Nutritionally Enhanced Rice to Combat Malnutrition Disorders of the Poor

Major deficiency disorders, including vitamin A deficiency, are especially common in countries in which rice is the staple food. In response to the devastating effects of vitamin A deficiency, which may include blindness and, even death, "Golden Rice” has been developed to deliver this nutrient to those populations who need it most. The case of Golden Rice is used to demonstrate the challenges of radical GMO opposition, consumer acceptance, and regulation of biotechnology-derived food

Genetic Modification and the Public Good

Genetic engineering (GMO-technology) offers great opportunities to contribute to the public good by improving public health, e.g. by improving the micro-nutrient status of poor populations, cost effectively and - therefore - sustainably. The prime example for such a project from the public domain for public good is ‘Golden Rice' (www.goldenrice.org). There are exclusive public funds involved (from altruistic organizations), no dependence from industry except for in-kind support and help in acquiring free licenses for humanitarian use. There is no financial reward for anyone involved. The only beneficiaries are the poor in developing countries. Theoretically, when considering the arguments of the anti-GMO lobby, this is an ideal application of GMO-technology. However, Golden Rice is considered a Trojan Horse, which must be prevented under all circumstances. The consequence: millions of avoidable blind and dead children. The author considers those who are responsible for this avoidable suffering of many innocent children (and mothers at childbirth) a crime to humanity. There are those who commit this deliberately and those who are participating passively, such as numerous ‘humanitarian organizations' and ‘decision makers' in politics and elsewhere. There is a wealth of scientific information and broad consensus that GMO-technology is at least as safe as any other technology involved in any context with our food or our environment. What we experience here is an example of ‘unreason' and a perfect example in the context of The March of Unreason. Our ‘enlightenment' and science-based successful European culture is on the verge of being replaced by unreason-based failure and lack of cultur

Geminivirus sequences as bidirectional transcription termination/polyadenylation signals for economic construction of stably expressed transgenes

Bidirectional, convergent transcription of transgenes in transgenic plants can occur due to leaky transcription termination of separate convergent genes or from genomic promoters. It might also be engineered with the purpose of generating double-stranded RNA to downregulate genes by RNA interference. We have tested the effects of convergent transcription on expression levels and analysed the potential of geminivirus derived DNA sequences to act as bidirectional transcription termination/polyadenylation signals in transgenes to counteract such negative effects. Convergent, overlapping transcription decreased expression, however, no increased propensity for induction of gene silencing was observed. The geminivirus terminators in both orientations supported efficient expression of single genes and of convergent genes, whereas a unidirectional terminator allowed only reduced expression in this latter situation. Geminivirus terminators could be used as efficient transcription control element for the expression of two genes simultaneously or, with single genes, could afford protection of the gene against unwanted anti-sense transcription from transcription units downstream of the gene, e.g. in the integration locus. Our results also suggest that flanking of a given sequence by two convergent promoters would not be an efficient way to generate double-stranded RNA and induce gene silencing by RNA

Lincomycin treatment: A simple method to differentiate primary and processed transcripts in rice ( Oryza sativa L.) chloroplasts

Visualizing full-length primary transcripts is helpful in identifying transcription initiation sites and mapping promoter regions of plastid genes and operons. Detection of primary unprocessed transcripts from certain regions of the plastid genome is difficult, and sometimes impossible, because of their rapid and extensive processing. We tested the effect of lincomycin, a prokaryotic protein synthesis inhibiter, on in vivo RNA processing activities in different types of rice plastid. Steady-state levels of RNA produced from the region of the rice plastid genome that includes thetrnV and 16s rRNA genes were analysed by using an RNase protection assay. Results show that sublethal lincomycin levels inhibit RNA processing in leaf chloroplasts and allow the accumulation of primary transcripts, easily distinguishable from processed and processing intermediates. These features were used to identify regions of the 16r andtrnV transcription start sites. This is the first report of the use of lincomycin for mapping plastidic transcript

High efficiency transient and stable transformation by optimized DNA microinjection into Nicotiana tabacum protoplasts

An efficient system has been established that allows well controlled DNA microinjection into tobacco (Nicotiana tabacum) mesophyll protoplasts with partially regenerated cell walls and subsequent analysis of transient as well as stable expression of injected reporter genes in particular targeted cells or derived clones. The system represents an effective tool to study parameters important for the successful transformation of plant cells by microinjection and other techniques. Protoplasts were immobilized in a very thin layer of medium solidified with agarose or alginate. DNA microinjection was routinely monitored by coinjecting FITC-dextran and aimed at the cytoplasm of target cells. The injection procedure was optimized for efficient delivery of injection solution into this compartment. Cells were found to be at the optimal stage for microinjection about 24 h after immobilization in solid medium. Embedded cells could be kept at this stage for up to 4 d by incubating them at 4 °C in the dark. Within 1 h successful delivery of injection, solution was routinely possible into 20-40 cells. Following cytoplasmic coinjection of FITC-dextran and pSHI913, a plasmid containing the neo (neomycin phosphotransferase II) gene, stably transformed, paromomycin-resistant clones could be recovered through selection. Transgenic tobacco lines have been established from such clones. Injection solutions containing pSHI913 at a concentration of either 50 μg ml−1 or 1 mg ml−1 have been tested. With 1 mg ml−1 plasmid DNA the percentage of resistant clones per successfully injected cell was determined to be about 3.5 times higher. Incubation of embedded protoplasts at 4°C before microinjection was found to reduce the percentage of resistant clones obtained per injected cell Protoplasts were immobilized above a grid pattern and the location of injected cells was recorded by Polaroid photography. The fate of particular targeted cells could be observed. Isolation and individual culture of clones derived from injected cells was possible. Following cytoplasmic coinjection of FITC-dextran and 1 mg ml−1 plasmid DNA on average about 20% of the targeted cells developed into microcalli and roughly 50% of these calli were stably transformed. Transient expression of the firefly luciferase gene (Luc) was nondestructively analysed 24 h after injection of pAMLuc. Approximately 50% of the injected cells that were alive at this time point expressed the Luc gene transiently. Apparently, stable integration of the injected genes occurred in essentially all transiently expressing cells that developed into clone

Two cassava promoters related to vascular expression and storage root formation

Cassava (Manihot esculenta Crantz) storage roots, organs accumulating large amounts of starch, develop from primary roots via secondary growth. The availability of promoters related to storage-root formation is a prerequisite for engineering root traits in cassava. Two cDNAs, c15 and c54, were identified from a storage-root cDNA library of cassava MCol1505 via differential screening. The transcripts of c15 and c54 were detected in storage roots but not in leaves by Northern analysis. Homology analysis of the deduced amino acid sequences showed that C15 is likely to be related to cytochrome P450 proteins, which are involved in the oxidative degradation of various compounds, while C54 may be related to Pt2L4, a cassava glutamic acid-rich protein. The promoter regions of c15 and c54 were isolated from the corresponding clones in a cassava genomic library. A 1,465-bp promoter fragment (p15/1.5) of c15 and a 1,081-bp promoter region (p54/1.0) of c54 were translationally fused to the uidA reporter gene, and introduced into cassava and Arabidopsis thaliana (L.) Heynh. The expression patterns of p15/1.5::uidA and p54/1.0::uidA in transgenic plants showed that both promoters are predominantly active in phloem, cambium and xylem vessels of vascular tissues from leaves, stems, and root systems. More importantly, strong β-glucuronidase activity was also detected in the starch-rich parenchyma cells of transgenic storage roots. Our results demonstrate that the two promoters are related to vascular expression and secondary growth of storage roots in cassav

Upstream and downstream sequence elements determine the specificity of the rice tungro bacilliform virus promoter and influence RNA production after transcription initiation

The contribution of sequences upstream and downstream of the transcription start site to the strength and specificity of the promoter of rice tungro bacilliform virus was analysed in transgenic rice plants. The promoter is strongly stimulated by downstream sequences which include an intron and is active in all vascular and epidermal cells. Expression in the vascular tissue requires a promoter element located between −100 and −164 to which protein(s) from rice nuclear extracts bind. Elimination of this region leads to specificity for the epidermis. Due to the presence of a polyadenylation signal in the intron, short-stop RNA is produced from the promoter in addition to full-length primary transcript and its spliced derivatives. The ratio between short-stop RNA and full-length or spliced RNA is determined by upstream promoter sequences, suggesting the assembly of RNA polymerase complexes with different processivity on this promote

Bioengineered ‘golden’ indica rice cultivars with β-carotene metabolism in the endosperm with hygromycin and mannose selection systems

Vitamin-A deficiency (VAD) is a major malnutrition problem in South Asia, where indica rice is the staple food. Indica-type rice varieties feed more than 2 billion people. Hence, we introduced a combination of transgenes using the biolistic system of transformation enabling biosynthesis of provitamin A in the endosperm of several indica rice cultivars adapted to diverse ecosystems of different countries. The rice seed-specific glutelin promoter (Gt-1 P) was used to drive the expression of phytoene synthase (psy), while lycopene β-cyclase (lcy) and phytoene desaturase (crtI), fused to the transit peptide sequence of the pea-Rubisco small subunit, were driven by the constitutive cauliflower mosaic virus promoter (CaMV35S P). Transgenic plants were recovered through selection with either CaMV35S P driven hph (hygromycin phosphotransferase) gene or cestrum yellow leaf curling virus promoter (CMP) driven pmi (phophomannose isomerase) gene. Molecular and biochemical analyses demonstrated stable integration and expression of the transgenes. The yellow colour of the polished rice grain evidenced the carotenoid accumulation in the endosperm. The colour intensity correlated with the estimated carotenoid content by spectrophotometric and HPLC analysis. Carotenoid level in cooked polished seeds was comparable (with minor loss of xanthophylls) to that in non-cooked seeds of the same transgenic line. The variable segregation pattern in T1 selfing generation indicated single to multiple loci insertion of the transgenes in the genome. This is the first report of using nonantibiotic pmi driven by a novel promoter in generating transgenic indica rice for possible future use in human nutrition