Xanthone compounds in shoot cultures of <Emphasis Type="Italic">Gentianella bulgarica</Emphasis>

Shoot cultures of Gentianella bulgarica established from seedling epicotyls were maintained on MS medium supplemented with BA 0.2 mg l⁻¹ + NAA 0.1 mg l⁻¹. Cultures were prone to precocious flowering requiring the use of small shoot buds for multiplication purposes. The contents of three xanthone compounds identified as DGL, BGL, and DMB, in different plant material were determined by HPLC. The analysis revealed that the production of xanthones was affected by different concentrations of BA in medium. Shoot cultures grown at higher BA concentrations contained more DGL than material grown in nature. The concentrations of other two xanthones were lower in shoot cultures than in plants from nature. The radical scavenging activity of plant extracts and xanthones was investigated by DPPH test. Samples from plants grown in nature showed the highest activity (IC₅₀ = 0.26 mg ml⁻¹), while the extracts of shoot cultures grown in media with higher concentrations of BA showed moderate activities (IC₅₀ from 1.6 to 4.4 mg ml⁻¹).     

Bioreactor application on adventitious root culture of <Emphasis Type="Italic">Astragalus membranaceus</Emphasis>

Astragalus membranaceus is one of the most widely used traditional medicinal herbs in China, but the time required to generate a useful product in the field production is long. The growth of adventitious root cultures was compared between cultures grown in solid, liquid, or a 5-L balloon-type bubble bioreactor. The maximum growth ratio (final dry weight/initial dry weight) was determined for adventitious roots grown in the bioreactor. Studies carried out to optimize biomass production of adventitious roots compared adventitious root growth from various inoculum root lengths, inoculum densities, and aeration volume in the bioreactors. The maximum growth ratio occurred in treatments with a 1.5-cm inoculum root length, with 30 g (fresh weight) of inoculum per bioreactor or with an aeration volume of 0.1 vvm (air volume/culture medium volume per min). The polysaccharide, saponin, and flavonoid content of roots from bioreactor-grown cultures were compared to roots from field-grown plants grown for 1 and 3 yr. Total polysaccharide content of adventitious roots in the bioreactor (30.0 mg g⁻¹ dry weight (DW)) was higher than the roots of 1-yr-old (13.8 mg g⁻¹ DW) and 3-yr-old (21.1 mg g⁻¹ DW) plants in the field. Total saponin (3.4 mg g⁻¹ DW) and flavonoid (6.4 mg g⁻¹ DW) contents were nearly identical to 3-yr-old roots and higher than that of 1-yr-old roots under field cultivation.     

Biotechnological production of <Emphasis Type="SmallCaps">l</Emphasis>-ribose from <Emphasis Type="SmallCaps">l</Emphasis>-arabinose

l-Ribose is a rare and expensive sugar that can be used as a precursor for the production of l-nucleoside analogues, which are used as antiviral drugs. In this work, we describe a novel way of producing l-ribose from the readily available raw material l-arabinose. This was achieved by introducing l-ribose isomerase activity into l-ribulokinase-deficient Escherichia coli UP1110 and Lactobacillus plantarum BPT197 strains. The process for l-ribose production by resting cells was investigated. The initial l-ribose production rates at 39°C and pH 8 were 0.46 ± 0.01 g g⁻¹ h⁻¹ (1.84 ± 0.03 g l⁻¹ h⁻¹) and 0.27 ± 0.01 g g⁻¹ h⁻¹ (1.91 ± 0.1 g l⁻¹ h⁻¹) for E. coli and for L. plantarum, respectively. Conversions were around 20% at their highest in the experiments. Also partially purified protein precipitates having both l-arabinose isomerase and l-ribose isomerase activity were successfully used for converting l-arabinose to l-ribose.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Efficient separation and purification of allophycocyanin from <Emphasis Type="Italic">Spirulina</Emphasis> (<Emphasis Type="Italic">Arthrospira</Emphasis>) <Emphasis Type="Italic">platensis</Emphasis>

Allophycocyanin (APC) is a minor component of phycobiliproteins in cyanobacteria and red algae. This paper describes a simple and inexpensive extracting method for isolating APC from Spirulina (Arthrospira) platensis with high efficiency. The crude phycobiliprotein extract was pretreated by ammonium sulfate fractionation. Then, by adding hydroxylapatite into crude phycobiliprotein extract dissolved in 20 mM phosphate buffer (pH 7.0), APC was selectively adsorbed by hydroxylapatite but C-phycocyanin (C-PC) was not. The hydroxylapatite was collected and APC was extracted from the crude phycobiliprotein extract. Then, the enriched APC was washed off from the hydroxylapatite using 100 mM phosphate buffer (pH 7.0). In this simple extracting method it was easy to remove C-PC and isolate APC in large amounts. The absorbance ratio A ₆₅₀/A ₂₈₀ of extracted APC reached 2.0. The recovery yield was 70%, representing 4.61 mg · g⁻¹ wet weight. The extracted APC could be further purified by a simple anion-exchange chromatography with a pH gradient from 5.6 to 4.0. The absorbance ratio A ₆₅₀/A ₂₈₀ of the purified APC reached 5.0, and the overall recovery yield was 43%, representing 2.83 mg · g⁻¹ wet weight. Its purity was confirmed by native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate-PAGE.     

<Emphasis Type="Italic">In vitro</Emphasis> culture of <Emphasis Type="Italic">Chrysanthemum</Emphasis> plantlets using light-emitting diodes

Effects of illumination spectrum on the morphogenesis of chrysanthemum plantlets (Chrysanthemum morifolium Ramat. ‘Ellen’) grown in vitro were studied using an illumination system consisting of four groups of light-emitting diodes (LEDs) in the following spectral regions: blue (450nm), red (640nm), red (660nm), and far-red (735nm). Taking into account all differences in shoot height, root length, and fresh and dry weight (FW and DW, respectively), observed while changing the total photon flux density (PFD), the optimal total PFD for growth of chrysanthemum plantlets in vitro was estimated. For 16 h photoperiod and typical fractions of the spectral components (14%, 50%, 28%, and 8%, respectively), the optimal total PFD was found to be 40 μmol m⁻² s⁻¹. Our study shows that the blue component in the illumination spectrum inhibits the plantlet extension and formation of roots and simultaneously increases the DW to FW ratio and content of photosynthetic pigments. We demonstrate photomorphogenetic effects in the blue region and its interaction with the fractional PFD of the far-red spectral component. Under constant fractional PFD of the blue component, the root number, length of roots and stems, and fresh weight of the plantlets have a correlated nonmonotonous dependence on the fractional PFD of the far-red component.     

Enhancement of polysaccharides production in <Emphasis Type="Italic">Ganoderma lucidum</Emphasis> by the addition of ethyl acetate extracts from <Emphasis Type="Italic">Eupolyphaga sinensis</Emphasis> and <Emphasis Type="Italic">Catharsius molossus</Emphasis>

To screen stimulators from Chinese medicinal insects for mycelial growth and polysaccharides production of Ganoderma lucidum, G. lucidum was inoculated into the media with and without supplementation of medicinal insect extracts. The ethyl acetate extract of Eupolyphaga sinensis at 55 mg l⁻¹ lead to significant increase in both biomass and intracellular polysaccharides (IPS) concentration from 8.53 ± 0.41 to 14.16 ± 0.43 and 1.28 ± 0.09 to 2.13 ± 0.11 g l⁻¹, respectively. In addition, the ethyl acetate extract of Catharsius molossus at 55 mg l⁻¹ significantly enhanced extracellular polysaccharides (EPS) production; the EPS yield increased from 350.9 ± 14.1 to 475.1 ± 15.3 mg l⁻¹. There were no new components in the two types of polysaccharides obtained by the addition of the insect extracts.     

Heterologous expression of polyhydroxyalkanoate depolymerase from <Emphasis Type="Italic">Thermobifida</Emphasis> sp. in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and catalytic analysis by surface plasmon resonance

A polyhydroxyalkanote depolymerase gene from Thermobifida sp. isolate BCC23166 was cloned and expressed as a C-terminal His₆-tagged fusion in Pichia pastoris. Primary structure analysis revealed that the enzyme PhaZ-Th is a member of a proposed new subgroup of SCL-PHA depolymerase containing a proline–serine repeat linker. PhaZ-Th was expressed as two glycosylated forms with apparent molecular weights of 61 and 70 kDa, respectively. The enzyme showed esterase activity toward p-nitrophenyl alkanotes with V _max and K _m of 3.63 ± 0.16 μmol min⁻¹ mg⁻¹ and 0.79 ± 0.12 mM, respectively, on p-nitrophenyl butyrate with optimal activity at 50–55°C and pH 7–8. Surface plasmon resonance (SPR) analysis demonstrated that PhaZ-Th catalyzed the degradation of poly-[(R)-3-hydroxybutyrate] (PHB) films, which was accelerated in (R)-3-hydroxyvalerate copolymers with a maximum degradation rate of 882 ng cm⁻² h⁻¹ for poly[(R)-3-hydroxybutyrate-co-3-hydroxyvalerate] (12 mol% V). Surface deterioration, especially on the amorphous regions of PHB films was observed after exposure to PhaZ-Th by atomic force microscopy. The use of P. pastoris as an alternative recombinant system for bioplastic degrading enzymes in secreted form and a sensitive SPR analytical technique will be of utility for further study of bioplastic degradation.     

Isolation of C-phycocyanin from <Emphasis Type="Italic">Synechococcus</Emphasis> sp., (<Emphasis Type="Italic">Anacystis nidulans</Emphasis> BD1)

We report a procedure for obtaining fairly pure phycocyanin from a local isolate of the cyanobacterium Synechococcus sp (Anacystis nidulans BD1). Cells were incubated with 1 mg∙mL⁻¹ of lysozyme at 37°C for 16 h with shaking. The cell-free extract was treated with activated charcoal and chitosan. The purity (A _620/280) of phycocyanin obtained after lysozyme treatment was up to 2.18, which could be improved to 4.72 after incubation with activated charcoal and chitosan. The yield of phycocyanin was 80–100 mg∙g⁻¹ dry weight of cells. The method reported here is a single-step and efficient procedure and has the potential to be adopted for large-scale production of phycocyanin.     

Kinetics of styrene biodegradation by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. E-93486

The research into kinetics of styrene biodegradation by bacterial strain Pseudomonas sp. E-93486 coming from VTT Culture Collection (Finland) was presented in this work. Microbial growth tests in the presence of styrene as the sole carbon and energy source were performed both in batch and continuous cultures. Batch experiments were conducted for initial concentration of styrene in the liquid phase changed in the range of 5–90 g m⁻³. The Haldane model was found to be the best to fit the kinetic data, and the estimated constants of the equation were: μ _m = 0.1188 h⁻¹, K _S = 5.984 mg l⁻¹, and K _i = 156.6 mg l⁻¹. The yield coefficient mean value Y_\textxs^\textapp Y_{\text{xs}}^{\text{app}} for the batch culture was 0.72 g_{dry cells weight} (g_substrate)⁻¹. The experiments conducted in a chemostat at various dilution rates (D = 0.035–0.1 h⁻¹) made it possible to determine the value of the coefficient for maintenance metabolism m _d = 0.0165 h⁻¹ and the maximum yield coefficient value Y_\textxs^\textM = 0.913 Y_{\text{xs}}^{\text{M}} = 0.913 . Chemostat experiments confirmed the high value of yield coefficient Y_\textxs^\textapp Y_{\text{xs}}^{\text{app}} observed in the batch culture. The conducted experiments showed high activity of the examined strain in the styrene biodegradation process and a relatively low sensitivity to inhibition of its growth at higher concentrations of styrene in the solution. Such exceptional features of Pseudomonas sp. E-93486 make this bacterial strain the perfect candidate for technical applications.     

Activity of antioxidant enzyme during <Emphasis Type="Italic">in vitro</Emphasis> organogenesis in <Emphasis Type="Italic">Crocus sativus</Emphasis>

The effect of various hormonal combinations on regeneration of shoots and roots from meristem-derived callus of Crocus sativus L. and activities of antioxidant enzymes have been studied. The most efficient regeneration occurred with 1.0 mg dm⁻³ 1-naphthaleneacetic acid (NAA) + 1.0 mg dm⁻³ thidiazuron and 1.0 mg dm⁻³ NAA + 2.0 mg dm⁻³ kinetin. For sprouting, regenerated shoot were subcultured on Murashige and Skoog medium containing 1.0 mg dm⁻³ NAA + 1.0 mg dm⁻³ benzylaminopurine (BAP). Protein content and superoxide dismutase activity decreased in regenerated shoots and roots and increased in sprouting shoots, while catalase (CAT), peroxidase (POX) and polyphenol oxidase (PPO) activities increased during organogenesis and decreased in sprouting shoots. High CAT and PPO activities were detected in regenerated roots, whereas high POX activity was observed in regenerated shoot.     

Simple and efficient expression of <Emphasis Type="Italic">Agaricus meleagris</Emphasis> pyranose dehydrogenase in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Pyranose dehydrogenase (PDH) is a fungal flavin-dependent sugar oxidoreductase that is highly interesting for applications in organic synthesis or electrochemistry. The low expression levels of the filamentous fungus Agaricus meleagris as well as the demand for engineered PDH make heterologous expression necessary. Recently, Aspergillus species were described to efficiently secrete recombinant PDH. Here, we evaluate recombinant protein production with expression hosts more suitable for genetic engineering. Expression in Escherichia coli resulted in no soluble or active PDH. Heterologous expression in the methylotrophic yeast Pichia pastoris was investigated using two different signal sequences as well as a codon-optimized sequence. A 96-well plate activity screening for transformants of all constructs was established and the best expressing clone was used for large-scale production in 50-L scale, which gave a volumetric yield of 223 mg L⁻¹ PDH or 1,330 U L⁻¹ d⁻¹ in space–time yield. Purification yielded 13.4 g of pure enzyme representing 95.8% of the initial activity. The hyperglycosylated recombinant enzyme had a 20% lower specific activity than the native enzyme; however, the kinetic properties were essentially identical. This study demonstrates the successful expression of PDH in the eukaryotic host organism P. pastoris paving the way for protein engineering. Additionally, the feasibility of large-scale production of the enzyme with this expression system together with a simplified purification scheme for easy high-yield purification is shown.     

Stimulation of saponin production in <Emphasis Type="Italic">Panax ginseng</Emphasis> hairy roots by two oligosaccharides from <Emphasis Type="Italic">Paris polyphylla</Emphasis> var. <Emphasis Type="Italic">yunnanensis</Emphasis>

Two oligosaccharides, a heptasaccharide (HS) and an octasaccharide (OS), isolated from Paris polyphylla var. yunnanensis, stimulated the growth and saponin accumulation of Panax ginseng hairy roots at 5–30 mg l⁻¹. HS and OS at 30 mg l⁻¹, fed separately to hairy root cultures at 10 days post-inoculation, increased the root biomass dry weight by more than 70% to ∼20 g l⁻¹ from 13 g l⁻¹ and the total saponin content of roots by more than 1-fold to ∼3.5% from 1.6% (w/w). The results suggest that the two oligosaccharides may have plant growth-regulatory activity in plant tissue cultures.     

Changes in variable charge and acidity of rye (<Emphasis Type="Italic">Secale cereale</Emphasis> L<Emphasis Type="Italic">.</Emphasis>) roots surface under Zn-stress

The plants of winter rye variety Rostockie grown in nutrient solution prepared according to Marschner and Romheld (1983) were stressed at shooting stage with different zinc (ZnCl₂) concentrations of 0, 20, 200 and 400 mgXdm⁻³ during 10 days at pH = 4.5. The control plants were grown continuously at pH = 7, without Zn²⁺. The roots of all plants were titrated with 0.1 molXdm⁻³ NaOH in 1 molXdm⁻³ NaCl solution with a rate 0.01 ml/min using auto-titrator Titrino 702 (Metrohm). The amount of the base consumed between pH 3 and 10 was recorded with a step of 0.1 pH unit. From the titration data root surface charge was characterized. Roots grown at pH = 4.5 without Zn²⁺ addition, had apparently the same charge properties as the control roots. Under 200 and 400 mgXdm⁻³ of zinc addition, variable charge, Q, and the cation exchange capacity, CEC, of the roots significantly decreased as did the fractions of surface acidic functional groups of strong and medium acidity i.e. the groups having apparent surface dissociation constants, Kapp, between 10^−3.5 and 10^−5.5. For 20 mgXdm⁻³ zinc stress the fraction of weaker surface groups (surface dissociation constants around 10^−6.5) decreased also. The surface charge density, SCD, of roots decreased under the Zn stress.     

Characterization of Cadmium-Binding Ligands from Roots of <Emphasis Type="Italic">Echinochloa crusgalli</Emphasis> var. <Emphasis Type="Italic">frumentacea</Emphasis>

Herbaceous Echinochloa crusgalli var. frumentacea is highly resistant to a wide range of heavy metal concentrations. However, its detoxification mechanism has not been reported yet. We exposed plants to 100 μM CdCl₂ for 7 days then examined cadmium accumulation and its subcellular distribution in binding to ligands. Cd concentration increased over the exposure period to a saturation point at day 5 when its level reached 1,732.41 μg g⁻¹ dry weight, an amount about 820 times greater than that found in the control. In the roots, most Cd was distributed in the insoluble fraction, perhaps because of an absorption mechanism at the root surface. Our profile for distribution revealed two Cd-binding ligand peaks: a high molecular weight of 60 kDa and a 2.5-kDa Cd-binding ligand. The latter increased with time and had a typical phytochelatin (PC) amino acid composition of predominantly cysteine, glutamate, and glycine (16.5%, 16.6%, and 11.9%, respectively). The ratio of glutamate/cysteine/glycine was 1.4:1.4:1.0, which is similar to that for other typical PCs.     

Acetic acid acts as an elicitor exerting a chitosan-like effect on xanthone biosynthesis in <Emphasis Type="Italic">Hypericum perforatum</Emphasis> L. root cultures

Key message

Acetic acid acts as a signal molecule, strongly enhancing xanthone biosynthesis in Hypericum perforatum root cultures. This activity is specific, as demonstrated by the comparison with other short-chain monocarboxylic acids.

Abstract

We have recently demonstrated that Hypericum perforatum root cultures constitutively produce xanthones at higher levels than the root of the plant and that they respond to chitosan (CHIT) elicitation with a noteworthy increase in xanthone production. In the present study, CHIT was administered to H. perforatum root cultures using three different elicitation protocols, and the increase in xanthone production was evaluated. The best results (550 % xanthone increase) were obtained by subjecting the roots to a single elicitation with 200 mg l^?1 CHIT and maintaining the elicitor in the culture medium for 7 days. To discriminate the effect of CHIT from that of the solvent, control experiments were performed by administering AcOH alone at the same concentration used for CHIT solubilization. Unexpectedly, AcOH caused an increase in xanthone production comparable to that observed in response to CHIT. Feeding experiments with ¹³C-labeled AcOH demonstrated that this compound was not incorporated into the xanthone skeleton. Other short-chain monocarboxylic acids (i.e., propionic and butyric acid) have little or no effect on the production of xanthones. These results indicate that AcOH acts as a specific signal molecule, able to greatly enhance xanthone biosynthesis in H. perforatum root cultures.

     

Energy Budget for the Cultured,Zooxanthellate Octocoral <Emphasis Type="Italic">Sinularia flexibilis</Emphasis>

The zooxanthellate octocoral Sinularia flexibilis is a producer of potential pharmaceutically important metabolites such as antimicrobial and cytotoxic substances. Controlled rearing of the coral, as an alternative for commercial exploitation of these compounds, requires the study of species-specific growth requirements. In this study, phototrophic vs. heterotrophic daily energy demands of S. flexibilis was investigated through light and Artemia feeding trials in the laboratory. Rate of photosynthetic oxygen by zooxanthellae in light (≈200 μmol quanta m⁻² s⁻¹) was measured for the coral colonies with and without feeding on Artemia nauplii. Respiratory oxygen was measured in the dark, again with and without Artemia nauplii. Photosynthesis–irradiance curve at light intensities of 0, 50, 100, 200, and 400 μmol quanta m⁻² s⁻¹ showed an increase in photosynthetic oxygen production up to a light intensity between 100 and 200 μmol quanta m⁻² s⁻¹. The photosynthesis to respiration ratio (P/R > 1) confirmed phototrophy of S. flexibilis. Both fed and non-fed colonies in the light showed high carbon contribution by zooxanthellae to animal (host) respiration values of 111–127%. Carbon energy equivalents allocated to the coral growth averaged 6–12% of total photosynthesis energy (mg C g ⁻ 1 buoyant weight day ⁻ 1) and about 0.02% of the total daily radiant energy. “Light utilization efficiency (ε)” estimated an average ε value of 75% 12 h ⁻ 1 for coral practical energetics. This study shows that besides a fundamental role of phototrophy vs. heterotrophy in daily energy budget of S. flexibilis, an efficient fraction of irradiance is converted to useable energy.     

Tissue culture of <Emphasis Type="Italic">Sinningia speciosa</Emphasis> and analysis of the <Emphasis Type="Italic">in vitro</Emphasis>-generated <Emphasis Type="Italic">tricussate whorled phyllotaxis</Emphasis> (<Emphasis Type="Italic">twp</Emphasis>) variant

Two protocols were developed for the efficient regeneration of Sinningia speciosa from leaf explants via two developmental pathways. The first method involved formation of callus and buds, followed by subsequent root growth, in Murashige and Skoog medium (MS) containing 2.0 mg l⁻¹ 6-benzylaminopurine (BA) and 0.2 mg l⁻¹ α-naphthalene acetic acid (NAA), with a regeneration efficiency of 99.0%. The second method involved producing callus and roots, followed by subsequent formation of buds, in MS medium supplemented with 1.0–5.0 mg l⁻¹ NAA, and resulted in a regeneration efficiency of 90.4%. Our experiments indicate that the root-first pathway resulted in a lower plant regeneration efficiency. Through five continual generations using the buds-first method, a total of 215 regenerated plants were obtained in the last generation, and eight exhibited a phenotype we named tricussate whorled phyllotaxis (twp). Six of the regenerated twp variant plants maintained their tricussate whorled phyllotaxis phenotype, showing no other abnormalities, while one reverted to a wild type before flowering and another formed two rounds of sepals. Physiological analysis revealed that the twp plants responded differently than wild type to exogenous NAA and 2,3,5-triiodobenzoic acid (TIBA), while high-performance liquid chromatography (HPLC) analysis showed that the levels of endogenous indole-3-acetic acid (IAA) and gibberellin (GA) were lower in twp than wild-type plants. These results suggest that the formation of the twp mutant may be related to phytohormones and that the twp variant could be an important material for investigating the molecular mechanism of plant phyllotaxis patterning.     

Highly efficient plant regeneration and <Emphasis Type="Italic">in vitro</Emphasis> polyploid induction using hypocotyl explants from diploid mulberry (<Emphasis Type="Italic">Morus multicaulis</Emphasis> Poir.)

Efficient plant regeneration is essential for successful transformation and in vitro polyploidy induction in mulberry. A high frequency (80%) of plant regeneration from hypocotyls occurred under in vitro conditions in mulberry (Morus multicaulis Poir.). We identified three key factors for enhancing successful regeneration based on earlier work: (1) hypocotyl position, (2) the combination and concentration of growth regulators, and (3) the addition of AgNO₃. The highest frequency of shoot regeneration was achieved using hypocotyl segments, which are proximal to apical meristems, and the optimal culture conditions were Murashige and Skoog’s (MS) (Murashige and Skoog, 1962) basal medium supplemented with 3.0 mg l⁻¹ 6-benzylamino purine, 0.3 mg l⁻¹ indole-3-acetic acid, 0.1% polyvinypyrrolidone, and 1.0 mg/l silver nitrate (AgNO₃) under subdued light at 25 ± 2°C. Treating the shoots with 0.2% colchicine (dipping for 72 h) resulted in a 14% tetraploid frequency, whereas a 20% tetraploid frequency resulted from using a 0.25% colchicine (dripping for 5 d) treatment, as determined by chromosome number counts. The diploid plant chromosome number was 28 (2n = 2x = 28) and that of tetraploid plants was 56 (2n = 4x = 56). Regenerated shoots rooted easily in 8–10 d using half-strength basal MS medium with 0.5 mg l⁻¹ indole-3-butyric acid and were successfully established in the soil.