Ingo Potrykus

Agarose plating and a bead type culture technique enable and stimulate development of protoplast-derived colonies in a number of plant species

AbstractTwo novel techniques improve division and colony formation from protoplasts:1)Plating in agarose stimulates colony formation of protoplasts from a wide range of species. Protoplasts from Nicotiana tabacum developed to colonies from lower initial population densities in agarose than in agar or liquid. Protoplasts from Hyoscyamus muticus which do not divide in agar divided and formed colonies in agarose at higher efficiencies than in liquid medium.2)Culture of gel embedded protoplasts in large volumes of liquid medium on a gyrotatory shaker (‘bead culture’) further improved plating efficiencies in some species (e.g. Lycopersicon esculentum and Crepis capillaris) and enabled sustained proliferation of protoplasts which had not previously developed beyond the few cell colony stage (Brassica rapa and a mutator gene variety of Petunia hybrida).nThe combination of ‘agarose plating’ and ‘bead culture’ dramatically improved plating efficiencies of protoplasts in all species tested.

Genetic engineering approaches to improve the bioavailability and the level of iron in rice grains

Abstractu2002Iron deficiency is the most widespread micronutrient deficiency world-wide. A major cause is the poor absorption of iron from cereal and legume-based diets high in phytic acid. We have explored three approaches for increasing the amount of iron absorbed from rice-based meals. We first introduced a ferritin gene from Phaseolus vulgaris into rice grains, increasing their iron content up to two-fold. To increase iron bioavailability, we introduced a thermotolerant phytase from Aspergillus fumigatus into the rice endosperm. In addition, as cysteine peptides are considered a major enhancer of iron absorption, we overexpressed the endogenous cysteine-rich metallothionein-like protein. The content of cysteine residues increased about seven-fold and the phytase level in the grains about 130-fold, giving a phytase activity sufficient to completely degrade phytic acid in a simulated digestion experiment. High phytase rice, with an increased iron content and rich in cysteine-peptide, has the potential to greatly improve iron nutrition in rice-eating populations.

Fighting Iron Deficiency Anemia with Iron-Rich Rice

Objective: Iron deficiency is estimated to affect about 30% of the world population. Iron supplementation in the form of tablets and food fortification has not been successful in developing countries, and iron deficiency is still the most important deficiency related to malnutrition. Here we present experiments that aim to increase the iron content in rice endosperm and to improve its absorption in the human intestine by means of genetic engineering. Methods: We first introduced a ferritin gene from Phaseolus vulgaris into rice grains, increasing their iron content up to twofold. To increase iron bioavailability, we introduced a thermo-tolerant phytase from Aspergillus fumigatus into the rice endosperm. In addition, as cysteine peptides are considered major enhancers of iron absorption, we over-expressed the endogenous cysteine-rich metallothionein-like protein. Results: The content of cysteine residues increased about sevenfold and the phytase level in the grains about one hundred and thirtyfold, giving a phytase activity sufficient to completely degrade phytic acid in a simulated digestion experiment. Conclusions: This rice, with higher iron content, rich in phytase and cysteine-peptide has a great potential to substantially improve iron nutrition in those populations where iron deficiency is so widely spread.

Herbicide-resistant Indica rice plants from IRRI breeding line IR72 after PEG-mediated transformation of protoplasts.

The commercially important Indica rice cultivar Oryza sativa cv. IR72 has been transformed using direct gene transfer to protoplasts. PEG-mediated transformation was done with two plasmid constructs containing either a CaMV 35S promoter/HPH chimaeric gene conferring resistance to hygromycin (Hg) or a CaMV 35S promoter/BAR chimaeric gene conferring resistance to a commercial herbicide (Basta) containing phosphinothricin (PPT). We have obtained so far 92 Hgr and 170 PPTr IR72 plants from protoplasts through selection. 31 Hgr and 70 PPTr plants are being grown in the greenhouse to maturity. Data from Southern analysis and enzyme assays proved that the transgene was stably integrated into the host genome and expressed. Transgenic plants showed complete resistance to high doses of the commercial formulations of PPT.

Genetic engineering of wheat for increased resistance to powdery mildew disease

Abstractu2002Fungal wheat (Triticum aestivum) diseases greatly affect crop productivity and require the economically and ecologically undesirable application of fungicides in wheat agriculture. We have generated transgenic wheat plants constitutively expressing an antifungal barley-seed class II chitinase. The transgene was stably expressed and the chitinase properly localized in the apoplast of the transgenic lines. The engineered wheat plants showed increased resistance to infection with the powdery mildew-causing fungus Erysiphe graminis.

Antifungal activity of a virally encoded gene in transgenic wheat

The cDNA encoding the antifungal protein KP4 from Ustilago maydis-infecting virus was inserted behind the ubiquitin promoter of maize and genetically transferred to wheat varieties particularly susceptible to stinking smut (Tilletia tritici) disease. The transgene was integrated and inherited over several generations. Of seven transgenic lines, three showed antifungal activity against U. maydis. The antifungal activity correlated with the presence of the KP4 transgene. KP4-transgenic, soil-grown wheat plants exhibit increased endogenous resistance against stinking smut.

Analysis of genetic stability of plants regenerated from suspension cultures and protoplasts of meadow fescue (Festuca pratensis Huds.).

A cytological and molecular analysis was performed to assess the genetic uniformity and true-to-type character of plants regenerated from 20 week-old embryogenic suspension cultures of meadow fescue (Festuca pratensis Huds.), and compared to protoplastderived plants obtained from the same cell suspension. Cytological variation was not observed in a representative sample of plants regenerated directly from the embryogenic suspensions and from protoplasts isolated therefrom. Similarly, no restriction fragment length polymorphisms (RFLPs) were detected in the mitochondrial, plastid and nuclear genomes in the plants analyzed. Randomly amplified polymorphic DNA markers (RAPDs) have been used to characterise molecularly a set of mature meadow fescue plants regenerated from these in vitro cultures. RAPD markers using 18 different short oligonucleotide primers of arbitrary nucleotide sequence in combination with polymerase chain reaction (PCR) allowed the detection of pre-existing polymorphisms in the donor genotypes, but failed to reveal newly generated variation in the protoplast-derived plants compared to their equivalent suspensionculture regenerated materials.The genetic stability of meadow fescue plants regenerated from suspension cultures and protoplasts isolated therefrom and its implications on gene transfer technology for this species are discussed.

Transgenic Italian ryegrass (Lolium multiflorum) plants from microprojectile bombardment of embryogenic suspension cells

Transgenic forage-type Italian ryegrass (Lolium multiflorum Lam.) plants have been obtained by microprojectile bombardment of embryogenic suspension cells using a chimeric hygromycin phosphotransferase (hph) gene construct driven by riceActl 5′ regulatory sequences. Parameters for the bombardment of embryogenic suspension cultures with the particle inflow gun were partially optimized using transient expression assays of a chimericβ-glucuronidase (gusA) gene driven by the maizeUbi1 promoter. Stably transformed clones were recovered with a selection scheme using hygromycin in liquid medium followed by a plate selection. Plants were regenerated from 33% of the hygromycin-resistant calli. The transgenic nature of the regenerated plants was demonstrated by Southern hybridization analysis. Expression of the transgene in transformed adult Italian ryegrass plants was confirmed by northern analysis and a hygromycin phosphotransferase enzyme assay.

Effects of combined expression of antifungal barley seed proteins in transgenic wheat on powdery mildew infection

Transgenicwheat plants (variety Frisal) constitutively expressing a number of potentialantifungal proteins alone or in combinations were generated and tested forincreased resistance to Blumeria graminis f.sp. tritici(powdery mildew) in a detached leaf infection assay. The most significativerateof protection was obtained with an apoplastic ribosome-inactivation proteinfrombarley seed. Apoplastic Barnase was less efficient and individual plant linesharbouring a barley seed chitinase and β-1,3-glucanase showed linespecificphenotypes from increased resistance to increased susceptibility. Combinationbycrossing of three barley seed proteins did not lead to significant improvementof protection.

Biotechnology in forage and turf grass improvement

1 Introduction.- 1.1 Agronomic Importance of the Festuca-Lolium Complex..- 1.1.1 Major Festuca Species.- 1.1.2 Major Lolium Species.- 1.2 Distribution of Fescues and Ryegrasses.- 1.3 Biotechnology in Festuca-Lolium Improvement: General Considerations.- References.- 2 Meristem Culture.- 2.1 Introduction.- 2.2 Culture of Vegetative Meristems in Festuca and Lolium.- 2.3 Culture of Floral Meristems in Festuca and Lolium.- 2.4 Meristem Culture in Other Grasses.- 2.5 Summary and Conclusions.- References.- 3 Callus Cultures and Somaclonal Variation.- 3.1 Introduction.- 3.2 Regeneration from Callus Cultures in Festuca and Lolium.- 3.3 Somaclonal Variation in Festuca and Lolium.- 3.4 Regeneration from Callus Cultures in Other Grasses.- 3.5 Summary and Conclusions.- References.- 4 Anther Culture and Production of Haploids.- 4.1 Introduction.- 4.2 Anther Culture and Haploids in Festuca.- 4.3 Anther Culture and Haploids in Lolium.- 4.4 Anther Culture in Other Grasses.- 4.5 Summary and Conclusions.- References.- 5 Plant Regeneration from Suspension Cells and Protoplasts.- 5.1 Introduction.- 5.2 Cell Suspension and Protoplast Cultures in Festuca.- 5.2.1 Plant Regeneration from Embryogenic Cell Suspensions in Festuca.- 5.2.2 Plant Regeneration from Protoplasts in Festuca.- 5.3 Cell Suspension and Protoplast Cultures in Lolium.- 5.3.1 Plant Regeneration from Embryogenic Cell Suspensions in Lolium.- 5.3.2 Plant Regeneration from Protoplasts in Lolium.- 5.4 Suspension and Protoplast Cultures in Other Grasses.- 5.5 Summary and Conclusions.- References.- 6 Somatic Hybridization.- 6.1 Introduction.- 6.2 Somatic Hybridization in Festuca and Lolium.- 6.3 Cybridization in Festuca and Lolium.- 6.4 Somatic Hybridization in Other Grasses.- 6.5 Summary and Conclusions.- References.- 7 Transgenic Plants from Protoplasts.- 7.1 Introduction.- 7.2 Direct Gene Transfer to Protoplasts in Festuca.- 7.3 Direct Gene Transfer to Protoplasts in Lolium.- 7.4 Direct Gene Transfer to Protoplasts in Other Grasses.- 7.5 Summary and Conclusions.- References.- 8 Protoplast-Independent Production of Transgenic Plants.- 8.1 Introduction.- 8.2 Protoplast-Independent Transformation in Festuca.- 8.3 Protoplast-Independent Transformation in Lolium.- 8.4 Protoplast-Independent Transformation in Other Grasses.- 8.5 Summary and Conclusions.- References.- 9 Molecular Markers.- 9.1 Introduction.- 9.2 Molecular Markers in Festuca and Lolium.- 9.2.1 Isozyme Markers.- 9.2.2 Species-Specific Repetitive DNA Sequences.- 9.2.3 RFLP Markers.- 9.2.4 RAPD Markers.- 9.3 Molecular Markers in Other Grasses.- 9.4 Summary and Conclusions.- References.- 10 Perspectives.- 10.1 Introduction.- 10.2 Forage Quality.- 10.2.1 Manipulation of Lignin Biosynthesis.- 10.2.2 Manipulation of Fructan Metabolism.- 10.2.3 Transgenic Expression of Rumen By-pass Proteins.- 10.3 Disease and Pest Resistance.- 10.3.1 Fungal Pathogens.- 10.3.2 Viruses.- 10.3.3 Pests.- 10.4 Growth and Development.- 10.4.1 Manipulation of Pollen Allergens.- 10.4.2 Manipulation of Flowering Time and Senescence.- 10.4.3 Manipulation of Apomixis.- 10.4.4 Manipulation of Self-Incompatibility and Cytoplasmic Male Sterility.- 10.4.5 Grasses as Bioreactors.- 10.5 Summary and Conclusions.- References.