Plant regeneration and transformation of Trachyspermum ammi using Agrobacterium tumefaciens and zygotic embryos

Background Trachyspermum ammi is one of the key medicinal plant species with many beneficial properties. Thymol is the most important substance in the essential oil of this plant. Thymol is a natural monoterpene phenol with high anti-microbial, anti-bacterial, and anti-oxidant properties. Thymol in the latest research has a significant impact on slowing the progression of cancer cells in human. In this research, embryos were employed as convenient explants for the fast and effectual regeneration and transformation of T. ammi. To regenerate this plant, Murashige and Skoog (MS) and Gamborg's B5 (B5) media were supplemented with diverse concentrations of plant growth regulators, such as 6-benzyladenine (BA), 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and kinetin (kin). Transgenic Trachyspermum ammi plants were also obtained using Agrobacterium-mediated transformation and zygotic embryos explants. Moreover, two Agrobacterium tumefaciens strains (EHA101 and LBA4404) harboring pBI121-TPS2 were utilized for genetic transformation to Trachyspermum ammi. Results According to the obtained results, the highest plant-regeneration frequency was obtained with B5 medium supplemented with 0.5 mg/l BA and 1 mg/l NAA. The integrated gene was also approved using the PCR reaction and the Southern blot method. Results also showed that the EHA101 strain outperformed another strain in inoculation time (30 s) and co-cultivation period (1 day) (transformation efficiency 19.29%). Furthermore, HPLC method demonstrated that the transformed plants contained a higher thymol level than non-transformed plants. Conclusions In this research, a fast protocol was introduced for the regeneration and transformation of Trachyspermum ammi, using zygotic embryo explants in 25–35 days. Our findings confirmed the increase in the thymol in the aerial part of Trachyspermum ammi. We further presented an efficacious technique for enhancing thymol content in Trachyspermum ammi using Agrobacterium-mediated plant transformation system that can be beneficial in genetic transformation and other plant biotechnology techniques.

Kanamycin in Cereal Biotechnology: A Screening or a Selectable Marker?

Kanamycin is a widely used selection agent in dicot-plant genetic transformation systems. In monocots, however, it does not seem to be effective as it has no or minimal effect on the normal growth of non-transformed plants. Kanamycin was previously demonstrated to bleach the pigments of the non-transgenic plants. This may yield the idea that kanamycin can be used as an effective screening marker rather than a selectable marker in monocots. Copyright (c) 2021 Malik Nawaz Shuja, Hasan Riaz, Muhsin Jamal, Muhammad Imran

Kanamycin in Cereal Biotechnology: A Screening or a Selectable Marker?

Kanamycin is a widely used selection agent in dicot-plant genetic transformation systems. In monocots, however, it does not seem to be effective as it has no or minimal effect on the normal growth of non-transformed plants. Kanamycin was previously demonstrated to bleach the pigments of the non-transgenic plants. This may yield the idea that kanamycin can be used as an effective screening marker rather than a selectable marker in monocots.

Kanamycin in Cereal Biotechnology: A Screening or a Selectable Marker?

Kanamycin is a widely used selection agent in dicot-plant genetic transformation systems. In monocots, however, it does not seem to be effective as it has no or minimal effect on the normal growth of non-transformed plants. Kanamycin was previously demonstrated to bleach the pigments of the non-transgenic plants. This may yield the idea that kanamycin can be used as an effective screening marker rather than a selectable marker in monocots.

Kanamycin in Cereal Biotechnology: A Screening or a Selectable Marker?

Kanamycin is a widely used selection agent in dicot-plant genetic transformation systems. In monocots, however, it does not seem to be effective as it has no or minimal effect on the normal growth of non-transformed plants. Kanamycin was previously demonstrated to bleach the pigments of the non-transgenic plants. This may yield the idea that kanamycin can be used as an effective screening marker rather than a selectable marker in monocots.

Characterization of Carnivorous Plants Sarracenia Purpurea L. Transformed with Agrobacterium Rhizogenes

Carnivorous plant of Sarracenia genus are used by human in folk medicine for centuries. The reason for this phenomenon is biochemical composition of Sarracenia plants, which possess many bioactive compounds with anti-inflammatory, antioxidant, antiviral and antibacterial properties.The subject of this research was genetic transformation of Sarracenia purpurea L. with Agrobacterium rhizogenes strain 15834, 9402 and A4 by using two alternatively methods of bacterial injection or co-culture the bacteria with plants explants. These studies confirmed the possibility of hairy roots induction in S. purpurea using the strain of A. rhizogenes 15834 and the injection method. Seven lines of transformed plants, exhibiting the integration of rolB gene, were obtained. The formed hairy roots showed morphological differences in comparison to the roots of unmodified plants. A mathematical model was used to optimize the conditions for the extraction of bioactive compounds. Extracts isolated under optimal conditions from transformed plants showed biochemical changes i.e., the increase in the accumulation of total polyphenols and triterpenes in comparison to untransformed plants, especially when induced roots were analyzed. HPLC analysis showed increase in the level of betulinic acid in some transformed Sarracenia lines. Betulinic acid remains pentacyclic triterpenoid compound with high pharmacological significance. The further work connected with isolation and identification of the other bioactive compounds will be done in the nearest future.

Effects associated with insertion of rol genes on morphogenic potential in explants derived from transgenic Bacopa monnieri (L.) Wettst

The present study deals with the establishment of rolA-transgenic and rolB-transgenic plants for the first time through Agrobacterium tumefaciens mediated transformation, exploiting the inherent morphogenic potential of an important medicinal plant, Bacopa monnieri (L.) Wettst. The rolA-transgenic and rolB-transgenic plants showed integration and expression of rolA and rolB genes respectively, whereas Ri-transformed plants showed integration and expression of rolA, rolB, rolC and rolD genes. Morphogenic potential of different types of explants derived from rolA-transgenic, rolB-transgenic and Ri-transformed plants on basal medium was evaluated. Shoot organogenesis was enhanced significantly in leaf (1.6-fold) and internode (1.4-fold) explants derived from rolA-transgenic plants, rolB-transgenic leaf (2.4-fold) and internode (1.6-fold) explants as well as leaf (5.2-fold) and internode (3.3-fold) explants derived from Ri-transformed plants compared to explants from non-transformed plants. Substantial increase in root organogenesis was also noticed in rolA-transgenic leaf (1.7-fold) explants, rolB-transgenic leaf (3.6-fold) and internode (1.4-fold) explants as well as leaf (4.1-fold) and internode (1.9-fold) explants derived from Ri-transformed plants compared to non-transformed ones. In addition to this, growth of root tip and shoot regeneration was also noticed from Ri-transformed root explants, but not in rolA-transgenic, rolB-transgenic and non-transformed roots. Clones of all three transgenic plants differed morphologically from non-transformed plants; however, rolB gene alone has a pronounced effect in alteration of plant phenotype in B. monnieri. Clones of rolB-transgenic plants were similar in shoot length, internode length, number of nodes/plant and number of leaves/plant when compared with Ri-transformed plant clones

SmFLS negatively regulates peel coloring of eggplant (Solanum melongena) under high temperature

The SmFLS gene was cloned from eggplant, with an ORF of 1014bp, encoding 337 amino acids. The deduced protein sequence of SmFLS was 88.07 % and 84.94 % identical to homologs encoded by StFLS in S. tuberosum and SlFLS in S. lycopersicumrespectively. SmFLS contains typical DIOX_N and 2OG-Fe (II)_Oxy functional domains, and 5 strictly conserved amino acid residues (H223, D225, H279, R289 and S291) related to FLS enzyme activity. Phylogenetic tree analysis showed that SmFLS has the closest genetic relationship with the FLSs in potato and tomato. At a high temperature of 35 °C , the expression level of SmFLS was higher than that of the control in the same period, and reached extremely significant levels on 15DAF and 20DAF, and the eggplant peel color became lighter accordingly. SmFLS was overexpressed in eggplant, the flavonol content of transformed plants was significantly higher than that of non-transformed plants, and the peel color was lighter than that of the control. The above results indicated that SmFLS negatively regulates eggplant peel coloration under high temperature.

An improved and efficient method of Agrobacterium syringe infiltration for transient transformation and its application in the elucidation of gene function in poplar

Background Forest trees have important economic and ecological value. As a model tree, poplar has played a significant role in elucidating the molecular mechanisms underlying tree biology. However, a lack of mutant libraries and time-consuming stable genetic transformation processes severely limit progress into the functional characterization of poplar genes. A convenient and fast transient transformation method is therefore needed to enhance progress on functional genomics in poplar. Methods A total of 11 poplar clones were screened for amenability to syringe infiltration. Syringe infiltration was performed on the lower side of the leaves of young soil-grown plants. Transient expression was evaluated by visualizing the reporters β-glucuronidase (GUS) and green fluorescent protein (GFP). The experimental parameters of the syringe agroinfiltration were optimized based on the expression levels of the reporter luciferase (LUC). Stably transformed plants were regenerated from transiently transformed leaf explants through callus-induced organogenesis. The functions of Populus genes in secondary cell wall-thickening were characterized by visualizing lignin deposition therein after staining with basic fuchsin. Results We greatly improved the transient transformation efficiency of syringe Agrobacterium infiltration in poplar through screening for a suitable poplar clone from a variety of clones and optimizing the syringe infiltration procedure. The selected poplar clone, Populus davidiana × P. bolleana, is amenable to Agrobacterium syringe infiltration, as indicated by the easy diffusion of the bacterial suspension inside the leaf tissues. Using this technique, we localized a variety of poplar proteins in specific intracellular organelles and illustrated the protein–protein and protein–DNA interactions. The transiently transformed leaves could be used to generate stably transformed plants with high efficiency through callus induction and differentiation processes. Furthermore, transdifferentiation of the protoxylem-like vessel element and ectopic secondary wall thickening were induced in the agroinfiltrated leaves via the transient overexpression of genes associated with secondary wall formation. Conclusions The application of P. davidiana × P. bolleana in Agrobacterium syringe infiltration provides a foundation for the rapid and high-throughput functional characterization of Populus genes in intact poplar plants, including those involved in wood formation, and provides an effective alternative to Populus stable genetic transformation.

Transformed tissue of Dionaea muscipula J. Ellis as a source of biologically active phenolic compounds with bactericidal properties

The Venus flytrap (Dionaea muscipula J. Ellis) is a carnivorous plant able to synthesize large amounts of phenolic compounds, such as phenylpropanoids, flavonoids, phenolic acids, and 1,4-naphtoquinones. In this study, the first genetic transformation of D. muscipula tissues is presented. Two wild-type Rhizobium rhizogenes strains (LBA 9402 and ATCC 15834) were suitable vector organisms in the transformation process. Transformation led to the formation of teratoma (transformed shoot) cultures with the bacterial rolB gene incorporated into the plant genome in a single copy. Using high-pressure liquid chromatography, we demonstrated that transgenic plants were characterized by an increased quantity of phenolic compounds, including 1,4-naphtoquinone derivative, plumbagin (up to 106.63 mg × g−1 DW), and phenolic acids (including salicylic, caffeic, and ellagic acid), in comparison to non-transformed plants. Moreover, Rhizobium-mediated transformation highly increased the bactericidal properties of teratoma-derived extracts. The antibacterial properties of transformed plants were increased up to 33% against Staphylococcus aureus, Enterococcus faecalis, and Escherichia coli and up to 7% against Pseudomonas aeruginosa. For the first time, we prove the possibility of D. muscipula transformation. Moreover, we propose that transformation may be a valuable tool for enhancing secondary metabolite production in D. muscipula tissue and to increase bactericidal properties against human antibiotic-resistant bacteria. Key points • Rhizobium-mediated transformation created Dionaea muscipula teratomas. • Transformed plants had highly increased synthesis of phenolic compounds. • The MBC value was connected with plumbagin and phenolic acid concentrations.