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1.  Multiple shoot induction from axillary bud cultures of the medicinal orchid, Dendrobium longicornu 
AoB Plants  2012;2012:pls032.
The present investigation was undertaken to propagate D. longicornu, a medicinally important orchid using axillary bud segments. This approach could also help in conserving other threatened orchids as well.
Background and aims
Dendrobium longicornu, commonly known as the ‘Long-horned Dendrobium’, is an endangered and medicinally important epiphytic orchid. Over-exploitation and habitat destruction seriously threaten this orchid in Northeast India. Our objective was to develop an efficient protocol for the mass propagation of D. longicornu using axillary bud segments.
Methodology and principal results
Axillary buds cultured in Murashige and Skoog semi-solid medium supplemented with α-naphthalene acetic acid (NAA), 2,4-dichlorophenoxy acetic acid (2,4-D) and 6-benzylaminopurine (BAP) readily developed into plantlets. These formed either directly from shoot buds or from intermediary protocorm-like bodies (PLBs). The maximum explant response (86.6 %) was obtained in medium supplemented with NAA at 30 µM, while the maximum number of shoots (4.42) and maximum bud-forming capacity (3.51) were observed in medium containing 15 µM BAP and 5 µM NAA in combination. Protocorm-like bodies were obtained when the medium contained 2,4-D. The maximum number of explants forming PLBs (41.48 %) was obtained in medium containing 15 µM BAP and 15 µM 2,4-D. Well-developed plantlets obtained after 20–25 weeks of culture were acclimatized and eventually transferred to the greenhouse. Over 60 % of these survived to form plants ∼3–4 cm tall after 90 days in glasshouse conditions using a substrate of crushed brick and charcoal, shredded bark and moss.
The method described can readily be used for the rapid and large-scale regeneration of D. longicornu. Its commercial adoption would reduce the collection of this medicinally important and increasingly rare orchid from the wild.
PMCID: PMC3491754  PMID: 23136638
2.  In Vitro Propagation and Reintroduction of the Endangered Renanthera imschootiana Rolfe 
PLoS ONE  2014;9(10):e110033.
Renanthera imschootiana Rolfe is an endangered tropical epiphytic orchid that is threatened with extinction due to over-collection and the loss of suitable habitats. In vitro propagation is a useful way to mass produce plants for re-establishment in the wild and for commercial propagation. Seeds collected 150 days after pollination (DAP) were the optimum stage for in vitro culture. Seed germination reached 93.1% on quarter-strength MS (i.e., MS containing a quarter of macro- and micronutrients) medium containing 0.5 mg l−1 α-naphthaleneacetic acid (NAA), 20% coconut water (CW), 1.0 g l−1 peptone, 10 g l−1 sucrose and 1.0 g l−1 activated charcoal (AC). Quarter-strength MS medium supplemented with 1.0 mg l−1 BA, 0.5 mg l−1 NAA, 1.0 g l−1 peptone, 10 g l−1 sucrose and 20% CW was suitable for the sub-culture of protocorm-like bodies (PLBs) in which the PLB proliferation ratio was 2.88. Quarter-strength MS medium containing 1.0 mg l−1 NAA, 1.0 g l−1 peptone, 100 g l−1 banana homogenate (BH), and 1.0 g l−1 AC was suitable for plantlet formation and 95.67% of plantlets developed from PLBs within 60 days of culture. Hyponex N016 medium supplemented with 0.5 mg l−1 NAA, 1.0 g l−1 peptone, 20 g l−1 sucrose, 150 g l−1 BH, and 1.0 g l−1 AC was suitable for the in vitro growth of plantlets about 2-cm in height. Plantlets 3-cm in height or taller were transplanted to Chilean sphagnum moss, and 95% of plantlets survived after 60 days in a greenhouse. Three hundred transplanted of seedlings 360-days old were reintroduced into three natural habitats. Highest percentage survival (79.67%) was observed in Yuanjiang Nature Reserve two years after reintroduction, followed by Huolu Mountain forest park (71.33%). This protocol is an efficient means for the large-scale propagation and in vitro and in vivo germplasm conservation of R. imschootiana.
PMCID: PMC4211678  PMID: 25350549
3.  Shoot differentiation from protocorm callus cultures of Vanilla planifolia (Orchidaceae): proteomic and metabolic responses at early stage 
BMC Plant Biology  2010;10:82.
Vanilla planifolia is an important Orchid commercially cultivated for the production of natural vanilla flavour. Vanilla plants are conventionally propagated by stem cuttings and thus causing injury to the mother plants. Regeneration and in vitro mass multiplication are proposed as an alternative to minimize damage to mother plants. Because mass production of V. planifolia through indirect shoot differentiation from callus culture is rare and may be a successful use of in vitro techniques for producing somaclonal variants, we have established a novel protocol for the regeneration of vanilla plants and investigated the initial biochemical and molecular mechanisms that trigger shoot organogenesis from embryogenic/organogenic callus.
For embryogenic callus induction, seeds obtained from 7-month-old green pods of V. planifolia were inoculated on MS basal medium (BM) containing TDZ (0.5 mg l-1). Germination of unorganized mass callus such as protocorm -like structure (PLS) arising from each seed has been observed. The primary embryogenic calli have been formed after transferring on BM containing IAA (0.5 mg l-1) and TDZ (0.5 mg l-1). These calli were maintained by subculturing on BM containing IAA (0.5 mg l-1) and TDZ (0.3 mg l-1) during 6 months and formed embryogenic/organogenic calli. Histological analysis showed that shoot organogenesis was induced between 15 and 20 days after embryogenic/organogenic calli were transferred onto MS basal medium with NAA (0.5 mg l-1). By associating proteomics and metabolomics analyses, the biochemical and molecular markers responsible for shoot induction have been studied in 15-day-old calli at the stage where no differentiating part was visible on organogenic calli. Two-dimensional electrophoresis followed by matrix-assisted laser desorption ionization time-of-flight-tandem mass spectrometry (MALDI-TOF-TOF-MS) analysis revealed that 15 protein spots are significantly expressed (P < 0.05) at earlier stages of shoot differentiation. The majority of these proteins are involved in amino acid-protein metabolism and photosynthetic activity. In accordance with proteomic analysis, metabolic profiling using 1D and 2D NMR techniques showed the importance of numerous compounds related with sugar mobilization and nitrogen metabolism. NMR analysis techniques also allowed the identification of some secondary metabolites such as phenolic compounds whose accumulation was enhanced during shoot differentiation.
The subculture of embryogenic/organogenic calli onto shoot differentiation medium triggers the stimulation of cell metabolism principally at three levels namely (i) initiation of photosynthesis, glycolysis and phenolic compounds synthesis; (ii) amino acid - protein synthesis, and protein stabilization; (iii) sugar degradation. These biochemical mechanisms associated with the initiation of shoot formation during protocorm - like body (PLB) organogenesis could be coordinated by the removal of TDZ in callus maintenance medium. These results might contribute to elucidate the complex mechanism that leads to vanilla callus differentiation and subsequent shoot formation into PLB organogenesis. Moreover, our results highlight an early intermediate metabolic event in vanillin biosynthetic pathway with respect to secondary metabolism. Indeed, for the first time in vanilla tissue culture, phenolic compounds such as glucoside A and glucoside B were identified. The degradation of these compounds in specialized tissue (i.e. young green beans) probably contributes to the biosynthesis of glucovanillin, the parent compound of vanillin.
PMCID: PMC3095354  PMID: 20444255
4.  Establishment of an Efficient In Vitro Regeneration Protocol for Rapid and Mass Propagation of Dendrobium chrysotoxum Lindl. Using Seed Culture 
The Scientific World Journal  2014;2014:740150.
An efficient in vitro regeneration protocol from seed culture has been established successfully for Dendrobium chrysotoxum, an epiphytic orchid having tremendous ornamental and medicinal values. Seed germination response was encouraging in Mitra (M) medium enriched with different combinations of auxins and cytokinins. Medium supplemented with 0.4% activated charcoal (AC), 2 mg/L 6-benzyl amino purine (BAP), and 2 mg/L indole-3-acetic acid (IAA) produced best seed germination percentage in 2 weeks of culture. Incorporation of higher concentration of kinetin (KN) or BAP in combination with low auxin in medium induced pronounced shooting and leaf formation. Reduction in leaf development was evident when cytokinins exist singly in medium indicating synergistic effect of auxin and cytokinin in leaf induction. Presence of elevated level of indole-3-butyric acid (IBA) or 1-naphthalene acetic acid (NAA) with low cytokinin content in medium generated more in vitro rooting, though IBA was found to be more effective in rooting induction as compared to NAA. The in vitro protocol for asymbiotic seed germination developed from the present investigation can be used for rapid mass propagation of this highly important Dendrobium orchid species.
PMCID: PMC4221871  PMID: 25401154
5.  Ex situ conservation of Cymbidium eburneum Lindl.: a threatened and vulnerable orchid, by asymbiotic seed germination 
3 Biotech  2012;2(4):337-343.
The population of many splendid orchids is reducing from their natural habitats at an alarming rate and their conservation is becoming a matter of global concern. Asymbiotic seed germination has been applied for ex situ conservation of rare, endangered and threatened orchid taxa and could provide rapid means their multiplication. In the present study reported here, seeds of an epiphytic and rare orchid, Cymbidium eburneum were germinated asymbiotically in different basal media viz., Murashige and Skoog (MS), Knudson C, Mitra et al. (Mitra), Gamborg et al. (B5) and Nitsch. The highest germination rate was observed in Mitra medium, whereas the development of the protocorms was found to be best in MS medium. Effects of growth regulators viz., indole-3 acetic acid (IAA), α-naphthalene acetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-d), thidiazuron (TDZ), 6-benzyl aminopurine (BAP) and kinetin (Kn) both singly and in combination incorporated in the MS medium were studied on growth and development of seedlings. It was observed that MS medium nourished with 15 μM each of BAP and NAA in combination was found to enhance shoot number and length, and root number and length in the seedlings. The rooted seedlings were successfully acclimatized.
PMCID: PMC3482442
Ex situ conservation; Endangered; Asymbiotic seed germination; Protocorms; Cymbidium eburneum
6.  Asymbiotic seed germination and in vitro conservation of Coelogyne nervosa A. Rich. an endemic orchid to Western Ghats 
Coelogyne nervosa is an epiphytic orchid endemic to Western Ghats, South India. The mature seeds of C. nervosa were cultured on ½ MS (Murashige and Skoog), MS, Kn (Knudson) and VW (Vacin and Went) media to evaluate the seed germination response. Of the four basal media used, MS medium supported maximum seed germination. Further experiments to enhance seed germination were done on MS medium supplemented with various concentrations (10, 20, 30 and 40 %) of coconut water (CW). Thirty percent CW gave the highest response in terms of percent seed germination (96), fresh weight (7.2 mg/seedling) and protocorm length (15.2 mm). Since CW containing medium did not support further seedling growth, each seedling was isolated and cultured on MS medium supplemented with either BA (6-benzylaminopurine) or Kin (kinetin) alone (1.0–4.0 mg/l each) or in combination with NAA (1-naphthaleneacetic acid; 0.2–1.0 mg/l). Maximum growth was observed on MS medium supplemented with BA (3.0 mg/l) and NAA (0.5 mg/l). On this medium, the seedlings reached an average length of 3.6 cm with 2.8 well expanded green leaves per seedling. Similarly optimum, healthy, white root induction (3.3 roots/seedlings) was also observed on the same medium. The rooted seedlings were successfully transplanted to pots with 91 % success. The 2-year-old tissue culture derived plants produced normal flowers and fruits.
PMCID: PMC3550512  PMID: 23814439
Coelogyne nervosa; Epiphytic orchid; Micropropagation; Seedlings; Tissue culture
7.  Mycorrhizal associations and reproductive isolation in three closely related Orchis species 
Annals of Botany  2010;107(3):347-356.
Background and Aims
The maintenance of species boundaries in sympatric populations of closely related species requires some kind of reproductive isolation that limits gene flow among species and/or prevents the production of viable progeny. Because in orchids mycorrhizal fungi are needed for seed germination and subsequent seedling establishment, orchid–mycorrhizal associations may be involved in acting as a post-mating barrier.
We investigated the strength of post-mating barriers up to the seed germination stage acting between three closely related Orchis species (Orchis anthropophora, O. militaris and O. purpurea) and studied the role of mycorrhizal fungi in hybridization by burying seed packets of pure and hybrid seeds. After retrieval and assessment of seed germination, the fungi associating with protocorms originating from hybrid and pure seeds were determined and compared with those associating with adult individuals using DNA array technology.
Whereas pre-zygotic post-mating barriers were rather weak in most crosses, post-zygotic post-mating barriers were stronger, particularly when O. purpurea was crossed with O. anthropophora. Germination trials in the field showed that seed germination percentages of hybrid seeds were in most cases lower than those originating from pure crosses. In all species pair combinations, total post-mating reproductive isolation was asymmetric. Protocorms associated with a smaller range of fungal symbionts than adult plants, but there was considerable overlap in mycorrhizal associations between protocorms and their respective parents.
Our results suggest that mycorrhizal associations contribute little to reproductive isolation. Pre-mating barriers are probably the main factors determining hybridization rates between the investigated species.
PMCID: PMC3043927  PMID: 21186239
DNA array; gene flow; hybrid zones; mycorrhizal associations; reproductive barriers; seed germination
8.  Micropropagation of an Exotic Ornamental Plant, Calathea crotalifera, for Production of High Quality Plantlets 
The Scientific World Journal  2014;2014:457092.
A successful protocol was established for micropropagation in two selected varieties of exotic ornamental plants, Calathea crotalifera. The effects of different sterilization techniques, explant type, and the combination and concentration of plant growth regulators on shoots induction were studied. The axillary shoot buds explants sprouted from rhizomes in soil free conditions showed high induction rate of shoots with lowest contamination percentage when treated with combination of 30% (v/v) NaOCl, 70% (v/v) ethanol, and 0.3% (w/v) HgCl2. In the present study, the highest number of multiple shoots was obtained in MS basal medium supplemented with 3.5 mg/L 6-Benzylaminopurine (BAP), 1.0 mg/L 1-Naphthaleneacetic acid (NAA), 3% sucrose, and 6 g/L plant agar for both varieties and was used as multiplication medium. Microshoots were highly induced when the young shoot bud explants were incised longitudinally prior subculture. Chlorophyll analysis was studied to test the effects of activated charcoal and L-glutamine on reduction of necrosis problem. The maximum roots induction was recorded on MS medium supplemented with 1.0 mg/L 1-Naphthaleneacetic acid (NAA) compared to indolebutyric acid (IBA). The complete regenerated plantlets were successfully acclimatized in the soilless medium under greenhouse condition. This is the first report of rapid mass propagation for C. crotalifera.
PMCID: PMC4127217  PMID: 25136669
9.  Genetic stability and phytochemical analysis of the in vitro regenerated plants of Dendrobium nobile Lindl., an endangered medicinal orchid 
Meta Gene  2014;2:489-504.
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.
•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.
PMCID: PMC4287867  PMID: 25606433
In vitro propagation; Genetic fidelity; RAPD; SCoT; Antioxidants; Secondary metabolites
10.  Auxin regulates first leaf development and promotes the formation of protocorm trichomes and rhizome-like structures in developing seedlings of Spathoglottis plicata (Orchidaceae) 
AoB Plants  2013;5:pls053.
This work demonstrated that auxin plays an important role in seedling establishment of the orchid S. plicata. In particular, normal first leaf formation required an appropriate distribution and concentration of auxin, as revealed by the use of polar auxin transport inhibitors and exogenously applied auxin. Moreover, auxin promoted the precocious formation of protocorm trichomes, which are an essential part of normal orchid seedling development. In addition, this work revealed that auxin can induce orchid seedlings to form propagative structures that are specific to a particular stage in seedling development.
Auxin flows in a polar manner to target tissues and exert its morphogenic effect. Preventing auxin movement, with polar auxin transport (PAT) inhibitors, or increasing auxin levels in tissues through exogenous application can provide a means for assessing the importance of appropriate tissue distribution and concentration of this hormone during development. The formulation of culture media for micropropagation has been the primary focus of most orchid tissue culture research, a goal that unveils seedling hormone responses at a single point in development. This study was unique because it evaluated the auxin response of orchids during three stages of seedling development. Seedlings were grown on standard culture media for 10, 35 and 85 days. Each group was sub-cultured onto auxin- and/or PAT inhibitor-containing media for an additional 10, 30 and 60 days, respectively. Data were collected on first leaf initiation, trichome formation and the appearance of propagative structures. In the 20-day seedlings, auxins and PAT inhibitors promoted precocious formation and random placement of protocorm hairs rather than in tufts, as seen in older, control seedlings. The 65-day seedlings formed protocorm-like bodies, rhizome-like growths from the stem, and fleshy leaves with trichomes. Seedlings cultured for 145 days developed microshoots or callus growth in the axils of older leaves and exhibited necrosis of original seedling roots and leaves. In general, exogenously applied auxin promoted the reversion of differentiated Spathoglottis plicata seedling tissue to a morphology that had propagative properties. Additionally, auxins commonly induced hair formation, which suggests that protocorm hairs may be root hair-like in nature. This work characterized three auxin growth responses in S. plicata seedlings that have not been reported in orchids: (i) the inhibition of first leaf initiation and abnormal first leaf morphology; (ii) the promotion of trichome formation; and (iii) the induction of rhizome-like structures and microshoots at a specific stage in seedling development.
PMCID: PMC4104637
Auxin; PAT inhibitors; propagation; protocorm trichomes; rhizome-like structures; seedling development; Spathoglottis.
11.  An effective nutrient medium for asymbiotic seed germination and large-scale in vitro regeneration of Dendrobium hookerianum, a threatened orchid of northeast India 
AoB Plants  2011;2012:plr032.
Submergence inhibits photosynthesis by terrestrial wetland plants, but less so in species that possess leaf gas films when submerged. Floodwaters are often supersaturated with dissolved CO2 enabling photosynthesis by submerged terrestrial plants, although rates remain well-below those in air. This important adaptation that enhances survival in submerged conditions is reviewed.
Background and aims
Dendrobium hookerianum is a rare and threatened epiphytic orchid of northeast India. Prospects for conservation would be strengthened by developing an in vitro method for mass propagation. Seeds are minute and difficult to use directly in the field for this purpose, being non-endospermous with a low nutrient content and dependent on a specific fungus for germination and early seedling development. Although produced in large numbers (2–3 million per capsule), <5 % germinate naturally in the wild. Our objective was to develop a rapid and successful method for in vitro propagation based on an initial in vitro asymbiotic seed germination step that achieved high percentages.
Effects of four different media, i.e. (i) Murashige and Skoog (MS), (ii) Mitra et al., (iii) Knudson (KC) and (iv) Gamborg et al. (B5), were evaluated for large-scale multiplication by asymbiotic seed germination. Seedling leaf number, shoot number, shoot length, root number and root length were scored. After 7–8 months, large numbers of well-rooted plantlets were transferred to a glasshouse in thermocol pots containing compost. Six different composts based on broken brick and charcoal were compared for their ability to support further development over 90 days of hardening.
Principal results
The fastest and highest percentage seed germination was achieved using MS medium. Seeds on MS medium germinated in 3–4 weeks compared with 7–8 weeks on B5 medium. Seedling development was also superior on MS medium. The inclusion of plant growth regulators was unnecessary. Compost comprising broken brick and charcoal with an upper layer of moss was found to be the most suitable for the survival of transferred plantlets. Ninety per cent survival of plantlets was achieved 90 days after transfer to a glasshouse.
The use of MS culture medium is well suited for the mass multiplication of D. hookerianum plants intended for re-introducing this threatened orchid into the wild.
PMCID: PMC3260561
12.  Effects of phytohormones on thermal denaturation profiles of Cymbidium DNA: indication of differential DNA replication. 
Nucleic Acids Research  1976;3(8):2033-2039.
Protocorm pieces of the orchid Cymbidium were aseptically cultured either without phytohormones, or with one of the growth promoting substances, auxin cytokinin, and gibberellin. The derivative melting profiles of the extracted DNA's differ from each other with respect to the size of various AT- and GC- rich fractions. Evidence has been obtained for the increase of the more AT--rich fractions in auxin-treated cultures, while gibberellin stimulated the expansion of the GC-rich fractions. These results are consistent with earlier cytological and cytochemical findings and indicate the involvement of hormone-controlled differential DNA replication in the development of Cymbidium protocorms in vitro.
PMCID: PMC343059  PMID: 967687
13.  The Effects of Different Media on Shoot Proliferation From the Shoot Tip of Aloe vera L. 
Aloe vera L. is an important pharmaceutical plant from which several medicinal and cosmetic compounds are extracted. Aloe is naturally propagated through offset, which is a slow and expensive labor cost method with low economical income.
In this study, the effect of different media on shoot proliferation of the shoot tip of Aloe vera L. was investigated.
Materials and Methods
In vitro techniques are some of the suggested methods for rapid propagation of Aloe. In this experiment, the shoot tips of mother plants were grown in a greenhouse. After surface sterilization of the explants, they were cultured on Murashige and Skoog (1962) (MS) medium containing different concentrations of kinetin and naphthaleneacetic acid (NAA). The experiment was carried out in the form of a randomized complete design with three replications.
The results showed that MS media containing 1.5 mg/L kinetin along with 0.15 or 0.3 mg/L NAA produced the highest percentage of proliferated shoots. In addition, the percentage of proliferated shoots in MS medium containing 2.0 or 2.5 mg/L benzylaminopurine (BAP) + 0.15 mg/L NAA was significantly higher than the other treatments.
Analysis of the interactive effects of NAA, kinetin and BAP on shoot proliferation showed that most of the proliferated shoots produced in MS medium containing 1.0 mg/L BAP + 1.0 mg/L kinetin + 0.15 mg/L NAA were significantly different from other treatments. Rooting quality was greater in MS media containing 1.0 mg/L IBA than a 1.0 mg/L NAA treatment.
PMCID: PMC3941910  PMID: 24624195
Plant Shoots; Benzylaminopurine; Plants, Medicinal
14.  In vitro callus induction and plantlet regeneration of Achyranthes aspera L., a high value medicinal plant 
To study callus induction from different explants (internode, leaf, root) and in vitro plantlets propagation from medicinally important plant Achyranthes aspera L.
Sterilized explants were prepared by using 0.1% HgCl2 and 0.5% Bavistin and callus was obtained when cultured onto Murashige Skoog's (MS) medium by using different concentrations and combination of 2,4-D, NAA, BAP, IAA, IBA with 3% sucrose and 0.8% agar. Induced callus was immediately transferred to MS medium containing at different concentrations of phytohormones for shootlets and rootlets induction respectively.
Sterilization treatment of 0.1% HgCl2 for 2-3 min and Bavistin 0.5% for 10-12 min showed the highest percentage of asepsis and survival rate. Maximum induction of callus was obtained from a combination of 2.0 mg/L 2,4-D and 0.5 mg/L NAA from leaf. Highest shootlets number (4.83±0.17) and length (3.8±0.16) cm were observed on full strength MS medium when fortified with BAP 4.0 mg/L and KIN 0.5 mg/L. Concerted efforts of BAP 2.0 mg/L and NAA 0.5 mg/L on full strength MS medium showed highest leaf number (6.77±0.94). In vitro raised shoots were allowed to root on different strengths of MS medium fortified with IAA and IBA at different concentrations. Experimentally, 3.0 mg/L IBA was enabled to induce maximum rootlets number (10.0±9.82) on full strength MS medium. Afterwards, regenerated shoots with well developed roots were successfully subjected to hardening process and were acclimatized. The survived plantlets showed 66.67% survival frequency without any morphological abnormality.
The results demonstrated that different explants were good source of callus induction, morphology analysis as well as indirect plantlets regeneration.
PMCID: PMC3819494  PMID: 24144129
Achyranthes aspera; Callus induction; Plantlets propagation; Murashige Skoog's; Micropropagation
15.  In Vitro Propagation of Muña-Muña (Clinopodium odorum (Griseb.) Harley) 
A micropropagation protocol was developed which may assist in the safeguarding and augmentation of dwindling natural populations of Clinopodium odorum (Griseb.) Harley, a critically and endangered medicinal plant. Factors affecting culture initiation bud sprouting and growth, rooting, and acclimatization were studied, using nodal segments of in vitro germinated seedling as primary explants on six media supplemented with different concentrations and combinations of 6-benzylaminopurine (BAP) (0.5–1.5 and 2-Naphthalene acetic acid (NAA) (0.5–1.5). Best results for culture initiation with sustainable multiplication rates (100%) were obtained on WP medium without any growth regulator. WP with the addition of 0.5 : 1 or 0.5 : 1.5) of BAP and NAA promoted a higher elongation; however, the optimum number of nodes were obtained in plantlets grown on 1/2 MS with the addition of 1 : 1.5 of BAP and NAA. Culture of sectioned individual nodes transferred to the media with different rates of BAP and NAA 1/2 MS-9 (1.5 : 1.5), SH-8 (1.5 : 1.0), and 1/2 B5-4 (1.0 : 0.5) media resulted in no proliferated shoots. The in vitro plants were successfully acclimatized garden soil and sand (2 : 1) in the greenhouse, with over 90% survival rate. The in vitro-grown plants could be transferred to ex vitro conditions and the efficacy in supporting ex vitro growth was assessed, with a view to develope longer-term strategies for the transfer and reintroduction into natural habitats.
PMCID: PMC3483647  PMID: 23119167
16.  Indirect shoot organogenesis from leaf explants of Adhatoda vasica Nees 
SpringerPlus  2014;3:648.
A novel protocol for indirect shoot organogenesis of Adhatoda vasica was developed using petiole explants derived from mature shrubby plants. Media with concentrations of cytokinins in combination with auxins were used to induce callus formation in two explants types: petiole and leaf segment. The frequency of callus formation from petiole and leaf segment explants on Murashige and Skoog (MS) basal medium supplemented with 0.25 mg l−1 thidiazuron (TDZ) and 0.25 mg l−1 α-naphthaleneacetic acid (NAA) was 100 ± 0.0 and 83.70 ± 0.52% respectively, while on this medium supplemented with 0.25 mg l−1 6-(γ-γ, dimethylallyamino purine) (2iP) and 0.25 mg l−1 NAA, the callus frequency was 100 ± 0.0 and 96.70 ± 0.67% respectively. The highest shoot regeneration (90.60 ± 0.52%) response and the maximum shoots (8.10 ± 0.28) per callus were achieved from petiole explants on MS medium containing 0.25 mg l−1 TDZ and 0.25 mg l−1 NAA. On the contrary, on Schenk & Hildebrandt (SH) basal medium supplemented with 0.25 mg l−1 TDZ and 0.25 mg l−1 NAA, the frequency of callus formation from petiole and leaf segment explants was 100 ± 0.0 and 90.50 ± 0.89% respectively while the callus frequency on this medium containing 0.25 mg l−1 2iP and 0.25 mg l−1 NAA was 100 ± 0.0 and 89.90 ± 0.72% respectively. The shoot regeneration frequency for petiole explants was 89.90 ± 0.46% producing 6.00 ± 0.21 shoots per callus on SH basal medium supplemented with 0.25 mg l−1 TDZ and 0.25 mg l−1 NAA. Whereas petiole explants could induce 83.70 ± 0.50% shoot regeneration and 7.3 ± 1.05 shoots per callus on SH medium containing 0.25 mg l−1 indole-3-butyric acid (IBA), 0.5 mg l−1 6-benzyladenine (BA) and 0.5 mg l−1 2iP. Elongation of regenerated shoot was obtained on MS basal medium supplemented with 0.25 mg l−1 TDZ. All regenerated shoots developed adventitious roots within 4 weeks when transferred to rooting medium containing SH medium supplemented with 0.5 mg l−1 IBA. Total nine rooted plantlets were transferred from in vitro to in vivo conditions and eight plants survived and successfully acclimatized in the shaded greenhouse 12 weeks after transplanting.
PMCID: PMC4230685  PMID: 25485191
Adhatoda vasica; Adventitious shoots; Callus induction; Organogenesis; Shoot regeneration
17.  In vitro clonal propagation of Achyranthes aspera L. and Achyranthes bidentata Blume using nodal explants 
To develop the reproducible in vitro propagation protocols for the medicinally important plants viz., Achyranthes aspera (A. aspera) L. and Achyranthes bidentata (A. bidentata) Blume using nodal segments as explants.
Young shoots of A. aspera and A. bidentata were harvested and washed with running tap water and treated with 0.1% bavistin and rinsed twice with distilled water. Then the explants were surface sterilized with 0.1% (w/v) HgCl2 solutions for 1 min. After rinsing with sterile distilled water for 3–4 times, nodal segments were cut into smaller segments (1 cm) and used as the explants. The explants were placed horizontally as well as vertically on solid basal Murashige and Skoog (MS) medium supplemented with 3% sucrose, 0.6% (w/v) agar (Hi-Media, Mumbai) and different concentration and combination of 6-benzyl amino purine (BAP), kinetin (Kin), naphthalene acetic acid (NAA) and indole acetic acid (IAA) for direct regeneration.
Adventitious proliferation was obtained from A. aspera and A. bidentata nodal segments inoculated on MS basal medium with 3% sucrose and augmented with BAP and Kin with varied frequency. MS medium augmented with 3.0 mg/L of BAP showed the highest percentage (93.60±0.71) of shootlets formation for A. aspera and (94.70±0.53) percentages for A. bidentata. Maximum number of shoots/explants (10.60±0.36) for A. aspera and (9.50±0.56) for A. bidentata was observed in MS medium fortified with 5.0 mg/L of BAP. For A. aspera, maximum mean length (5.50±0.34) of shootlets was obtained in MS medium augmented with 3.0 mg/L of Kin and for A. bidentata (5.40±0.61) was observed in the very same concentration. The highest percentage, maximum number of rootlets/shootlet and mean length of rootlets were observed in 1/2 MS medium supplemented with 1.0 mg/L of IBA. Seventy percentages of plants were successfully established in polycups. Sixty eight percentages of plants were well established in the green house condition. Sixty five percentages of plants were established in the field.
The results have shown that use of nodal buds is an alternative reproducible and dependable method for clonal propagation of A. aspera and A. bidentata. The high rate of direct shoot-root multiplication and their high rate of post-hardening survival indicate that this protocol can be easily adopted for commercial large scale cultivation.
PMCID: PMC3609210  PMID: 23569824
In vitro; Plant regeneration; Tissue culture; Nodal culture; Achyranthes aspera; Achyranthes bidentata; Clonal propagation; Nodal explant; Reproducible; MS medium; Shoots; BAP; NAA; IAA; Kinetin; Nodal segment; Shootlet; Rootlet
18.  An efficient and rapid regeneration via multiple shoot induction from mature seed derived embryogenic and organogenic callus of Indian maize (Zea mays L.) 
Plant Signaling & Behavior  2013;8(10):e25891.
An efficient method for in vitro micro propagation and genetic transformation of plants are crucial for both basic and applied research. Maize is one of the most important cereal crops around the world. Regeneration from immature embryo is hampered due to its unavailability round the year. On the contrary mature embryo especially tropical maize is recalcitrant toward tissue culture. Here we report a highly efficient regeneration (90%) system for maize by using 2 different approaches i.e., embryogenic and organogenic callus cultures. Seeds were germinated on MS medium supplemented with 5 mg/l 2,4-D and 3 mg/l BAP. Nodal regions of 2 wks old seedlings were longitudinally split upon isolation and subsequently placed on callus initiation medium. The maximum frequency of embryogenic callus formation (90%) was obtained on MS medium supplemented with 2 mg/l 2,4-D and 1 mg/l BAP in the dark conditions. The compact granular organogenic callus formation (85% frequency) was obtained on MS medium supplemented with 2.5 mg/l 2,4-D and 1.5 mg/l BAP at light conditions. MS medium supplemented with 2 mg/l BAP, 1 mg/l Kinetin and 0.5 mg/l NAA promoted the highest frequency of shoot induction. The highest frequency of root formation was observed when shoots were grown on MS medium. The regenerated plants were successfully hardened in earthen pots after adequate acclimatization. The important advantage of this improved method is shortening of regeneration time by providing an efficient and rapid regeneration tool for obtaining more stable transformants from mature seeds of Indian tropical maize cultivar (HQPM-1).
PMCID: PMC4091112  PMID: 23921544
embryogenic callus; immature embryo; maize; mature seeds; multiple shoots induction; organogenic callus; regeneration
19.  Tissue culture of Sophora tonkinensis Gapnep. and its quality evaluation 
Pharmacognosy Magazine  2013;9(36):323-330.
Sophora tonkinensis Gapnep. is an important rare medicinal plant in China. There were only a few papers on the rapid propagation of S. tonkinensis through in vitro tissue culture, and still no report focuses on the quality analysis of in vitro tissue culture plantlets.
Materials and Methods:
The different concentrations of 6-benzylaminopurine (BAP), kinetin (KT), and indole-3-acetic acid (IAA) were used to establish and screen the optimal rapid propagation technology of S. tonkinensis by orthogonal test; the different concentrations of a-naphthalene acetic acid (NAA), indole-3-butyric acid (IBA), and ABT rooting power (ABT) were used to screen the optimal rooting technology. For quality evaluation of tissue culture plants, three different sites were chose to finish planting experiment. The leaf characteristics, radix ex rhizoma yield, and contents of matrine and oxymatrine were evaluated, respectively, to provide evidence of high yield and good qualities of tissue culture plants.
A large number of buds could be induced directly from epicotyl and hypocotyl explants on the Murashige and Skoog (MS) medium supplemented with 1.5 mg/l BAP, 0.5 mg/l IAA, and 0.5 mg/l KT; the best root induction medium was solid MS medium at half the macronutrient concentration supplemented with 1.0 mg/l NAA, 0.4 mg/l IBA, and 0.1 mg/l ABT. The rooting rate was 98%. All tissue culture plants showed normal leaf characteristics. Tissue culture plants from two sites possessed higher radix ex rhizoma yield and overall productivity of matrine and oxymatrine than those of seed plants.
Tissue culture is a rapid, effective, and convenient propagation method for S. tonkinensis, and the quality of S. tonkinensis tissue culture plants meets the requirement of quality standard of China Pharmacopoeia (edition 2010), the crude drug from S. tonkinensis tissue culture plants will be suitable for substituting the crude drug from seed plants.
PMCID: PMC3793337  PMID: 24124284
Matrine and oxymatrine; micropropagation; quality evaluation; Sophora tonkinensis Gapnep.; tissue culture plant
20.  In vitro regeneration of Eucalyptus camaldulensis 
An efficient in vitro regeneration protocol enables mass multiplication, genetic modification and germplasm conservation of desired plants. In vitro plant regeneration was achieved from nodal segments of 18-months-old superior genotypes of Eucalyptus camaldulensis trees through direct organogenesis (DO) and direct somatic embryogenesis (DSE) pathways. Initial bud break (BB) stage occurred via DO while shoot multiplication phase followed both DO and DSE pathways. Interestingly, both BB and shoot multiplication stages were achieved on shoot induction and multiplication (SIM) media composed of Murashige and Skoog (MS) basal medium supplemented with 2 mg l−1 benzyl aminopurine (BAP) and 0.1 mg l−1 naphthalene acetic acid (NAA). Best shoot elongation response was observed on half strength MS fortified with 0.5 mg l−1 BAP, while root induction and elongation was superior in 1/2 MS + 1 mg l−1 Indole butyric acid (IBA). Full strength MS fortified with cytokinins (BAP) and weak auxin (NAA) in the ratio of 20:1 favored direct regeneration pathways. Further, half strength MS supported shoot and root development. The absence of intervening callus phase in this protocol can help in minimizing the chance occurrence of somaclones. When compared to other compositions tried, hardening in 100 % coco peat resulted in maximum survival (80 %) of the in vitro raised plantlets. For mass multiplication, fortnight subculturing of a single nodal explants for eight passages on SIM medium resulted in 60–148 shoot initials. Repeated subculturing in SIM medium induced the formation of direct somatic embryos which in turn improved the turnover capacity and enabled large scale clonal multiplication of elite and desirable trees of E. camaldulensis. Following this protocol, it takes a minimum time period of four-months between in vitro explant inoculation to hardening stage. In the present study, DO and DSE pathway of plant regeneration was reported occurring simultaneously in the same nodal explants of E. camaldulensis.
PMCID: PMC3550532  PMID: 23573043
Direct organogenesis; Direct somatic embryogenesis; In vitro regeneration; Nodal stem cuttings; Eucalyptus camaldulensis
21.  Cytokinin induced shoot regeneration and flowering of Scoparia dulcis L. (Scrophulariaceae)-an ethnomedicinal herb 
To develop an improved protocol for micropropagation of ethnomedicinally important Scoparia dulcis (S. dulcis) L.
Explants were inoculated on MS basal medium supplemented with kinetin and 6-benzylaminopurine for shoot bud induction. To enhance the shoot induction, various auxins like 3-indoleacetic acid or 3-indolebutyric acid or α-naphthylacetic acid were tested along with 2.32 M KI and 4.44 µM BAP. The regenerated shoots were rooted in half strength MS medium supplemented with various concentrations of IAA, IBA or NAA. After roots were developed, the plantlets were transplanted to pots filled with vermiculate and sand and kept in growth chamber with 70%–80% humidity under 16 h photoperiod. After acclimatization, the plantlets were transferred to the garden and survival percentage was calculated. Data were statistically analyzed and means were compared using Duncan's multiple range test (P<0.05).
An in vitro method was developed to induce high frequency shoots regeneration from stem, mature leaf and young leaf explants of S. dulcis. Shoot induction on young leaf explants was most successful in MS medium supplemented with combination of two cytokinins (2.32 µM KI and 4.44 µM BAP) 2.85 µM IAA, 10% CM and 1 483.79 µM adenine sulfate. A single young leaf explant was capable of producing 59 shoots after 13 days of culture. Flower was induced in medium supplemented with combination of KI and BAP.
Cytokinins are the key factor to induce the direct shoot regeneration and flowering of S. dulcis.
PMCID: PMC3609182  PMID: 23569752
Ethnomedicinal herb; Scoparia dulcis; Shoot induction; Regeneration; Cytokinin; Micropropagation; Explant; Flowering; Auxin; Survival percentage; Bud induction
22.  In vitro regeneration in Sarcostemma acidum (Roxb.) –an important medicinal plant of semi-arid ecosystem of Rajasthan, India 
An efficient regeneration protocol for Sarcostemma acidum – an important medicinal plant has been established. Callus initiated from nodal explant on MS medium with 2.0 mg L−1 of NAA + additives. Callus initiated was subcultured on MS medium containing various concentrations of NAA or 2,4-D. Out of these combinations, MS medium +1.0 mg L−1 of NAA + additives was found to be effective for the multiplication of callus. Subculture was done after an interval of 20–22 days. For differentiation of callus BAP or Kinetin alone was found to be less effective. Maximum frequency of shoot regeneration recorded on MS medium +1.0 mg L−1 of BAP + 0.5 mg L−1 of Kinetin and 0.1 mg L−1 of NAA + additives. The in vitro differentiated shoots were excised and inoculated on 1/4 strength MS medium +2.0 mg L−1 of IBA + 0.02 % activated charcoal for in vitro rooting. Maximum response (90 %) was recorded on this medium. In vitro differentiated shoots were inoculated on autoclaved soilrite® after treatment with root inducing auxins. Ex vitro rooting in this plant species has been reported for the first time. Eighty five percent of the shoots rooted under ex vitro conditions. Both in vitro and ex vitro rooted plantlets were hardened in a green house.
PMCID: PMC3656190  PMID: 24431495
Adenine sulphate; Ex vitro; Hardening; In vitro; Micropropagation; Sarcostemma acidum
23.  Asymbiotic Germination Response to Photoperiod and Nutritional Media in Six Populations of Calopogon tuberosus var. tuberosus (Orchidaceae): Evidence for Ecotypic Differentiation 
Annals of Botany  2008;102(5):783-793.
Background and Aims
Ecotypic differentiation has been explored in numerous plant species, but has been largely ignored in the Orchidaceae. Applying a specific germination protocol for widespread seed sources may be unreliable due to inherent physiological or genetic differences in localized populations. It is crucial to determine whether ecotypic differentiation exists for restoration and conservation programmes. Calopogon tuberosus var. tuberosus, a widespread terrestrial orchid of eastern North America, is a model species to explore ecotypic differences in germination requirements, as this species occupies diverse habitats spanning a wide geographical range.
Mature seeds were collected from south Florida, north central Florida, three locations in South Carolina, and the upper Michigan peninsula. Effects of three photoperiods (8/16, 12/12, 16/8 h L/D) were examined on asymbiotic in vitro seed germination and seedling development of C. tuberosus. Germination and early development was monitored for 8 weeks, while advanced development was monitored for an additional 8 weeks. In an additional experiment, asymbiotic seed germination and development was monitored for 8 weeks on six culture media (BM-1 terrestrial orchid medium, Knudson C, Malmgrem, half-strength MS, P723, and Vacin and Went). A tetrazolium test for embryo viability was performed.
Key Results
Short days promoted the highest germination among Florida populations, but few differences among photoperiods in other seed sources existed. Different media had little effect on the germination of Michigan and Florida populations, but germination of South Carolina seeds was higher on media with higher calcium and magnesium. Tetrazolium testing confirmed that South Carolina seeds exhibited low viability while viability was higher in Florida seeds. Seed germination and corm formation was rapid in Michigan seeds across all treatments. Michigan seedlings allocated more biomass to corms compared with other seed sources.
Rapid germination and corm formation may be a survival mechanism in response to a compressed growing season in northern populations. Ecotypic differentiation may be occurring based on seed germination and corm formation data.
PMCID: PMC2712386  PMID: 18757881
Asymbiotic germination; corm development; Calopogon tuberosus; ecotypic differentiation; native orchid; orchid seed germination; seedling development
24.  Shoot Organogenesis and Plant Regeneration from Leaf Explants of Lysionotus serratus D. Don 
The Scientific World Journal  2013;2013:280384.
The gesneriaceous perennial plant, Lysionotus serratus, has been used in traditional Chinese medicine. It also has a great development potential as an ornamental plant with its attractive foliage and beautiful flowers. An efficient propagation and regeneration system via direct shoot organogenesis from leaf explant was established in this study. High active cytokinin (6-benzyladenine (BA) or thidiazuron (TDZ)) was effective for direct organogenesis of initial induction. Murashige and Skoog (MS) growth media containing 0.5 mg L−1 BA alone or with combination of 0.1 mg L−1  α-Naphthaleneacetic acid (NAA) were the most effective for shoot proliferation. High BA concentration (1.0 mg L−1) in the media caused high percentage of vitrified shoots though they introduced high shoot proliferation rate. Histological observation indicated that adventitious shoot regeneration on the medium containing 0.5 mg L−1 BA alone occurred directly from leaf epidermal cells without callus formation. Regenerated shoots rooted well on medium containing half-strength MS medium with 0.5 mg L−1 indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA), and the plantlets successfully acclimatized and grew vigorously in the greenhouse with a 94.2% and 92.1% survival rate.
PMCID: PMC3747412  PMID: 23983626
25.  In vitro direct shoot regeneration from proximal, middle and distal segment of Coleus forskohlii leaf explants 
Coleus forskohlii is an endangered multipurpose medicinal plant that has widespread applications. In spite of this, there have been few attempts to propagate its cultivation in India. The present communication presents an in vitro rapid regeneration method using leaf explants of Coleus forskohlii through direct organogenesis. Leaf explants that were excised into three different segments i.e. proximal (P), middle (M) and distal (D) were cultured on Murashige and Skoog (MS) basal medium supplemented with cytokinins. MS Media containing 5.0 mg L−1 BAP (6-Benzylaminopurine) promoted regeneration of multiple shoots through direct organogenesis from the leaf, which were further elongated on MS media augmented with 0.1 mg L-1 BAP and 0.1 mg L−1 IAA (Indole-3-acetic acid), cytokinin and auxin combination. Regenerated and elongated shoots, when transferred to ose resulted in profuse rooting plants that were transferred to soil after acclimatization and maintained in a green house. The current protocol offers a direct, mass propagation method bypassing the callus phase of C. forskohlii and is suitable for conservation, large-scale commercial cultivation, and genetic transformation with agronomically desirable traits.
PMCID: PMC3550600  PMID: 23572969
6-benzylaminopurine; Coleus forskohlii; cytokinin; tissue culture; multiple shoots

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