Effect of carbon dioxide on cell growth and saponin production in suspension cultures of Panax ginseng

The effects of carbon dioxide supply within the range of 1–5 % (along with purified air), on cell culture of Panax ginseng were investigated in a balloon type bubble bioreactor containing 4 dm³ of Murashige and Skoog (MS) medium supplemented with 7.0 mg dm⁻³ indolebutyric acid, 0.5 mg dm⁻³ kinetin and 30 g dm⁻³ sucrose. A 1 % CO₂ supply was found beneficial for the production of cell mass; however, increasing CO₂ concentration to 2.5 and 5 % decreased the biomass accumulation. CO₂ enrichment was not beneficial for saponin production and 1, 2.5, and 5 % CO₂ supply resulted in decrease in saponin accumulation up to 11.6, 19.5, and 50.6 %, respectively.     

Effects of sucrose concentration and exogenous hormones on growth and periplocin accumulation in adventitious roots of Periploca sepium Bunge

Periploca sepium adventitious roots were cultured on 0.5 Murashige and Skoog solid media supplemented with exogenous hormones of different types and various concentrations, and with sucrose of different concentrations. Auxins (indole butyric acid (IBA) and naphthalene acetic acid (NAA)) and cytokinins (6-benzylaminopurine (BA) and kinetin (KT)) were selected as exogenous hormones for adventitious root proliferation. Compared with other hormones, IBA was the suitable auxin for adventitious root proliferation. Under this circumstance, every root explant generates 10?C15 adventitious roots (1- to 2-cm long) after 30?days. However, nothing but callus was induced on the root explants when NAA was added into the medium and the same result was achieved when auxins (IBA or NAA) were added into the media together with cytokinins (BA or KT). The suitable concentration of IBA for adventitious root proliferation was 1?C2?mg/l, when every root explant generated 10?C20 adventitious roots (1- to 2-cm long). The optimum concentration of IBA for periplocin accumulation was 1?mg/l, when the periplocin content reached 95.46???g/g. With regard to the investigation of sucrose concentration, 2?C3% (w/v) sucrose was favorable for adventitious root proliferation as every root explant in this concentration generated 10?C20 adventitious roots (1- to 2-cm long). The highest periplocin content (101.56???g/g) was achieved at 5% (w/v) sucrose, whereas the periplocin content at 5% (w/v) sucrose did not show significant difference from the periplocin content (95.38 and 98.47???g/g, respectively) at 3% (w/v) or 4% (w/v) sucrose.     

Efficient and repetitive production of leaf-derived somatic embryos of Oncidium

Oncidium cultivars gave different embryogenic responses of leaf explants when affected by auxins (2,4-D, IAA, IBA and NAA), cytokinins (2iP, BA, kinetin, TDZ and zeatin), sucrose, NaH₂PO₄, casein hydrolysate, peptone, and glutamine. The best embryogenic responses of cv. Sweet Sugar were at 20 g dm⁻³ sucrose, 85 mg dm⁻³ NaH₂PO₄ and 3 mg dm⁻³ kinetin, respectively. The development of somatic embryos on leaf explants of cv. Sweet Sugar was delayed for about 10 – 20 d in comparison with cv. Gower Ramsey. On growth regulator-free medium, about 40 % of leaf derived embryos of cv. Gower Ramsey were fused together in their basal parts and so called multiple-state embryos. However, under the same condition, the embryos of cv. Sweet Sugar were all in multiple-state form.     

Achillea millefolium (yarrow) cell suspension cultures: Establishment and growth conditions

Summary Friable calli were obtained fromAchillea millefolium L. hypocotyls, in Gamborg B5 medium, supplemented with 1.5mg.1^–1 2,4-D / 0.1mg.1^–1 Kin, and used for the production of cell suspension cultures in the same liquid medium. The growth pattern of the cultures was determined in permanent light or dark conditions and with different inoculum densities, basal media, growth regulators and sucrose concentrations. Different sources and nitrogen amounts were assayed to study the effect on yarrow cell growth. The conditions found to be optimal for growth of yarrow cell suspension cultures were: 70g (f.w.).1^–1 of initial inoculum in Gamborg B5 medium, supplemented with 1.5mg. 1^–1 2,4-D / 0.1mg.1^–1 Kin, NO₃ ^–/NH₄ ⁺ (30/lmM), and 2% sucrose, in darkness. In these culture conditions the cell suspensions showed a doubling time of 35–40h.Abbreviations 2,4-D dichlorophenoxyacetic acid - NAA naphtalenacetic acid - BA benzyladenine - Kin Kinetin     

Culture medium optimization for camptothecin production in cell suspension cultures of Nothapodytes nimmoniana (J. Grah.) Mabberley

The effect of culture medium nutrients on growth and alkaloid production by plant cell cultures of Nothapodytes nimmoniana (J. Grah.) Mabberley (Icacinaceae) was studied with a view to increasing the production of the alkaloid camptothecin, a key therapeutic drug used for its anticancer properties. Amongst the various sugars tested with Murashige and Skoog (MS) medium, such as glucose, fructose, maltose, and sucrose, maximum accumulation of camptothecin was observed with sucrose. High nitrate in the media supports the biomass, while high ammonium enhances the camptothecin content. Selective feeding of 60 mM total nitrogen with a NH₄ ⁺/NO₃ ^? balance of 5/1 on day 15 of the culture cycle results in a 2.4-fold enhancement in the camptothecin content over the control culture (28.5 μg/g DW). Furthermore, the sucrose feeding strategy greatly stimulated cell biomass and camptothecin production. A modified MS medium was developed in the present study, which contained 0.5 mM phosphate, a nitrogen source feeding ratio of 50/10 mM NH₄ ⁺/NO₃ ^? and 3 % sucrose with additional 2 % sucrose feeding (added on day 12 of the cell culture cycle) with 10.74 μM naphthaleneacetic acid and 0.93 μM kinetin. Finally, the selective medium has 1.7- and 2.3-fold higher intracellular and extracellular camptothecin content over the control culture (29.2 and 8.2 μg/g DW), respectively.     

Characteristics of transformedPanax ginseng C.A. Meyer hairy roots: Growth and nutrient profile

Ginseng (Panax ginseng C.A. Meyer) hairy root cultures, which are established via the infection of ginseng root discs withRhizobium rhizogenes, have been used to construct profiles of both biomass growth and nutrient consumption in flask cultures. In a 250 mL shake flask culture, the maximum biomass was observed on the 59th day of the culture period, at 216.8 g (fresh wt) per liter or 11.4 g (dry wt) per liter. The hairy roots were determined to have a growth rate of 0.355 g-DW/g cells/day during the exponential growth phase and a maximum specific growth rate on day 7. Total ginseng saponin and phenolic compound contents were noted to have increased within the latter portion of the culture period. Linear correlations between increases in biomass weight and nutrient uptake were used to imply the conductivity yield 2.60 g-DW/(L·mS) and carbon yield 0.45 g-DW/(g sugar) in the 250 mL flask cultures. The biomass yield when two different nitrogen sources were used (ammonia and nitrate) was shown to remain approximately constant, at 0.47 g-DW/(l·mM NH₄) and 0.33 g-DW/(L·mM NO₃); it remained at these levels for 16 days with the ammonia, and for 24 days with the nitrate. The biomass yield when a phosphate source was used was also shown to remain approximately constant for 9 days, at 3.17 g-DW/(L·mM PO₄), with an R² of 0.99.     

Effects of Nitrogen, Sucrose, IAA and Kinetin on Explants of Beta vulgaris Grown in vitro

Hypocotyl explants of Beta vulgaris L. were grown on defined agar media with different combinations of IAA and kinetin at varying concentrations of nitrogen or sucrose. The cultures were kept in light (18 h a day) at 27°C for 5 weeks. Root initiation and callus growth were recorded and the callus tissue was analysed for N and K. Root formation was found to increase with increasing nitrogen concentration (from 5 mM to 23.3 mM) in the medium at 10.0 mg/1 of IAA, whereas no stimulation was found at 0.1 mg/1 of IAA. When raising the sucrose level from 20 g/1 to 100 mg/1 at 10.0 mg/1 of IAA and 1.0 mg/1 of kinetin, root initiation was also stimulated. At a lower kinetin and auxin level, however, no increase was recorded. Callus growth was affected by changes in the nitrogen or sucrose concentration of the culture media. The nitrogen content of the callus tissue increased with rising nitrogen concentration of the media. When raising the sucrose level instead of the nitrogen level, the nitrogen content of the tissue decreased.     

Effect of oxygen supply on cell growth and saponin production in bioreactor cultures of Panax ginseng

The effects of oxygen supply within the range 20.8–50% (using pure oxygen and air), on cell cultures of Panax ginseng were investigated in a balloon-type bubble bioreactor (5 L capacity, containing 4 L Murashige and Skoog medium, supplemented with 7.0 mg L⁻¹ indolebutyric acid, 0.5 mg L⁻¹ kinetin and 30 g L⁻¹ sucrose). A 40% oxygen supply was found to be optimal for the production of both cell mass and saponin yielding values of 12.8 g (DW) L⁻¹, 4.5 mg (g DW)⁻¹ on day 25, respectively. Low (20.8%, 30%) and high (50%) oxygen concentration supplies were unfavorable to cell growth and saponin accumulation. The results indicate that oxygen supplementation to bioreactor-based ginseng cultures was beneficial for biomass accumulation and saponin production.     

Effects of source of macronutrients and plant growth regulator concentrations on shoot proliferation of Cornus florida

Nodal stem segments of flowering dogwood (Cornus florida L.) were cultured on media containing seven different sources of macronutrients including full- and half-strength Murashige & Skoog (MS) macrosalts, N6, Anderson's (AND), Quoirin & Lepoivre's (LP), Nitsch & Nitsch (NIT), and Woody Plant Medium (WPM). All media contained MS micronutrients, Staba vitamins, 20 g l^-1 sucrose, and 6.5 g l^-1 Difco Bacto-agar, and were supplemented with 2.2 M 6-benzyladenine (BA) and 0.49 M indole-3-butyric acid (IBA). Following three subcultures, the best shoot proliferation was supported on media containing WPM macronutrients. To optimize the proliferation rate, shoots were cultured on WPM macronutrients supplemented with eight combinations of BA and IBA, and 3.3 M BA without IBA was determined to be the best.Abbreviations BA 6-benzyladenine - IBA indole-3-butyric acid     

Effect of medium acidity on growth and rooting of different plant species growing in vitro

Micropropagated Choisya, Daphne, Delphinium, Hemerocallis, Hosta, Iris and Photinia were found to adjust the pH of Murashige and Skoog's plant tissue culture medium (initial pH 5.6 or 3.5) to different values depending on the species. When plant growth and rooting rates were determined after plants had been grown on media initially adjusted or buffered to values between 2.6 and 5.7 the different plant species were also found to have distinct pH requirements for optimal growth and/or rooting rates.Abbreviations MS Murashige & Skoog's (1962) medium - MS19 MS with additionally 10 g l^–1 sucrose - 80 mg l^–1 adenine sulphate and 130.9 mg l^–1 NaH₂PO₄ - BA 6-benzyladenine - NAA 1-naphthyl-acetic acid - IBA 3-indole-butyric acid - IAA 3-indole-acetic acid - 2iP N₆(2-isopentyl) adenine     

Rapid and efficient regeneration in soybean [<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merrill] from whole cotyledonary node explants

A rapid and efficient regeneration protocol was established for soybeans [Glycine max (L.) Merrill]. Whole cotyledonary node explants were collected from aseptic seedlings cultured on MSB₅ medium containing 0.4 mg l⁻¹ N⁶-benzyladenine (BA). The effects of the plant growth regulators BA, kinetin (KT), indole-3-butyric acid (IBA) as well as the explant genotype on shoot regeneration were evaluated by the orthogonal design [L₁₆(4⁵)]. The process of shoot development was also observed. The regenerated shoots were elongated on MSB₅ medium and sufficiently elongated shoots were rooted on MSB₅ medium containing 0.5 mg l⁻¹ IBA. The results showed that all three of the plant growth regulators significantly affected shoot regeneration, with BA exhibiting the greatest benefit. The best combination for shoot regeneration was MSB₅ medium supplemented with 3.0 mg l⁻¹ BA , 0.2 mg l⁻¹ IBA and 0.5 mg l⁻¹ KT on Hefeng 25 genotype. Under these most favorable conditions, one explant could regenerate 30–35 shoots. Plantlets could be obtained within 2 months. The result of histocytological analysis indicated that protein accumulated gradually and reached to peak at late shoot bud formation.     

Somatic embryogenesis and plant regeneration from immature cotyledons of European chestnut (Castanea sativa Mill.)

Here, we established a protocol for induction of somatic embryogenesis and plant regeneration from immature cotyledons of open-pollinated seeds of European chestnut (Castanea sativa Mill.) cultivars ‘Osmano?lu’ and ‘Sar?a?lama’. Basal media, Murashige and Skoog medium (MS), Driver and Kuniyuki Walnut medium (DKW), and Woody Plant Medium (WPM) supplemented with l-glutamine or casein hydrolysate, with or without silver nitrate, agar or gelrite, and various plant growth regulator (PGR) combinations were tested in initial cultures for induction of somatic embryos. The effects of initial cultures on the percentage of somatic embryos and average number of embryos per cotyledon explant, subcultured monthly, were determined at the end of 4 mo. Interactions were observed among the different treatments for ‘Osmano?lu’ cultivar, with the highest rates of somatic embryogenesis (4.7–9.7%) being obtained in MS, DKW, or WPM basal media supplemented with (1) 6-benzyladenine (BA; 1 mg/L)?+?kinetin (KIN; 2 mg/L)?+?indole-3-butyric acid (IBA; 0.01 mg/L); (2) BA (1 mg/L)?+?1-phenyl-3-(1,2,3-thiadiazol-5-yl; TDZ 0.1 mg/L)?+?IBA (0.01 mg/L), and (3) KIN (2 mg/L)?+?TDZ (0.1 mg/L)?+?IBA (0.01 mg/L) PGR combinations plus l-glutamine or casein hydrolysate, with or without silver nitrate, and with either gelrite or agar. The highest percentages (12.0% and 11.2%) of somatic embryogenesis for ‘Sar?a?lama’ were obtained in DKW supplemented with PGR combinations of (1) BA (1 mg/L)?+?KIN (2 mg/L)?+?IBA (0.01 mg/L), (2) BA (1 mg/L)?+?TDZ (0.1 mg/L)?+?IBA (0.01 mg/L), respectively. The average number of somatic embryos ranged between 0 and 0.65 per explant for ‘Osmano?lu’ and between 0 and 0.49 per ‘Sar?a?lama’ explant. For germination of somatic embryos, root, shoot, and plantlet regeneration, different treatments included desiccation, cold and gibberellic acid (GA₃), and BA alone or in combination with auxins (IBA or α-naphthaleneacetic acid, NAA; 0.1 mg/L). The highest rate of somatic embryos regeneration (27.5%) occurred using MS basal media with half-strength microelements containing 0.1 mg/L BA?+?0.1 mg/L NAA, after treatments of desiccation, or desiccation plus cold or GA₃ (3 mg/L).     

In vitro somatic embryogenesis from callus culture of the timber yielding treeHardwickia binata Roxb.

Somatic embryogenesis was achieved in callus cultures derived from immature cotyledonary explants ofHardwickia binata Roxb., a multipurpose leguminous tree, on semisolid modified Murashige and Skoog's (mMS) medium containing 2900 mg/l potassium nitrate (KNO₃) supplemented with 4.64 µM kinetin (Kn) and 5.37µM a-naphthaleneacetic acid (NAA). Somatic embryos proliferated rapidly after transfer to MS basal medium supplemented with 2052.6 µM L-glutamine and 0.084 µM gibberellic acid (GA₃). Maturation of somatic embryos was achieved on half-strength MS basal medium supplemented with 1.23 µM IBA and 2% (w/v) sucrose. Histological studies confirmed different developmental stages of somatic embryogenesis inHardwickia binata. Abbreviations BA N⁶-benzyladenine - Kn kinetin - NAA a-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - GA₃ gibberellic acid - MS Murashige and Skoog (1962) medium - mMS modified Murashige and Skoog (1962) medium     

Anthocyanin production in cultured cells of Aralia cordata Thunb.

High anthocyanin-producing cell lines, which were grown in a dark or in a light-dark regime, were selected from callus cultures initiated from stem and leaf tissues of Aralia cordata Thunb. by small-cell-aggregate selection. To verify the optimum culture conditions for anthocyanin production, cells were tested by changing the various basal media, sucrose concentration and nitrogen source and concentration. Good growth was obtained in the dark on Linsmaier-Skoog's basal medium containing 1.0 mg l^-1 2,4-d and 0.1 mg l^-1 kinetin, 2% (w/v) sucrose and full strength of nitrogen concentration. However, the highest anthocyanin yield (10.3% dry wt) was obtained in the dark on B5 medium containing 1.0 mg l^-1 2,4-d and 0.1 mg l^-1 kinetin. Our results suggested that it has became feasible to find the most effective conditions for cell growth and anthocyanin production by optimizations of the nitrogen concentration and the ratio of NH₄ ⁺ to NO₃ ^- in the medium.Abbreviations B5 Gamborg (Gamborg et al. 1968) - 2,4-d 2,4-dichlorophenoxyacetic acid - LS Linsmaier and Skoog (Linsmaier & Skoog 1965) - MS Murashige and Skoog (Murashige & Skoog 1962) - NN Nitsch and Nitsch (Nitsch & Nitsch 1967) - WH White (White 1963) This paper is part 81 in the series Studies on Plant Tissue Cultures. For Part 80 see Furuya T, Sakamoto K, Iida K, Asada Y, Yoshikawa T, Sakai S & Aimi N (1992) Phytochemistry 31: 3065–3068.     

Statistical media optimization for cell growth and azadirachtin production in Azadirachta indica (A. Juss) suspension cultures

Azadirachtin is one of the most widely used biopesticide originating from a plant source. Its production from plant cell cultivation was viewed to overcome constraints associated with its regular supply from the seed kernels. In order to select the effective carbon and nitrogen source, different concentrations of carbon (sucrose/glucose) and nitrogen (NO₃⁻/NH₄⁺ ratio) were studied in A. indica suspension culture. Glucose turned out to be a better carbon source over sucrose yielding high biomass (6.32 g/L) and azadirachtin (11.12 mg/L) content. Nitrate alone as nitrogen source was favorable for both biomass and azadirachtin accumulation. Plackett–Burman design was adopted to select the most important nutrients influencing the growth and azadirachtin accumulation in suspension culture. After identifying effective nutrients, Central Composite Design (CCD) was used to develop mathematical model equations, study responses and establish the optimum concentrations of the key nutrients for higher growth and azadirachtin production. A maximum of 15.02 g/L biomass and 2.98 mg/g azadirachtin was produced using optimum nutrient concentrations representing 99 and 96% validity of the model prediction with respect to biomass and azadirachtin, respectively. This optimized media can be used for cultivation of A. indica cells in bioreactor for mass production of azadirachtin.     

Micropropagation of zedoary (<Emphasis Type="Italic">Curcuma zedoaria</Emphasis> Roscoe) – a valuable medicinal plant

Tissue culture propagation system was developed for zedoary (Curcuma zedoaria Roscoe), a valuable medicinal plant, using rhizome sprout cultures. Shoots were induced from rhizomes on basal MS medium containing 20 g l^–1 sucrose and 5 g l^–1 agar, supplemented with 20 (v/v) coconut water (CW) and benzylaminopurine (BA) concentrations from 0.5 to 5.0 m g l^–1. The excised shoots were subcultured on Murashige-Skoog (MS) medium with 20 (v/v) CW and different concentrations of BA and kinetin (Kin), either alone or in combination with indolebutyric acid (IBA) or naphthaleneacetic acid (NAA). MS medium with 20 (v/v) CW, 3 mg l^–1 BA, and 0.5 mg l^–1 IBA resulted in a multiplication rate per shoot; 5.6 shoots per explant were obtained on average after 30 days of culture. Well-developed shoots (30–40 mm in length) were rooted on MS medium containing 20 g l^–1 sucrose and 8 g l^–1 agar, supplemented with 20 (v/v) CW and 2 mg l^–1 NAA. More than 95 of the rooted plants were established in pots after hardening.     

Effects of macro elements and nitrogen source on adventitious root growth and ginsenoside production in ginseng (Panax ginseng C. A. Meyer)

We investigated the effects on ginseng adventitious root growth and ginsenoside production when macro-element concentrations and nitrogen source were manipulated in the culture media. Biomass growth was greatest in the medium supplemented with 0.5-strength NH₄PO₃, whereas ginsenoside accumulation was highest (9.90 mg g^-1 DW) in the absence of NH₄PO₃. At levels of 1.0-strength KNO₃, root growth was maximum, but a 2.0 strength of KNO₃ led to the greatest ginsenoside content (9.85 mg g^-l). High concentrations of MgSO₄ were most favorable for both root growth and ginsenoside accumulation (up to 8.89 mg g^-1 DW). Root growth and ginsenoside content also increased in proportion to the concentration of CaCI₂ in the medium, with the greatest accumulation of ginsenoside (8.91 mg g^-1 DW) occurring at a 2.0 strength. The NH₄/NO₃ ^-- ratio also influenced adventitious root growth and ginsenoside production; both parameters were greater when the NO₃ ^- concentration was higher than that of NH₄ ⁺. Maximum root growth was achieved at an NH₄ ⁺/NO₃ ^- ratio of 7.19/18.50, while ginsenoside production was greatest (83.37 mg L^-1) when NO₃ ^- was used as the sole N source.     

Efficient propagation and rooting of three citrus rootstocks using different plant growth regulators

The influence of various basal medium and plant growth regulators on the efficient micropropagation of nodal explants from mature trees of alemow, sour orange, and ??Cleopatra?? mandarin citrus rootstocks was studied. All three citrus rootstock shoot cultures showed a preference for high-salt media, like Murashige and Skoog or Driver and Kuniyuki Walnut medium. Several combinations of N ⁶-benzyladenine (BA) and adenine (AD), kinetin (KIN) or gibberellic acid (GA) were tested to optimize the shoot proliferation phase. BA/GA combinations improved the proliferation of all the rootstocks studied, especially alemow. The addition of BA and AD to the culture medium improved shoot proliferation in sour orange and ??Cleopatra?? mandarin in the same way as BA and GA. The addition of different combinations of BA/KIN did not result in further improvement of any of the studied variables. The transfer of in vitro shoots to rooting media, containing different concentrations of indolebutyric acid (IBA) and indoleacetic acid (IAA), resulted in regeneration of complete plantlets. Alemow and ??Cleopatra?? mandarin shoots rooted well using these plant growth regulators; however, all combinations of IBA and IAA tested resulted in very low rooting percentages in sour orange. To improve rooting in sour orange and ??Cleopatra?? mandarin, different combinations of naphthaleneacetic acid (NAA) and IBA were tested. All NAA/IBA combinations produced higher rooting percentages than did the IBA/IAA combinations, and in sour orange nearly 100 % of explants developed roots. An efficient and simple protocol for the micropropagation of three citrus rootstocks, alemow, ??Cleopatra?? mandarin, and sour orange, by culturing nodes from mature plants, has been established.     

Effectiveness of indoleacetic acid,indolebutyric acid and naphthaleneacetic acid during adventitious root formation in vitro in Malus ‘Jork 9’

The production of an antifungal spirostanol saponin designated SC-1 has been detected in cell suspension cultures of the Mexican species Solanum chrysotrichum. Batch cultures of a cell suspension obtained from hypocotyl derived calluses of this species were grown for 25 days in shake flasks containing Murashige & Skoog (MS) medium. Throughout the growth cycle, fresh and dry weight, SC-1 yield, and uptake of sucrose, glucose and fructose were determined. The effects of inoculum size and sucrose concentration on the biomass accumulation and synthesis of the active metabolite, were studied. The maximum SC-1 production, above 14 mg.g⁻¹ (which was fifty times that of field grown plants), was reached after 20 days using a 2% inoculum and complete MS medium supplemented with 2 mgl⁻¹ 2,4-D, 2 mg l⁻¹kinetin, and sucrose between 30 and 45 gl⁻¹. . This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of growth regulators on rapid micropropagation and psoralen production in <Emphasis Type="Italic">Psoralea corylifolia</Emphasis> L.

A rapid and efficient micropropagation system was developed for Psoralea corylifolia, an endangered, valuable medicinal plant. Multiple shoot buds were obtained in half-strength liquid Phillips–Collins (L2) medium supplemented with 5 μM benzylaminopurine (BA) and 5 μM thidiazuron (TDZ) from apical bud explants of 1-week-old cultures. The shoot buds were subcultured on enriched solid L2 medium supplemented with different concentrations and combinations of BA, kinetin (KIN), 2-isopentenyladenine (2iP), TDZ, bavistin (BVN) and trimethoprim (TMP). Enriched solid L2 medium supplemented with 2 μM BA, 1 μM TDZ and 100 mg l⁻¹ BVN were more effective in producing greater number of shoots per explant (85.2 ± 0.9 shoots/explant) after 4 weeks of culture. The regenerated shoots (40–50 mm in length) rooted and accompanied by hardening upon transfer to 50 μM indole-3-butyric acid (IBA) for 15 min and followed by planting in sterile soil mixture and vermiculate (3:1 v/v), with 50 ml of one-eight strength L2 basal salt solution devoid of sucrose and inositol, supplemented with 5 μM IBA and 100 mg l⁻¹ BVN. The plants achieved 100% rooting with hardening. Subsequently the rooted plants were successfully established in the field. The survival percentage differed with seasonal variations. The concentration of psoralen was evaluated in different tissues of ex vitro and in vivo grown plants by high-performance liquid chromatography (HPLC). Psoralen content was increased in leaves (2.97%), roots (2.38%), stems (5.40%) and seeds (1.63%) of ex vitro plants than the in vivo plants. This system facilitates for commercial and rapid propagation of P. corylifolia for conservation strategies and phytomedicine production.