Baker´s yeast Saccharomyces cerevisiae has been widely used to understand mitochondrial biology for decades. This model has provided knowledge about essential, conserved mitochondrial pathways among eukaryotes, and fungi or yeast-specific pathways. One of the many abilities of S. cerevisiae is the capacity to manipulate the mitochondrial genome, which so far is only possible in S. cerevisiae and the unicellular algae Chlamydomonas reinhardtii. The biolistic transformation of yeast mitochondria allows us to introduce site-directed mutations, make gene rearrangements, and introduce reporters. These approaches are mainly used to understand the mechanisms of two highly coordinated processes in mitochondria: translation by mitoribosomes and assembly of respiratory complexes and ATP synthase. However, mitochondrial transformation can potentially be used to study other pathways. In the present work, we show how to transform yeast mitochondria by high-velocity microprojectile bombardment, select and purify the intended transformant, and introduce the desired mutation in the mitochondrial genome.

CONCLUSIONS: A stable genetic transformation system for Erigeron breviscapus was developed. We cloned the EbYUC2 gene and genetically transformed it into Arabidopsis thaliana and E. breviscapus. The leaf number, YUC2 gene expression, and the endogenous auxin content in transgenic plants were significantly increased. Erigeron breviscapus is a prescription drug for the clinical treatment of cardiovascular and cerebrovascular diseases. The rosette leaves have the highest content of the major active compound scutellarin and are an important component in the yield of E. breviscapus. However, little is known about the genes related to the leaf number and flowering time of E. breviscapus. In our previous study, we identified three candidate genes related to the leaf number and flowering of E. breviscapus by combining resequencing data and genome-wide association study (GWAS). However, their specific functions remain to be characterized. In this study, we cloned and transformed the previously identified full-length EbYUC2 gene into Arabidopsis thaliana, developed the first stable genetic transformation system for E. breviscapus, and obtained the transgenic plants overexpressing EbYUC2. Compared with wild-type plants, the transgenic plants showed a significant increase in the number of leaves, which was correlated with the increased expression of EbYUC2. Consistently, the endogenous auxin content, particularly indole-3-acetic acid, in transgenic plants was also significantly increased. These results suggest that EbYUC2 may control the leaf number by regulating auxin biosynthesis, thereby laying a foundation for revealing the molecular mechanism governing the leaf number and flowering time of E. breviscapus.

Transient in planta transformation is a fast and cost-effective alternative for plant genetic transformation. Most protocols for in planta transformation rely on the use of Agrobacterium-mediated transformation. However, the protocols currently in use are standardized for small-sized plants due to the physical and economic constraints of submitting large-sized plants to a vacuum treatment. This work presents an effective protocol for localized vacuum-based agroinfiltration customized for large-sized plants. To assess the efficacy of the proposed method, we tested its use in cacao plants, a tropical plant species recalcitrant to genetic transformation. Our protocol allowed applying up to 0.07 MPa vacuum, with repetitions, to a localized aerial part of cacao leaves, making it possible to force the infiltration of Agrobacterium into the intercellular spaces of attached leaves. As a result, we achieved the Agrobacterium-mediated transient in planta transformation of attached cacao leaves expressing for the RUBY reporter system. This is also the first Agrobacterium-mediated in planta transient transformation of cacao. This protocol would allow the application of the vacuum-based agroinfiltration method to other plant species with similar size constraints and open the door for the in planta characterization of genes in recalcitrant woody, large-size species.

CONCLUSIONS: A highly efficient transformation procedure to generate transgenic Stylosanthes roots was established. SgEXPB1 is involved in Stylosanthes root growth under phosphorus deficiency. Stylo (Stylosanthes spp.) is an important forage legume widely applied in agricultural systems in the tropics. Due to the recalcitrance of stylo genetic transformation, functional characterization of candidate genes involved in stylo root growth is limited. This study established an efficient procedure for Agrobacterium rhizogenes-mediated transformation for generating transgenic composite plants of S. guianensis cultivar \'Reyan No. 5\'. Results showed that composite stylo plants with transgenic hairy roots were efficiently generated by A. rhizogenes strains K599 and Arqual, infecting the residual hypocotyl at 1.0 cm of length below the cotyledon leaves of 9-d-old seedlings, leading to a high transformation efficiency of > 95% based on histochemical β-glucuronidase (GUS) staining. Notably, 100% of GUS staining-positive hairy roots can be achieved per composite stylo plant. Subsequently, SgEXPB1, a β-expansin gene up-regulated by phosphorus (P) deficiency in stylo roots, was successfully overexpressed in hairy roots. Analysis of hairy roots showed that root growth and P concentration in the transgenic composite plants were increased by SgEXPB1 overexpression under low-P treatment. Taken together, a highly efficient A. rhizogenes-mediated transformation procedure for generating composite stylo plants was established to study the function of SgEXPB1, revealing that this gene is involved in stylo root growth during P deficiency.

The stable genetic transformation of soybean is time-consuming and inefficient. As a simple and practical alternative method, hairy root transformation mediated by Agrobacterium rhizogenes is widely applied in studying root-specific processes, nodulation, biochemical and molecular functions of genes of interest, gene editing efficiency of CRISPR/Cas9, and biological reactors and producers. Therefore, many laboratories have developed unique protocols to obtain hairy roots in composite plants composed of transgenic roots and wild-type shoots. However, these protocols still suffer from the shortcomings of low efficiency and time, space, and cost consumption. To address this issue, we developed a new protocol efficient regeneration and transformation of hairy roots (eR&T) in soybean, by integrating and optimizing the main current methods to achieve high efficiency in both hairy root regeneration and transformation within a shorter period and using less space. By this eR&T method, we obtained 100% regeneration of hairy roots for all explants, with an average 63.7% of transformation frequency, which promoted the simultaneous and comparative analysis of the function of several genes. The eR&T was experimentally verified Promoter:GUS reporters, protein subcellular localization, and CRISPR/Cas9 gene editing experiments. Employing this approach, we identified several novel potential regulators of nodulation, and nucleoporins of the Nup107-160 sub-complex, which showed development-dependent and tissue-dependent expression patterns, indicating their important roles in nodulation in soybean. Thus, the new eR&T method is an efficient and economical approach for investigating not only root and nodule biology, but also gene function.

The aim of the study was to evaluate the long-term effect of Agrobacterium rhizogenes genes transfer on plant antioxidant system by the study of superoxide dismutase (SOD) activity in \"hairy\" roots of Artemisia and Althaea spp plants. PCR analyses revealed stability of the transformation and presence of bacterial rol B and rol C genes in the \"hairy\" roots after 4-6 years from the transformation event. SOD activity in the roots of untransformed in vitro cultivated plants used for the initiation of \"hairy\" roots growth was in the range of 45.8 ± 8.7 U/μg (Althaea officinalis) and 275 ± 97.1 U/μg (Artemisia ludoviciana). After a long-term in vitro cultivation more than half of tested \"hairy\" root lines (54%) showed a significant increase in SOD activity values compared to untransformed roots. The highest SOD activity values of \"hairy\" root lines (24-fold increase) were founded in A. officinalis (1105 ± 174 U/μg) and A. dracunculus (1356 ± 402 U/μg). The increase of the activity was found also in \"hairy\" roots of A. vulgaris (up to 375 ± 28.2 U/μg, sevenfold increase), A. ludoviciana (1001 ± 191 U/μg, 3.6-fold increase), and A. tilesii (438 ± 104 U/μg, 1.6-fold increase). The results of our study indicate that transformation by wild-type A. rhizogenes not harboring any foreign genes implementing in SOD activity regulation can often stably activate plant antioxidant enzyme system. This effect, observed in the \"hairy\" roots of five plant species in 4-6 years of the transformation event, obviously, should be taken into account in works aimed at creating transgenic plants by Agrobacterium-mediated transformation.

Ion channels play crucial roles in cell physiology, and are a major class of targets for clinically relevant pharmaceuticals. Because they carry ionic current, the function and pharmacology of ion channels can be studied using electrophysiological approaches that range in resolution from the single molecule to many millions of molecules. This chapter describes electrophysiological approaches for the study of one representative ion channel that is defective in a genetic disease, and that is the target of so-called highly effective modulator therapies now used in the clinic: the cystic fibrosis transmembrane conductance regulator (CFTR). Protocols are provided for studying CFTR expressed heterologously, for CFTR expressed in situ in airway epithelial cells, and for purified or partially purified CFTR protein reconstituted into planar lipid bilayers.

Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive organs/gametes. Recent advances in CRISPR-Cas9 genome editing technology allow for high editing efficacy and timesaving knockout mutations of endogenous candidate genes at specific sites. Additionally, Agrobacterium-mediated genetic transformation of rice is also a key method for gene modification, which has been widely adopted by many public and private laboratories. In this study, we applied CRISPR-Cas9 genome editing tools and successfully generated three male sterile mutant lines by targeted genome editing of OsABCG15 in a japonica cultivar. We used a modified Agrobacterium-mediated rice transformation method that could provide excellent means of genetic emasculation for hybrid seed production in rice. Transgenic plants can be obtained within 2-3 months and homozygous transformants were screened by genotyping using PCR amplification and Sanger sequencing. Basic phenotypic characterization of the male sterile homozygous line was performed by microscopic observation of the rice male reproductive organs, pollen viability analysis by iodine potassium iodide (I2-KI) staining semi-thin cross-sectioning of developing anthers.

Auxin transport mediates the asymmetric distribution of auxin that determines the fate of cell development. Agrobacterium-mediated genetic transformation is an important technical means to study gene function. Our previous study showed that the expression levels of LpABCB21 and LpPILS7 are significantly up-regulated in the somatic embryogenesis (SE) of Lilium pumilum DC. Fisch. (L. pumilum), but the functions of both genes remain unclear. Here, the genetic transformation technology previously developed by our team based on the L. pumilum system was improved, and the genetic transformation efficiency increased by 5.7-13.0%. Use of overexpression and CRISPR/Cas9 technology produced three overexpression and seven mutant lines of LpABCB21, and seven overexpression and six mutant lines of LpPILS7. Analysis of the differences in somatic embryo induction of transgenic lines confirmed that LpABCB21 regulates the early formation of the somatic embryo; however, excessive expression level of LpABCB21 inhibits somatic embryo induction efficiency. LpPILS7 mainly regulates somatic embryo induction efficiency. This study provides a more efficient method of genetic transformation of L. pumilum. LpABCB21 and LpPILS7 are confirmed to have important regulatory roles in L. pumilum SE thus laying the foundation for subsequent studies of the molecular mechanism of Lilium SE.

An essential gene is defined as a gene that cannot be completely removed from the genome. Investigation of an essential gene function is limited because its deletion strain cannot be readily created. Here we describe a protocol called plasmid shuffling that can be conveniently employed in yeast to study essential gene functions. The essential gene is first cloned into a YCp-based plasmid with URA3 as a selectable marker and then transformed into host cells. The transformed cells can then be used to delete the chromosomal copy of the essential gene. The gene is then cloned into another YCp-based plasmid with a different selectable marker, and the gene sequence can be altered in vitro. Plasmids carrying the mutated gene sequences are transformed into the above cells, resulting in carrying two plasmids. These cells are grown in medium containing 5-FOA that selects ura3 cells. The 5-FOA-resistant cells are expected to only carry the plasmid containing the mutated essential gene, whose functions can be assessed.