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Despite major breeding efforts by various national and international agencies, yields for the ~40 million hectares of maize, the major food crop in sub‐Saharan Africa, have stagnated at <2 tons/ha/year for the past decade, one‐third the global average. Breeders have succeeded in breeding increased yield with a modicum of tolerance to some single‐weed or pathogen stresses. There has been minimal adoption of these varieties because introgressing polygenic yield and tolerance traits into locally adapted material is very challenging. Multiple traits to deal with pests (weeds, pathogens, and insects) are needed for farmer acceptance, because African fields typically encounter multiple pest constraints. Also, maize has no inherent resistance to some of these pest constraints, rendering them intractable to traditional breeding. The proposed solution is to simultaneously engineer multiple traits into one genetic locus. The dominantly inherited multi‐pest resistance trait single locus can be bred simply into locally adapted, elite high‐yielding material, and would be valuable for farmers, vastly increasing maize yields, and allowing for more than regional maize sufficiency. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

The insertion of genes into mitochondria by biolistic transformation is currently only possible in the yeast Saccharomyces cerevisiae and the algae Chlamydomonas reinhardtii. The fact that S. cerevisiae mitochondria can exist with partial (ρ− mutants) or complete deletions (ρ0 mutants) of mitochondrial DNA (mtDNA), without requiring a specific origin of replication, enables the propagation of exogenous sequences. Additionally, mtDNA in this organism undergoes efficient homologous recombination, making it well‐suited for genetic manipulation. In this review, we present a summarized historical overview of the development of biolistic transformation and discuss iconic applications of the technique. We also provide a detailed example on how to obtain transformants with recombined foreign DNA in their mitochondrial genome. [ABSTRACT FROM AUTHOR]

Chymosin is an important milk clotting enzyme, massively used in the dairy industry. Due to the limited amount of natural chymosin, the recombinant enzyme produced in different organisms is the main source of chymosin. Plants have several advantages for large scale cost-effective production of recombinant chymosin. Here, we used a synthetic codon-optimized version of bovine prochymosin gene for ideal expression in the tobacco host. In this study, we utilized biolistic co-transformation method to introduce the synthetic bovine prochymosin gene into the tobacco genome. Several transgenic plants were regenerated on a selective medium. Molecular analysis confirmed successful integration and expression of prochymosin gene in several transgenic candidates. Total soluble protein extracts from transgenic plants were successfully applied for milk coagulation experiments, demonstrating the production of functional prochymosin in transgenic lines. In conclusion, here, we report successful expression of functional chymosin in tobacco plants from a novel synthetic version of the bovine prochymosin gene. Our experimental data support that plants could serve as a reliable source for safe and cost-effective production of recombinant chymosin enzyme for industrial usage. [ABSTRACT FROM AUTHOR]

Biolistics is one of the widely used methods to deliver nucleic acids into plant cells. In this work, we optimized the protocol for biolistic transformation of Nicotiana tabacum with the gene gun PDS-1000/He Biolistic Particle Delivery System. The Green Fluorescent Protein (GFP) gene was used as a marker. The optimal parameters for the transformation of N. tabacum leaf cells were determined as: pressure, 1350 psi; tungsten particle size, 1.3 μm; plasmid DNA purification method, ethanol re-precipitation. The results should be useful for the development of biolistic transformation protocols for plant cells, including application for plant genome editing of agricultural species. [ABSTRACT FROM AUTHOR]

Background: Sudangrass (Sorghum sudanense) is a major biomass producer for livestock feed and biofuel in many countries. It has a wide range of adaptations for growing on marginal lands under biotic and abiotic stresses. The immature inflorescence is an explant with high embryogenic competence and is frequently used to regenerate different sorghum cultivars. Caffeic acid O-methyl transferase (COMT) is a key enzyme in the lignin biosynthesis pathway, which limits ruminant digestion of forage cell walls and is a crucial barrier in the conversion of plant biomass to bioethanol. Genome editing by CRISPR/Cas9-mediated mutagenesis without a transgenic footprint will accelerate the improvement and facilitate regulatory approval and commercialization of biotech crops. Methods and results: We report the overcome of the recalcitrance in sudangrass transformation and regeneration in order to use genome editing technique. Hence, an efficient regeneration system has been established to induce somatic embryogenesis from the immature inflorescence of two sudangrass cultivars on four MS-based media supplemented with different components. Our results indicate an interaction between genotype and medium composition. The combination of Giza-1 cultivar and M4 medium produces the maximum frequency of embryogenic calli of 80% and subsequent regeneration efficiency of 22.6%. Precise mutagenesis of the COMT gene is executed using the CRISPR/Cas9 system with the potential to reduce lignin content and enhance forage and biomass quality in sudangrass. Conclusion: A reliable regeneration and transformation system has been established for sudangrass using immature inflorescence, and the CRISPR/Cas9 system has demonstrated a promising technology for genome editing. The outcomes of this research will pave the road for further improvement of various sorghum genotypes to meet the global demand for food, feed, and biofuels, achieving sustainable development goals (SDGs). [ABSTRACT FROM AUTHOR]

Chromoblastomycosis (CBM) is a disease caused by several dematiaceous fungi from different genera, and Fonsecaea is the most common which has been clinically isolated. Genetic transformation methods have recently been described; however, molecular tools for the functional study of genes have been scarcely reported for those fungi. In this work, we demonstrated that gene deletion and generation of the null mutant by homologous recombination are achievable for Fonsecaea pedrosoi by the use of two approaches: use of double-joint PCR for cassette construction, followed by delivery of the split-marker by biolistic transformation. Through in silico analyses, we identified that F. pedrosoi presents the complete enzymatic apparatus required for tryptophan (trp) biosynthesis. The gene encoding a tryptophan synthase trpB —which converts chorismate to trp—was disrupted. The ΔtrpB auxotrophic mutant can grow with external trp supply, but germination, viability of conidia, and radial growth are defective compared to the wild-type and reconstituted strains. The use of 5-FAA for selection of trp- phenotypes and for counter-selection of strains carrying the trp gene was also demonstrated. The molecular tools for the functional study of genes, allied to the genetic information from genomic databases, significantly boost our understanding of the biology and pathogenicity of CBM causative agents. [ABSTRACT FROM AUTHOR]

Several biotic and abiotic stresses adversely affect sugarcane crop. Amongst the abiotic stresses, salinity causes substantial losses to sugarcane yield. Lack of conducive environment for sugarcane flowering and unstable genome are the main constraints in sugarcane crop improvement via conventional breeding. Therefore, genetic engineering of sugarcane using Oryza sativa L. H+-PPase gene (OVP1) to confer salt tolerance is the preferred alternative strategy. In the present research, tissue culture and biolistic transformation protocols for sugarcane were optimized. For callus induction, Dichlorophenoxyacetic acid (2,4-D) was most effective. Maximum callus induction (50.22%) was achieved on CM-3 augmented with 2.5 mgL-1 2,4-D. The induced calli was subjected to biolistic transformation. For biolistic transformation, the gold particles (0.6 and 1.0 µm) and plasmid DNA (100 and 50 ng) were used for 1X and 2X bombardment. Significant differences (P≤0.05) were observed amongst the gold particles and plasmid DNA concentrations. Maximum transformation frequencies were observed when calli were bombarded 2X with 0.6 µm gold particles coated with 100 ng of DNA (23.60%). Selection and regeneration of transgenic calli were performed on MS media augmented with BAP (2.5 mgL-), NAA (1 mgL-1) hygromycin (25 mgL-1) and 2% coconut water. Regenerated plants after root induction on MS media containing NAA (2 mgL-1) and 2% coconut water were transferred to loam and clay (1:1) and acclimatized to greenhouse condition. The transgenic plants were confirmed through PCR and RT-PCR.

Plastid engineering has several unique advantages such as high expression of transgenes due to high polyploidy of plastid genomes and environmental biosafety because of maternal inheritance of transgenes, and has become a promising tool for molecular farming, metabolic engineering, and genetic improvement. However, there are no standard vectors available for plastid transformation. Moreover, the construction of plastid transformation vectors containing long operons or genes encoding proteins that are toxic to Escherichia coli was tedious or difficult. Here, we developed a simple plastid transformation technology without the need for in vitro vector construction by using multiple linear DNA fragments which share homologous sequences (HSs) at their ends. The strategy is based on homologous recombination between HSs of DNA fragments via endogenous recombination machinery in plastids, which subsequently are integrated into the plastid genome. We found that HSs of 200 bp or longer were sufficient for mediating the integration into the plastid genome with at least similar efficiency to that of plasmid DNA-based plastid transformation. Furthermore, we successfully used this method to introduce a phage lysin-encoding gene and a long operon into a tobacco plastid genome. The establishment of this technology simplifies the plastid transformation procedure and provides a novel solution for expressing proteins, which are either toxic to the cloning host or large operons in plastids, without need of vector cloning. [ABSTRACT FROM AUTHOR]

The method of biological ballistics (biolistic transformation, genetic bombardment) of plants is one of the most modern methods used for direct gene transfer into plant cells. The main advantages of this method include the ability to simultaneously incorporate several target genes into the plant genome, carry out transfer without unnecessary agrobacterial parts and plasmid DNA sequences, and the short time needed to produce transgenic cells. For different plant objects, the efficiency of obtaining transgenic plants by the ballistic method varies from 1 to 3 %. For potato plants, the transformation efficiency is quite low at the moment and the selection of optimal conditions for biolistics is one of the pressing issues of practical biotechnology. This article presents a successful experience of introducing two genes of interest into two potato varieties using the biolistic approach. The results of biolistic transformation experiments are presented for two types of explants: potato internodes and calli of the varieties Aksor and Nevskiy. Of the 862 explants used for transformation, 56 regenerated plants were obtained. PCR screening of transformants revealed one plant with the insertion of the chitinase gene, one with the insertion of the endo-β-1,3-glucanase gene, and co-transformation by both genes was confirmed in four regenerants. The average transformation efficiency for potato explants was 0.7 %. A high number of regenerants (56) as opposed to a low number of transformants (6) reflects an attempt to increase the number of regenerants by using a lower concentration of the selective agent (kanamycin). Although this approach requires more effort, it can be used to produce potato lines with integrated genes of interest for further use in crop breeding. The lines of potato obtained in the current study by introducing two genes associated with the plant response to fungal pathogens will be further assessed for their resistance to fungal diseases and, if successful, will be used in potato crop breeding.

Maize is an important crop for billions of people globally. The existing immature embryo-based regeneration protocol of maize has major limitations due to the non-availability of explants throughout the year, limited durability for culturing, and its laborious nature. Mature embryos, especially in tropical maize, are considered recalcitrant towards tissue culture. Therefore, standardization of a robust regeneration and transformation protocol in tropical maize using mature embryos or seeds as starting material is long envisaged. Considering this, in this study, 28 diverse tropical maize genotypes were evaluated for their embryogenic callus induction potential using two different explants (nodal explants and split embryo region) under two different callusing media. Out of 28 genotypes, better callus induction was achieved in four genotypes (BML 6, DHM 117, DMRH 1301, and DMRH 1308) from nodal explants. Further, in vitro regeneration was standardized using 22 different combinations of various auxins and cytokinins. Out of 28 genotypes, two recently commercialized and high-yielding cultivars (DMRH 1301 and DMRH 1308) demonstrated the best callusing and regeneration capability with an average regeneration percentage of 60.4% and 53.6%, respectively. Using the nodal explants-derived embryogenic calli, the genetic transformation was successfully carried out using the 'Biolistic' approach, and up to ~ 5% transformation efficiency was achieved. This efficient regeneration and transformation protocol can overcome the major limitations associated with the existing immature embryo-based protocol in tropical maize as mature seeds can be obtained easily in ample quantity round the year. Such a generalized and reproducible protocol has the potential to be a major tool for maize improvement using transgenic and genome-edited techniques. Key message: The standardized protocol not only overcomes the major limitations associated with the existing and predominately used immature embryo-based protocol but it is easier, reproducible, and has either higher or comparable callusing, regeneration, and transformation efficiency. [ABSTRACT FROM AUTHOR]

Wheat is one of three most important cereals worldwide. Its production rises every year. There is a possibility to improve quantitative and qualitative parameters by biolistic method of transformation. The process of introduction of desired gene into the wheat genome and plant regeneration is affected by many factors. To identify the suitable conditions, selection system, the influence of donor, plant environment and the regeneration capacity of wheat genotypes were tested. The scutella of immature embryos served as the initial explants. Plant regeneration was achieved by 6 out of 11 genotypes tested. The highest values were reached by the cultivar Ilias. The effect of donor, plant environment was evaluated by two genotypes. Plants from growth chamber appeared to be better source of explants compared to plants grown in the natural conditions. The selection system was optimized as follows: regeneration medium in the dark and subsequently in photoperiod conditions (DR) with 5 mg/l of phosphinotricin (PPT), shoot induction medium (DS) with 7 mg/l of PPT.

Abstract Background The relatively low efficiency of biolistic transformation and subsequent integration of multiple copies of the introduced gene/s significantly complicate the genetic modification of wheat (Triticum aestivum) and other plant species. One of the key factors contributing to the reproducibility of this method is the uniformity of the DNA/gold suspension, which is dependent on the coating procedure employed. It was also shown recently that the relative frequency of single copy transgene inserts could be increased through the use of nanogram quantities of the DNA during coating. Results A simplified DNA/gold coating method was developed to produce fertile transgenic plants, via microprojectile bombardment of callus cultures induced from immature embryos. In this method, polyethyleneglycol (PEG) and magnesium salt solutions were utilized in place of the spermidine and calcium chloride of the standard coating method, to precipitate the DNA onto gold microparticles. The prepared microparticles were used to generate transgenics from callus cultures of commercial bread wheat cv. Gladius resulting in an average transformation frequency of 9.9%. To increase the occurrence of low transgene copy number events, nanogram amounts of the minimal expression cassettes containing the gene of interest and the hpt gene were used for co-transformation. A total of 1538 transgenic wheat events were generated from 15,496 embryos across 19 independent experiments. The variation of single copy insert frequencies ranged from 16.1 to 73.5% in the transgenic wheat plants, which compares favourably to published results. Conclusions The DNA/gold coating procedure presented here allows efficient, large scale transformation of wheat. The use of nanogram amounts of vector DNA improves the frequency of single copy transgene inserts in transgenic wheat plants.

Genetic transformation plays an imperative role in our understanding of the biology in unicellular yeasts and filamentous fungi, such as Saccharomyces cerevisiae, Aspergillus nidulans, Cryphonectria parasitica, and Magnaporthe oryzae. It also helps to understand the virulence and drug resistance mechanisms of the pathogenic fungus Cryptococcus that causes cryptococcosis in health and immunocompromised individuals. Since the first attempt at DNA transformation in this fungus by Edman in 1992, various methods and techniques have been developed to introduce DNA into this organism and improve the efficiency of homology-mediated gene disruption. There have been many excellent summaries or reviews covering the subject. Here we highlight some of the significant achievements and additional refinements in the genetic transformation of Cryptococcus species. [ABSTRACT FROM AUTHOR]

Haematococcus pluvialis has high commercial value, yet it displays low development of genetic transformation systems. In this research, the endogenous 5′ and 3′ flanking sequences of the constitutive alpha tubulin (tub) gene were cloned along with its encoding region in H. pluvialis, in which some putative promoter elements and polyadenylation signals were identified, respectively. Three selection markers of tub/aadA, tub/hyr and tub/ble with three different antibiotic-resistance genes fused between the endogenous tub promoter (Ptub) and terminator (Ttub) were constructed and utilized for biolistic transformation of H. pluvialis. Stable resistant colonies with introduced aadA genes were obtained after bombardments of either H. pluvialis NIES144 or SCCAP K0084 with the tub/aadA cassette, the efficiency of which could reach up to 3 × 10–5 per μg DNA through an established manipulation flow. Two key details, including the utilization of culture with motile flagellates dominant and controlled incubation of them on membrane filters during bombardments, were disclosed firstly. In obtained transformants, efficient integration and transcription of the foreign tub/aadA fragments could be identified through genome PCR examination and qPCR analysis, nonetheless with random style instead of homologous crossover in the H. pluvialis genome. The presented selection marker and optimized transforming procedures in this report would strengthen the platform for genetic manipulation and modification of H. pluvialis.

Haematococcus pluvialis has high commercial value, yet it displays low development of genetic transformation systems. In this research, the endogenous 5′ and 3′ flanking sequences of the constitutive alpha tubulin (tub) gene were cloned along with its encoding region in H. pluvialis , in which some putative promoter elements and polyadenylation signals were identified, respectively. Three selection markers of tub/aadA, tub/hyr and tub/ble with three different antibiotic-resistance genes fused between the endogenous tub promoter (Ptub) and terminator (Ttub) were constructed and utilized for biolistic transformation of H. pluvialis. Stable resistant colonies with introduced aadA genes were obtained after bombardments of either H. pluvialis NIES144 or SCCAP K0084 with the tub/aadA cassette, the efficiency of which could reach up to 3 × 10–5 per μg DNA through an established manipulation flow. Two key details, including the utilization of culture with motile flagellates dominant and controlled incubation of them on membrane filters during bombardments, were disclosed firstly. In obtained transformants, efficient integration and transcription of the foreign tub/aadA fragments could be identified through genome PCR examination and qPCR analysis, nonetheless with random style instead of homologous crossover in the H. pluvialis genome. The presented selection marker and optimized transforming procedures in this report would strengthen the platform for genetic manipulation and modification of H. pluvialis. [ABSTRACT FROM AUTHOR]

Summary: Emerging genome editing technologies hold great promise for the improvement of agricultural crops. Several related genome editing methods currently in development utilize engineered, sequence‐specific endonucleases to generate DNA double strand breaks (DSBs) at user‐specified genomic loci. These DSBs subsequently result in small insertions/deletions (indels), base substitutions or incorporation of exogenous donor sequences at the target site, depending on the application. Targeted mutagenesis in soybean (Glycine max) via non‐homologous end joining (NHEJ)‐mediated repair of such DSBs has been previously demonstrated with multiple nucleases, as has homology‐directed repair (HDR)‐mediated integration of a single transgene into target endogenous soybean loci using CRISPR/Cas9. Here we report targeted integration of multiple transgenes into a single soybean locus using a zinc finger nuclease (ZFN). First, we demonstrate targeted integration of biolistically delivered DNA via either HDR or NHEJ to the FATTY ACID DESATURASE 2‐1a (FAD2‐1a) locus of embryogenic cells in tissue culture. We then describe ZFN‐ and NHEJ‐mediated, targeted integration of two different multigene donors to the FAD2‐1a locus of immature embryos. The largest donor delivered was 16.2 kb, carried four transgenes, and was successfully transmitted to T1 progeny of mature targeted plants obtained via somatic embryogenesis. The insertions in most plants with a targeted, 7.1 kb, NHEJ‐integrated donor were perfect or near‐perfect, demonstrating that NHEJ is a viable alternative to HDR for gene targeting in soybean. Taken together, these results show that ZFNs can be used to generate fertile transgenic soybean plants with NHEJ‐mediated targeted insertions of multigene donors at an endogenous genomic locus. [ABSTRACT FROM AUTHOR]

Site-directed mutagenesis enables researchers to switch a gene of interest off for functional characterization of the gene. In the pathogenic yeasts, Cryptococcus neoformans and sister species C. deneoformans, this is almost exclusively achieved by introducing DNA into cells through either biolistic transformation or electroporation. The targeted gene is then disrupted by homologous recombination (HR) between the gene and the transforming DNA. Both techniques have downsides; biolistic transformation equipment is very expensive, limiting the use thereof to well-resourced laboratories, and HR occurs at extremely low frequencies in electroporated cryptococcal cells, making this method unappealing for gene targeting when not making use of additional modifications or methods to enhance HR in these cells. One approach to increase the frequency of HR in electroporated cryptococcal cells have recently been described. In this approach, CRISPR-Cas9 technology is utilized to form a double strand break in the targeted gene where after the occurrence of HR seems to be higher. The less expensive electroporation technique can therefore be used to deliver the CRISPR-Cas9 components into cells to disrupt a gene of interest, but only if the CRISPR components can be maintained for long enough in cells to enable their expression. Maintenance of episomal DNA occurs readily in C. deneoformans, but only under certain conditions in C. neoformans. In addition, CRISPR-Cas9 allows for gene complementation in order to fulfill Falkow’s molecular Koch’s postulates and adds other novel methods for studying genes as well, such as the addition of a fluorophore to an inactive Cas9 enzyme to highlight the location of a gene in a chromosome. These developments add less expensive alternatives to current methods, which could lead to more research on this yeast in developing countries where cryptococcal infections are more prevalent and researchers have access to more clinical isolates.

Genome modifications in microalgae have emerged as a crucial and indispensable tool for research in fundamental and applied biology. In particular, CRISPR/Cas9 has gained significant recognition as a highly effective method for genome engineering in these photosynthetic organisms, enabling the targeted induction of mutations in specific regions of the genome. Here, we present a comprehensive protocol for generating knock-out mutants in the model diatom Phaeodactylum tricornutum using CRISPR/Cas9 by both biolistic transformation and bacterial conjugation. Our protocol outlines the step-by-step procedures and experimental conditions required to achieve successful genome editing, including the design and construction of guide RNAs, the delivery of CRISPR/Cas9 components into the algae cells, and the selection of the generated knockout mutants. Through the implementation of this protocol, researchers can harness the potential of CRISPR/Cas9 in P. tricornutum to advance the understanding of diatom biology and explore their potential applications in various fields., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Biolistic transformation of Cryptococcus neoformans is used as a molecular tool to genetically alter or delete targeted genes. The DNA is introduced into the yeast on DNA-coated gold beads by a helium shock wave produced using a biolistic particle system. The procedure often involves insertion of a dominant selectable marker into the desired site by homologous recombination. To increase the likelihood of homologous recombination, large fragments of overlapping DNA are used. The two most used dominant selectable markers are nourseothricin and Geneticin. With the need to generate multiple gene deletions in the same strain, there are recyclable marker systems, such as the bacteriophage P1 Cre-loxP system or CRISPR that provide additional useful molecular tools. While newer strategies exist to generate deletions and introduce markers and other gene modifications, biolistic transformation has remained a viable tool to facilitate the construction of genetically modified yeast strains. This chapter provides a working protocol on how to delete and restore a gene in C. neoformans., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Sugarcane and energycane (Saccharum spp. hybrids) are prominent sources of sugar, ethanol, as well as high-value bioproducts globally. Genetic analysis for trait improvement of sugarcane is greatly hindered by its complex genome, limited germplasm resources, long breeding cycle, as well as recalcitrance to genetic transformation. Here, we present a biolistic-based transformation and bioreactor-based micro-propagation system that has been utilized successfully to transform twelve elite cane genotypes, yielding transformation efficiencies of up to 39%. The system relies on the generation of embryogenic callus from sugarcane and energycane apical shoot tissue, followed by DNA bombardment of embryogenic leaf roll discs (approximately one week) or calli (approximately 4 weeks). We present optimal criteria and practices for selection and regeneration of independent transgenic lines, molecular characterization, as well as a bioreactor-based micro-propagation technique, which can aid in rapid multiplication and analysis of transgenic lines. The cane transformation and micro-propagation system described here, although built on our previous protocols, has significantly accelerated the process of producing and multiplying transgenic material, and it is applicable to other varieties. The system is highly reproducible and has been successfully used to engineer multiple commercial sugarcane and energycane varieties. It will benefit worldwide researchers interested in genomics and genetics of sugarcane photosynthesis, cell wall, and bioenergy related traits. [ABSTRACT FROM AUTHOR]

Plant infection with Agrobacterium rhizogenes leads to the development of a hairy root disease notable for the rapid agravitropic growth of roots on hormone-free nutrient media. In order to look into the interaction of A. rhizogenes with plants and assess opportunities of practical application of hairy root culture, new approaches to their production are elaborated. A method of bacterium-free and plasmid-free production of genetically modified roots (hairy roots) by means of biolistic transformation of leaf explants with a DNA fragment (size of 5461 bp) consisting of genes rolA, rolB, rolC, and rolD are proposed. In most cases, such transformation resulted in the emergence of only adventitious roots with transient expression of rol-genes, and the growth of such roots on hormone-free media ceased in 2-3 months in contrast to genuine hairy roots capable of unrestricted growth. Molecular analysis of different systems of target genes’ expression showed an important role of transgene rolC and host gene of cyclin-dependent protein kinase CDKB1-1 in the maintenance of rapid growth of hairy roots in vitro (in isolated cultures). [ABSTRACT FROM AUTHOR]

Described in humans in 1894, Cryptococcus neoformans , a medically-important basidiomycete fungus, has since been studied in order to identify the factors that enable it to cause disease. Large-scale collections of mutants have been created, and mutant strains deposited in biorepositories. Studying these collections provides deeper functional insights for genes controlling biological processes, and ultimately complements a wealth of genomics and transcriptomics information. Therefore, to many laboratories use of these resources is profoundly insightful to study the molecular basis of cryptococcal pathobiology. The available collections of C. neoformans mutants also aid the molecular target identification and drug discovery efforts. To facilitate access to information, large-scale gene discoveries made from screening C. neoformans collections for over the past decade and a half are hereby compiled in a single document, and their key findings presented in brief. Cryptococcus neoformans is potentially the only basidiomycete yeast with extensively analyzed collections of mutants, and this makes it a plausible model for the creation of important collections for other basidiomycete fungi. Indeed, the genome-wide collections discussed in this present review represent a large sum of genes with associated phenotypes in virulence of this species on which prospective screens can rely as a reference point. Therefore, it is desired that studies underway will draw from these genome-wide screens to identify management strategies against fungal diseases. [ABSTRACT FROM AUTHOR]

Cryptococcus neoformans is a fungal pathogen that claims hundreds of thousands of lives annually. Targeted genetic manipulation through biolistic transformation in C. neoformans drove the investigation of this clinically important pathogen at the molecular level. Although costly and inefficient, biolistic transformation remains the major method for editing the Cryptococcus genome as foreign DNAs introduced by other methods such as electroporation are predominantly not integrated into the genome. Although the majority of DNAs introduced by biolistic transformation are stably inherited, the transformation efficiency and the homologous integration rate (~1-10%) are low. Here, we developed a Transient CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 coupled with Electroporation (TRACE) system for targeted genetic manipulations in the C. neoformans species complex. This method took advantages of efficient genome integration due to double-strand breaks created at specific sites by the transient CRISPR-Cas9 system and the high transformation efficiency of electroporation. We demonstrated that TRACE can efficiently generate precise single-gene deletion mutants using the ADE2 locus as an example. This system can also effectively delete multiple genes in a single transformation, as evident by the successful generation of quadruple mfα1Δ2Δ3Δ4Δ mutants. In addition to generating gene deletion mutants, we complemented the ade2Δ mutant by integrating a wild-type ADE2 allele at the "safe haven" region (SH2) via homologous recombination using TRACE. Interestingly, introduced DNAs can be inserted at a designated genetic site without any homologous sequences, opening up numerous other applications. We expect that TRACE, an efficient, versatile, and cost-effective gene editing approach, will greatly accelerate research in this field. [ABSTRACT FROM AUTHOR]

DNA-protein interactions are critical to almost all cellular functions, and identification of the proteins that bind to an DNA site of interest (gene-centered approach) is an important investigation area. However, gene-centered methods are mainly based on DNA hybridization to isolate target proteins, which is complex and inefficient. Here, we built a gene-centered approach involving direct isolation of target DNA, termed protein capture based on biolistic transformation (PCaB). The target DNA was labeled with biotin and cyanine 3 (Cy3) at its 5′ and 3′ DNA ends, respectively, and introduced into the host plants through biolistic transformation. The DNA and its binding proteins were crosslinked using formaldehyde. The labeled DNAs were obtained using gel excision and biotin-Streptavidin affinity according to the indication of Cy3 fluorescence, which make harvest of target DNA with a low background. The DNA-binding proteins were identified using mass spectrometry analysis. The PCaB method allowed us to identify and confirm 16 putative upstream regulators of the BpERF3 gene from Betula platyphylla. Theoretically, PCaB could be adapted to all plant species that can be transformed using biolistic bombardment, and captures DNA-binding proteins quickly with a low background. Therefore, PCaB will provide a powerful tool to discover DNA-protein interactions. • A method captures DNA-binding protein efficiently with low background. • The DNA-binding proteins are captured using gel excision and biotin-Streptavidin affinity. • This method studies DNA-protein interactions in all plants with biolistic transformation. [ABSTRACT FROM AUTHOR]

Polyunsaturated fatty acids (PUFAs) affect diverse physiological processes and human health. Most cereals are poor in n-3 and n-6 PUFAs. Using biolistics, barley (Hordeum vulgare L. cv. Golden Promise) was transformed with an artificial gene encoding Δ6-desaturase (D6D) under an endosperm-specific promoter. This artificial gene was designed from the sequence of D6D of the filamentous fungus Thamnidium elegans, but codon usage was optimised for cereals. A signal sequence from the gene encoding for high molecular mass glutenin Dx5 was added to a destinate mature protein. Successful transformation was confirmed in T0 plants at the genomic level and in T1 seeds at the transcriptomic and metabolomic levels. Transformed plants produced up to 0.141 % of γ-linolenic acid (GLA) and 0.294 % of stearidonic acid (SDA) of the total amount of fatty acids in their grains. Although the content of these fatty acids was relatively low, the current study provides the first evidence that transgenic barley can be a source of GLA/SDA.

This research utilized the E. coli manA gene encoding phosphomannose isomerase (PMI) selection on sucrose/mannose medium to increase transformation efficiencies after biolistic transformation of two immature citrus rootstock cultivars. Plasmid DNA, containing the manA gene and the enhanced green fluorescent protein (egfp) reporter gene, was bombarded into epicotyl explants of immature Carrizo citrange and Swingle citrumelo. GFP positive shoots were micro-grafted onto in vitro grown immature Carrizo rootstocks. Nineteen transgenic Carrizo shoots were obtained from ten paired shots, and eight Swingle shoots from five paired shots. The mean transformation efficiency of Carrizo was 1.9 transgenics/paired shot while the transformation efficiency of Swingle was comparable at 1.6 transgenics/paired shot. The transformants were analyzed by PCR for the presence of transgenes. Southern blot analysis of eight representative Carrizo transgenic events and four Swingle transgenic events showed that all transgenics had one to three copies of the manA gene. The PMI enzyme activity in the transgenic lines was confirmed using the chlorophenol red assay.

A few methods only enable to follow the fate of plant cells in vivo. One of the most promising is using the Green Fluorescent Protein (GFP). In our preliminary study we set up the experimental system enabling labelling of Anagallis arvensis cells with this marker. We prepared an expression plasmid containing red-shifted gfp with optimised translation start site context, under the control of CaMV 35S transcription promoter. The construct was introduced into A. arvensis cells by particle bombardment. We developed two methods of material preparation for this transformation: in vitro cultured stem internodes with regenerating adventitious shoots (the earliest stages of regeneration); and shoot tips with temporarily exposed apices. The reflected light fluorescence microscope Olympus with the set of filters U-MNB designed for fluorescein detection enables the observation of GFP fluorescence. Both ordinary epidermal cells and stomata guard cells were transformed. Their fluorescence was observed for up to 14 days. Artefacts (autofluorescence of glandular trichomes and faint green glowing of meristematic tissue) could be overcome by the optimisation of the filter set.

Here, we report the optimized conditions for biolistic particle delivery-mediated genetic transformation of bitter melon using petiole segments. In this study, DNA-coated gold particles of 0.6 µm were used for optimizing the parameters of transformation and eventually regeneration of bitter melon putative transgenics. Initially, biolistic parameters namely helium pressure and macrocarrier to target tissue distance, were optimized using binary vector pBI121 carrying both β-glucuronidase gene ( GUS) and neomycin phosphotransferase II gene ( npt II) as a reporter and as a selectable marker gene, respectively. The effect of optimized physical parameters on the frequency of transient (79.2 ± 1.52%) and stable (41.9%) expressions has been investigated. The optimized biolistic parameters for petiole segments of Momordica charantia L. were determined as follows: 650 psi helium pressure and 6 cm target distance. Using the optimized parameters, transformation of bitter melon was carried out for generation of putative transformants from bombarded tissues on SRM-K medium, with a mean number of 50.3 explants surviving at the end of the final selection (50 mg l kanamycin) round. Finally, the transformants produced were subjected to GUS histochemical assay, and integration of the transgenes ( GUS and npt II) into the nuclear genome was confirmed by PCR analysis. DNA blot analysis confirmed the transgene integration in the transformed plantlet genomes. The present study may be used for developing transplastomic technology in this valuable medicinal plant for enhanced metabolic engineering pathways and production of biopharmaceuticals. [ABSTRACT FROM AUTHOR]

Key message: A large, 53-kbp, intact DNA fragment was inserted into the wheat ( Triticum aestivum L.) genome. FISH analyses of individual transgenic events revealed multiple insertions of intact fragments. Abstract: Transferring large intact DNA fragments containing clusters of resistance genes or complete metabolic pathways into the wheat genome remains a challenge. In a previous work, we showed that the use of dephosphorylated cassettes for wheat transformation enabled the production of simple integration patterns. Here, we used the same technology to produce a cassette containing a 44-kb Arabidopsis thaliana BAC, flanked by one selection gene and one reporter gene. This 53-kb linear cassette was integrated in the bread wheat ( Triticum aestivum L.) genome by biolistic transformation. Our results showed that transgenic plants harboring the entire cassette were generated. The inheritability of the cassette was demonstrated in the T1 and T2 generation. Surprisingly, FISH analysis performed on T1 progeny of independent events identified double genomic insertions of intact fragments in non-homoeologous positions. Inheritability of these double insertions was demonstrated by FISH analysis of the T1 generation. Relative conclusions that can be drawn from molecular or FISH analysis are discussed along with future prospects of the engineering of large fragments for wheat transformation or genome editing. [ABSTRACT FROM AUTHOR]

The transgenic wheat cell lines were obtained via biolistic transformation of the callus cultures initiated from the 3-day-old sterile seedling shoot apexes. The pAHC25 vector construction used for 14- and 28-day-old callus cultures transformation carried the selective phosphinothricin-N-acetyltransferase (bar) gene and reporter ?-glucuronidase gene. The cell line selection was carried out on the media with phosphinothricin by means of graduated cell selection. The transgenic status of the obtained forms was proved by PCR-analysis. The presence of new relatively high molecular (more than 1 000 bp) amplicons were found out for three transformed lines by means of IRAP PCRanalysis with the primers coding for long termainal repeats sequences of SIRE 1 retrotransposon. This fact may prove transposition of this mobile genetic element. The new DNA fragments were detected for three of the seven analyzed lines but for the control callus. It is possible to assume at induction of SIRE 1 transposition bis probably caused by the genomic stress of foreign DNA inserting or associated with the transformation process (mechanical wounding, cultivation on selective media).

The mature embryos of fourteen elite winter wheat cultivars have been transformed by a biolistic approach. The gene coding for γ-glutamylcysteine synthetase (EC 6.3.2.2) was used as a transgene in order to obtain stable transformants resistant to drought stress. A binary vector, pBinarUTRECS, was used. The gene was under the control of the CaMV35S promoter region. GUS::GFP gene fusion was used as a reporter system and nptII served as a selectable marker gene. A high regeneration capacity of callus tissue under the selective pressure and successful GUS assay of transformed tissue were an indication of successful insertion of a transgene into mature embryo derived wheat tissue. .

An efficient genetic transformation system for oil palm using particle bombardment was established. The transformation was performed using the pCAMBIA 1302 DNA which contains the green fluorescent protein (mgfp5) reporter gene and the selectable marker hygromycin phosphotransferase (hph) gene. Oil palm explants were bombarded under the following conditions: rupture disk to macrocarrier distance, 11 mm; macrocarrier to target tissue, 90 mm and using 1 μm gold particles as microcarrier. Four different pressures of helium were tested with three types of target tissues (mature embryo, embryogenic callus and young seedlings). From the transformation efficiency, calli were much more efficiently transformed in the biolistic process compared with mature embryos and seedlings. A 100% transformation efficiency for DNA delivery into callus oil palm explants was obtained at 850 psi helium pressures, for embryos a maximum 81.8% efficiency required 850 psi and for seedlings a maximum 75.9% efficiency required 1,550 psi. Using a confocal laser scanning microscope, and appropriate filters to block out the red fluorescence of chlorophyll, expression of the GFP gene was observed in all three bombarded explant types by a bright-green fluorescence. The mgfp5 gene was still present more than 8 months after bombardment, hence it indicated the stability of transgene in those transformants.

Virus-mediated transient gene overexpression and gene expression silencing can be used to screen gene functions in plants. Sugarcane mosaic virus (SCMV) is a positive strand RNA virus in the Potyviridae family that has been modified to be used as vector to infect monocots, including maize ( Zea mays ), for transient gene overexpression and gene expression silencing. Relative to stable transformation, SCMV-mediated transient expression in maize has the advantages of being faster and less expensive. Here, we describe a protocol for cloning constructs into the plasmid vector pSCMV-CS3. After maize seedlings are transformed with pSCMV-CS3 constructs by particle bombardment, the virus replicates and spreads systemically in the plants. Subsequent infections of maize seedlings can be accomplished by rub inoculation with sap from SCMV-infested plants. As an example of a practical application of the method, we also describe virus-induced gene silencing (VIGS) of fall armyworm ( Spodoptera frugiperda ) gene expression. Transgenic viruses are created by cloning a segment of the fall armyworm target gene into pSCMV-CS3 prior to maize transformation. Caterpillars are fed on the virus-infected maize plants, which make dsRNA to silence the expression of the fall armyworm target gene after ingestion. This use of SCMV for plant-mediated VIGS in insects allows rapid screening of gene functions when caterpillars are feeding on their host plants. Graphical overview., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (©Copyright : © 2023 The Authors; This is an open access article under the CC BY-NC license.)