Development of a shoot multiplication system for hop (Humulus lupulus L.)

Summary Nodal explants from hop were exposed to plant growth regulators to determine suitable media for initiation from axillary buds and subsequent micropropagation. Efficient culture establishment (96.6% of explants) was achieved on Murashige and Skoog (MS) medium (modified to contain 1 mg l⁻¹ thiamine hydrochloride) supplemented with 0.57 μM indoleacetic acid (IAA) and 2.22 μM 6-benzylaminopurine (BA). Subsequent transfer of explants to treatments containing an auxin ([1-naphthaleneacetic acid], NAA or IAA) and BA, 6-[γ,γ-dimethylallylamino]purine (2iP), kinetin (KIN) or thidiazuron (N-phenyl-N′-1,2,3-thidiazol-5-ylurea [TDZ]) resulted in significantly different amounts of multiplication. Optimal TDZ-supplemented media elicited a greater than threefold increase in the number of shoots and nodes generated per explant compared to optimal media containing BA, 2iP and KIN. Shoots were successfully rooted using half-strength MS supplemented with 5.71 μM IAA and 4.9 μM indolebutyric acid (IBA), and regenerated plants were successfully transferred to soi. An overall micropropagation schedule, which can be implemented into hop commercialization programs, includes: (i) establishment in MS with 0.57 μM IAA and 2.22 μM BA; (ii) multiplication in MS with 0.57 μM IAA and 2.27 μM TDZ; (iii) elongation in MS; and (iv) rooting in half-strength MS with 5.71 μM IAA and 4.9 μM IBA.     

Petal: a reliable explant for direct bulblet regeneration of endangered wild populations of <Emphasis Type="Italic">Fritillaria imperialis</Emphasis> L.

Wild populations of Fritillaria imperialis L. are facing extinction and need urgent conservation. This paper presents an efficient system for in vitro direct bulblet regeneration of these populations by petal culturing of flower buds. Petals at different developmental stages, green-closed flower bud (before nectar secretion) and red-closed flower bud (beginning of nectar secretion), were used as explants, and the effects of various proportions of cytokinin to auxin on direct bulblet regeneration pathway were evaluated. More explants switched on direct regeneration pathway in combination of auxins (0.6 mg l⁻¹ NAA + 0.4 mg l⁻¹ IAA) with higher level of cytokinin (1 mg l⁻¹ BAP). In contrast, auxins (0.6 mg l⁻¹ NAA + 0.4 mg l⁻¹ IAA) with lower level of cytokinin (0.1 mg l⁻¹ BAP) produced more bulblets per regenerated explant. In green-closed flower bud stage, direct bulblets regenerated from the end of petal where it was connected to the receptacle, while nectar secretion site was the place of bulblet formation in red-closed flower bud stage. In addition, genotype-dependency of direct bulblet regeneration pathway was investigated by using two different wild populations of Fritillaria imperialis. This plant regeneration procedure was applicable to different Fritillaria genotypes and regenerated bulblets were normal.     

Effects of Auxins and Cytokinins on Embryo Formation from Root-derived Callus of Oncidium ‘Gower Ramsey’

In an attempt to optimize somatic embryo formation in Oncidium ‘Gower Ramsey’, the effects of five auxins (2,4-D, IAA, IBA, NAA and picloram) and five cytokinins (2iP, BA, kinetin, TDZ and zeatin), used alone, was tested in vitro using root-derived callus. In general, kinetin (0.5 and 2 mg l⁻¹) and zeatin (0.5 mg l⁻¹) were found to be more effective than other auxin and cytokinin treatments to induce somatic embryogenesis from root-derived callus.     

TDZ-induced high frequency plant regeneration through multiple shoot formation in witloof chicory (<Emphasis Type="Italic">Cichorium intybus</Emphasis> L.)

A very efficient and rapid regeneration system via multiple shoot formation was developed for Cichorium intybus L. when leaf explants excised from sterile seedlings were cultured on medium supplemented with different concentrations and combinations of various plant growth regulators. In a comparison of leaf lamina and petiole explants, lamina explants produced over three times more shoots than petiole explants, with a mean of 7.5 shoots compared to 2.4. Of the combinations of KIN/IAA, KIN/NAA, BAP/IAA, or BAP/NAA, 0.5 mg l⁻¹ KIN combined with 0.3 mg l⁻¹ IAA was the most effective, producing a mean of 19.7 shoots per lamina explant while the control treatment involving no plant growth regulators produced no shoots at all. When either cytokinin was used alone, BAP was found nearly twice more successful than KIN. However, the most effective treatment of all was the combination of 0.01 mg l⁻¹ TDZ and 1.0 mg l⁻¹ IAA, producing as many as 35.8 shoots per lamina explant. This rate of shoot regeneration is remarkably higher than those previously reported for C. intybus, most likely due to the highly inductive effect of TDZ, which was tested for the first time in this species. Rooting of the shoots was readily achieved on medium containing different concentrations of IAA or IBA. IAA was more effective than IBA and resulted in the highest frequency of shoots that rooted (100%) and mean number of roots per shoot (4.2) when used at 0.5 mg l⁻¹. Hardening off process resulted in a production of more than 80% healthy plantlets.     

High Frequency of Shoot Regeneration from Hypocotyls and Stem Segments of Antirrhinum majus (Snapdragon)

A broadly applicable direct shoot regeneration method from hypocotyls and stem explants has been developed for six cultivars of Antirrhinum majus L. In order to establish a stable and high frequency of shoot regeneration system, leaves, hypocotyls and stem explants of six cultivars were tested with 72 combinations of auxin (naphthaleneacetic acid (NAA) or 3-indoleacetic acid (IAA)) and cytokinin (6-benzylaminopurine (BA) or zeatin (Z)). A few adventitious shoots were directly regenerated from hypocotyl segments of cv. Orchid on MS medium with NAA + BA, IAA + BA, NAA + Z and IAA + Z. High frequency of direct shoot regeneration was obtained from hypocotyl segments on MS medium with 0.05, 0.1 or 0.25 mg l⁻¹ NAA + 2 mg l⁻¹ Z and 0.5 mg l⁻¹ IAA + 2 mg l⁻¹ Z. Finally, stable and high frequency (92–100%) of shoot regeneration with more than 10 adventitious shoots per explant was achieved from the hypocotyls and stem explants of all six cultivars on MS medium with 0.25 mg l⁻¹ NAA + 2 mg l⁻¹ Z. The shoots emerged directly from the hypocotyls and stem segments 4 weeks after culture initiation.     

In Vitro Propagation Of Four Watsonia Species

The genus Watsonia has a number of species with potential to be developed as new ornamental crop plants, but to date there are no reports on in vitro propagation of any member of this genus. Seeds from four Watsonia species, Watsonia gladioloides, Watsonia lepida, Watsonia laccata, and Watsonia vanderspuyiae were decontaminated and germinated on one-tenth strength MS media without hormones or sucrose. Shoots were induced from seedling hypocotyl segments when both an auxin [α-naphthaleneacetic acid (NAA)] and cytokinin [N⁶−benzylaminopurine (BA)] were present in the medium, while root and leaf explants failed to produce shoots. Multiplication of axillary shoots was greatest when only BA (0.5 mg l⁻¹) was added to the medium. Shoot explants propagated in a ‘liquid-shake’ culture exhibited greater growth rates than those on agar-solidified medium, but shoot production varied between species. Meristemoids were induced in all species, but no significant trend was found between growth index (GI) and meristemoid formation, suggesting that reduction in GI may not necessarily be a prerequisite for producing meristemoids. Corm formation was inconsistent and storage organs could only be induced in one of the four species, W. vanderspuyiae. This occurred best at 25°C with 3% sucrose and an agar level of 15%. Indole-3-acetic acid (IAA) and NAA at 1 mg l⁻¹ significantly increased mean number of roots per shoot explant on all four species, while indole-3-butyric acid (IBA) was more effective when applied at 0.1 mg l⁻¹. Plantlet survival ex vitro was negatively affected when NAA and 2,4-dichlorophenoxyacetic acid (2,4-D) were used to root shoot explants for all species. In W. laccata, all auxin treatments [IAA, IBA, NAA, phenylacetic acid (PAA), and 2,4-D] at a concentration of 1 mg l⁻¹ significantly reduced ex vitro survival of plantlets. Successful micropropagation of Watsonia is an important step in the further development of this genus as a horticultural crop.     

An efficient in vitro shoot propagation of cranberry (Vaccinium macrocarpon ait.) by axillary bud proliferation

Summary Cultures of two eranberry (Vaccinium macrocarpon Ait.) cultivars, ‘Ben Lear’ and ‘Pilgrim’, and three eranberry clones from natural stands in Newfoundland were established in a nutrient medium containing N⁶[2-isopentenyl]adenine (2iP) from nodal and/or shoot-tip explants obtained under aseptic conditions. The cultivars differed in shoot regeneration in terms of shoot number per explant with various concentrations of 2iP over two culture periods. Best total shoot production was obtained when nodal segments were cultured in the medium supplemented with 2.5–5.0 mg 2iP l⁻¹ (12.3–24.6 μM). With higher 2iP levels, shoots did not expand and had a high mortality rate. Nodal explants of the three clones cultured in the same nutrient medium supplemented with 2.5 mg 2iP l⁻¹ (12.3 μM) produced three to five healthy axillary shoots per explant. In another experiment, nodal explants were more productive than shoot tips. In all experiments with subculture, there was an increase in shoot multiplication rate for all genotypes. Shoots were rooted in vitro in the same media used for shoot proliferation, but without any growth regulators. After their transfer to potting medium, almost all of the rooted plants survived. Cranberry genotypes can be efficiently propagated and maintained through nodal culture in a nutrient medium without auxin that contains 2.5–5 mg 2iP l⁻¹ (12–25 μM).     

Micropropagation of <Emphasis Type="Italic">Penthorum chinense</Emphasis> through axillary bud

This study reports a protocol for successful micropropagation of Penthorum chinense using nodal explants on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA) or kinetin (Kn). The presence of BA promoted a higher rate of shoot multiplication than Kn. Maximum multiple shoot formation was observed in 59.2% of nodal explants cultured on MS medium supplemented with 2.0 mg l⁻¹ BA after 6 wk. After subculture for 4 wk, the maximum number of shoots (6.4) was obtained on a medium with 2.0 mg l⁻¹ BA, but shoots were too short and not suitable for micropropagation. The taller shoots that regenerated in the presence of lower BA concentration (1.0 mg l⁻¹) were selected for root induction study. Most shoots (98.8%) rooted in the presence of 0.5 mg l⁻¹ indole-3-acetic acid after 3 wk, with each shoot forming an average of 10.0 roots. Plantlets were transferred to soil and successfully acclimatized.     

Micropropagation with 3-methyleneoxindole: Its obligatory role in indole-3-acetic acid-mediated auxin action

Summary 3-Methyleneoxindole (MO), a metabolite of the plant auxin indole-3-acetic acid (IAA), was more active than IAA in supporting Stage II and III micropropagation of several plant species. In Stage II micropropagation, characterized by the rapid numerical increase of shoots, the optimal IAA concentration was 0.01 mM compared to 0.1 mM MO for most plants. In Stage III micropropagation where auxin is required for the rhizogenic response, 0.1 μM MO was more effective than 0.01 mM IAA. Inhibition analysis of plant growth with chlorogenic acid (CGA) suggested an obligatory role for MO in IAA-mediated auxin reactions: CGA, which blocks the enzymatic oxidation of IAA to MO, in vivo, completely abolished IAA's ability to support the growth of explants during micropropagation. In contrast, CGA did not inhibit the auxin activity of MO, the product of the blocked reaction. The growth rate and rooting efficiency of tobacco propagules in Stage III medium was improved substantially if these were first exposed to a high concentrations of MO and subsequently transferred to media containing low or no MO.     

In vitro germination and development of western hemlock dwarf mistletoe

A procedure for in vitro culture of the parasitic flowering plant western hemlock dwarf mistletoe, Arceuthobium tsugense (Rosend.) G.N. Jones subsp. tsugense, is described. A factorial experiment evaluated the effects of media (Harvey's medium (HM) and modified White's medium (WM)), temperatures (15 °C and 20 °C), presence or absence of light, and plant growth regulators (the auxin 2,4-dichlorophenoxyacetic acid (2,4-D) and the cytokinin 6-benzylaminopurine (BAP) at varying concentrations (0.001 mg l⁻¹ to 1 mg l⁻¹)). Seed explants germinated in less than one week in culture and produced radicles. Optimal conditions for radicle elongation were WM at 20 °C in the presence of light and without plant growth regulators. Some of the radicles split at the tip to yield callus while others swelled to become spherical holdfasts. Holdfasts were also produced at the tips of radicles, and callus arose from split holdfasts. Factors that promoted holdfast production were Harvey's medium, light, and 2,4-D at 1 mg l⁻¹. Callus development from split radicles and split holdfasts was optimal on WM with 0.5 mg l⁻¹ 2,4-D and 1 mg l⁻¹ BAP at 20 °C in the dark. This revised version was published online in June 2006 with corrections to the Cover Date.     

The role of GA,IAA and BAP in the regulation of <Emphasis Type="Italic">in vitro</Emphasis> shoot growth and microtuberization in potato

The effect of auxin, GA and BAP on potato shoot growth and tuberization was investigated under in vitro condition. The shoot length of potato explants increased with the increasing of concentrations (0.5 – 10 mg dm⁻³) of IAA treatment especially with the addition of GA₃ (0.5 mg dm⁻³), but was inhibited by BAP (5 mg dm⁻³). The root number and root fresh weight of potato explants increased with the increasing of IAA levels either in the presence of GA₃ (treatment IAA+GA) or not (IAA alone). However, no root was observed in the treatment IAA+BAP, instead there were brown swollen calli formed around the basal cut surface of the explants. The addition of GA₃ remarkably increased the fresh weight and diameter of calli. Microtubers were formed in the treatments of IAA+BAP and IAA + GA + BAP but not observed in the treatments of IAA alone or IAA + GA. IAA of higher concentrations (2.5 – 10 mg dm⁻³) was helpful to form sessile tubers. With the increasing of IAA levels, the fresh weight and diameter of microtubers increased progressively. At 10 mg/L IAA, the fresh weight and diameter of microtubers in the treatment of IAA + GA + BAP were 409.6 % and 184.4 % of that in the treatment of IAA + BAP respectively, indicating the interaction effect of GA and IAA in potato microtuberization.     

Micropropagation of Lepidium virginicum (Brassicaceae), a plant with antiprotozoal activity

Summary Micropropagation is a technique to ensure a constant and uniform source of medicinal plants. In this report, we describe the micropropagation of Lepidium virginicum L. (Brassicaceae), a wild plant used as an antiamoebic in traditional Mexican medicine. In vitro-germinated seeds were cultured in Murashige and Skoog (MS) medium to obtain pathogen-free cotyledons, hypocotyls, and apical bud (AB) explants. For induction of morphogenesis, the effect of cytokinins, benzyladenine (BA) and kinetin (KN), combined with auxin, indole-3-acetic acid (IAA) was evaluated. The best rate of shoot proliferation was induced 15 d after culture on MS mineral medium supplemented with IAA∶KN (0.57∶13.94 μM) from AB explants. Maximum shoot elongation was achieved without plant growth regulators. The effect of indole-3-butyric acid (IBA) (14.76 μM) was evaluated for in vitro root induction; 60 d after culture all the shoots had developed roots. All rooted plants were successfully transferred to pots and 100% acclimatized in ex vitro conditions. The methanol extracts from the micropropagated active explants of L. virginicum showed and IC₅₀ antiprotozoal value between 141.90 and 268.53 μg ml⁻¹.     

Morphactin influences guggulsterone production in callus cultures of <Emphasis Type="Italic">Commiphora wightii</Emphasis>

Guggulsterone, a hypolipidemic natural agent, is produced in resin canals of the plant Commiphora wightii. In this study, the efficacy of different plant growth regulators was evaluated for optimizing its production. Morphactin was found to be effective in enhancing the accumulation of guggulsterones in callus cultures. Maximum callus growth was recorded on medium containing morphactin (0.1 mg l⁻¹) and 2iP (2.5 mg l⁻¹), whereas maximum guggulsterone production occurred when the calluses were cultured on medium containing 0.1 mg l⁻¹ morphactin and 1.0 mg l⁻¹ 2iP. Morphactin and 2iP interacted significantly to enhance the callus growth and guggulsterone production by about 8-folds in one-year-old cultures. However, the effect of morphactin on callus growth and guggulsterone production was not uniform over the levels of 2iP tested. Such an effect of morphactin has never been reported on the production of secondary metabolites.     

Induction and growth characteristics of embryogenic avocado cultures

Embryogenic cultures were induced from immature avocado zygotic embryos representing different botanical races and complex hybrids. The optimum induction medium consisted of B5 major salts, MS minor salts, 0.4 mg l⁻¹ thiamine HCl, 100 mg l⁻¹ myo-inositol, 30 g l⁻¹ sucrose, 0.41 μM picloram and 8 g l⁻¹ TC agar. Somatic embryogenesis occurred directly from the explants on induction medium, and secondary embryos and proembryonic masses proliferated in liquid and on semisolid maintenance medium. Embryogenic culture maintainance was optimized in liquid, filter-sterilized MS medium, supplemented with 30–50 mg l⁻¹ sucrose, 4 mg l⁻¹ thiamine HCl and 0.41 μM picloram. Two types of embryogenic cultures were recognized: –genotypes that proliferated as proembryonic masses in the presence of auxin (PEM-type) and; –genotypes in which the heart stage and later stages of somatic embryos developed in the presence of auxin(SE-type). Embryogenic suspension cultures became increasingly disorganized over time, and this was associated with progressive loss of embryogenic potential. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of N6-isopentenyladenine concentration on growth initiation in vitro and rooting of bilberry and lingonberry microshoots

Buds and shoot tips of wild bilberry (Vaccinium myrtillus L.) and lingonberry (V. vitis-idaea L.) plants were cultured on a modified MS medium containing N⁶-isopentenyladenine (2iP), 9.8–78.4 μM, in order to study the effect of the 2iP-concentration on the initiation of growth. The experiment was first performed in the autumn and repeated in the spring to determine the influence of season on growth initiation. To optimise rooting, three different rooting treatments were tested for the bilberry and lingonberry microshoots. Shoots were rooted either in vitro with 0.49 μM IBA (indole-3-butyric acid) or ex vitro, incubating microshoots in 2.07 mM KIBA-solution (potassium salt of IBA) before planting, or microshoots were planted directly on peat without exogenous auxin. The best 2iP concentration for the initiation of the growth for bilberry was 49.2 μM and for lingonberry 24.6 μM. It was observed that increasing the 2iP concentration at the growth initiation stage increased the number of brownish explants both in bilberry and in lingonberry microcultures. Spring was a considerably better time than autumn for the initiation of new growth, for both species. The results of the rooting test showed that the KIBA-treatment before planting on peat increases rooting efficiency in both bilberry and lingonberry. This revised version was published online in June 2006 with corrections to the Cover Date.     

An efficient protocol for micropropagation of lemon (<Emphasis Type="Italic">Citrus limon</Emphasis>) from mature nodal segments

The influence of the basal medium and different plant growth regulators on micropropagation of nodal explants from mature trees of lemon cultivars was investigated. Although the basal medium did not affect any of the variables, explants on DKW medium were greener. Several combinations of 6-benzyladenine (BA) and gibberellic acid (GA) were used to optimise the proliferation phase. The number of shoots was dependent on the BA and GA concentrations and the best results were obtained with 2 mg l⁻¹ BA and 1 or 2 mg l⁻¹ GA. Explants length was shorter with the higher BA concentrations and, in all genotypes, shoot length was greater with 2 mg l⁻¹ GA. The best results for productivity (number of shoots × the average shoot length) were obtained with 2 mg l⁻¹ BA and 2 mg l⁻¹ GA, although explants with chlorosis and narrow leaves were observed. The presence of BA and GA in the proliferation medium was essential for the explant multiplication but GA had a greater influence. The transfer of in vitro shoots to rooting media, containing different concentrations of indole butyric acid (IBA) and indole acetic acid (IAA) produced complete plantlets. Lemon shoots rooted well in all rooting combinations. The highest rooting percentages were obtained on media containing 3 mg l⁻¹ IBA alone or IBA in combination with 1 mg l⁻¹ IAA and on these media the highest numbers of roots were produced. The average root length was affected significantly by the IBA and IAA concentrations. Root length was greater when only 3 mg l⁻¹ IBA was used, and in this rooting medium explants had a better appearance, with greener and larger leaves. The success during the acclimatisation was close to 100% and the plantlets exhibited normal growth in soil under greenhouse conditions.     

Thidiazuron-induced <Emphasis Type="Italic">in vitro</Emphasis> shoot organogenesis of the medicinal plant <Emphasis Type="Italic">Arnebia euchroma</Emphasis> (Royle) Johnst

Summary An efficient in vitro propagation system was developed for Arnebia euchroma, an important Chinese traditional medicinal plant. The present study utilized thidiazuron (TDZ) for the induction of shoot organogenesis on cotyledon and hypocotyl explants. The maximal number of shoots was obtained on the modified Linsmaier and Skoog (LS) medium supplemented with 1.0 mgl⁻¹ (4.5 μM) TDZ for 12d on cotyledon explants (8.6 shoots per cotyledon explant). Other cytokinins (kinetin and 6-benzyladenine) and auxin (α-naphthaleneacetic acid) were not efficient in inducing regeneration on cotyledon explants. Browning of the basal portion of the subcultured shoots could be significantly reduced when they were cultured on the modified LS medium supplemented with 100 mgl⁻¹ (33.3 μM) polyvinylpyrrolidone. Well-developed shoots formed roots on the same medium containing 1.0 mgl⁻¹ (4.9 μM) indole-3-butyric acid. The efficient regeneration protocol reported here provides an important means of micropropagation of this plant. Furthermore, this protocol is essential to future genetic improvement of plants via transformation protocols.     

Effects of low nitrogen medium on endogenous changes in ethylene, auxins, and cytokinins in in vitro shoot formation from rhizomes of Cymbidium kanran

Summary Shoot formation from rhizome explants of Cymbidium kanran was promoted on Murashige and Skoog (MS) medium: (1) with 1 mgl⁻¹ (4.4μM) 6-benzyladenine (BA) and 0.1 mgl⁻¹ (0.54μM) α-naphthaleneacetic acid (NAA); (2) with ethylene inhibitor (silver nitrate, AgNO₃); or (3) by reducing ammonium nitrate (NH₄NO₃) and potassium nitrate (KNO₃) to 25 and 50%, respectively, of their original concentrations. Shoot formation by BA and NAA was strongly inhibited with the application of ethephon, an ethylene releaser. The ethylene production from the rhizome explants was reduced 30–55% on low nitrogen medium after 1–3 mo. of culture and 52% on BA and NAA medium after 1 mo. of culture compared with explants on standard MS medium. No difference in endogenous auxin (indole-3-acetic acid, IAA) and cytokinin (isopentenyl adenosine, iPA) contents in the rhizomes was found between the treatments. Low ethylene levels were correlated with higher frequency of shoot formation from the rhizomes.     

Micropropagation ofCereus peruvianus mill. (cactaceae) by areole activation

Summary Currently,Cereus peruvianus plants can be rapidly clonedin vitro via adventitious organogenesis using callus cultures; however, somaclonal variation is a problem. A method is described herein using lateral bud explants to produce multiple shoots for clonal propagation. Apical and lateral explants were cultured on MS (Murashige and Skoog, 1962) media with factorial combinations of the auxins indole-3-acetic acid (IAA), 1-naphthaleneacetic acid (NAA), and cytokinins 6-ben-zyladenine (BA) and N-(2-furanyl-methyl)-1-purine-6 amine (kinetin) at the concentrations 0.0, 0.01, 0.1, 1.0 mg“l⁻¹. Positive results were obtained from the lateral explants in all conditions tested, but apical explants did not respond toin vitro multiplication ofC. peruvianus cactus at all growth regulator combinations tested. Formation of axillary shoots inC. peruvianus seems most frequent in medium containing BA at 1.0 mg·l⁻¹ (4.44 μM) and IAA or NAA at 1.0 mg·l⁻¹ (5.71 μM or 5.37 μM respectively), but the frequency of shoot formation in the BA or kinetin and NAA or IAA combinations indicated that any of the combinations tested can be used for multiplication ofC. peruvianus plants regenerated from callus tissue culture. Root formation occurred in all (100%) of the cactus shoots after 9 wk in the same culture medium. All the cacti that developed at the different auxin and cytokin combinations continued growth after transfer to a potting mix of red earth (Paleudult) and ground river sand (1∶1).     

Axillary multiplication of <Emphasis Type="Italic">Swertia chirayita</Emphasis> (Roxb. Ex Fleming) H. Karst., a critically endangered medicinal herb of temperate Himalayas

The present study describes the micropropagation of Swertia chirayita, an endangered medicinal herb of the temperate Himalayas, through axillary multiplication from 4-wk-old seedling-derived nodal explants. We obtained 4.5-fold multiplication every 4 wk on Murashige and Skoog (MS) medium supplemented with 4 μM benzyl amino purine (BA) and 1.5 μM 6-(γ,γ-dimethylallylamino)purine (2iP). Rooting was optimized on modified MS medium supplemented with 1 μM naphthalene acetic acid (NAA) and 500 mg l⁻¹ of activated charcoal (AC). A success rate of 94 % was obtained by in vitro hardening in the growth-room and by ex vitro hardening in greenhouse conditions. The present study can serve as a tool for the mass multiplication of elite genotypes of this critically endangered species and can also be utilized for multiplying large numbers of quality planting material for the replantation in their natural habitat. This approach can also help meet the requirements of the growing pharmaceutical industry by ensuring the regular supply of authentic planting material.