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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.  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
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.  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
5.  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
6.  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
7.  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
8.  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
9.  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
10.  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
11.  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
12.  TDZ-induced direct shoot organogenesis and somatic embryogenesis on cotyledonary node explants of lentil (Lens culinaris Medik.) 
An efficient and simple procedure for inducing high frequency direct shoot organogenesis and somatic embryogenesis in lentil from cotyledonary node explants (without both the cotyledons) in response to TDZ alone is reported. TDZ at concentration lower than 2.0 μM induced shoot organogenesis whereas at higher concentration (2.5–15 μM) it caused a shift in regeneration from shoot organogenesis to somatic embryogenesis. The cotyledonary node and seedling cultures developed only shoots even at high concentrations of BAP and TDZ, respectively. TDZ at 0.5 and 5.0 μM was found to be optimal for inducing an average of 4–5 shoots per cotyledonary node in 93 % of the cultures and 55 somatic embryos in 68 % of the cultures, respectively. The somatic embryos were germinated when transferred to lower TDZ concentration (0.5–1.0 μM). The shoots were rooted on MS basal medium containing 2.5 μM IBA. The plantlets were obtained within 8 weeks from initiation of culture and were morphologically similar to seed-raised plants. The possible role of stress in thidiazuron induced somatic embryogenesis is discussed.
PMCID: PMC3550644  PMID: 23572901
Thidiazuron; Lens culinaris; Somatic embryogenesis; Organogenesis
13.  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
14.  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
15.  Induced Expression of AtLEC1 and AtLEC2 Differentially Promotes Somatic Embryogenesis in Transgenic Tobacco Plants 
PLoS ONE  2013;8(8):e71714.
Arabidopsis LEAFY COTYLEDON (LEC) genes, AtLEC1 and AtLEC2, are important embryonic regulators that play key roles in morphogenesis and maturation phases during embryo development. Ectopic expression of AtLEC1 and AtLEC2 in tobacco caused abnormality in transgenic seedling. When transgenic seeds germinated on medium containing 30 µM DEX, LEC1 transgenic seedlings were ivory and fleshy, with unexpanded cotyledons, stubby hypocotyls, short roots and no obvious callus formation at the shoot meristem position. While LEC2 transgenic seedlings formed embryonic callus on the shoot apical meristem and somatic embryo-like structures emerged from the surface of the callus. When callus were transferred to hormone free MS0 medium more shoots were regenerated from each callus. However, shoot formation was not observed in LEC1 overexpressors. To investigate the mechanisms of LEC2 in somatic embryogenesis, we studied global gene expression by digital gene expression profiling analysis. The results indicated that ectopic expression of LEC2 genes induced accumulation of embryo-specific proteins such as seed storage proteins, late embryogenesis abundant (LEA) proteins, fatty acid biosynthetic enzymes, products of steroid biosynthesis related genes and key regulatory genes of the embryo development. Genes of plant-specific transcription factors such as NAC domain protein, AP2 and GRAS family, resistance-related as well as salicylic acid signaling related genes were up-regulated in LEC2 transgenic seedlings. Ectopi c expression of LEC2 induced large number of somatic embryo formation and shoot regeneration but 20 d DEX induction of LEC1 is not sufficient to induce somatic embryogenesis and shoot formation. Our data provide new information to understand the mechanisms on LEC2 gene’s induction of somatic embryogenesis.
PMCID: PMC3741171  PMID: 23951228
16.  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
17.  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
18.  Embryo Development in the Lady's Slipper Orchid, Paphiopedilum delenatii, with Emphasis on the Ultrastructure of the Suspensor 
Annals of Botany  2006;98(6):1311-1319.
• Background and Aims Owing to large-scale collecting, the lady's slipper orchid, Paphiopedilum delenatii, is under threat of extinction. Asymbiotic germination provides a useful way to re-establish plants in the wild and for commercial propagation. A detailed study of embryo development would provide information on subsequent germination events and aid in the propagation of the species.
• Methods Developing capsules were collected for histochemical and ultrastructural studies by using both light and transmission electron microscopy.
• Key Results The suspensor of this species consists of three vacuolated cells. During the early globular stage of embryo development, structural differentiation occurs, revealing an abundance of smooth endoplasmic reticulum cisternae and wall ingrowths within the suspensor cells. These features are not present in cells of the embryo proper. Furthermore, the results of Nile red staining demonstrate that a cuticular layer is present only in the embryo proper, but absent from the suspensor. Cuticular material is also present in the inner walls of the seed coat, and persists through seed maturation.
• Conclusions The morphological features of the transfer cell and the absence of cuticular material in the suspensor cell wall corroborate the hypothesis that the suspensor is the major nutrient uptake site for the developing embryo in the lady's slipper orchid. The absence of an endosperm and presence of cuticular material in the inner walls of the seed coat enclosing the embryo proper further support the notion that nutrient uptake by the embryo is confined to the micropylar end of the seed through the suspensor.
PMCID: PMC2803589  PMID: 17056612
Cuticular material; embryology; lady's slipper orchid; Paphiopedilum; suspensor
19.  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
20.  Carry-over of Differential Salt Tolerance in Plants Grown from Dimorphic Seeds of Suaeda splendens 
Annals of Botany  2008;102(1):103-112.
Background and Aims
Halophytic species often show seed dimorphism, where seed morphs produced by a single individual may differ in germination characteristics. Particular morphs are adapted to different windows of opportunity for germination in the seasonally fluctuating and heterogeneous salt-marsh environment. The possibility that plants derived from the two morphs may also differ physiologically has not been investigated previously.
Experiments were designed to investigate the germination characteristics of black and brown seed morphs of Suaeda splendens, an annual, C4 shrub of non-tidal, saline steppes. The resulting seedlings were transferred to hydroponic culture to investigate their growth and photosynthetic (PSII photochemistry and gas exchange) responses to salinity.
Key Results
Black seeds germinated at low salinity but were particularly sensitive to increasing salt concentrations, and strongly inhibited by light. Brown seeds were unaffected by light, able to germinate at higher salinities and generally germinated more rapidly. Ungerminated black seeds maintained viability for longer than brown ones, particularly at high salinity. Seedlings derived from both seed morphs grew well at high salinity (400 mol m−3 NaCl). However, seedlings derived from brown seeds performed poorly at low salinity, as reflected in relative growth rate, numbers of branches produced, Fv/Fm and net rate of CO2 assimilation.
The seeds most likely to germinate at high salinity in the Mediterranean summer (brown ones) retain a requirement for higher salinity as seedlings that might be of adaptive value. On the other hand, black seeds, which are likely to delay germination until lower salinity prevails, produce seedlings that are less sensitive to salinity. It is not clear why performance at low salinity, later in the life cycle, might have been sacrificed by the brown seeds, to achieve higher fitness at the germination stage under high salinity. Analyses of adaptive syndromes associated with seed dimorphism may need to take account of differences over the entire life cycle, rather than just at the germination stage.
PMCID: PMC2712427  PMID: 18463109
Chlorophyll fluorescence; germination; growth rate; halophyte; photosynthesis; photosystem II; salt tolerance; seed dimorphism; seed viability; Suaeda splendens
21.  Germination and Plantlet Regeneration of Encapsulated Microshoots of Aromatic Rice (Oryza sativa L. Cv. MRQ 74) 
The Scientific World Journal  2012;2012:578020.
Plant tissues such as somatic embryos, apical shoot tips, axillary shoot buds, embryogenic calli, and protocom-like bodies are potential micropropagules that have been considered for creating synthetic seeds. In the present study, 3–5 mm microshoots of Oryza sativa L. Cv. MRQ 74 were used as explant sources for obtaining synthetic seeds. Microshoots were induced from stem explants on Murashige and Skoog (MS) medium supplemented with 1.5 mg/L benzylaminopurine (BAP). They were encapsulated in 3% (w/v) sodium alginate, 3% sucrose, 0.1 mg/L BAP, and 0.1 mg/L α-Naphthalene acetic acid (NAA). Germination and plantlet regeneration of the encapsulated seeds were tested by culturing them on various germination media. The effect of storage period (15–30 days) was also investigated. The maximum germination and plantlet regeneration (100.0%) were recorded on MS media containing 3% sucrose and 0.8% agar with and without 0.1 mg/L BAP. However, a low germination rate (6.67%) was obtained using top soil as a sowing substrate. The germination rate of the encapsulated microshoots decreased from 93.33% to 3.33% after 30 days of storage at 4°C in the dark. Therefore, further research is being done to improve the germination rate of the synthetic seeds.
PMCID: PMC3419424  PMID: 22919338
22.  Improvement of somatic embryogenesis and plantlet conversion in Oplopanax elatus, an endangered medicinal woody plant 
SpringerPlus  2013;2:428.
Oplopanax elatus is a medicinal plant on the verge of extinction because of overexploitation. In the present study, the effects of various factors on enhancing somatic embryogenesis and plantlet conversion were studied. Mature seeds were collected from a total of 13 plants from 4 mountains in South Korea, and the genetic distances were calculated to analyze the effect of genotype on somatic embryogenesis. Results of cluster analysis and the unweighted-pair-group method with arithmetic mean of 13 genotypes indicated the presence of 3 main groups. Both genotype and explant type affected the induction of somatic embryos (SEs). Sorak 2 and root were found to be the most suitable genotype and explant type, respectively, for SE induction in O. elatus. Among the different types of carbon sources tested, 5% sucrose induced the maximum number of SEs. The formation and development of SEs were significantly influenced by culture density; thus, 10 mg embryonic callus was found to be the most suitable for SE induction. The highest rates of germination and SE conversion were obtained in a germination medium containing 1.8 gelrite and 3.2 g·l-1 agar. In addition, 80% of the plantlets that were transplanted into artificial soil acclimatized successfully. Thus, our results showed that the percentage survival of O. elatus during in vitro proliferation could be increased by optimizing to the somatic embryogenesis system.
PMCID: PMC3766505  PMID: 24024109
Micropropagation; Oplopanax elatus; Conservation; Somatic embryogenesis; Regeneration
23.  Alginate Encapsulation of Begonia Microshoots for Short-Term Storage and Distribution 
The Scientific World Journal  2013;2013:341568.
Synthetic seeds were formed from shoot tips of two in vitro grown Begonia cultivars using 3% sodium alginate in Murashige and Skoog medium (MS) salt solution as the gel matrix and 100 mM calcium chloride for complexation. Synthetic seed formation was achieved by releasing the sodium alginate/explant combination into 100 mM calcium chloride (CaCl2·H2O) solution for 30 or 45 min. Both control and encapsulated shoots were transferred into sterile Petri dishes and stored at 4°C or 22°C for 0, 2, 4, 6, or 8 weeks. Conversion of synthetic seeds into plantlets for both storage environments was assessed in MS medium or peat-based substrate. No significant difference was found between the 30 and 45 min CaCl2·H2O treatments or the two cultivars. Encapsulation of explants improved survival rate over time irrespective of the medium type or storage environment. Survival rates of 88, 53, 28, and 11% for encapsulated microshoots versus 73, 13, 0, and 0% for control explants were achieved in microshoots stored for 2, 4, 6, and 8 weeks, respectively. The best results were obtained when synthetic seeds were stored at 4°C and germinated on MS medium. Regenerated plantlets were successfully established in potting soil.
PMCID: PMC3875128  PMID: 24396296
24.  Role of growth regulators on in vitro regeneration and histological analysis in Indian ginseng (Withania somnifera L.) Dunal 
An efficient, rapid and improved in vitro plant regeneration protocol has been established for Withania somnifera L. using shoot tip and nodal explants, excised from 15 days old aseptic seedlings. A range of cytokinins were investigated for multiple shoot regeneration. Of the three cytokinins, 6-benzyladenine (BA), Kinetin (Kin) and 2-isopentenyl adenine (2-iP) evaluated as supplement to Murashige and Skoog (MS) medium, BA at an optimal concentration of 2.5 μM was most effective in proliferating apical and axillary buds. The highest regeneration frequency (95 %) and number of shoots (36.1 ± 0.33) were obtained on MS medium fortified with BA (2.5 μM) and NAA (0.5 μM) from nodal segments. High frequency of rooting (100 %) was obtained in in vitro raised shoots when transferred to half-strength MS medium supplemented with NAA (0.5 μM). Histological sections revealed that additional shoot bud primordia were differentiated within the explants just underneath the suberized cells which appeared to be arrested in their development. The presence of additional bud primordia within the explants is thereby helpful to maximize the potential of this system. The regenerated plantlets with well developed shoots and roots were hardened successfully, established in earthen pots containing garden soil and maintained in greenhouse with 95 % survival rate.
PMCID: PMC3550525  PMID: 23573041
Withania somnifera; Multiple shoots; Shoot tips; Nodal explants; Plant growth regulators; Histology
25.  Germination Response of MR 219 Rice Variety to Different Exposure Times and Periods of 2450 MHz Microwave Frequency 
The Scientific World Journal  2013;2013:408026.
Germination is a key process in plants' phenological cycles. Accelerating this process could lead to improvment of the seedling growth as well as the cultivation efficiency. To achieve this, the effect of microwave frequency on the germination of rice seeds was examined. The physiological feedbacks of the MR 219 rice variety in terms of seed germination rate (GR), germination percentage (GP), and mean germination time (MGT) were analyzed by exposing its seeds to 2450 MHz of microwave frequency for one, four, seven, and ten hours. It was revealed that exposing the seeds to the microwave frequency for 10 hours resulted in the highest GP. This treatment led to 100% of germination after three days with a mean germination time of 2.1 days. Although the other exposure times of microwave frequency caused the moderate effects on germination with a GPa3 ranged from 93% to 98%, they failed to reduce the MGTa3. The results showed that ten-hour exposure times of microwave frequency for six days significantly facilitated and improved the germination indices (primary shoot and root length). Therefore, the technique is expected to benefit the improvement of rice seed germination considering its simplicity and efficacy in increasing the germination percentage and rate as well as the primary shoot and root length without causing any environmental toxicity.
PMCID: PMC3836452  PMID: 24307869

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