylamino)-propane - GA₄-O-ASA = GA₄-17-yl-1-(1-thia)propane-3-ol-4-azidosalicylate - [¹²⁵I]GA₄-O-ASA = GA₄-17-yl-1-(1-thia)propan-3-ol-4-azido-5-[¹²⁵I]iodosalicylate - NPA = 1-Naphthylphthalmic acid - PAGE = Polyacrylamide gel electrophoresis - PMSF = phenylmethylsulfonyl fluoride - SDS = Sodium dodecyl sulphate - TLCK = L-1-Chloro-3-(4-tosylamido)-7-amino-2-heptanone-HCl     

Water uptake and distribution in non-dormant and dormant wild oat (Avena fatua L.) caryopses

The influence of seed coat modification and light quality onwater uptake and distribution in caryopses of dormant and non-dormantlines of wild oat (Avena fatua L.) was determined using NMRmicroimaging. Non-dormant seeds absorbed water more rapidlythan dormant seeds during imbibition on distilled water. Thiseffect was detected first in the embryo-scutellar region (8h) and later in the proximal endosperm (12 h). Cutting the testaand pericarp close to the embryo or scarification with KOH promotedrapid embryo/scutellum hydration and germination. Cutting atthe middle part of the caryopsis did not enhance embryo hydrationnor did it greatly improve germination. The sensitivity of waterdistribution to the phytochrome germination effect was examined.Significant differences in imbibitional water uptake by embryos-scutellumtissue were detected by 18 h following red-light (germinationpromoter) compared with far-red (germination inhibitor) treatment.The results indicated that both the rate and the sequence ofembryo/scutellum hydration were important in initiating germinationin dormant seeds. A refinement of the model that describes waterimbibition in wild oat seeds during the early stages of germinationis discussed. Key words: Water uptake, water distribution, Avena fatua, seed coat modification, light quality, dormant and non-dormant seeds     

A genetic model and molecular markers for wild oat (Avena fatua L.) seed dormancy

Seed dormancy allows weed seeds to persist in agricultural soils. Wild oat (Avena fatua L.) is a major weed of cereal grains and expresses a range of seed dormancy phenotypes. Genetic analysis of wild oat dormancy has been complicated by the difficulty of phenotypic classification in segregating populations. Therefore, little is known about the nature of the genes that regulate dormancy in wild oat. The objectives of our studies were to develop methods to classify the germination responses of segregating wild oat populations and to find molecular markers linked to quantitative trait loci (QTL) that regulate seed dormancy in wild oat. RAPD markers OPX-06 and OPT-04 explained 12.6% and 6.8% respectively, of the F₂ phenotypic variance. OPF-17 was not significant in a simple regression model, but it was linked in repulsion to OPT-04. A three-locus model of seed dormancy in wild oat is presented based on the 41-day germination profiles of F₁, F₂, F₃, BC₁P₁F₁, BC₁P₁F₂, and BC₁P₂F₁ generations, and the 113 day germination profile of 126 F₇ recombinant inbred lines. Loci G ₁ and G ₂ promote early germination, and the D locus promotes late germination. If at least one copy of the dominant G ₁ or G ₂ alleles are present regardless of the genotype at D locus, then the individual will be nondormant. If the genotype is g ₁ g ₁ g ₂ g ₂ D_, then the phenotype will be dormant. Received: 1 December 1998 / Accepted: 1 February 1999     

Growth, anatomy and morphology of the mesocotyl and the growth of appendages of the wild oat (Avena fatua L.) seedling

Morphological and histological studies were made on the mesocotyl and the emergence of seedlings of a nondormant strain (CS40) of wild oats (Avena fatua L.). The elongation of the mesocotyl was primarily responsible for the emergence of seedlings from deeper levels of soil. The mesocotyl of the seedling is here interpreted as the hypocotyl. The functionally suctorial scutellum together with coleoptile constitutes the first cotyledon and the first true-leaf is regarded as the second cotyledon. The development of tillers from scutellar and first-leaf buds depends on the depth at which level the seeds (caryopses) germinated and the seedlings emerged above the soil surface. The first-leaf axillary buds, regradless of depths, develop into dominant tillers. The scutellar buds, especially at greater depths, remain inhibited. At shallower levels, however, they develop into tillers. The scutellar buds, at deeper levels, behave as reserve ramets which feature adds to the success of the species as a weed in the agricultural prairies.     

Growth and development of embryo parts during the germination of caryopses of the wild oat (Avena fatua L.)

Wild oat (Avena fatua L.) caryopses were germinated on moist filter paper and under water in the presence and absence of hydrogen peroxide (H₂O₂). The sequential growth and development of embryo parts were studied. Germination, as indicated by radicle emergence, was least and slowest in caryopses submerged in deoxygenated water. The coleorhiza in such caryopses elongated much earlier than the root, in contrast to the other treatments where the coleorhiza and the root emerged at about the same time. In caryopses incubated on moist filter paper all embryo parts showed considerable growth. In H₂O₂ treated caryopses only the epicotyl showed substantial growth over the experimental period. In all treatments the first mitotic peaks were noticed at the same period. The occurrence of these early nuclear divisions may be due to release of 4 C nuclei from inhibition by the uptake of water during caryopsis imbibition. The mitosis continued in the radicle of the embryo in those caryopses germinating on moist filter paper, indicating occurrence of DNA synthesis. In the other two treatments, however, few divisions were detected. Here the early growth of the root, causing caryopsis germination, was due to cell elongation, especially in the proximal part of the root.     

Protoplasts isolated from aleurone layers of wild oat (Avena fatua L.) exhibit the classic response to gibberellic acid

Viable, long-lived, gibberellic acid (GA₃)-responsive protoplasts have, for the first time, been isolated from aleurone layers of mature wild oat (Avena fatua L.) grain. More than 90% of the cells of aleurone layers are recovered as protoplasts, and these respond to treatment with GA₃ in essentially the same manner as the tissue from which they were derived. Protoplasts become vacuolate during incubation in vitro and, although not dependent upon GA₃, vacuolation is markedly stimulated by the hormone. Amylase and ribonuclease (RNase) are produced and secreted only in the presence of GA₃ and only after lag periods of 3 d and 4 d respectively. The amounts of amylase produced and secreted are proportional to GA₃ concentrations as low as 1.61·10^-13 M. With increasing concentrations of mannitol in the culture medium both vacuolation and the GA₃-induced production and secretion of enzymes are inhibited progressively, the latter being precluded by 0.6 M to 0.7 M mannitol.Abbreviations GA₃ gibberellic acid₃ - RNase ribonuclease     

Effect of ammonium sulfate on the phytotoxicity, foliar uptake, and translocation of imazamethabenz in wild oat

Experiments were conducted in greenhouse, growth chamber, and laboratory conditions to determine the effect of ammonium sulfate [(NH₄)₂SO₄] on the phytotoxicity, foliar uptake, and translocation of imazamethabenz on wild oat. Rates of (NH₄)₂SO₄ up to 5% (w/v) applied with a greenhouse sprayer did not affect the phytotoxicity of the herbicide when the mix was applied at the one- to two-leaf stage. However, inclusion of 1 and 2% (NH₄)₂SO₄ increased the phytotoxicity of the herbicide when the mix was sprayed at the two- to three-leaf, or the three- to four-leaf stage. At 10%, (NH₄)₂SO₄ decreased the phytotoxicity of the sublethal dosage of the herbicide. When the herbicide was applied as individual drops to the growth chamber-grown plants, inclusion of (NH₄)₂SO₄ at 1% did not affect phytotoxicity as measured by shoot growth. The presence of (NH₄)₂SO₄ did not affect the amount of imazamethabenz retained by wild oat foliage, but it decreased [¹⁴C]imazamethabenz absorption, slightly antagonized acropetal translocation, and increased the basipetal translocation of [¹⁴C]imazamethabenz. It was concluded that application methods greatly modify the effect of (NH₄)₂SO₄ on imazamethabenz phytotoxicity. Herbicide absorption and translocation as determined by one method do not necessarily represent the absorption and translocation patterns when different application methods are used. Absorption and translocation were not the factors that were responsible for the observed effect of (NH₄)₂SO₄ on the herbicide phytotoxicity.Abbreviations SC suspension concentrate     

After-ripening and phytochrome action in seeds of dormant lines of wild oat (Avena fatua)

The effects of a short exposure to red, far-red or alternate red/far-red light on the germination of seeds after-ripened for different periods of time were studied in dormant lines of wild oat ( Avena fatua L.). Three stages were distinguishable in the after-ripening period in the response of germination to light. Seeds stayed dormant and showed no response to light during stage I. Phytochrome-mediated germination was observed in seeds during stage II. The phytochrome action disappeared during stage III, i.e. seeds fully germinated following treatments of all light qualities. When the seeds were imbibed in polyethylene glycol solutions, dark germination was reduced and phytochrome again had an effect, which suggested the involvement of phytochrome in water uptake of the seed.     

Aluminum resistance mechanisms in oat (Avena sativa L.)

Background and aims

Enhanced aluminum (Al) resistance has been observed in dicots over-expressing enzymes involved in organic acid synthesis; however, this approach for improving Al resistance has not been investigated in monocots. Among the cereals, oat (Avena sativa L.) is considered to be Al resistant, but the basis of resistance is not known.

Methods

A hydroponic assay and hematoxylin staining for Al accumulation in roots were used to evaluate Al resistance in 15 oat cultivars. Malate and citrate release from roots was measured over a 24?h period. A malate dehydrogenase gene, neMDH, from alfalfa (Medicago sativa L.) was used to transform oat.

Results

Oat seedlings were highly resistant to Al, as a concentration of 325?μM AlK(SO₄)₂ was needed to cause a 50% decrease in root growth. Most oat cultivars tested are naturally resistant to high concentrations of Al and effectively excluded Al from roots. Al-dependent release of malate and Al-independent release of citrate was observed. Al resistance was enhanced in a transgenic oat line with the highest accumulation of neMDH protein. However, overall root growth of this line was reduced and expression of neMDH in transgenic oat did not enhance malate secretion.

Conclusions

Release of malate from oat roots was associated with Al resistance, which suggests that malate plays a role in Al resistance of oat. Over-expression of alfalfa neMDH enhanced Al resistance in some lines but was not effective alone for crop improvement.     

Callus formation and plant regeneration from somatic embryos of oat (Avena sativa L.)

Globular-stage somatic embryos were isolated by vortexing friable, embryogenic callus of oat (Avena sativa L.) followed by fractionation based on size. Somatic embryos were most frequently found in the 300–380 m size fraction. Friable, embryogenic callus was reinitiated from 55% of isolated somatic embryos. Fertile plants were regenerated from 22% of isolated somatic embryos. Reinitiation of callus from somatic embryos and growth of friable, embryogenic callus was inhibited by the selective agents G418 and methotrexate. These results suggest that somatic embryos isolated from friable, embryogenic callus of oat may be useful totipotent targets for particle acceleration-mediated transformation.     

The Influence of Light and Potassium Nitrate on the Dormancy and Germination of Avena fatua L. (wild oat) Seed Stored Buried Under Natural Conditions

The influence of light and potassium nitrate on the dormancyand germination of Avena fatua L. seed stored buried under naturalconditions was studied over a 13 month period. Natural emergenceof seedlings from buried seed in relation to rainfall and temperatureregimes and fluctuation was also monitored. The present findingsshow that potassium nitrate is stimulatory to germination onlyin the light and, further, that seeds from two positions inthe spikelet respond differently. Key words: Dormancy, Light, Nitrate     

Effects of light and regulators on senescence-related changes in soluble proteins in detached oat (Avena sativa L.) leaves

The rate of senescence and the two-dimensional pattern of soluble proteins from detached oat leaves senescing in either darkness or light were analyzed, and compared to those of leaves in which senescence was delayed by application of the cytokinin benzyladenine or enhanced through the action of abscisic acid.Senescence of detached leaves in light did not differ significantly from senescence in attached leaves on intact plants. In darkness, protein was lost at a higher rate than in light, but several individual proteins showed relative increases. Notably, proteins previously characterized as high-molecular-weight proteins and senescence-associated proteins (Klerk et al., 1992) increased. Changes observed during incubation in light or darkness appeared to be related to this condition rather than the rate or progress of senescence. Cytokinins delayed and abscisic acid accelerated the changes in protein pattern compared to water. Beside changes previously identified in leaves senescing on the plant, detached leaves show alterations that reflect their condition of incubation rather than their developmental progress.Abbreviations 2D-PAG two-dimensional polyacrylamide gel electrophoresis - ABA abscisic acid - BA N⁶-benzyladenine - BSA bovine serum albumin - EDTA ethylenediamine tetraacetic acid - IEF isoelectric focusing - Rubisco ribulosebisphosphate carboxylase/oxygenase - SDS sodium dodecyl sulfate - Tris tris (hydroxymethyl) aminomethane     

Production and dormancy of wild oat (Avena fatua) seed from plants grown under soil waterstress

In 1974 wild oat plants grown from very dormant seed types fA, fB, fC originating from a single field, and in 1976 plants of type fB derived from another location were waterstressed from the time when the spikelets were just beginning to emerge until the seed was fully ripe. The seed production and dormancy of the seed were determined. Waterstress reduced the number of viable seeds per plant by 42% in 1974 and 49% in 1976. The number of viable seed produced on the tillers of the plants were reduced to a greater extent by waterstress than the number produced on their main stems. Some of the differences between the number of viable seeds produced by the three wild oat types were significant. In 1974, 78% of seed from stressed plants was dormant as compared with 90% of that from unstressed plants. Under a given soil moisture, the dormancy of the three types differed little, although seed dormancy was less in tiller seed than in main stem seed. In 1976 seed from stressed plants was 80% dormant, immediately after collection, whereas that from unstressed plants was totally dormant. Storage for 6 months at 25°C decreased these percentages to 17% and 90% and at 5°C to 72% and 98% respectively. Buried in soil immediately after collection, 66% of viable seed from stressed plants gave seedlings in the first autumn after burial as compared with 4% of seed from unstressed plants. Most of the seed from waterstressed plants gave rise to seedlings in the first autumn, and seed from non-stressed plants in the second spring. The α-amylase content was four times greater in seeds from stressed compared with non-stressed plants.     

Preparation and polypeptide composition of plasma membrane and other subcellular fractions from wild oat (Avena fatua) aleurone

Plasma membranes can be isolated from a variety of plant tissues by first preparing a post-mitochondrial membrane fraction enriched in plasma membranes, by differential centrifugation, and partitioning this on a dextran-polyethylene glycol two-phase system. With wild oat aleurone, however, we observed that differential centrifugation could not be used to produce a microsomal fraction enriched in plasma membrane. Approximately 70% of the plasma membrane in aleurone homogenates was pelleted by sequential centrifugation at 100 g× 10 min and 1000 g× 10 min. The remainder sedimented at 112 000 g× 1 h. All the material that was pelletable by centrifugation was, therefore, subjected to dextran-polyethylene glycol two-phase partitioning. The plasma membrane marker enzymes glucan synthase II (GSII, EC 2. 4. 1. 34) and UDP-glucose:sterol glucosyltransferase (SGT, EC 2. 4. 1.) were enriched in the upper phase, whereas cytochrome c oxidase activity (EC 1. 9. 3. 1), a mitochondrial marker enzyme, was depleted. The presence of endoplasmic reticulum (ER) and protein body membranes in the phase system was assessed by probing western blots, of SDS-PAGE separated proteins, with polyclonal antiserum either to binding protein (BiP, an ER marker) or to tonoplast intrinsic protein (TIP, a protein body membrane marker). BiP and TIP were present in the lower phase, but were not detected in the upper phase. In addition, the polypeptide patterns of material in the upper and lower phases were very different. These observations suggested that high purity aleurone plasma membrane had been isolated. Although the procedure for isolating plasma membranes was applicable to both aleurone protoplasts and layers, the polypeptide patterns of plasma membranes prepared from these sources were very different. The major protein components of wild oat aleurone were 7 S and 12 S storage globulins. These proteins were present in the lower phase, but not in the plasma membrane enriched upper phase, after aqueous two-phase partitioning. Differential centrifugation studies showed that it was necessary to homogenise aleurone in a buffer of pH 6. 0 or less if a soluble protein fraction, essentially devoid of storage globulins, was to be obtained. The use of these fractionation techniques is discussed in relation to photoaffinity labelling of gibberellin (GA)-binding proteins in aleurone.     

Immunodetection and immunolocalization of tryptophanins in oat (Avena sativa L.) seeds

Tryptophanins (TRPs) are low molecular weight, tryptophan-rich, basic proteins found in oat (Avena sativa L.) seeds. Like their counterpart puroindolines (PINs) from wheat (Triticum aestivum L.), TRPs are thought to be involved in flour softness as well as disease resistance against phytopathogenic fungi. PINs are known to be the major components of ‘friabilin’ associated with the surface of water washed starch grains and possess lipid binding properties. Two polyclonal antisera against puroindoline-a (PIN-a), and puroindoline-b (PIN-b) respectively; and a monoclonal antiserum raised against ‘friabilin’ were used as primary antibodies in immunoblotting experiments. All antisera detected immunoreactive polypeptides, with approximate relative masses of 15–16 kDa, in oat, wheat, and barley (Hordeum vulgare L.) seed extracts but not in rice (Oryza sativa L.), maize (Zea mays L.), bean (Phaseolus vulgaris L.), pea (Pisum sativum L.) and lentil (Lens culinaris Medic.) seed extracts. Immunoreactive polypeptides were detected in aqueous ethanol [52% (v/v) ethanol] seed extracts. Both anti-‘friabilin’ monoclonal and anti-PIN-b polyclonal antisera recognized 15 as well as 16 kDa tryptophanins in oat seeds from different cultivars. On the other hand, anti-PIN-a polyclonal antiserum strongly cross-reacted with 16 kDa TRP and weakly with 15 kDa TRP. Tryptophanins were found to be associated with the surface of starch grains in oat endosperm tissue using both fluorescence and confocal laser scanning microscopies with anti-‘friabilin’ monoclonal antiserum. SDS-PAGE and immunoblotting assays revealed a gradual synthesis of TRPs as early as milk stage in developing oat seeds. On the other hand, TRPs tend to undergo degradation during seed germination.     

A simulation model of Avena fatua L. (wild-oat) growth and development

An eco-physiological simulation model of the growth and development of Avena fatua was parameterised and tested. The model simulates growth ofA. fatua, in kg dry matter ha^-1 day^-1 from sowing to maturity as a function of irradiance, temperature and various species characteristics. Parameter values were derived from the literature and from field experiments, including both autumn and spring sowings of A. fatua over three years at two sites in southern England. With two exceptions, a single set of parameter values was sufficient to accurately simulate the emergence, growth and development of both autumn and spring cohorts over all years and sites. The two exceptions were the result of differences between autumn and spring cohorts of A. fatua in the rate of early leaf area growth and in the relationship between specific leaf area and developmental stage.     

Putrescine-enhanced somatic embryos and plant numbers from elite oat (Avena spp. L.) and reciprocal crosses

Summary Mature embryos from hulled, regenerable GP-1 (A. sativa L.), hull-less, recalcitrant Tibor (A. nuda L.) and reciprocal crosses were cultured in vitro on a putrescine- (Put) containing medium. Hormone-free Murashige and Skoog medium (MS-0) or shoot proliferation medium (SPM) [2.0 mgl⁻¹ (9.0 μM) 2,4-dichlorophenoxyacetic acid (2,4-D)], with and without 0.5 mM Put or 1 mM Put, were tested for effects on somatic embryogenesis and plant regeneration. Put/SPM (0.5mM) was the best medium for both somatic embryos (SEs) and plant numbers per gram of callus, regardless of genotype. This effect was most evident in Tibor, which produced no somatic embryos or plants on SPM, a previously published regeneration medium, and in Tibor ×GP-1, which produced reduced numbers of SE and plants on the remaining media. The number of SEs per gram of callus for GP-1 and GP-1× Tibor showed little significant differences between the different media. Put treatments produced plants from the four genotypes but the regeneration efficiency on Put-containing medium was similar or even better than on SPM for explants containing maternal GP-1 germplasm. This suggests that Put-containing MS-0 medium can be used for testing regeneration of other oat lines. In addition, SPM containing 0.5 mM Put can be used to induce significant regeneration of plants from normally recalcitrant genotypes. This improvement greatly increases the number of potential germplasms for further transformation efforts.     

Influence of temperature and light intensity on absorption, translocation, and phytotoxicity of fenoxaprop-ethyl and imazamethabenz-methyl in Avena fatua

The absorption and translocation of fenoxaprop-ethyl and imazamethabenz-methyl were investigated in wild oat (Avena fatua L.) plants grown under different temperature and light intensity conditions by using ¹⁴C tracer techniques. The phytotoxicity of both herbicides, applied as individual droplets, was also determined under similar environments. The absorption of fenoxaprop-ethyl and imazamethabenz-methyl was increased by high temperature (30/20°C) and to a lesser extent by 70% shading; low temperature (10/5°C) had limited effect on the absorption. The basipetal translocation of fenoxaprop-ethyl was not affected by high temperature, and the increase in imazamethabenz-methyl translocation at high temperature was likely the result of the increased absorption. Low temperature decreased total translocation and translocation efficiency in both fenoxaprop-ethyl and imazamethabenz-methyl. Low light intensity tended to reduce the efficiency of basipetal translocation of both herbicides. Fenoxaprop-ethyl phytotoxicity was reduced by high temperature but not by low temperature. Temperature had little effect on imazamethabenz-methyl effectiveness. Under 70% shading, the phytotoxicity of both herbicides was enhanced.Abbreviation S.E.D. standard errors of difference     

Influence of some plant growth regulators on the growth and organogenesis ofCymbidium aloifolium (L.) Sw. seed-derived rhizomesin vitro

Summary An efficient procedure is outlined forin vitro regeneration of an epiphytic orchid,Cymbidium aloifolium (L.) Sw. using rhizomes developed from seeds. Murashige and Skoog's (1962) medium (MS) containing indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), or 1-naphthaleneacetic acid (NAA) stimulated growth and proliferation of rhizomes with NAA being most effective at 5.0 mg.l⁻¹ (27.0 μM). Shoot bud differentiation was induced in the apical portions of the rhizomes on MS medium containing kinetin (Kn) or N⁶-benzyladenine (BA). The highest frequency of shoot regeneration (91.5%) and the maximum number of shoot buds formed (3.5 shoots/rhizome) were recorded with BA at 1.0 mg.l⁻¹ (4.4 μM). NAA (0.1 mg.l⁻¹, 0.54 μM), whenever added to the medium in conjunction with BA (1.0 mg.l⁻¹, 4.4 μM), slightly enhanced the frequency of shoot bud regeneration (92.6%) and the number of shoot buds formed (5.2 shoots/rhizome). Moreover, an NAA-BA combination induced rooting in regenerated shoots thereby producing complete plantlets in one step. Shoots developed on cytokinin-supplemented medium were rooted on MS containing NAA at 1.0 mg.l⁻¹ (5.4 μM). Regenerated plantlets were acclimated and eventually established in a garden.