Over-expression of α-galactosidase in pea seeds to reduce raffinose oligosaccharide contentThis paper is one of a selection of papers published in a Special Issue from the National Research Council of Canada – Plant Biotechnology Institute.

Botany ◽  
2009 ◽  
Vol 87 (6) ◽  
pp. 526-532 ◽  
Author(s):  
Patricia L. Polowick ◽  
David S. Baliski ◽  
Cheryl Bock ◽  
Heather Ray ◽  
Fawzy Georges
Keyword(s):  
Intestinal Flora ◽  
National Research Council ◽  
Plant Biotechnology ◽  
Single Copy ◽  
Over Expression ◽  
Transgenic Lines ◽  
Pea Seeds ◽  
Plant Families ◽  
Coffea Arabica L ◽  
Selection Of

The raffinose family of oligosaccharides (RFO) is a series of complex carbohydrates stored in seeds of many plant families, especially in legumes. The digestive system of nonruminant animals, including that of humans, cannot break down all of the chemical bonds in these carbohydrates; therefore, catabolism is achieved anaerobically by intestinal flora. The resulting digestive problems reduce acceptance and limit the widespread consumption of these otherwise nutritious seeds. To demonstrate a solution to this problem, transgenic lines of pea ( Pisum sativum  L.) expressing the α-galactosidase gene from coffee ( Coffea arabica L.) were developed. Plants with a single copy of the inserted gene were selected, and two of these lines showed significant reductions of up to 40% in oligosaccharide content (raffinose, stachyose). Quantitative RT-PCR confirmed the presence of the α-galactosidase RNA in both leaves and cotyledons. Sugars were analyzed using whole seeds or only a portion of a seed; in the latter case, germination rates for each of the seeds analyzed were determined. The reduced raffinose contents did not affect germination rates, which remained very high (96%). The relative oligosaccharide contents of tissues within a seed also were determined; these were highest in the embryo axis, lower in the cotyledon and lowest in the seed coat.

Molecular cloning and characterization of Dbr1, a 2-alkenal reductase from Artemisia annuaThe nucleotide sequence reported in this article has been deposited in the GenBank database under accession No. FJ750460.This paper is one of a selection of papers published in a Special Issue from the National Research Council of Canada – Plant Biotechnology Institute.

Botany ◽  
2009 ◽  
Vol 87 (6) ◽  
pp. 643-649 ◽  
Author(s):  
Yansheng Zhang ◽  
Keat H. Teoh ◽  
Darwin W. Reed ◽  
Patrick S. Covello
Keyword(s):  
Double Bond ◽  
Artemisia Annua ◽  
Sequence Similarity ◽  
National Research Council ◽  
Plant Biotechnology ◽  
In Planta ◽  
Carbon Double Bond ◽  
Expressed Sequence ◽  
Selection Of

The molecular genetics of carbon–carbon double bond reduction in the plant Artemisia annua  L. was studied. Expressed sequence tags from this plant were investigated for sequences with similarity to known double-bond reductases. This resulted in the isolation of a cDNA, corresponding to the gene A. annua Dbr1 (Double bond reductase1), encoding a member of the medium chain dehydrogenase/reductase protein superfamily with sequence similarity to tobacco allyl alcohol dehydrogenase. Recombinant A. annua Dbr1 protein was purified from Escherischia coli and shown to catalyze the reduction of the carbon–carbon double bond of 2-alkenals. This activity included the reduction of the double bond at C11–C13 in the artemisinin precursor artemisinic aldehyde, albeit with unnatural stereochemistry. The substrate specificity, product stereochemistry, and expression pattern of A. annua Dbr1 point to its involvement in planta in the detoxification of 2-alkenals, which may be generated under oxidative stress conditions.

Adaptive epigenetic memory of ancestral temperature regime in Arabidopsis thalianaThis paper is one of a selection of papers published in a Special Issue from the National Research Council of Canada – Plant Biotechnology Institute.

Botany ◽  
2009 ◽  
Vol 87 (6) ◽  
pp. 650-657 ◽  
Author(s):  
C. A. Whittle ◽  
S. P. Otto ◽  
M. O. Johnston ◽  
J. E. Krochko
Keyword(s):  
Temperature Regime ◽  
National Research Council ◽  
Plant Biotechnology ◽  
Environmental Parameters ◽  
Adaptive Responses ◽  
Temperature Regimes ◽  
Heat Treated ◽  
Epigenetic Phenomenon ◽  
Selection Of ◽  
Classical Genetics

Although certain acquired nongenetic (i.e., epigenetic) traits are known to be heritable in plants, little is known currently about whether environmental parameters can induce adaptive epigenetic responses in plants and whether such effects can persist through generations. We used an experimental design based on classical genetics principles to assess whether plants respond to the environmental conditions of their ancestors in an adaptive epigenetic manner. An extensive examination of genetically identical Arabidopsis thaliana (L.) Heynh lines exposed to mild heat (30 °C) or cold (16 °C) treatments in the parental and F1 generations revealed that the prior elevated temperature regime lead to a greater than fivefold improvement in fitness (seed production per individual) for plants exposed to heat in a later generation (F3). The heat-specific fitness improvements among F3 plants were observed even though the heat-treated parental and F1 generations were followed by a generation grown at a normal temperature (F2) and point towards a temperature-induced adaptive epigenetic phenomenon. No such adaptive responses were detected for cold-treated plants, indicating that there are distinctive biological processes inherent to these two temperature regimes. Overall, the data are consistent with the existence of an environmentally induced epigenetic and heritable adaptive response in plants.

Molecular modification of triacylglycerol accumulation by over-expression of DGAT1 to produce canola with increased seed oil content under field conditionsThis paper is one of a selection of papers published in a Special Issue from the National Research Council of Canada – Plant Biotechnology Institute.

Botany ◽  
2009 ◽  
Vol 87 (6) ◽  
pp. 533-543 ◽  
Author(s):  
David C. Taylor ◽  
Yan Zhang ◽  
Arvind Kumar ◽  
Tammy Francis ◽  
E. Michael Giblin ◽  
...  
Keyword(s):  
Field Trial ◽  
Oil Content ◽  
Seed Oil ◽  
National Research Council ◽  
Plant Biotechnology ◽  
Field Trials ◽  
Substantial Effect ◽  
Oilseed Crop ◽  
Seed Oil Content ◽  
Over Expression

The final step in the Kennedy pathway for seed oil synthesis is catalyzed by an acyl-CoA-dependent diacylglycerol acyltransferase, DGAT1 (EC. 2.3.1.20). We have cloned DGAT1 genes from both Arabidopsis thaliana (L.) Heynh ecotype Columbia and Brassica napus ‘Jet Neuf’ and over-expressed them in canola under the control of the seed-specific promoter, napin. DGAT1 from A. thaliana was inserted into B. napus ‘Quantum,’ whereas DGAT1 from B. napus was introduced into the B. napus double haploid breeding line DH12075. Both sets of transgenic plants exhibited increased seed oil content in both greenhouse and in field trial settings, ranging from 2.5% to 7% of dried mass on an absolute basis. The ‘Quantum’ transgenic lines were field-tested in plots at Watrous, Saskatchewan, in 2006 and 2007. Larger scale field trials of the DH12075 transgenics were carried out in 2007 at Ellerslie and Vegreville, Alberta. This is the first study wholly dedicated to DGAT1 over-expression and the resultant oil-content increases in transgenic canola under field conditions. Collectively, the field trial results strongly support the hypothesis that the level of DGAT1 activity during seed development in an oilseed crop can have a substantial effect on the flow of carbon into seed oil. Therefore, the over-expression of DGAT1 is a positive strategy for increasing oil content and cultivar performance in canola.

Down-regulation of galactinol synthesis in oilseed Brassica napus leads to significant reduction of antinutritional oligosaccharidesThis paper is one of a selection of papers published in a Special Issue from the National Research Council of Canada – Plant Biotechnology Institute.

Botany ◽  
2009 ◽  
Vol 87 (6) ◽  
pp. 597-603 ◽  
Author(s):  
Cheryl Bock ◽  
Heather Ray ◽  
Fawzy Georges
Keyword(s):  
Brassica Napus ◽  
National Research Council ◽  
Plant Biotechnology ◽  
Antinutritional Factors ◽  
Mrna Levels ◽  
Brassica Napus L ◽  
Gene Encoding ◽  
Galactinol Synthase ◽  
Important Intermediate ◽  
Selection Of

The utility of defatted seed meal from many crops such as canola ( Brassica napus  L.) is limited by the presence of antinutritional factors, including sucrose galactosides, raffinose, and stachyose. Anaerobic breakdown of these sugars in the digestive tract of livestock is a major source of production of farm gases. In this report, the gene encoding galactinol synthase was isolated from B. napus and reintroduced into the same species in an antisense orientation to limit the production of galactinol, an important intermediate in the biosynthesis of raffinose and stachyose. This approach substantially reduced the accumulation of galactinol and stachyose in mature transgenic canola seed. Substantial changes in the mRNA levels of galactinol synthase and several sugar-related genes were also observed.

Over-expression of the rice OsAMT1-1 gene increases ammonium uptake and content, but impairs growth and development of plants under high ammonium nutrition

2006 ◽  
Vol 33 (2) ◽  
pp. 153 ◽  
Author(s):  
Mohammad S. Hoque ◽  
Josette Masle ◽  
Michael K. Udvardi ◽  
Peter R. Ryan ◽  
Narayana M. Upadhyaya
Keyword(s):  
Transgenic Plants ◽  
Ammonium Assimilation ◽  
Ammonium Transporter ◽  
Ammonium Uptake ◽  
Vegetative Stage ◽  
Over Expression ◽  
Transgenic Lines ◽  
Ammonium Nutrition ◽  
Maize Ubiquitin Promoter

A transgenic approach was undertaken to investigate the role of a rice ammonium transporter (OsAMT1-1) in ammonium uptake and consequent ammonium assimilation under different nitrogen regimes. Transgenic lines overexpressing OsAMT1-1 were produced by Agrobacterium-mediated transformation of two rice cultivars, Taipei 309 and Jarrah, with an OsAMT1-1 cDNA gene construct driven by the maize ubiquitin promoter. Transcript levels of OsAMT1-1 in both Taipei 309 and Jarrah transgenic lines correlated positively with transgene copy number. Shoot and root biomass of some transgenic lines decreased during seedling and early vegetative stage compared to the wild type, especially when grown under high (2 mm) ammonium nutrition. Transgenic plants, particularly those of cv. Jarrah recovered in the mid-vegetative stage under high ammonium nutrition. Roots of the transgenic plants showed increased ammonium uptake and ammonium content. We conclude that the decreased biomass of the transgenic lines at early stages of growth might be caused by the accumulation of ammonium in the roots owing to the inability of ammonium assimilation to match the greater ammonium uptake.

A novel T-DNA vector design for selection of transgenic lines with simple transgene integration and stable transgene expression

2005 ◽  
Vol 32 (8) ◽  
pp. 671 ◽  
Author(s):  
Song Chen ◽  
Christopher A. Helliwell ◽  
Li-Min Wu ◽  
Elizabeth S. Dennis ◽  
Narayana M. Upadhyaya ◽  
...  
Keyword(s):  
Transgene Expression ◽  
Marker Gene ◽  
Direct Repeat ◽  
Transgene Silencing ◽  
Selective Marker ◽  
Hairpin Rna ◽  
Transgenic Lines ◽  
Flanking Sequences ◽  
Dna Vector ◽  
Selection Of

Plants transformed with Agrobacterium frequently contain T-DNA concatamers with direct-repeat (d / r) or inverted-repeat (i / r) transgene integrations, and these repetitive T-DNA insertions are often associated with transgene silencing. To facilitate the selection of transgenic lines with simple T-DNA insertions, we constructed a binary vector (pSIV) based on the principle of hairpin RNA (hpRNA)-induced gene silencing. The vector is designed so that any transformed cells that contain more than one insertion per locus should generate hpRNA against the selective marker gene, leading to its silencing. These cells should, therefore, be sensitive to the selective agent and less likely to regenerate. Results from Arabidopsis and tobacco transformation showed that pSIV gave considerably fewer transgenic lines with repetitive insertions than did a conventional T-DNA vector (pCON). Furthermore, the transgene was more stably expressed in the pSIV plants than in the pCON plants. Rescue of plant DNA flanking sequences from pSIV plants was significantly more frequent than from pCON plants, suggesting that pSIV is potentially useful for T-DNA tagging. Our results revealed a perfect correlation between the presence of tail-to-tail inverted repeats and transgene silencing, supporting the view that read-through hpRNA transcript derived from i / r T-DNA insertions is a primary inducer of transgene silencing in plants.

scholarly journals Peculiarities of the Transformation of Asteraceae Family Species: The Cases of Sunflower and Lettuce

2021 ◽  
Vol 12 ◽  
Author(s):  
Flavia Soledad Darqui ◽  
Laura Mabel Radonic ◽  
Valeria Cecilia Beracochea ◽  
H. Esteban Hopp ◽  
Marisa López Bilbao
Keyword(s):  
Genetic Transformation ◽  
Molecular Farming ◽  
Plant Biotechnology ◽  
Transformation Techniques ◽  
Single Compound ◽  
Complete Sequencing ◽  
Plant Families ◽  
Transformation Vectors ◽  
Asteraceae Family

The Asteraceae family is the largest and most diversified family of the Angiosperms, characterized by the presence of numerous clustered inflorescences, which have the appearance of a single compound flower. It is estimated that this family represents around 10% of all flowered species, with a great biodiversity, covering all environments on the planet, except Antarctica. Also, it includes economically important crops, such as lettuce, sunflower, and chrysanthemum; wild flowers; herbs, and several species that produce molecules with pharmacological properties. Nevertheless, the biotechnological improvement of this family is limited to a few species and their genetic transformation was achieved later than in other plant families. Lettuce (Lactuca sativa L.) is a model species in molecular biology and plant biotechnology that has easily adapted to tissue culture, with efficient shoot regeneration from different tissues, organs, cells, and protoplasts. Due to this plasticity, it was possible to obtain transgenic plants tolerant to biotic or abiotic stresses as well as for the production of commercially interesting molecules (molecular farming). These advances, together with the complete sequencing of lettuce genome allowed the rapid adoption of gene editing using the CRISPR system. On the other hand, sunflower (Helianthus annuus L.) is a species that for years was considered recalcitrant to in vitro culture. Although this difficulty was overcome and some publications were made on sunflower genetic transformation, until now there is no transgenic variety commercialized or authorized for cultivation. In this article, we review similarities (such as avoiding the utilization of the CaMV35S promoter in transformation vectors) and differences (such as transformation efficiency) in the state of the art of genetic transformation techniques performed in these two species.

Transgenic Rice Expressing Isoflavone Synthase Gene from Soybean Shows Resistance against Blast Fungus (Magnaporthe oryzae)

Plant Disease ◽  
2021 ◽  
Author(s):  
Suresh Pokhrel ◽  
Sathish K Ponniah ◽  
Yulin Jia ◽  
Oliver Yu ◽  
Muthusamy Manoharan
Keyword(s):  
Disease Resistance ◽  
Transgenic Rice ◽  
Magnaporthe Oryzae ◽  
Plant Disease Resistance ◽  
Rice Blast ◽  
Plant Disease ◽  
Plant Secondary Metabolites ◽  
Single Copy ◽  
Transgenic Lines ◽  
Isoflavone Synthase

The isoflavones are a group of plant secondary metabolites primarily synthesized in legumes and are known for their role in improving human health and plant disease resistance. The isoflavones, especially genistein, act as precursors for the production of phytoalexins, which may induce broad-spectrum disease resistance in plants. In the present study, we screened transgenic rice lines expressing the isoflavone synthase (GmIFS1) gene from soybean for rice blast (Magnaporthe oryzae) resistance. Two homozygous transgenic lines (I2 and I10), based on single copy gene integration, were identified. The expression of GmIFS1 in transgenic lines was confirmed by qRT-PCR. Genistein was detected in the transgenic lines using LC-MS/MS. Subsequently, the transgenic lines were evaluated against the rice blast pathogen, isolate YJ54 (race IB-54). The results indicated that more than 60% of the plants in both the lines (I2 and I10) showed resistance against the blast pathogen. The progenies of one of the resistant transgenic lines (I10) also showed more than 65% resistance against rice blast. The resistance of these transgenic lines against rice blast may be attributed to the synthesis of isoflavone (genistein) in rice.

scholarly journals Expression of thaumatin, a new type of alternative sweetener in rice

2020 ◽  
Vol 48 (3) ◽  
pp. 1276-1291
Author(s):  
Shahina AKTER ◽  
Md. Amdadul HUQ ◽  
Yu-Jin JUNG ◽  
Kwon-Kyoo KANG
Keyword(s):  
Transgenic Rice ◽  
Oryza Sativa L ◽  
Plasmid Vector ◽  
Single Copy ◽  
Cauliflower Mosaic Virus ◽  
Pcr Analysis ◽  
35S Promoter ◽  
Alternative Sweetener ◽  
Sweet Proteins ◽  
Transgenic Lines

  Sweet proteins are the natural alternative to the artificial sweeteners as well as flavor enhancers. Among other sweet protein, thaumatin protein was isolated from Thaumatococcus daniellii Benth plant fruit. In this study, pinII Ti plasmid vector was constructed with thaumatin gene, where thaumatin was placed under the control of the duel cauliflower mosaic virus 35S promoter into rice (Oryza sativa L. var. japonica cv. ‘Dongjinbyeo’) by Agrobacterium-mediated transformation to generate transgenic plants. Thirteen plant lines were regenerated and the transgenic rice lines were confirmed by different molecular analysis. The genomic PCR result revealed that all of the plant lines were transgenic. The single copy and intergenic plant lines were selected by Taqman PCR analysis and FST analysis, respectively. Expression of thaumatin gene in transgenic rice resulted in the accumulation of thaumatin protein in the leave. Thaumatin protein was also accumulated in leave of T1 generation. Sensory analysis result suggested that the thaumatin protein expressing transgenic lines exerted sweet tasting activity. These results demonstrated that thaumatin was expressed in transgenic rice plants.

NPR1 enhances the DNA binding activity of the Arabidopsis bZIP transcription factor TGA7This paper is one of a selection of papers published in a Special Issue from the National Research Council of Canada – Plant Biotechnology Institute.

Botany ◽  
2009 ◽  
Vol 87 (6) ◽  
pp. 561-570 ◽  
Author(s):  
Heather L. Shearer ◽  
Lipu Wang ◽  
Catherine DeLong ◽  
Charles Despres ◽  
Pierre R. Fobert
Keyword(s):  
Salicylic Acid ◽  
Dna Binding ◽  
National Research Council ◽  
Plant Biotechnology ◽  
Plant Genome ◽  
Bzip Transcription Factor ◽  
Resistance Mutations ◽  
Promoter Elements ◽  
Tga Factors

Pathogen-induced transcriptional reprogramming of the plant genome is mediated predominantly by the cofactor NPR1 (NON-EXPRESSOR OF PATHOGENESIS-RELATED GENES1). NPR1 lacks any known DNA-binding domain and is proposed to regulate transcription through interactions with TGA transcription factors that bind to as-1-like promoter elements. Previous studies have focused on the interaction of NPR1 with subgroup I (TGA1, TGA4) or subgroup II (TGA2, TGA5, TGA6) factors. Using the yeast two-hybrid system, we showed that a member of subgroup III (TGA7) interacts with wild-type NPR1 but not with mutants in the ankyrin repeats that are important for disease resistance. Mutations in the NPR1 BTB/POZ domain also greatly reduced interaction with TGA7. NPR1 substantially increased the binding of TGA7 to cognate promoter elements in vitro, including a salicylic-acid-inducible element of the PR-1 promoter. While TGA7 interacted with all TGA factors tested, interactions were not observed between TGA2 and subgroup I factors, indicating that cross-clade interaction is not a general property of the family. Transcripts from subgroup III TGA factors were weakly inducible by salicylic acid and pathogens, but only TGA3 expression was dependent on NPR1. These results suggest that NPR1-mediated DNA binding of TGA7 could regulate the activation of defense genes.

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