Induction of direct shoot organogenesis and in vitro flowering from shoot tip explants of cucumber (Cucumis sativus L. cv. ‘Green long’)

In vitro flowering is an alternative breeding tool for generating hybrid Cucumis spp. as it is able to overcome limitations caused by interspecific incompatibility. The present study describes an efficient method for induction of multiple shoots and in vitro flowering from shoot tip explants of cucumber (Cucumis sativus L.). Shoot tip explants were excised from 7-day-old seedlings and cultured on Murashige and Skoog (MS) medium fortified with different concentrations of 6-benzylaminopurine (BAP; 0.5–2.5 mg/L) alone or in combination with 0.5 mg/L kinetin (KIN). The highest frequency (93.1%) of multiple shoot formation with maximum number of shoots (15.2 shoots/explant) was achieved on MS medium supplemented with 1.0 mg/L BAP. For in vitro flowering, shoots were cultured on MS medium supplemented with 0.5 mg/L BAP and different concentrations of sucrose. Flowering occurred on about 95% of in vitro shoots cultured on MS medium fortified with 6% (w/v) sucrose and 0.5 mg/L BAP after 15 d. For rooting, shoots (>2 cm) were cultured on MS medium augmented with various concentrations of indole-3-butyric acid (IBA; 0.5–2.5 mg/L) alone or in combination with 0.5 mg/L KIN. Among the combinations tested, supplementation with IBA (1.5 mg/L) and KIN (0.5 mg/L) induced maximum rooting rates (95.4%) with 7.8 roots/shoot. Rooted plantlets were successfully transferred into plastic cups containing a mixture of soil and sand (1:1), established in the greenhouse, and subsequently acclimatized in the field. The in vitro flowering reported in this study may facilitate rapid hybridization in Cucumis species and offers a model system for studying the physiological mechanisms involved in flowering.     

The role of in vitro cultivation on asymbiotic trait variation in a single species of arbuscular mycorrhizal fungus

Cultivating arbuscular mycorrhizal (AM) fungi in vitro is an efficient way to produce material for industry and research. However, such artificial growing conditions may impose selective pressure on fungi grown in vitro over many generations. We hypothesized that isolates subjected to long term propagation in vitro may develop increasingly ruderal traits. We proposed a predictive framework for the effect of in vitro cultivation on asymbiotic AM fungal traits. Using photomicrography and image processing, we analyzed morphology and growth traits for 14 isolates representing an in vitro cultivation gradient from 0 to >80 generations in vitro. We investigated the range of trait variation among asymbiotic growth of arbuscular mycorrhizal (AM) fungus isolates (Rhizoglomus irregulare). Spore dormancy was strongly associated with in vitro cultivation. We observed extremely high levels of inter-isolate variation for most fungal traits, but this was not related to time in vitro. Our results indicate that intra-specific diversity may have a strong ecological role in AM fungal communities.     

Flow cytometry distinction between species and between landraces within Lathyrus species and assessment of true-to-typeness of in vitro regenerants

The genus Lathyrus includes a number of neglected wild relatives of pea with potential as genetic resources for acquisition of stress resistance traits, but, due to little breeding, genotypes under culture are mainly landraces and seldom true varieties. Development of in vitro approaches for Lathyrus is also limited, and assessments of nuclear DNA content, for taxonomical or breeding purposes, are sparse. Genome size and AT/GC ratio were determined by flow cytometry, allowing for distinction between protein and forage L. sativus, L. cicera, L. ochrus and L. clymenum and the ornamental sweet pea (L. odoratus), and also between landraces within L. sativus L. and L. cicera L. In addition, explants from in vitro seedlings of eight genotypes from the five Lathyrus species above were cultivated in vitro, plant regeneration was achieved for all landraces and species, and the nuclear DNA content of the regenerants was compared with that of their mother plants, whereby the true-to-typeness of such regenerants was confirmed.     

Effect of methyl jasmonate on gummosis in petioles of culinary rhubarb (<Emphasis Type="Italic">Rheum rhabarbarum</Emphasis> L.) and the determination of sugar composition of the gum

This study aimed to know the key chemical compound influencing gummosis in petioles of intact growing culinary rhubarb (Rheum rhabarbarum L.) with special emphasis on its sugar composition. The application of methyl jasmonate (JA-Me, 0.5 and 1% in lanolin, w/w) in the middle of intact petiole of growing rhubarb substantially induced gummosis in the entire petioles, below and above the treatment, within several days. JA-Me at 0.5% in lanolin greatly stimulated ethylene production in intact petiole of growing rhubarb, on the 3rd day after JA-Me treatment, ethylene level being increased five times or more. However, an ethylene-releasing compound, ethephon (2-chloroethylphosphonic acid, 1 and 2% in lanolin, w/w) alone had no effect on gummosis. Analysis of gum polysaccharides by a gel permeation chromatography with a Tosho TSK-gel G5000PW gel permeation column revealed that almost all of rhubarb gum polysaccharides were eluted near the void in this gel chromatography system, suggesting that molecular mass of rhubarb gum polysaccharides are more than 500 kDa, while precise mass has not been decided in this study. Analysis of gum sugar composition after hydrolysis revealed that rhubarb gums is rich in galactose (ca. 30%), arabinose (ca. 20%), and galacturonic acid (15–20%), although other sugars also existed in small quantities. These results suggest that the key chemical compound of gummosis in petioles of rhubarb is jasmonates rather than ethylene, and gum polysaccharides consist of not only pectic arabinogalactans but also homogalacturonans.     

Ethephon-Induced Gummosis in Sour Cherry (Prunus cerasus L.) : I. Effect on Xylem Function and Shoot Water Status

Ethephon, (2-chloroethyl)phosphonic acid, was sprayed at concentrations up to 69.2 millimolar to enhance gum formation in 1-year-old shoots of mature Prunus cerasus L. cv Montmorency trees. Gum accumulation caused rupturing of the shoot periderm, followed by gum extrusion. Lower ethephon concentrations were required to induce gum formation in spring and early summer (1.7-3.5 millimolar) then in late summer and fall (13.8-69.2 millimolar). The number of functional vessels, shoot hydraulic conductance, and water potential of both leaf and internode tissue decreased as gum content of shoots increased. Nontreated control shoots also contained small quantities of gum. There was no difference in neutral sugar composition of gum exuded by the tree, obtained from aqueous shoot extracts, or flushed from the vessels of shoots, whether induced by ethephon or not. Severe decrease in shoot and leaf water potential was associated with shoot die-back. Recovery of xylem function may occur where gummosis is less severe. Discrepancy between measured and predicted hydraulic conductance increased as shoot gum content increased, suggesting that decrease in number of functional vessels alone was not sufficient to explain the effects of gum on loss of shoot hydraulic conductance. Increased gum content in those vessels remaining functional would increase vessel sap viscosity and further reduce hydraulic conductance. The viscosities necessary to account for discrepancy between measured and predicted hydraulic conductance were calculated. Gum concentration less than 1.0% (w/v) would produce these viscosities.     

Regulation of a novel Fusarium cytokinin in Fusarium pseudograminearum

Fusarium pseudograminearum is an agronomically important fungus, which infects many crop plants, including wheat, where it causes Fusarium crown rot. Like many other fungi, the Fusarium genus produces a wide range of secondary metabolites of which only few have been characterized. Recently a novel gene cluster was discovered in F. pseudograminearum, which encodes production of cytokinin-like metabolites collectively named Fusarium cytokinins. They are structurally similar to plant cytokinins and can activate cytokinin signalling in vitro and in planta. Here, the regulation of Fusarium cytokinin production was analysed in vitro. This revealed that, similar to deoxynivalenol (DON) production in Fusarium graminearum, cytokinin production can be induced in vitro by specific nitrogen sources in a pH-dependent manner. DON production was also induced in both F. graminearum and F. pseudograminearum in cytokinin-inducing conditions. In addition, microscopic analyses of wheat seedlings infected with a F. pseudograminearum cytokinin reporter strain showed that the fungus specifically induces its cytokinin production in hyphae, which are in close association with the plant, suggestive of a function of Fusarium cytokinins during infection.     

In vitro and in vivo antifungal activates of the essential oils of various plants against strawberry grey mould disease agent Botrytis cinerea

In order to investigate the effects of antifungal essential oils on postharvest decay and some quality factors of strawberry fruit, experiments were conducted under in vitro and in vivo conditions. The antifungal activates of essential oils obtained from fennel, anis, peppermint and cinnamon at concentrations 0, 200, 400, 600 and 800 μL L^?1 were investigated against Botrytis cinerea with four replications. In vitro results showed that the growth of B. cinerea was completely inhibited by fennel, cinnamon and anis essential oils at relatively low concentrations (400–800 μL L^?1). In vivo results showed that all the used essential oils at all applied concentrations caused an increase in the shelf life and inhibited of B. cinerea growth on strawberry fruits completely in comparison to the controls. The results of this study confirmed the antifungal effect of four essential oils in both in vitro and on fruit postharvest.     

Improved micropropagation of Gypsophila paniculata with bioreactor and factors affecting ex vitro rooting in microponic system

Studies on the mass production of high-quality plantlets in Gypsophila paniculata L. using a bioreactor and microponic system (a hydroponic system in which micropropagation shoots are planted) indicated that both aeration treatments, in which bioreactors were aerated from the top of explants by sparger (AS) and by tub (AT), were more effective than unaerated treatment for shoot proliferation and growth, and the maximum shoots (15.7 shoots per explant) with low hyperhydricity rate (2.9%) were found in the AS group. The ex vitro culture was more efficient for rooting when compared to the in vitro culture; the better shoot and root growth was obtained in the ex vitro culture, with rooting rate reaching 100% after 20 d of culture, but only 65% of in vitro shoots rooted; all stomata of ex vitro shoots closed, and their length was more than their width, but the stomata in in vitro shoots were all opened, the length close to the width. Furthermore, the stomata numbers were less in ex vitro (67.8) than in vitro (267.2). The survival rate of ex vitro plants reached 83.3% when plantlets derived in vitro and ex vitro were transferred to pots, while only 23.3% of in vitro plantlets survived. During ex vitro rooting with the microponic system, foam as the supporter material, 90 μmol?m^?2?s^?1 of light, and 80 shoots of planting density were favorable for shoot and root growth. The combination of bioreactor and microponic systems is an efficient way to produce high-quality plantlets of G. paniculata. Their application can reduce costs during large-scale industrial production.     

High-Frequency Regeneration by Abscisic Acid (ABA) from Petiole Callus of Jatropha curcas

Jatropha curcas L. is attaining worldwide interest as an important biofuel crop. Experiments were conducted to improve the prevailing micropropagation technique as well as to develop a new ex vitro rooting method for J. curcas plant regeneration. Regeneration and ex vitro rooting efficiency was enhanced by augmenting the culture medium with abscisic acid (ABA). Different concentrations of 6-benzylaminopurine (BAP) and indole-3-butyric acid (IBA) were tested for callus generation from both in vitro and in vivo explants (leaf and petiole) on Murashige and Skoog (MS) medium. The best regenerative callus was achieved on MS medium supplemented with BAP (4.44 μM) and IBA (2.45 μM) from in vitro-cultured petioles. Highest regeneration (91%) was achieved by culturing petiole callus on MS medium supplemented with BAP (8.88 μM), IBA (0.49 μM), and ABA (1.9 μM), whereas 61% regeneration was obtained from in vitro leaf callus. Shoot proliferation and elongation was achieved on BAP (2.22 μM) and IAA (8.56 μM) with 10–13 shoots per explants. Highest rooting (65%) was achieved from M1 shoots (BAP, IAA, and ABA) on MS medium supplemented with IBA (2.45 μM), naphthaleneacetic acid NAA (0.54 μM), and 0.02% activated charcoal. Ex vitro rooting of 1-mo-old M1 shoots obtained from the charcoal-containing medium resulted optimum rooting (>72%) when transferred to polybags containing sterile sand. The plantlets were successfully acclimatized in soil with more than 98% survival rate in the greenhouse.     

Genetic variability among populations of Fusicladium species from different host trees and geographic locations in the USA

Peach and almond scab caused by Venturia carpophila and pecan scab caused by Fusicladium effusum result in yield loss, downgrading of fruit, defoliation and subsequent decline of an orchard. To understand the levels of genetic diversity and divergence of pathogens from different hosts and locations 51 isolates were genotyped and analyzed using 10 RAPD and 5 UP-PCR markers, including 18 isolates of V. carpophila from peach trees in the southeastern United States, 12 isolates of V. carpophila from almond trees in California, and 21 isolates of F. effusum (a related species) from pecan trees in the southeastern United States. The combined marker results showed a low incidence of polymorphisms among the peach isolates (4.2 % of markers), but a higher incidence of polymorphisms among the almond isolates (42.0 %) and the pecan isolates (61.0 %). The Dice coefficient of similarity ranged from 0.932 to 1.000 for the peach V. carpophila isolates, 0.214 to 0.976 for the almond V. carpophila isolates, and 0.528 to 0.920 for the pecan F. effusum isolates. UPGMA bootstrap values indicated that UP-PCR data were slightly more robust and, based on the combined data, the UPGMA bootstrap analysis (1,000 runs) gave a high node value (100 %) differentiating all the isolates of V. carpophila from F. effusum and a moderate node value differentiating the peach and almond isolates of V. carpophila (68 %). The results suggest some divergence between the V. carpophila populations of almond trees in California and peach tree populations in the southeastern United States, and different levels of genetic diversity within the two populations.     

Potential of kenaf (Hibiscus cannabinus L.) and corn (Zea mays L.) for phytoremediation of dredging sludge contaminated by trace metals

The potential of kenaf (Hibiscus cannabinus L.) and corn (Zea mays L.) for accumulation of cadmium and zinc was investigated. Plants have been grown in lysimetres containing dredging sludge, a substratum naturally rich in trace metals. Biomass production was determined. Sludge and water percolating from lysimeters were analyzed by atomic absorption spectrometry. No visible symptoms of toxicity were observed during the three- month culture. Kenaf and corn tolerate trace metals content in sludge. Results showed that Zn and Cd were found in corn and kenaf shoots at different levels, 2.49 mg/kg of Cd and 82.5 mg/kg of Zn in kenaf shoots and 2.1 mg/kg of Cd and 10.19 mg/kg in corn shoots. Quantities of extracted trace metals showed that decontamination of Zn and Cd polluted substrates is possible by corn and kenaf crops. Tolerance and bioaccumulation factors indicated that both species could be used in phytoremediation.     

Isolation and identification of a plant growth inhibitor from Tinospora tuberculata Beumee

Tinospora tuberculata Beumee has been used widely as a folk medicine and several bioactive compounds have been isolated. However, no herbicidal compound has been reported in this species. Therefore, we investigated the presence of phytotoxins in T. tuberculata. The aqueous methanol extracts of T. tuberculata inhibited the growth of roots and shoots of cress (Lepidum sativum L.), lettuce (Lactuca sativa L.), timothy (Phleum pratense L.) and barnyard grass (Echinochloa crus-galli (L.) Beauv.). The extract was then purified by several chromatographic runs with monitoring the inhibitory activity and the main phytotoxic substance was isolated. The chemical structure of the compound was determined by spectral data as syringin (4-[(1E)-3-Hydroxyprop-1-en-1-yl]-2,6-dimethoxyphenyl β-d-glucopyranoside). It inhibited the root and shoot growth of all test plant species at concentrations >10 µM. The concentrations required for 50 % inhibition of root and shoot growth of cress and lettuce ranged from 78.2 to 412 μM, and that of timothy and barnyard grass renged from 9.8 to 73.2 µM. Effectiveness of syringin on monocotyledonous (timothy and barnyard grass) plants was greater than that on dicotyledonous (cress and lettuce) plants. These results suggest that syringin may contribute to the allelopathic effect caused by the T. tuberculata extract.     

Efficient direct plant regeneration from immature leaf roll explants of sugarcane (<Emphasis Type="Italic">Saccharum officinarum</Emphasis> L.) using polyamines and assessment of genetic fidelity by SCoT markers

A rapid and efficient method for in vitro direct plant regeneration from immature leaf roll explants of Saccharum officinarum L. (sugarcane) cv. Co 86032 was developed by the application of exogenous polyamines (PA). The effect of explant source from apical meristems and pre-culture of explants in the dark on shoot regeneration was studied. Adventitious shoot regeneration occurred on the proximal regions of immature leaf roll explants when pre-incubated in the dark for 2 wk and the regeneration response was decreased from the middle to distal end. A higher number of direct shoots (130 primary shoots explant^?1) and multiple shoots (657 secondary shoots explant^?1), were obtained with a combination of spermidine (103.27 μM), spermine (49.42 μM), and putrescine (31.04 μM) along with plant growth regulators. Shoot induction was increased up to twofold and multiplication was increased up to threefold in the medium supplemented with PA. Profuse rooting was observed in putrescine (93.12 μM), spermidine (68.84 μM), and spermine (24.71 μM), with mean number of 57 roots. A twofold increase in the number of roots was observed in medium supplemented with PA with respect to control cultures, which facilitated the successful transplantation and acclimatization process of in vitro propagated sugarcane plants. Histology and scanning electron microscopy analyses supported adventitious direct shoot regeneration from immature leaf roll explants. The genetic stability of in vitro regenerated plants was confirmed using start codon targeted polymorphism marker system.     

Temporary immersion systems for Amaryllidaceae alkaloids biosynthesis by Pancratium maritimum L. shoot culture

The alkaloid patterns of sea daffodil (Pancratium maritimum L.) shoot culture, cultivated in a temporary immersion cultivation system were investigated. The shoots accumulated maximal amounts of biomass (0.8 g dry biomass/L and Growth Index?=?1.6) at immersion frequency with 15 min flooding and 12 h stand-by periods. At this regime P. maritimum shoots achieved the highest degree of utilization of carbon source. Twenty-two alkaloids, belonging to narciclasine, galanthamine, haemanthamine, lycorine, montanine, tazettine, homolycorine and tyramine types were identified in intracellular and extracellular alkaloid extracts. The immersion frequency affected strongly the capacity of alkaloid biosynthesis in P. maritimum shoots and at the optimum conditions of cultivation, the total intracellular alkaloid content reached up to 3,469 μg/g dry biomass. The main biosynthesized alkaloids were haemanthamine (900.1 μg/g) and lycorine (799.9 μg/g). The obtained results proved that temporary immersion technology, as a cultivation approach, and P. maritimum shoots, as a biological system, are prospective for producing wide range bioactive alkaloids.     

Lithium chloride contributes to blood–spinal cord barrier integrity and functional recovery from spinal cord injury by stimulating autophagic flux

Blood–spinal cord barrier (BSCB) disruption following spinal cord injury (SCI) significantly compromises functional neuronal recovery. Autophagy is a potential therapeutic target when seeking to protect the BSCB. We explored the effects of lithium chloride (LiCl) on BSCB permeability and autophagy-induced SCI both in a rat model of SCI and in endothelial cells subjected to oxygen–glucose deprivation. We evaluated BSCB status using the Evans Blue dye extravasation test and measurement of tight junction (TJ) protein levels; we also assessed functional locomotor recovery. We detected autophagy-associated proteins in vivo and in vitro using both Western blotting and immunofluorescence staining. We found that, in a rat model of SCI, LiCl attenuated the elevation in BSCB permeability, improved locomotor recovery, and inhibited the degradation of TJ proteins including occludin and claudin-5. LiCl significantly induced the extent of autophagic flux after SCI by increasing LC3-II and ATG-5 levels, and abolishing p62 accumulation. In addition, a combination of LiCl and the autophagy inhibitor chloroquine not only partially eliminated the BSCB-protective effect of LiCl, but also exacerbated TJ protein degradation both in vivo and in vitro. Together, these findings suggest that LiCl treatment alleviates BSCB disruption and promotes locomotor recovery after SCI, partly by stimulating autophagic flux.     

Characterization of extracellular proteins in members of the Paracoccidioides complex

Paracoccidioides is a thermodimorphic fungus that causes Paracoccidioidomycosis (PCM) – an endemic systemic mycosis in Latin America. The genus comprises several phylogenetic species which present some genetic and serological differences. The diversity presented among isolates of the same genus has been explored in several microorganisms. There have also been attempts to clarify differences that might be related to virulence existing in isolates that cause the same disease. In this work, we analyzed the secretome of two isolates in the Paracoccidioides genus, isolates Pb01 and PbEpm83, and performed infection assays in macrophages to evaluate the influence of the secretomes of those isolates upon an in vitro model of infection. The use of a label-free proteomics approach (LC-MS^E) allowed us to identify 92 proteins that are secreted by those strains. Of those proteins, 35 were differentially secreted in Pb01, and 36 in PbEpm83. According to the functional annotation, most of the identified proteins are related to adhesion and virulence processes. These results provide evidence that different members of the Paracoccidioides complex can quantitatively secrete different proteins, which may influence the characteristics of virulence, as well as host-related processes.     

Salvianolic acid B inhibits myofibroblast transdifferentiation in experimental pulmonary fibrosis via the up-regulation of Nrf2

Salvianolic acid B (SalB) is one of the most bioactive components extracted from Salvia miltiorrhiza, and its antioxidant capacity corresponds with its protective effects against cell injury from oxidative stress. The aim of the present study was to evaluate the effect of SalB on experimental pulmonary fibrosis and its ability to ameliorate the oxidative/antioxidative imbalance during fibrosis pathogenesis. The anti-fibrotic activity of SalB was first confirmed in Transforming growth factor β1(TGF-β1)-stimulated MRC-5 cells. The protection of SalB against oxidative stress during fibrogenesis in vitro was verified by detecting ROS production, the levels of glutathione (GSH) and malondialdehyde (MDA). The Western blot and PCR results indicated that SalB could up-regulate nuclear factor erythroid-derived 2-like 2 (Nrf2) at both the protein and mRNA levels and induce Nrf2 nuclear translocation in vitro, which may be the mechanism underlying the anti-fibrotic capacity of SalB. Furthermore, the anti-fibrotic and antioxidant capacities of SalB in vivo were confirmed in rats with BLM-induced pulmonary fibrosis. The immunohistochemistry results showed that Nrf2 was absent in fibroblastic foci (FF) areas, while the SalB treatment could increase the expression of Nrf2 in lung tissues, especially in FF areas.     

The effect of glutamate-induced excitotoxicity on DNA methylation in astrocytes in a new in vitro neuron-astrocyte-endothelium co-culture system

Glutamate-induced excitotoxicity is a contributer to many neurological diseases. Astrocytes may represent a new target for treating glutamate-induced excitotoxicity. However, the in vitro culture system that mimics the in vivo microenvironment is lacking. This study aimed to establish a new in vitro co-culture system including neurons, astrocytes, and endothelial cells (NAE), and to investigate the effect of glutamate-induced excitotoxicity on DNA methylation in astrocytes. A NAE co-culture method was created using a Transwell chamber, in which neurons were seeded on the bottom of the lower chamber, endothelial cells were plated on the top membrane, and astrocytes were plated on the bottom membrane of the insert. Glutamate-induced toxicity was induced using glutamate and glycine, and examined using immunofluorescence and lactate dehydrogenase release assay. Global methylation in astrocytes was analyzed, and the expression of DNMT1 and DNMT3a was examined using Western blot analysis. Glutamate treatment induced less neuronal damage in the NAE system compared with the control group in which neurons and astrocytes were cultured alone. Global DNA methylation was increased and the expression of DNMT1 and DNMT3a in astrocytes was increased after glutamate treatment, which was blocked by application of the NMDAR inhibitor MK-801 and the DNMT inhibitor 5-azaC from the endothelial cells. The in vitro ANE culture system is effective for studying glutamate-induced excitotoxicity, and may be used for testing the passage of drugs across the blood–brain barrier. Inhibition of DNA methylation in astrocytes may be a new therapeutic strategy for treating glutamate-induced excitotoxicity.     

Shoot organogenesis from leaf explants of Dayaoshania cotinifolia W. T. Wang

Dayaoshania cotinifolia W. T. Wang is a rare and endangered member of the Gesneriaceae family which is endemic to China. To conserve this species, an efficient in vitro propagation and regeneration system via shoot organogenesis was established from young leaf explants. Adventitious shoot induction was possible within 50–60 d on basal Murashige and Skoog medium supplemented with 1–3 μM 6-benzyladenine, although 5 μM 6-benzyladenine induced hyperhydricity. Basal medium containing 1–5 μM thidiazuron induced fewer shoots, while 1–5 μM α-naphthaleneacetic acid induced numerous adventitious roots and a few adventitious shoots. However, when thidiazuron and α-naphthaleneacetic acid were combined, both the induction percentage and number of shoots increased. Leaf explants cultured on induction medium supplemented with 1–5 μM 2,4-dichlorophenoxyacetic acid become necrotic and died. Induction medium supplemented with 1 μM α-naphthaleneacetic acid and 1–3 μM 6-benzyladenine was optimal for inducing adventitious shoots as was the combination of 1–3 μM thidiazuron and 1 μM α-naphthaleneacetic acid. Induction medium containing 2.0 μM 6-benzyladenine and 0.5 μM indole-3-acetic acid was optimal for the multiplication of adventitious shoots. Rooting was achieved on half-strength MS medium supplemented with 3.0 μM indole-3-acetic acid or α-naphthaleneacetic acid and 0.1% activated charcoal. Plantlets were transplanted to a mixture of sand, vermiculite, and humus (1:1:1); 92% survived. This protocol is a unique and effective means to micropropagate this rare and important plant and could serve as a solution for in vitro and ex vitro conservation.