Ruth Wingender

Gene-transfer and plant-regeneration (techniques)

The production of transgenic plants depends on the stable introduction of foreign DNA into the plant genome, followed by regeneration to produce intact plants, and the subsequent expression of the introduced gene(s). A variety of transformation methods have been developed, and this has allowed many of the worlds important crop plants to be transformed. Transformation mediated by Agrobacterium has provided a reliable means of creating transgenics in a wide variety of species that are amenable to tissue culture and regeneration. Vectorless gene-transfer systems include particle bombardment (biolistics), electroporation and membrane permeabilization using chemicals. Of these, biolistics has proved to be particularly successful with plants that are less amenable to tissue culture, such as cereals and legumes.

Rapid response reactions of roots to boron deprivation

Upon B removal from the nutrient solution, several response reactions of root cells can be measured within minutes. These include: reduction of cell wall elasticity modulus e, increase of hydraulic conductivity, reduced activity of plasmalemma-bound inducible (NADH) reductase, (smaller) changes of the membrane potential, and liberation of Ca2+ (apoplastic and membrane-bound). The B most demanding (root) tissues are epidermal and outer cortical cells of the extension zone, xylem vessels, and root hair tips. Deprivation of B leads to morphological changes which can be noticed within hours to days, including browning of tissues, growth inhibition, death of apical meristems, and lack of root hairs. How the primary response reaction(s) lead to the expression of visible symptoms, however, is not yet clear. The present review summarizes rapid responses to B deprivation and shows several possibilities how primary might be linked to secondary reactions, including cytoskeleton-mediated responses. Schnelle Reaktionen von Wurzeln auf Bor-Mangel Nach Umsetzen auf ein B-Mangelmedium konnen verschiedene Reaktionen von Wurzelzellen innerhalb von Minuten beobachtet werden, wie z.B. Verringerung des Zellwandelastizitatsmoduls e, Zunahme der hydraulischen Leitfahigkeit, verringerte Plasmalemma-gebundene induzierbare (NADH) Reduktaseaktivitat, (kleinere) Anderungen des Membranpotenzials und eine Freisetzung von apoplastischem und membrangebundenem Ca2+. Die Wurzelgewebe mit dem hochsten B-Bedarf scheinen die epidermalen und auseren Rindenzellen der Streckungszone, Xylem und Wurzelhaarspitzen zu sein. B-Entzug fuhrt zu morphologischen Anderungen, die innerhalb von Stunden und Tagen sichtbar werden, wie Gewebeverbraunung, Wachstumshemmung und Absterben apikaler Meristeme, sowie Fehlen von Wurzelhaaren. Wie die raschen Reaktionen zur (sekundaren) Auspragung von Mangelsymptomen fuhren, ist jedoch noch nicht klar. In dieser Ubersicht werden rasche Reaktionen auf B-Entzug zusammengefasst und Moglichkeiten zur Verknupfung primarer und sekundarer Reaktionen diskutiert.

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

Abstract 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.

A regeneration protocol for sunflower (Helianthus annuus L.) protoplasts

SummaryHypocotyl protoplasts of four different Helianthus annuus genotypes were cultivated for 22–28 days in agarose droplets covered with liquid medium. In the first week, supplementation of the medium with plant growth regulators was at a 0.8/1 ratio of cytokinin and auxin followed by a high auxin concentration in the second week and a cytokinin to auxin ratio of 8/1 in the third and fourth week. Following transfer onto solid medium containing cytokinin and auxin in a proportion of 40/1 morphogenic callus started to form globular structures that developed into leaf primordia. Subsequent shoot elongation and rooting were obtained on hormone free medium after dipping the cut shoots into high auxin solution. Thirteen weeks after protoplast isolation, plantlets could be transferred to the greenhouse. Shoot regeneration was obtained for all four cultivars (Florom-328, Cerflor, Euroflor, Frankasol) at different rates reflecting their regenerative potential.

Regeneration of fertile plants from Helianthus nuttallii T&G and Helianthus giganteus L. mesophyll protoplasts

Abstract Interspecific hybridisation in the genus Helianthus via somatic cell fusion is thought to play an important role in future sunflower breeding programs. The establishment of this technique requires, however, the development of single-cell-regeneration protocols. For this purpose, we applied a regeneration protocol recently developed for Helianthus annuus L. to mesophyll protoplasts of two wild sunflowers (H. nuttallii T&G, H. giganteus L). Protoplasts of both species were embedded in agarose droplets and covered by liquid mKM medium. After 4–5 weeks, callus was transferred onto solid differentiation medium yielding plating efficencies of 1.5% (H. nuttallii) and 2.5% (H. giganteus). Emerging shoots were elongated on hormone-free medium, and root formation was induced by an NAA treatment. Regenerated plants were transferred to the greenhouse where they grew up to a height of 2 m and flowered after 3 months. Seeds were harvested from regenerated plants of both species.

Rapid Responses of Plants to Boron Deprivation

B deficiency has been described to affect a wide range of processes in plants, ranging from metabolism of nucleic acids, protein synthesis, metabolism and transport of carbohydrates, chemistry and physics of cell walls, synthesis and transport of plant hormones (especially IAA), regulation of plasma membrane-bound ATPase and oxido-reductase activities, as well as synthesis and metabolism of phenolics and incidence of oxidative damage of membranes, (for reviews see e.g. Goldbach, 1997; Blevins and Lucaszewski, 1998; Bell et al., 2001, this volume). Considering that many observations started at least several hours or even days and weeks after B deprivation, and taking into account that recent reports point to very rapid reactions within minutes (Findeklee and Goldbach, 1996; Findeklee et al., 1997; Muhling et al., 1998; Wimmer and Goldbach, 1999), it is intriguing to explore the sequence of responses to B deprivation. Below, we summarise effects which have been observed within the first minutes to few hours of B deficiency and discuss how these early responses may be linked to the expression of secondary reactions or symptoms in the light of recent observations.

Ultrastructure of sunflower protoplast derived ealluses differing in their regenerative potential

The ultrastructural properties of microcalluses derived from mesophyll protoplasts of commercial sunflower (Helianthus annuus L.) cultivars were investigated by light and electron microscopy. Two culture regimes were chosen: Regime A giving rise to callus formation but of little embryogenic potential and regime B resulting in higher embryogenicity. Bipolar colonies that developed during early stages of regime A were found to be composed of mostly degenerated structures. No differentiation or embryonal organization as suggested by the compactness and shape of the microcalluses could be observed. Amorphous calluses obtained at later stages of the same regime consisted of small groups of meristematic as well as vacuolated cells. Incomplete cellular divisions occurred in almost all colonies grown under the regime A, causing most probably the lack of further callus organization. In contrast calluses of irregular shape, cultivated under regime B, mostly lacked incomplete cell partitioning but showed the formation of organized regions. These structural investigations can give us a tool to identify and characterize the quality of embryogenic calluses.

INTRACELLULAR MOVEMENTS AND REORGANIZATION OF ELECTRICALLY FUSED SUNFLOWER PROTOPLASTS

Early events during the fusion process of sunflower protoplasts have been investigated using videoenhanced microscopy and fine structure analysis. Electrical pulses applied for membrane breakdown induced an immediate cessation of cytoplasmic streaming, which resumed 0.5-5 min after pulsing. This observation may provide a marker for fusion product viability and an indicator to optimize fusion parameters. Correlated with the cessation of cytoplasmic streaming, internal vesicles formed probably from the ER. Two types of fusion events were observed: (1) protoplasts interconnected by cytoplasmic bridges showing no rounding up and subsequent individual cell divisions and (2) protoplasts connected by a broad fusion plane rounding up in ca. 1 h. This process was accompanied by accumulation of cytoplasm in the fusion plane, formation of cytoplasmic strands connecting the fused protoplasts, and dislocation of the nuclei. The creation of viable fusion products required not only membrane fusion but also complete reorganization of the cytoplasmic material.

Fusion of Sunflower Protoplasts: Isoenzyme Analysis and Regeneration Capacity

ABSTRACTHypocotyl protoplasts isolated from two sunflower cultivars were electrically fused and subsequently cultivated. After about six weeks of culture, crude extracts of the generated calli were analyzed by starchgel electrophoresis. The gels were stained for the isoenzyme activities of glucosephosphate isomerase and malate dehydrogenase, which allow discrimination between the cultivars used for the fusion experiment. About 26% of the calli generated from pulsed protoplasts showed isoenzyme patterns of putative binary heterokaryocytes. The isoenzyme pattern of the nonfused control revealed less than 5% of possible chimeric colonies. Additionally, in a similar type of experiment, different culture regimes of the protoplasts were investigated. Calli treated with relatively high auxin concentrations generated somatic embryos, however, mostly those originating from electrostimulated protoplasts.