scholarly journals An increased proportion of transgenic plants in the progeny of rapeseed (Brassica napus L.) transformants

2021 ◽  
Vol 25 (2) ◽  
pp. 147-156
Author(s):  
G. N. Raldugina ◽  
T. Z. Hoang ◽  
H. B. Ngoc ◽  
I. V. Karpichev
Keyword(s):  
Transgenic Plants ◽  
Marker Gene ◽  
Leaf Explants ◽  
Media Analysis ◽  
Mendelian Segregation ◽  
Genetic Construct ◽  
Self Pollination ◽  
Original Plant ◽  
Nodal Cuttings ◽  
Transgenic Cells

Cotyledon and leaf explants of two spring rapeseed varieties were transformed with Agrobacterium tumefaciens harboring a genetic construct with the gfp marker gene. In order to reduce the proportion of hyperhydrated shoots, which appeared during regenerant formation, we optimized sucrose content in the regeneration media. Analysis of the progeny obtained from T0 regenerants showed that in a number of lines the distribution of the gfp marker did not follow Mendelian segregation of a monogenic trait in self-pollinated plants, while in the progeny of the other lines of transgenic plants, the gfp marker was completely absent, although its presence had been confirmed in all selected T0 plants. We also found that in individual transformants gfp is randomly inherited throughout the central peduncle; its presence in the genome of seedlings does not depend on the location of the pod. Thus, both transformed and non-transformed cells were involved in the formation of gametes in T0 plants. In addition, marker segregation was different in plants of the T1 line obtained by nodal cuttings of a primary transformant, depending on the location of the cuttings on the stem of the original plant, indicating that the nature of T1 generation plants was also chimeric. Furthermore, we showed that propagation of plants by cutting followed by propagation by seeds formed as a result of self-pollination led to an increase in the proportion of transgenic plants in subsequent generations. The results obtained during the course of this study show that the transformants were chimeric, i. e. their tissues contained both transgenic and non-transgenic cells, and this chimeric nature was passed on to subsequent generations. We found that, in addition to nutrient media composition, other factors such as plant genotype and explant type also contribute to the rising of chimeric plants during transformation. Based on these results, we developed a simplified method, which consists of several rounds of a combination of cutting, seed production by self-pollination, and subsequent culling of wild-type plants, which significantly enriched descendent populations of the original rapeseed transformants with plants transgenic for the gfp marker.

scholarly journals Aminoglycoside antibiotics affect the in vitro morphogenic response of chrysanthemum and tobacco

2002 ◽  
Vol 8 (2) ◽  
Author(s):  
Da Silva Teixeira ◽  
S. Fukai
Keyword(s):  
Marker Gene ◽  
Adventitious Shoots ◽  
Leaf Explants ◽  
Aminoglycoside Antibiotics ◽  
Tobacco Callus ◽  
Tobacco Stem ◽  
Cell Layers ◽  
Transgenic Cells ◽  
Selection Of

Broadly the success of genetic transformation of plants requires non-chimeric selection of transformed tissues and its subsequent regeneration. With rare exceptions, most plant transformation protocols still heavily utilize antibiotics for the selection of transgenic cells containing an antibiotic-degrading selectable marker gene. The morphogenic capacity of in vitro chrysanthemum and tobacco stem and leaf explants change with the addition of aminoglycoside antibiotics (AAs). Of 6 antibiotics tested, phytotoxicity occurred at 10-25 and 50-100 pgml-I in chrysanthemum and tobacco explants, respectively, depending on the size of the explant and the timing of application. The presence of light or darkness also had a significant effect. The use of transverse thin cell layers (tTCLs) in conjunction with high initial AA selection levels supported the greatest regeneration of transgenic material (adventitious shoots or callus) and the lowest number of escapes. Flow cytometric analyses demonstrate that regeneration can be predicted in both species, depending on the ploidy level of the callus. Endoreduplication was not observed in chrysanthemum, even at high AA levels, but occurred (8C or more) in tobacco callus, even at low AA concentrations (5-10 pgml-1). The higher the AA level, the greater the DNA degradation and the lower the 2C and 4C values.

scholarly journals Receiving of genetic-modified wheat plants with heterolous ornitin-δ-aminotransferase gene

2018 ◽  
Vol 22 ◽  
pp. 222-227
Author(s):  
O. M. Honcharuk ◽  
O. V. Dubrovna
Keyword(s):  
Triticum Aestivum ◽  
Transgenic Plants ◽  
Bread Wheat ◽  
Binary Vector ◽  
Triticum Aestivum L ◽  
Callus Cultures ◽  
Pcr Analysis ◽  
Genetic Construct ◽  
Agrobacterium Mediated Transformation

Aim. Receiving of genetically modified plants of bread wheat with heterologous ornithine‑δ‑aminotransferase gene. Methods. Agrobacterium-mediated transformation of callus cultures in vitro, PCR-analysis. Results. By Agrobacterium-mediated transformation of the morphogenic calluses of bread wheat (Triticum aestivum L.) using the AGLO strain containing the binary vector pBi-OAT with the target ornithine-δ-aminotransferase (oat) and selective neomycinphosphotransferase II (nptII), transgenic plants-regenerators have been obtained. Conclusions. As a result of the genetic transformation of Zimoyarka variety, 12 wheat regenerants were obtained in the genome which revealed a complete integration of the genetic construct containing the oat and nptII transgenes. Keywords: Triticum aestivum L., Agrobacterium-mediated transformation, ornithine‑δ‑aminotransferase gene, PCR-analysis.

scholarly journals A Traceable DNA-Replicon Derived Vector to Speed Up Gene Editing in Potato: Interrupting Genes Related to Undesirable Postharvest Tuber Traits as an Example

Plants ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 1882
Author(s):  
Giovana Acha ◽  
Ricardo Vergara ◽  
Marisol Muñoz ◽  
Roxana Mora ◽  
Carlos Aguirre ◽  
...  
Keyword(s):  
Gene Editing ◽  
Fluorescent Protein ◽  
Solanum Tuberosum L ◽  
Marker Gene ◽  
Leaf Explants ◽  
Analysis Tool ◽  
Targeted Deletion ◽  
Potato Genome ◽  
Guide Rna ◽  
Yellow Dwarf Virus

In potato (Solanum tuberosum L.), protoplast techniques are limited to a few genotypes; thus, the use of regular regeneration procedures of multicellular explants causes us to face complexities associated to CRISPR/Cas9 gene editing efficiency and final identification of individuals. Geminivirus-based replicons contained in T-DNAs could provide an improvement to these procedures considering their cargo capability. We built a Bean yellow dwarf virus-derived replicon vector, pGEF-U, that expresses all the editing reagents under a multi-guide RNA condition, and the Green Fluorescent Protein (GFP) marker gene. Agrobacterium-mediated gene transfer experiments were carried out on ‘Yagana-INIA’, a relevant local variety with no previous regeneration protocol. Assays showed that pGEF-U had GFP transient expression for up to 10 days post-infiltration when leaf explants were used. A dedicated potato genome analysis tool allowed for the design of guide RNA pairs to induce double cuts of genes associated to enzymatic browning (StPPO1 and 2) and to cold-induced sweetening (StvacINV1 and StBAM1). Monitoring GFP at 7 days post-infiltration, explants led to vector validation as well as to selection for regeneration (34.3% of starting explants). Plant sets were evaluated for the targeted deletion, showing individuals edited for StPPO1 and StBAM1 genes (1 and 4 lines, respectively), although with a transgenic condition. While no targeted deletion was seen in StvacINV1 and StPPO2 plant sets, stable GFP-expressing calli were chosen for analysis; we observed different repair alternatives, ranging from the expected loss of large gene fragments to those showing punctual insertions/deletions at both cut sites or incomplete repairs along the target region. Results validate pGEF-U for gene editing coupled to regular regeneration protocols, and both targeted deletion and single site editings encourage further characterization of the set of plants already generated.

scholarly journals Novel Marker Gene for Assessment of Behavior of Transgenic Plants in the Field

2003 ◽  
Vol 20 (3) ◽  
pp. 225-227
Author(s):  
Masanori TAMAOKI ◽  
Yumio TODA ◽  
Nobuyoshi NAKAJIMA ◽  
Akihiro KUBO ◽  
Mitsuko AONO ◽  
...  
Keyword(s):  
Transgenic Plants ◽  
Marker Gene

scholarly journals Using Morphogenic Genes to Improve Recovery and Regeneration of Transgenic Plants

Plants ◽  
2019 ◽  
Vol 8 (2) ◽  
pp. 38 ◽  
Author(s):  
Bill Gordon-Kamm ◽  
Nagesh Sardesai ◽  
Maren Arling ◽  
Keith Lowe ◽  
George Hoerster ◽  
...  
Keyword(s):  
Gene Expression ◽  
Tissue Culture ◽  
Transgenic Plants ◽  
Plant Genome ◽  
Inducible Promoters ◽  
Genome Modification ◽  
Meristem Development ◽  
Ectopic Overexpression ◽  
Transgenic Cells ◽  
Culture Strategies

Efficient transformation of numerous important crops remains a challenge, due predominantly to our inability to stimulate growth of transgenic cells capable of producing plants. For years, this difficulty has been partially addressed by tissue culture strategies that improve regeneration either through somatic embryogenesis or meristem formation. Identification of genes involved in these developmental processes, designated here as morphogenic genes, provides useful tools in transformation research. In species from eudicots and cereals to gymnosperms, ectopic overexpression of genes involved in either embryo or meristem development has been used to stimulate growth of transgenic plants. However, many of these genes produce pleiotropic deleterious phenotypes. To mitigate this, research has been focusing on ways to take advantage of growth-stimulating morphogenic genes while later restricting or eliminating their expression in the plant. Methods of controlling ectopic overexpression include the use of transient expression, inducible promoters, tissue-specific promoters, and excision of the morphogenic genes. These methods of controlling morphogenic gene expression have been demonstrated in a variety of important crops. Here, we provide a review that highlights how ectopic overexpression of genes involved in morphogenesis has been used to improve transformation efficiencies, which is facilitating transformation of numerous recalcitrant crops. The use of morphogenic genes may help to alleviate one of the bottlenecks currently slowing progress in plant genome modification.

Stable transformation ofphaC2 gene in tobacco chloroplast genome

2006 ◽  
Vol 3 (3) ◽  
pp. 201-207
Author(s):  
Wang Yu-Hua ◽  
Wu Zhong-Yi ◽  
Zhang Xiu-Hai ◽  
Wang Yong-Qin ◽  
Huang Cong-Lin ◽  
...  
Keyword(s):  
Transgenic Plants ◽  
Chloroplast Genome ◽  
Marker Gene ◽  
Chloroplast Transformation ◽  
Single Copy ◽  
Stable Transformation ◽  
Pha Synthase ◽  
Type Ii ◽  
Tobacco Chloroplast ◽  
Chloroplast Genomes

AbstractMedium-chain-length polyhydroxyalkanoates (mcl-PHAs) belong to the group of microbial polyesters. The key enzyme for mcl-PHA biosynthesis is type II PHA synthase. The genephaC2 encoding type II PHA synthase was placed under the control ofpsbA-pro andpsbA-ter of rice (Oryza sativa) to construct aphaC2 cassette, which was ligated with the screening marker geneaadAcassette (prrn–aadA–TpsbA-ter). These recombined fragments were cloned between the plastidrbcLandaccDgenes for targeting to the large single copy region of the chloroplast genome. A chloroplast transformation vector, pTC2, was constructed and introduced into the tobacco (Nicotiana tobacum) chloroplast genome by particle bombardment. PCR and Southern blot analysis confirmed stable integration ofphaC2 into the chloroplast genomes of T0and T1transgenic plants, and T1transgenic plants exhibited homoplasmy. The expression ofphaC2 at transcription level was detected by reverse transcriptase–polymerase chain reaction (RT-PCR). Recombinant transgenes in the tobacco chloroplast genome were maternally inherited and were not transmitted via pollen when out-crossed with untransformed female plants. To our knowledge, this is the first report on the stable transformation ofphaC2 encoding type II PHA synthase in tobacco via chloroplast genetic engineering.

scholarly journals Aminoglycoside Antibiotics Inhibit Shoot Regeneration from Siberian Elm Leaf Explants

HortScience ◽  
1999 ◽  
Vol 34 (4) ◽  
pp. 727-729 ◽  
Author(s):  
James A. Kapaun ◽  
Zong-Ming Cheng
Keyword(s):  
Shoot Regeneration ◽  
Marker Gene ◽  
Leaf Explants ◽  
Shoot Formation ◽  
Aminoglycoside Antibiotics ◽  
Gelling Agent ◽  
Direct Selection ◽  
Selective Agent ◽  
Selection Scheme ◽  
Stock Solutions

Four aminoglycoside antibiotics were evaluated for their effects on shoot regeneration from leaf explants of Siberian elm (Ulmus pumila L.) seedlings and their potential use as selective agents in genetic transformation with the neomycin phosphotransferase II gene as the selective marker gene. Kanamycin at 100 mg·L–1 or higher concentration reduced shoot regeneration, with complete inhibition at 225 mg·L–1, and was considered a suitable selective agent. Neomycin completely inhibited shoot regeneration at 450 mg·L–1, but all explants remained green; therefore, it may also be used as a selective agent. Geneticin significantly inhibited shoot formation at 1 mg·L–1 and completely killed the explants at 4 mg·L–1 after 1 week. Geneticin was too toxic for direct selection, but may be useful in a delayed selection scheme or for confirmation of transformation. Paromomycin was least effective in inhibiting shoot formation; 13% of explants still regenerated shoots on the medium with the highest concentration tested (400 mg·L–1). Both neomycin and paromomycin precipitated in media containing Phytagel as a gelling agent if antibiotic stock solutions were added to the medium without adjusting their pH. Precipitation was prevented by adjusting the pH of the stock solutions from 6.2 (neomycin) or 6.9 (paromomycin) to above 9, or by using agar as a gelling agent. The precipitation was not affected by the concentrations of salts in the media.

Expression of the DREB1A gene in lentil (Lens culinaris Medik. subsp. culinaris) transformed with the Agrobacterium system

2011 ◽  
Vol 62 (6) ◽  
pp. 488 ◽  
Author(s):  
Fateh Khatib ◽  
Antonios Makris ◽  
Kasuko Yamaguchi-Shinozaki ◽  
Shiv Kumar ◽  
Ashtuosh Sarker ◽  
...  
Keyword(s):  
Transgenic Plants ◽  
Lens Culinaris ◽  
Marker Gene ◽  
Single Copy ◽  
Plant Genome ◽  
Direct Organogenesis ◽  
Complete Resistance ◽  
Protocol Optimisation ◽  
Gusa Gene ◽  
Rd29a Promoter

Until now three publications have reported the development of transgenic lentil plants through protocol optimisation using the gusA gene, but there are no reports of the introduction of a gene with agronomic importance. In the present study we report the introduction of the DREB1A gene into lentil to enhance drought and salinity tolerance. Decapitated embryos were immersed in Agrobacterium suspension and then co-cultivated for 4 days. Direct organogenesis was induced from the apical meristems and cotyledonary buds. Subsequently, the explants were subjected to selection in medium containing 10 mg/L phosphinothricin for nine rounds with 2-week intervals. The putative transgenic explants were micro-grafted onto non-transformed rootstocks to establish transgenic plants. The PCR results confirmed the insertion and stable inheritance of the gene of interest and bar marker gene in the plant genome. The Southern blot analysis revealed the integration of a single copy of the transgenes. T0 plants and progeny up to T2 generations showed complete resistance to the herbicide Basta. The DREB1A gene driven by the rd29A promoter was induced in transgenic plants by salt stress from sodium chloride solution. The total RNA was extracted and cDNA synthesised. The results showed that DREB1A mRNA was accumulated and thus the DREB1A transgene was expressed in the transgenic plants, whereas no expression was detected in the non-transformed parents.

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