Selection Of Transgenic Plants Research Articles

A novel hybrid seed production technology based on a unilateral cross-incompatibility gene in maize.

Hybrid seed production technology (SPT) using genic recessive male sterility is of great importance in maize breeding. Here, we report a novel SPT based on a maize unilateral cross-incompatibility gene ZmGa1F with an extremely low transgene transmission rate (TTR). Proper pollen-specific ZmGa1F expression severely inhibits pollen tube growth leading to no fertilization. The maintainer line harbors a transgene cassette in an ipe1 male sterile background containing IPE1 to restore ipe1 male fertility, ZmGa1F to prevent transgenic pollen escape, the red fluorescence protein encoding gene DsRed2 for the separation of male sterile and fertile seeds, and the herbicide-resistant gene Bar for transgenic plant selection. When the maintainer line is selfed, gametes of ipe1/transgene and ipe1/- genotypes are produced, and pollen of the ipe1/transgene genotype is not able to fertilize female gametes due to pollen tube growth inhibition by ZmGa1F. Subsequently, seeds of ipe1/ipe1 and ipe1/transgene genotypes are produced at a 1:1 ratio and could be separated easily by fluorescence-based seed sorting. Not a single seed emitting fluorescence is detected in more than 200,000 seeds examined demonstrating that the pollen-tube-inhibition (PTI)-based TTR is lower than what has been reported for similar technologies to date. This PTI-based SPT shows promising potential for future maize hybrid seed production.

CRISPR/Cas9-mediated knockout of a prolyl-4-hydroxylase subfamily in Nicotiana benthamiana using DsRed2 for plant selection.

The properties of host plants used for molecular farming can be modified by CRISPR/Cas9 genome editing to improve the quality and yield of recombinant proteins. However, it is often necessary to target multiple genes simultaneously, particularly when using host plants with large and complex genomes. This is the case for Nicotiana benthamiana, an allotetraploid relative of tobacco frequently used for transient protein expression. A multiplex genome editing system incorporating the DsRed2 fluorescent marker for the identification and selection of transgenic plants was established. As proof of principle, NbP4H4 was targeted encoding a prolyl-4-hydroxylase involved in protein O-linked glycosylation. Using preselected gRNAs with efficiencies confirmed by transient expression, transgenic plant lines with knockout mutations in all four NbP4H4 genes were obtained. Leaf fluorescence was then used to screen for the absence of the SpCas9 transgene in T1 plants, and transgene-free lines with homozygous or biallelic mutations were identified. The analysis of plant-produced recombinant IgA1 as a reporter protein revealed changes in the numberof peptides containing hydroxyproline residues and pentoses in the knockout plants. The selection of efficient gRNAs combined with the DsRed2 marker reduces the effort needed to generate N. benthamiana mutants and simplifies the screening processes to obtain transgene-free progeny.

Chili Pepper AN2 (CaAN2): A Visible Selection Marker for Nondestructive Monitoring of Transgenic Plants.

Selecting transformed plants is generally time consuming and laborious. To develop a method for transgenic plant selection without the need for antibiotics or herbicides, we evaluated the suitability of the R2R3 MYB transcription factor gene CaAN2 from purple chili pepper (Capsicum annuum) for use as a visible selection marker. CaAN2 positively regulates anthocyanin biosynthesis. Transient expression assays in tobacco (Nicotiana tabacum) leaves revealed that CaAN2 actively induced sufficient pigment accumulation for easy detection without the need for a basic helix-loop-helix (bHLH) protein as a cofactor; similar results were obtained for tobacco leaves transiently co-expressing the anthocyanin biosynthesis regulators bHLH B-Peru from maize and R2R3 MYB mPAP1D from Arabidopsis. Tobacco plants harboring CaAN2 were readily selected based on their red color at the shoot regeneration stage due to anthocyanin accumulation without the need to impose selective pressure from herbicides. Transgenic tobacco plants harboring CaAN2 showed strong pigment accumulation throughout the plant body. The ectopic expression of CaAN2 dramatically promoted the transcription of anthocyanin biosynthetic genes as well as regulators of this process. The red coloration of tobacco plants harboring CaAN2 was stably transferred to the next generation. Therefore, anthocyanin accumulation due to CaAN2 expression is a useful visible trait for stable transformation, representing an excellent alternative selection system for transgenic plants.

Expression of apple MdMYB10 transcription factor in sugar beet with a screenable marker role and antimicrobial activity.

The online version contains supplementary material available at 10.1007/s13205-022-03120-7.

Improved cotton transformation protocol mediated by Agrobacterium and biolistic combined-methods

The combined Agrobacterium- and biolistic-mediated methods of cotton transformation provide a straightforward and highly efficient protocol for obtaining transgenic cotton. Cotton (Gossypium spp.) is the most important crop for natural textile fiber production worldwide. Nonetheless, one of the main challenges in cotton production are the losses resulting from insect pests, pathogens, and abiotic stresses. One effective way to solve these issues is to use genetically modified (GM) varieties. Herein, we describe an improved protocol for straightforward and cost-effective genetic transformation of cotton embryo axes, merging biolistics and Agrobacterium. The experimental steps include (1) Agrobacterium preparation, (2) seed sterilization, (3) cotton embryo excision, (4) lesion of shoot-cells by tungsten bombardment, (5) Agrobacterium-mediated transformation, (6) embryo co-culture, (7) regeneration and selection of transgenic plants in vitro, and (8) molecular characterization of plants. Due to the high regenerative power of the embryonic axis and the exceptional ability of the meristem cells for plant regeneration through organogenesis in vitro, this protocol can be performed in approximately 4-10weeks, with an average plant regeneration of about 5.5% (± 0.53) and final average transformation efficiency of 60% (± 0.55). The transgene was stably inherited, and most transgenic plants hold a single copy of the transgene, as desirable and expected in Agrobacterium-mediated transformation. Additionally, the transgene was stably expressed over generations, and transgenic proteins could be detected at high levels in the T2 generation of GM cotton plants. The T2 progeny showed no phenotypic or productivity disparity compared to wild-type plants. Collectively, the use of cotton embryo axes and the enhanced DNA-delivery system by combining particle bombardment and Agrobacterium infection enabled efficient transgenic plant recovery, overcoming usual limitations associated with the recalcitrance of several cotton genotypes subjected to somatic embryogenesis. The improved approach states this method's success for cotton genetic modification, allowing us to obtain GM cotton plants carrying traits, which are of fundamental relevance for the advancement of global agribusiness.

A GRF-GIF chimeric protein improves the regeneration efficiency of transgenic plants.

The potential of genome editing to improve the agronomic performance of crops is often limited by low plant regeneration efficiencies and few transformable genotypes. Here, we show that expression of a fusion protein combining wheat GROWTH-REGULATING FACTOR 4 (GRF4) and its cofactor GRF-INTERACTING FACTOR 1 (GIF1) substantially increases the efficiency and speed of regeneration in wheat, triticale and rice and increases the number of transformable wheat genotypes. GRF4-GIF1 transgenic plants were fertile and without obvious developmental defects. Moreover, GRF4-GIF1 induces efficient wheat regeneration in the absence of exogenous cytokinins, which facilitates selection of transgenic plants without selectable markers. We also combined GRF4-GIF1 with CRISPR-Cas9 genome editing, generating 30 edited wheat plants with disruptions in the gene Q (AP2L-A5). Finally, we show that a dicot GRF-GIF chimera improves regeneration efficiency in citrus, suggesting that this strategy can be applied to dicot crops.

Glyphosate selection of maize transformants containing CP4epsps gene

Aim. To study the selection conditions of maize transformants containing the CP4epsps gene using glyphosate as a selective agent. Methods. Tissue culture in vitro, Agrobacterium-mediated transformation, selection of transgenic plants, isolation of total plant DNA, analysis of plant DNA by polymerase chain reaction (PCR). Results. The morphogenic maize callus of immature embryos of the hybrid (PLS61)R2×PLS61 was produced, which had a high regeneration rate (up to 95%), that persisted over long cultivation. Agrobacterium mediated transformation of the morphogenic callus and selection of the transgenic material using glyphosate yielded maize transformants containing the CP4epsps gene at a frequency of 1%. Conclusions. Maize genotype (PLS61)R2×PLS61 is promising for studies on the maize genetic transformation, in particular for the production of transgenic maize resistant to glyphosate herbicide. The use of morphogenic maize callus (PLS61)R2×PLS61 and glyphosate as a selective agent at a concentration of 0.1 mM and 0.25 mM in media for callusogenesis and 0.01 mM in the medium for regeneration was effective for the selection of transgenic plants with the gene CP4epsps.
 Keywords: Zea mays L., morphogenic callus, Agrobacterium-mediated transformation, PCR, genetic engineering.

Soybean Embryonic Axis Transformation: Combining Biolistic and Agrobacterium-Mediated Protocols to Overcome Typical Complications of In Vitro Plant Regeneration.

The first successful attempt to generate genetically modified plants expressing a transgene was preformed via T-DNA-based gene transfer employing Agrobacterium tumefaciens-mediated genetic transformation. Limitations over infectivity and in vitro tissue culture led to the development of other DNA delivery systems, such as the biolistic method. Herein, we developed a new one-step protocol for transgenic soybean recovery by combining the two different transformation methods. This protocol comprises the following steps: agrobacterial preparation, seed sterilization, soybean embryo excision, shoot-cell injury by tungsten-microparticle bombardment, A. tumefaciens-mediated transformation, embryo co-cultivation in vitro, and selection of transgenic plants. This protocol can be completed in approximately 30–40 weeks. The average efficiency of producing transgenic soybean germlines using this protocol was 9.84%, similar to other previously described protocols. However, we introduced a more cost-effective, more straightforward and shorter methodology for transgenic plant recovery, which allows co-cultivation and plant regeneration in a single step, decreasing the chances of contamination and making the manipulation easier. Finally, as a hallmark, our protocol does not generate plant chimeras, in contrast to traditional plant regeneration protocols applied in other Agrobacterium-mediated transformation methods. Therefore, this new approach of plant transformation is applicable for studies of gene function and the production of transgenic cultivars carrying different traits for precision-breeding programs.

Obtaining Wheat (Triticum aestivum L.) Lines with Yeast Genes for Trehalose Biosynthesis

Yeast (Saccharomyces cerevisiae) genes for trehalose biosynthesis (TPS1 and TPS2) were transferred into genomes of several common wheat cultivars using two methods of Agrobacterium-mediated transformation (in vitro and in planta) to enhance drought tolerance. For this purpose, vectors pBract214-TPS1 and pBract214-TPS2 were constructed using the Gateway-cloning technique. Both the vectors contained TPS1 and TPS2 genes under control of the constitutive maize ubiquitin promoter (PUbi) and selectable marker hygromycin-phosphotransferase (hpt) gene. Three- to five-day-old calluses obtained from immature wheat embryos were used as explants for the transformation in vitro. Selection of transgenic plants was carried out on nutrient medium supplemented with 30 mg/L hygromycin (as a selectable agent). Seeds of wheat (transgenic generation T1) were obtained by the in planta method of transformation. Integration and the presence of yeast genes in wheat genomic DNA isolated from transgenic plants were confirmed by PCR analysis using primers specific to TPS1 and TPS2 genes.

Plumular meristem transformation system for chickpea: an efficient method to overcome recalcitrant tissue culture responses

Major biotic and abiotic stresses have led to the reduction of chickpea productivity, creating a strong barrier for its utilization as major food legume. Genetic transformation has revolutionized the chickpea improvement programs for biotic stress tolerance. Recent knowledge on genomic resources offers a range of approaches for genetic improvement of chickpea with a greater resolution, but relies on the transgenic establishment which is the major bottleneck in this process. Poor tissue culture responses along with insensitivity to in vitro rooting hindered the transgenic chickpea development through tissue culture based transformation methods. In the present report, Agrobacterium-mediated plumular meristem transformation was adapted in chickpea to bypass the recalcitrancy of regenerable tissue. Embryonic axis of immature seedlings of chickpea cultivar was decapitated followed by pricking of plumular meristem present in shoot apex and cotyledonary nodes. Pricking is followed by infection by Agrobacterium tumefaciens carrying binary vector pBI121. Three days co-cultivation was performed with infected explants in 6-benzylaminopurine and α-naphthaleneacetic acid supplemented modified Murashige and Skoog medium. Transient GUS expression was observed in co-cultivated explants and PCR based screening strategy allowed the establishment of primary transgenic events with a frequency of 60%. Kanamycin mediated stringent selection of T1 events helped to eliminate the chimeric plants and 44% of T1 progenies were confirmed through PCR. Further, Southern hybridization was performed to identify transgene integration in T1 events. Additionally, prominent GUS activity in T1 events confirmed the expression of transgene. Plumular transformation method, based on culture dependent Agrobacterium-infection and culture independent plant selection as well as establishment, was reported for the first time in chickpea. This transformation method will boost the recovery of novel genotypes with improved agronomic traits and mutant development for functional genomics in chickpea. Agrobacterium-mediated transformation of plumular meristem in chickpea is established, following in vitro co-cultivation of explants and culture independent selection of transgenic plants, at higher frequency.

The influence of bacteria acdS-gene of Pseudomonas putida B-37 on Nicotiana tabacum transgenic plants under abiotic stress conditions

In the present work was carried out selection of transgenic plants of Nicotiana tabacum carrying acdS-gene of bacteria Pseudomonas putida B-37. Using polymerase chain reaction with specific primers, the presence of the target gene was proved. Transgenic plants selected on a selective medium were planted in the ground and subjected to abiotic stress caused by soil contamination with heavy metal salts (15 mg / kg for Cu2+ and 30 mg / kg for Cr6+ and Pb2+ ) and salinization of the soil (200 mmol/L NaCl). Reverse transcription polymerase chain reaction and real-time polymerase chain reaction were conducted and confirmed the transcriptional activity of the bacterial acdS-gene in transgenic plant cells at a level with the reference gene Ef-1a. Determination of activity of ACC-deaminase, the product of expression of the acdS-gene, confirmed the formation of the active enzyme in the leaf tissues of transgenic tobacco plants. The beneficial effect of the acdS-gene of the bacteria P. putida B-37 on transgenic N. tabacum plants under abiotic stress has been proven.

A Procedure for Agrobacterium tumefaciens Mediated Genetic Transformation of Sweet Potato

Many sweet potato plants have been successfully transformed but the transgenic plants regenerated, however, have been limited to a few genotypes. Reported in this paper is a procedure in which several explants of three sweet potato genotypes from Papua New Guinea (PNG) were used to transform and regenerate transgenic plants. To achieve stable transformation, an efficient shoot regeneration system for different explants was developed. The shoot regeneration protocol developed enabled for a reproducible stable transformation mediated by Agrobacterium tumefaciens strain 1065. The plasmid pVDH65 contains the npt II gene for kanamycin (km) resistance, hpt gene for hygromycin resistance and Gus-intron reporter gene (GUS) for β-glucuronidase. Explants inoculated with the bacterial strain were co-cultured for 3, 5 and 7 days (d) in the dark on Murashige and Skoog (MS) medium without growth hormones. After co-cultivation, the explants were washed in liquid MS medium containing 500 mg L-1 cefotaxime, rinsed in sterile, deionised water for 10 mins and cultured on km selection medium containing 100 mg L-1 km followed by transfer of explants to 125 mg L-1 km after 14 d of culture. The km-resistant shoots selected on the former km concentration were transferred to the latter for double selection. Km-resistant shoots obtained at 125 mg L-1 were rooted on MS based medium also containing 0.008 mg L-1 IAA, 0.03 mg L-1 kinetin and 0.001 mg L-1 folic acid. This double selection method led to effective elimination of escapes (up to 75%) and successful recovery of transgenic plants from stem explants at more than 25%, leaf discs 10% and petioles 13.3% of each sweet potato cultivar. Polymerase chain reaction (PCR) analysis of the three km-resistant and GUS-positive plants revealed the presence of the expected fragment for npt II. This is the first report of successfully transforming sweet potato plants with bacterial strain 1065 and selection of transgenic plants at km concentrations higher than 100 mg L-1.

Targeted expression of a cysteine protease (AdCP) in tapetum induces male sterility in Indian mustard, Brassica juncea.

The development of male sterile plants is a prerequisite to developing hybrid varieties to harness the benefits of hybrid vigor in crops and enhancing crop productivity for sustainable agriculture. In plants, cysteine proteases have been known for their multifaceted roles during programmed cell death, and in ubiquitin- and proteasome-mediated proteolysis. Here, we showed that Arachis diogoi cysteine protease (AdCP) expressed under the TA-29 promoter induced complete male sterility in Indian mustard, Brassica juncea. The herbicide resistance gene bar was used for the selection of transgenic plants. Mustard transgenic plants exhibited male sterile phenotype and failed to produce functional pollen grains. Irregularly shaped aborted pollen grains with groove-like structures were observed in male sterile plants during scanning electron microscopy analysis. The T1 progeny plants obtained from the seed of primary transgenic male sterile plants crossed with the wild-type plants exhibited segregation of the progeny into male sterile and fertile plants with normal seed development. Further, male sterile plants exhibited higher transcript levels of AdCP in anther tissues, which is consistent with its expression under the tapetum-specific promoter. Our results clearly suggest that the targeted expression of AdCP provides a potential tool for developing male sterile lines in crop plants by the malfunction of tapetal cells leading to male sterility as shown earlier in tobacco transgenic plants (Shukla et al.2014, Funct Integr Genomics 14:307-317).

Pollen Tube Pathway-Mediated Cotton Transformation.

Although many gene transfer methods have been employed for successfully obtaining transgenic cotton, the major constraint in cotton improvement is the limitation of genotype because the majority of transgenic methods require plant regeneration from a single transformed cell which is limited by cotton tissue culture. Comparing with other plant species, it is difficult to induce plant regeneration from cotton; currently, only a limited number of cotton cultivars can be cultured for obtaining regenerated plants. Thus, developing a simple and genotype-independent genetic transformation method is particularly interested for cotton. In this chapter, we present a simple, cost-efficient, and genotype-independent cotton transformation method - pollen tube pathway-mediated transformation. This method uses pollen tube pathway to deliver transgene into cotton embryo sacs and then insert foreign genes into cotton genome. There are three major steps for pollen tube pathway-mediated genetic transformation, which include injection of foreign genes into pollen tube, integration of foreign genes into plant genome, and selection of transgenic plants.

Efficient plant regeneration and Agrobacterium-mediated transformation via somatic embryogenesis in purslane (Portulaca oleracea L.): an important medicinal plant

Portulaca oleracea is an important medicinal plant, which is a source of pharmacologically active molecules such as β-Carotene, ascorbic acid, and Omega-3 fatty acids. The present research focuses on the development of an efficient protocol for micropropagation and Agrobacterium-mediated genetic transformation of P. oleracea. Callus induction, somatic embryogenesis, and plant regeneration from stem and leaf explants were investigated at various concentrations of kinetin (Kin) and 6-Benzylaminopurine (BAP) alone or in combination with indole-3-acetic acid, 1-Naphthaleneacetic acid and 2,4-Dichlorophenoxyacetic acid (2,4-D). Direct differentiation of somatic embryos from leaf explants occurred on the MS medium supplemented with 1.5 mg/L BAP under dark conditions. The embryos were transferred to the same medium without growth regulators under 16 h light/8 h dark cycles. In this medium, germinated somatic embryos rapidly developed into healthy plantlets with shoots and roots. Several parameters such as pre-culture of explants, co-cultivation period, wounding of explants, type of explants and bacterial strains were studied to optimize transformation efficiency. Different kanamycin concentrations were assessed for the selection of transgenic plants. Agrobacterium tumefaciens strains LBA4404 and GV3101, harbouring the GUS gene on pBI121 binary vector, were used for plant transformation and strain LBA4404 was found to be more efficient. The results indicated that use of leaf as explant, pre-culture of explants for 7 days, co-cultivation period for 4 days at 25 ± 2 °C and wounding of leaf explants produced the best transformation results. Expression, integration and inheritance of GUS reporter gene were confirmed by histochemical and molecular analyses.

Agrobacterium-mediated in planta genetic transformation of winter wheat (Triticum aestivum L.)

Aim. To optimize the agrobacterium-mediated method of winter wheat transformation (Triticum aestivum L.); to select the conditions and period of inoculation to effectively transfer the genes during pollination. Methods. Agrobacterium-mediated in planta genetic transformation of winter wheat (Triticum aestivum L.) during pollination. Results. The conditions for agrobacterium-mediated transformation method of winter wheat during natural (frequency pollination was 1 %) and non-natural (frequency pollination was 4 %) pollination were defined. Conclusions. The possibility of integrating transgenes into the genome of winter wheat plants by the method of Agrobacterium-mediated transformation in planta in the process of forced and natural pollination is demonstrated. It is found that the transformation efficiency to a large extent depends on the plant genotype and the method of carrying out the transformation procedure. The selection of transgenic plants under water deficit conditions allowed to identify the plants with functional transgene. The signs of functioning transgene have been remaining in the next generation of genetically modified winter wheat.
 Keywords: Triticum aestivum L., Agrobacterium-mediated transformation in planta, transgenic plants, seeds.

Comparison of a kanamycin versus hygromycin resistance gene in transgenic plant selection of Arabidopsis thaliana L.

Comparison of a kanamycin versus hygromycin resistance gene in transgenic plant selection of Arabidopsis thaliana L.

Small serine recombination systems ParA-MRS and CinH-RS2 perform precise excision of plastid DNA.

Selectable marker genes (SMGs) are necessary for selection of transgenic plants. However, once stable transformants have been identified, the marker gene is no longer needed. In this study, we demonstrate the use of the small serine recombination systems, ParA‐MRS and CinH‐RS2, to precisely excise a marker gene from the plastid genome of tobacco. Transplastomic plants transformed with the pTCH‐MRS and pTCH‐RS2 vectors, containing the visual reporter gene DsRed flanked by directly oriented MRS and RS2 recognition sites, respectively, were crossed with nuclear‐genome transformed tobacco plants expressing plastid‐targeted ParA and CinH recombinases, respectively. One hundred per cent of both types of F1 hybrids exhibited excision of the DsRed marker gene. PCR and Southern blot analyses of DNA from F2 plants showed that approximately 30% (CinH‐RS2) or 40% (ParA‐MRS) had lost the recombinase genes by segregation. The postexcision transformed plastid genomes were stable and the excision events heritable. The ParA‐MRS and CinH‐RS2 recombination systems will be useful tools for site‐specific manipulation of the plastid genome and for generating marker‐free plants, an essential step for reuse of SMG and for addressing concerns about the presence of antibiotic resistance genes in transgenic plants.

Comparison of a kanamycin versus hygromycin resistance gene in transgenic plant selection of Arabidopsis thaliana L.

Comparison of a kanamycin versus hygromycin resistance gene in transgenic plant selection of Arabidopsis thaliana L.

Assessment of the mannose inhibition on tobacco regeneration for establishment of a mannose selection system for transgenic plants

Recently, the application of environmentally friendly methods in transgenic-plant selection has been exploited with an interest to contribute to the commercialization of transgenic products. In this study, mannose was chosen in selecting transgenic tobacco. The ability of mannose to inhibit tobacco regeneration was assessed through three stages: creating multi-shoot, elongating shoot, and rooting of tobacco cultivars K326 and C9-1. The results showed that mannose concentrations at 30 g/l and 20 g/l are suitable for creating multi-shoot of K326 and C9-1, respectively. In addition, mannose concentration at 30 g/l in combination with 7,5 g/l sucrose is fit for elongating shoot while its concentration at 30 g/l is appropriate for rooting transgenic tobacco in both K326 and C9-1 cultivars. The mannose selection of transgenic tobacco using gus gene showed that the transgenic effectiveness is ranging 16-17% of K326 and 9-11% of C9-1 cultivars. The successful establishment of mannose selection system in transgene tobacco contributes to generate bio-safety transgenic plants.