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1.  A simple and efficient protocol for the mass propagation of Cymbidium mastersii: an ornamental orchid of Northeast India 
AoB Plants  2012;2012:pls023.
The present investigation was undertaken to mass propagate Cymbidium mastersii, an ornamental orchid of Northeast India by in vitro propagation method. This approach could also help for the conservation as well as commercialization of C. mastersii and other threatened and ornamental orchids.
Background and aims
Cymbidium mastersii is an epiphytic orchid distributed mainly in Northeast India. Owing to its high commercial value in the floricultural industry, natural populations are under threat from over-exploitation. Mass propagation provides an alternative means of satisfying the demand. Unfortunately, conventional propagation is slow and difficult, suggesting in vitro methods for mass multiplication may be more appropriate. The objective of this study was to develop an efficient protocol.
Methodology and principal results
Four nutrient media were evaluated for seed germination and early protocorm development: Murashige and Skoog (MS), half-strength MS, Knudson ‘C’ (KC), and Vacin and Went (VW). In addition, the effects of plant growth regulators 6-benzylaminopurine (BAP), kinetin (KN), α-naphthalene acetic acid (NAA) and indole-3-butyric acid (IBA) were studied alone and in combination. The maximum percentage seed germination (93.58 ± 0.56) was obtained in MS basal medium after 8–9 weeks of culture. Secondary protocorms (protocorm-like bodies) were developed from primary protocorms on MS medium fortified with different concentrations and combinations of cytokinins (BAP and KN) and auxins (NAA and IBA). The highest numbers of secondary protocorms (20.55 ± 0.62)/primary protocorms were obtained in MS medium supplemented with 5.0 µM BAP and 2.5 µM NAA. The most effective auxin source promoting root production (7.46 ± 0.09 per shoot) was 10.0 µM IBA. The plants were acclimatized effectively (survival percentage 88 %) in a greenhouse using a rooting medium of crushed sterile brick and charcoal (1 : 1 v/v) and vermicompost (leaf litter + cow dung, 1 : 1 v/v).
An efficient protocol was established for in vitro propagation of C. mastersii using seed as the starting material. The percentage seed germination varied with the composition of the nutrient media and was highest in full-strength MS basal medium. The number of secondary protocorms that developed from primary protocorms was increased by the addition of 5.0 µM BAP and 2.5 µM NAA. In vitro raised plantlets acclimatized in a greenhouse were closely similar to the mother plants in morphology.
PMCID: PMC3447538  PMID: 22997547
2.  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
3.  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
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.  In vitro Asymbiotic Germination of Immature Seed and Formation of Protocorm by Cephalanthera falcata (Orchidaceae) 
Annals of Botany  2006;98(6):1197-1206.
• Background and Aims Many Orchidaceous species are threatened globally by development and over-collection from their natural habitats for horticultural purposes. Artificial propagation from seeds is difficult in most terrestrial orchids native to temperate regions. Seed production is another limiting factor in the artificial propagation for these species because of the lessened probability of pollination and the destruction of fruit by insect larvae. Members of the genus Cephalanthera are distributed across Europe, Asia and North America. C. falcata is a temperate species of East Asia and an endangered species in Japan. As successful propagation from seeds of this species has never been reported, a reproducible method is described here for seed production in situ and propagation using immature seeds in asymbiotic culture in vitro.
• Methods Effects of hand-pollination and bagging treatment of ovaries were examined. Young capsules were collected every 10 d from 50 d after pollination until 120 d after pollination. Immature seeds obtained from these capsules were cultured asymbiotically on modified Kano medium and ND medium. Seed viability was examined within TTC (2,3,5-triphenyl tetrazolium chloride) test solution and histological observations were made on viable seeds by paraffin embedding at each collection stage.
• Key Results and Conclusions Hand-pollination followed by bagging treatment of ovaries with aluminium foil was effective for insect control during fruit development, and successfully yielded capsules. Of the capsules, 74·5 % survived to full maturity. The highest frequency (39·8 %) of seed germination was obtained with seeds harvested 70 d after pollination. The frequency declined with progress of seed maturity on the mother plant. Minimal germination was observed with seeds harvested 100 d or later after pollination. Histological observation suggests that accumulation of such substances as lignin in the inner integument surrounding the embryo during seed maturation plays an important role in induction of dormancy.
PMCID: PMC3292276  PMID: 17071633
Orchidaceae; Cephalanthera falcata; seed dormancy; seed germination; seed production; immature seed; inner integument
7.  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
8.  A genetically stable rooting protocol for propagating a threatened medicinal plant—Celastrus paniculatus 
AoB Plants  2012;5:pls054.
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.
PMCID: PMC4455238
Celastrus paniculatus; ex vitro rooting; genetic fidelity; molecular marker; shoot multiplication.
9.  Mycorrhizal preference promotes habitat invasion by a native Australian orchid: Microtis media 
Annals of Botany  2012;111(3):409-418.
Background and Aims
Mycorrhizal specialization has been shown to limit recruitment capacity in orchids, but an increasing number of orchids are being documented as invasive or weed-like. The reasons for this proliferation were examined by investigating mycorrhizal fungi and edaphic correlates of Microtis media, an Australian terrestrial orchid that is an aggressive ecosystem and horticultural weed.
Molecular identification of fungi cultivated from M. media pelotons, symbiotic in vitro M. media seed germination assays, ex situ fungal baiting of M. media and co-occurring orchid taxa (Caladenia arenicola, Pterostylis sanguinea and Diuris magnifica) and soil physical and chemical analyses were undertaken.
Key Results
It was found that: (1) M. media associates with a broad taxonomic spectrum of mycobionts including Piriformospora indica, Sebacina vermifera, Tulasnella calospora and Ceratobasidium sp.; (2) germination efficacy of mycorrhizal isolates was greater for fungi isolated from plants in disturbed than in natural habitats; (3) a higher percentage of M. media seeds germinate than D. magnifica, P. sanguinea or C. arenicola seeds when incubated with soil from M. media roots; and (4) M. media–mycorrhizal fungal associations show an unusual breadth of habitat tolerance, especially for soil phosphorus (P) fertility.
The findings in M. media support the idea that invasive terrestrial orchids may associate with a diversity of fungi that are widespread and common, enhance seed germination in the host plant but not co-occurring orchid species and tolerate a range of habitats. These traits may provide the weedy orchid with a competitive advantage over co-occurring orchid species. If so, invasive orchids are likely to become more broadly distributed and increasingly colonize novel habitats.
PMCID: PMC3579446  PMID: 23275632
Terrestrial orchid; mycorrhizal fungi; disturbed habitats; south-western Australia; invasive species; Microtis media
10.  Growth of in vitro Oncidesa plantlets cultured under cold cathode fluorescent lamps with super-elevated CO2 enrichment 
AoB Plants  2013;5:plt044.
How can one increase the biomass of plants, particularly orchids, in an effective way? Photoautotrophic micropropagation is an effective means of increasing the biomass of Oncidesa orchids. Using 10,000 µmol mol−1 CO2 and a photosynthetic photon flux density of 60 µmol m−2 s−1, the number of leaves and roots and shoot and root fresh and dry weights could be considerably increased, but only when cultured on Kyoto medium rather than Vacin and Went medium. Super-elevated CO2 enrichment and growth under cold cathode fluorescent lamps can positively affect the efficiency and quality of commercial production of clonal Oncidesa plantlets.
As interest in how to increase biomass production through biotechnological means gains traction, focus is turning towards the use of photoautotrophic micropropagation under elevated levels of carbon dioxide (CO2) to maximize plant growth and productivity. The effect of super-elevated CO2 with cold cathode fluorescent lamps (CCFLs) on the photoautotrophic growth of Oncidesa in vitro has been studied using a gas-permeable film culture vessel, the ‘Vitron’. The growth of Oncidesa (formerly Oncidesa Gower Ramsey ‘U-1’) plantlets on Vacin and Went (VW) medium was stimulated by 10 000 μmol mol−1 CO2. In particular, increasing the photosynthetic photon flux density (PPFD) from 45 to 60 μmol m−2 s−1 under 10 000 μmol mol−1 CO2 in the growth chamber remarkably increased the number of leaves and roots, and shoot and root fresh and dry weights compared with plantlets under the same level of CO2 under low PPFD (45 μmol m−2 s−1). However, there was a remarkable decrease in photosynthetic capacity, and chlorosis and browning of leaves. In stark contrast, plantlets grown on Kyoto medium at 10 000 μmol mol−1 CO2 under high PPFD had a higher photosynthetic rate than plantlets grown on VW medium, and no chlorosis or browning was observed. Furthermore, shoot growth was remarkably enhanced. Therefore, super-elevated CO2 (10 000 μmol mol−1) enrichment and growth under CCFLs can positively affect the efficiency and quality of commercial production of clonal Oncidesa plantlets.
PMCID: PMC4455684
CCFL; Oncidesa (formerly Oncidesa Gower Ramsey ‘U-1’); photoautotrophic growth; single-leaf photosynthesis; super-elevated CO2
11.  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
12.  Seed germination and seedling development in response to submergence in tree species of the Central Amazonian floodplains 
AoB Plants  2015;7:plv041.
This study shows that Central Amazonian floodplain trees can cope with long-term flooding during the early life-history stages. Seven of the eight studied species germinated and formed seedlings under water that endured submersion without any apparent injury for periods of 20 to 115 days, depending on the species. Only one of the seven did not survive re-exposure to air. The ability to germinate and form seedlings in water that subsequently are able to thrive in aerated soils would allow the most use of the short terrestrial phase available for seedling establishment in the lower portions of the flood-level gradient.
Successful germination and seedling establishment are crucial steps for maintenance and expansion of plant populations and recovery from perturbations. Every year the Amazon River and its tributaries overflow and flood the adjacent forest, exerting a strong selective pressure on traits related to seedling recruitment. We examined seed characteristics, stored reserves, germination, seedling development and survival under water of eight representative tree species from the lower portions of the flood-level gradient to identify adaptive strategies that contribute to their regeneration in this extreme ecosystem. Submerged seedlings were assessed for longevity and survival until they showed symptoms of injury. At this point, the remaining healthy seedlings were planted in unsaturated soil to monitor recovery after re-exposure to air over 30 days. All small (seed mass ≤0.17 g) seeds had epigeal phanerocotylar-type germination, a trait that would allow plants to acquire light and CO2 in the shortest time. Cell wall storage polysaccharide was a major component of all seeds, suggesting plant investment in structural reserves. Seven of the eight species germinated and formed healthy seedlings under water that endured submersion without any apparent injury for periods of 20–115 days, depending on the species. Seedlings of some species changed the direction of root growth and grew towards the surface of the water, which might have increased the uptake of oxygen to the tissues. Only one of the seven species did not survive re-exposure to air. Species able to germinate and produce seedlings under submersion, which subsequently are able to establish in aerated soils, would have more time available for terrestrial growth. This is critical for colonization of lower portions of the flood-level gradient where establishment is constrained by the short terrestrial phase that precedes the next flood.
PMCID: PMC4480040  PMID: 25922297
Carbohydrate reserves; cell wall storage polysaccharides; flood tolerance; seed germination in water; submergence tolerance; tropism
13.  Micropropagation of Ilex khasiana, a critically endangered and endemic holly of Northeast India 
AoB Plants  2011;2011:plr012.
The paper describes in vitro techniques for mass propagation of IIex khasiana, a rare and critically endangered holly endemic to Khasi Hills Hills of Meghalaya, India. The approach will help conserve I. khasiana and other endangered species.
Background and aims
Ilex khasiana is a rare and critically endangered holly endemic to the Khasi Hills of Meghalaya, India, and confined to a small number of pocket areas. In addition to conventional methods of propagation, endemic and threatened plants such as this could be more effectively multiplied and conserved using in vitro methods. Such techniques have the additional advantage of having a low impact on wild populations because they require a minimum of starting material. Our objective was to develop methodologies for the successful in vitro mass propagation of I. khasiana.
Seedlings were germinated in vitro under sterile conditions and nodal explants from these were transferred to Murashige and Skoog (MS) medium supplemented with 8.88 µM 6-benzyladenine and 4.64 µM kinetin.
Principal results
This generated ∼10 shoots per explant. In a second approach, callus was obtained from seedling-derived leaf discs cultured on MS medium supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzyladenine. Approximately 12 adventitious shoots per callus were regenerated from 83.33 % of the calli after transfer to MS medium supplemented with 6.63 µM 6-benzyladenine. The most effective treatment for inducing root formation on the shoots was transfer of shoots to half-strength MS medium with 9.84 µM indole-3-butyric acid. Regenerated plantlets with well-developed shoots and roots were hardened and transferred to open soil with 70 % survival after 4 weeks.
Both the methods described here are well suited for the mass multiplication of this critically endangered tree species.
PMCID: PMC3129536  PMID: 22476482
14.  An efficient in vitro propagation methodology for Annatto (Bixa orellana L.) 
Efficient methods were developed for both in vitro seed germination and micropropagation of an economically important dye yielding multipurpose tree, Bixa orellana L. Mature seeds were inoculated onto Murashige and Skoog (MS) medium supplemented with different concentrations of gibberellic acid (GA3). Highest frequency of germination (93.3 %) was recorded on medium supplemented with 3 μM GA3 against 13.33 % in control. Nodal explants cultured on MS medium fortified with 5 μM isopentanyl adenine (2-iP) produced maximum explants response (93.3 %) and highest number of shoots (35.71). Addition of relatively higher concentration (15 μM) of benzyl adenine (BA) resulted in the production of significantly (P < 0.05) reduced number of shoots (12.66). Sucrose at 87.6 mM was found to be the best carbohydrate source for multiple shoot induction compared to glucose and fructose. Regenerated shoots (3–4 cm) were rooted (95.5 %) on agar gelled MS medium supplemented with 10 μM indole-3-butyric acid (IBA). In vitro developed plantlets with well-developed roots were potted and acclimatized initially in the growth chamber and then moved to a green house with 83.3 % survival. The present protocol avoids the use of auxins in shoot multiplication medium, which will lower the cost, avoid callus formation and thus reduces the possibility of somaclonal variation in the regenerated plants. The method is efficient to produce over 32,000 hardened plants within a 10-month culture period starting from a single nodal explant.
PMCID: PMC3550577  PMID: 23573018
Carbohydrate source; Nodal segment; 2-iP; Multiple shoots; Rhizogenesis
15.  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
16.  Micropropagation and non-steroidal anti-inflammatory and anti-arthritic agent boswellic acid production in callus cultures of Boswellia serrata Roxb. 
Micropropagation through cotyledonary and leaf node and boswellic acid production in stem callus of a woody medicinal endangered tree species Boswellia serrata Roxb. is reported. The response for shoots, roots and callus formation were varied in cotyledonary and leafy nodal explants from in vitro germinated seeds, if inoculated on Murshige and Skoog’s (MS) medium fortified with cytokinins and auxins alone or together. A maximum of 8.0 ± 0.1 shoots/cotyledonary node explant and 6.9 ± 0.1 shoots/leafy node explants were produced in 91 and 88 % cultures respectively on medium with 2.5 μM 6-benzyladenine (BA) and 200 mg l−1 polyvinylpyrrolidone (PVP). Shoots treated with 2.5 μM IBA showed the highest average root number (4.5) and the highest percentage of rooting (89 %). Well rooted plantlets were acclimatized and 76.5 % of the plantlets showed survival upon transfer to field conditions. Randomly amplified polymorphic DNA (RAPD) analysis of the micropropagated plants compared with mother plant revealed true-to-type nature. The four major boswellic acid components in calluses raised from root, stem, cotyledon and leaf explants were analyzed using HPLC. The total content of four boswellic acid components was higher in stem callus obtained on MS with 15.0 μM IAA, 5.0 μM BA and 200 mg l−1 PVP. The protocol reported can be used for conservation and exploitation of in vitro production of medicinally important non-steroidal anti-inflammatory metabolites of B. serrata.
PMCID: PMC3550693  PMID: 24381442
Anti-arthritic; Anti-inflammatory; Boswellia serrata; Boswellic acid; Burseraceae; Callus; Indian olibanum; Micropropagation; Salai guggul; Steroids
17.  Comparison of Somatic Embryogenesis‐derived Coffee (Coffea arabica L.) Plantlets Regenerated in vitro or ex vitro: Morphological, Mineral and Water Characteristics 
Annals of Botany  2002;90(1):77-85.
Coffea arabica L. plantlets obtained ex vitro after sowing somatic embryos produced in a bioreactor in horticultural substrate were compared with those obtained in vitro from the same embryo population under conventional culturing conditions on semi‐solid media. The intensity and quality of aerial and root system development were compared. Shoot emergence was more efficient in vitro but rooting frequencies were low. In contrast, all ex vitro‐regenerated embryos rooted. The cotyledon area of mature embryos produced in a bioreactor positively affected plantlet development when regeneration was carried out ex vitro. Embryos with an intermediate cotyledon area (0·86 cm2) had the highest rates of plant conversion ex vitro (63 %), and also resulted in vigorous plantlets. Mortality was higher in nursery conditions, but better plant development was obtained. The quality of plantlets produced under ex vitro conditions was reflected in better growth of the aerial and root systems, and also by similar morphological, mineral and water status characteristics to seedlings. Unlike roots formed on semi‐solid media, those produced in soil were branched, fine (30–50 % had a diameter of less than 0·5 mm) and they bore root hairs. Leaves of plantlets regenerated ex vitro had a histological structure similar to that of seedling leaves, and a lower stomatal density (100 vs. 233 mm–2). Moreover, they were more turgid, as indicated by higher pressure potential (ψP) (0·91 vs. 0·30 MPa) and relative water content values (97 vs. 93 %). Furthermore, under in vitro conditions, leaves had larger stomata which were abnormally round and raised. Direct sowing of germinated somatic embryos resulted in the rapid production of vigorous plantlets under ex vitro conditions, whilst removing the need for problematical and costly conventional acclimatization procedures.
PMCID: PMC4233853  PMID: 12125775
Acclimatization; histology; in vitro; plant regeneration; rooting; somatic embryogenesis; water characteristics
18.  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
19.  Mechanisms associated with tolerance to flooding during germination and early seedling growth in rice (Oryza sativa) 
Annals of Botany  2008;103(2):197-209.
Background and Aims
Flooding slows seed germination, imposes fatalities and delays seedling establishment in direct-seeded rice. This study describes responses of contrasting rice genotypes subjected to flooding or low oxygen stress during germination and discusses the basis of tolerance shown by certain cultivars.
In one set of experiments, dry seeds were sown in soil and either watered normally or flooded with 10 cm of water. Seedling survival and shoot and root growth were assessed and seed portions of germinating seedlings were assayed for soluble sugars and starch concentrations. The whole germinating seedlings were assayed for amylase and peroxidase activities and for ethylene production. Activities of enzymes associated with anaerobic respiration were examined and gene expression was analysed separately with seeds germinating under different amounts of dissolved oxygen in dilute agar.
Key Results
Flooding during germination reduced survival but to a lesser extent in tolerant genotypes. Starch concentration in germinating seeds decreased while sugar concentration increased under flooding, but more so in tolerant genotypes. Amylase activity correlated positively with elongation (r = 0·85 for shoot and 0·83 for root length) and with plant survival (r = 0·92). Tolerant genotypes had higher amylase activity and higher RAmy3D gene expression. Ethylene was not detected in seeds within 2 d after sowing, but increased thereafter, with a greater increase in tolerant genotypes starting 3 d after sowing. Peroxidase activity was higher in germinating seeds of sensitive genotypes and correlated negatively with survival.
Under low oxygen stress, tolerant genotypes germinate, grow faster and more seedlings survive. They maintain their ability to use stored starch reserves through higher amylase activity and anaerobic respiration, have higher rates of ethylene production and lower peroxidase activity as germinating seeds and as seedlings. Relevance of these traits to tolerance of flooding during germination and early growth is discussed.
PMCID: PMC2707318  PMID: 19001425
Amylase; anoxia; crop establishment; direct-seeded rice; ethylene; flooding; germination; hypoxia; Oryza sativa
20.  Isolation and Characterization of Actinomycete Antagonists of a Fungal Root Pathogen † 
Applied and Environmental Microbiology  1993;59(11):3899-3905.
By use of selective media, 267 actinomycete strains were isolated from four rhizosphere-associated and four non-rhizosphere-associated British soils. Organic media with low nutrient concentrations were found to be best for isolating diverse actinomycetes while avoiding contamination and overgrowth of isolation media by eubacteria and fungi. While all isolates grew well at pHs 6.5 to 8.0, a few were unable to grow at pH 6.0 and a significant number failed to grow at pH 5.5. Eighty-two selected isolates were screened for in vitro antagonism towards Pythium ultimum by use of a Difco cornmeal agar assay procedure. Five isolates were very strong antagonists of the fungus, four were strong antagonists, and ten others were weakly antagonistic. The remaining isolates showed no antagonism by this assay. Additional studies showed that several of the P. ultimum antagonists also strongly inhibited growth of other root-pathogenic fungi. Twelve isolates showing antifungal activity in the in vitro assay were also tested for their effects on the germination and short-term growth of lettuce plants in glasshouse pot studies in the absence of pathogens. None of the actinomycetes prevented seed germination, although half of the isolates retarded seed germination and outgrowth of the plants by 1 to 3 days. During 18-day growth experiments, biomass yields of some actinomycete-inoculated plants were reduced in comparison with untreated control plants, although all plants appeared healthy and well rooted. None of the actinomycetes significantly enhanced plant growth over these short-term experiments. For some, but not all, actinomycetes, some correlations between delayed seed germination and reduced 18-day plant biomass yields were seen. For others, plant biomass yields were not reduced despite an actinomycete-associated delay in seed germination and plant outgrowth. Preliminary glasshouse experiments indicated that some of the actinomycetes protect germinating lettuce seeds against damping-off caused by P. ultimum.
PMCID: PMC182547  PMID: 16349093
21.  Interaction between seed size and NaCl on germination and early seedling growth of some Turkish cultivars of chickpea (Cicer arietinum L.) 
Chickpea is an important food legume crop of Turkey and is largely grown for human consumption on low moisture or salt-affected soils. The objective of the study was to find the effects of NaCl stress at electrical conductivities of 4.5, 8.6, 12.7 and 16.3 dS/m and seed sizes (7, 8 and 9 mm) on germination and early seedling growth of three popular chickpea cultivars (AKN-97, Gokce and Uzunlu-99). Mean frequency of germination, germination time, germination index, root length, shoot length and seedling fresh weight showed seed size-dependent responses of cultivars to salt stress. In general, small seeds germinated and grew more rapidly compared to medium and large seeds of the same cultivars against all levels of salt stress, with the best results in cultivar Uzunlu-99. No effect of NaCl treatments was observed on frequency of germination; however, a drastic decrease in early seedling growth was recorded at increased NaCl concentrations. Regression analysis results showed a significantly positive relationship (P<0.01) between seed size and mean germination time, whereas a significantly negative relationship was recorded between seed size and germination index, root length, shoot length. Moreover, linear regression values apparently confirmed that increased seed size in each cultivar affected decreased germination index, root and shoot lengths with enhanced mean germination time. Thus, it was concluded that the use of small seeds could considerably reduce the production costs of chickpea in salt-affected soils.
PMCID: PMC2367375  PMID: 18500776
Chickpea (Cicer arietinum L.); NaCl stress; Cultivar; Seed size; Germination
22.  Responses of Seed Germination, Seedling Growth, and Seed Yield Traits to Seed Pretreatment in Maize (Zea mays L.) 
The Scientific World Journal  2014;2014:834630.
A series of seed priming experiments were conducted to test the effects of different pretreatment methods to seed germination, seedling growth, and seed yield traits in maize (Zea mays L.). Results indicated that the seeds primed by gibberellins (GA), NaCl, and polyethylene glycol (PEG) reagents showed a higher imbibitions rate compared to those primed with water. The final germination percentage and germination rate varied with different reagents significantly (P < 0.05). The recommended prime reagents were GA at 10 mg/L, NaCl at 50 mM, and PEG at 15% on account of germination experiment. 15% PEG priming reagent increased shoot and root biomass of maize seedling. The shoot biomass of seedlings after presoaking the seeds with NaCl reagent was significantly higher than the seedlings without priming treatment. No significant differences of plant height, leaf number, and hundred-grain weight were observed between control group and priming treatments. Presoaking with water, NaCl (50 mM), or PEG (15%) significantly increased the hundred-grain weight of maize. Therefore, seed pretreatment is proved to be an effective technique to improve the germination performance, seedling growth, and seed yield of maize. However, when compared with the two methods, if immediate sowing is possible, presoaking is recommended to harvest better benefits compared to priming method.
PMCID: PMC4100373  PMID: 25093210
23.  Microbial Properties of Composts That Suppress Damping-Off and Root Rot of Creeping Bentgrass Caused by Pythium graminicola 
Composts prepared from a variety of feedstocks were tested for their ability to suppress seedling and root diseases of creeping bentgrass caused by Pythium graminicola. Among the most suppressive materials in laboratory experiments were different batches of a brewery sludge compost and a biosolids compost from Endicott, N.Y. Batches of these composts that were initially not suppressive to Pythium damping-off became more suppressive with increasing compost age. Leaf, yard waste, food, and spent mushroom composts as well as certain biosolids, cow manure, chicken-cow manure, and leaf-chicken manure composts were not suppressive to Pythium damping-off. In some cases, turkey litter, chicken manure, chicken-leaf, and food waste composts were inhibitory to creeping bentgrass seed germination in laboratory experiments. Microbial populations varied among all of the composts tested. Bacterial populations were high in all composts except the turkey litter compost, in which populations were 1,000- to 10,000-fold lower than in the other composts tested. Among the highest populations of heterotrophic fungi and antibiotic-producing actinomycetes were those found in all batches of the brewery sludge compost, whereas the lowest populations were found in turkey litter, chicken manure, and food waste composts. Heat treatment of suppressive composts reduced populations of bacteria, fungi, and actinomycetes in all composts tested. Disease suppressiveness was also reduced or eliminated in heated composts. Amending heated composts with small amounts of nonheated compost restored suppressive properties and partially restored microbial populations to wild-type levels. A strong negative relationship between compost microbial activity (as measured by the hydrolysis of fluorescein diacetate) and Pythium damping-off severity was observed. When composts were applied to creeping bentgrass in field experiments, a significant level of suppressiveness was evident with some composts when disease pressure was high (i.e., disease ratings high in uninoculated plots). A 1991 batch of turkey litter compost and the 1990 batch of Endicott biosolids were consistently suppressive to foliar symptoms of Pythium root rot on creeping bentgrass. This study indicates that suppression of Pythium diseases of creeping bentgrass in batches of brewery sludge and Endicott biosolids composts, and possibly in other suppressive composts examined in less detail in this study, is related directly to the microbial activities in the composts. On the other hand, the mechanisms of Pythium suppression in turkey litter and perhaps other poultry-based composts is not related directly to the compost microbial activity. Although turkey litter showed a lack of suppressiveness in laboratory bioassays and low microbial populations and activity, it resulted in a significant and consistent level of suppressiveness in field experiments. Therefore, the microbiological properties of Pythium-suppressive composts may differ substantially, and measurements of microbial populations and activity may not be predictive of the level of disease suppression in all composts.
PMCID: PMC1388845  PMID: 16535307
24.  Selection of suitable propagation method for consistent plantlets production in Stevia rebaudiana (Bertoni) 
Stevia rebaudiana (Bert.) is an emerging sugar alternative and anti-diabetic plant in Pakistan. That is why people did not know the exact time of propagation. The main objective of the present study was to establish feasible propagation methods for healthy biomass production. In the present study, seed germination, stem cuttings and micropropagation were investigated for higher productivity. Fresh seeds showed better germination (25.51–40%) but lost viability after a few days of storage. In order to improve the germination percentage, seeds were irradiated with 2.5, 5.0, 7.5 and 10 Gy gamma doses. But gamma irradiation did not show any significant change in seed germination. A great variation in survival of stem cutting was observed in each month of 2012. October and November were found the most suitable months for stem cutting survival (60%). In order to enhance survival, stem cuttings were also dipped in different plant growth regulators (PGRs) solution. Only indole butyric acid (IBA; 1000 ppm) treated cutting showed a higher survival (33%) than control (11.1%). Furthermore, simple and feasible indirect regeneration system was established from leaf explants. Best callus induction (84.6%) was observed on MS-medium augmented with 6-benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid (2,4-D; 2.0 mg l−1). For the first time, we obtained the highest number of shoots (106) on a medium containing BA (1.5 mg l−1) and gibberellic acid (GA3; 0.5 mg l−1). Plantlets were successfully acclimatized in plastic pots. The current results preferred micropropagation (85%) over seed germination (25.51–40%) and stem cutting (60%).
PMCID: PMC4250518  PMID: 25473365
Stevia rebaudiana; Seed germination; Seed radiation; Stem cuttings; Micropropagation
25.  An efficient in vitro shoot regeneration from immature inflorescence and ex vitro rooting of Arnebia hispidissima (Lehm). DC. - A red dye (Alkannin) yielding plant 
Arnebia hispidissima, which belongs to the family Boraginaceae, is an important medicinal and dye yielding plant. The alkannin, a red dye, are root-specific secondary metabolites of A. hispidissima. Shoots were regenerated from callus derived from immature inflorescence explants obtained from field grown plants. MS medium containing 4.52 μM 2, 4-D and 3.33 μM BAP was found to be most effective for the proliferation of callus, induced on medium containing 4.52 μM 2, 4-D. Maximum number (43.1 ± 0.25) with average length (5.2 ± 0.23) of shoots regenerated when callus was transferred to MS medium supplemented with 1.11 μM BAP, 1.16 μM Kin and 0.57 μM IAA. About 75.5 % of in vitro regenerated shoots were rooted on half-strength MS medium supplemented with 9.84 μM of IBA and 200 mg l−1 of activated charcoal. In comparison to in vitro, higher percent (90.2 %) of shoots were rooted under ex vitro conditions when treated with IBA (0.98 mM) for 5 min. Plantlets rooted in vitro as well as ex vitro were acclimatized successfully under the green house conditions. Ex vitro rooted plants exhibited higher survival percentage (75 %) as compared to in vitro rooted plantlets (60 %). Present study may be applicable in the large-scale root-specific red dye (alkannin) production via root induction under ex vitro condition.
PMCID: PMC3715650  PMID: 24431511
Alkannin; Organogenesis; Secondary metabolites

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