Differential biosynthesis and accumulation of picrosides in an endangered medicinal herb Picrorhiza kurroa

Picrorhiza kurroa Royle ex Benth (Family: Scrophulariaceae) is a medicinal herb, mainly found in the North-Western Himalayas. Extensive harvesting for pharmaceutical purposes, lack of organized cultivation and unorganized methods of uprooting the plants because of unawareness has brought an endangered status to this important herb in nature. The medicinal property of this plant is attributed to monoterpenoid picrosides. The influence of developmental status of different growth stages on picrosides content is poorly understood in Picrorhiza kurroa. Picroside-I (P-I) content increased from 0.05 % to 0.76 % in different growth stages of shoots. Significant increase in the contents of P-I (0.15–0.50 %) and Picroside-II (P-II) (0.1–0.45 %) was observed in rhizomes of different developmental stages. Highest amounts of P-I (8.7 %) and P-II (5.3 %) was detected in uppermost part of mature dried rhizomes compared to bottom part with 2.9 % and 2.2 % of P-I and P-II, respectively. P. kurroa grown at high altitude (Sairopa, 4,500 amsl) showed 1.75-folds increase in P-I in leaves whereas exponential increase in the P-I content was detected (0.05–1.7 %) in the leaves of different developmental stages (L1-L5) of P. kurroa grown at lower altitude (Jagatsukh, 1,900 m). Variable amounts of P-I and P-II in different growth and developmental stages of P. kurroa imply importance of selection of plant material (rhizomes and roots). The study undertaken explored the status of metabolites accumulation and biosynthesis in the field grown plants of P. kurroa where not only environmental parameters but different morphogenetic stages of its developmental cycles, different age groups and different parts of plantlets were extensively analysed and estimated for medicinally important picrosides.     

Modulation of Picroside-I Biosynthesis in Grown Elicited Shoots of <Emphasis Type="Italic">Picrorhiza kurroa</Emphasis> In Vitro

Elicitors are considered as biostimulants for growth improvement and enhancement of secondary metabolite content. To date, only seaweed extract (SWE) powder has been studied for its effect on picroside-I (P-I) production in in vitro grown Picrorhiza kurroa plants. However, little is known at the molecular level about P-I production in P. kurroa plants upon SWE treatment. Here, we investigated the relative effects of supplying different elicitors including methyl jasmonate (MeJa), sodium nitroprusside (SNP), and abscisic acid (ABA) with SWE on plant growth and P-I production in addition to their effects at the molecular level reflecting the metabolic status of P-I biosynthesis. Our results indicated that only SWE, ABA, and SNP stimulated P-I production by 2.60-, 2.01-, and 1.35-fold, respectively, whereas MeJa decreased P-I content. Interestingly, SWE modulated all four integrating secondary metabolic pathways, covering almost all critical steps in the methylerythritol phosphate (MEP), mevalonate (MVA), iridoid, and phenylpropanoid pathways to stimulate P-I biosynthesis. SNP targeted the MVA/MEP pathways in conjunction with the iridoid pathway, whereas ABA modulated the phenylpropanoid pathway to increase the P-I content in P. kurroa. This is apparently the first report on treatment of different elicitors in in vitro grown P. kurroa plants for eliciting P-I content and exploring the role of different elicitors at the molecular level.     

High frequency multiple shoot induction from nodal segments and rhinacanthin production in the medicinal shrub Rhinacanthus nasutus (L.) Kurz

High frequency multiple shoots have been induced from nodal segments of Rhinacanthus nasutus (L.) Kurz., a potent anticancerous ethnomedicinal plant. For initiation of cultures, nodal segments were cultured on MS medium supplemented with various concentrations (1.0–5.0 μM) of 6-benzyladenine or thidiazuron (TDZ) alone or in combination with α-naphthalene acetic acid (NAA 0.5–1.0 μM). The optimum frequency of response (85 %) and shoot number (3.3) was observed on MS medium supplemented with 4.0 μM TDZ and 0.8 μM NAA. The shoots developed on initiation media were excised and nodal segments were subcultured on MS medium supplemented with TDZ (4.0 μM) and NAA (0.5–1.0 μM). This subculturing process was repeated thrice, each with 45 days of duration and the multiple shoot formation was recorded at the end of every subculture stage. The highest frequency of response (100 %) and number of multiple shoots (24.1) per explant were recorded at the end of the third subculture passage on MS medium supplemented with 4.0 μM TDZ and 0.8 μM NAA. The optimum rooting of shoots was observed on ½ MS medium fortified with 3.0 μM indole-3-butyric acid. The rooted plants were successfully transplanted to soil. The estimation of rhinacanthin (RC) content in shoots and roots was carried out in 6-month-old ex vitro plants (i.e., plants regenerated via in vitro culture) and field grown natural plants by high performance liquid chromatography. Both shoots and roots of naturally grown plants showed slightly higher RC content than ex vitro grown plants. The highest RC content (4.6 mg/g DW RC-C, 0.14 mg/g DW RC-D and 0.10 mg/g DW RC-N) was recorded in roots of naturally grown plants.     

Isolation and HPLC assisted quantification of two iridoid glycoside compounds and molecular DNA fingerprinting in critically endangered medicinal Picrorhiza kurroa Royle ex Benth: implications for conservation

Picrorhiza kurroa is a medicinally important, high altitude perennial herb, endemic to the Himalayas. It possesses strong hepato-protective bioactivity that is contributed by two iridoid picroside compounds viz Picroside-I (P-I) and Picroside-II (P-II). Commercially, many P. kurroa based hepato-stimulatory Ayurvedic drug brands that use different proportions of P-I and P-II are available in the market. To identify genetically heterozygous and high yielding genotypes for multiplication, sustained use and conservation, it is essential to assess genetic and phytochemical diversity and understand the population structure of P. kurroa. In the present study, isolation and HPLC based quantification of picrosides P-I and P-II and molecular DNA fingerprinting using RAPD, AFLP and ISSR markers have been undertaken in 124 and 91 genotypes, respectively. The analyzed samples were collected from 10 natural P. kurroa Himalayan populations spread across four states (Jammu & Kashmir, Sikkim, Uttarakhand and Himachal Pradesh) of India. Genotypes used in this study covered around 1000 km geographical area of the total Indian Himalayan habitat range of P. kurroa. Significant quantitative variation ranging from 0.01 per cent to 4.15% for P-I, and from 0.01% to 3.18% in P-II picroside was observed in the analyzed samples. Three molecular DNA markers, RAPD (22 primers), ISSR (15 primers) and AFLP (07 primer combinations) also revealed a high level of genetic variation. The percentage polymorphism and effective number of alleles for RAPD, ISSR and AFLP analysis varied from 83.5%, 80.6% and 72.1%; 1.5722, 1.5787 and 1.5665, respectively. Further, the rate of gene flow (Nm) between populations was moderate for RAPD (0.8434), and AFLP (0.9882) and comparatively higher for ISSR (1.6093). Fst values were observed to be 0.56, 0.33, and 0.51 for RAPD, ISSR and AFLP markers, respectively. These values suggest that most of the observed genetic variation resided within populations. Neighbour joining (NJ), principal coordinate analysis (PCoA) and Bayesian based STRUCTURE grouped all the analyzed accessions into largely region-wise clusters and showed some inter-mixing between the populations, indicating the existence of distinct gene pools with limited gene flow/exchange. The present study has revealed a high level of genetic diversity in the analyzed populations. The analysis has resulted in identification of genetically diverse and high picrosides containing P. kurroa genotypes from Sainj, Dayara, Tungnath, Furkia, Parsuthach, Arampatri, Manvarsar, Kedarnath, Thangu and Temza in the Indian Himalayan region. The inferences generated in this study can be used to devise future resource management and conservation strategies in P. kurroa.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12298-021-00972-w.     

Differences in hypericin synthesis between experimentally induced seedling shoot cultures of Hypericum hookerianum Wight & Arn.

During seed germination trials of Hypericum hookerianum, seedlings of Lake View accession from Palni hills of Southern India segregated into green- (97.44 %) and red-pigmented (2.56 %) types. Seedlings cultured in Murashige and Skoog (1962) basal medium developed into fast growing green and slow growing red plant types in 6 weeks, the latter showing increased concentrations of total phenols, anthocyanins and flavonoids and 19-fold higher concentration of hypericin. Hypocotyls/cotyledons of red seedlings cultured using 2.325 μM kinetin (KIN) produced hypericin-rich (4.38 ± 0.18 mg/g DW), stunted (0.5–1.2 cm) shoots which ceased to grow in 8 weeks. Segments (4–6 mm) of these shoots sub-cultured in the dark for 4 weeks followed by 2-week light exposure and repeated subculture enabled mass multiplication of productive (3.93 ± 0.06 mg hypericin/g DW) shoots. Green hypocotyls and cotyledons subjected to 4 + 2 weak dark–light treatment also produced 9.18 ± 2.44 and 4.25 ± 0.96 comparable hypericin-rich (3.73 ± 0.21 mg/g DW) shoots. Red and green seedling explants cultured in basal medium in the dark produced 6.82 ± 0.75 cm etiolated shoots with reduced leaves which synthesized 2.27 ± 0.15 mg hypericin/g DW on illumination. Green cotyledons cultured in the dark using 2.45 μM indole-3 butyric acid (IBA) formed calluses which on illumination formed 12.64 ± 3.8 productive (3.86 ± 0.31 mg hypericin/g DW) 0.5- to 1.5-cm-long shoot clusters. Phenotypic segregation of seedlings, the ability of both red and green seedling explants to multiply in the dark and produce hypericin on illumination, and IBA-induced indirect shoots producing significant amounts of metabolite compared to wild plants (0.35 ± 0.09 mg/g DW) and green shoot cultures (0.91 ± 0.03 mg/g DW) are new to Hypericum.     

An improved method for micropropagation and regeneration of date palm (Phoenix dactylifera L.)

We developed a novel large-scale micropropagation pathway for date palm (Phoenix dactylifera L.) based on organogenesis. We obtained organogenic stems from shoot tip explants of the Moroccan date palm cultivar Najda, and investigated shoot proliferation from these organogenic stems in vitro on various media; Beauchesne medium (BM) and Murashige and Skoog medium (MS) at full-strength, half-strength, and one-third-strength, containing various concentrations (0, 0.25, 0.5, and 1 mg/L) of 2-naphthoxyacetic acid (NOAA) and kinetin. The optimal medium during the multiplication phase was half-strength Murashige and Skoog medium (MS/2) supplemented with 0.5 mg/L NOAA and 0.5 mg/L kinetin (23.5 morphologically superior shoots per explant, with low vitrification rates). For the shoot elongation phase, shoots were transferred to the same proliferation medium, or to MS or MS/2 media without plant growth regulators (PGRs). Shoots elongated rapidly and showed a high rate of root formation on media supplemented with PGRs. For example, on MS/2 medium containing 1 mg/L NOAA and 1 mg/L kinetin, the average shoot length was 15.1 cm, the average number of roots per shoot was 6.2, and their average length was 3.4 cm. On PGR-free media, shoots were shorter with wider and greener leaves, and had fewer roots. The plantlets were transferred to a greenhouse for acclimation. The survival rate after 2 months was related to the medium used during the elongation phase; >90 % of shoots that were cultured on PGR-free media survived, while there was a poor survival rate of shoots that had been cultured on media containing PGRs.     

Isolation and partial characterization of chromoprotein from the larval hemolymph of the Japanese oak silkworm (Antheraea yamamai)

A total of two different hemolymph proteins (designated P-I and P-II) of the Japanese oak silkworm, Antheraea yamamai, were purified from the hemolymph of the fifth instar larvae using four chromatographic steps: (a) hydrophobic interaction chromatography; (b) ion exchange chromatography; (c) gel-filtration; and (d) reverse-phase high performance liquid chromatography (HPLC). These two proteins were separated by TSKgel Phenyl-5PW RP column chromatography. P-I has an apparent molecular weight of 31 000 or 35 000, as determined by gel-filtration and SDS-PAGE, respectively. P-II shows a molecular weight of 22 000 or 25 000, by gel-filtration and SDS-PAGE, respectively. The molecular weight of P-I and P-II were determined to be 31 076 and 21 500 by MALDI-TOF MS, respectively. These results suggest that both P-I and P-II are monomers. The N-terminal sequence analysis suggests that P-I is closely related to the ommochrome-binding protein (OBP) from the hemolymph of Manduca sexta, with 40% identity in the first 30 residues, while P-II is similar to the biliproteins (BPs) from other lepidopteran insects (50% identity). Spectroscopic analysis shows that the blue chromophore of A. yamamai BP is not biliverdin IX, which is present in the biliproteins of most insects.     

In vitro plantlet regeneration and Agrobacterium tumefaciens-mediated genetic transformation of Indian Kino tree (Pterocarpus marsupium Roxb.)

The objective of the present study was to develop a protocol for in vitro plantlet regeneration and Agrobacterium tumefaciens-mediated genetic transformation using immature cotyledon explants of Indian Kino tree (Pterocarpus marsupium Roxb.). Immature cotyledon explants excised from 9-day-old axenic seedlings produced optimal callus on Murashige and Skoog (MS) medium supplemented with 1.07 μM α-naphthalene acetic acid (NAA), after 2 weeks of culture. When the above said callus was incubated on MS + 8.90 μM 6-benzylaminopurine (BAP) + 1.07 μM NAA, a regeneration frequency of 60.41 % with shoot number and length 12.2 ± 0.85 and 1.4 ± 0.13, respectively, was observed. For further shoot multiplication and elongation, these cultures were transferred onto MS + 4.40 μM BAP. Elongated shoots dipped in 19.60 μM indole-3-butyric acid (IBA) for 24 h and then cultured on ½MS + 2.85 μM IBA, 75 % shoots developed roots and 95 % of plantlets survived in field condition. Organogenic callus was co-cultivated with the A. tumefaciens strain LBA4404 harboring the binary plasmid pCAMBIA1301with ß-glucuronidase (uidA) and hygromycin phosphotransferase (hpt) genes and grown on MS + 8.90 μM BAP + 1.07 μM NAA (RM) + 200 μM acetosyringone for 2 days and then transferred to MS + 8.90 μM BAP + 1.07 μM NAA + 20 mg/l hygromycin + 250 mg/l cefotaxime (SIM) and 4.40 μM BAP + 15 mg/l hygromycin + 200 mg/l cefotaxime (SEM). The putatively transformed shoots were subsequently rooted on ½MS + 2.85 μM IBA + 20 mg/l hygromycin (SRM), after pulse treatment for 24 h with 19.60 μM IBA. Successful gene transfer into putatively transformed plantlets was confirmed by histochemical GUS assay, PCR and RT-PCR analysis. Southern blot analysis of regenerated plantlets confirmed the integration of hpt gene in transgenic plantlets. In the present study, a rate of 20.92 % transformation frequency was achieved and the genetic transformation protocol presented here may pave way for genetic manipulation of this multipurpose legume tree.     

In vitro morphogenesis in Selaginella microphylla (Kunth.) Spring

Selaginella, an extant genus of primitive vascular plants, has survived over 400 million years of evolution. In vitro morphogenesis in Selaginella microphylla is considered for the first time to establish a well-documented aseptic culture on half- strength Murashige and Skoog’s basal medium with 2ip (4.92–49.21 μM), or Kn (4.65–46.47 μM) or GA₃ (2.89–28.90 μM) for shoot multiplication, and with different concentrations of IBA (4.9–49 μm) to initiate root cultures. GA₃ was instrumental for shoot multiplication as well as induction of reproductive structures in each and every leaf axil. On the other hand, it is observed that IBA alone in S. microphylla can act as signal molecules for induction of enormous numbers of root masses from a few existing roots. An interesting pattern of re-differentiation has also been observed where apical portions of large numbers of roots were converted to green shoot apical meristems. Further differentiation produced tiny green shoots. Distinct bipolarity was noted in shoots when they were isolated from root masses and appeared as embryo-like structures. Chromosome analysis from in vitro sporophytic plants revealed 2n = 16 chromosomes, indicating chromosomal stability. The interesting in vitro pattern of morphogenesis obtained in S. microphylla may provide new insights into totipotency of plants.     

NiO(s) (bunsenite) is not available to Alyssum species

To determine if the Ni-hyperaccumulator Alyssum corsicum can absorb Ni from the kinetically inert crystalline mineral NiO(s) (bunsenite). A. corsicum and A. montanum plants were grown for 30 days in a serpentine Hoagland solution. NiO was provided at 0 or 0.1 g L^?1 (1.34 mmol L^?1) as reagent grade NiO particles <1 μm diameter, continuously mixed by aeration. A. corsicum and A. montanum shoots contained 19.4 and 5.2 mg Ni kg^?1 DW with NiO, and 3.0 and 1.1 mg Ni kg^?1 DW shoots, respectively in the control treatment. A. corsicum normally absorbs over 12,000 mg Ni kg^?1 shoots when supplied 300 μM soluble Ni. Roots were coated with the NiO particles and contained 3–5% Ni at harvest. Despite the small Ni accumulation in the NiO treatment above control, A. corsicum is not capable of dissolving and hyperaccumulating Ni from NiO.     

In vitro propagation and field establishment of Eulophia cullenii (Wight) Bl., a critically endangered orchid of Western Ghats, India through culture of seeds and axenic seedling-derived rhizomes

An efficient protocol has been devised for the propagation and field establishment of Eulophia cullenii (Wight) Bl., a terrestrial orchid having ornamental potentialities, and is critically endangered in Western Ghats, India. Seeds extracted from 60–90-d-old capsules germinated in ½ MS, ¼ MS, Knudson C, or Mitra liquid medium developed into 1.4–2.5-mm-diameter protocorms in 60 d. Supplementation of organic additives like coconut water, peptone, yeast extract, and casein acid hydrolysate (CH) significantly enhanced protocorm growth. Upon subculture onto agar-gelled Mitra medium fortified with 0.05% CH, 56% of protocorms regenerated into shoots through the formation of linear mini-rhizomes. The regenerated shoots grew vigorously in ½ MS, producing new rhizomes. Mature rhizomes from axenic seedlings produced maximum (13?±?1.4) shoots/whole rhizome in ½ MS fortified with 44.4 μM 6-benzylaminopurine (BAP), in 120–150 d. Horizontal and longitudinal halves of the rhizome also gave multiple shoots (6–8.5) in the presence of 44.4 μM BAP. Shoots or shoot clumps sub-cultured onto ½ MS basal medium produced roots followed by rhizomes in 60–150 d. Seedlings with mature rhizomes showed 70% establishment in the nursery and added a new rhizome at the end of one growth cycle. An average of 70.6% of the rhizomes originating from seedlings during the second growth cycle sprouted to produce new shoots, when planted in the native localities. Asymbiotic germination and cloning through rhizomes thus can provide a large number of vigorous plants of E. cullenii for ornamental exploitation as well as eco-restoration, if rhizome as storage organ is ensured in the propagule.     

Micropropagation and salt tolerance of in vitro grown Crithmum maritimum L.

Crithmum maritimum (Apiaceae), a perennial halophyte native in Greece, could be used as an alternative culture at problematic soils. It presents significant economical potentials as its essential oils are in high demand from the medicinal and cosmetic industry. The response of the species on in vitro conditions was studied. MS proved to be the most effective of the basal media tested for in vitro adventitious shoot production, resulting in significantly increased number of new microshoots/explant and higher shoots. 6-Benzyladenine (BA) at 2.5 μM increased shoot proliferation. The combination of α-Naphthaleneacetic acid (NAA) (1–2.5 μM) with BA (2.5 μM) had a positive influence at simultaneous proliferation and rooting resulting in high rooting percentage (82.5–95%) and increased number of roots. Rooting percentage reached 100% and number of roots increased significantly when 0.5 μM and 1 μM IBA was combined with ½MS and full strength MS. The in vitro response to salinity stress (0–300 mM NaCl) was also tested. Shoot proliferation was gradually reduced at higher concentrations of NaCl but shoot height was enhanced. Acclimatization procedure was successful.     

Aflatoxigenic strains of Aspergillus section Flavi isolated from marketed peanuts (Arachis hypogaea) in Algiers (Algeria)

The present study reports on the natural mycobiota occurring in Chinese peanuts marketed in Algiers, paying special attention to the incidence of Aspergillus section Flavi species that are potential producers of aflatoxins. The mean value counts of fungi ranged from 155 to 577 CFU/g dry matter (DM) and the predominant fungi were different species of the genus Penicillium (83.81–93.85 %) and Aspergillus belonging to section Flavi (2.73–73.96 %). Results indicated that 82 isolates (100 %) were aflatoxigenic. The Aspergillus section Flavi strains revealed that 65 isolates (79.27 %) were highly aflatoxigenic, producing four kinds of aflatoxins [AFB1 (0.846–3.330 μg/g), AFB2 (0.005–0.007 μg/g), AFG1 (0.008–1.595 μg/g), and AFG2 (0.005–0.010 μg/g)], whereas 17 isolates (20.73 %) synthetized low levels of one or two aflatoxins (AFB1 and AFG2). Aflatoxin production was also screened on Coconut Agar Medium (CAM), and the results were consistent with the HPLC analysis. Based on the combination of mycotoxins produced, five Aspergillus section Flavi chemotypes were established. Sclerotia production expressed a correlation to aflatoxigenicity. The total aflatoxins were detected in four analyzed samples at levels ranging from 0.71 to 25.50 μg/kg. Furthermore, the amplicons corresponding to the ITS1-5.8 S-ITS2 rDNA of six representative strains showed that four strains belonged to Aspergillus flavus, one to A. minisclerotigenes, and one to A. caelatus. The results obtained indicate that there is a possible risk factor posed by aflatoxins contamination of peanuts marketed in Algiers.     

Recovery of avocado (<Emphasis Type="Italic">Persea americana</Emphasis> Mill.) plants transformed with the antifungal plant defensin gene PDF1.2

Embryogenic avocado cultures derived from ‘Hass’ protoplasts were genetically transformed with the plant defensin gene (pdf1.2) driven by the CaMV 35S promoter in pGPTV with uidA as a reporter gene and bar, the gene for resistance to phosphinothricin, the active ingredient of the herbicide Finale® (Basta) (Bayer Environmental Science, Research Triangle Park, Durham, NC ). Transformation was mediated by Agrobacterium tumefaciens strain EHA105. Transformed cultures were selected in the presence of 3.0 mg l^?1 phosphinothricin in liquid maintenance medium for 3–4 mo. Liquid maintenance medium consisted of modified MS medium containing (per liter) 12 mg NH₄NO₃ and 30.3 mg KNO₃ and supplemented with 0.1 mg l^?1 thiamine HCl, 100 mg l^?1 myo-inositol, 30 g l^?1 sucrose, 3.0 mg l^?1 phosphinothricin, and 0.41 μM picloram. Somatic embryo development from transformed cultures was initiated on MS medium supplemented with 45 g l^?1 sucrose, 4 mg l^?1 thiamine HCl, 100 mg l^?1 myo-inositol, 10% (v/v) filter-sterilized coconut water, 3.0 mg l^?1 phosphinothricin, and 6.0 g l^?1 gellan gum. Limited plant recovery occurred from somatic embryos on semi-solid MS medium supplemented with 3.0 mg l^?1 phosphinothricin, 4.44 μM 6-benzylaminopurine (BA), and 2.89 μM GA₃; transformed shoots were micrografted on in vitro-grown seedling rootstocks. Approximately 1 yr after acclimatization in the greenhouse, transformed shoots were air-layered to recover transformed roots. Genetic transformation of embryogenic cultures, somatic embryos, and regenerated plants was confirmed by polymerase chain reaction (PCR), Southern blot hybridization, the XGLUC reaction for uidA, and application of the herbicide Finale® to regenerated plants.     

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.