Search

Ardisia crenata var. bicolor is a multi-purpose plant and has important ornamental and medicinal properties. Conventional methods of propagating the species from seeds and cuttings have low efficiency because of the recalcitrant properties of seeds and low survival rate of high-quality cuttings. This work aims to study the in vitro regeneration protocol for direct organogenesis from nodal segments of A. crenata var. bicolor on Murashige and Skoog (MS) medium, with different combinations and concentrations of plant growth regulators (PGRs). The treatments used for the establishment and proliferation of shoots included MS medium supplemented with different concentrations of Benzyl-aminopurine (BAP) and indole-3-butyric acid (IBA). For rooting, IBA was used in combination with naphthaleneacetic acid (NAA) in full- and half-strength MS media. Maximum shoot establishment (76.67%) and the highest shoot length (6.6 cm) were observed on MS medium with 1.0 mg·L−1 BAP with 0.5 mg·L−1 IBA, while BAP at 1.0 mg·L−1 with 0.25 mg·L−1 IBA obtained the highest shoot proliferation (4.5 ± 1.53). The best rooting response (83.33%) was achieved on half-strength MS including 1.0 mg·L−1 IBA with 0.25 mg·L−1 NAA, and the maximum survival rate of 84.4% was observed after acclimatization under 75% shading. To define their genetic stability, using eleven primers of ISSR markers to assess the genetic stability of the unstable leaf color samples compared with their mother plant, the ISSR markers demonstrated a level of genetic polymorphism in plantlets, but without other morphological variations. This indicates the genetic resemblance to the mother plant and the reliability of this protocol for the efficient micropropagation of A. crenata var. bicolor.

Manglietiastrum sinicum Y.W. Law is a critically endangered species with great ornamental and commercial value, which urgently requires protection. We tested different combinations of basal media and plant growth regulators to determine (i) the optimal conditions for bud induction and proliferation of explants and (ii) optimal rooting conditions. RAPD- and ISSR-PCR were used to assess the genetic fidelity of regenerated plantlets. Murashige and Skoog medium (MS) supplemented with 0.5 mg/L 6-benzyladenine (BA) and 0.05 mg/L indole-3-butyric acid (IBA) is the optimal medium for bud induction (100% induction). MSM medium (a special basal medium for M. sinicum) was more suitable for the efficient proliferation and rooting of M. sinicum. Maximum bud proliferation rate (446.20%) was obtained on MSM, with 0.4 mg/L BA, 0.5 mg/L kinetin, and 0.06 mg/L IBA, while maximum root induction rate (88.89%) was obtained on MSM supplemented with 0.4 mg/L 1-naphthylacetic acid and 1.0 mg/L IBA with a 7-day initial darkness treatment. The rooted plantlets were transferred to a substrate containing peat soil, perlite, coconut chaff, and bark (volume ratio 2:1:1:1), with a resulting survival rate of 92.2%. RAPD and ISSR markers confirmed the genetic uniformity and stability of regenerated plants.

The species belonging to the Rhododendron genus are well-known for their colorful corolla. Molecular marker systems have the potential to elucidate genetic diversity as well as to assess genetic fidelity in rhododendrons. In the present study, the reverse transcription domains of long terminal repeat retrotransposons were cloned from rhododendrons and used to develop an inter-retrotransposon amplified polymorphism (IRAP) marker system. Subsequently, 198 polymorphic loci were generated from the IRAP and inter-simple sequence repeat (ISSR) markers, of which 119 were derived from the IRAP markers. It was shown that in rhododendrons, IRAP markers were superior to the ISSRs in some polymorphic parameters, such as the average number of polymorphic loci (14.88 versus 13.17). The combination of the IRAP and ISSR systems was more discriminative for detecting 46 rhododendron accessions than each of the systems on their own. Furthermore, IRAP markers demonstrated more efficiency in genetic fidelity detection of in-vitro-grown R. bailiense Y.P.Ma, C.Q.Zhang and D.F.Chamb, an endangered species recently recorded in Guizhzhou Province, China. The available evidence revealed the distinct properties of IRAP and ISSR markers in the rhododendron-associated applications, and highlighted the availability of highly informative ISSR and IRAP markers in the evaluation of genetic diversity and genetic fidelity of rhododendrons, which may facilitate preservation and genetic breeding of rhododendron plants.

Background Anarrhinum pubescens Fresen. (Plantaginaceae) is a rare plant, endemic to the Saint Catherine area, of South Sinai, Egypt. Earlier studies have reported the isolation of cytotoxic and anti-cholinesterase iridoid glucosides from the aerial parts of the plant. The present study aimed to investigate the chemical profiling of the wild plant shoots as well as establish efficient protocols for in vitro plant regeneration and proliferation with further assessment of the genetic stability of the in vitro regenerated plants. Results Twenty-seven metabolites have been identified in wild plant shoots using the Nuclear Magnetic Resonance (NMR) spectroscopy. The metabolites include alkaloids, amino acids, carbohydrates, organic acids, vitamins, and a phenol. In vitro propagation of the plant was carried out through nodal cutting-micropropagation and leaf segment-direct organogenesis. The best results were obtained when nodal cutting explants were cultured on Murashige and Skoog medium with Gamborg B5 vitamins supplemented with 6-benzylaminopurine (BAP) (1.0 mg/L) and naphthaleneacetic acid (NAA) (0.05 mg/L), which gave a shoot formation capacity of 100% and a mean number of shoots of 27.67 ± 1.4/explant. These shoots were successfully rooted and transferred to the greenhouse and the survival rate was 75%. Genetic fidelity evaluation of the micropropagated clones was carried out using random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) molecular markers. Jaccard’s similarity coefficient indicated a similarity as high as 98% and 95% from RAPD and ISSR markers, respectively. Conclusions This study provides the chemical profiling of the aerial part of Anarrhinum pubescens. Moreover, in vitro regeneration through different tissue culture techniques has been established for mass propagation of the plant, and the genetic fidelity of the in vitro regenerated plants was confirmed as well. Our work on the in vitro propagation of A. pubescens will be helpful in ex situ conservation and identification of bioactive metabolites.

Simple sequence repeat (SSR) markers were used to evaluate the genetic stability of the acclimatized micropropagated and regenerated plants of a high cannabidiol (H-CBD) and a high cannabigerol (H-CBG) variety of Cannabis sativa L. Shoot regeneration and proliferation were achieved by culturing calli in Murashige and Skoog basal medium (MS) supplemented with several concentrations of 6-benzyladenine (BA) or thidiazuron (TDZ). Calli derived mostly from stem explants, rather than leaves, cultured on MS supplemented with 2,4-Dichlorophenoxyacetic acid (2,4-D) or combination of kinetin (KIN) with 1-Naphthaleneacetic acid (NAA) or 2,4-D. Rooting of the regenerated plantlets accomplished on half-strength MS medium supplemented with indole-3-butyric acid (IBA). Previous studies performed have developed an efficient in vitro micropropagation protocol for mass production. Both in vitro methodologies can be employed in genetic breeding via molecular techniques. The genetic stability of micropropagated and regenerated plants was accomplished using twelve SSR primer pairs that produced reproducible and clear bands, ranging from 90 to 330 bp in size, and resulted in amplification of one or two alleles, corresponding to homozygous or heterozygous individuals. The SSR amplification products were monomorphic across all the micropropagated and regenerated plants and comparable to mother plants. The monomorphic banding pattern confirmed the genetic homogeneity of the in vitro cultured acclimatized and mother plants as no somaclonal variation was detected in clones for these specific SSRs. Our results evidently suggest that the developed culture protocols for in vitro multiplication is appropriate and applicable for clonal mass propagation of the C. sativa varieties and demonstrate the reliability of this in vitro propagation system.

A high throughput regeneration protocol has been developed for Lagerstroemia speciosa through node explants under the regime of various plant growth regulators (PGRs). This protocol can provide an alternative mode to seed-grown plants and minimize the cost–time of regeneration, significantly. Murashige and Skoog (MS) medium containing various combinations of PGRs exhibited a marked stimulatory effect on morphogenesis. Of the various combinations tried, node explant pretreated with thidiazuron (TDZ; 5.0 µM) for 4 weeks and followed with transfer into MS medium containing 1.0 μM 6-benzyladenine (BA) and 0.25 μM α-naphthalene acetic acid (NAA) was reported to be the best treatment as it resulted in a maximum number of 24.5 shoots with an average shoot length of 7.1 cm per explant in 90% of cultures after 12 weeks of incubation. The in vitro-generated shoots rooted satisfactorily in the adopted ex vitro method of rooting, which saves time and cost. Among the different treatments, the greatest rooting percentage (85%) was observed in the 200 μM IBA-treated shoots, with the highest root number (8.7) and length (3.4 cm) occurring after 4 weeks. Four months after being transferred to ex vitro, some of the physiological attributes of the in vitro-propagated plants were examined and compared to the ex vitro plants. Further, analysis of the genetic integrity in tissue culture-raised plantlets along with the parental tree was accomplished through DNA-based RAPD technique. The monomorphic banding pattern obtained by the RAPD primers resulted in a high level of genetic uniformity in regenerated plants.

A new and efficient method of direct shoot proliferation was developed here in two muskmelon genotypes (Cucumis melo L.), including ‘Khatooni’ and ‘Sooski Sabz’ (100%) using 4-day-old cotyledonary explants in vitro. The regeneration of muskmelon was evaluated on Murashige and Skoog (MS) medium supplemented separately with 17 treatments as combinations of two auxins (1-Naphthaleneacetic acid [NAA] and Naphthoxyacetic acid [NOA]) and four cytokinins (Benzylaminopurine [BAP], Thidiazuron [TDZ], Kinetin [KIN], and N-(2-Chloro-4-pyridyl)-N′-phenylurea [CPPU]). The treatments were experimented on 4- and 8-day-old cotyledon explants. A significant degree of organogenesis occurred in 4-day-old cotyledons (P

Panax ginseng Meyer grows in east Russia and Asia. There is a high demand for this crop due to its medicinal properties. However, its low reproductive efficiency has been a hindrance to the crop’s widespread use. This study aims to establish an efficient regeneration and acclimatization system for the crop. The type of basal media and strength were evaluated for their effects on somatic embryogenesis, germination, and regeneration. The highest rate of somatic embryogenesis was achieved for the basal media MS, N6, and GD, with the optimal nitrogen content (≥35 mM) and NH4+/NO3− ratio (1:2 or 1:4). The full-strength MS medium was the best one for somatic embryo induction. However, the diluted MS medium had a more positive effect on embryo maturation. Additionally, the basal media affected shooting, rooting, and plantlet formation. The germination medium containing 1/2 MS facilitated good shoot development; however, the medium with 1/2 SH yielded outstanding root development. In vitro-grown roots were successfully transferred to soil, and they exhibited a high survival rate (86.3%). Finally, the ISSR marker analysis demonstrated that the regenerated plants were not different from the control. The obtained results provide valuable information for a more efficient micropropagation of various P. ginseng cultivars.

Tetraclinis articulata (Vahl) Masters is an endangered tree growing in coastal and arid environments that is widely exploited by the timber and resin industry, among other applications. In this context, the use of in vitro techniques is highly encouraged for its propagation. We present a protocol for micropropagation using twigs from adult trees as a source of explants. The Schenk and Hildebrandt basal medium (SH) supplemented with 30 g L−1 sucrose, 6.5 g L−1 plant agar, 4.0 mg L−1 6-benzyladenine (BA), and 0.05 mg L−1 1-naphthaleneacetic acid (NAA) provided the optimum multiplication rate (90.48 ± 9.52 explants with basal shoots and 2.58 ± 0.29 basal shoots per explant). Application of activated charcoal (AC) or ½ Knop solution in a liquid overlay produced significantly longer shoots. Supplementation of solid media with indole-3-butyric acid (IBA) or NAA gave low rooting percentages (

Muntingia calabura L. is a potent medicinal plant. Because of that several researchers reported phytochemical and pharmacological studies, but only minimal information is obtainable on tissue culture and genetic stability evaluation studies. Therefore, in the present investigation we attempted to establish a reliable direct and indirect regeneration study through leaf and node explants on MS medium containing cytokinins BAP and KN (0.5-3.0 mg l-1) in combination with auxins IAA and NAA (0.5 mg l-1). The breakage of bud and shoot initiation were noticed with the initiation of callus at lower concentrations of auxin alone at 0.5 mg l-1. 2,4-D was found to be good in callus induction from leaf and nodal explants. A problem of browning callus was prevented by regular subculturing of callus cultures. Leaf explants exhibited maximum number of shoots (32±0.88a) with shoot length (7.6±0.17a) and nodal explants obtained optimal number of shoots (26±0.88a) with shoot length (9.6±0.30a) on MS medium supplemented with BAP (2 mg l-1) and IAA (0.5 mg l-1). Half strength MS medium fortified with IBA (2.0 mg l-1) was effective and achieved 70% of rooting. These well-developed plantlets were shifted to pots containing soil and vermicompost in 1:1 ratio for acclimatization. The acclimatized plants were field transferred with survival rate of 85%. The ISSR and RAPD markers analysis revealed the genetic stability of in vitro regenerated plants with the mother plant.

Ruta chalepensis L., an evergreen shrub in the citrus family, is well-known around the world for its essential oils and variety of bioactivities, indicating its potential medicinal applications. In this study, we investigated the effect of different culture conditions, including plant growth regulators, media types, pH of the medium, and carbon sources, on in vitro regeneration from nodal explants of R. chalepensis. Following 8 weeks of culture, the highest percentage of regeneration (96.3%) and maximum number of shoots (40.3 shoot/explant) with a length of 4.8 cm were obtained with Murashige and Skoog (MS) medium at pH 5.8, supplemented with 3.0% sucrose and 5.0 µM 6-Benzyladenine (BA) in combination with 1.0 µM 1-naphthaleneacetic acid (NAA). For rooting, individually harvested shootlets were transferred on ½ MS (half-strength) supplemented with IAA (indole-3-acetic acid), IBA (indole 3-butyric acid), or NAA, and the best response in terms of root induction (91.6%), number of roots (5.3), and root mean length (4.9 cm) was achieved with 0.5 µM IBA after 6 weeks. An average of 95.2 percent of healthy, in vitro regenerated plantlets survived after being transplanted into potting soil, indicating that they were effectively hardened. DNA assays (PCR-based markers) such as random amplification of polymorphic DNA (RAPD) and directed amplification of minisatellite-region (DAMD) were employed to assess in vitro cultivated R. chalepensis plantlets that produced a monomorphic banding pattern confirming the genetic stability. Additionally, no changes in the flow cytometric profile of ploidy between regenerated plantlets and donor plants were detected. Regeneration of this valuable medicinal plant in vitro will open up new avenues in pharmaceutical biotechnology by providing an unconventional steadfast system for mass multiplication and might be effectively used in genetic manipulation for enhanced bioactive constituents.

Tetraclinis articulata (Vahl) Masters is an endangered tree growing in coastal and arid environments that is widely exploited by the timber and resin industry, among other applications. In this context, the use of in vitro techniques is highly encouraged for its propagation. We present a protocol for micropropagation using twigs from adult trees as a source of explants. The Schenk and Hildebrandt basal medium (SH) supplemented with 30 g L−1 sucrose, 6.5 g L−1 plant agar, 4.0 mg L−1 6-benzyladenine (BA), and 0.05 mg L−1 1-naphthaleneacetic acid (NAA) provided the optimum multiplication rate (90.48 ± 9.52 explants with basal shoots and 2.58 ± 0.29 basal shoots per explant). Application of activated charcoal (AC) or ½ Knop solution in a liquid overlay produced significantly longer shoots. Supplementation of solid media with indole-3-butyric acid (IBA) or NAA gave low rooting percentages (

Banana (Musaspp.), one of the most important economical fruit crops, belongs family Musaceae. It is a major staple food crop that globally occupied the fourth position next to rice, wheat, and maize and in addition, it serves as a cash crop in form of the cornerstone to a number of country’s economy. An accelerated, innovative and efficientin vitrosingle-step concurrent multiple shoot and root development protocol was successfully developed inMusa acuminatacv. Grand Nain, popularly called as “Chiquita banana” which has eminent potential for export market. Shoot tip explants wereinoculated in Murashige and Skoog (MS) medium individually fortified with 0.5—1.5 mg/l of 6-benzyladenine (BA), kinetin (KIN),meta-Topolin (mT), thidiazuron (TDZ) and zeatin. The maximum number of shoots was obtained in 0.5 mg/l TDZ and the same medium resulted in substantially higher number of true roots as well. The minimum time to fresh shoot initiation and maximum shoot elongation was found in MS medium supplemented with 1.5 mg/l KIN. However, earliest root induction, multiple root formation and elongation was observed in MS medium with 0.5 mg/l KIN.By and large, TDZ (0.5 mg/l) was found to be optimum medium in terms of accelerated simultaneous shooting and rooting. During acclimatization, these plantlets survived and proliferated best in cocopeat in contrast to other substrates (such as sand, soil—sand, vermicompost—soil, perlite and vermiculite). Eventually, the clonal fidelity assessment of thein vitroregenerants using ISSR primers produced monomorphic bands and thus no genetic variation was detected withinin vitroregenerants as well as with their mother plant.

Ruta chalepensis L., an evergreen shrub in the citrus family, is well-known around the world for its essential oils and variety of bioactivities, indicating its potential medicinal applications. In this study, we investigated the effect of different culture conditions, including plant growth regulators, media types, pH of the medium, and carbon sources, on in vitro regeneration from nodal explants of R. chalepensis. Following 8 weeks of culture, the highest percentage of regeneration (96.3%) and maximum number of shoots (40.3 shoot/explant) with a length of 4.8 cm were obtained with Murashige and Skoog (MS) medium at pH 5.8, supplemented with 3.0% sucrose and 5.0 µM 6-Benzyladenine (BA) in combination with 1.0 µM 1-naphthaleneacetic acid (NAA). For rooting, individually harvested shootlets were transferred on ½ MS (half-strength) supplemented with IAA (indole-3-acetic acid), IBA (indole 3-butyric acid), or NAA, and the best response in terms of root induction (91.6%), number of roots (5.3), and root mean length (4.9 cm) was achieved with 0.5 µM IBA after 6 weeks. An average of 95.2 percent of healthy, in vitro regenerated plantlets survived after being transplanted into potting soil, indicating that they were effectively hardened. DNA assays (PCR-based markers) such as random amplification of polymorphic DNA (RAPD) and directed amplification of minisatellite-region (DAMD) were employed to assess in vitro cultivated R. chalepensis plantlets that produced a monomorphic banding pattern confirming the genetic stability. Additionally, no changes in the flow cytometric profile of ploidy between regenerated plantlets and donor plants were detected. Regeneration of this valuable medicinal plant in vitro will open up new avenues in pharmaceutical biotechnology by providing an unconventional steadfast system for mass multiplication and might be effectively used in genetic manipulation for enhanced bioactive constituents.

This review article presents a consolidated explanation and provides a comprehensive description of various studies, carried out on in vitro culture and hairy root cultures of S. marianum which can be consider an alternative source of flavonolignans. To overcome the constrains of conventional propagation of silybum plant, tissue culture and advanced biotechnology proved to be an influential tool that can complement conventional breeding and accelerate silybum development. The present review is focused on biotechnological tools like in vitro culture, hairy root cultures and genetic fidelity of S. marianum which can be a potent tool for production of secondary metabolites from these cultures. Keywords: S. marianum, In vitro culture, Hairy roots, Genetic fidelity

Multiplication of banana cvs. Grand Naine (Musa AAA, Cavendish-sub group) and Rasthali (Musa AAB, Silk-sub group) were attempted through somatic embryogenesis. The influence of position of male flower buds, amino acid supplements in the induction of somatic embryogenesis and field performance of embryogenic cell suspension (ECS) derived banana plants were studied. Differentiated immature male flower buds positioned at 6–8 th bract whorl as explants showed better callus induction and somatic embryogenesis. Supplementation with glutamine at 400 mg L−1 along with 20:20 g L−1sucrose: maltose in maturation media induced a 10-fold increase in somatic embryo formation compared to control. Cotyledonary stage somatic embryos desiccated for 2 h showed higher germination compared to non-desiccated embryos. The plantlets generated were hardened, and the genetic fidelity of the plantlets was confirmed using ISSR marker. To check the field performance of ECS derived plants, plantlets were hardened and planted in the field along with meristem and sucker. During the field growth, these ECS derived plants were morphologically similar to those of control plants. In this experiment, it was observed that ECS derived banana plants displayed normal phenotype as that of plants grown from meristem and sucker. The protocol developed could be useful highly for large-scale micropropagation or genetic manipulation studies in these commercially important banana cultivars. Keywords: Banana, Immature male flower bud, Somatic embryogenesis, Genetic fidelity

Background: We have developed a micropropagation technique methodology to clonal micro-garden formation of Bambusa vulgaris selected adult plant. Collection site (i.e., stock plant cultivated in different environments) and seasonality of shoot collection (i.e., months of the year) effects on in vitro culture were evaluated. Explants that showed normal development were ex vitro rooted in mini-incubator system, and three culture media (WPM, MS and deionized water + agar) supplemented with plant growth regulators (IBA, NAA and BAP) and antibiotic (streptomycin sulphate and culture medium free - control) were evaluated. Micropropagated plants were acclimatized in a shadow house and transferred to a semi-hydroponic system for establishment of a clonal micro-garden. The efficacy of the cloning protocol was determined with genetic fidelity analysis by ISSR molecular markers. Results: Considering all inoculations, 21.9% of nodal segments were in vitro established in nine shoot collections. The rooting percentage was 36.6%, and no interactions were observed between the use of culture medium and antibiotic. Culture medium free antibiotic resulted in 80.0% of survival and 50.0% of adventitious rooting. Micropropagated plants presented adequate growth and adaptation to ex vitro conditions in the clonal micro-garden. Molecular analyses by ISSR markers indicated the absence of genetic variations, and histological analyses revealed normal adventitious root formation originating from meristematic cells. Conclusion: The formation of a clonal micro-garden was demonstrated, proving the feasibility of the tested technique. Our results contribute to the development of a clonal propagation protocol for B. vulgaris selected adult plants.

Prunus africana is an endangered medicinal plant and hence new propagation methods are urgently required to increase its populations. Unfortunately, propagation through seeds is challenging due to its long flowering cycle and recalcitrant seeds. We developed a protocol for micropropagation using nodal segment explants. A woody plant medium supplemented with vitamins, 15 g L−1 sucrose, and 1.0 mg L−1 6-benzylaminopurine (BAP) supported the optimum rate (100%) of axillary shoot initiation. Supplementation with 15 g L−1 sucrose and 1.5 mg L−1 indole-3-acetic acid (IAA) provided the optimum rate (75%) of root initiation. Rooted plantlets were successfully planted in sterilized horticultural soil containing perlite (2:1 v/v) and the survival rate was 98% following acclimatization. The photosynthetic rate assessed using FlourPen FP110 series showed that the ratio of variable fluorescence to maximum fluorescence mean value for in vitro regenerated P. africana (0.830 ± 0.0008) was similar to that of the maternal P. africana plant (0.825 ± 0.005), indicating similarity in their photosynthetic performance; a pivotal process for growth and development. The Fourier transform near-IR (FT-NIR) spectrometer analysis of the in vitro regenerated and the maternal P. africana plant samples exhibited homogeneity in the absorbance peaks at 8,273, 6,344, and 4,938–4,500 cm−1 associated with lipids, starch, and proteins. The genetic fidelity of regenerated plants was confirmed using the randomly amplified polymorphic DNA (RAPD) technique. Our protocol is suitable for use in large-scale P. africana to meet the increasing demands for it in the global market.

A droplet-vitrification cryopreservation protocol has been successfully developed for Aranda Broga Blue orchid hybrid using protocorm-like bodies (PLBs). However, maximum growth regeneration percentage was recorded at 5% only based on previous report. Thus, to improve growth recovery of cryopreserved PLBs, cryopreservation stages were supplemented with ascorbic acid, tested at 50, 100 and 150 mg/L. However, results demonstrated that exogenous ascorbic acid was not favorable in regeneration of cryopreserved explants (maximum value of 1.67 % with 50 mg/L ascorbic acid supplementation). Total soluble protein and various antioxidant enzyme activities such as catalase (CAT), superoxide dismutase (SOD) and ascorbate peroxidase (APX) were evaluated after each cryopreservation stages in conjunction with the application of exogenous ascorbic acid. Addition of antioxidant must be carefully evaluated and its application may not guarantee successful growth recovery. RAPD and SCoT molecular analysis confirmed the genetic stability of regenerated cryopreserved PLBs as no polymorphism was detected compared to control PLBs culture.

Highlights • Exogenous ascorbic acid does not guarantee improvement of growth recovery in cryopreserved PLBs. • Exogenous ascorbic acid and cryopreservation stages has a significant interaction effect to total soluble protein content, SOD and APX activities. • Changes in total protein content, SOD and CAT activities do not correlated with growth recovery of cryopreserved PLBs. • High APX activities are associated with growth recovery of cryopreserved explants. • Genetic stability of regenerants recovered from cryopreserved Aranda PLBs was confirmed based on RAPD and SCoT molecular markers., A droplet-vitrification cryopreservation protocol has been successfully developed for Aranda Broga Blue orchid hybrid using protocorm-like bodies (PLBs). However, maximum growth regeneration percentage was recorded at 5% only based on previous report. Thus, to improve growth recovery of cryopreserved PLBs, cryopreservation stages were supplemented with ascorbic acid, tested at 50, 100 and 150 mg/L. However, results demonstrated that exogenous ascorbic acid was not favorable in regeneration of cryopreserved explants (maximum value of 1.67 % with 50 mg/L ascorbic acid supplementation). Total soluble protein and various antioxidant enzyme activities such as catalase (CAT), superoxide dismutase (SOD) and ascorbate peroxidase (APX) were evaluated after each cryopreservation stages in conjunction with the application of exogenous ascorbic acid. Addition of antioxidant must be carefully evaluated and its application may not guarantee successful growth recovery. RAPD and SCoT molecular analysis confirmed the genetic stability of regenerated cryopreserved PLBs as no polymorphism was detected compared to control PLBs culture.

Orthosiphon stamineus has been widely used as traditional remedy for various illnesses and diseases, such as cardiovascular diseases and epileptic seizures. In this study, direct regeneration through nodal segment of this species was attempted using Kinetin (6-Furfurylaminopurine) and IAA (indole-3-acetic acid). Optimum regeneration media was identified as MS media supplemented with 2.0 mg L&minus, 1 Kin plus 0.5 mg L&minus, 1 IAA. This yielded the highest number of shoots (5.57 &plusmn, 0.42) and leaves (20.53 &plusmn, 1.91) per explant. Acclimatization of the resulting in vitro regenerants was successful in all potting mixtures tested. However, potting mixture PF (1:1:1 ratio of black soil/red soil/compost) was identified as the best medium for acclimatization of this species, as it yielded 100% survival percentage after 90 days of acclimatization. Ten in vitro regenerants of O. stamineus were randomly collected after the third subculture and subjected to genetic variation analysis using inter-simple sequence repeat (ISSR) markers. Out of 20 ISSR markers tested, 10 working primers were observed to produce satisfactory amplification of bands, with an average of 7.11 bands per primer. A total of 610 bands were produced by the 10 primers. The percentage of polymorphism was observed to be very low, yielding only 7.32% polymorphism among all samples. Jaccard dissimilarity analysis was also conducted and very low genetic distance (about 0.1) was found among the in vitro regenerants and between the regenerants with the mother plant, thus ascertaining the clonal nature of the plantlets produced in this study.

Diplocyclos palmatus (L.) C. Jeffrey, commonly referred to as “Shivalingi” or “Lollipop climber” is a valuable medicinal plant with a climbing growth habit used in traditional medicine. It is reputed to have antiarthritic, anti-diabetic properties and to be useful in various skin and reproductive problems. Overexploitation of wild plants and low seed germination have resulted in the decline of the species in the wild. Thus, the present investigation was aimed to establish an effective in vitro propagation procedure for its large-scale production and conservation. Nodal explants, obtained from an established mother plant were grown on MS basal medium augmented with various cytokinins, alone or in combination with auxins, to study the morphogenic response. A maximum of 8.3 shoots/explants with an average shoot length of 7.2 cm were produced after six weeks on MS containing benzylaminopurine 5.0 µM + 1-naphthaleneacetic acid 2.0 µM. After 4 weeks of transfer, microshoots rooted well on a low nutrient medium of ½ MS + 1.0 µM indole-3-butyric acid, with a maximum of 11.0 roots/microshoot and an average root length of 7.4 cm. With an 80% survival rate, the regenerated plantlets were effectively acclimatized to natural conditions. DNA-based molecular markers were used to investigate the genetic uniformity. Scanning Electron Microscopic examination of leaves indicated the adaptation of the plantlets to natural, as evidenced by the formation of normal stomata. Gas chromatography-mass spectrometry analyses of mother and micropropagated plants were performed to identify essential secondary metabolites. The results obtained show that the in vitro propagation system can be adopted for preservation, large-scale production and secondary metabolites’ production in D. palmatus.

A micropropagation protocol via direct shoot organogenesis from Streptocarpus x hybridus Voss. leaf explants was established in this study. The shoot induction of three Streptocarpus cultivars (‘Snow White’, ‘Black Panther’ and ‘Slumber Song’) was successfully achieved on Murashige and Skoog (MS) medium supplemented with 0.2 mg L-1 -indole-3-acetic acid (IAA) and 0.2 mg L-1 thidiazuron (TDZ). In proliferation stage, the effects of two combinations of plant growth regulators -PGR- (V1-0.2 mg/L-1 IAA + 0.5 mg/L-1 BAP and V2-1.0 mg L-1 NAA + 0.2 mg L-1 TDZ) on shoot number and length were examined. The results suggest that PGRs combinations significantly influenced shoot proliferation and root induction in all Streptocarpus cultivars. Among the treatments, 0.2 mg L-1 (IAA) in combination with 0.5 mg L-1 6-benzylaminopurine (BAP) were the most effective for in vitro shoot multiplication and rooting. The in vitro rooting percentage was also determined before subjecting the plantlets to the acclimatization process. Due to acclimatization, Streptocarpus plantlets showed a very high rate of survival (90%). The generated PCR-RAPD profiles for the selected in vitro-raised plants and donor plants were similar which indicates the clonal or true-to-type nature of the progenies.

An efficient in vitro direct shoot regeneration system has been described for Lycium chinense Miller using leaf explants. Influence of various parameters such as growth regulator concentration, explant type, effect of basal salt type, Murashige and Skoog (1962) medium (MS), Schenk and Hildebrandt (1972) medium (SH), Gamborg et al. (1968) medium (B5), and carbon sources (sucrose, maltose, and fructose) on the regenerating shoots has been studied. Micromorphological studies and genetic fidelity of regenerated shoots were assessed and compared with those of the donor plants. Among the different concentrations of plant growth regulator (PGRs) tested, MS supplemented with lower concentration of 6-benzylaminopurine (BAP) (0.5 mgL−1) and thidiazuron (TDZ) (0.5 mgL−1) increased the frequency of shoot. Comparatively, indole-3-butyric acid (IBA) was more effective in the regeneration and growth of the root system. A higher number of root formation (6.67 ± 1.25) was observed when the rooting medium comprised half-strength MS salts supplemented with 3% sucrose. The surviving plantlets were gradually transferred to the greenhouse and natural soil. More than 90% of the plantlets survived and matured within 85 days. Similarity in the band patterns produced by inter simple sequence repeat (ISSR) primers confirmed the genetic stability and uniformity between the regenerated and donor plants. The present optimized direct shoot regeneration system may be useful for mass propagation and improving the genetic traits in L. chinense.

Embryogenic cultures have been used in cryopreservation, genetic transformation, propagation, virus elimination, induced mutagenesis and in many other biotechnological applications, providing excellent opportunities for biotechnology advances in grapevine. Unfortunately the efficiency of somatic embryogenesis (SE) is genotype-dependent in addition to showing interaction with explant type used and the plant growth regulator (PGR) composition. In order to identify the interaction of these parameters in SE, we tested eight wine grapevine cultivars, three explant types (ovary, anther/filament and stigma/style) and four PGR combinations in a statistically designed experiment. The genotype was the major determining factor, with embryogenic response varying from 0.1 to 5.1% (about 50-fold difference). For PGR composition of the medium, embryogenesis ranged between 0.5 and 3.3% (a ≈7-fold difference). The explant type was the least important factor with embryogenesis ranging between 0.8% (anther/filament) and 2.3% (ovary)—only a ≈3-fold change. Anther/filament, that had generally been considered to be the most promising explant, surprisingly gave the lowest embryogenesis percentage. Genetic homogeneity of plants developed from in vitro SE was assessed in comparison to mother plants using six inter-simple sequence repeat and ten random amplified polymorphic DNA primers that produced reproducible and clear bands ranging from 150 to 3500 bp. The amplification products were monomorphic across all the regenerated plants and their respective mother plants confirming the genetic homogeneity of the regenerants and demonstrating the suitability of SE for in vitro grapevine germplasm conservation and propagation.

An efficient micropropagation protocol for Rheum rhabarbarum L. was developed in this study. The in vitro rhubarb plants obtained in the multiplication stage (proliferation rate: 5.0 &plusmn, 0.5) were rooted in vitro (96% rooting percentage) and acclimatized ex vitro in floating perlite, with 90% acclimatization percentage. To assess the genetic fidelity between the mother plant and in vitro propagated plants, sequence-related amplified polymorphism (SRAP) markers were used. All banding profiles from the micropropagated plants were monomorphic and similar to those of the mother plant indicating 100% similarity. Regarding the polyphenolic profile, gallic, protocatechuic, p-hydroxybenzoic, vanillic, chlorogenic, caffeic, syringic, p-coumaric and ferulic acid were present in different amounts (2.3&ndash, 2690.3 &mu, g g&minus, 1 dry plant), according to the extracted matrix. Aglicons and glycosides of different classes of flavonoids were also identified. The rhizome extracts (both from in vitro and field grown plants) contained resveratrol, a stilbene compound with high antioxidant properties, ranging between 229.4 to 371.7 &mu, 1 plant. Our results suggest that in vitro propagation of Rheum rhabarbarum L. represents a reliable alternative to obtain a large number of true-to-type planting material with high bioactive compound content of this valuable nutritional and medicinal species.

Determination of the genetic characters of the plants obtained using plant tissue culture methods is important. In this study, the genetic fidelity of the plants, which are obtained by the micropropagation of the shoot tip explants of the Origanum majorana L. plant of medicinal and economical value in Murashige and Skoog (MS) medium which contains 1.0 mg/L Benzil Amino Purin (BAP), 0.1 mg/L naphthaleneacetic acid (NAA), 3% sucrose and 0.7% agar, has been investigated by using random amplified polymorphic DNA (RAPD-PCR) technique. Monomorphic bands were obtained as a result of all of the RAPD-PCR analyses performed. According to the results obtained, no polymorphism was detected among the micropropagated plants.

An efficient genetically stable regeneration protocol with increased phytochemical production has been established for Dendrobium nobile, a highly prized orchid for its economic and medicinal importance. Protocorm like bodies (PLBs) were induced from the pseudostem segments using thidiazuron (TDZ; 1.5 mg/l), by-passing the conventional auxin–cytokinin complement approach for plant regeneration. Although, PLB induction was observed at higher concentrations of TDZ, plantlet regeneration from those PLBs was affected adversely. The best rooting (5.41 roots/shoot) was achieved in MS medium with 1.5 mg/l TDZ and 0.25% activated charcoal. Plantlets were successfully transferred to a greenhouse with a survival rate of 84.3%, exhibiting normal development. Genetic stability of the regenerated plants was investigated using randomly amplified polymorphic DNA (RAPD) and start codon targeted (SCoT) polymorphism markers which detected 97% of genetic fidelity among the regenerants. The PIC values of RAPD and SCoT primers were recorded to be 0.92 and 0.76 and their Rp values ranged between 3.66 and 10, and 4 and 12 respectively. The amplification products of the regenerated plants showed similar banding patterns to that of the mother plant thus demonstrating the homogeneity of the micropropagated plants. A comparative phytochemical analysis among the mother and the micropropagated plants showed a higher yield of secondary metabolites. The regeneration protocol developed in this study provides a basis for ex-situ germplasm conservation and also harnesses the various secondary metabolite compounds of medicinal importance present in D. nobile., Highlights • An efficient micropropagation protocol for Dendrobium nobile, has been developed. • Genetic stability evaluated by ScoT and RAPD markers. • Higher antioxidant activity within the micropropagated plants over the mother plant. • Efficiency of TDZ as a potent growth regulator.

Flow cytometry and microsatellite markers were used to determine a genetic fidelity of micropropagated plants from the two Eucalyptus urophylla x E. globulus clones and a Eucalyptus grandis x E. globulus clone derived from adult material. Clones were repeatedly subcultured for 25 subcultures on MS medium supplemented with BA (2.22 µM) and ANA (0.05 µM) for in vitro shoot multiplication. The elongation was performed in MS culture medium supplemented with AIB (2.46 µM) and BA(0.22 µM). The ex vitro rooting and acclimatization phases were lead at the same time. The micropropagated clones showed genetic stability by flow cytometry and microsatellite markers. The results proved that micropropagation, for purposes of rejuvenation, can be a viable technique to generate genetically stable or identical E. globulus hybrid clones. RESUMO A citometria de fluxo e os marcadores microssatélites foram utilizados para avaliar a fidelidade genética de plantas micropropagadas de dois clones de Eucalyptus urophylla x E. globulus e um clone de Eucalyptus grandis x E. globulus, provenientes de material adulto. Os clones foram multiplicados in vitro em 25 subcultivos sucessivos em meio de cultura MS suplementado com BA (2,22 µM) e ANA (0,05 µM). O alongamento das brotações foi obtido no meio MS acrescido de AIB (2,46 µM) e BA (0,22 µM). O enraizamento ex vitro e a aclimatização foram realizados conjuntamente. Os clones micropropagados demonstraram estabilidade genética através das análises de citometria de fluxo e de marcadores microssatélites. Os resultados revelaram que a micropropagação com fins de rejuvenescimento clonal, corresponde a uma técnica viável quanto à obtenção de clones híbridos de E. globulus, geneticamente estáveis.

24 p.-4 fig.-6 tab., In the present study, we develop an efficient and reproducible in vitro regeneration system for two cultivars viz., Jamila and Tomaland of Solanum lycopersicum L., an economically important vegetable crop throughout the world. Sterilization of seeds with 2.5 % (v/v) NaOCl was found to be most effective, about 97 % of seeds germinated on cotton in magenta box moistened with sterile half strength (½)Murashige and Skoog (MS) medium. Regeneration efficiency of cotyledonary leaf (CL) and cotyledonary node (CN) explants derived from 08 days old aseptic seedling were assessed on MS medium supplemented with different concentrations of auxins and cytokinin. CL explants were found more responsive in comparison to CN in both the cultivars. Types of basal media were also assessed and found to have a significant effect on shoot regeneration. Highest regeneration frequency and maximum number of shoots were standardized from CL explants on MS medium supplied with 6- benzyl adenine (BA; 5.0 µM), indole-3-butyric acid (IBA; 2.5 µM) and Kinetin (Kin; 10.0 µM). In vitro regenerated microshoots were rooted on ½MS medium containing 0.5 µM indole-3-butyric acid (IBA). Regenerated plantlets with well-developed roots and shoot system were successfully acclimated to ex vitro condition. Genetic uniformity of tissue culture raised plantlets was first time evaluated using flow cytometry and single primer amplification reaction (SPAR) methods viz., DAMD and ISSR. No significant changes in ploidy level and nuclear DNA content profile were observed between in vitro propagated plants and normal plants of both the cultivars. Similarly, the SPAR analysis also revealed monomorphic banding patterns in regenerated plantlets of S. lycopersicum verifying their genetic uniformity and clonal fidelity. This efficient regeneration system can be used as a fast and reproducible method for genetic transformation of this important vegetable crop., This project was funded by the National Plan for Science, Technology and Innovation (MAARIFAH), King Abdul Aziz City for Science and Technology, Kingdom of Saudi Arabia, Award Number 12-BIO2919-02.

In vitro clonal propagation of small fruit species has been one of the most broadly exploited approaches in biotechnology. However, the most critical aspect of this propagation technology has been the maintenance of genetic fidelity of tissue culture-raised plants. In the present study, three techniques were deployed in the assessment of genetic fidelity of micropropagated plants (TC plants) of blackberry cultivar ‘Cacanska Bestrna’ and raspberry cultivar ‘Meeker’ in the second year after planting in the open field, in comparison to plants originating from the standard planting material (ST plants). Flow cytometry analysis deployed to estimate the DNA ploidy level and relative nuclear DNA content in TC and ST plants revealed no significant differences in nuclear DNA content between plants of different origin. Chromosome counting in root/shoot tip meristems also showed a normal tetraploid chromosome number (2 n = 4 x = 28) in TC blackberry plants and a normal diploid chromosome number (2 n = 2 x = 14) in TC raspberry plants. Polyacrylamide gel electrophoresis of peroxidase (POX) extracted from the leaf tissue revealed no differences in POX profiles between TC and ST plants either. The results obtained in this study verify the suitability of micropropagation by axillary branching for commercial exploitation in blackberry and raspberry.

An in vitro protocol has been established for clonal propagation of Nothapodytes nimmoniana which is an important source of Camptothecin (CPT). Elite source was identified based on the chemical potency to accumulate the optimum level of CPT. Different types and concentrations of plant growth regulators were used to study their effect on inducing multiple shoots from the explants regenerated from embryos of N. nimmoniana. Of these, a combination of N6-benzyladenine (0.2 mg L−1) and Indole-3-butyric acid (IBA) (0.1 mg L−1) proved optimum for differentiating multiple shoots in 90.6 % of the cultures with an average of 10.24 shoots per explant obtained within 8 weeks of inoculation. Nearly, 92 % of the excised in vitro shoots rooted on half strength Murashige and Skoog (MS) medium containing 0.05 % activated charcoal, supplemented with 1-naphthaleneacetic acid and IBA at 0.1 mg L−1 each. The micropropagated plants were evaluated for their genetic fidelity by employing inter simple sequence repeats (ISSR) markers. Ten individuals, randomly chosen from a population of 145 regenerants, were compared with the donor plant. The regenerated plants were also evaluated for their chemical potency using high-performance liquid chromatography (HPLC) analysis of CPT content. The true-to-type nature of the micropropagated plants was confirmed based on their monomorphic banding profiles with that of the mother plants using ISSR markers. Besides, HPLC evaluation of the CPT content confirmed the existence of chemical uniformity among the regenerated plants and the elite mother plant. Electronic supplementary material The online version of this article (doi:10.1007/s13205-016-0490-y) contains supplementary material, which is available to authorized users.

The aim of this study was to produce sugarcane plantlets from cell suspension culture and study its genetic fidelity using molecular markers. The study was carried out using sugarcane varieties Co 86032 and Q117. Callus cultures of both the varieties were optimized using six different callus induction media. After screening the growth response of callus on six different callus induction media, it was observed that medium no. VI supplemented with 500 mg l−1 of each PVP, Casein hydrolysate and MES buffer showed high amounts of callus in Co 86032 (79.66 ± 0.44%) and Q117 (82.83 ± 1.69%). Addition of PEG 8000 at 2.5% to this medium had a profound impact on inducing somatic embryogenesis in Co 86032 (54.66 ± 1.76%) and Q117 (66.66 ± 2.60%) as compare to control (24.33 ± 1.76%) and (27.33 ± 2.73%), respectively. Cell suspension cultures were established by culturing embryogenic calli in liquid medium showed well established suspension cultures with fever cell aggregates. There was negligible cell division during initial 2 days of incubation and cell count increased rapidly between 2 and 8 days. Further incubation beyond 8 days resulted in a decrease in cell viability. Enhanced callus proliferation in Q117 while enhanced shoot regeneration in Co 86032 was observed from cell suspension culture. The clonal fidelity of in vitro regenerated plants was assessed by using RAPD and ISSR markers. Analysis of the ten RAPD markers indicated that 90.48 and 86.95% true-to-type regenerated plantlets in Co 86032 and Q117, respectively. However, in the ISSR markers, Co 86032 did not show any polymorphism and in the Q117, 92.18% true-to-type plantlets were found. These results confirmed that somaclonal variation occurs during the process of indirect organogenesis and RAPD and ISSR marker based molecular analysis is a suitable method for an early detection of variation in sugarcane. Electronic supplementary material The online version of this article (doi:10.1007/s13205-016-0579-3) contains supplementary material, which is available to authorized users.

In vitro plant propagation and maintenance could face genetic mutation occurring during in vitro phases. The objective of the present study was to evaluate the genetic fidelity in micropropagated plantlets of globe artichoke. Leaves from both in vivo (mother plants in the field) and from in vitro material (multiplicated shoots) were analyzed over two years to determine intraclonal variation and to evaluate the influence of the number of subcultures on the generation of somaclonal variants. In this study, the usefulness of two different DNA-based techniques, inter simple sequence repeats (ISSR) and simple sequence repeat (SSR) markers was evaluated, in assessing the genetic stability of micropropagated plants of six ecotypes. Eight primers were successfully used to amplify the DNA demonstrating a different ability to detect genetic variation. ISSR techniques are efficient markers in showing the occurrence of the genetic changes that occur during the micropropagation process of globe artichoke. We conclude that when the tissue culture technique is used, the analysis of somaclonal variability could require more than one DNA-based technique; different markers detect different genome regions and have different polymorphic ability, as our results showed for SSR and ISSR markers. The consequences on breeding activities of present results are discussed.

Nodal segments, obtained from 12 years-old mature plant, were used as explants for in vitro propagation of Celastrus paniculatus, an important medicinal plant of India. Shoot multiplication was achieved by repeated transfer of mother explants and subculturing of in vitro produced shoot clumps on MS medium supplemented with various concentrations of BAP alone or in combination with auxin (IAA or NAA). In vitro raised shoots were rooted under ex vitro condition. Genetic fidelity of the regenerated plants was assessed using random amplified polymorphic DNA (RAPD)., Celastrus paniculatus, belonging to the family Celastraceae, is an important medicinal plant of India. Owing to the ever-increasing demand from the pharmaceutical industry, the species is being overexploited, thereby threatening its stock in the wild. Poor seed viability coupled with low germination restricts its propagation through sexual means. Thus, alternative approaches such as in vitro techniques are highly desirable for large-scale propagation of this medicinally important plant. Nodal segments, obtained from a 12-year-old mature plant, were used as explants for multiple shoot induction. Shoot multiplication was achieved by repeated transfer of mother explants and subculturing of in vitro produced shoot clumps on Murashige and Skoog's (MS) medium supplemented with various concentrations of 6-benzylaminopurine (BAP) alone or in combination with auxin (indole-3-acetic acid (IAA) or α-naphthalene acetic acid (NAA)). The maximum number of shoots (47.75 ± 2.58) was observed on MS medium supplemented with BAP (0.5 mg L−1) and IAA (0.1 mg L−1). In vitro raised shoots were rooted under ex vitro conditions after treating them with indole-3-butyric acid (300 mg L−1) for 3 min. Over 95 % of plantlets acclimatized successfully. The genetic fidelity of the regenerated plants was assessed using random amplified polymorphic DNA. No polymorphism was detected in regenerated plants and the mother plant, revealing the genetic fidelity of the in vitro raised plantlets. The protocol discussed could be effectively employed for large-scale multiplication of C. paniculatus. Its commercial application could be realized for the large-scale multiplication and supply to the State Forest Department.

This study reports on the cryostorage of embryogenic lines derived from selected mature Quercus robur trees, following application of the PVS2-vitrification based procedure. In seven oak genotypes, embryo recovery levels ranging from 57-92% were obtained when 4-6 mg embryo clumps were precultured for 3 days on 0.3 M sucrose basal medium, treated with PVS2 solution for 60 min at 24ºC, and then immersed in liquid nitrogen (LN). Embryos of six out of seven lines were cryostored for one week and one year and used to evaluate cryopreservation tolerance, germination ability and to assess genetic fidelity by random amplified polymorphic DNA (RAPD) markers. There were no significant differences between the recovery frequencies of samples retrieved from LN after 1 week and 1 year of cryostorage. In five out of six lines, RAPD profiles of cryopreserved somatic embryos and regenerated plantlets were identical to those of the controls. Although polymorphisms were detected in only one cryostored embryo of one genotype, no genetic instability was found in the regenerated plantlets. This methodology appears to be suitable for long-term storage of this valuable germplasm, as the recovered plantlets were found to be genetically stable., Xunta de Galicia project: PGIDIT03RFO40001PR MEC (Spain) project: AGL2006-01387/FOR

Multiple shoots emerged from the nodal shoot segments of the field-grown candidate plus clump explants of Bambusa pallida Munro when cultured on Murashige and Skoog (MS) liquid medium with additives (ascorbic acid 50 mg/L + citric acid 25 mg/L + cysteine 25 mg/L) and combined use of α-naphthalene acetic acid (NAA) 1.34 μM + thiodiozuron 1.125 μM in a 2-week period. Further shoot multiplication was achieved in MS liquid medium with additives + NAA 1.34 μM + 6-benzylaminopurine 4.4 μM at 25 ± 2 °C and 33.78 μmol photons m−2 s−1 light illumination for a 12-h photoperiod. These shoots were rooted within four weeks in MS/2 basal salt medium with additives +2% sucrose +1% glucose, and 0.6% agar by pulse treatment of shoots with indole 3 butyric acid 0.5 mg/mL for 30 min prior to inoculation. Rooted plants were successfully hardened in the mist chamber. Survival rate during hardening was more than 95%. Micropropagated plants achieved a height of 25–30 cm with 3–4 tillers (shoots) with miniature rhizome in a 4-month period. Genetic stability was observed in the micropropagated plants.