Günther Hahne

Transformation of sunflower (Helianthus annuus L.): a reliable protocol.

SummaryA reliable protocol for the transformation of cultivated sunflower (Helianthus annuus L.) has been established, based on microprojectile bombardment of half shoot apices in combination with Agrobacterium tumefaciens coculture. Transgenic shoots have been obtained from 5 inbred lines, although transformation efficiencies varied with the genotype. Plants expressing the transgenes could be recovered from up to 7% of the explants. A minority of plants was shown to be chimaeric for expression of ß-glucuronidase activity while most appeared to be uniformly transformed. Genetic segregation was 3∶1 for both ß-glucuronidase and neomycine phospho transferase in some plants, indicating that the respective mother plants were uniformly transformed. Integration of the foreign genes was also shown by Southern analysis.

Plant regeneration at high frequency from mature sunflower cotyledons

Methods for sunflower (Helianthus annuus L.) tissue culture and transformation are to date characterized by a low degree of efficiency, reliability and reproducibility. The most widely used approach for regeneration of plants from somatic cells makes use of immature embryos, a material laborious to obtain. We describe here an efficient regeneration system using cotyledons of young plantlets, yielding up to ten shoots per cotyledon. Factors found to influence quantitative aspects (frequency of response) and qualitative aspects (response type) include hormonal balance and nitrogen supply in the medium, as well as explant type, age and genotype. Physical culture conditions, while exerting a certain effect, appear to be of minor importance. A number of regenerated plants have been transferred to the greenhouse and seeds were obtained from each plant. The total time required from isolation of the explant to harvest of seeds was 4–6 months.

Immuno-cytochemical localization of indole-3-acetic acid during induction of somatic embryogenesis in cultured sunflower embryos

Abstract. Immature zygotic embryos of sunflower (Helianthus annuus L.) produce somatic embryos when cultured on medium supplemented with a cytokinin as the sole source of exogenous growth regulators. The timing of the induction phase and subsequent morphogenic events have been well characterized in previous work. We address here the question of the role of endogenous indole-3-acetic acid (IAA), since auxins are known to have a crucial role in the induction of somatic embryogenesis in many other culture and regeneration systems. The fact that in the sunflower system no exogenous auxin is required for the induction of somatic embryos makes this system very suitable for the study of the internal dynamics of IAA. We used an immuno-cytochemical approach to visualize IAA distribution within the explants before, during and after the induction phase. IAA accumulated transiently throughout cultured embryos during the induction phase. The detected signal was not uniform but certain tissues, such as the root cap and the root meristem, accumulated IAA in a more pronounced manner. IAA accumulation was not restricted to the reactive zone but the kinetics of endogenous variations strikingly mimic the pulse of IAA that is usually provoked by exogenous IAA application. The direct evidence presented here indicates that an endogenous auxin pulse is indeed among the first signals leading to the induction of somatic embryogenesis.

Use of green fluorescent protein for detection of transformed shoots and homozygous offspring

Abstract Using tobacco as a model system, the data obtained demonstrated that the green fluorescent protein (GFP) can be used as a visual selection marker for transformed tissues. Based on differences in the intensity of GFP fluorescence, homozygous and hemizygous states could be easily visualized in seeds and seedlings of the T1 generation. These results were confirmed by genetic analysis. Optimized conditions for GFP analysis of stable transformants are presented.

Somatic embryogenesis and organogenesis induced on the immature zygotic embryo of sunflower (Helianthus annum L.) cultivated in vitro: role of the sugar.

SummaryImmature zygotic embryos of sunflower constitute an experimental system where the change of a single key factor (sucrose concentration) conditions the in vitro morphogenesis to either organogenesis (87 mM sucrose) or somatic embryogenesis (350 mM sucrose). Experiments with a variety of culture media differing in the sugar type and concentration, as well as osmotic pressure, indicate that a minimal threshold level of both, sugar supply and osmotic pressure, are required for somatic embryogenesis, but not organogenesis, to occur. The nature of the sugar used, though, was less important.

The potential impact of plant biotechnology on the Millennium Development Goals

The eight Millennium Development Goals (MDGs) are international development targets for the year 2015 that aim to achieve relative improvements in the standards of health, socioeconomic status and education in the world’s poorest countries. Many of the challenges addressed by the MDGs reflect the direct or indirect consequences of subsistence agriculture in the developing world, and hence, plant biotechnology has an important role to play in helping to achieve MDG targets. In this opinion article, we discuss each of the MDGs in turn, provide examples to show how plant biotechnology may be able to accelerate progress towards the stated MDG objectives, and offer our opinion on the likelihood of such technology being implemented. In combination with other strategies, plant biotechnology can make a contribution towards sustainable development in the future although the extent to which progress can be made in today’s political climate depends on how we deal with current barriers to adoption.

Release of Phytotoxic Factors from Plant Cell Walls During Protoplast Isolation

Isolation of protoplasts from plant cells involves a number of steps, the impact of which on the physiological response of the resulting protoplasts is not well understood. In particular, the usual treatment of cells with hydrolytic enzymes of fungal origin has received little attention, except for empirical selection of appropriate commercial preparations and, in some cases, their purification via gel filtration.

Different Pathogenesis-Related-Proteins are Expressed inSunflower (Helianthus annuus L.) in Response to Physical, Chemical and Stress factors

Summary PR proteins are induced by a broad range of stresses experienced by plants, from pathogen attacks toabnormal concentrations of plant hormones, or the presence of pollutants such as heavy metals. We have studied the effect of different treatments on the production of PR proteins of class 1 (PR1), 2 (PR2), 3 (PR3) and S (PRS) in sunflower leaf disks. Auxins and ethylene applications, but not cytokinins, induced high levels of sunflower PR proteins. Oxalic acid, the major toxin of Sclerotinia sclerotiorum , as well as citric acid were found to be efficient inducers. Manganese chloride induced all PR proteins, whereas mercuric chloride slightly induced only two classes of sunflower PR proteins. UV light exposure of the leaf disks provoked a strong induction of sunflower PR3 and PRS. Phenotypic variability between batches of sunflower plants was manifest at the level of their PR proteinproduction. Greenhouse-grown sunflower plants often presented necrotic symptoms of unknown origins, and we have shown here that they produced high levels of PR proteins of all 4 types characterized in sunflower. Plants grown in the more controlled conditions of an incubator never presented such a high level of PR protein production. However, comparison of β-1,3-glucanase activities between individual plants of the same batch showed significant differences between plants, indicating that the small variations of growth conditions may provoke different physiological states. This may be one of the causes of the often observed variability in experiments using explants excised from whole plants.

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

SummaryCotyledon 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.

Transient gene expression in sunflower (Helianthus annuus L.) following microprojectile bombardment

Abstract Transient expression of the uidA gene, coding for β-glucuronidase (GUS) and driven by different promoters, has been induced in sunflower cotyledonary explants and immature zygotic embryos of different developmental stages using two particle delivery systems. Explants were evaluated for expression of β-glucuronidase activity 3 days, 2, and 4 weeks post bombardment. Immature embryos were more suitable for transformation than cotyledons derived from mature seeds. The highest level of transient GUS expression after 3 and 14 days was obtained with embryos (≤1.5 mm) precultured for 3 days in the presence of NAA and BAP. After 2 and 4 weeks of culture, increased GUS activities were determined following bombardment with plasmids harboring a doubled CaMV 35S and a stress inducible promoter, respectively. In cotyledons, GUS-expressing cells were mostly observed in the epidermal layer, while in immature embryos they were located between the epidermis and the fourth mesophyll layer. The performance of the two biolistic equipments was comparable. Under any condition, GUS expression declined with increasing culture time.