scholarly journals Haploidy in Tobacco Induced by PsASGR-BBML Transgenes via Parthenogenesis

Genes ◽  
2020 ◽  
Vol 11 (9) ◽  
pp. 1072 ◽  
Author(s):  
Zhifen Zhang ◽  
Joann Conner ◽  
Yinping Guo ◽  
Peggy Ozias-Akins
Keyword(s):  
Flow Cytometry ◽  
Transgenic Tobacco ◽  
Marker Gene ◽  
Hybrid Vigor ◽  
Egg Cell ◽  
Apomictic Species ◽  
Haploid Production ◽  
Transgenic Lines ◽  
Pennisetum Squamulatum ◽  
Parthenogenetic Embryos

Background: Engineering apomixis in sexually reproducing plants has been long desired because of the potential to fix hybrid vigor. Validating the functionality of genes originated from apomictic species that contribute to apomixis upon transfer to sexually reproducing species is an important step. The PsASGR-BABYBOOM-like (PsASGR-BBML) gene from Pennisetum squamulatum confers parthenogenesis in this apomict, and its functionality was demonstrated in several sexually reproducing monocots but not in any dicots. Methods: We introduced the PsASGR-BBML gene regulated by egg cell-specific promoters, either AtDD45 or AtRKD2, into tobacco, and analyzed progeny of the transgenic lines resulting from self-pollination and crossing by flow cytometry. Results: We identified haploid progeny at a frequency lower than 1% in the AtDD45pro lines, while at a frequency of 9.3% for an octoploid (2n = 8x) AtRKD2pro line. Haploid production in the T2 generation, derived from the tetraploid T1 offspring of this original octoploid AtRKD2pro line, was also observed. Pollinated by homozygous transgenic tobacco carrying a DsRed marker gene, 4x progeny of the AtRKD2pro line yielded parthenogenetic embryos identified as DsRed negative. We verified that the DsRed negative seedlings recovered were haploid (2x). Conclusion: The PsASGR-BBML gene regulated by egg cell-specific promoters could enable parthenogenesis in tobacco, a dicotyledon species.

Co-expression of AGPS and AGPL genes encoded for AGPase from cassava to enhance starch accumulation in transgenic tobacco

2016 ◽  
Vol 14 (2) ◽  
pp. 287-293
Author(s):  
Nguyễn Văn Đoài ◽  
Nguyễn Minh Hồng ◽  
Lê Thu Ngọc ◽  
Nguyễn Thị Thơm ◽  
Nguyễn Đình Trọng ◽  
...  
Keyword(s):  
Transgenic Tobacco ◽  
Higher Plants ◽  
Starch Content ◽  
Genetically Modified Crops ◽  
Starch Accumulation ◽  
35S Promoter ◽  
Effective Strategies ◽  
Plant Expression Vector ◽  
Transgenic Lines ◽  
Cassava Varieties

The AGPase (ADP-Glucose pyrophosphorylase) is one of the ubiquitous enzymes catalyzing the first step in starch biosynthesis. It plays an important role in regulation and adjusts the speed of the entire cycle of glycogen biosynthesis in bacteria and starch in plants. In higher plants, it is a heterotetramer and tetrameric enzyme consisting two large subunits (AGPL) and two small subunits (AGPS) and encoded by two genes. In this paper, both AGPS and AGPL genes were sucessfully isolated from cassava varieties KM140 and deposited in Genbank with accession numbers KU243124 (AGPS) and KU243122 (AGPL), these two genes were fused with P2a and inserted into plant expression vector pBI121 under the control of 35S promoter. The efficient of this construct was tested in transgenic N. tabacum. The presence and expression of AGPS and AGPL in transgenic plants were confirmed by PCR and Western hybridization. The starch content was quantified by the Anthrone method. Transgenic plant analysis indicated that that two targeted genes were expressed simultaneously in several transgenic tobacco lines under the control of CaMV 35S promoter.  The starch contents in 4 analyzed tobacco transgenic lines displays the increase 13-116%  compared to WT plants. These results indicated that the co-expression of AGPS and AGPL is one of effective strategies for enhanced starch production in plant. These results can provide a foundation for developing other genetically modified crops to increase starch accumulation capacity.

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.

Early diagnosis of ploidy status in doubled haploid production of maize by stomata length and flow cytometry measurements

2019 ◽  
Vol 138 (3) ◽  
pp. 266-276 ◽  
Author(s):  
Willem S. Molenaar ◽  
Evellyn Giselly Oliveira Couto ◽  
Hans‐Peter Piepho ◽  
Albrecht E. Melchinger
Keyword(s):  
Flow Cytometry ◽  
Early Diagnosis ◽  
Doubled Haploid ◽  
Haploid Production ◽  
Ploidy Status

scholarly journals Chitin Biosynthesis Inhibition of Meloidogyne incognita by RNAi-Mediated Gene Silencing Increases Resistance to Transgenic Tobacco Plants

2020 ◽  
Vol 21 (18) ◽  
pp. 6626
Author(s):  
Vimalraj Mani ◽  
Chinreddy Subramanyam Reddy ◽  
Seon-Kyeong Lee ◽  
Soyoung Park ◽  
Hyoung-Rai Ko ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Transgenic Tobacco ◽  
Meloidogyne Incognita ◽  
Chitin Synthase ◽  
Egg Mass ◽  
Transgenic Tobacco Plants ◽  
Tobacco Plants ◽  
Wide Range ◽  
Transgenic Lines ◽  
Plant Parasitic

Meloidogyne incognita is a devastating plant parasitic nematode that causes root knot disease in a wide range of plants. In the present study, we investigated host-induced RNA interference (RNAi) gene silencing of chitin biosynthesis pathway genes (chitin synthase, glucose-6-phosphate isomerase, and trehalase) in transgenic tobacco plants. To develop an RNAi vector, ubiquitin (UBQ1) promoter was directly cloned, and to generate an RNAi construct, expression of three genes was suppressed using the GATEWAY system. Further, transgenic Nicotiana benthamiana lines expressing dsRNA for chitin synthase (CS), glucose-6-phosphate isomerase (GPI), and trehalase 1 (TH1) were generated. Quantitative PCR analysis confirmed endogenous mRNA expression of root knot nematode (RKN) and revealed that all three genes were more highly expressed in the female stage than in eggs and in the parasitic stage. In vivo, transformed roots were challenged with M. incognita. The number of eggs and root knots were significantly decreased by 60–90% in RNAi transgenic lines. As evident, root galls obtained from transgenic RNAi lines exhibited 0.01- to 0.70-fold downregulation of transcript levels of targeted genes compared with galls isolated from control plants. Furthermore, phenotypic characteristics such as female size and width were also marginally altered, while effect of egg mass per egg number in RNAi transgenic lines was reduced. These results indicate the relevance and significance of targeting chitin biosynthesis genes during the nematode lifespan. Overall, our results suggest that further developments in RNAi efficiency in commercially valued crops can be applied to employ RNAi against other plant parasitic nematodes.

scholarly journals Over-expression of poplar NAC15 gene enhances wood formation in transgenic tobacco

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Wenjing Yao ◽  
Dawei Zhang ◽  
Boru Zhou ◽  
Jianping Wang ◽  
Renhua Li ◽  
...  
Keyword(s):  
Transgenic Tobacco ◽  
Wood Formation ◽  
Potential Candidate ◽  
Rna Seq ◽  
Populus Simonii ◽  
Over Expression ◽  
Potential Candidate Gene ◽  
Relative Expression Level ◽  
Transgenic Lines ◽  
Highly Expressed Genes

Abstract Background NAC (NAM/ATAF/CUC) is one of the largest plant-specific transcription factor (TF) families known to play significant roles in wood formation. Acting as master gene regulators, a few NAC genes can activate secondary wall biosynthesis during wood formation in woody plants. Results In the present study, firstly, we screened 110 differentially expressed NAC genes in the leaves, stems, and roots of di-haploid Populus simonii×P. nigra by RNA-Seq. Then we identified a nucleus-targeted gene, NAC15 gene, which was one of the highly expressed genes in the stem among 110 NAC family members. Thirdly, we conducted expression pattern analysis of NAC15 gene, and observed NAC15 gene was most highly expressed in the xylem by RT-qPCR. Moreover, we transferred NAC15 gene into tobacco and obtained 12 transgenic lines overexpressing NAC15 gene (TLs). And the relative higher content of hemicellulose, cellulose and lignin was observed in the TLs compared to the control lines containing empty vector (CLs). It also showed darker staining in the culms of the TLs with phloroglucinol staining, compared to the CLs. Furthermore, the relative expression level of a few lignin- and cellulose-related genes was significantly higher in the TLs than that in the CLs. Conclusions The overall results indicated that NAC15 gene is highly expressed in the xylem of poplar and may be a potential candidate gene playing an important role in wood formation in transgenic tobacco.

scholarly journals Production of Stress Tolerant Plants by Overproduction of Enzymatic Oxyradical Scavengers

1993 ◽  
Author(s):  
Barbara A. Zilinskas ◽  
Doron Holland ◽  
Yuval Eshdat ◽  
Gozal Ben-Hayyim
Keyword(s):  
Oxidative Stress ◽  
Glutathione Peroxidase ◽  
Transgenic Tobacco ◽  
Ascorbate Peroxidase ◽  
Biochemical Characterization ◽  
Monodehydroascorbate Reductase ◽  
Saline Stress ◽  
Tobacco Plants ◽  
Transgenic Lines ◽  
Antisense Construct

Most of the objectives that were outlined in the original proposal have been met with two exceptions. Briefly, our goals were to: (1) constract transgenic tobacco plants which overproduce one or more of the enzymatic oxyradical scavengers and associated ancillary enzymes, including superoxide dismutase, ascorbate peroxidase, glutathione peroxidase, glutathione reductase, and monodehydrascorbate reductase; (2) evaluate the tolerance of these transgenic plants to oxidative stress; and (3) extend these studies to an agronomically important crop such as citrus. As can be seen i the following pages, our objectives (1) and (2) have been achieved, although transgenic lines overexpressing phospholipid hydroperoxidase glutathione peroxidase (PHGPX) were not obtained and our evidence to date suggests that constitutive overexpressing of the enzyme is probably lethal. Howeever, transgenic tobacco expressing the antisense construct for PHGPX were obtained. Tobacco plants overexpressing ascorbate peroxidase and those sensesuppressing monodehydroascorbate reductase are more tolerant to oxidative stress, as mediated by the redox-cycling agent paraquant; in contrast, plants expressing the PHGPX-antisense construct are more sensitive to paraquat. Additional research is warranted on each of the six types of transgenic lines which we generated with regard to their tolerance to saline stress. Until recently, attempts to transform citrus were not very successful, and thus additional attention is currently being directed at objective (3). We are optimistic that use of the plant transformation vector, pBIN, will lead to stable transgenic citrus, as preliminary experiments demonstrate stable expression of the GUS reporter gene. Other important contributions resulting from this BARD project include the biochemical characterization of the first plant phospholipid glutathione peroxidase and the biochemical and molecular analysis of another key antioxidant enzyme, monodehydroascorbate reductase. Overall this BARD-supported project was quite successful, and the biological resource of numerous transgenic lines which have altered levels of antioxidant enzymes should be valuable for years to come.

scholarly journals 635 Stability of Herbicide Resistance and GUS Expression in Transgenic Hybrid Poplars during Several Years of Field Trials and Vegetative Propagation

HortScience ◽  
1999 ◽  
Vol 34 (3) ◽  
pp. 557B-557
Author(s):  
Richard Meilan ◽  
Caiping Ma ◽  
Steven H. Strauss
Keyword(s):  
Herbicide Resistance ◽  
Transgene Expression ◽  
Marker Gene ◽  
Field Trials ◽  
Gus Expression ◽  
Gene Encoding ◽  
Hybrid Poplars ◽  
Transgenic Lines ◽  
Single Field ◽  
The Stability

We assessed the stability of transgene expression in 79 transgenic lines (i.e., transformation events) of hybrid poplars during several years of field trials. The transgenic lines were comprised of 40 lines of hybrid cottonwoods (P. trichocarpa × P. deltoides) that were grown at three field sites, and 39 lines of hybrid aspens (section Leuce, P. alba × P. tremula) that were grown at a single field site. All the lines were transformed with a binary construct that included two genes that confer tolerance to glyphosate (GOX and CP4), a gene encoding resistance to the antibiotic kanamycin (nptII), and a visible marker gene (GUS). Agrobacterium tumefaciens was used for transformation; callogenesis and organogenesis occurred under kanamycin selection. In addition to repeated applications of herbicide to test stability of transgene expression, for the first time, we challenged ramets of 40 lines that had not previously been tested for herbicide resistance in their fourth season of vegetative growth. We report on the stability of herbicide resistance and GUS expression and evidence for somaclonal variation in growth and leaf morphology.

scholarly journals Over-expression of poplar NAC15 gene enhances wood formation in transgenic tobacco

2019 ◽  
Author(s):  
Wenjing Yao ◽  
Dawei Zhang ◽  
Boru Zhou ◽  
Jianping Wang ◽  
Renhua Li ◽  
...  
Keyword(s):  
Transgenic Tobacco ◽  
Wood Formation ◽  
Potential Candidate ◽  
Rna Seq ◽  
Populus Simonii ◽  
Over Expression ◽  
Potential Candidate Gene ◽  
Relative Expression Level ◽  
Transgenic Lines ◽  
Highly Expressed Genes

Abstract Background NAC (NAM/ATAF/CUC) is one of the largest plant-specific transcription factor (TF) families known to play significant roles in wood formation. Acting as master gene regulators, a few NAC genes can activate secondary wall biosynthesis during wood formation in woody plants. Results In the present study, firstly, we screened 110 differentially expressed NAC genes in the leaves, stems, and roots of di-haploid Populus simonii×P. nigra by RNA-Seq. Then we identified a nucleus-targeted gene, NAC15 gene, which was one of the highly expressed genes in the stem among 110 NAC family members. Thirdly, we conducted expression pattern analysis of NAC15 gene, and observed NAC15 gene was most highly expressed in the xylem by RT-qPCR. Moreover, we transferred NAC15 gene into tobacco and obtained 12 transgenic lines overexpressing NAC15 gene (TLs). And the relative higher content of hemicellulose, cellulose and lignin was observed in the TLs compared to the control lines containing empty vector (CLs). It also showed darker staining in the culms of the TLs with phloroglucinol staining, compared to the CLs. Furthermore, the relative expression level of a few lignin- and cellulose-related genes was significantly higher in the TLs than that in the CLs. ConclusionsThe overall results indicated that NAC15 gene is highly expressed in the xylem of poplar and may be a potential candidate gene playing an important role in wood formation in transgenic tobacco.

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