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1.  A perspective on underwater photosynthesis in submerged terrestrial wetland plants 
AoB Plants  2011;2011:plr030.
Submergence inhibits photosynthesis by terrestrial wetland plants, but less so in species that possess leaf gas films when submerged. Floodwaters are often supersaturated with dissolved CO2 enabling photosynthesis by submerged terrestrial plants, although rates remain well-below those in air. This important adaptation that enhances survival in submerged conditions is reviewed.
Background and aims
Wetland plants inhabit flood-prone areas and therefore can experience episodes of complete submergence. Submergence impedes exchange of O2 and CO2 between leaves and the environment, and light availability is also reduced. The present review examines limitations to underwater net photosynthesis (PN) by terrestrial (i.e. usually emergent) wetland plants, as compared with submerged aquatic plants, with focus on leaf traits for enhanced CO2 acquisition.
Floodwaters are variable in dissolved O2, CO2, light and temperature, and these parameters influence underwater PN and the growth and survival of submerged plants. Aquatic species possess morphological and anatomical leaf traits that reduce diffusion limitations to CO2 uptake and thus aid PN under water. Many aquatic plants also have carbon-concentrating mechanisms to increase CO2 at Rubisco. Terrestrial wetland plants generally lack the numerous beneficial leaf traits possessed by aquatic plants, so submergence markedly reduces PN. Some terrestrial species, however, produce new leaves with a thinner cuticle and higher specific leaf area, whereas others have leaves with hydrophobic surfaces so that gas films are retained when submerged; both improve CO2 entry.
Submergence inhibits PN by terrestrial wetland plants, but less so in species that produce new leaves under water or in those with leaf gas films. Leaves with a thinner cuticle, or those with gas films, have improved gas diffusion with floodwaters, so that underwater PN is enhanced. Underwater PN provides sugars and O2 to submerged plants. Floodwaters often contain dissolved CO2 above levels in equilibrium with air, enabling at least some PN by terrestrial species when submerged, although rates remain well below those in air.
PMCID: PMC3249690  PMID: 22476500
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.  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
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.  Aquatic adventitious root development in partially and completely submerged wetland plants Cotula coronopifolia and Meionectes brownii 
Annals of Botany  2012;110(2):405-414.
Background and Aims
A common response of wetland plants to flooding is the formation of aquatic adventitious roots. Observations of aquatic root growth are widespread; however, controlled studies of aquatic roots of terrestrial herbaceous species are scarce. Submergence tolerance and aquatic root growth and physiology were evaluated in two herbaceous, perennial wetland species Cotula coronopifolia and Meionectes brownii.
Plants were raised in large pots with ‘sediment’ roots in nutrient solution and then placed into individual tanks and shoots were left in air or submerged (completely or partially). The effects on growth of aquatic root removal, and of light availability to submerged plant organs, were evaluated. Responses of aquatic root porosity, chlorophyll and underwater photosynthesis, were studied.
Key Results
Both species tolerated 4 weeks of complete or partial submergence. Extensive, photosynthetically active, aquatic adventitious roots grew from submerged stems and contributed up to 90 % of the total root dry mass. When aquatic roots were pruned, completely submerged plants grew less and had lower stem and leaf chlorophyll a, as compared with controls with intact roots. Roots exposed to the lowest PAR (daily mean 4·7 ± 2·4 µmol m−2 s−1) under water contained less chlorophyll, but there was no difference in aquatic root biomass after 4 weeks, regardless of light availability in the water column (high PAR was available to all emergent shoots).
Both M. brownii and C. coronopifolia responded to submergence with growth of aquatic adventitious roots, which essentially replaced the existing sediment root system. These aquatic roots contained chlorophyll and were photosynthetically active. Removal of aquatic roots had negative effects on plant growth during partial and complete submergence.
PMCID: PMC3394642  PMID: 22419759
Adventitious roots; aquatic plants; aquatic roots; Cotula coronopifolia; flooding; Meionectes brownii; Haloragis brownii; root porosity; root photosynthesis; submergence tolerance; underwater photosynthesis; wetland plants
7.  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
8.  Recovery dynamics of growth, photosynthesis and carbohydrate accumulation after de-submergence: a comparison between two wetland plants showing escape and quiescence strategies 
Annals of Botany  2010;107(1):49-63.
Background and Aims
The capacity for fast-growth recovery after de-submergence is important for establishment of riparian species in a water-level-fluctuation zone. Recovery patterns of two wetland plants, Alternanthera philoxeroides and Hemarthria altissima, showing ‘escape’ and ‘quiescence’ responses, respectively, during submergence were investigated.
Leaf and root growth and photosynthesis were monitored continuously during 10 d of recovery following 20 d of complete submergence. Above- and below-ground dry weights, as well as carbohydrate concentrations, were measured several times during the experiment.
Key Results
Both species remobilized stored carbohydrate during submergence. Although enhanced internode elongation depleted the carbohydrate storage in A. philoxeroides during submergence, this species resumed leaf growth 3 d after de-submergence concomitant with restoration of the maximal photosynthetic capacity. In contrast, some sucrose was conserved in shoots of H. altissima during submergence, which promoted rapid re-growth of leaves 2 d after de-submergence and earlier than the full recovery of photosynthesis. The recovery of root growth was delayed by 1–2 d compared with leaves in both species.
Submergence tolerance of the escape and quiescence strategies entails not only the corresponding regulation of growth, carbohydrate catabolism and energy metabolism during submergence but also co-ordinated recovery of photosynthesis, growth and carbohydrate partitioning following de-submergence.
PMCID: PMC3002471  PMID: 21041230
Alternanthera philoxeroides; carbohydrate; flooding; Hemarthria altissima; leaf growth; root growth; shade; submergence; wetland plant
9.  Rice germination and seedling growth in the absence of oxygen 
Annals of Botany  2008;103(2):181-196.
Higher plants are aerobic organisms which suffer from the oxygen deficiency imposed by partial or total submergence. However, some plant species have developed strategies to avoid or withstand severe oxygen shortage and, in some cases, the complete absence of oxygen (tissue anoxia) for considerable periods of time.
Rice (Oryza sativa) is one of the few plant species that can tolerate prolonged soil flooding or complete submergence thanks to an array of adaptive mechanisms. These include an ability to elongate submerged shoot organs at faster than normal rates and to develop aerenchyma, allowing the efficient internal transport of oxygen from the re-emerged elongated shoot to submerged parts. However, rice seeds are able to germinate anaerobically by means of coleoptile elongation. This cannot be explained in terms of oxygen transport through an emerged shoot. This review provides an overview of anoxic rice germination that is mediated through coleoptile rather than root emergence.
Although there is still much to learn about the biochemical and molecular basis of anaerobic rice germination, the ability of rice to maintain an active fermentative metabolism (i.e. by fuelling the glycolytic pathway with readily fermentable carbohydrates) is certainly crucial. The results obtained through microarray-based transcript profiling confirm most of the previous evidence based on single-gene studies and biochemical analysis, and highlight new aspects of the molecular response of the rice coleoptile to anoxia.
PMCID: PMC2707302  PMID: 18660495
Anoxia; coleoptile; fermentative metabolism; germination; hypoxia; Oryza sativa; rice
10.  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
11.  Cellular basis of growth suppression by submergence in azuki bean epicotyls 
Annals of Botany  2008;103(2):325-332.
Background and Aims
Complete submergence severely reduces growth rate and productivity of terrestrial plants, but much remains to be elucidated regarding the mechanisms involved. The aim of this study was to clarify the cellular basis of growth suppression by submergence in stems.
The effects of submergence on the viscoelastic extensibility of the cell wall and the cellular osmotic concentration were studied in azuki bean epicotyls. Modifications by submergence to chemical properties of the cell wall; levels of osmotic solutes and their translocation from the seed to epicotyls; and apoplastic pH and levels of ATP and ethanol were also examined. These cellular events underwater were compared in etiolated and in light-grown seedlings.
Key Results
Under submergence, the osmotic concentration of the cell sap was substantially decreased via decreased concentrations of organic compounds including sugars and amino acids. In contrast, the viscoelastic extensibility of the cell wall was kept high. Submergence also decreased ATP and increased the pH of the apoplastic solution. Alcoholic fermentation was stimulated underwater, but the resulting accumulated ethanol was not directly involved in growth suppression. Light partially relieved the inhibitory effects of submergence on growth, osmoregulation and sugar translocation.
A decrease in the levels of osmotic solutes is a main cause of underwater growth suppression in azuki bean epicotyls. This may be brought about by suppression of solute uptake via breakdown of the H+ gradient across the plasma membrane due to a decrease in ATP. The involvement of cell wall properties in underwater growth suppression remains to be fully elucidated.
PMCID: PMC2707313  PMID: 18940853
Apoplastic pH; cell wall extensibility; growth suppression; osmoregulation; osmotic concentration; submergence; sugar translocation; Vigna angularis
12.  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
13.  Perspectives on MADS-box expression during orchid flower evolution and development 
The diverse morphology of orchid flowers and their complex, often deceptive strategies to become pollinated have fascinated researchers for a long time. However, it was not until the 20th century that the ontogeny of orchid flowers, the genetic basis of their morphology and the complex phylogeny of Orchidaceae were investigated. In parallel, the improvement of techniques for in vitro seed germination and tissue culture, together with studies on biochemistry, physiology, and cytology supported the progress of what is now a highly productive industry of orchid breeding and propagation. In the present century both basic research in orchid flower evo-devo and the interest for generating novel horticultural varieties have driven the characterization of many members of the MADS-box family encoding key regulators of flower development. This perspective summarizes the picture emerging from these studies and discusses the advantages and limitations of the comparative strategy employed so far. I address the growing role of natural and horticultural mutants in these studies and the emergence of several model species in orchid evo-devo and genomics. In this context, I make a plea for an increasingly integrative approach.
PMCID: PMC3779858  PMID: 24065980
Orchidaceae; evo-devo; MADS-box gene; peloric mutant; gene family; transcriptome; model species
14.  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
15.  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
16.  Photosynthetic acclimation is important for post-submergence recovery of photosynthesis and growth in two riparian species 
Annals of Botany  2009;104(7):1435-1444.
Background and Aims
Concomitant increases in O2 and irradiance upon de-submergence can cause photoinhibition and photo-oxidative damage to the photosynthetic apparatus of plants. As energy and carbohydrate supply from photosynthesis is needed for growth, it was hypothesized that post-submergence growth recovery may require efficient photosynthetic acclimation to increased O2 and irradiance to minimize photo-oxidative damage. The hypothesis was tested in two flood-tolerant species: a C3 herb, Alternanthera philoxeroides; and a C4 grass, Hemarthria altissima. The impact of low O2 and low light, typical conditions in turbid floodwater, on post-submergence recovery was assessed by different flooding treatments combined with shading.
Experiments were conducted during 30 d of flooding (waterlogging or submergence) with or without shading and subsequent recovery of 20 d under growth conditions. Changes in dry mass, number of branches/tillers, and length of the longest internodes and main stems were recorded to characterize growth responses. Photosynthetic parameters (photosystem II efficiency and non-photochemical quenching) were determined in mature leaves based on chlorophyll a fluorescence measurements.
Key Results
In both species growth and photosynthesis recovered after the end of the submergence treatment, with recovery of photosynthesis (starting shortly after de-submergence) preceding recovery of growth (pronounced on days 40–50). The effective quantum yield of photosystem II and non-photochemical quenching were diminished during submergence but rapidly increased upon de-submergence. Similar changes were found in all shaded plants, with or without flooding. Submerged plants did not suffer from photoinhibition throughout the recovery period although their growth recovery was retarded.
After sudden de-submergence the C3 plant A. philoxeroides and the C4 plant H. altissima were both able to maintain the functionality of the photosynthetic apparatus through rapid acclimation to changing O2 and light conditions. The ability for photosynthetic acclimation may be essential for adaptation to wetland habitats in which water levels fluctuate.
PMCID: PMC2778401  PMID: 19854720
Aerenchyma; Alternanthera philoxeroides; flooding; growth; Hemarthria altissima; low light; photosynthesis; shade; submergence; waterlogging; wetland plant
17.  Molecular Adaptation of rbcL in the Heterophyllous Aquatic Plant Potamogeton 
PLoS ONE  2009;4(2):e4633.
Heterophyllous aquatic plants show marked phenotypic plasticity. They adapt to environmental changes by producing different leaf types: submerged, floating and terrestrial leaves. By contrast, homophyllous plants produce only submerged leaves and grow entirely underwater. Heterophylly and submerged homophylly evolved under selective pressure modifying the species-specific optima for photosynthesis, but little is known about the evolutionary outcome of habit. Recent evolutionary analyses suggested that rbcL, a chloroplast gene that encodes a catalytic subunit of RuBisCO, evolves under positive selection in most land plant lineages. To examine the adaptive evolutionary process linked to heterophylly or homophylly, we analyzed positive selection in the rbcL sequences of ecologically diverse aquatic plants, Japanese Potamogeton.
Principal Findings
Phylogenetic and maximum likelihood analyses of codon substitution models indicated that Potamogeton rbcL has evolved under positive Darwinian selection. The positive selection has operated specifically in heterophyllous lineages but not in homophyllous ones in the branch-site models. This suggests that the selective pressure on this chloroplast gene was higher for heterophyllous lineages than for homophyllous lineages. The replacement of 12 amino acids occurred at structurally important sites in the quaternary structure of RbcL, two of which (residue 225 and 281) were identified as potentially under positive selection.
Our analysis did not show an exact relationship between the amino acid replacements and heterophylly or homophylly but revealed that lineage-specific positive selection acted on the Potamogeton rbcL. The contrasting ecological conditions between heterophyllous and homophyllous plants have imposed different selective pressures on the photosynthetic system. The increased amino acid replacement in RbcL may reflect the continuous fine-tuning of RuBisCO under varying ecological conditions.
PMCID: PMC2646136  PMID: 19247501
18.  Effect of Light on Seed Germination of Eight Wetland Carex Species 
Annals of Botany  2006;98(4):869-874.
• Background and Aims In wetland plant communities, species-specific responses to pulses of white light and to red : far-red light ratios can vary widely and influence plant emergence from the seed bank. Carex species are the characteristic plants of sedge meadows of natural prairie wetlands in mid-continental USA but are not returning to restored wetlands. Little is known about how light affects seed germination in these species—information which is necessary to predict seed bank emergence and to develop optimal revegetation practices. The effects of light on germination in eight Carex species from prairie wetlands were investigated.
• Methods Non-dormant seeds of eight Carex species were used to determine the influence of light on germination by examining: (a) the ability of Carex seeds to germinate in the dark; (b) the effect of different lengths of exposures to white light on germination; (c) whether the effect of white light can be replaced by red light; and (d) whether the germination response of Carex seeds to white or red light is photoreversible by far-red light.
• Key Results Seeds of C. brevior and C. stipata germinated >25 % in continuous darkness. Germination responses after exposure to different lengths of white light varied widely across the eight species. Carex brevior required <15 min of white light for ≥50 % germination, while C. hystericina, C. comosa, C. granularis and C. vulpinoidea required ≥8 h. The effect of white light was replaced by red light in all species. The induction of germination after exposure to white or red light was reversed by far-red light in all species, except C. stipata.
• Conclusions The species-specific responses to simulated field light conditions suggest that (a) the light requirements for germination contribute to the formation of persistent seed banks in these species and (b) in revegetation efforts, timing seed sowing to plant community development and avoiding cover crops will improve Carex seed germination.
PMCID: PMC2806167  PMID: 16905568
Carex; far-red light; seed germination ecology; photomorphogenesis; phytochrome; prairie wetland; red light; sedge; white light
19.  Novel Seed Adaptations of a Monocotyledon Seagrass in the Wavy Sea 
PLoS ONE  2013;8(9):e74143.
Returning to the sea, just like invasion of land, has occurred in many groups of animals and plants. For flowering plants, traits adapted to the terrestrial environments have to change or adopt a new function to allow the plants to survive and prosper in the sea where water motion tends to rotate and move seeds. In this investigation, how seeds of the seagrass Thalassia hemprichii (Hydrocharitaceae), a common monocotyledon in the Indo-Pacific, adapt to the wavy environment was studied. Mature seeds were collected from Dongsha Atoll in South China Sea. The effects of light qualities on seed germination, the seed morphology, the unipolar distribution of starch granules in the endosperms and growth of root hair-like filamentous cells from basal surface of the seeds were all found to differ from those of terrestrial monocotyledons. Physiologically, germination of the seeds was stimulated by blue light rather than red light. Morphologically, the bell-shaped seeds coupled with the unipolar distribution of starch granules in the enlarged bases helped maintain their upright posture on the tidal seafloor. Growth of root hair-like filamentous cells from the basal surface of the seeds prior to primary root growth served to attach onto sediments, providing leverage and attachment required by the primary roots to insert into sediments. These filamentous cells grasped coral sand but not silicate sand, demonstrating a habitat preference of this species.
PMCID: PMC3767646  PMID: 24040188
20.  Gibberellic acid-mediated salt signaling in seed germination 
Plant Signaling & Behavior  2008;3(10):877-879.
Seed germination initiates the postembryonic development of plants, which determines successful seedling establishment and plant propagation. It is therefore tightly regulated by diverse environmental conditions, including high salinity and drought, as well as by intrinsic developmental programs, among which gibberellic acid (GA) is best understood. Regulatory roles of GA in seed germination have been extensively studied. It is also known that high salinity inhibits germination by repressing genes encoding GA biosynthetic enzymes. However, it is still unclear how salt signals are coordinately incorporated into the GA signaling pathway at the molecular level. We recently demonstrated that a membrane-bound NAC transcription factor, NTL8, mediates salt signaling, primarily through a RGL2-independent GA pathway, in regulating seed germination. High salinity promotes NTL8 transcription and proteolytic activation of NTL8. Notably, the NTL8-mediated salt signaling is independent of abscisic acid (ABA). These observations indicate that membrane-mediated transcription control is an important component of salt signaling during seed germination.
PMCID: PMC2634403  PMID: 19704528
Arabidopsis; gibberellic acid (GA); salt stress; membrane-bound transcription factor; NAC; NTL8; RGL2
21.  Analysis of ADP-glucose pyrophosphorylase expression during turion formation induced by abscisic acid in Spirodela polyrhiza (greater duckweed) 
BMC Plant Biology  2012;12:5.
Aquatic plants differ in their development from terrestrial plants in their morphology and physiology, but little is known about the molecular basis of the major phases of their life cycle. Interestingly, in place of seeds of terrestrial plants their dormant phase is represented by turions, which circumvents sexual reproduction. However, like seeds turions provide energy storage for starting the next growing season.
To begin a characterization of the transition from the growth to the dormant phase we used abscisic acid (ABA), a plant hormone, to induce controlled turion formation in Spirodela polyrhiza and investigated their differentiation from fronds, representing their growth phase, into turions with respect to morphological, ultra-structural characteristics, and starch content. Turions were rich in anthocyanin pigmentation and had a density that submerged them to the bottom of liquid medium. Transmission electron microscopy (TEM) of turions showed in comparison to fronds shrunken vacuoles, smaller intercellular space, and abundant starch granules surrounded by thylakoid membranes. Turions accumulated more than 60% starch in dry mass after two weeks of ABA treatment. To further understand the mechanism of the developmental switch from fronds to turions, we cloned and sequenced the genes of three large-subunit ADP-glucose pyrophosphorylases (APLs). All three putative protein and exon sequences were conserved, but the corresponding genomic sequences were extremely variable mainly due to the invasion of miniature inverted-repeat transposable elements (MITEs) into introns. A molecular three-dimensional model of the SpAPLs was consistent with their regulatory mechanism in the interaction with the substrate (ATP) and allosteric activator (3-PGA) to permit conformational changes of its structure. Gene expression analysis revealed that each gene was associated with distinct temporal expression during turion formation. APL2 and APL3 were highly expressed in earlier stages of turion development, while APL1 expression was reduced throughout turion development.
These results suggest that the differential expression of APLs could be used to enhance energy flow from photosynthesis to storage of carbon in aquatic plants, making duckweeds a useful alternative biofuel feedstock.
PMCID: PMC3268088  PMID: 22235974
Duckweed; Spirodela; Starch; Turion; ADP-glucose pyrophosphorylase
22.  Atractiellomycetes belonging to the ‘rust’ lineage (Pucciniomycotina) form mycorrhizae with terrestrial and epiphytic neotropical orchids 
Distinctive groups of fungi are involved in the diverse mycorrhizal associations of land plants. All previously known mycorrhiza-forming Basidiomycota associated with trees, ericads, liverworts or orchids are hosted in Agaricomycetes, Agaricomycotina. Here we demonstrate for the first time that Atractiellomycetes, members of the ‘rust’ lineage (Pucciniomycotina), are mycobionts of orchids. The mycobionts of 103 terrestrial and epiphytic orchid individuals, sampled in the tropical mountain rainforest of Southern Ecuador, were identified by sequencing the whole ITS1-5.8S-ITS2 region and part of 28S rDNA. Mycorrhizae of 13 orchid individuals were investigated by transmission electron microscopy. Simple septal pores and symplechosomes in the hyphal coils of mycorrhizae from four orchid individuals indicated members of Atractiellomycetes. Molecular phylogeny of sequences from mycobionts of 32 orchid individuals out of 103 samples confirmed Atractiellomycetes and the placement in Pucciniomycotina, previously known to comprise only parasitic and saprophytic fungi. Thus, our finding reveals these fungi, frequently associated to neotropical orchids, as the most basal living basidiomycetes involved in mycorrhizal associations of land plants.
PMCID: PMC2842812  PMID: 20007181
Orchid mycorrhiza; simple-septate Basidiomycota; Pucciniomycotina; Atractiellales; Helicogloea; neotropical mountain rainforest
23.  In vitro propagation of an endangered medicinal herb Chlorophytum borivilianum Sant. et Fernand. through somatic embryogenesis 
Tuberous roots of Chlorophytum borivilianum Sant. et Fernand. which are a source of steroidal saponins, possess immunomodulatory, adaptogenic, aphrodisiac, antipyretic, diuretic, hemostatic and anti-tumour properties. Poor seed setting and germination and slow growth in conventional vegetative propagation are major constraints in the large-scale cultivation of this commercially important medicinal plant. In the present study, a procedure for in vitro propagation of this endangered herb through somatic embryogenesis has been established. Seeds of Chlorophytum borivilianum were germinated on MS medium supplemented with 57.74 μM gibberellic acid and hypocotyl portion from germinated seedling was used as explant for callus induction. Moderate to good callus induction was observed on MS medium containing 1.16 μM kinetin and 1.13–2.26 μM 2,4-dichlorophenoxyacetic acid. Regular subculturing of callus on kinetin (1.16 μM) and 2,4-dichlorophenoxyacetic acid (1.13 μM) supplemented medium induced somatic embryogenesis. In modified MS medium, 1.79 mM NH4NO3 and 10.72 mM KNO3 was optimal for somatic embryogenesis. 7.38 μM 2-isopentenyladenine supplemented to modified MS medium, showed best response for somatic embryogenesis while proline (0.76 mM) as an amino acid supplement gave better response than glutamine. 30% germination of mature somatic embryos was achieved on MS medium supplemented with 15.54 μM 6-benzylaminopurine. Multiplication of C. borivilianum through somatic embryogenesis may offer a better approach compared to organogenesis for developing scale-up technology employing bioreactors for its mass propagation.
PMCID: PMC3550677  PMID: 23572975
Liliaceae; Safed musli; Saponins; Somatic embryogenesis; Proline
The Journal of Cell Biology  1970;46(3):435-454.
Structural changes in endosperm cells of germinating castor beans were examined and complemented with a cytochemical analysis of staining with diaminobenzidine (DAB). Deposition of oxidized DAB occurred only in microbodies due to the presence of catalase, and in cell walls associated with peroxidase activity. Seedling development paralleled the disappearance of spherosomes (lipid bodies) and matrix of aleurone grains in endosperm cells. 6 to 7 days after germination, a cross-section through the endosperm contained cells in all stages of development and senescence beginning at the seed coat and progressing inward to the cotyledons. Part of this aging process involved vacuole formation by fusion of aleurone grain membranes. This coincided with an increase in microbodies (glyoxsomes), mitochondria, plastids with an elaborate tubular network, and the formation of a new protein body referred to as a dilated cisterna, which is structurally and biochemically distinct from microbodies although both apparently develop from rough endoplasmic reticulum (ER). In vacuolate cells microbodies are the most numerous organelle and are intimately associated with spherosomes and dilated cisternae. This phenomenon is discussed in relation to the biochemical activities of these organelles. Turnover of microbodies involves sequestration into autophagic vacuoles as intact organelles which still retain catalase activity. Crystalloids present in microbodies develop by condensation of matrix protein and are the principal site of catalase formerly in the matrix.
PMCID: PMC2107873  PMID: 4121486
25.  The role of carbohydrates in seed germination and seedling establishment of Himatanthus sucuuba, an Amazonian tree with populations adapted to flooded and non-flooded conditions 
Annals of Botany  2009;104(6):1111-1119.
Background and Aims
In the Amazonian floodplains plants withstand annual periods of flooding which can last 7 months. Under these conditions seedlings remain submerged in the dark for long periods since light penetration in the water is limited. Himatanthus sucuuba is a tree species found in the ‘várzea’ (VZ) floodplains and adjacent non-flooded ‘terra-firme’ (TF) forests. Biochemical traits which enhance flood tolerance and colonization success of H. sucuuba in periodically flooded environments were investigated.
Storage carbohydrates of seeds of VZ and TF populations were extracted and analysed by HPAEC/PAD. Starch was analysed by enzyme (glucoamylase) degradation followed by quantification of glucose oxidase. Carbohydrate composition of roots of VZ and TF seedlings was studied after experimental exposure to a 15-d period of submersion in light versus darkness.
Key Results
The endosperm contains a large proportion of the seed reserves, raffinose being the main non-structural carbohydrate. Around 93 % of the cell wall storage polysaccharides (percentage dry weight basis) in the endosperm of VZ seeds was composed of mannose, while soluble sugars accounted for 2·5%. In contrast, 74 % of the endosperm in TF seeds was composed of galactomannans, while 22 % of the endosperm was soluble sugars. This suggested a larger carbohydrate allocation to germination in TF populations whereas VZ populations allocate comparatively more to carbohydrates mobilized during seedling development. The concentration of root non-structural carbohydrates in non-flooded seedlings strongly decreased after a 15-d period of darkness, whereas flooded seedlings were less affected. These effects were more pronounced in TF seedlings, which showed significantly lower root non-structural carbohydrate concentrations.
There seem to be metabolic adjustments in VZ but not TF seedlings that lead to adaptation to the combined stresses of darkness and flooding. This seems to be important for the survival of the species in these contrasting environments, leading these populations to different directions during evolution.
PMCID: PMC2766203  PMID: 19770164
Carbohydrate reserves; Himatanthus sucuuba; submergence tolerance; floodplains; galactomannans; raffinose; population differentiation; Amazon; storage

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