Regeneration of plantlets from the callus of stem segments of adult plants of Ficus religiosa L.

Stem segments of adult plants of Ficus religiosa L. cultured on MS medium containing 1.0 mg/l 2,4-D produced callus. Shoots were regenerated when the induced calli were transferred to medium supplemented with 0.05 to 2.0 mg/l BAP. Callus derived shoots produced roots and developed into plantlets when transferred to medium supplemented with 1.0 mg/l NAA.Abbreviations MS Murashige and Skoog (1962) - BAP 6-benzylaminopurine - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Plantlets from somatic callus tissue of the woody legume Sesbania bispinosa (Jacq.) W. F. Wight

In vitro regeneration of plantlets and multiplication of Sesbania bispinosa (Jacq.) W.F. Wight plants from cultured callus tissue were demonstrated. Callus was established from both cotyledons and mature leaflets on Murashige and Skoog (MS) basal medium supplemented with BAP (0.5 mg/l) and 2,4-D (2 mg/l). Callus mediated shoot bud differentiation was studied under defined nutritional, hormonal and cultural conditions. Various concentrations of BAP or kinetin (Kn) with coconut milk (CM) in MS media induced different levels of shoot bud differentiation as well as multiplication. Multiple shoot bud differentiation occurred in most of the primary calli. The best medium for shoot bud differentiation from cotyledon derived callus, contained BAP (2 mg/l) and 15% CM (V/V). More efficient shoot bud organogenesis was recorded with BAP than Kn. Supplementation with CM in MS media accelerated shoot bud organogenesis in differentiating callus tissue. Rooting of differentiated shoots was achieved by a three step culture procedure involving (a) MS solid medium containing IBA (2 mg/l), (b) growth regulator free half strength MS medium with 1% charcoal, and (c) half strength MS liquid medium free of vitamins, growth regulators and charcoal.Abbreviations IAA indoleacetic acid - IBA indole-3-butyric acid - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - Kn kinetin - CM coconut milk - MS Murashige and Skoog's medium - SBI shoot bud inducing medium     

Plantlet regeneration through indirect shoot organogenesis and somatic embryogenesis in Justicia gendarussa Burm. f., a medicinal plant

A protocol for the regeneration of a large number of plantlets via indirect shoot organogenesis and somatic embryogenesis has been developed from the stem and leaf explants of Justicia gendarussa Burm. f. The callus was efficiently induced from the explants using Murashige and Skoog (MS) medium supplemented with α-Naphthalene acetic acid (NAA) + Benzyl amino purine (BAP) (1.0?+?0.1 mg/l). The highest number of plantlets through indirect shoot organogenesis was obtained when the callus was subcultured to MS medium with BAP + NAA (0.1?+?1.0 mg/l). The maximum number of plantlets via somatic embryos was obtained in the medium with BAP + NAA (1.0?+?0.1 mg/l) for stem derived calli and Kinetin (Kn) + NAA (2.0?+?0.1 mg/l) for leaf derived calli. The in vitro developed shoots were rooted well in half strength MS medium supplemented with 0.5 mg/l of Indole-3-acetic acid (IAA). The in vitro regenerated plantlets were hardened using a mixture of sterile sand:soil:manure (1:1:1). The present study is the first report on the regeneration of plants through somatic embryogenesis from stem and leaf derived calli of J. gendarussa.     

In vitro plant regeneration through direct organogenesis in Populus deltoides clone G48 from petiole explants

A rapid and efficient protocol is developed for in vitro plantlet regeneration of Populus deltoides clone G48 using petiole explants. The highest frequency of shoot regeneration (74.75%) from petiole was obtained on MS medium supplemented with 0.50?mg/l BAP and 0.20?mg/l IAA. The regenerated shoots started turning brown and necrotic after 10?C15?days in culture. To overcome the browning problem, the explants along with the developing shoot buds were transferred to modified MS medium containing 0.50?mg/l BAP, 0.20?mg/l IAA, 15?mg/l AdS, 0.1% PVP, 100?mg/l casein hydrolysate, 50?mg/l L-glutamine, 250?mg/l (NH₄)₂SO₄ and 0.5% agar. Shoot multiplication and elongation took place on the same medium. Indole-3-acetic acid at 0.10?mg/l was most effective for root regeneration. Using the current protocol, it took 2?months to regenerate plantlets. The in vitro regenerated plantlets were successfully acclimatized and established in greenhouse conditions. This regeneration system using petiole explants provides a foundation for Agrobacterium-mediated genetic transformation of P. deltoides clone G48 for incorporation of various silviculturally important traits.     

In vitro regeneration and Agrobacterium mediated genetic transformation of Artemisia aucheri Boiss

In the present study, we developed an efficient protocol for in vitro plant regeneration and genetically transformed root induction in medicinal plant Artemisia aucheri Boiss. Leaf explants were cultivated in MS medium supplemented by combination of plant growth regulators including α-naphthalene-acetic acid, 6-benzyl-aminopurine, indole-3-acetic acid and 2, 4-dichlorophenoxyaceticacid. The highest frequency of shoot organogenesis occurred on MS medium supplemented with 0.05 mg/l NAA plus 2 mg/l BA (96.3 %) and MS medium supplemented with 0.5 mg/l IAA plus 2 mg/l BA (88.3 %). Root induction was obtained on MS medium supplemented with 0.5 mg/l IBA. This is a simple, reliable, rapid and high efficient regeneration system for A. aucheri Boiss in short period via adventitious shoot induction approach. Also, an efficient genetically transformed root induction for A. aucheri was developed through Agrobacterium rhizogenes-mediated transformation by four bacterial strains, A4, ATCC15834, MSU440, and A13 (MAFF-02-10266). The maximum frequency of hairy root induction was obtained using MSU440 (93 %) and ATCC15834 (89 %) bacterial strains. Hairy root lines were confirmed by PCR using the rolB gene specific primers and Southern blot analysis.     

Establishment of Plantlets and Evaluation of Differentiated Roots for Alkaloids in Rauwolfia serpentha

Different explants of Rauwolfia serpentina were tested for their capacity and root differentiation. MS sedium containing 2.0 mg I^-1 BAP or 1.5 mg I^-1 BAP with 0.5 mg I-* NAA gave the best shoot proliferation from shoot apices (84.12%). Multiple shoots subcultured on the same media gave higher number of shoots (6–9). Callus formed at the cut ends of explants produced shoots when subcultured on the above media. Rooting was achieved after transfer of shoots either to hormone free or half strength or full strength MS medium with different concentrations of IAA (0.5,1.0 mg I^-1 and IBA (0.5,1.0 mg I^-1).The complete regenerated plantlets have been successfully transferred to earthen pots in green house. Maximum root differentiation from leaves, stem and nodal cuttings derived calli was obtained on MS medium supplemented with NAA (2.0 mg I^-1) and BAP (1.5 mg I^-1). Identification of reserpine and other alkaloids from differentiated roots holds an interesting alternative for controlled production of alkaloids from this endangered, overexploited medicinal plant species.     

Regeneration of Heracleum candicans Wall Plants from Callus Cultures through Organogenesis

Callus was initiated from petiole explants of Heracleum candicans on MS medium fortified with BAP and 2,4-D ( 0.5 mg I^-1 each). Maximum shoot differentiation from callus occurred on MS medium containing 1 mg I^-1 BAP and 0.2 mg I^-1 NAA. The regenerated shoots were rooted on MS medium supplemented with 1 mg I^-1 IBA. The rooted plants were transferred to the field after successful hardening in pots containing vermiculite. All regenerated plants were diploid with 2n=22 chromosomes in their root tip cells.     

An efficient in vitro regeneration of Ceropegia noorjahaniae: an endemic and critically endangered medicinal herb of the Western Ghats

An efficient protocol was developed for the rapid in vitro multiplication of an endemic and critically endangered medicinal herb, Ceropegia noorjahaniae Ans., via enhanced axillary bud proliferation from nodal explants. The effects of phytohormones [6-benzylaminopurine (BAP), kinetin (Kin) thidiazuron (TDZ), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) or α-naphthalene acetic acid (NAA)] on in vitro regeneration were investigated. The highest number of shoots (18.3 ± 1.3), maximum shoot length (10.1 ± 0.8 cm) and the highest response of shoot induction (95 %) were recorded on MS medium supplemented with 2.0 mg/l BAP. Rooting was best achieved on half-strength MS medium augmented with IBA (1.0 mg/l). Half-strength MS medium supplemented with BAP (4 mg/l) and sucrose (5 %, w/v) produced an average of 5.6 flower buds per microshoots with highest (90 %) flower bud induction response. The plantlets regenerated in vitro with well-developed shoot and roots were successfully established in pots containing sterile sand and coco peat (1:1) and grown in a greenhouse with 85 % survival rate. The regenerated plants did not show any detectable morphological variation. The developed method can be successfully employed for large-scale multiplication and conservation of C. noorjahaniae.     

In vitro propagation and assessment of genetic integrity of Talinum triangulare (Jacq.) Willd: a valuable medicinal herb

Talinum triangulare is a medicinally important herb and various parts of the plant are used pharmaceutically for the treatment of different diseases. In our study, a rapid and efficient protocol for micropropagation has been developed from shoot tip and nodal explants of T. triangulare. High shooting frequency (93.33?%) was achieved with shoot tip explants when cultured on Murashige and Skoog??s (MS) medium supplemented with 1.0?mg/L 6-benzyl amino purine (BAP) producing an average of 12.50?±?0.23 shoots and 5.07?±?0.02?cm shoot length per explant. A combination of 0.5?mg/L BAP and 0.5?mg/L kinetin was found to be more effective by producing 15.67?±?0.25 shoots and 6.22?±?0.02?cm shoot length per explant. The microshoots were excised and cultured on half-strength MS and full-strength MS medium containing different concentrations of indole-3-acetic acid and indole-3-butyric acid (IBA) for root induction. More number of roots (45.10?±?0.96) with an average length of 5.46?±?0.08?cm was obtained on half-strength MS medium supplemented with 0.5?mg/L IBA. The rooted shoots were successfully transplanted from different planting substrates to the field with a 100?% survival rate. Random amplified polymorphic DNA analysis was carried out using four random decamer primers. The amplification products were monomorphic in the micropropagated plants and were similar to the mother plant. Absence of polymorphism revealed that no variation was induced, thus maintaining the genetic integrity of the micropropagated plants of T. triangulare.     

High-frequency direct shoot regeneration from <Emphasis Type="Italic">Drymaria cordata</Emphasis> Willd. leaves

An efficient and reproducible procedure is described for direct shoot regeneration in Drymaria cordata Willd. using leaf explants cultured on Murashige and Skoog (MS) medium supplemented with α-naphthalene acetic acid (NAA) and 6-benzyladenine. The regeneration frequency varied with the plant growth regulator concentrations, orientation of the explants, and the carbon source and basal salts present in the regeneration medium. The highest mean number of shoots per explant (10.65 ± 1.03) was recorded on MS plates containing 3% sucrose and 0.8% agar supplemented with 0.1 mg/l NAA and 1.0 mg/l BAP. Shoot buds were induced in the basal parts of the leaf explants. Concentrations of NAA exceeding 1 mg/l suppressed shoot regeneration. Explants bearing the entire lamina and petiole were much more responsive than those having only the lamina. The plantlets that regenerated from the leaf explants were rooted successively on MS medium alone or in combination with indole butyric acid (IBA). The highest mean number of root organogenesis, with 25.67 ± 3.68 roots per leaf segment, was obtained in the presence of 1 mg/l IBA. Histological investigations of the regenerating shoots showed that the shoot buds had emerged from epidermal cells without callus formation. More than 90% of the in vitro-propagated plants survived when transferred to a greenhouse for acclimatization. Thus, this optimized regeneration system may be used for rapid shoot proliferation and genetic transformation.     

Callus-mediated organogenesis and effect of growth regulators on production of different valepotriates in Indian valerian (Valeriana jatamansi Jones.)

A reproducible and efficient callus-mediated shoot regeneration system was developed for the large-scale production of Valeriana jatamansi Jones., a highly medicinal plant species of global pharmaceutical importance. Effect of Murashige and Skoog (MS) medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthaleneacetic acid (NAA) and indole-3-butyric acid (IBA) on callus induction and production of valepotriates accumulation was studied by using different explants. In V. jatamansi, the degree of callus induction varied significantly depending on explants type and the growth regulators used. Among different explants used, rhizomes have the highest callus induction potential followed by leaf. The callus induction frequency was found to be optimum in rhizome explants on media supplemented with 0.5 mg/l 2,4-D. The regenerative ability of proliferated compact calli was studied by the application of cytokinins alone and in combination with auxin. MS medium fortified with 0.75 mg/l thidiazuron in combination with 0.5 mg/l NAA showed the highest regeneration frequency (88.6 %) and produced the maximum number of shoot buds (15.20 ± 0.20) capable of growing into single plants. Vigorous callus obtained from MS medium supplemented with different concentrations of 2,4-D, NAA and IBA were used for industrially important valepotriates [acevaltrate (ACE), valtrate (VAL) and didrovaltrate (DID)] analysis. High performance liquid chromatography analysis of callus revealed that medium with 2,4-D (1 mg/l) was found responsible for increasing ACE and DID yield, whereas VAL production was higher in case of medium supplemented with NAA (1 mg/l). However, the accumulation of valepotriates in callus decreased in logarithmic phase after 8 weeks. IBA was not beneficial for the valepotriate synthesis, as it helped to accumulate significantly lower concentration of ACE, VAL and DID. Micropropagated plantlets with well-developed root system were successfully acclimatized in greenhouse condition, in root trainers containing garden soil with a survival frequency of 100 %. As Indian valerian is a highly traded medicinal plant due to extensive use of its industrially important secondary metabolites, the present system can be utilized to obtain mass multiplication of the species as well as for the stable biomass and continuous valepotriate production for the pharmaceutical industries.     

Rapid in vitro multiplication and ex vitro rooting of Rotula aquatica Lour., a rare rhoeophytic woody medicinal plant

Single medium-based efficient protocols for large-scale multiplication of the rare woody aromatic medicinal plant Rotula aquatica Lour. by means of axillary bud multiplication and indirect organogenesis were established using Murashige and Skoog (MS) medium. There were no significant differences with respect to the induction of shoots per node or callus and roots per shoot on media prepared either with tap water and commercial sugar or those prepared with double distilled water and tissue culture-grade sucrose. The most effective medium for axillary bud proliferation was MS medium fortified with 1.0 mg l(-1 )N(6)-benzylaminopurine (BAP) and 0.5 mg l(-1 )indole-3-butyric acid (IBA), on which shoots were induced at the rate of 15 per node. The excision of node segments from the in vitro-derived shoots and their subsequent culture on medium supplemented with same concentrations of BAP and IBA facilitated enhanced axillary bud proliferation. Callus that developed from the lower cut end of the node explants induced shoots during subculture on half-strength MS medium with 1.0 mg l(-1 )BAP and 0.5 mg l(-1 )kinetin. The shoots developed rooted best on half-strength MS medium supplemented with 0.5 mg l(-1 )naphthaleneacetic acid (NAA). Rooted shoots, following acclimation in the greenhouse, were successfully transferred to field conditions, and 80% of the plantlets survived. When the basal ends of shoots harvested from multiplication medium were dipped in an NAA (0.5 mg l(-1)) solution for 25 days, a mean of 5.6 roots per shoot developed; the transfer to small pots facilitated the survival of 75% of the rooted shoots. Ex vitro rooting by direct transfer of the shoots from the multiplication medium to the greenhouse resulted in a 65% survival. Commercial sugar and tap water and ex vitro rooting make the protocol economically advantageous. About 750 plantlets were procured in a 3-month period starting from a single node explant.     

Efficient protocols for in vitro regeneration of Pennisetum glaucum (L) Br

A system was developed for in vitro regeneration of Pennisetum glaucum through organogenesis and somatic embryogenesis. Mature embryo and leaf base explants of Pennisetum glaucum (L) Br. cv HH B60 (Poaceae) were cultured on Murashige and Skoog agar medium supplemented with 11.3 microM of 2,4-D for callus induction. Embryogenic calli were induced within eight weeks. Percentage of callus induction and somatic embryogenesis was significantly higher in mature embryo than leaf base explants. Maximum shoot regeneration was obtained via organogenesis on MS medium supplemented with 4.43 microM of BAP and 4.64 microM of kinetin from the calli of both the explants. The frequency of plant regeneration through somatic embryogenesis was comparatively lower than organogenesis. Regeneration frequency was higher in mature embryo explants than leaf base explants. The shoots regenerated via organogenesis were elongated and rooted efficiently on MS medium supplemented with IBA (0.49 microM). The rooted plantlets were hardened and transferred to soil.     

Plant regeneration from leaf-derived callus cultures of the tropical pasture legume Stylosanthes scabra Vog.

Callus cultures of 5 genotypes of S. scabra Vog. were optimally established from leaf tissue on Murashige and Skoog (MS) basal medium supplemented with 0.5–2.0 mg l^-1 2, 4-Dichlorophenoxy acetic acid (2, 4-D) and 1.0–2.0 mg l^-1 6-benzylaminopurine (BAP). On media containing 2, 4-D only, calli were soft, and rhizogenesis occurred on calli of 4 genotypes. Calli formed on media containing BAP only, but not with kinetin only. In the presence of 2, 4-D, BAP inhibited rhizogenesis and promoted better callus growth than kinetin. High frequency shoot induction was achieved for 3 genotypes on MS +2.0 mg l^-1 BAP. Roots formed on shoots when sub-cultured on half-strenght MS without growth regulators. The form of cytokinin used in the callus induction media appeared to affect subsequent shoot organogenesis. Genotypic differences were observed for shoot organogenesis. There was some morphological variation evident among regenerants.     

High frequency organogenesis in hypocotyl,cotyledon, leaf and petiole explants of broccoli (Brassica oleracea L. var. italica), an important vegetable crop

Broccoli (Brassica oleracea L. var. italica) is an important, nutritionally rich vegetable crop, but severely affected by environmental stresses, pests and diseases which cause massive yield and quality losses. Genetic manipulation is becoming an important method for broccoli improvement. In the present study, a reproducible and highly efficient protocol for obtaining organogenesis from hypocotyl, cotyledon, leaf and petiole explants of broccoli (Brassica oleracea L. var. italica cv. Solan green head) has been developed. Hypocotyl and cotyledon explants were used from 10 to 12 days old aseptically grown seedlings whereas leaf and petiole explants were excised from 18 to 20 days old green house grown seedlings and surface sterilized. These explants were cultured on shoot induction medium containing different concentration and combination of BAP and NAA. High efficiency shoot regeneration has been achieved in hypocotyl (83.33 %), cotyledon (90.11 %), leaf (62.96 %) and petiole (91.10 %) explants on MS medium supplemented with 3.5 mg/l BAP + 0.019 mg/l NAA 2.5 mg/l BAP + 0.5 mg/l NAA, 4.0 mg/l BAP + 0.5 mg/l NAA and 4.5 mg/l BAP + 0.019 mg/l NAA respectively. Petiole explants showed maximum shoot regeneration response as compared to other explants. MS medium supplemented with 0.10 mg/l NAA was found best for root regeneration (100 %) from in vitro developed shoots. The regenerated complete plantlets were transferred to the pots containing cocopeat and successfully acclimatized. This optimized regeneration protocol can be efficiently used for genetic transformation in broccoli. This is the first comparative report on multiple shoot induction using four different types of explants viz. hypocotyl, cotyledon, leaf and petiole.     

Micropropagation protocol for Antigonon leptopus an important ornamental and medicinal plant

The effect of some factors on in vitro consecutive micropropagation behavior of Antigonon leptopus was examined including those of culture establishment, shootlets multiplication, rooting and acclimatization stages. The highest percent of aseptic cultures and survival of explants (100%) were obtained as a result of using Clorox 10% for 3?min followed by MC 0.1% for 2?min while, using each of them individually (Clorox 20% or MC 0.1%) for 5?min caused the highest percent of shoot formation. During the multiplication stage, the highest percent of shoot formation was reached to 100% with repeating culture of explants (two times) on MS medium supplemented with 2ip at 1.0 and IBA at 0.2?mg/l. The highest numbers of shootlets/explant were obtained when 2.0?mg/l of BAP or 0.5?mg/l BA?+?0.2?mg/l of IBA were added to MS culture medium. Culturing the explants on MS medium supplemented with 2ip at 0.5 or 1.0?mg/l each combined with 0.2?mg/l of IBA showed the longest shootlets. Reducing the strength of culture media to ½ or ¾ had promotion effect on rooting formation of shootlets. The best results of plant acclimatization (survival percent, plant height and root length) were obtained by using sand or peat moss soil. The amplified DNA fragments using B7, B9 and C19 primers for mother and micropropagated plants showed that the produced pattern by primer B7 had a maximum number of 10 bands of DNA fragments with molecular size ranging between 1025.57 and 176.36?bp, micropropagated plants showed 95.2% similarity in relation to mother plant.     

High frequency shoot regeneration from nodal and shoot tip explants in Holarrhena antidysenterica L

Shoot tip and nodal segment explants of Holarrhena antidysenterica when cultured on MS medium containing BAP (1.0-3.0 mg/l) with NAA (0.2-1.0 mg/l) and BAP (1.0-3.0 mg/l) with Kn. (0.2-1.0 mg/l) produced multiple shoots. Maximum multiple shoots was found in MS medium supplemented with BAP (2.0 mg/l) and NAA (0.5 mg/l). Subculture on the same medium resulted in rapid shoot multiplication at an average rate of 16 new shoots per subculture. Addition of urea (100 mg/l) in the medium increased the number of shoots up to 22 per culture. For best rooting, the shoots were excised from the culture flask and implanted individually on half strength MS medium with 0.5 mg/l each of IBA, IAA and NAA. After 20 days of transfer on root induction medium 95% rooting was achieved. Regenerated plantlets were successfully acclimatized and established in soil. About 90% of plantlets survived under open field conditions.     

In vitro organogenesis and plant regeneration from unpollinated ovary cultures of Azadirachta indica

A novel method of organogenesis in neem (Azadirachta indica A. Juss.) from unfertilized ovaries is described. The Murashige and Skoog’s (MS) medium with 9 % sucrose, 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 5 μM 6-benzylaminopurine (BAP) was the best for callus induction from unfertilized ovaries. However, further proliferation of callus occurred better on MS medium supplemented with 0.5 μM 2,4-D either alone or in combination with 4.5 μM kinetin. Maximum shoot regeneration (78 %) was observed when calli, induced from ovaries of 4 mm size flower buds and proliferating on MS + 0.5 μM 2,4-D, were subcultured to MS medium containing 5 μM BAP. Histological analysis revealed that 4 mm sized flower bud corresponds to a 2-nucleate stage of embryo sac. The shoots were then multiplied by forced axillary branching on MS medium supplemented with 1.0 μM BAP and 250 mg dm⁻³ casein hydrolysate. The shoots could be rooted on 1/4 strength MS medium supplemented with 0.5 μM indole-3-butyric acid (IBA) at a frequency of 79 %. Cytological analysis by root tip squash preparations revealed that all the plantlets were diploids. These plants were subsequently hardened and established in soil with transplantation rate of 81.8 %.     

Micropropagation of Ajuga bracteosa, a medicinal herb

For conservation and genetic transformation, a successful in vitro micropropagation protocol for Ajuga bracteosa, a medicinal herb has been established for the first time. MS medium supplemented with IAA (2 mg/L) and BA (5 mg/L) induced 100 % shoot regeneration with an average of 41.4 shoots of 8.4 cm per culture. Excised in vitro shoots when transferred to MS + IBA (0.5 mg/L) produced 20 roots/shoot of 20.2 cm average length in 100 % cultures. Of the three explants, leaf, petiole and root, leaf displayed quickest response followed by petiole while root was the slowest. Hardening of plantlets was achieved with 82 % survival. The hardened plants were maintained in pots with garden soil under controlled (Temp. 25?±?2 °C) conditions. RAPD exhibited genetic fidelity with 100 % monomorphism in regenerants.     

In vitro propagation of an endemic and endangered medicinal plant <Emphasis Type="Italic">Notopterygium incisum</Emphasis>

An efficient system in vitro propagation for Notopterygium incisum Ting ex H. T. Chang, an endemic and endangered medicinal plant, was established to address increased demand and germplasm conservation goals. Optimum response in callus induction (CI) was observed on Murashige and Skoog (MS) medium supplemented with 1.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.2 mg/l 6-benzylaminopurine (BAP), which the induction rate and growth of callus were 84.44% and 0.67 g respectively. The highest shoot regeneration frequency (76.97%) and maximum number of shoots (3.6 shoots per callus) were achieved on MS medium with 1.5 mg/l BAP and 0.2 mg/l naphthalene acetic acid (NAA). Half-strength MS medium supplemented with 0.6 mg/l indole-3-butyric acid (IBA) was determined to be the best rooting medium, resulting in the maximum number of roots (18.6 roots per shoot) and the highest rooting frequency (92.28%). An approximate 83.8% survival rate among the regenerated plantlets was recorded after they were transplanted in the field at an altitude of 3200 m. An HPLC analysis showed that the content of two main chemical constituents, notopterol and isoimperatorin, in the rhizomes of 3-year-old regenerated plantlets was higher (3.84 mg/g and 4.05 mg/g, respectively) than that in commercially marketed crude drugs. This first report of complete regeneration in vitro could provide an alternative method for the rapid, large-scale production and conservation of this valuable, rare, and endangered medicinal plant.