Anaerobic biodegradation of phenol by <Emphasis Type="Italic">Candida albicans</Emphasis> PDY-07 in the presence of 4-chlorophenol

Biodegradation of phenol and 4-chlorophenol (4-cp) using pure culture of Candida albicans PDY-07 under anaerobic condition was studied. The results showed that the strain could completely degrade up to 1,800 mg/l phenol within 68 h. The capacity of the strain to degrade phenol was higher than that to degrade 4-cp. In the dual-substrate system, 4-cp intensely inhibited phenol biodegradation. Comparatively, low-concentration phenol from 25 to 150 mg/l supplied a carbon and energy source for Candida albicans PDY-07 in the early phase of biodegradation and accelerated the assimilation of 4-cp, which resulted in that 50 mg/l 4-cp was degraded within less time than that without phenol. While the biodegradation of 50 mg/l 4-cp was inhibited in the presence of 200 mg/l phenol. In addition, the intrinsic kinetics of cell growth and substrate degradation were investigated with phenol and 4-cp as single and dual substrates in batch cultures. The results demonstrated that the models adequately described the dynamic behaviors of biodegradation by Candida albicans PDY-07.     

Isolation and characterization of phenol-degrading yeasts from an oil refinery wastewater in Brazil

This study investigated the aerobic degradation of phenol by yeast strains isolated from an oil refinery wastewater from the Northeast of Brazil. The samples displayed low fungal diversity, as only yeast colonies were detected on Sabouraud dextrose agar containing chloramphenicol 0.05% (w/v). Among the isolates, three yeast strains were selected to be evaluated for their potential for degrading high phenol concentrations. These species were identified through morphological and biochemical characteristics as Candida tropicalis, C. rugosa, and Pichia membranaefaciens. Although the strains were able to degrade the phenol concentration present in the wastewater, which was 7 mg l⁻¹, only C. tropicalis was capable of growing at high concentrations of phenol such as 500 mg l⁻¹ and 1,000 mg l⁻¹ in a mineral medium containing this pollutant as the only carbon source. C. rugosa and P. membranaefaciens were inhibited in the presence of 500 mg l⁻¹ of phenol. However, a longer incubation time was needed for C. tropicalis strain to degrade 1,000 mg l⁻¹ of phenol compared to the time required to degrade 500 mg l⁻¹. Moreover, the strain released a significant amount of polysaccharide biosurfactant in the medium probably to minimize the toxic effect of the high phenol concentration. When challenged with 1,500 and 2,000 mg l⁻¹ of phenol, C. tropicalis was unable to grow at the tested conditions. The results indicate that this strain of C. tropicalis can be considered both a good phenol-degrader and biosurfactant-producer. Application of this strain might be useful in bioremediation activities or treatment of phenol-polluted wastewater.     

Single-culture aerobic granules with <Emphasis Type="Italic">Acinetobacter calcoaceticus</Emphasis>

Aerobic granules are cultivated by a single bacterial strain, Acinetobacter calcoaceticus, in a sequencing batch reactor (SBR). This strain presents as a good phenol reducer and an efficient auto coagulator in the presence of phenol, mediated by heat-sensitive adhesins proteins. Stable 2.3-mm granules were formed in the SBR following a 7-week cultivation. These granules exhibit excellent settling attributes and degrade phenol efficiently at concentrations of 250–2,000 mg l⁻¹. The corresponding phenol degradation rate reached 993.6 mg phenol g⁻¹ volatile suspended solids (VSS) day⁻¹ at 250 mg l⁻¹ phenol and 519.3 mg phenol g⁻¹ VSS day⁻¹ at 2,000 mg l⁻¹ phenol concentration. Meanwhile, free A. calcoaceticus cells were fully inhibited at phenol >1,500 mg l⁻¹. Denaturing gradient gel electrophoresis fingerprint profile demonstrated no genetic modification in the strain during aerobic granulation. The present single-strain granules showed long-term structural stability and performed high phenol degrading capacity and high phenol tolerance. The confocal laser scanning microscopic test revealed that live A. calcoaceticus cells principally distributed at 200–250 μm beneath the outer surface, with an extracellular polymeric substance layer covering them to defend phenol toxicity. Autoaggregation assay tests demonstrated the possibly significant role of secreted proteins on the formation of single-culture A. calcoaceticus granules.     

Biodegradation of phenol by immobilized Aspergillus awamori NRRL 3112 on modified polyacrylonitrile membrane

Covalent immobilization of Aspergillus awamori NRRL 3112 was conducted onto modified polyacrylonitrile membrane with glutaraldehyde as a coupling agent. The polymer carrier was preliminarily modified in an aqueous solution of NaOH and 1,2-diaminoethane. The content of amino groups was determined to be 0.58 mgeq g⁻¹. Two ways of immobilization were used—in the presence of 0.2 g l⁻¹ phenol and without phenol. The capability of two immobilized system to degrade phenol (concentration—0.5 g l⁻¹) as a sole carbon and energy source was investigated in batch experiments. Seven cycles of phenol biodegradation were conducted. Better results were obtained with the immobilized system prepared in the presence of phenol, regarding degradation time and phenol biodegradation rate. Scanning electron micrographs of the polyacrylonitrile membrane/immobilized Aspergillus awamori NRRL at the beginning of repeated batch cultivation and after the 7th cycle were compared. After the 7th cycle of cultivation the observations showed large groups of cells. The results from the batch experiments with immobilized system were compared to the results produced by the free strain. Phenol biodegradation experiments were carried out also in a bioreactor with spirally wound membrane with bound Aspergillus awamori NRRL 3112 in a regime of recirculation. 10 cycles of 0.5 g l⁻¹ phenol biodegradation were run consecutively to determine the degradation time and rate for each cycle. The design of the bioreactor appeared to be quite effective, providing large membrane surface to bind the strain.     

Enrichment, isolation, and characterization of 4-chlorophenol-degrading bacterium Rhizobium sp. 4-CP-20

The objectives of this research were to monitor the variations of species in mixed cultures during the enrichment period, isolate species and identify and characterize the pure 4-chlorophenol (4-CP) degrading strains from enriched mixed cultures. Strain Rhizobium sp. 4-CP-20 was isolated from the acclimated mixed culture. The DGGE result indicated that strain Rhizobium sp. 4-CP-20 was undetectable at the beginning but detectable after 2 weeks of enrichment. The optimum growth temperatures for Rhizobium sp. 4-CP-20 were both 36°C using 350 mg l⁻¹ glucose or sodium acetate as the substrate. The optimum pH range for degrading 100 mg l⁻¹ 4-CP was between 6.89 and 8.20. Strain Rhizobium sp. 4-CP-20 could degrade 4-CP completely within 3.95 days, as the initial 4-CP concentration was 100 mg l⁻¹. If the initial 4-CP concentration was higher than 240 mg l⁻¹, the growth of bacterial cells and the activity of degrading 4-CP were both inhibited.     

Xylitol conversion by fermentation using five yeast strains and polyelectrolyte-assisted ultrafiltration

Batch fermentations for xylitol production were conducted using Candida boidinii (BCRC 21432), C. guilliermondii (BCRC 21549), C. tropicalis (BCRC 20520), C. utilis (BCRC 20334), and P. anomala (BCRC 21359) together with a mixture of sugars simulating lignocellulosic hydrolysates as the carbon source. C. tropicalis had the highest bioconversion yield (Y_P/S) of 0.79 g g⁻¹ (g xylitol·g xylose⁻¹) over 48 h. Additional fermentations with C. tropicalis achieved Y_P/S values of 0.6 and 0.39 g g⁻¹ after 96 and 72 h using urea and soybean meal as the nitrogen sources, respectively. Ethanol and arabitol were also produced in all fermentation. Xylitol in the fermentation broth was recovered by cross-flow ultrafiltration. With prior application of 2 mg polydiallyl dimethylammonium chloride l⁻¹ on the membrane surface, protein in the permeate was reduced from 7.1 to 1.5 mg l⁻¹ after 2 h.     

Mineralization of diuron [3-(3,4-dichlorophenyl)-1, 1-dimethylurea] by co-immobilized <Emphasis Type="Italic">Arthrobacter</Emphasis> sp. and <Emphasis Type="Italic">Delftia acidovorans</Emphasis>

Mineralization of diuron has not been previously demonstrated despite the availability of some bacteria to degrade diuron into 3,4-dichloroaniline (3,4-DCA) and others that can mineralize 3,4-DCA. A bacterial co-culture of Arthrobacter sp. N4 and Delftia acidovorans W34, which respectively degraded diuron (20 mg l⁻¹) to 3,4-DCA and mineralized 3,4-DCA, were able to mineralize diuron. Total diuron mineralization (20 mg l⁻¹) was achieved with free cells in co-culture. When the bacteria were immobilized (either one bacteria or both), the degradation rate was higher. Best results were obtained with free Arthrobacter sp. N4 cells co-cultivated with immobilized cells of D. acidovorans W34 (mineralization of diuron in 96 h, i.e., 0.21 mg l⁻¹ h⁻¹ vs. 0.06 mg l⁻¹ h⁻¹ with free cells in co-culture).     

Concentration dependent growth/non-growth linked kinetics of endosulfan biodegradation by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

The rates of biodegradation of endosulfan by P. aeruginosa were determined with different initial endosulfan concentrations (10, 50, 100, 150, 200 and 250 mg l⁻¹) and different growth linked kinetic models were fitted at these concentrations. At 10 mg endosulfan l⁻¹, Monod no growth model was well fitted. Monod with growth model described the biodegradation pattern at an initial concentration of 50, 100 and 150 mg endosulfan l⁻¹. Significant increases of P. aeruginosa MN2B14 density in broth culture during incubation further support this result. Conversely, zero order kinetic model was well fitted into the biodegradation data if initial endosulfan concentration was ≥200 mg endosulfan l⁻¹. The kinetics of endosulfan biodegradation by P. aeruginosa MN2B14 in liquid broth was highly dependent upon its initial concentration. The results of this study could be employed for predicting the persistence of endosulfan in water environment containing P. aeruginosa as an endosulfan degrading bacterium.     

Continuous phenol biodegradation in a simple packed bed bioreactor of calcium alginate-immobilized <Emphasis Type="Italic">Candida tropicalis</Emphasis> (NCIM 3556)

Phenol biodegradation in a continuous system of immobilized Candida tropicalis NCIM 3556 was studied. The bioreactor was simple, it had a feed inlet from the bottom and the effluent outlet from top, no supplementary oxygen was supplied, the reactor was operated continuously for 116 days. Initially the column was run continuously with a feed concentration of 2 g l⁻¹ for 42 days whence a degradation of >97% was achieved. The feed concentration was then increased to 3 g l⁻¹, for which a ~80% biodegradation was sustained for 90 days after which there was a steady decrease in the performance. When the phenol degradation was reduced to ~50% in 116 days, the reactor was stopped. The efficiency of free cells recycled every 24 h and immobilized cells were compared; it was estimated that repeated reuse of free cells in batch mode gave an overall efficiency of 0.102 g phenol degradation g⁻¹ cell wet weight in 12 days. In contrast, the immobilized system of the same biomass had a longer working lifetime of ~4 months indicating an efficiency of 3.72 g phenol g⁻¹ cell wet wt.     

Enhanced degradation of phenol by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. CP4 entrapped in agar and calcium alginate beads in batch and continuous processes

Phenol is one of the major toxic pollutants in the wastes generated by a number of industries and needs to be eliminated before their discharge. Although microbial degradation is a preferred method of waste treatment for phenol removal, the general inability of the degrading strains to tolerate higher substrate concentrations has been a bottleneck. Immobilization of the microorganism in suitable matrices has been shown to circumvent this problem to some extent. In this study, cells of Pseudomonas sp. CP4, a laboratory isolate that degrades phenol, cresols, and other aromatics, were immobilized by entrapment in Ca-alginate and agar gel beads, separately and their performance in a fluidized bed bioreactor was compared. In batch runs, with an aeration rate of 1 vol⁻¹ vol⁻¹ min⁻¹, at 30°C and pH 7.0 ± 0.2, agar-encapsulated cells degraded up to 3000 mg l⁻¹ of phenol as compared to 1500 mg l⁻¹ by Ca-alginate-entrapped cells whereas free cells could tolerate only 1000 mg l⁻¹. In a continuous process with Ca-alginate entrapped cells a degradation rate of 200 mg phenol l⁻¹ h⁻¹ was obtained while agar-entrapped cells were far superior and could withstand and degrade up to 4000 mg phenol l⁻¹ in the feed with a maximum degradation rate of 400 mg phenol l⁻¹ h⁻¹. The results indicate a clear possibility of development of an efficient treatment technology for phenol containing waste waters with the agar-entrapped bacterial strain, Pseudomonas sp. CP4.     

Incorporation of granular activated carbon in an immobilized membrane bioreactor for the biodegradation of phenol by <Emphasis Type="Italic">Pseudomonas putida</Emphasis>

Granular activated carbon (GAC) was incorporated into hollow fiber membrane bioreactors for the biodegradation of 1,000 mg phenol l⁻¹ through immobilization of Pseudomonas putida. The phenol was removed within 25 h in the hybrid bioreactor, comparing with 31 h for a GAC-free bioreactor. Sorption, biodegradation, desorption, and bioregeneration were four steps for the phenol removal during batch operation.     

Isolation and Characterization of a Pseudomonas Oleovorans Degrading the Chloroacetamide Herbicide Acetochlor

To date, no pure bacterial cultures that could degrade acetochlor have been described. In this study, one strain of microorganism capable of degrading acetochlor, designated as LCa2, was isolated from acetochlor-contaminated soil. The strain LCa2 is Pseudomonas oleovorans according to the criteria of Bergey’s manual of determinative bacteriology and sequence analysis of the partial 16S rRNA gene. Optimum growth temperature and pH were 35 °C and 8.0, respectively. The strain could degrade 98.03% of acetochlor treated at a concentration of 7.6 mg l⁻¹ after 7 days of incubation and could tolerate 200 mg l⁻¹ of acetochlor. When the acetochlor concentration became higher, the degradation cycle became longer. The acetochlor biodegradation products were identified by GC–MS based on mass spectral data and fragmentation patterns. The main plausible degradative pathways involved dechlorination, hydroxylation, N-dealkylation, C-dealkylation and dehydrogenation.     

Establishment of an <Emphasis Type="Italic">Agrobacteriuim</Emphasis>-mediated cotyledon disc transformation method for <Emphasis Type="Italic">Jatropha curcas</Emphasis>

Jatropha curcas contains high amounts of oil in its seed and has been considered for bio-diesel production. A transformation procedure for J. curcas has been established for the first time via Agrobacterium tumefaciens infection of cotyledon disc explants. The results indicated that the efficiency of transformation using the strain LBA4404 and phosphinothricin for selection was an improvement over that with the strain EHA105 and hygromycin. About 55% of the cotyledon explants produced phosphinothricin-resistant calluses on Murashige and Skoog (MS) medium supplemented with 1.5 mg l⁻¹ benzyladenine (BA), 0.05 mg l⁻¹ 3–indolebutyric acid (IBA), 1 mg l⁻¹ phosphinothricin and 500 mg l⁻¹ cefotaxime after 4 weeks. Shoots were regenerated following transfer of the resistant calli to shoot induction medium containing 1.5 mg l⁻¹ BA, 0.05 mg l⁻¹ IBA, 0.5 mg l⁻¹ gibberellic acid (GA3), 1 mg l⁻¹ phosphinothricin and 250 mg l⁻¹ cefotaxime, and about 33% of the resistant calli differentiated into shoots. Finally, the resistant shoots were rooted on 1/2 MS media supplemented with 0.3 mg l⁻¹ IBA at a rate of 78%. The transgenic nature of the transformants was demonstrated by the detection of β-glucuronidase activity in the primary transformants and by PCR and Southern hybridization analysis. 13% of the total inoculated explants produced transgenic plants after approximately 4 months. The procedure described will be useful for both, the introduction of desired genes into J. curcas and the molecular analysis of gene function.     

Biodegradation of nicotine from tobacco waste extract by <Emphasis Type="Italic">Ochrobactrum intermedium</Emphasis> DN2

Ochrobactrum intermedium DN2 was used to degrade nicotine in tobacco waste extracts. The optimal temperature and pH of nicotine degradation by strain DN2 was 30–37 °C and 7.0, respectively. Under these optimal conditions, the average degradation rate of nicotine in a 30L fed-batch culture was 140.5 mg l⁻¹ h⁻¹. The results of this study indicate that strain DN2 may be useful for reducing the nicotine content of reconstituted tobacco.     

Degrading high-strength phenol using aerobic granular sludge

Aerobic granules were adopted to degrade high-strength phenol wastewater in batch experiments. The acclimated granules effectively degraded phenol at a concentration of up to 5,000 mg l⁻¹ without severe inhibitory effects. The biodegradation of phenol by activated sludge was inhibited at phenol concentrations >3,000 mg l⁻¹. The granules were composed of cells embedded in a compact extracellular matrix. After acid or alkaline pretreatment, the granules continued to degrade phenol at an acceptable rate. The polymerase chain reaction-denaturing gradient gel electrophoresis technique was employed to monitor the microbial communities of the activated sludge and the aerobic granules following their being used to treat high concentrations of phenol in batch tests.     

Agrobacterium tumefaciens-mediated transformation of Lesquerella fendleri L., a potential new oil crop with rich lesquerolic acid

A protocol was developed for regeneration and Agrobacterium-mediated genetic transformation of Lesquerella fendleri. Calli were first induced from hypocotyls and cotyledons on MS plus 0.5 mg l⁻¹ BA, 1 mg l⁻¹ NAA and 1 mg l⁻¹ 2,4-D, then co-cultivated for 2–3 days in darkness on MS supplemented with 0.5 mg l⁻¹ BA, 0.2 mg l⁻¹ NAA and 100 μmol l⁻¹As together with Agrobacterium tumefaciens strain EHA105/pCAMBIA1301 that harbored genes for uidA (GUS) and hygromycin resistance. Following co-cultivation, calli transfected by A. tumefaciens were transferred to MS with 0.5 mg l⁻¹ BA, 0.2 mg l⁻¹NAA, 500 mg l⁻¹ Cef and 10 mg l⁻¹ hygromycin and cultured for 10 days, then the hygromycin was increased to 20 mg l⁻¹ on the same medium. After 4 weeks the resistant regenerants were transferred to MS with 0.5 mg l⁻¹BA, 0.2 mg l⁻¹ NAA, 500 mg l⁻¹ Cef and 25 mg l⁻¹ hygromycin for further selections. Transgenic plants were confirmed by polymerase chain reaction analysis, GUS histochemical assay and genomic Southern blot hybridization. With this approach, the average regeneration frequency from transfected calli was 22.70%, and the number of regenerated shoots per callus was 6–13. Overall results described in this study demonstrate that Agrobacterium-mediated transformation is a promising approach for improvement of this Lesquerella species.     

Interspecific hybridization of <Emphasis Type="Italic">Prunus persica</Emphasis> with <Emphasis Type="Italic">P. armeniaca</Emphasis> and <Emphasis Type="Italic">P. salicina</Emphasis> using embryo rescue

Embryo rescue technique was used successfully to produce interspecific hybrids by crossing peach (P. persica) as a female parent with apricot (P. armeniaca) and plum (P. salicica). In those crosses that had ‘Yuhualu’ or ‘Zhonghuashoutao’ as female parents, hybrid embryos aborted from the 7th or 8th week after pollination mainly due to post-pollination incompatibility. An embryo rescue protocol was established to rescue such embryos and recover hybrid plants. Modified half-strength MS medium containing 4 mg l⁻¹ 6-BA and 0.5 mg l⁻¹ IBA produced up to 90% germination in the embryos. Modified MS medium with 1.0 mg l⁻¹ 6-BA and 1.0 mg l⁻¹ IBA gave the highest bud induction and multiplication whereas modified MS medium containing 0.5 mg l⁻¹ IAA and 0.2 mg l⁻¹ NAA gave the best rooting percentage. All the hybrids obtained using this embryo rescue technique were verified using simple sequence repeat (SSR) markers. A series of pollen treatments were carried out to partially overcome pre-pollination incompatibility, and it was found accidentally that pollen treatment with electrostatic field not only improved pollen germination but also increased the multiplication coefficient of embryo-induced shoots.     

A reliable protocol for plant regeneration from callus culture of <Emphasis Type="Italic">Phalaenopsis</Emphasis>

Summary Callus of Phalaenopsis Nebula was induced from seed-derived protocorms on 1/2 Murashige and Skoog (MS) basal medium plus 0–1.0 mg l⁻¹ (0–4.52 μM) N-phenyl-N′-1,2,3,-thiadiazol-5-yl urea (TDZ) and/or 0–10 mg l⁻¹ (0–45.24 μ M) 2,4-dichlorophenoxyacetic acid (2,4-D). Protocorms 2 mo. old performed better than 1-mo.-old protocorms for callus induction. More calluses formed on 1/2 MS basal medium supplemented with 0.1–1.0 mg l⁻¹ (0.45–4.52 μM) TDZ. These calluses could be maintained by subculturing every month with basal medium supplemented with 0.5 mg l⁻¹ (2.27 μM) TDZ and 0.5 mg l⁻¹ (2.26 μM) 2,4-D. Protocorm-like bodies were formed, and plants regenerated from these calluses on 1/2 MS basal medium alone or supplemented with 0.1–1.0 mg l⁻¹ (0.45–4.52 μM) TDZ. Plantlets were then potted on sphagnum moss in the greenhouse and grew well. No chromosomal abnormalities were found among the root-tip samples of 21 of the regenerated plantlets that were successfully acclimatized.     

Establishment of a highly efficient <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated leaf disc transformation method for <Emphasis Type="Italic">Broussonetia papyrifera</Emphasis>

Broussonetia papyrifera is well-known for its bark fibers, which are used for making paper, cloth, rope etc. This is the first report of a successful genetic transformation protocol for B. papyrifera using Agrobacterium tumefaciens. Callus was initiated at a frequency of about 100% for both leaf and petiole explants. Shoots formed on these calli with a success rate of almost 100%, with 14.08 and 8.36 shoots regenerating from leave-derived and petiole-derived callus, respectively. For genetic transformation, leaf explants of B. papyrifera were incubated with A. tumefaciens strain LBA4404 harboring the binary vector pCAMBIA 1301 which contains the hpt gene as a selectable marker for hygromycin resistance and an intron-containing β-glucuronidase gene (gus-int) as a reporter gene. Following co-cultivation, leaf explants were cultured on Murashige and Skoog (Physiol Plant 15:473, 1962) (MS) medium supplemented with 1.5 mg l⁻¹ benzyladenine (BA) and 0.05 mg l⁻¹ indole-3-butyric acid (IBA) (CI medium) containing 5 mg l⁻¹ hygromycin and 500 mg l⁻¹ cefotaxime, in the dark. Hygromycin-resistant calli were induced from leaf explants 3 weeks thereafter. Regenerating shoots were obtained after transfer of the calli onto MS medium supplemented with 1.5 mg l⁻¹ BA, 0.05 mg l⁻¹ IBA, and 0.5 mg l⁻¹ gibberellic acid (GA3) (SI medium), 5 mg l⁻¹ hygromycin and 250 mg l⁻¹ cefotaxime under fluorescent light. Finally, shoots were rooted on half strength MS medium (1/2 MS) supplemented with 10 mg l⁻¹ hygromycin. Transgene incorporation and expression was confirmed by PCR, Southern hybridisation and histochemical GUS assay. Using this protocol, transgenic B. papyrifera plants containing desirable new genes can be obtained in approximately 3 months with a transformation frequency as high as 44%.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of the chinese medicinal plant, <Emphasis Type="Italic">Dendrobium candidum</Emphasis> wall. ex lindl., from axenic nodal segments

Summary Dendrobium candidum Wall. Ex Lindl. is an important species used in the formulation of Shih-hu, a Chinese traditional medicine. An efficient protocol for in vitro propagation of D. candidum using the axenic nodal segments of the shoots, originated from the in vitro germinated seedlings, was developed. The seeds from 120-d-old capsules after pollination were first germinated on half-strength Murashige and Skoog (MS) basal medium supplemented with 30 g l⁻¹ sucrose. After 4 mo., the seedlings were subcultured on a similar medium supplemented with 1 ml l⁻¹ HYPONeX, 80 g l⁻¹ potato homogenate and 2 g l⁻¹ activated charcoal for further growth. Axenic nodal segments excised from 9-mo.-old seedlings were cultured on the medium in the presence of 2 mg l⁻¹ benzyladenine (BA) and 0.1 mg l⁻¹ naphthaleneacetic acid (NAA). After 75 d, 73.2% of the explants gave rise to buds/shoots. The elongated shoots were rooted on the medium containing 0.2 mg l⁻¹ NAA and the plantlets were successfully acclimatized in soil.