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PubMed2006

An efficient protocol for genetic transformation and shoot regeneration of turmeric (Curcuma longa L.) via particle bombardment.

Turmeric (Curcuma longa L.) is an important spice crop plant that is sterile and cannot be improved by conventional breeding. An efficient method for stable transformation for turmeric, C. longa L., was developed using particle bombardment. Callus cultures initiated from shoots were bombarded with gold particles coated with plasmid pAHC25 containing the bar and gusA genes each driven by the maize ubiquitin promoter. Transformants were selected on medium containing glufosinate. Transgenic lines were established on selection medium from 50% of the bombarded calluses. Transgenic shoots regenerated from these were multiplied and stably transformed plantlets were produced. Polymerase chain reaction (PCR) and histochemical GUS assay confirmed the stable transformation. Transformed plantlets were resistant to glufosinate.

Aminobutyrates↗
PubMed2005

Codon-modifications and an endoplasmic reticulum-targeting sequence additively enhance expression of an Aspergillus phytase gene in transgenic canola.

Transgenic plants offer advantages for biomolecule production because plants can be grown on a large scale and the recombinant macromolecules can be easily harvested and extracted. We introduced an Aspergillus phytase gene into canola (Brassica napus) (line 9412 with low erucic acid and low glucosinolates) by Agrobacterium-mediated transformation. Phytase expression in transgenic plant was enhanced with a synthetic phytase gene according to the Brassica codon usage and an endoplasmic reticulum (ER) retention signal KDEL that confers an ER accumulation of the recombinant phytase. Secretion of the phytase to the extracellular fluid was also established by the use of the tobacco PR-S signal peptide. Phytase accumulation in mature seed accounted for 2.6% of the total soluble proteins. The enzyme can be glycosylated in the seeds of transgenic plants and retain a high stability during storage. These results suggest a commercial feasibility of producing a stable recombinant phytase in canola at a high level for animal feed supplement and for reducing phosphorus eutrophication problems.

6-Phytase↗
PubMed2006

Tailoring tropane alkaloid accumulation in transgenic hairy roots of Atropa baetica by over-expressing the gene encoding hyoscyamine 6beta-hydroxylase.

Atropa baetica hairy roots, over-expressing cDNA from Hyoscyamus niger encoding the gene for hyoscyamine 6beta-hydroxylase (H6H), were produced by Agrobacterium rhizogenes infection. The transgenic roots over-expressing h6h had an altered alkaloid profile in which hyoscyamine was entirely converted into scopolamine. In the best h6h clone, scopolamine accumulation increased 9-fold compared to plants, amounting to 5.6 mg g dry wt(-1), some of which was released into the liquid medium. Only negligible amounts of hyoscyamine were detected. In contrast, the gus control culture contained a much higher amount of hyoscyamine than scopolamine, mimicking the situation in the plant. At the molecular level, a higher conversion of hyoscyamine into scopolamine was related to a higher level of h6h mRNA; in some instances this was 5 - 10-fold higher.

Alkaloids↗
PubMed2005

Analysis of RNA silencing in agroinfiltrated leaves of Nicotiana benthamiana and Nicotiana tabacum.

In this study we analyse several aspects of cytoplasmic RNA silencing by agroinfiltration of DNA constructs encoding single- and double-stranded RNAs derived from a GFP transgene and from the endogenous Virp1 gene. Both types of inductors resulted after 2-4 days in much higher concentration of siRNAs in the agroinfiltrated zone than normally seen during systemic silencing. More specifically, infiltration of two transgene hairpin constructs resulted in elevated levels of siRNAs. However, differences between the two constructs were observed: the antisense-sense arrangement was more effective than the sense-antisense order. For both double-stranded forms, we observed a relative increase of the 24-mer size class of siRNAs. When a comparable hairpin construct of the endogenous Virp1 gene was assayed, the portion of the 24-mer siRNA class remained low as observed for all kinds of single-stranded inducers. The lack of increase of Virp1-derived 24-mers was independent of the expression level, as demonstrated by agroinfiltration into a transgenic plant that overexpressed Virp1 and showed the same pattern. Using transducer constructs, we could detect within a week transitive silencing from GFP to GUS sequences in the infiltrated zone and in either direction 5'-3' and 3'-5'. Conversely, for the endogenous Virp1 gene neither transitive silencing nor the induction of systemic silencing could be observed. These results are discussed in view of the current models of RNA silencing.

Agrobacterium tumefaciens↗
PubMed2005

Biotechnology and the domestication of forest trees.

Wood is one of the major renewable materials. To compensate for the ever-increasing demand for wood and to reduce pressure on native forests, more wood of higher quality will need to be produced on less land by planting highly productive trees. Biotechnology has shown great promise for forest tree improvement and over the past 10 years this field has flourished. Not only has the potential of transgenic trees with optimized yield and quality traits been demonstrated in field trials, but progress in genetical genomics and association genetics promise quantum leaps forward for tree improvement.

Alleles↗
PubMed2002

Plant biotechnology in agriculture.

Knowledge on plant genomes has progressed during the past few years. Two plant genomes, those of Arabidopsis thaliana and rice, have been sequenced. Our present knowledge of synteny also indicates that, despite plasticity contributing to the diversity of the plant genomes, the organization of genes is conserved within large sections of chromosomes. In parallel, novel plant transformation systems have been proposed, notably with regard to plastid transformation and the removal of selectable marker genes in transgenic plants. Furthermore, a number of recent works considerably widen the potential of plant biotechnology.

Agriculture↗
PubMed2003

Plant cell factories and mucosal vaccines.

Many advances continue to be made in the field of plant-derived vaccines. Plants have been shown capable of expressing a multicomponent vaccine that when orally delivered induces a T-helper cell subset 1 response and enables passive immunization. Furthermore, a plant-derived vaccine has been shown to protect against challenge in the target host. Increased antigen expression levels (up to 4.1% total soluble protein) have been obtained through transformation of the chloroplast genome. In view of these findings, plant-derived vaccines have been proved as valuable commodities to the world's health system; however, before their application, studies need to focus on optimization of immunization strategies and to investigate antigen stability.

Administration, Oral↗
PubMed2003

From plant genomics to breeding practice.

New alleles are constantly accumulated during intentional crop selection. The molecular understanding of these alleles has stimulated new genomic approaches to mapping quantitative trait loci (QTL) and haplotype multiplicity of the genes concerned. A limited number of quantitative trait nucleotides responsible for QTL variation have been described, but an acceleration in their rate of discovery is expected with the adoption of linkage disequilibrium and candidate gene strategies for QTL fine mapping and cloning. Additional layers of regulatory variation have been studied that could also contribute to the molecular basis of quantitative genetics of crop traits. Despite this progress, the role of marker-assisted selection in plant breeding will ultimately depend on the genetic model underlying quantitative variation.

Breeding↗
PubMed1999

Down-regulation of cinnamyl alcohol dehydrogenase in transgenic alfalfa (Medicago sativa L.) and the effect on lignin composition and digestibility.

To improve the digestibility of the forage crop alfalfa (Medicago sativa L.), cinnamyl alcohol dehydrogenase (CAD), which catalyses the last step in the biosynthesis of the lignin monomers, was down-regulated by using an antisense approach. A subset of six transgenic lines with reduced CAD activity and control lines were analysed when grown in the greenhouse and in the field. The down-regulation of the CAD enzyme was associated with a red coloration of the stem. The lignin quantity remained unchanged, but the lignin composition, as determined by thioacidolysis, was altered. The highest reduction of CAD activity was associated with a lower syringyl/guaiacyl (S/G) ratio and a lower S+G yield, mainly because of a decreased amount of S units. An increase in in situ disappearance of dry matter and of cell wall residue was detected in one of the transgenic lines grown in the greenhouse, and for two of the lines grown in the field the rate of disappearance of dry matter slightly improved. Furthermore, these two lines had a higher solubility in alkali as shown by the lower yield of saponified residue. This study opens perspectives for improving forage crop digestibility by the modulation of enzymes involved in lignin biosynthesis.

Alcohol Oxidoreductases↗
PubMed2002

Starch content and yield increase as a result of altering adenylate pools in transgenic plants.

Starch represents the most important carbohydrate used for food and feed purposes. With the aim of increasing starch content, we decided to modulate the adenylate pool by changing the activity of the plastidial adenylate kinase in transgenic potato plants. As a result, we observed a substantial increase in the level of adenylates and, most importantly, an increase in the level of starch to 60% above that found in wild-type plants. In addition, concentrations of several amino acids were increased by a factor of 2-4. These results are particularly striking because this genetic manipulation also results in an increased tuber yield. The modulation of the plastidial adenylate kinase activity in transgenic plants therefore represents a potentially very useful strategy for increasing formation of major storage compounds in heterotrophic tissues of higher plants.

Adenylate Kinase↗
PubMed2001

Transgenic peanut plants obtained by particle bombardment via somatic embryogenesis regeneration system.

After pre-culture and treatment of osmosis, cotyledons of immature peanut (Arachis hypogaea L.) zygotic embryos were transformed via particle bombardment with a plasmid containing a chimeric hph gene conferring resistance to hygromycin and a chimeric intron-gus gene. Selection for hygromycin resistant calluses and somatic embryos was initiated at 10th d post-bombardment on medium containing 10-25 mg/L hygromycin. Under continuous selection, hygromycin resistant plantlets were regenerated from somatic embryos and were recovered from nearly 1.6% of the bombarded cotyledons. The presence and integration of foreign DNA in regenerated hygromycin resistant plants was confirmed by PCR (polymerase chain reaction) for the intron-gus gene and by Southern hybridization of the hph gene. GUS enzyme activity was detected in leaflets from transgenic plants but not from control, non-transformed plants. The production of transgenic plants are mainly based on a newly improved somatic embryogenesis regeneration system developed by us.

Anti-Bacterial Agents↗
PubMed1998

Development and application of a selective detection method for genetically modified soy and soy-derived products.

A method has been developed to distinguish between traditional soy beans and transgenic Roundup Ready soy beans, i.e. the glyphosate ('Roundup') resistant soy bean variety developed by Monsanto Company. Glyphosate resistance results from the incorporation of an Agrobacterium-derived 5-enol-pyruvyl-shikimate-3-phosphatesynthase (EPSPS) gene. The detection method developed is based on a nested Polymerase Chain Reaction (PCR) procedure. Ten femtograms of soy bean DNA can be detected, while, starting from whole soy beans, Roundup Ready DNA can be detected at a level of 1 Roundup Ready soy bean in 5000 non-GM soy beans (0.02% Roundup Ready soy bean). The method has been applied to samples of soy bean, soy-meal pellets and soy bean flour, as well as a number of processed complex products such as infant formula based on soy, tofu, tempeh, soy-based desserts, bakery products and complex meat and meat-replacing products. The results obtained are discussed with respect to practical application of the detection method developed.

DNA, Plant↗
PubMed2007

Tetrapyrrole biosynthesis in higher plants.

Tetrapyrroles play vital roles in various biological processes, including photosynthesis and respiration. Higher plants contain four classes of tetrapyrroles, namely, chlorophyll, heme, siroheme, and phytochromobilin. All of the tetrapyrroles are derived from a common biosynthetic pathway. Here we review recent progress in the research of tetrapyrrole biosynthesis from a cellular biological view. The progress consists of biochemical, structural, and genetic analyses, which contribute to our understanding of how the flow and the synthesis of tetrapyrrole molecules are regulated and how the potentially toxic intermediates of tetrapyrrole synthesis are maintained at low levels. We also describe interactions of tetrapyrrole biosynthesis and other cellular processes including the stay-green events, the cell-death program, and the plastid-to-nucleus signal transduction. Finally, we present several reports on attempts for agricultural and horticultural applications in which the tetrapyrrole biosynthesis pathway was genetically modified.

Apoproteins↗