Origin and development in vitro of shoot buds and somatic embryos from intact roots of Helianthus annuus x H. tuberosus

A variant clone of the tetraploid (2n = 4x = 68) interspecific hybrid Helianthus annuus x H. tuberosus derived by in vitro tissue culture showed a deviation from the usual pattern of organization of the plant body. This variant developed shoot-like structures and somatic embryos from intact adventitious roots of in vitro-grown plantlets. The morphogenetic structures were not normally able to differentiate complete plants. They did show cellular proliferation with the inception of additional secondary embryos, leaf-like structures and unorganized masses of callus. Nevertheless, some ectopic structures isolated from roots and transferred onto fresh basal medium without growth regulators were able to produce plantlets that exhibited the same phenotype as the original clone. Histological analyses demonstrate that they originate from cortical cells in association with the development of lateral root primordia.     

Paratingia wudensis sp. nov., a whole noeggerathialean plant preserved in an earliest Permian air fall tuff in Inner Mongolia, China

Noeggerathiales are a little known group of Carboniferous and Permian plants of uncertain systematic position that have been variously considered to be ferns, sphenopsids, progymnosperms, or a separate group. These heterosporous plants carry adaxial sporangia on leaf-like or disk-shaped sporophylls that form cones. Leaves are pinnate with a rather stiff appearance, and pinnules can be attached in either two or four rows. In the present report, we present the top of a noeggerathialean plant with leaves and strobili attached, Paratingia wudensis Wang, Pfefferkorn et Bek sp. nov., from an earliest Permian volcanic ash fall tuff in Inner Mongolia. The excellent preservation allows the reconstruction of the whole plant, the complex three-dimensional leaves with anisophyllous pinnules, the heterosporous strobili, and the spores in situ. The homology of leaves and strobili can be elucidated and contributes to an understanding of the debated taxonomic position of Noeggerathiales. The "anisophyllous" leaves carry pinnules arranged in four rows. The strobili are bisporangiate and have disk-shaped sporophylls, each with one ring of 10-14 adaxial sporangia around the strobilus axis. Megaspores have an equatorial bulge. This new species expands the known diversity of Noeggerathiales. It grew in a peat-forming forest, thus changing earlier interpretations of the growth of noeggerathialean plants with anisophyllous pinnules.     

New evidence of the reproductive organs of Glossopteris based on permineralized fossils from Queensland,Australia. II: pollen-bearing organ Ediea gen. nov

Ediea homevalensis H. Nishida, Kudo, Pigg & Rigby gen. et sp. nov. is proposed for permineralized pollen-bearing structures from the Late Permian Homevale Station locality of the Bowen Basin, Queensland, Australia. The taxon represents unisexual fertile shoots bearing helically arranged leaves on a central axis. The more apical leaves are fertile microsporophylls bearing a pair of multi-branched stalks on their adaxial surfaces that each supports a cluster of terminally borne pollen sacs. Proximal to the fertile leaves there are several rows of sterile scale-like leaves. The pollen sacs (microsporangia) have thickened and dark, striate walls that are typical of the Arberiella type found in most pollen organs presumed to be of glossopterid affinity. An examination of pollen organs at several developmental stages, including those containing in situ pollen of the Protohaploxypinus type, provides the basis for a detailed analysis of these types of structures, which bear similarities to both compression/impression Eretmonia-type glossopterid microsporangiate organs and permineralized Eretmonia macloughlinii from Antarctica. These fossils demonstrate that at least some Late Permian pollen organs were simple microsporophyll-bearing shoot systems and not borne directly on Glossopteris leaves.     

Histological analysis of somatic embryogenesis induced in leaf explants ofHelianthus smithii Heiser

Summary According to the hormonal conditions, after one month of culture shoots or somatic embryos could be obtained from leaf explants ofHelianthus smithii. Well-shaped embryos developed on media containing a combination of auxin and cytokinin, while on media containing only cytokinin shoots were observed. The primary leaves of these shoots resembled cotyledons. A detailed histological study of the regeneration process on three media, containing only cytokinin or auxin, or a combination of both, allowed the origin and development of the observed structures to be determined. All three conditions induced somatic embryos, which then developed differently and, within one month, finally gave rise to the two types of structures which were initially observed.Abbrevations BAP 6-benzylaminopurine - MES 2-(N-morpholino)ethane-sulfonic acid - MS medium of Murashige and Skoog - NAA 1 -naphthaleneacetic acid - TDZ thidiazuron     

Morphogenesis in Helianthus Tuberosus: Genotypic Influence and Increased Totipotency in Previously Regenerated Plants

Leaf tissues of 38 genotypes, derived from four accessions, of the hexaploid species Helianthus tuberosus (2n=6x=102) responded to growth regulators (BA, NAA) chiefly by forming callus, while aventitious organogenesis or somatic embryogenesis were induced occasionally. A remarkable regeneration frequency (about 30 %) was achieved only from leaves of genotype HTPI-15. Explants of many regenerated plants of HTPI-15 subjected to a second culture cycle in vitro displayed a high morphogenetic potential (regeneration frequency > 90 %). White globular structures were initiated on the adaxial surface of these leaves without a callus phase. Somatic embryogenesis was asynchronous and embryoids, of different developmental stage, were simultaneously detected on each explant. Although many embryos developed single or malformed cotyledons or germinated precocciously, without the differentiation of a complete root system, phenotypically normal plants were regenerated after rooting on regulator-free half-strength MS medium. This revised version was published online in July 2006 with corrections to the Cover Date.     

Structure and development of epiphylly in <Emphasis Type="Italic">knox</Emphasis>-transgenic tobacco

Anatomical modifications and developmental patterns of tobacco (Nicotiana tabacum L.) plants transgenic for the barley (Hordeum vulgare L.) homeo box genes bkn-1 or bkn-3 were analysed and the morphogenetic processes interpreted. No appreciable difference between bkn-1 and bkn-3 transgenic tobacco was observed in the examined transgenic plants. They were dwarfed and developed epiphyllous appendages on all leaves except the cotyledons. Two major categories of epiphyllous structure were recognised: (i) vegetative shoots on juvenile heart-shaped leaves and (ii) inflorescences or flowers on adult lanceolate leaves. Both types of epiphyllous structure originated in the subepidermal layers, either directly from individual meristems or from an expanded meristematic "cushion". Structural analysis confirmed that the occurrence of epiphylls in the tobacco transformants represented a switch from determinate to indeterminate leaf growth and reproduced the phenotype of the barley Hooded mutant, which had earlier been shown to be associated with the bkn-3 gene [K.J. Muller et al. (1995) Nature 374:727-730].     

Agrobacterium-mediated transformation of sunflower (Helianthus annuus L.) shoot apices: transformation patterns

Apical segments of embryonic axes of sunflower (Helianthus annuus L.) embryos were submitted to co-culture experiments with a disarmed strain of Agrobacterium tumefaciens, harbouring a plasmid coding for the marker enzyme -glucuronidase. The expression patterns of this marker were analysed at different developmental stages of the regenerated shoots. The results are consistent with the hypothesis that transformed shoots originate from transformation events that have occurred within the existing meristems. Two of the resulting chimaeric plants have been analysed in detail, and some representative gene integration patterns are presented.     

Ectopic expression of class 1 KNOX genes induce adventitious shoot regeneration and alter growth and development of tobacco (Nicotiana tabacum L) and European plum (Prunus domestica L)

Transgenic plants of tobacco (Nicotiana tabacum L) and European plum (Prunus domestica L) were produced by transforming with the apple class 1 KNOX genes (MdKN1 and MdKN2) or corn KNOX1 gene. Transgenic tobacco plants were regenerated in vitro from transformed leaf discs cultured in a medium lacking cytokinin. Ectopic expression of KNOX genes retarded shoot growth by suppressing elongation of internodes in transgenic tobacco plants. Expression of each of the three KNOX1 genes induced malformation and extensive lobbing in tobacco leaves. In situ regeneration of adventitious shoots was observed from leaves and roots of transgenic tobacco plants expressing each of the three KNOX genes. In vitro culture of leaf explants and internode sections excised from in vitro grown MdKN1 expressing tobacco shoots regenerated adventitious shoots on MS (Murashige and Skoog 1962) basal medium in the absence of exogenous cytokinin. Transgenic plum plants that expressed the MdKN2 or corn KNOX1 gene grew normally but MdKN1 caused a significant reduction in plant height, leaf shape and size and produced malformed curly leaves. A high frequency of adventitious shoot regeneration (96%) was observed in cultures of leaf explants excised from corn KNOX1-expressing transgenic plum shoots. In contrast to KNOX1-expressing tobacco, leaf and internode explants of corn KNOX1-expressing plum required synthetic cytokinin (thidiazuron) in the culture medium to induce adventitious shoot regeneration. The induction of high-frequency regeneration of adventitious shoots in vitro from leaves and stem internodal sections of plum through the ectopic expression of a KNOX1 gene is the first such report for a woody perennial fruit trees.     

Ectopic divisions in vascular and ground tissues of Arabidopsis thaliana result in distinct leaf venation defects

Leaf venation patterns vary considerably between species and between leaves within a species. A mechanism based on canalization of auxin transport has been suggested as the means by which plastic yet organized venation patterns are generated. This study assessed the plasticity of Arabidopsis thaliana leaf venation in response to ectopic ground or procambial cell divisions and auxin transport inhibition (ATI). Ectopic ground cell divisions resulted in vascular fragments between major veins, whereas ectopic procambial cell divisions resulted in additional, abnormal vessels along major veins, with more severely perturbed lines forming incomplete secondary and higher-order venation. These responses imply limited vascular plasticity in response to unscheduled cell divisions. Surprisingly, a combination of ectopic ground cell divisions and ATI resulted in massive vascular overgrowth. It is hypothesized that the vascular overproduction in auxin transport-inhibited wild-type leaves is limited by simultaneous differentiation of ground cells into mesophyll cells. Ectopic ground cell divisions may negate this effect by providing undifferentiated ground cells that respond to accumulated auxin by differentiation into vascular cells.     

Anatomically preserved leaves of the conifer Notophytum krauselii (Podocarpaceae) from the Triassic of Antarctica

Permineralized leaves of the Triassic podocarpaceous conifer Notophytum krauselii are described from the Fremouw Formation of Antarctica. The leaves are elongate and apetiolate with 8-12 parallel veins. The adaxial epidermis consists of rows of rectangular to pentagonal cells; the abaxial epidermis is papillate. Longitudinally oriented stomata occur on both surfaces. An adaxial palisade layer is present and auxiliary sclereids are common in the mesophyll. The vascular bundles have a weakly defined sheath and are flanked by transfusion tracheids. Bundles in the basipetal area of the leaf are capped by sclerotic tissue and subtended by resin canals. These leaves are superficially similar to those of the extant podocarp genus Nageia, but probably represent a distinct acquisition of this leaf type within the Podocarpaceae. Notophytum leaves are similar to the common compression fossil Heidiphyllum elongatum and may be closely related or even conspecific. Evidence from Antarctica suggests that Heidiphyllum and the seed cone Telemachus were produced by the same plant, and may be closely related to several other early Mesozoic conifers with multiveined leaves.     

Tuber morphology and starch accumulation are independent phenomena: Evidence from ipt-transgenic potato lines

Tuber formation and carbohydrate metabolism in potatoes were studied using transgenic potato plants carrying the Agrobacterium tumefaciens ipt gene, involved in cytokinin biosynthesis. Three independent transformants, viz. clones 1, 11 and 13, whose cytokinin and auxin content had previously been shown to be different from each other and from the wild-type, were analysed in vitro. Clones 11 and 13 showed a higher ability to form stolons and tubers, as evident from: (1) stolon development in whole plants grown under non-inductive conditions, (2) total number and weight of tubers formed by cuttings of this clone in darkness, (3) tubers appeared earlier than tubers of wild-type plants and at a lower sucrose concentration in the medium. Clone 1 did not form stolons or tubers under any conditions tested, but rather formed short shoots. A series of metabolic changes, known to be characteristic for tubers, were analysed in leaves, stems and developing buds. It was found that the short type of shoots, formed by clone 1, had metabolic characteristics very similar to tubers formed in wild-type or clones 11 and 13, including glucose, fructose, sucrose, and starch levels, and activities of invertase, sucrose synthase and fructokinase. It is concluded that the regulation of the stolon swelling and of carbohydrate metabolism, normally occurring simultaneously, can be uncoupled, and are thus, at least partly independent phenomena. The present data obtained with a high-cytokinin line indicate that cytokinins (probably in concert with auxins) might be mainly involved in the regulation of tuber morphology.     

Silica Deposition in Some C3 and C4 Species of Grasses, Sedges and Composites in the USA

We report new information on silica deposition in 15 plant species,including nine grasses, two sedges and four composites. Thesilica depositional patterns found in seven of the grass speciesindicate that they are C₄ plants. However the festucoid grassCortaderia selloana is a C₃ plant with long leaf trichomes andoval silica structures in the leaves. In contrast the panicoidC₄ grasses Chasmathium latifolium, Chasmathium sessiflorum,Imperata cylindrica, Panicum repens, Panicum commutatum andSetaria magna, all produce dumb-bell-shaped silica structuresin the leaves. The chloridoid grasses Spartina patens and Spartinacynosuroides have saddle-shaped structures and no dumb-bellor oval shaped ones. The sedges Rhynchospora plumosa and Scirpuscyperinus were found to have oval phytoliths and may be C₃ plants.Our examination of these and other grasses strongly suggeststhat C₄ grasses tend to produce the same type of silica cells.Grasses and sedges with C₃ type photosynthesis tend to produceoval silica structures. The composite Grindelia squarrosa andsunflowers Helianthus angustifolia, Helianthus atrorubens andHelianthus tuberosus absorb relatively small amounts of siliconand larger amounts of calcium, where both elements deposit inleaf trichomes. We found no clear indicator for the C₃ sunflowersor C₄ types in the Asteraceae. Helianthus tuberosus leaves havemany trichomes on the adaxial surface. These trichomes havea higher concentration of silica than the surrounding leaf surface.Helianthus tuberosus leaves had much higher ash and silica contentsthan those of Helianthus angustifolia and Helianthus atrorubens.The composite Grindelia squarrosa has a usual deposition ofsilica in the basal cells around the guard cells. Silica depositionoften reflects the surface features of a leaf. An exceptionis Scripus cyperinus where the silica structures are deep inthe tissue and do not reflect the surface configurations. Theinforescence of Setaria magna had a 14.64 silica content. Thetufts of white, silky hairs characteristic of Imperata cylindricainflorescence have no silica. C₃ and C₄ plants, silica and ash content, scanning electron microscopy, energy-dispersive X-ray analysis, silicon distribution, spectra of elements in plants, trichomes, silica fibres, phytoliths     

Leaf dimorphism in Thuja plicata and Platycladus orientalis (thujoid Cupressaceae s. str., Coniferales): the changes in morphology and anatomy from juvenile needle leaves to mature scale leaves

Thuja plicata and Platycladus orientalis initially produce only bifacial needle leaves. When the first lateral shoots develop, the leaf morphology and anatomy changes dramatically. Subsequently, only greatly reduced, bifacial scale leaves are developed. A new kind of “superimposed bifaciality” occurs with the change from juvenile needle leaves to mature scale leaves. Anatomical dorsiventrality affects not only the individual leaf, but also the complete plagiotropic lateral shoots of Thuja, which have a sun- and shade-exposed side. The upper light-exposed median leaves show adaxial leaf anatomy, contrary to the lower shaded median leaves showing abaxial leaf anatomy. Due to their mixed exposure, the lateral leaves show a lateral differentiation. At vertical lateral shoots of Platycladus, a predominant light-exposed side is absent. Thus, the anatomical dorsiventrality does not affect the complete shoot. Here the morphological abaxial side of a scale leaf becomes functionally and physiologically adaxial by reorientation of the palisade parenchyma and stomata. In juvenile needle leaves, the palisade parenchyma is located adaxial, with the majority of stomata being located abaxial. Conversely, in mature scale leaves, the palisade parenchyma is abaxial and the majority of stomata are adaxial.     

Cytokinin receptors are involved in alkamide regulation of root and shoot development in Arabidopsis

Alkamides and N-acilethanolamides are a class of lipid compounds related to animal endocannabinoids of wide distribution in plants. We investigated the structural features required for alkamides to regulate plant development by comparing the root responses of Arabidopsis (Arabidopsis thaliana) seedlings to a range of natural and synthetic compounds. The length of the acyl chain and the amide moiety were found to play a crucial role in their biological activity. From the different compounds tested, N-isobutyl decanamide, a small saturated alkamide, was found to be the most active in regulating primary root growth and lateral root formation. Proliferative-promoting activity of alkamide treatment was evidenced by formation of callus-like structures in primary roots, ectopic blades along petioles of rosette leaves, and disorganized tumorous tissue originating from the leaf lamina. Ectopic organ formation by N-isobutyl decanamide treatment was related to altered expression of the cell division marker CycB1:uidA and an enhanced expression of the cytokinin-inducible marker ARR5:uidA both in roots and in shoots. The involvement of cytokinins in mediating the observed activity of alkamides was tested using Arabidopsis mutants lacking one, two, or three of the putative cytokinin receptors CRE1, AHK2, and AHK3. The triple cytokinin receptor mutant was insensitive to N-isobutyl decanamide treatment, showing absence of callus-like structures in roots, the lack of lateral root proliferation, and absence of ectopic outgrowths in leaves under elevated levels of this alkamide. Taken together our results suggest that alkamides and N-acylethanolamides may belong to a class of endogenous signaling compounds that interact with a cytokinin-signaling pathway to control meristematic activity and differentiation processes during plant development.