Ecological studies on five species of endangered Himalayan poppy, Meconopsis (Papaveraceae)

Ecological studies were carried out on 19 endangered populations of Meconopsis horridula, M. paniculata, M. simplicfolia, M. sinuata and M. villosa from the Sikkim Himalayas. The populations of M. horridula, M. paniculata and M. sinuata inhabit open alpine slopes, those of M. simplicfolia are restricted to alpine meadows, while those of M. villosa are confined to the interiors of temperate forests and along forest fringes. The habitats of these species remain snow-hound from October to February. Analyses suggest that all the species except M. horridula , have a broad area of distribution, but narrow habitats and niches. All five species are confined to moist, clayey to silty soils with acidic pH, high concentrations of organic matter, nitrate, phosphate, potassium and calcium. M. paniculata is a 'generalist', whereas all other species investigated are 'specialists'.     

Random amplified polymorphic DNA (RAPD) markers reveal genetic homogeneity in the endangered Himalayan species Meconopsis paniculata and M. simplicifolia

Random amplified polymorphic DNA (RAPD) marker-based analysis was carried out to study the extent of genetic polymorphism between populations of the two endangered Himalayan poppy species, Meconopsis paniculata and M. Simplicifolia. Of the 90 primers tested, 38 revealed marked inter-species genetic polymorphism between individuals of the two species from geographically isolated populations. However, intra-species genetic homogeneity was also evident with respect to a number of primers both within and between populations. A comprehensive analysis incorporating data from RAPDs, DNA fingerprinting and isozyme pattern was carried out and, based on the presence or absence of bands, three matrices of similarity indices were estimated. These matrices were subsequently utilized in cluster analysis. In order to compare the three clusters generated using these three different marker systems, a Mantel matrix-correspondence test was carried out on the basis of comparisons of co-phenetic values. The overall representation of relationships by cluster analysis was similar for all three marker systems and this was substantiated by high correlations among the three analyses revealed by the Mantel matrix-correspondence test. Our results point to very low or absence of, genetic polymorphism in M. paniculata and M. simplicifolia, and are in broad agreement with our previous observations on genetic diversity of Meconopsis species which point to a genetic basis for the possible extinction of this economically important genus.     

Seed Coat Dormancy in Two Species of Grevillea(Proteaceae)

The role played by the seed coat in seed dormancy of Grevillealinearifolia(Cav.) Druce and G. wilsonii(A. Cunn.) was testedby a series of manipulations in which the seed coat was dissectedand removed, dissected and returned to the decoated seed, ordissected, removed and given a heat shock, and returned to thedecoated seed. Germination of intact seeds of both species wasalso examined after exposure to heat shock, smoke, or heat shockand smoke combined. Water permeability of the seed coat wasinvestigated by examining imbibition. For intact seeds, virtuallyno germination occurred under any treatment (G. wilsonii), orgermination was increased by exposure to either heat or smoke(G. linearifolia). Removal of the seed coat led to germinationof all decoated seeds for G. linearifolia, or a proportion ofdecoated seeds for G. wilsonii. Inclusion of smoked water inthe incubation medium led to a higher proportion of decoatedseeds germinating for G. wilsonii. Returning the seed coat,either with or without heat shock to the seed coat, did notsignificantly affect germination in either species. Seed coatswere permeable to water in both species. For the two Grevilleaspecies, there were different dormancy mechanisms that werecontrolled by the seed coat (G. linearifolia) or by both theseed coat and embryo (G. wilsonii). Copyright 2000 Annals ofBotany Company Grevillea linearifolia, Grevillea wilsonii, dormancy, seed coat dormancy, seed coat permeability, smoke, heat shock, germination     

Seed Coat Microsculpturing in Brassica and Allied Genera (Subtribes Brassicinae, Raphaninae, Moricandiinae)

To obtain new information on the taxonomy of Brassica and alliedgenera, seed surfaces of 44 species (78 accessions) belongingto 11 genera of subtribes Brassicinae, Raphaninae and Moricandiinaewere examined using a scanning electron microscope. Ten typesof basic ornamentation patterns were recognized. While fourtypes were represented by only one species each, six types hadmore than one species either representing one genus (two types)or more (four types). The different species in each of the sixtypes could generally be distinguished from each other on thebasis of differences in microsculpturing features. This analysisprovides evidence for the close relationship among the variousgenera within the subtribe Brassicinae, and also the closenessof Raphanus, Enarthrocarpus and Moricandia of subtribe Raphaninaeand Moricandiinae, respectively, with the Brassicinae. Copyright2000 Annals of Botany Company Brassicinae, Moricandiinae, Raphaninae, SEM, seed coat microsculpturing, taxonomic implications     

Seed Surface Architecture and Random Amplified Polymorphic DNA Profiles of Paulownia fortunei, P. tomentosa and their Hybrid

The surface patterns of winged seeds of Paulownia fortunei,P. tomentosa and P. fortuneixP. tomentosa were examined by scanningelectron microscopy. The pattern of reticulation on the wingsand seed coat of P. fortunei and the hybrid are comparable,while that on P. tomentosa is different and more elongated.Also, the wings are more extended at the oblong ends of theseeds in the former when compared to the wings of P. tomentosa.Distinct random amplified polymorphic DNA (RAPD) patterns wereobtained for the three taxa and P. kawakamii with five differentrandom oligonucleotide primers, suggesting that the method canyield genetic markers for differentiating the taxa. Also, Southernblot analyses of the RAPD products of the hybrid and the twoparent species revealed shared (inherited) genetic polymorphisms.Copyright 1999 Annals of Botany Company Paulownia species and hybrid, seed surface architecture, reticulated thickening, RAPD markers, Scrophulariaceae.     

Characterization of 13 microsatellite loci developed from Meconopsis horridula

Meconopsis horridula is one of the eight most famous flowers in Chinese province of Yunnan. In this study, a modified biotin-streptavidin capture method was used to detect 13 microsatellite markers in the genome of M. horridula. The polymorphism of each locus was assessed in 24 samples collected from four populations. The number of alleles per locus ranged from 2 to 7 (mean: 3.2). The observed and expected heterozygosities ranged from 0.0833 to 0.9167 and 0.0816 to 0.8050, respectively. Additionally, nine of the 13 microsatellite markers were successfully amplified in three other congeneric species. These polymorphic SSR markers could be useful for studying the population genetics of M. horridula and for assessing genetic variation in this and congenerc species in conservation programs.     

Structural Diversity and its Adaptive Significancein Seeds of Vigna minima (Roxb.) Ohwi Ohashi and its Allies (Leguminosae-Papilionoideae)

The surface features of seed coat, hilum and strophiole, histologicalaspects of hilum and strophiole, and mechanism of seed dormancyinVigna minima, V. umbellata, V. dalzelliana and V. calcarataauct. pi. have been investigated. Scanning electron microscopyhas revealed a substantial variability in seed coat microtopography,hilum and strophiole, which is of taxonomic and evolutionarysignificance. The histology of the hilum is uniform in all fourtaxa, but the structure of the strophiole shows differencesbetween wild and cultivated species. All four taxa have hardseeds; those ofV. umbellata have no dormancy but those of wildspecies have water-impermeable dormancy. Seed germination experimentsusing araldite-coated, and hilum-micropyle- and strophiole-pluggedhard seeds of V. umbellata and water-impermeable seeds of V.minima, which were pin-pricked through the strophiolar groove,demonstrate that the zone of weakness to the permeability ofwater in hard seeds of these species is the strophiolar region,and not the seed coat, hilum or micropyle. The parenchymatoustissue of the strophiolar plug in the seeds ofV. umbellataisassociated with the lack of dormancy, whereas the sclerenchymatoustissue of the strophiolar plug in the seeds ofV. minima is responsiblefor the water-impermeable seed dormancy. Under natural conditions,the water-impermeable dormancy of wild species breaks down asa result of the degradation of the hard seed coat, by microbialaction, at the strophiolar groove.     

Molecular Phylogeny of Asian Meconopsis Based on Nuclear Ribosomal and Chloroplast DNA Sequence Data

The taxonomy and phylogeny of Asian Meconopsis (Himalayan blue poppy) remain largely unresolved. We used the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA) and the chloroplast DNA (cpDNA) trnL-F region for phylogenetic reconstruction of Meconopsis and its close relatives Papaver, Roemeria, and Stylomecon. We identified five main clades, which were well-supported in the gene trees reconstructed with the nrDNA ITS and cpDNA trnL-F sequences. We found that 41 species of Asian Meconopsis did not constitute a monophyletic clade, but formed two solid clades (I and V) separated in the phylogenetic tree by three clades (II, III and IV) of Papaver and its allies. Clade V includes only four Asian Meconopsis species, with the remaining 90 percent of Asian species included in clade I. In this core Asian Meconopsis clade, five subclades (Ia–Ie) were recognized in the nrDNA ITS tree. Three species (Meconopsis discigera, M. pinnatifolia, and M. torquata) of subgenus Discogyne were imbedded in subclade Ia, indicating that the present definition of subgenera in Meconopsis should be rejected. These subclades are inconsistent with any series or sections of the present classifications, suggesting that classifications of the genus should be completely revised. Finally, proposals for further revision of the genus Meconopsis were put forward based on molecular, morphological, and biogeographical evidences.     

Ecological Consequences of Altering the Timing Mechanism for Metamorphosis in Anural Ascidians

SYNOPSIS. In the present study the timing of metamorphosis inan anural ascidian, Molgula pacifica, was compared to metamorphosisin a urodele species Boltenia villosa. Metamorphosis in M. pacificawas triggered at a fixed time in development (32–36 hoursafter fertilization), just prior to hatching. In contrast, metamorphosiswas triggered in B. villosa after the hatched larvae respondedto substrate cues. The timing of metamorphosisin B. villosawas often delayed for up to four days, whereas delays in M.pacifica were not observed. An antibody, termed Epi-3, was foundto cross-react exclusively with epidermal cells in both species.The binding of FITC-labelled Epi-3 was very low prior to metamorphosisand then it increased dramatically after metamorphosis was triggered.The cytoplasm of ampulla tip cells and the tunic immediatelysurrounding each ampulla showed the highest levels of Epi-3fluorescence. The histological and ultrastructural featuresof the ampulla cells suggest that Epi-3 antibody recognizesgranules localized in the apical cytoplasm. How the evolution of an internal "clock" mechanism responsiblefor initiating metamorphosis may be beneficial to anural speciesis discussed. One possibility is that the anural type of timingmechanism reduces mortality rates during this critical phaseof its life cycle.     

Impact of cytomixis on meiosis, pollen viability and pollen size in wild populations of Himalayan poppy (Meconopsis aculeata Royle)

We report the occurrence of cytomixis in wild populations of Himalayan poppy (Meconopsis aculeata Royle), which is considered to be an important and threatened medicinal plant growing in the high hills of the Himalayas. The impact of cytomixis on meiotic behaviour, reduced pollen viability and heterogeneous-sized pollen grains was also studied. Cytological studies in the seven wild populations from the high hills of Himachal Pradesh revealed that all the Himalayan populations exist uniformly at the tetraploid level (2n=56) on x=14. The phenomenon of chromatin transfer among the proximate pollen mother cells (PMCs) in six populations caused various meiotic abnormalities. Chromatin transfer also resulted in the formation of coenocytes, aneuploid, polyploid and anucleated PMCs. Among individuals that showed chromatin transfer, chromosome stickiness and interbivalent connections were frequently observed in some PMCs. The phenomenon of cytomixis in the species seems to be directly under genetic control; it affects the meiotic course considerably and results in reduced pollen viability.     

SYSTEMATIC IMPLICATIONS OF SEED COAT MORPHOLOGY IN NAMA (HYDROPHYLLACEAE)

Seeds from 37 species of Nama (Hydrophyllaceae) were examined by scanning electron microscopy in order to assess systematic implications of seed coat structure. Generally, seed coat morphology is species specific. Nevertheless, similarities among species in seed coat organization, particularly, outer testa anatomy and wall thickening-pitting patterns allow the recognition of six groups among examined taxa; furthermore, seed coat features often suggest both intra-group and inter-group relationships. Recognized groupings do not correspond well with the more classicial treatments of Nama; rather, they suggest that a restructuring of the infrageneric and even the generic taxonomy of Nama is in order.     

SEED COAT MORPHOLOGY IN CORDYLANTHUS (SCROPHULARIACEAE) AND ITS TAXONOMIC SIGNIFICANCE

A study of seed coat sculpturing in Cordylanthus (Scrophulariaceae—Rhinantheae) using the scanning electron microscope (SEM) shows that seed surface patterns are characteristic and constant for a given species or a group of species. Seeds of 23 species were examined and classified into four types (irregularly crested, deeply reticulate, shallowly reticulate, and irregularly striate) based on differences in the reticulated seed coat. Anatomical studies of sections with the light microscope show that the reticular patterns result from enlargement of the epidermal cells of the integument, followed by formation of characteristic wall patterns through lignification. Seed characters furnish useful data for formulating the taxonomy of Cordylanthus both on the sectional and subsectional level and for the delimitation of certain species. Seed coat morphology offers evidence for including the genus Dicranostegia in Cordylanthus and for maintaining C. hispidus and C. palmatus as separate species. The distinctness of two seed coat types within section Cordylanthus suggests two major phylogenetic lines within this section.     

Differentiation of a Functional Aleurone Layer Within the Seed Coat of Sinapis alba L.

The hitherto unresolved ontogenetic origin of the aleurone layerin mustard (Sinapis alba L.) seeds was investigated with lightand electron microscopy. Contrary to previous views, this layerof storage cells is neither derived from the endosperm nor fromthe nucellus, but from a particular cell layer within the innerintegument of the seed coat. These cells differentiate and becomefilled with storage protein and fat concurrently with the maturationof the embryo. They survive seed desiccation and become depletedof storage materials during seed germination. Temporally correlatedwith the germinating embryo, the aleurone cells produce microbodyenzymes, which are controlled by light in a similar fashionin both tissues. Sinapis alba L., mustard, aleurone layer, seed coat, seed formation, germination     

Silica and Ash in Seeds of Cultivated Grains and Native Plants

Silica and ash contents and the depositional patterns of opalinesilica have been determined in the seeds of 31 plant species.Included were 13 monocotyledons, eight dicotyledons and theseeds of eight common cereal grains. The cereal grains, exceptfor Oryza sativa L. (3.2%) and Avena sativa L. (1.4%), werequite low in silica. The silica in these cereals was in thelemma. In seeds with high silica content it often makes up morethan 50% of the ash. Silica in seeds occurs largely in the outercoating of the seed. Dicotyledon seeds tend to have less silicathan those of monocotyledons. Energy-dispersive X-ray analysisshows that the distribution of the element silicon is clearlyrelated to certain epidermal structures such as ridges, raisedareas, trichomes and hairs. It also occurs in cell walls. Membersof a specific plant family tend to have very similar silicadepositional patterns in their seeds. Small amounts of K, S,Cl and Ca are also found in seeds. Light-microscopy studiesshow that the silica in the lemma of seeds such as Oryza sativaL. is deposited in cellular sheet-like structures with crenateedges. Silica in seeds also occurs in fibres and in other cellularstructures (silica cells) that become phytoliths. Seeds, epidermis, seed coat, silica and ash content, scanning electron microscopy, energy-dispersive X-ray analysis, silica depositional patterns, trichomes     

Mikromorphologie der Samenoberflächen der GattungEuphorbia

Seed coat surfaces of 127 species, representing 23 sections of the genusEuphorbia L. (Euphorbiaceae) have been examined with the scanning electron microscope (SEM). Various surface patterns and cell wall structures are described. In some groups the seed coat is rather uniform (e.g. in sect.Euphorbium), in other sections even closely related species can be separated by seed coat morphology (e.g.Tithymalus). The taxonomic application of testa micromorphology and the possible systematic position of certain taxa are briefly discussed. The seed coat cells of all species in sect.Tithymalus show remarkable intercellulares filled with small particles, which are described in detail for the first time.

     

Seed Coat Retention and Hypocotyl Hook Development in Mutants of Arabidopsis thaliana L.

Germination and growth of wild-type and two mutant strains (aux-1and Dwf) of Arabidopsis thaliana L. have been examined. Seedlingsof aux-1 exhibit agravitropic roots whereas Dwf display bothagravitropic roots and shoots. Wild-type seedlings retained the seed coat at the root-hypocotyltransition zone and developed hypocotyl hooks. In contrast,aux-I and Dwf seedlings did not retain their seed coats andlacked hypocotyl hooks. A positive gravitropic response of theroots was essential for the retention of the seed coat at theroot—hypocotyl transition zone by the attachment of roothairs to the seed coat. The development of the hypocotyl hookwas aided by the retention of the seed coat. The apical regionof the hypocotyl apparently remained agravitropic during formationand maintenance of the hypocotyl hook. Arabidopsis thaliana L., auxins, gravitropism, hypocotyl hook, mutants, peg formation, germination     

Characteristics of Sugar, Amino Acid and Phosphate Release from the Seed Coat of Developing Seeds of Vicia faba and Pisum sativum

After removal of the embryo from developing seeds of Vicia fabaL. and Pisum sativum L., the ‘empty’ ovules werefilled with a standard solution (pH 5.5). Seed coat exudatesof both species were collected during relatively long experiments(up to about 12 h) and the concentration of sugar (mainly sucrose),amino acids and phosphate in the exudate measured. A discussionis presented on the amino acid/sugar ratio and the phosphate/sugarratio in the seed coat exudate. A pretreatment (15 min) withp-chloromercuribenzenesulphonic acid (PCMBS) reduced the releaseof sugar, amino acids and phosphate from broad bean seed coats.After excision of ‘empty’ ovules of Vicia faba andPisum sativum from the maternal plant, 2–4 h after thistreatment a strong difference became visible between sucroserelease from excised seed coats and sucrose release from attachedseed coats. Similarly, when the rate of phloem transport ofsucrose into an ‘empty’ ovule of Vicia faba or Pisumsativum was reduced by a sub-optimal mannitol concentrationin the solution, a reduced rate of sugar release from the seedcoat could be observed. Excision and treatment with a sub-optimalmannitol concentration reduced the release of amino acids toa lesser extent than for sucrose. These treatments did not reducethe rate of phosphate release from the seed coat. Key words: Seed development, Seed coat exudate, Phloem transport     

A mutation at the rb gene, lowering ADPGPPase activity, affects storage product metabolism of pea seed coats

Seed coat development was studied on two nearisogenic linesof peas (Pisum sativum L.): RbRb (wild type, round seed) andrbrb (wrinkled seed). A mutation at the rb locus modifies thedry seed shape and reduces the starch content of the embryo.This mutation is now known to affect the activity of ADPGlucosepyrophosphorylase, a key enzyme in the starch biosynthetic pathway.We have investigated the effects of the rb mutation on seedcoat development and found that the mutation reduces the growthrate and starch content in this organ. However, experimentson the kinetics of ¹⁴C-sucrose loading showed that starch synthesisfrom unloaded sucrose occurred in the seed coat for both mutantand wild-type lines. In addition, the sucrose concentrationwas increased and amino acid concentration decreased such thatthe nutritional balance of the embryos was affected. However,osmolality of the seed coat cells was not affected, suggestinga regulatory process which allows the maintenance of the importof assimilates in the seeds of either line. Key words: ADPGlucose pyrophosphorylase, seed coat, seed development, starch metabolism, wrinkled seed     

Cryopreservation of Shoot Tips from Six Endangered Australian Species using a Modified Vitrification Protocol

An efficient vitrification procedure was developed and successfullyapplied to cryopreserve six endangered West Australian species(family Haemodoraceae: Anigozanthos humilis ssp. chrysanthusHopper;A. kalbarriensis Hopper;A. viridis ssp. terraspectansHopper;Conostylis dielsia ssp.teres Hopper;C. micrantha Hopperand C. wonganensis Hopper). Species were initially evaluatedfor cryostorage using a basic vitrification protocol involving:culturing plantlets in vitro for 21 d; excision of shoot apices;preculture of apical tips on 0.4 M sorbitol for 2 d, followedby incubation in PVS2 (plant vitrification solution 2) for 25min at 0 °C, then direct immersion in liquid nitrogen (LN).Warming of retrieved material was for 1 min in a 40 °C waterbath. Using this protocol five of the six species exhibitedlow post-storage survival, while the sixth species, A. viridisssp. terraspectans posted higher survival (61.1%). Using A.viridis ssp. terraspectans as an indicator species, the initialprotocol was modified to include: 3 d preculture on 0.80 M glycerol,loading treatment with 2.0 M glycerol plus 0.4 M sucrose solutionfor 20 min, followed by 25 min exposure to a modified PVS2.Survival was significantly improved in the test species, andin further experiments three other species also showed significantimprovements with the new protocol. Key findings include: effectivenessof glycerol in the preculture medium; the effect of precultureduration; the importance of a loading stage for these species;and the successful use of modified PVS2 solutions with reducedor zero dimethyl sulfoxide (DMSO). Copyright 2001 Annals ofBotany Company A. humilis ssp. chrysanthus, A. kalbarriensis, A. viridis ssp.terraspectans , Conostylis dielsia ssp. teres, C. micrantha, C. wonganensis, kangaroo paws, Haemodoraceae, vitrification, cryopreservation, rare and endangered, conservation