Regeneration of fertile interspecific hybrids from protoplast fusions between Helianthus annuus L. and wild Helianthus species

The use of interesting characteristics from wild Helianthus species in sunflower breeding is limited by poor crossability or sterility of interspecific hybrids. To overcome this barrier, mesophyll protoplasts of Sclerotinia sclerotiorum-resistant clones of Helianthus maximiliani, H. giganteus and H. nuttallii were fused with hypocotyl protoplasts of H. annuus in the presence of polyethyleneglycol and dimethylsulfoxide. Fusion products were embedded in agarose and subjected to a regeneration protocol developed for sunflower protoplasts. Organogenic calli were transferred onto solid medium and emerging shoots were elongated in the absence of plant growth regulators. Rooting of shoots was induced by a 1-naphthaleneacetic acid treatment and putative hybrid plants from fusions between H. annuus + H. maximiliani and H. annuus + H. giganteus were transferred into the greenhouse. All of them exhibited a hybrid phenotype with a high percentage of rhizome producing plants. Their hybrid origin was confirmed by random amplified polymorphic DNA analysis. Plants flowered after 3–4 months and set seeds, of which 70–80% germinated. Received: 18 February 1997 / Revision received: 25 March 1997 / Accepted: 15 May 1997     

Production of fertile somatic hybrid plants of sunflower and Helianthus giganteus L. by protoplast fusion

Summary Protoplasts of Helianthus giganteus and Helianthus annuus were fused using polyethylene glycol. Before fusion H.giganteus protoplasts were subjected to iodoacetic acid treatment to render them unable to divide. Fused protoplasts were cultured in V-KM medium containing benzylaminopurine and naphtaleneacetic acid. Hybrid calli were identified on the basis of their ability of embryogenic development contributed by the Helianthus giganteus parent. Fifty embryogenic calli were cultured on MS based medium without growth regulators to induce further development of somatic embryos. Elongated shoots were removed, rooted and transferred into growth chambers. Overall morphology of the plants was intermediate between the two parents. Their hybrid nature was confirmed by chromosome counting and by the analysis of esterase isozymes. The plants flowered within two to three months and later died. Thus the perennial nature of H.giganteus is a recessive trait in this interspecific hybrid. Seeds were obtained from two of the regenerated plants. From these seeds normal fertile F₂ plants could be grown.Abbreviations PEG polyethylene glycol - NAA naphtaleneacetic acid - BA benzyladenine - MS Murashige and Skoog medium - V-KM protoplast culture medium of Binding and Nehls     

Plant regeneration from callus and protoplast cultures of Helianthus giganteus L.

Summary Methods of plant regeneration from callus and protoplasts of Helianthus giganteus L. are described. Embryogenic callus was obtained from leaf explants and plants were regenerated from these calli on MS media with different combinations of benzyladenine and naphtaleneacetic acid. Leaf protoplasts isolated from in vitro grown plants formed somatic embryos when cultured in agarose solidified droplets of V-KM medium containing benzyladenine and naphtaleneacetic acid. Embryos developed into plantlets on media with reduced auxin contents. Regenerated plants were successfully planted in soil.Abbreviations BA benzyladenine - IAA indoleacetic acid - MS Murashige and Skoog medium - NAA naphtaleneacetic acid - V-KM protoplast culture medium of Binding and Nehls     

Protocols for efficient repetitive and secondary somatic embryogenesis in Helianthus maximiliani (Schrader)

 Indirect somatic embryogenesis was induced on leaf explants of greenhouse-grown Helianthus maximiliani plants. Leaves of the regenerated plants were used as starting explants for the induction of direct somatic embryogenesis. Another cycle of somatic embryogenesis was induced on the leaves of regenerated plants. In both cases, leaf explants were cultured on media containing different auxin/cytokinin ratios. The auxin/cytokinin ratio had an influence on the intensity of embryo formation, germination and the capability to regenerate plants. Somatic embryogenesis was generally more intensive on the medium with lower concentrations of 6-benzylamino-purine. Further, the percentage of regenerated plants was higher when embryos were induced on high-cytokinin, low-auxin medium. Secondary somatic embryogenesis was induced on embryos by culture in liquid hormone-free medium. Similar to direct embryogenesis the efficiency of secondary embryogenesis depended on the medium used for the induction of the primary embryos. In contrast to the mostly low frequencies of conversion of secondary embryos into plants that has been observed in other species, the percentage of regenerated plants from secondary embryos of H. maximiliani was quite high, although slightly lower than that obtained in primary embryos. Received: 28 March 2000 / Revision received: 1 September 2000 / Accepted: 2 October 2000     

Phylogenetic analysis of Helianthus (Asteraceae) based on chloroplast DNA restriction site data

 Chloroplast DNA (cpDNA) restriction site data were used to analyze phylogenetic relationships within Helianthus. Data from new samples were consistent with previous results and showed the genus to be a well-defined (11 site changes) lineage within subtribe Helianthinae. The affinities of 2 species, Viguiera similis and V. phenax (V. ludens) that have sometimes been included in Helianthus were shown to lie outside the genus. The two species of Phoebanthus, however, formed a phylogenetic ingroup to the perennial Helianthus. Within the genus, cpDNA data indicated that there are four distinct phylogenetic lineages. Two of the lineages each contained a single, annual species (H. agrestis and H. porteri, respectively). The remaining annual species collectively formed a third lineage (sect. Helianthus). The fourth lineage contained all of the perennial species, including those of Phoebanthus. Within the perennial lineage, there was little variation in restriction site characters. The 3 species of series Pumili formed a paraphyletic group at the base of the perennial lineage, and the 3 species of series Ciliares formed a group that was supported by a single restriction site character. Received: 2 September 1996 / Accepted: 25 October 1996     

Competition between Invasive and Indigenous Species: Impact of Spatial Pattern and Developmental Stage

Elodea canadensis (indigenous) and Elodea nuttallii (invasive) were grown in experimental tanks in monocultures and mixtures in two spatial patterns (aggregated or mixed) and two developmental stages (small or large plants of E. canadensis, and small plants of E. nuttallii). Competitive interactions between the two species were assessed by monitoring the area colonised by each species, the number of rootings and biomass after 10 weeks. In monocultures the growth of E. canadensis was significantly lower than that of E. nuttallii. In mixtures the number of rootings and biomass of E. canadensis were always significantly less than those of E. nuttallii. The tank surface area colonised by E. canadensis was always significantly less than that occupied by E. nuttallii, but it was higher in the aggregated treatment, where the colonisation of E. nuttallii was lower. Therefore both spatial pattern and developmental stage of an indigenous species (E. canadensis) may influence the outcome of competition with potential invaders (E. nuttallii).     

Plant regeneration from different explants in Helianthus smithii Heiser

Leaf, rhizome, and stem explants of Helianthus smithii, a wild relative of the cultivated sunflower, have been tested for their morphogenic potential on media containing a range of hormonal combinations including α-naphthaleneacetic acid and 6-benzylaminopurine (0–2 mg/l). Somatic embryos appeared on the majority of the tested media incorporating auxins, while shoots were observed on media containing exclusively 6-benzylaminopurine. Fertile plants were recovered from all explant types under a wide range of conditions, albeit with different efficiencies. The induction mechanism of the observed shoots is discussed. Received: 12 July 1996 / Revision received: 13 September 1996 / Accepted: 5 October 1996     

Influence of flow turbulence on growth and indole acetic acid and H<Subscript>2</Subscript>O<Subscript>2</Subscript> metabolism of three aquatic macrophyte species

This study aimed to determine the influence of different turbulence levels on morphologically different aquatic macrophyte species. The variations of growth and the alterations of indole acetic acid (IAA) and H₂O₂ metabolism of three morphologically distinct aquatic macrophyte species, Elodea nuttallii, Potamogeton crispus and Vallisneria spiralis were studied in the exposure to three different turbulence conditions. Plants were subjected to turbulence generated by vertically oscillating horizontal grids at three different frequencies: 1, 2 and 4 Hz. A no-turbulence condition was used as a control. The growth of V. spiralis and E. nuttallii was suppressed by exposure to greater turbulence, though increased turbulence enhanced the growth of P. crispus. The growth rate of plants was positively correlated with IAA concentration. IAA concentration was negatively correlated with turbulence level in V. spiralis and E. nuttallii, while it was positively correlated in P. crispus. H₂O₂ concentration was positively correlated with turbulence level in the former species, while it had no correlation in the latter. P. crispus showed more resistance to turbulence-induced mechanical stress and growth suppression than the other two species. The observed variations of growth and IAA and H₂O₂ metabolism showed that plant morphology has a significant influence on the response to flow turbulence. The results from this study provide a basis for understanding the mechanism and consequential morphological changes in plants that grow in moving water.     

Protoplasts from cotyledon and hypocotyl of sunflower (Helianthus annuus L.): shoot regeneration and seed production

Summary Cotyledon and hypocotyl protoplasts of Helianthus annuus inbred line 47 302 bcd were embedded in alginate and plated on L4 medium (Lenée and Chupeau 1986). After one month, the calli were transferred on MSSH regeneration medium (Murashige and Skoog 1962; Schenk and Hildebrandt 1972) where they regenerated shoots (overall efficiency 10^–2%). The shoots were elongated on B5 (Gamborg et al. 1968) medium first without hormones, then supplemented with GA3 and BAP (both 0.05 mg/l). In order to overcome the difficulty to induce rooting by classical methods, the elongated shoots were grafted on a sunflower rootstock. The grafted shoots produced flowers and seeds. Different factors have been shown to have an important influence on the capacity to regenerate shoots: the genotype, the physical culture conditions at the callus regeneration step (e.g. protoplasts embedded in alginate), and the media composition.Abbreviations BAP 6-benzylaminopurine - GA3 gibberellic acid - IBA indole-3-butanoic acid - IAA indole acetic acid - MES 2-N-morpholinoethane sulfonic acid - NAA 1-naphthalene acetic acid - 2,4D 2,4 dichlorophenoxyacetic acid     

Transfer of a grapevine stilbene synthase gene to rice (Oryza sativa L.)

A gene derived from grapevine (Vitis vinifera) coding for stilbene synthase has been transferred into protoplasts of the commercially important japonica rice cultivar Nipponbare using PEG-mediated direct gene transfer. Transgenic plants were regenerated from calli selected on kanamycin. Southern blot analysis of genomic DNA isolated from regenerants and progeny plants demonstrated that the stilbene synthase gene is stably integrated in the genome of transgenic rice plants and inherited in the offspring. The transient formation of stilbene-synthase-specific mRNA shortly after inoculation with the fungus of the rice blast Pyricularia oryzae has demonstrated that the grapevine stilbene synthase promoter is also active in monocotyledonous plants. Preliminary results indicate an enhanced resistance of transgenic rice to P. oryzae. Received: 1 July 1996 / Revision received: 5 November 1996 / Accepted: 30 November 1996     

Temporal changes and vertical distribution of macrophytes in Lake Kawaguchi

 The distribution of macrophytes in Lake Kawaguchi, Japan, was surveyed in August 1999 using a sampling anchor from a boat. The survey revealed that the present aquatic vegetation was composed of 17 submerged species, and no floating-leaved plants were present. The diversity of submerged plants and their vertical growth limits decreased progressively from the west to the east end of the lake. At the time of the survey, the dominant species was Elodea nuttallii (Planch.) St. John, which had once grown explosively in the mid-1980s in the lake, and except for Vallisneria asiatica Miki, all of the species commonly observed in the lake seem to have diminished their growth greatly. Among them, Potamogeton compressus L., which was the most dominant species in the lake before the invasion of Elodea nuttallii, had decreased most severely. It is suggested that the present state of aquatic vegetation in this lake was much influenced by the persistent growth of Elodea nuttallii as well as by environmental factors. Received: August 7, 2001 / Accepted: February 9, 2002     

Root hydraulic conductivity of Larrea tridentata and Helianthus annuus under elevated CO2

While investigations into shoot responses to elevated atmospheric CO₂ are extensive, few studies have focused on how an elevated atmospheric CO₂ environment might impact root functions such as water uptake and transport. Knowledge of functional root responses may be particularly important in ecosystems where water is limiting if predictions about global climate change are true. In this study we investigated the effect of elevated CO₂ on the root hydraulic conductivity (L_p) of a C₃ perennial, Larrea tridentata, and a C₃ annual, Helianthus annuus. The plants were grown in a glasshouse under ambient (360 μmol mol^–1) and elevated (700 μmol mol^–1) CO₂. The L_p through intact root systems was measured using a hydrostatic pressure-induced flow system. Leaf gas exchange was also determined for both species and leaf water potential (ψ_leaf) was determined in L. tridentata. The L_p of L. tridentata roots was unchanged by an elevated CO₂ growth environment. Stomatal conductance (g_s) and transpiration (E) decreased and photosynthetic rate (A_net) and Ψ_leaf increased in L. tridentata. There were no changes in biomass, leaf area, stem diameter or root : shoot (R : S) ratio for L. tridentata. In H. annuus, elevated CO₂ induced a nearly two-fold decrease in root L_p. There was no effect of growth under elevated CO₂ on A_net, g_s, E, above- and below-ground dry mass, R : S ratio, leaf area, root length or stem diameter in this species. The results demonstrate that rising atmospheric CO₂ can impact water uptake and transport in roots in a species-specific manner. Possible mechanisms for the observed decrease in root L_p in H. annuus under elevated CO₂ are currently under investigation and may relate to either axial or radial components of root L_p.     

Response of <Emphasis Type="Italic">Elodea Nuttallii</Emphasis> (Planch.) H. St. John to Manual Harvesting in the North-East of France

Elodea nuttallii (Planch). H. St John is an introduced aquatic macrophyte which was first observed in France in the early 1950s. The impact of two frequencies of harvesting on the biomass and regrowth strategy of this invasive species was evaluated by assessment of morphological traits monthly from February to October 2003. The effect of this management on the floristic biodiversity was also analysed. Harvesting caused a drastic reduction of biomass of E. nuttallii. Two harvests caused almost total disappearance of E. nuttallii. Furthermore, no significant difference was observed in the architecture of E. nuttallii between an unharvested site and harvested site. In one year, harvest did not allow the development of native aquatic plants.     

Somatic embryogenesis and shoot regeneration from transgenic roots of the cherry rootstock Colt (Prunus avium×P. pseudocerasus) mediated by pRi 1855 T-DNA of Agrobacterium rhizogenes

Hairy roots were obtained after inoculation with Agrobacterium rhizogenes strain NCPPB 1855 of the in-vitro-grown shoots of the cherry rootstocks Colt (Prunus avium×P. pseudocerasus) and Mazzard F12/1 (P. avium L.). Not all putatively transgenic roots were able to grow in hormone-free medium. Mazzard F12/1 roots, induced with A. rhizogenes, did not differentiate any shoot or embryo, while both somatic embryos and shoots differentiated from the transgenic roots of Colt in medium containing 1 mg/l 6-benzylaminopurine and 1 mg/l 1-naphthaleneacetic acid. Somatic embryos were capable of secondary embryogenesis, but few developed into whole plants. DNA hybridization showed both a different number of bands and signal intensity in each of the five transgenic shoot clones and embryos examined. In a morphogenetic in vitro test, leaf explants of the transgenic shoot clones showed an increased capacity to differentiate roots, although clones differed in their sensitivity to the hormone ratio. Clones from the transgenic shoots had not only an increased rooting ability when grown in vitro but also exhibited various hairy root phenotypes when cultured in vitro and when transferred into the greenhouse. Received: 12 December 1996 / Revision received: 21 March 1997 / Accepted: 10 May 1997     

Cytogenetic analysis of interspecific sunflower hybrids and molecular evaluation of their progeny

Meiotic cells of transgenic asymmetric somatic hybrid (ASH) plants obtained by fusion of microprotoplasts of the donor species Helianthus giganteus or Helianthus maximiliani and recipient protoplasts of Helianthus annuus were investigated. Over 85% of the ASH meiocytes showed regular bivalent chromosome pairing; however, several anomalies like anaphase bridges, laggard chromosomes, univalent and multivalent pairing were also observed. Pollen viability of the ASH plants ranged from 79.2 to 95% with a strong negative correlation to chromosome number which varied between 34 and 42. Molecular investigation of ASH progeny using RAPD markers revealed the presence of donor genotype markers in 68% of the offspring. These results suggest that asymmetric somatic hybridization offers an efficient alternative method to overcome sexual barriers for gene flow and the genetic improvement of H. annuus by introgression of economical important traits from wild Helianthus species. Received: 16 July 2000 / Accepted: 25 September 2000     

Protoplast culture and plant regeneration in Lycium barbarum L.

Protoplasts were isolated from leaves of the woody plant Lycium barbarum L. and cultured in liquid nutrient medium TM-2 at a density of 10⁴–10⁵ cells ml^-1. After ten days of culture, regenerated colonies were transferred to the agar-solidified medium TM-3, and 5–7 days later to regeneration media PRM or TM-4. Formation of shoots was observed after 30–40 days. Completely formed and rooted plants were transferred to the soil. Cytological and morphological analysis of the regenerated plants revealed relative genetic stability of this species in the process protoplast — plant. The results obtained allow us to conclude that L. barbarum can be used in the experiments on somatic hybridization or direct gene transfer.     

Food preference of freshwater invertebrates: comparing fresh and decomposed angiosperm and a filamentous alga

1. Fresh and decomposed Mougeotia sp. (a filamentous green alga) and Elodea nuttallii (a vascular plant) were offered as food to three species of aquatic macroinvertebrates (Lymnaea peregra, Asellus meridianus and Endochironomus albipennis) to test: (i) if filamentous algae are preferred to aquatic higher plants (hereafter, called ‘macrophytes’) and (ii), as is known for higher plants, if decomposition also results in greater palatability of filamentous algae. 2. Compared with the alga, the macrophyte in both states was of higher nutritional value. Conditioning improved the nutritional value of both food types, but especially of the macrophyte. 3. Both fresh alga and fresh macrophyte were eaten little by all animals except A. meridianus feeding upon the macrophyte. Consumption was higher for both plants in their decomposed state. However, L. peregra consumed more decomposed macrophyte than the decomposed alga. Both decomposed plants were eaten most by E. albipennis followed by A. meridianus and L. peregra. 4. Digestibility of both plants, but especially of the macrophyte, increased significantly after decomposition. The assimilation efficiencies of all animals on the fresh E. nuttallii were higher than on fresh Mougeotia sp. After decomposition, the efficiency increased significantly only on the alga. Consequently, both decomposed plants were assimilated with similar efficiency by all test animals. 5. Amongst aquatic macrophytes, the increase of their consumption and digestibility upon decomposition has hitherto been known only for vascular plants but not for filamentous algae.     

Phenotypic plasticity as a clue for invasion success of the submerged aquatic plant Elodea nuttallii

• Two closely related alien submerged aquatic plants were introduced into Europe. The new invader (Elodea nuttallii) gradually displaced E. canadensis even at sites where the latter was well established. The aim of the study was to evaluate the combined effects of environmental factors on several phenotypic characteristics of the two Elodea species, and to relate these phenotypic characteristics to the invasion success of E. nuttallii over E. canadensis.
• In a factorial design, Elodea plants were grown in aquaria containing five different nitrogen concentrations and incubated at five different light intensities. We used six functional traits (apical shoot RGR), total shoot RGR, relative elongation, root length, lateral spread, branching degree) to measure the environmental response of the species. We calculated plasticity indices to express the phenotypic differences between species.
• Light and nitrogen jointly triggered the development of phenotypic characteristics that make E. nuttallii a more successful invader in eutrophic waters than E. canadensis. The stronger invader showed a wider range of phenotypic plasticity. The apical elongation was the main difference between the two species, with E. nuttallii being more than two times longer than E. canadensis. E. canadensis formed dense side shoots even under high shade and low nitrogen levels, whereas E. nuttallii required higher light and nitrogen levels.
• We found that under more eutrophic conditions, E. nuttallii reach the water surface sooner than E. canadensis and through intensive branching outcompetes all other plants including E. canadensis. Our findings support the theory that more successful invaders have wider phenotypic plasticity.

     

Evidence for several genomes in Helianthus

Forty taxa belonging to 36 species and four unclassified accessions of Helianthus were studied using RAPD technology. Single ten-mer primers were screened for those amplifying fragments common to several species. We found that when several species shared a common fragment, they belong to the same section of the genus. Moreover, we also found that some fragments are common to all species of the Helianthus. Most of the fragments were found to be of the same size in these species and to share the homology indicated by molecular hybridization. Out of 118 retained fragments, 33 were common to all Helianthus species, 56 were unique to perennial species of sects. Atrorubentes and Ciliares, 24 were unique to sect. Atrorubentes, 29 were unique to sect. Helianthus, whereas 0 were unique to sect. Ciliares. Each set of common or specific fragments was assumed to belong to a genome: (1)?the C genome carrying the fragments common to all species of the three sections, (2)?the H genome unique to sect. Helianthus, (3)?the P genome common to perennial species (sects. Atrorubentes and Ciliares), and (4)?the A genome unique to sect. Atrorubentes. The genomic structure was therefore HC for sect. Helianthus, CPA for sect. Atrorubentes, and CP? for sect. Ciliares. Molecular hybridizations with amplification products revealed homologies between Helianthus genomes and several other genera in the Helianthinae sub-tribe. The simple method used to characterize these fragments led to powerful tools for recognizing genomes which reconcile the section organization of the genus and the degree of difficulty in crossing perennial and annual forms.     

Morphological and Morphometrical Variations of Selected Rotifer Species in Response to Predation: a Seasonal Study of Selected Brachionid Species from Lake Xochimilco (Mexico)

Allelopathy of filamentous green algae (FGA) has been less studied than that of macrophytes. Little Budworth Pool, Cheshire, UK is a small, shallow, clear-water lake with high TP concentrations, very high NO_3-N concentrations, only moderate phytoplankton density, high FGA growth (mainly Spirogyra sp.) and no submerged plants. Experiments were carried out to test the possible allelopathic effects of Spirogyra on the phytoplankton of this lake and on a submerged plant Elodea nuttallii. Changes in phytoplankton growth, phytoplankton species dynamics and species composition were apparently not influenced by allelopathy of live or decaying Spirogyra. A shift from diatom (Cyclotella sp) – cryptomonad (Chroomonas acuta and Cryptomonas erosa) dominance to Chlorococcales (Micractinium pusillum, Monoraphidium contortum and Scenedesmus opoliensis) – Volvocales (Chlorogonium elongatum and Pandorina morum) dominance was recorded in both control and FGA treatments, suggesting an effect of nutrient enrichment. Nutrient concentrations and differences in competitiveness among phytoplankton species can also explain differences in their growth rates in Spirogyra filtrate. Spirogyra also did not influence apex number per plant, shoot length or growth rate of E. nuttallii. This FGA species probably cannot control phytoplankton or E. nuttallii growth in nutrient rich conditions through allelopathy.