Influence of 6-diazo-5-oxo-L-norleucine (DON), a glutamine analogue, on cartilaginous differentiation in mouse limb buds in vitro.

The antibiotic, 6-diazo-5-oxo-L-norleucine (DON), an analogue of L-glutamine, causes limb malformations in several species, including mice. This report shows that DON also interferes with differentiation of cartilaginous rudiments of mouse limb buds grown as organ cultures for 3 to 8 days in medium containing the teratogen. DON (5 mug/ml) inhibits growth of the explants and interferes with production of normal matrix by chondrocytes. The cartilage of DON-treated cultures exhibits a striking lack of matrix, compared with that of control explants which contains abundant metachromatic matrix. Differentiation of osteoblasts, and secretion of osteoid around the scapula and humerus are enhanced by DON. The direct effects of DON on growth and chondrogenesis, which can be prevented by addition of L-glutamine (1 mg/ml) to the medium, can be attributed to the known interference of DON in L-glutamine-dependent steps in metabolism. The possible relationships between these effects of DON in vitro and the malformations produced in vivo, are discussed.     

The effects of 6-diazo-5-oxo-L-norleucine, a glutamine analogue, on the structure of the major cartilage proteoglycan synthesized by cultured chondrocytes

Incubation of embryonic chick chondrocytes with 6-diazo-5-oxo-L-norleucine (DON), a glutamine analogue, led to a dose-dependent inhibition of [35S]sulfate incorporation into proteoglycan. In the absence of exogenous L-glutamine, a maximal inhibition of 50-60% was achieved with DON concentrations greater than or equal to 1 microgram/ml (6 microM); the ED50 was approximately 0.2 microM. This inhibitory effect could be partially restored by the addition of 100-fold molar excess of either exogenous L-glutamine or M-glucosamine. The quantitative changes were due neither to inhibition of protein core synthesis nor to undersulfation of glycosaminoglycan chains. Rather, the proteoglycan synthesized in the presence of DON contained substantially fewer (approximately 50% of control) and smaller (10-15% of control, on the average) chondroitin sulfate chains as well as a paucity of keratan sulfate chains. The result of these structural changes was a proteoglycan with significantly lower molecular weight, buoyant density, and anionic charge. In spite of these modifications, the altered proteoglycan synthesized in the presence of DON was secreted normally and retained the ability to interact with exogenous hyaluronic acid and link proteins. The results of our experiments also indicate that DON substantially diminished the pool of hexosamine precursors required for glycosaminoglycan synthesis. We conclude that this decrease was responsible for the molecular alterations described above; and these, in turn, can account for the morphological changes previously seen in cartilage matrix synthesized in the presence of DON.     

Stimulation of embryonic mouse limb bud mesenchymal cell growth by peptide growth factors

The possible role of peptide growth factors in mammalian intrauterine cell growth has been investigated using primary cultures of undifferentiated mesenchymal cells from 11-day mouse embryo limb buds. When grown as monolayer cultures, proliferation is greatly favored by high cell densities. In medium containing 0.2% serum, purified epidermal growth factor (EGF), fibroblast growth factor (FGF), multiplication stimulating activity (MSA), insulin, and somatomedin-C (Sm-C) do not increase cell growth, but a 30-40,000 molecular weight component of mouse fetal liver conditioned medium is stimulatory. On the other hand, when limb bud cells are grown as high density or micromass cultures, a method which better approximates in vivo growth conditions, all of the purified growth factors tested stimulate cell growth significantly. These growth factors have additive effects when used in combination, the best stimulation being observed with liver medium (10% v/v), EGF (10 ng/ml), FGF (200 ng/ml), and either insulin (1 microgram/ml) or Sm-C (20 ng/ml). We conclude that the response of limb bud cells to growth stimulation is influenced by the manner in which the cells are cultured and that at least four different growth factors are required for optimal in vitro proliferation. One of these, the active component of liver medium, appears to be a previously uncharacterized growth factor.     

Long-term culture of renin containing tissue

Thin cortical tissue explants from kidneys of hydronephrotic mice were excised and incubated in different culture media containing growth and proliferation factors. Over a period of several months the content of renin in the explants and in the culture medium was repeatedly measured, to define the conditions necessary for the maintenance of renin production in a long-term culture. The best results were obtained when culturing the renal tissue in Dulbecco's medium (DMEM) with 10% fetal calf serum, 6 units/100 ml platelet-derived growth factor and 200 ng/ml glycylhistidyllysine. Renin was still present within the cells and in the culture medium after more than six months. Prevention of dedifferentiation, as evidenced in this case by the maintenance of renin production, seemed to be dependent on specific extracellular matrix proteins of renal origin. If the explants were dissociated from their matrix components by collagenase, a gradual loss of renin production was observed within 5 days. Complementation of the collagenase-digested cell suspension with different nonrenal extracellular matrix materials did not afford the stabilizing effect of the original pericellular matrix.     

Clonal growth of primary cultures of human hyaline chondrocytes in a defined medium

Rapid clonal growth of primary cultures of human costal chondrocytes in a defined medium has been achieved. The basal nutrient medium used for such growth is MCDB 104. It is prepared without linoleic acid and supplemented with 1 microgram/ml insulin, 100 ng/ml fibroblast growth factor, 1.0 x 10(-7) M dexamethasone, and 5 micrograms/ml mixed lipids, presented to the cells in the form of liposomes. The lipid supplement contains soybean lecithin, cholesterol, sphingomyelin, vitamin E, and vitamin E acetate. No expression of cartilage-like differentiation occurs in the defined medium. However, colonies grown for several days in the defined medium and then grown for an additional period of time in medium F12 supplemented with fetal bovine serum and chicken embryo extract synthesize large amounts of refractile matrix that is stained intensely by acidified alcian green, thus demonstrating that the cells growing in the defined medium are capable of expressing cartilage matrix in a permissive environment. Good clonal growth and expression of differentiation can also be obtained by inoculating primary cultures of human chondrocytes directly into the F12-serum-embryo extract medium.     

Long-term culture of renin containing tissue

Summary Thin cortical tissue explants from kidneys of hydronephrotic mice were excised and incubated in different culture media containing growth and proliferation factors. Over a period of several months the content of renin in the explants and in the culture medium was repeatedly measured, to define the conditions necessary for the maintenance of renin production in a long-term culture.The best results were obtained when culturing the renal tissue in Dulbecco's medium (DMEM) with 10% fetal calf serum, 6 units/100 ml platelet-derived growth factor and 200 ng/ml glycylhistidyllysine. Renin was still present within the cells and in the culture medium after more than six months. Prevention of dedifferentiation, as evidenced in this case by the maintenance of renin production, seemed to be dependent on specific extracellular matrix proteins of renal origin. If the explants were dissociated from their matrix components by collagenase, a gradual loss of renin production was observed within 5 days. Complementation of the collagenase-digested cell suspension with different nonrenal extracellular matrix materials did not afford the stabilizing effect of the original pericellular matrix.These studies were supported by the Deutsche Forschungsgemeinschaft-SFB 90-Cardiovasculäres System     

Growth limitation in hybridoma cell cultures: The role of inhibitory or toxic metabolites

Hybridoma cells usually grow to fairly low cell densities in batch cultures (1–3×10⁶ cells/ml). The reason for this is either that essential nutritional components of the medium are consumed, or that the cells produce some kind of inhibitory or toxic metabolite. This investigation presents evidence for the latter. Spent medium from cultures of hybridoma cells did not support growth of cells at lower cell densities (1–3×10⁵ cells/ml). The ability to support cell growth could not be restored by adding additional serum, energy sources (glucose, pyruvate) or L-glutamine. Furthermore, the consumption of amino acids could not account for this growth inhibition. On the contrary, the spent medium contained a substance that inhibited cell growth. This substance or metabolite was found in a fraction eluted from a gel filtration column when spent medium was applied to the column. This substance was found in the spent medium from all hybridoma and myeloma cell lines that were tested. The molecular weight of the substance was about 5 kD. Spent medium from two hybridoma cell lines also contained a substance that was eluted in the same fraction as albumin (67 kD). It is likely that this (or these) substance(s) is responsible for the growth limitation in hybridoma cell cultures.Abbreviations PBS phosphate buffered saline     

Muscle and cartilage differentiation in axolotl limb regeneration blastema cultures

A tissue culture system is described for explants of mesenchyme from Ambystoma mexicanum limb regeneration blastemas. Explants were cultured on collagen substrate for 3 weeks in minimal essential medium supplemented with the hormones insulin, thyroxine, somatotropin, and hydrocortisone, plus beef embryo extract (EE), 2%. This medium supported extensive cell migration onto the substrate followed by cell proliferation and differentiation of both cartilage matrix and myotubes. Cultures on plastic substrate, rather than on collagen, displayed similar cell outgrowth and cartilage formation, but relatively little myotube formation. Differentiation in EE-supplemented medium was compared with that in two defined media: Explants in medium containing only the hormones showed little outgrowth or cartilage development and never formed myotubes; medium containing the hormones plus fibroblast growth factor, 50 ng/ml, supported an intermediate degree of outgrowth and cartilage development and occasional myotube formation. Explant size was also a factor: Smaller explants survived and formed myotubes less frequently, even when on collagen in EE-supplemented medium.     

in Vitro Effect of Deoxynivalenol on the Differentiation of Human Colonic Cell Lines Caco-2 and t84

The effects of low concentrations of deoxynivalenol (DON) on structural and functional characteristics of human colonic adenocarcinoma cell lines Caco-2 and T84 were examined. Scanning electron microscopic (SEM) analysis of the apical surfaces of Caco-2 cells revealed reduction or abnormal formation of brush borders in the presence of 50, 100 and 200 ng/ml of DON. Monolayer integrity of Caco-2 and T84 cells was studied using cells which were cultured on permeable membranes. The transepithelial electrical resistance (TEER) of Caco-2 cells was significantly reduced at 50, 100 and 200 ng/ml of DON, significant increase in lucifer yellow (LY) permeability was also observed in these cells at 100 ng/ml of DON. The TEER of T84 cells was significantly reduced at 100 and 200 ng/ml of DON. LY permeability significantly increased at 200 ng/ml of DON in T84 cells. Enzyme activities in Caco-2 cells were also examined. Alkaline phosphatase activity was reduced from the 6th to 15th day of culture in the presense of 100 or 200 ng/ml of DON, whereas sucrase- isomaltase activity was significantly decreased by adding 50 or 100 ng/ml of DON for 15 or 20 days. Protein content was attenuated only by treatment with 200 ng/ml of DON thoughout the experimental period. The results indicate that DON interferes with structural and functional characteristics of differentiation in enterocytes at low doses. This revised version was published online in June 2006 with corrections to the Cover Date.     

Characterization of bone-derived chondrogenesis-stimulating activity on embryonic limb mesenchymal cells in vitro

Demineralized bone matrix contains factors which stimulate chondrogenesis and osteogenesis in vivo. A water-soluble extract of bone has been shown to stimulate chondrogenesis in vitro in embryonic limb mesenchymal cells (Syftestad, Lucas & Caplan, 1985). The aim of this study was to analyse the cellular mechanism of the bone-derived chondrogenesis-stimulating activity, with particular attention on how normal requirements for chondrogenesis may be altered. The effects of bovine bone extract (BBE) on chondrogenesis in vitro were studied using micromass cultures of chick limb bud mesenchyme isolated from embryos at Hamburger-Hamilton (HH) stage 23/24, an experimental system which is capable of undergoing chondrogenic differentiation. Bovine diaphyseal long bones were demineralized and extracted with guanidine-HCl to prepare BBE (Syftestad & Caplan, 1984). High-density mesenchyme cultures (30 x 10(6) cells/ml) were exposed to different doses of BBE (0.01-1.0 mg ml-1) and chondrogenesis was quantified based on cartilage nodule number and [35S]sulphate incorporation. BBE was tested on micromass cultures of varying plating densities (2-30 x 10(6) cells/ml), on cultures of 'young' limb bud cells (HH stage 17/18), and on cultures enriched with chondroprogenitor cells obtained from subridge mesoderm. Since poly-L-lysine (PL) has recently been shown (San Antonio & Tuan, 1986) to promote chondrogensis, PL and BBE were introduced together in different doses, in the culture medium, to determine if their actions were synergistic. Our results show that BBE stimulates chondrogenesis in a dose-dependent manner and by a specific, direct action on the chondroprogenitor cells but not in normally non-chondrogenic, low density or 'young' limb bud cell cultures. The effects of PL and BBE are additive and these agents appear to act by separate mechanisms to stimulate chondrogenesis; PL primarily enhances nodule formation, and BBE appears to promote nodule growth.     

Glutamine: A Suitable Nitrogen Source for Enhanced Shoot Multiplication in Cucumis sativus L.

Shoot tip explants of cucumber (Cucumis sativus L. cv. Poinsett 76) were cultured in vitro on Murashige-Skoog medium with L-glutamine, ammonium nitrate, adenine sulphate, asparagine, ammonium succinate, potassium nitrate and sodium nitrate as the nitrogen sources along with optimal concentration of 0.044 mM benzyladenine to study their effects on in vitro morphogenesis. The explants grown with 0.068 mM L-glutamine displayed the highest culture response (74.6 %) and greatest shoot number per explant (13.6) at the end of two subcultures. The explants cultured with other nitrogen sources resulted in low culture frequency and low number of shoots per explant accompanied by basal callusing and necrosis.     

Growth kinetics and differentiation in vitro of normal human uroepithelial cells on collagen gel substrates in defined medium

Conditions have been described for the selective growth, serial cultivation, and postconfluent morphological differentiation in vitro of normal adult human uroepithelial cells (HUC) on collagen gel substrates in a serum-free medium without the deliberate addition of undefined components and without a requirement for a polypeptide growth factor. The culture medium used (F12) was the standard Ham's F12 medium (0.3 mM calcium) supplemented with 1 microgram/ml hydrocortisone, 5 micrograms/ml transferrin, 10 micrograms/ml insulin, 0.1 mM nonessential amino acids, 2.0 mM L-glutamine, 2.7 mg/ml D-glucose, 10(-4) M ethanolamine or 10(-4) M phosphoethanolamine, and 5 X 10(-8) M selenium. HUC grown in F12 on Type I collagen gel substrates had a generation time of 33 hours and could be serially passed 3-5 times during log phase of growth (20-25 population doublings) before spontaneously senescing. Transmission electron microscopy showed that cultures of HUC grown entirely in serum-free F12 on collagen gel substrates morphologically differentiate postconfluence to resemble in some respects the stratified uroepithelium in vivo, although neither a basal lamina nor an asymmetric unit membrane develop. The addition of epidermal growth factor (EGF) to the F12 did not improve either the growth rate or the lifespan in vitro of HUC. In contrast, the addition of fetal bovine serum (FBS) to F12 was mitogenic to HUC in a dose-dependent manner in the concentration range 0.01-1.00% (4-400 micrograms/ml protein), but higher concentrations of FBS did not improve growth further. The generation time of HUC in 1% FBS-F12 decreased to 21 hours, and the potential population doublings in vitro increased to 31-36. Small amounts (140 micrograms/ml) of bovine pituitary extract (BPE) were similarly mitogenic to HUC in F12. Altering the calcium concentration in the standard Ham's F12 medium (0.3 mM), however, did not improve the growth of HUC in serum-containing or serum-free medium. Higher calcium concentrations (0.30-0.90 mM) were neither mitogenic nor inhibitory to HUC growth, but resulted in decreasing viability of HUC in growing cultures, suggesting an accelerating rate of cellular differentiation. In contrast HUC in low calcium, serum-free F12 (0.1 mM) failed to stratify and morphologically differentiate even in postconfluent cultures. This failure of HUC to differentiate in low calcium F12 medium did not confer a long-term growth advantage.(ABSTRACT TRUNCATED AT 400 WORDS)     

Factors controlling the proliferative rate, final cell density, and life span of bovine vascular smooth muscle cells in culture

Low density vascular smooth muscle (VSM) cell cultures maintained on extracellular-matrix(ECM)-coated dishes and plated in the presence of either plasma or serum will proliferate actively when serum-containing medium is replaced by a synthetic medium supplemented with three factors: high density lipoprotein (HDL, 250 micrograms protein/ml); insulin (2.5 micrograms/ml) or somatomedin C (10 ng/ml); and fibroblast growth factor (FGF, 100 ng/ml) or epidermal growth factor (EGF, 50 ng/ml). The omission of any of these three factors from the synthetic medium results in a lower growth rate of the cultures, as well as in a lower final cell density once cultures reach confluence. When cells are plated in the total absence of serum, transferrin (10 micrograms/ml) is also required to induce optimal cell growth. The effects of the substrate and medium supplements on the life span of VSM cultures have also been analyzed. Cultures maintained on plastic and exposed to medium supplemented with 5% bovine serum underwent 15 generations. However, when maintained on ECM-coated dishes the serum-fed cultures had a life span of at least 88 generations. Likewise, when cultures were maintained in a synthetic medium supplemented with HDL and either FGF or EGF, an effect on the tissue culture life span by the substrate was observed. Cultures maintained on plastic underwent 24 generations, whereas those maintained on ECM-coated dishes could be passaged repeatedly for 58 generations. These experiments demonstrate the influence of the ECM-substrate only in promoting cell growth but also in increasing the longevity of the cultures.     

Glucocorticoids regulate the secretion of a 21kDa-IGF-binding protein by sheep adipose tissue explants

Using a solution phase assay we have demonstrated that sheep adipose tissue explants secrete insulin-like growth factor binding proteins (IGFBPs) when cultured in serum-free medium over a 24 h period. Further, we demonstrate that secretion of IGFBP(s) is inhibited (up to 50%) by incubation of the cultures in the presence of 10^–8M dexamethasone. This inhibitory effects is overcome when insulin (10 ng/ml) and ovine growth hormone (100 ng/ml) are incubated together (but not separately) with glucocorticoid. Further characterisation of this IGF binding activity by high performance size exclusion chromatography and Western ligand blot analysis indicated that under our culture conditions sheep adipose tissue explants secrete one predominant 21 kDa IGFBP and it is this BP which is hormonally regulated as described above. We discuss our results in the context of endocrine/paracrine/autocrine control of adipose tissue metabolism and differentiation.Abbreviations IGF insulin-like growth factor - IGF-BP insulin-like growth factor binding proteins - DX dexamethasone - GH ovine growth hormone - CM conditioned medium     

Factors involved in the control of proliferation of bovine corneal endothelial cells maintained in serum-free medium

Experimental conditions have been defined that allow bovine corneal endothelial (BCE) cells to grow in the complete absence of serum. Low density BCE cell cultures maintained on extracellular matrix (ECM)-coated dishes and plated in the total absence of serum proliferate actively when exposed to a synthetic medium supplemented with high density lipoprotein (HDL 500 μg protein/ml), transferrin (10 μg/ml), insulin (5 μg/ml), and fibroblast (FGP) or epidermal growth factor (EGF) added at concentrations of 100 or 50 ng/ml, respectively. Omission of any of these components results in a lower growth rate and/or final cell density of the cultures. BCE cell cultures plated on plastic dishes and exposed to the same synthetic medium grow very poorly. The longevity of BCE cultures maintained on plastic versus ECM and exposed to serum-free versus serum-containing medium has been studied. The use of ECM-coated dishes extended the life span of BCE cultures maintained in serum-supplemented medium to over 120 generations, as compared to less than 20 generations for cultures maintained on plastic. Likewise, BCE cells maintained on ECM and exposed to a synthetic medium supplemented with optimal concentrations of HDL, transferrin, insulin, and FGF underwent 85 generations, whereas control cultures maintained on plastic could not be passaged. The enhancing effect of ECM on BCE cell growth and culture longevity clearly illustrates the importance of the cell substrate in the control of proliferation of these cells.     

Exogenous glycosaminoglycans modulate chondrogenesis, cyclic AMP level and cell growth in limb bud mesenchyme cultures

Effects of hyaluronate, heparin and chondroitin-6-sulfate were studied on micromass cultures of chick limb bud mesenchyme (Hamburger and Hamilton stages 23-24). Histochemical, electron microscopical, biochemical and radiochemical investigations of day 4 cultures revealed dose-dependent inhibitory effects of these glycosaminoglycans on chondrogenesis, cyclic AMP level and growth of cells. In addition, hyaluronate with 100 micrograms/ml dose caused a displacement of newly formed proteoglycan from cultures into the medium. It is supposed that exogenous glycosaminoglycans influence ionic equilibrium in the immediate vicinity of cells and disturb the organization of the prechondrogenic extracellular matrix resulting in alterations of cell membrane--cytoskeleton associations. These alterations may provoke a reduction in cyclic AMP level and DNA synthesis. It is suggested that a reduction in cyclic AMP level preceding the expression of cartilage phenotype results in the inhibition of chondrogenesis.     

In vitro effects of fetal bovine serum and glucose on multiplication of Cryptobia salmositica

Cryptobia salmositica multiplied rapidly at 10 C in a minimum essential medium (containing 1.0 mg glucose/ml, Hanks' salts and L-glutamine) supplemented with heat-inactivated fetal bovine serum (FBS) and HEPES buffer (25 mM). The multiplication rate of C. salmositica was related to the amount of FBS; the peak number (approximately 9 x 10(6) parasites/ml) was attained in about 6 wk when the medium contained 25% final concentration of FBS. Glucose utilization was related to the number of parasites; the maximum utilization was reached before peak numbers. Formation of rosette colonies was correlated with multiplication rate and numbers of parasites in cultures. Degenerate round forms found in old cultures probably were caused by the accumulation of metabolic wastes in the medium.     

Regulation of the proportion of insulin cells in embryonic chick pancreas: effect of a growth factor-reduced extracellular matrix in combination with retinoic acid

Both retinoic acid (RA) and transforming growth factor (TGF)-beta1 are known to be influential in the development of insulin cells. Respectively, they increase and decrease the proportion of insulin cells when added to cultures of embryonic chick dorsal pancreatic buds. The aim of this study was to define the action of RA in the presence of decreased levels of TGF-beta1, as are found in growth factor-reduced Matrigel (GFRM), on the proportion of insulin cells. The endodermal component of 5-d chick dorsal pancreatic buds was explanted on to GFRM. Retinoic acid (10(-6) M) was added to Ham's F12 culture medium containing insulin (5 microg/ml), transferrin (5 microg/ml), and selenium (10(-10) M) (F12.ITS). Control explants were cultured in F12.ITS alone or in F12.ITS containing dimethyl sulfoxide (DMSO). After 7 d in culture, insulin and glucagon cells were localized immunocytochemically; changes in numbers of insulin cells were expressed as a percentage of insulin plus glucagon cells. Medium containing RA or DMSO increased the proportion of insulin cells significantly compared with the proportion in the explants cultured in F12.ITS medium alone.     

Humic substances failed to prevent the systemic absorption of deoxynivalenol (DON) and its adverse effects on piglets

The aim of this study was to examine the effects of a control diet (CON, 0.25?mg DON/kg diet) or a Fusarium toxin-contaminated diet (FUS, 4.49?mg DON/kg diet) without and with humic substances (HS) (CON-HS and FUS-HS, 0.23 and 4.56?mg DON/kg diet, respectively) on piglets during a 5-week growth trial starting after weaning (6.7?±?0.9?kg live weight, n?=?20/group). Feed intake was significantly reduced by feeding the FUS containing diets by approximately 21% compared with the CON diet irrespective of HS supplementation. The decrease in live weight gain paralleled the feed intake depression and amounted to approximately 26%. Feeding the FUS diet was clearly reflected by the DON levels in blood. While only traces of DON with median concentrations of 3?ng/ml (2?C5?ng/ml) and 2?ng/ml (0?C3?ng/ml) were detected in piglets fed the CON and CON-HS diets, respectively, significantly higher levels of 22.5?ng/ml (7?C30?ng/ml) and 23.5?ng/ml (15?C32?ng/ml) were found in piglets fed the FUS and FUS-HS diet, respectively. The urinary excretion of DON and its metabolite de-epoxy-DON as percentage of DON intake was not significantly influenced by HS supplementation and amounted to 24.1 and 20.2% for groups FUS and FUS-HS, respectively. In conclusion, the tested HS preparation cannot be recommended as a DON inactivating feed supplement for pigs.     

A method for large-scale multiplication of Curculigo orchioides through bulbil formation from leaf explant in shake flask culture

An efficient method has been developed for large-scale multiplication of Curculigo orchioides (Hypoxidaceae), an endangered medicinal plant, through direct bulbil formation from leaf explants in shake flask cultures. Leaf-segments (7 x 10 mm) were cultured in B5 liquid medium containing KNO3 (200 mgNL-1), (NH4)2SO4 (50 mgNL-1), benzyl adenosine (2.2 microM), adenine (0.11 mM), indole butyric acid (1.0 microM) and polyvinyl pyrrolidone (250 mgL-1). About 95% of explants produced maximum number of bulbils (546/flask at 6 weeks growth) in the medium. Shake flask cultures yielded 2737 bulbils/L medium whereas static cultures yielded 624 bulbils/L medium. Germination of bulbils was maximum (90.62%) on agar-gelled B5 medium containing benzyl adenosine (2.2 microM) and gibberellic acid (3.5 microM). Plantlets developed in vitro were successfully transferred to soil with a high rate of survivability (90%) and were comparable to natural population in growth and vigour.