Concentrations Of Benzyladenine Research Articles

Influence of preculture treatment and types of explants on shoot growth and in vitro flowering of feathered amaranth (Celosia argentea var. plumose)

The shoots developed from both the shoot tip and nodal explants of feathered amaranth (Celosia argentea var. plumosa—feathered cockscomb or plumed cockscomb) after 8 weeks of culture in the presence of either paclobutrazol or benzyladenine (BA) were shorter than those developed on basal Murashige and Skoog (MS) medium (Physiol Plant, 15:473–497, 1962) alone. However, this retarding effect was more pronounced in the nodal explant culture. Shoot tip explants from 2-week-old seedlings were more adversely affected by 0.85 or 1.7 μM paclobutrazol than those from older seedlings. In contrast, regardless of preculture duration investigated nodal explants did not exhibit different response to three different concentrations of paclobutazol. The response to 2.2 or 4.4 μM BA appeared to be largely independent of the age of the shoot tip explants or preculture treatment of nodal explants. Shoots developed from nodal explants produced a higher number of terminal inflorescence than those from shoot tip explants. Moreover, only lateral shoots from nodal explant culture formed inflorescence. Increased preculture duration on basal MS medium could generally lessen the inhibitory effect of lower concentrations of paclobutazol or BA on terminal or lateral inflorescence formation in nodal explant culture.

Embryo rescue and plant regeneration in banana (Musa spp.)

An efficient regeneration protocol for zygotic embryos at varying maturity stages was developed for wild banana (Pisang Jajee (AA)). Embryo ontogeny was studied to determine the best maturity stage for embryo rescue, suitable media and culture conditions (light and dark) for germination and regeneration. The conversion of endosperm from transparent fluid into a semi-solid state was followed by visible embryo development, which commenced only after 70% embryo maturity. Zygotic embryos of Pisang Jajee at different maturity levels were excised and cultured on medium fortified with different concentrations of 6-benzyl adenine (BA) and indole acetic acid (IAA). Zygotic embryos produced callus or plantlets 25 days after initiation. The frequency of callus induction was greater in immature embryos irrespective of the media composition and decreased with increasing maturity. Fully matured embryos regenerated directly into plantlets without producing callus. Immature embryos required medium supplemented with plant growth regulators (PGRs) for successful regeneration. Although the culture conditions had no influence, dark conditions favoured callus induction and plant regeneration.

In vitro PROPAGATION OF Sprekelia formosissima Herbert., A WILD PLANT WITH ORNAMENTAL POTENTIAL

Vegetative propagation of Sprekelia (Sprekelia formosissima Herbert.) in natural conditions is limited because it produces only one bulb per year or none. The objective of this research was to generate an in vitro propagation protocol for this species to increase its commercial propagation rate without extracting the species from its natural habitat. Bulbs of 4 to 5 cm in diameter were used as disinfested donor explant material; 1 cm2 explants were obtained from the cataphyll leaves with and without a portion of basal disc; these explants were established in MS medium supplemented with 8.87 μM of N6 benzyl adenine (BA) and 0.98 μM of indole-3- butyric acid (IBA). For shoot multiplication, bulblets obtained from the previous phase were used as explants and cultivated in MS medium with 2.5, 5, 10, 15 and 20 μM of BA combined with IBA at a 10:1 ratio (BA: IBA). Shoots obtained from multiplication were established in MS medium supplemented with 1, 2, 3, 4, and 5 % (w/v) sucrose to promote growth. Bulblets were rooted in MS medium supplemented with 0, 0.49, 0.98, 1.96, 3.93 and 7.8 μM of IBA. Once roots formed, they were transferred to soil to assess their acclimation. We obtained 89.1 % of aseptic explants, of which 86 % formed two shoots on the average. Multiplication of shoots increased as BA concentration increased in culture medium, and the best results (75 % of bulblets with shoots, 2.66 shoots per bulblet and 2.0 mm diameter shoots) were obtained with 20 μM BA. The best bulb growth in diameter (4.2 mm) and number of bulblet leaves (3.5) was obtained with 5 % sucrose. The use of 0.98 μM IBA resulted in greater rooting percentage (93.7) and number of roots per bulblet (2.0), which were 2.4 cm long on average. Up to 83 % of the bulblets survived acclimation. This protocol to micropropagate Sprekelia formosissima allowed the production of at least 96 bulblets from one mother bulb in a six months period of in vitro culture.

In Vitro Optimization of Phytohormones on Micropropagation in Butterfly Pea (Clitoria ternatea L.)

An efficient protocol has been developed for plant regeneration from cotyledonary nodes of an in vitro grown Clitoria ternatea seedling. Proliferation of shoots was achieved on MS medium supplemented with various concentrations of 6-benzyladenine (BA), kinetin (Kin) and 2-isopentenyl adenine (2-iP) either singly or in combination with α-naphthalene acetic acid (NAA). Of all the cytokinins, BA, Kin, and 2-iP tested on the MS medium, BA (2.5 μM) was optimum for inducing the maximum number of shoots (9.8 ± 0.86). The highest efficacy for shoot proliferation (13.2 ± 0.31) was achieved in a combination of 2.5 μM BA and 1.0 μM NAA. The best condition for rooting was observed on half-strength MS medium augmented with 1.5 μM indole-3-butyric acid. After rooting, plantlets transferred to plastic cups filled with sterile soilrite media exhibited normal development, with more than 90% growing well even after transfer to field conditions.

Trichomes, essential oils and in vitro propagation of Lavandula pedunculata (Lamiaceae)

Lavanders ( Lavandula spp.) are aromatic plants with high industrial and commercial value, used in food industry, perfumery and pharmaceutical preparations. Micropropagation can be used for large-scale multiplication of essential oil producing plants thus avoiding an overexploitation of natural resources. This work aims to develop a reliable protocol for the in vitro propagation of Lavandula pedunculata and to evaluate the potential of these plants for essential oil production with industrial application. In vitro cultures were established on media with different concentrations of benzyladenine. Trichome morphology was examined by SEM and their secretion analyzed by histochemical tests. Essential oils were isolated by hydrodistillation and analyzed by GC and GC-MS. The essential oils and trichomes of in vitro plants were compared with those of field-growing plants. Best propagation rates were achieved with 0.25 mg/l BA. Rooting occurred without treatment with auxins thus preventing callus growth. Trichomes and essential oils of the in vitro propagated plants were similar to those of the parent plants. Two chemotypes were characterized: 1,8-cineole/camphor type and fenchone type. Since trichomes and essential oils of L. pedunculata micropropagated plantlets are identical to those produced by field-growing plants, in vitro cultures can be used for essential oil production without affecting natural resources. Moreover, the quality of the essential oils is assured and this protocol can be further applied to the propagation of selected chemotypes for industrial purposes.

In vitro plant regeneration system for Cassia siamea Lam., a leguminous tree of economic importance

A method for rapid in vitro propagation of Cassia siamea Lam. using cotyledonary node explants, excised from 14-day old aseptic seedlings, has been established. Murashige and Skoog (MS) medium supplemented with different concentrations of 6-benzyladenine (BA), kinetin (Kn) and thidiazuron (TDZ) singly or in combination with auxins was used for regeneration studies. Among the single treatment of three cytokinins BA at 1.0 µM was found to be optimum for direct shoot regeneration as it induced an average of 8.20 ± 0.66 shoots per explant. The regeneration frequency further enhanced with the application of auxin along with optimal BA concentration. The highest frequency for shoot regeneration (90%), the maximum number of shoots per explant (12.20 ± 0.73) and the maximum shoot length (6.40 ± 0.07) cm were obtained on the medium consisted of MS + 1.0 µM BA + 0.5 µM NAA. Successful in vitro rooting was induced from cut end of the microshoots when placed on half-strength MS + IBA (2.5 µM). The regenerated shoots with well developed root system were successfully acclimatized and established in pots containing sterilized garden soil and garden manure (1:1) and grown under greenhouse conditions with 85% survival rate.

Changes of antioxidant enzyme activities and isoenzyme profiles duringin vitroshoot formation in saffron (Crocus sativusL.)

Among the different concentrations of Thidiazuron (TDZ) and between the two media Gamborg (B5) and Murashige and Skoog (MS), the highest frequency of shoot formation could be seen in the MS medium with TDZ concentration of 4.54 microM. Among the different concentrations of Naphtalene acetic acid (NAA) and Benzyl adenine (BA) in the two aforementioned media, the maximum proliferation and rooting of saffron shoots were obtained in a B5 medium containing 2.22 microM NAA and 2.68 microM BA. Peroxidase (POD), catalase (CAT), superoxide dismutase (SOD), esterase (EST) and polyphenoloxidase (PPO) measurements proved that all the enzymes had a similar pattern of changes, according to which their concentrations increased in the first stages of development and then decreased. The same pattern was observed for polyphenoloxidase in a B5 medium while in the MS medium a reverse pattern was observed. The enzyme concentration decreased and then increased during shoot formation. The results show the principal role of antioxidant enzymes in the complicated process of organogenesis.

Adventitious shoot induction from cultured internodal explants of Malaxis acuminata D. Don, a valuable terrestrial medicinal orchid

Malaxis acuminata is a terrestrial orchid that grows in shady areas of semi-evergreen to shrubby forests. It is highly valued for its medicinal properties as dried pseudo-bulbs are important ingredients of several Ayurvedic preparations. In this study, adventitious shoot buds were induced from internodal explants of M. acuminata grown on Murashige and Skoog (MS) medium supplemented with different concentrations of 6-benzyladenine (BA), kinetin (Kn), and thidiazuron (TDZ). Of the three cytokinins used, TDZ at 3 mg l−1 induced the highest frequency (82%) of organogenic explants. However, all responding explants produced only a single adventitious shoot irrespective of the type and concentration of the cytokinin. Adding 0.5 mg l−1 α naphthaleneacetic acid (NAA) to the medium enhanced adventitious shoot formation. In the presence of 3 mg l−1 TDZ and 0.5 mg l−1 NAA, frequency of organogenesis was 96% with a mean number of 6.1 shoots per explant. Prolonged culture or subculture on the same medium did not promote further shoot production. However, transfer of these cultures to MS medium supplemented with 3 mg l−1 TDZ and 0.5 mg l−1 NAA and various concentrations of different polyamines (PAs), including spermine, spermidine, and putrescine, significantly increased mean shoot number per explant. The highest frequency of shoot induction (100%) and mean shoot number per explant (14.6) was observed on MS medium with 3 mg l−1 TDZ, 0.5 mg l−1 NAA, and 0.4 mM spermidine. Regenerated shoots were excised and subcultured on an elongation medium consisting of MS medium with 3 mg l−1 BA. Moreover, the highest frequency of rooting (96%) and mean number of roots per shoot (3.3) was observed on MS medium with 4 mg l−1 indole-3-butyric acid (IBA) and 1.5 mg l−1 activated charcoal (AC). Almost 90% of rooted shoots were successfully acclimatized and established ex vitro.

Micropropagation of Ocimum kilimandscharicum Guerke (Labiatae)

An efficient plant regeneration protocol has been developed from nodal explants of Ocimum kilimandscharicum Guerke, a medicinally important herbaceous plant species belonging to the family Lamiaceae. Axillary shoot bud proliferation was initiated from nodal explants cultured on MS medium supplemented with various concentrations of 6-benzyladenine (BA) (0.5–3.0 mg/l), kinetin (KN) (0.5–3.0 mg/l) and 2-isoPentenyladenine (2-iP) (0.5–3.0 mg/l). The maximum number of shoots (6.09±0.05), with average length 3.83±0.11 cm, was achieved with medium containing 1.0 mg/l BA. Shoot culture was established by repeated subculturing of the original nodal explants on shoot multiplication medium after each harvest of newly formed shoots. In this way, 20–30 shoots were obtained from a single nodal explant after 5 months. Rooting of shoots was achieved on half-strength MS medium supplemented with 1.5 mg/1 Indole-3-butyric acid (IBA) and 2% sucrose. Well-developed plantlets transferred to plastic pots containing soil and vermiculite (1:1) showed 81.13% survival. The genetic fidelity of in-vitro-raised field-grown plants to the donor plant was ascertained from random amplified polymorphic DNA (RAPD) markers. This protocol can be used for commercial propagation and for future genetic improvement studies.

Regeneration and histological analysis of snake melon (Cucumis melo var. flexuosus (L.) Naudin) by direct organogenesis

In vitro morphogenesis of Cucumis melo var. flexuosus (L.) Naudin was studied by direct organogenesis. Induction of adventitious buds was obtained from distal and proximal parts of the cotyledon incubated on Murashige and Skoog (1962) medium containing different concentrations of 6-benzyladenine (BA) (0.0, 0.5, 1.0, and 2.0 mg L^{-1}) and indole-3-acetic acid (IAA) (0.0, 0.1, and 0.5 mg L^{-1}). The best organogenesis was obtained from the proximal part of the cotyledon on medium containing 0.5 mg L^{-1} BA and 0.5 mg L^{-1} IAA (88%). This concentration was used for histological analysis. Less regeneration was obtained on media containing 1.0 mg L^{-1} BA (75%) or 1.0 mg L^{-1} BA and 0.1 mg L^{-1} IAA (60%). Plant recovery was much greater from proximal rather than distal explants. A callus formed on almost every explant on media supplemented with BA. Histological analyses showed that the formation of early shoot apical meristems was observed in 14-day-old tissues and meristematic structures were seen in 17-day-old tissues. The first shoot was formed after 21 days. By this time, the surface was covered with leaf primordia and leaves, mostly without accompanying buds.

Adventitious shoot regeneration from stem internode explants of Verbena officinalis L., a medicinal plant

Verbena officinalis L. (vervain) is a medicinal plant that shows several biological activities, including antimicrobial, antiinflammatory, analgesic, neuroprotective, hypnotic/sedative, gastroprotective, hepatoprotective, anticancer, cicatrizant, and antioxidant. This report describes an efficient plant regeneration system for V. officinalis via adventitious shoot development from stem internode and petiole explants. Stem internode explants were more effective than petiole explants in terms of both the number of shoots per explant and the percentages of explants forming shoots. Increasing benzyladenine (BA) concentrations steadily increased shoot formation up to 13.32 µM, but 22.22 µM BA caused a sharp decrease. The highest number of shoots (17.1 shoots per explant at 100% frequency) was obtained when 13.32 µM BA was combined with 5.71 µM indole-3-acetic acid (IAA). Rooting of the regenerated shoots was readily achieved when multiple shoots were singled out and cultured on medium containing varying concentrations of different auxins [IAA, indole-3-butyric acid (IBA), 2,4-dichlorophenoxyacetic acid (2,4-D), or naphthalene acetic acid (NAA)]. IBA was more effective in terms of the number of roots developed, producing a mean of 7.1 roots per shoot at 4.92 µM, whereas IAA was more effective for the frequency of shoots developing roots: 100% of the shoots rooted at 5.71 µM IAA. More than 90% of the regenerants survived through the hardening off process, when they were transferred to foam cups and kept under growth room conditions for 2 weeks before finally being moved to room conditions at low humidity. Approximately 3 months after the transfer to room conditions, the flowering of the regenerated plants could be observed.

In vitro propagation of the wild carrot Daucus carota L. subsp. halophilus (Brot.) A. Pujadas for conservation purposes

Daucus carota subsp. halophilus, is a wild crop relative of domestic carrot. It is an aromatic plant widely used in folk medicine due to recognized therapeutic properties of its essential oils. Experiments were carried out to evaluate the potential of in vitro propagation techniques to the conservation of this endemic and endangered taxon. The results showed that shoot tips of in vitro germinated seeds were able to proliferate in the presence of benzyladenine, with the best results being achieved using 4.4 µM, both in the first and second cultures. Shoots rooted after being transferred to 1/2-Murashige and Skoog basal medium. The results indicated that the concentration of benzyladenine used during the multiplication phase did not interfere with the rate of root formation. The obtained plantlets were morphologically and anatomically identical to those obtained by seeds. Some of the in vitro produced shoots developed flowers that produced viable pollen. Plant regeneration was also achieved by somatic embryogenesis induction in cotyledons and root segments cultured in the presence of 4.5 µM 2,4-dichlorophenoxyacetic acid. Somatic embryos converted into plantlets in a medium without growth regulators. Plants obtained either by shoot proliferation or somatic embryogenesis were acclimatized and are now growing at the Coimbra Botanical Garden. The first attempts to reintroduce these plants in the original habitat were successful. It can be concluded that the protocols developed are a useful approach to the conservation of this endemic species.

Relative water content, in vitro proliferation, and growth of Actinidia deliciosa plantlets are affected by benzyladenine

Relative water content in Actinidia deliciosa explants cultured in the presence of three benzyladenine (BA) concentrations (4.4, 44, and 440 μM) during different incubation periods were measured at different stages of the micropropagation process. BA incubation period during the multiplication phase determined the quality of A. deliciosa plantlets and affected both water content or retention capacity. Therefore, exogenous cytokinin application affected the quality and survival of plantlets in ex vitro conditions. In this sense, when 4.4 μM BA concentration was used, values higher than 20% for the dry weight/water saturated weight ratio at the end of the rooting phase can be indicative of low plantlet growth. Besides, the exposure period to cytokinins is of more affect than concentration.

In vitro propagation of the endangered orchid Laelia speciosa

Laelia speciosa is an endangered epiphytic orchid. The effects of various media components on germination of L. speciosa were evaluated. Pods were collected at 4, 7, and 9 months following hand-pollination, and seeds were germinated on Murashige and Skoog (MS) media with 30 g l−1 sucrose and five concentrations (0.0, 0.04, 0.22, 0.44, and 2.22 μM) of benzyladenine (BA) under light and dark conditions. Gibberellic acid (GA3; 0.0, 0.29, 1.44, 2.89, 14. 43 and 28.87 μM) with naphthaleneacetic acid (NAA; 0.0, 0.54, 1.34, 2.69, and 5.37 μM) were evaluated for in vitro subcultivation. MS medium with 30 g l−1 sucrose was effective for germination. The effects of BA and light on germination of L. speciosa seeds differed with pod maturity. All mature seeds germinated using 0.44 μM BA and light. The highest frequency of germinated seedlings (60%) was obtained using mature seeds grown on MS medium without BA and under light conditions. For subculture, MS with 30 g l−1 sucrose, 2.69 μM NAA, and 0.29 μM GA3 was effective. Plantlets of 5 cm in length were transplanted to the greenhouse, and a 77.5% of survival rate was obtained. A successful protocol for micropropagation by seed germination will contribute to the development of a sustainable management program for L. speciosa.

The influence of plant growth regulators on explant performance, bud break, and shoot growth from large stem segments of Acer saccharinum L

Benzyladenine (BA) and/or gibberellic acid (GA3) were applied in 20% white exterior latex paint separately at 0, 0.3, 1, 3, 10, or 30 mM; and at 1, 10, or 30 mM of each plant growth regulator (PGR) in a 3 × 3 factorial to 40 cm long stem segments of Acer saccharinum L. Softwood shoots were forced from these stem segments at various times of the year in a greenhouse and in a laboratory, these resulting shoots were surface disinfested and used as explants in vitro on Driver and Kuniyuki Walnut medium with 0 or 0.01 μM thidiazuron (TDZ). There was some response to the plant growth regulators applied in paint for shoot production from the stem segments and in vitro. Explants from softwood shoots forced from stems painted with 3 mM BA and cultured on medium with 0.01 μM TDZ produced more shoots than explants taken from softwood shoots forced with other BA concentrations or controls. Callus also grew significantly more on explants from stems treated with 3 mM BA cultured on 0.01 μM TDZ than explants harvested from stems painted with other concentrations of BA excluding 10 mM BA. When stem segments treated with BA plus GA3 were compared as a group to controls, more and longer softwood shoots grew on the stems painted with PGRs when all four runs were pooled (Sept. 2005 through Feb. 2006). Application of PGRs in paint extends the season of production of softwood shoots that may be used as explant materials and their subsequent performance in vitro.

In vitro conservation of Ceropegia intermedia - an endemic plant of south India

The purpose of this study was developing in vitro techniques for conserving wild and endemic species of Ceropegia intermedia by axillary shoot multiplication. Murashige and Skoog (MS) medium with 6.66 μM N 6 benzyladenine (BA) was best for axillary bud proliferation inducing a mean of 5.51 shoots per node. Excision and culture of the nodal segments from the in vitro shoots on fresh medium with same concentration of BA facilitated development of more than 5 shoots per node. Subsequent subculture slightly enhanced up to third subcultures and decreased thereafter. Shoots developed were rooted best on half strength MS with 5.37 μM NAA. Plantlets established in pots exhibited 75% survival. Plantlets were successfully established in field and morphological characters were identical to mother plants.

Benzyladenine and Cyclanilide Promote Shoot Development and Flowering of ‘Moonbeam’ Coreopsis

Abstract Effects of cyclanilide (CYC) and benzyladenine (BA) applied as foliar sprays or substrate drenches on the growth and flowering of ‘Moonbeam’ coreopsis (Coreopsis verticillata L. ‘Moonbeam’) during greenhouse production were determined. A foliar spray or substrate drench of 250 to 2000 ppm BA promoted increased numbers of vegetative shoots, reproductive shoots, and flower buds that increased with concentration. With BA concentrations of 250 or 500 ppm, flowering was delayed by 7 days or less and any foliar chlorosis was transient. Flowering delays of up to 19 days and persistent foliar chlorosis occurred in plants treated with 1000 or 2000 ppm BA. CYC applied at 25 to 100 ppm promoted fewer vegetative and reproductive shoots and flower buds than did BA; however, compared to control plants, plants developed more flower buds with no flowering delay, except when applied as a 100 ppm drench. Plants treated with CYC foliar sprays or substrate drenches developed twisted and horizontal growing shoots that persisted until full flower and detracted from the plants' appearance.

IN VITRO ROOTING IMPROVEMENT OF ADULT PISTACHIO, PISTACIA VERA L. 'ATLI'

Axenic cultures of Pistacia vera L. 'Atli' initiated on agar solidified Murashige and Skoog (MS) medium with Gamborg vitamins supplemented with different concentrations of 6-benzyladenine (BA) and indole-3-acetic-acid (IAA) were proliferated and maintained on an MS medium supplemented with 4.4 μM BA. Using these proliferated cultures, effects of: i) washing of the basal-cut-ends in sterile distilled water; ii) dipping of the basal-cut-ends in the different indole butyric acid (IBA) solutions; iii) cloned shoots; and iv) explant sizes (1.0, 2.0, 3.0 and 4.0 cm long) were assessed for root induction. The highest rooting frequency (92%) of microshoots was recorded at 4.0 cm long cloned and twice washed microshoots. Rooting was apparent within 14 days. The present study also aimed to reduce the occurrence of basal callus production. The microshoots cultured from the cloned shoots produced less calli than the mixed population, and developed roots that resemble the seedling plantlets. Those plantlets that had been rooted and exposed to sterile compost were able to acclimatize readily. Major improvement of the present study over previously published protocols has been achieved with the improvement of the in vitro rooting percentages of adult pistachio plantlets.

A new time-saving transformation system for Brassica napus

By optimizing different parameters for infection, an efficient Agrobacterium-mediated transformation system for Brassica napus was developed. A series of combinations of optical densities, infection durations, concentrations of acetosyringone and silwet L77 were evaluated. Maximum transformation efficiency reached 18.93% when cotyledonary nodes were infected for 10 min with an Agrobacterium suspension of 0.8 OD and 200 iM acetosyingone and 0.02% silwet L77 in the culture medium. In addition, the concentrations of 6-benzyladenine and naphthalene acetic acid were also optimized. Meanwhile, maximum transformation efficiency for green plantlet was 94.38% when cotyledonary nodes were cultivated in the medium containing 4 mg/L 6-benzyladenine and 0.04 mg/L naphthalene acetic after co-cultivation. Transformation was confirmed by polymerase chain reaction analysis, southern blotting analysis and green fluorescent protein assay.

An Efficient Procedure for Regeneration from Leaf-derived Calluses of Lonicera macranthoides ‘Jincuilei’, an Important Medicinal Plant

‘Jincuilei’ is a mutant selected from Lonicera macranthoides Hand.-Mazz. It produces abundant flowers that never open with a chlorogenic acid (CGA) content up to 6.0%. Propagation through rooting or grafting has only a 30% survival rate. This study was undertaken to establish an efficient protocol for rapidly regenerating this mutant. Leaf explants were inoculated on Gamborg's B5 medium supplemented with different concentrations of 6-benzyladenine (BA) and 2,4-dichlorophenozyacetic acid (2,4-D). The optimal combination for callus induction was 4.4 μm BA with 2.26 μm 2,4-D, which resulted in 86.7% of leaf explants producing calluses in 4 weeks. Calluses produced from this optimal medium were cultured on B5 medium containing different concentrations of kinetin (KT) and α-naphthalene acetic acid (NAA). The best formulation for shoot induction was B5 medium containing 0.9 μm KT and 5.4 μm NAA in which 73.4% of cultured calluses produced shoots in 8 weeks, and shoot numbers ranged from three to six per callus piece (1 cm3). Adventitious shoots were cut and rooted in half-strength Murashige and Skoog medium supplemented with 14.8 μm 3-indolebutyric acid. Roots initiated 10 d after culture, and rooting percentages ranged from 98% to 100%. Plantlets grown in a container substrate in a shaded greenhouse had over a 95% survival rate. During the last 6 years, over four million plantlets were regenerated using this established procedure, and there was no somaclonal variation. Fresh and dry weights of 1000 flowers, CGA contents, and dry flower yields of the regenerated plants were not significantly different from those of the stock ‘Jincuilei’ propagated by cutting, indicating that plants regenerated from this established procedure were stable. This established in vitro culture method has led to rapid commercial production of this medicinal plant on more than 1500 ha of production field.