Biolistic Method Research Articles

Advances in Soybean Genetic Improvement.

The soybean (Glycine max) is a globally important crop due to its high protein and oil content, which serves as a key resource for human and animal nutrition, as well as bioenergy production. This review assesses recent advancements in soybean genetic improvement by conducting an extensive literature analysis focusing on enhancing resistance to biotic and abiotic stresses, improving nutritional profiles, and optimizing yield. We also describe the progress in breeding techniques, including traditional approaches, marker-assisted selection, and biotechnological innovations such as genetic engineering and genome editing. The development of transgenic soybean cultivars through Agrobacterium-mediated transformation and biolistic methods aims to introduce traits such as herbicide resistance, pest tolerance, and improved oil composition. However, challenges remain, particularly with respect to genotype recalcitrance to transformation, plant regeneration, and regulatory hurdles. In addition, we examined how wild soybean germplasm and polyploidy contribute to expanding genetic diversity as well as the influence of epigenetic processes and microbiome on stress tolerance. These genetic innovations are crucial for addressing the increasing global demand for soybeans, while mitigating the effects of climate change and environmental stressors. The integration of molecular breeding strategies with sustainable agricultural practices offers a pathway for developing more resilient and productive soybean varieties, thereby contributing to global food security and agricultural sustainability.

TRANSGENE MANUFACTURING: FROM TOOLS TO APPLICATIONS

Genetic modification generation is an important intersection of genetic engineering and biotechnology and involves the integration of foreign genes into the genome of organisms to produce specific proteins or repair genetic abnormalities. The machine has many applications in medicine, agriculture and biotechnology. The evolution of genetically modified production technologies from traditional methods to advanced genetic engineering tools demonstrates their evolution. This review discusses various methods incorporating modern and advanced technologies and explores their applications and future prospects. Early models of genetic modification generally involved organisms modified to produce human insulin, followed by the evolution of animals and plants. Traditional methods such as microinjection, retrovirus-mediated gene transfer and embryonic stem cell -mediated gene transfer are important for the production of transgenic animals. Similarly, Agrobacterium-mediated transformation and biolistic transformation methods are used to produce genetically modified plants. Although useful, these methods often result in synergistic and differential gene expression. Made with genetic modification. This technology allows modification of the target with minimal impact on the target, thus increasing the predictability and efficiency of genetic modification. Additionally, CRISPR/Cas9's ability to alter multiple genes simultaneously and adaptability to various organisms expands its applications in medicine and agriculture. Synthetic biology continues to advance genetic engineering by designing and building new biological materials and systems, enabling innovations such as genetic engineering, metabolic processes, and minimal genomes. Viral vectors such as adeno-associated virus (AAV), lentivirus, and adenovirus have unique advantages and clinical challenges. Nonviral vectors, including lipid nanoparticles, electroporated, and polymeric vectors, provide alternative delivery methods with varying efficacy and specificity. Painkiller. It can improve crop growth, livestock and disease resistance in agriculture. Applications of biotechnology include biofuels, biopharmaceuticals, and bioremediation. Future directions aim to improve delivery, expand applications, explore synthetic genomics, advance personalized medicine, and develop regenerative agriculture.In summary, with the influence of advances in genetic engineering and technology, genetically modified production has become a complex field with many applications. Its future promises transformative solutions to global challenges in health, food security and environmental sustainability. To reach its full potential, continuous innovation and ethical thinking are essential.

Enhanced antifungal activity of sugarcane cv. NCo310 expressing chimeric chitinase 42

The development of sustainable and environmentally safe strategies for crop protection against fungal pathogens is a significant challenge in agriculture. This paper discusses the successful transfer of a chimeric chitinase 42 gene into sugarcane using the biolistic method, resulting in an enhanced antifungal activity. Chitinases possess the enzymatic capability to degrade chitin, a key component of the cell walls in various fungal phytopathogens. In this research, we employed a chimeric chitinase 42 that combines the 42 kDa chitinase from Trichoderma atroviridae with a chitin binding domain from Serratia marcescens chitinase B. This chimeric chitinase exhibits higher antifungal activity against phytopathogenic fungi. PCR analysis confirmed the integration of the transgene into the genome of transformants, while the expression of the transgene was assessed using reverse transcription-PCR. Transgenic lines showed to have different chitinase activity and a high increase in chitinase activity was detected in lines T3, T5 and T6 with about 18 U/mg chitinase activity compared to the control plants (2.8 U/mg). Antifungal activity of transgenic lines T3, T5 and T6 on sugarcane fungal pathogens was shown in radial diffusion and detached leaf assays. The enhanced antifungal activity observed in the transformed sugarcane lines indicates the potential of chimeric chitinase 42 as an effective antifungal agent.

Biolistics-mediated transformation of hornworts and its application to study pyrenoid protein localization.

Hornworts are a deeply diverged lineage of bryophytes and a sister lineage to mosses and liverworts. Hornworts have an array of unique features that can be leveraged to illuminate not only the early evolution of land plants, but also alternative paths for nitrogen and carbon assimilation via cyanobacterial symbiosis and a pyrenoid-based CO2-concentrating mechanism (CCM), respectively. Despite this, hornworts are one of the few plant lineages with limited available genetic tools. Here we report an efficient biolistics method for generating transient expression and stable transgenic lines in the model hornwort, Anthoceros agrestis. An average of 569 (±268) cells showed transient expression per bombardment, with green fluorescent protein expression observed within 48-72 h. A total of 81 stably transformed lines were recovered across three separate experiments, averaging six lines per bombardment. We followed the same method to transiently transform nine additional hornwort species, and obtained stable transformants from one. This method was further used to verify the localization of Rubisco and Rubisco activase in pyrenoids, which are central proteins for CCM function. Together, our biolistics approach offers key advantages over existing methods as it enables rapid transient expression and can be applied to widely diverse hornwort species.

Efficient transformation of the isolated microspores of Chinese cabbage (Brassica rapa L. ssp. pekinensis) by particle bombardment

BackgroundThe low efficiency of genetic transformation in Chinese cabbage (Brassica rapa L. ssp. pekinensis) is the key problem affecting functional verification. Particle bombardment is a widely used method along with the Agrobacterium-mediated method. As a physical means, it has almost no restrictions on the type of host and a wide range of receptor types, which largely avoids the restriction of explants. The bombardment parameters, which include the number of bombardments, the bombardment pressure, and the bombardment distance, may affect the microspores' genetic transformation efficiency.ResultsThe transformation efficiency was improved using the particle bombardment method under the combination of bombardment shot times (3, 4, 5) × bombardment pressure (900, 1100, 1350 psi) × bombardment distance (3, 6, 9 cm). The average viability of microspores in the treatment group ranged from 74.76 to 88.55%, while the control group was 88.09%. When the number of shot times was 4, the number of embryos incubated in the treatment group ranged from 16 to 236 per dish, and the control group had 117 embryos per dish. When the bombardment parameters of the biolistic method were 4 shot times—1350 psi—3 cm, 4 times—1100 psi—3 cm, and 4 times—900 psi—3 cm, they had high transient expression efficiency, and the average number of transformed microspores was 21.67, 11.67, and 11.67 per dish (3.5 mL), respectively. When the bombardment parameters were 4 times, 900 psi, and 6 cm, the highest genetically transformed embryos were obtained, and the transformation efficiency reached 10.82%.ConclusionA new genetic transformation system with proper parameters for Chinese cabbage microspores was established using particle bombardment. This proper transformation system could provide a useful tool for the improvement of cultivar quality and the investigation of functional genes in Chinese cabbage.

Combined biolistic and cell penetrating peptide delivery for the development of scalable intradermal DNA vaccines

Physical-based gene delivery via biolistic methods (such as the Helios gene gun) involve precipitation of nucleic acids onto microparticles and direct transfection through cell membranes of exposed tissue (e.g. skin) by high velocity acceleration. The glycosaminoglycan (GAG)-binding enhanced transduction (GET) system exploits novel fusion peptides consisting of cell-binding, nucleic acid condensing, and cell-penetrating domains, which enable enhanced transfection across multiple cell types. In this study, we combined chemical (GET) and physical (gene gun) DNA delivery systems, and hypothesized the combination would generate enhanced distribution and effective uptake in cells not initially transfected by biolistic penetration. Physicochemical characterization, optimization of bullet contents and transfection experiments in vitro in cell monolayers and engineered tissue demonstrated these formulations transfected efficiently, including DC2.4 dendritic cells. We incorporated these formulations into a biolistic format for gene gun by forming fireable dry bullets obtained via lyophilization (freeze drying). This system is simple and with enhanced scalability compared to conventional methods to generate bullets. Flushed GET bullet contents retained their ability to mediate transfection (17-fold greater and 13-fold greater reporter gene expression than standard spermidine bullets in the absence and presence of serum, respectively). Fired GET bullets in vitro (in cells and collagen gels) and in vivo (mice) showed increased reporter gene transfection compared to untreated controls, whilst maintaining cell viability in vitro and having no obvious toxicity in vivo. Lastly, a SARS-CoV-2 plasmid DNA vaccine with spike (S) protein-receptor binding domain (S-RBD) was delivered by gene gun using GET bullets. Specific T cell and antibody responses comparable to the conventional system were generated. The non-physical and physical combination of GET‑gold-DNA carriers using gene gun shows potential as an alternative DNA delivery method that is scalable for mass deployable vaccination and intradermal gene delivery.

Genetic transformation methods for diatom Nitzschia captiva: New tools to better understand dinotom endosymbiosis

Endosymbiosis is a widespread and ecologically significant phenomenon in the marine environment. How these endosymbiotic partners evolve into an organism with a new organelle is still mostly unknown and requires investigation into modern symbioses. Dinotoms, dinoflagellates with evolutionarily intermediate diatom plastids, are considered excellent models for studying organellogenesis as they remain at three successive but distinct stages. Efforts to understand the host dinoflagellate-endosymbiotic diatom relationship has been limited by the lack of genetic transformation methods for either member of the symbiosis. To address this absence, we modified existing diatom biolistic and conjugation transformation methods and cryopreservation protocols for the diatom Nitzschia captiva, an essential prey for the kleptoplastic dinotom Durinskia capensis. Through the use of Phaeodactylum tricornutum, Cylindrotheca fusiformis, and native Nitzschia captiva diatom designed plasmids, we successfully express and target EGFP to the cytosol, mitochondria, and plastids of N. captiva, and visualize these organelles inside D. capensis in vivo, allowing specific labeling and tracking of organelles and proteins after ingestion. Furthermore, we attempt to utilize CRISPR/Cas9 to target the introduced EGFP gene but find no evidence of successful gene editing.

Hyper-expression of GFP-fused active hFGF21 in tobacco chloroplasts

Human fibroblast growth factor 21 (hFGF21) is a promising candidate for metabolic diseases. In this study, a tobacco chloroplast transformation vector, pWYP21406, was constructed that consisted of codon-optimized encoding gene hFGF21 fused with GFP at its 5’ terminal; it was driven by the promoter of plastid rRNA operon (Prrn) and terminated by the terminator of plastid rps16 gene (Trps16). Spectinomycin-resistant gene (aadA) was the marker and placed in the same cistron between hFGF21 and the terminator Trps16. Transplastomic plants were generated by the biolistic bombardment method and proven to be homoplastic by Southern blotting analysis. The expression of GFP was detected under ultraviolet light and a laser confocal microscope. The expression of GFP-hFGF21 was confirmed by immunoblotting and quantified by enzyme-linked immunosorbnent assay (ELISA). The accumulation of GFP-hFGF21 was confirmed to be 12.44 ± 0.45% of the total soluble protein (i.e., 1.9232 ± 0.0673 g kg−1 of fresh weight). GFP-hFGF21 promoted the proliferation of hepatoma cell line HepG2, inducing the expression of glucose transporter 1 in hepatoma HepG2 cells and improving glucose uptake. These results suggested that a chloroplast expression is a promising approach for the production of bioactive recombinant hFGF21.

Cell-Penetrating Peptides for Use in Development of Transgenic Plants.

Genetically modified plants and crops can contribute to remarkable increase in global food supply, with improved yield and resistance to plant diseases or insect pests. The development of biotechnology introducing exogenous nucleic acids in transgenic plants is important for plant health management. Different genetic engineering methods for DNA delivery, such as biolistic methods, Agrobacterium tumefaciens-mediated transformation, and other physicochemical methods have been developed to improve translocation across the plasma membrane and cell wall in plants. Recently, the peptide-based gene delivery system, mediated by cell-penetrating peptides (CPPs), has been regarded as a promising non-viral tool for efficient and stable gene transfection into both animal and plant cells. CPPs are short peptides with diverse sequences and functionalities, capable of agitating plasma membrane and entering cells. Here, we highlight recent research and ideas on diverse types of CPPs, which have been applied in DNA delivery in plants. Various basic, amphipathic, cyclic, and branched CPPs were designed, and modifications of functional groups were performed to enhance DNA interaction and stabilization in transgenesis. CPPs were able to carry cargoes in either a covalent or noncovalent manner and to internalize CPP/cargo complexes into cells by either direct membrane translocation or endocytosis. Importantly, subcellular targets of CPP-mediated nucleic acid delivery were reviewed. CPPs offer transfection strategies and influence transgene expression at subcellular localizations, such as in plastids, mitochondria, and the nucleus. In summary, the technology of CPP-mediated gene delivery provides a potent and useful tool to genetically modified plants and crops of the future.

Multiplex CRISPR/Cas9 gene-editing platform in oil palm targeting mutations in EgFAD2 and EgPAT genes

BackgroundCRISPR/Cas9 is the most powerful and versatile genome-editing tool that permits multiplexed-targeted gene modifications for the genetic enhancement of oil palm. Multiplex genome-editing has recently been developed for modifying multiple loci in a gene or multiple genes in a genome with high precision. This study focuses on the development of high-oleic oil palm, the primary target trait for healthy low-saturated oil. To achieve this, the fatty acid desaturase 2 (FAD2) and palmitoyl-acyl carrier protein thioesterase (PAT) genes, both of which are associated with fatty acid metabolism biosynthesis pathways in oil palm, need to be knocked out. The knockout of FAD2 and PAT leads to an accumulation of oleic acid content in oil palms. ResultsA total of four single-guide RNAs (sgRNAs) were designed in silico based on the genomic sequences of EgFAD2 and EgPAT. Using robust plant CRISPR/Cas9 vector technology, multiple sgRNA expression cassettes were efficiently constructed into a single-binary CRISPR/Cas9 vector to edit the EgFAD2 and EgPAT genes. Each of the constructed transformation vectors was then delivered into oil palm embryogenic calli using the biolistic, Agrobacterium-mediated, and PEG-mediated protoplast transformation methods. Sequence analysis of PCR products from 15 samples confirmed that mutations were introduced at four target sites of the oil palm EgFAD2 and EgPAT genes. Single- and double-knockout mutants of both genes were generated, with large and small deletions within the targeted regions. Mutations found at EgFAD2 and EgPAT target sites indicate that the Cas9/sgRNA genome-editing system effectively knocked out both genes in oil palm. ConclusionThis technology is the first in oil palm to use CRISPR/Cas9 genome-editing to target high-oleic-associated genes. These findings showed that multiplex genome-editing in oil palm could be achieved using multiple sgRNAs. Targeted mutations detected establish that the CRISPR/Cas9 technology offers a great potential for oil palm.

Progress in Soybean Genetic Transformation Over the Last Decade.

Soybean is one of the important food, feed, and biofuel crops in the world. Soybean genome modification by genetic transformation has been carried out for trait improvement for more than 4 decades. However, compared to other major crops such as rice, soybean is still recalcitrant to genetic transformation, and transgenic soybean production has been hampered by limitations such as low transformation efficiency and genotype specificity, and prolonged and tedious protocols. The primary goal in soybean transformation over the last decade is to achieve high efficiency and genotype flexibility. Soybean transformation has been improved by modifying tissue culture conditions such as selection of explant types, adjustment of culture medium components and choice of selection reagents, as well as better understanding the transformation mechanisms of specific approaches such as Agrobacterium infection. Transgenesis-based breeding of soybean varieties with new traits is now possible by development of improved protocols. In this review, we summarize the developments in soybean genetic transformation to date, especially focusing on the progress made using Agrobacterium-mediated methods and biolistic methods over the past decade. We also discuss current challenges and future directions.

Biolistic Inoculation of Fruit Trees with Full-Length Infectious cDNA Clones of RNA Viruses.

Long life cycle and lack of efficient and robust virus inoculation technique are the major technical challenges for studying virus infection in perennial woody plants such as fruit trees. Biolistic technology also called particle bombardment is a physical approach that can directly introduce virions or viral full-length cDNA infectiousclones into target cells and tissues by high velocity microcarrier particles. The flexibility and high efficiency of the biolistic inoculation method facilitate research on fruit tree virology and the screening and identification of fruit tree germplasms resistant to viruses. Here, we describe a detailed protocol for the biolistic inoculation of peach with of a cDNA infectious clone of Plum pox virus (PPV) using the Helios gene gun, a biolistic particle delivery system.

Transfer and Expression of Native Human Insulin-Like Growth Factor-1 in Tobacco Chloroplasts.

Background: Insulin-like growth factor-1 (IGF-1), in addition to having insulin-like effects, has boosting effects on all cells in human body. Most of the recombinant IGF-1 required for patients suffering from its deficiency is currently produced by bacterial and yeast systems. Plant systems, especially chloroplasts, have many benefits for producing human blood proteins. Production costs are low in these systems, and their side effects are less than other systems.Objectives: In this study, the transfer and expression of mature IGF-1 protein cDNA in tobacco chloroplasts under the control of strong plastid transcription and translation elements was evaluated.Materials and Methods: The biolistic transformation method was used to transfer the IGF-1 gene cloned into the pRB94-IGF1 chloroplast vector. Homoplasmic transplastomic plants were produced through four selection rounds on the selective medium. Transfer of foreign genes to chloroplast genome was confirmed by PCR, Southern blotting and seed progeny test. RT-PCR and SDS-PAGE methods were used to evaluate the expression of IGF-1 gene in transgenic line.Results: A truly transformed line was identified from selected seedlings by PCR method. The seed progeny test of 4th-regeneration-round transgenic plants of this line showed maternal inheritance and homoplasmic level for the selectable marker gene, which confirms the transfer and expression of the marker gene in the chloroplast genome. The Southern blot test also confirmed the transfer of the IGF-1 gene into the chloroplast genome. RT-PCR test showed that IGF-1 gene transcription is performed correctly in transgenic plants. Finally, accumulation of IGF-1 protein in transgenic plants was detected by SDS-PAGE.Conclusions: Successful transfer and expression of the native human IGF-1 gene in tobacco chloroplast genome is reported.

Inheritance of NmDef02 synthetic defensin gene and hpt selectable marker in four successive generations of transgenic rice (Oryza sativa, cv. J-104)

The evaluation of the inheritance and stability of the transgenes is essential for the application of transgenic plants in agriculture. In this work, we studied the inheritance of two transgenes in T1, T2, T3 and T4 rice progenies. Transgenic rice plants (cv. J-104) was obtained by biolistic method using a synthetic defensin gene (NmDef02) and hpt as selectable marker gene, co-transformed in a 4:1 proportion, respectively. Regenerated plants were acclimated under natural conditions. The study started from the primary transformants that fulfilled the agronomic characters reported by the experts for the J-104 rice cultivar in the maturation stage. The integration and relative expression of NmDef02 in T1 plants was verified by Southern blot and qRT-PCR, respectively. The inheritance of transgenes over four generations was analyzed by PCR. The following transgene combinations were identified: NmDef02(+)hpt(+), NmDef02(+)hpt(-) and NmDef02(-)hpt(-). The most advantageous combination was NmDef02(+)hpt(-), which corresponded to the marker-free plants harboring the gene of interest. The inheritance of NmDef02 was confirmed in T1 and T2 progenies, but in some T3 and T4 lines the loss of this gene was verified. This inheritance analysis provided information concerning the transgenic population, but the mechanisms that destabilize the inheritance of the gene of interest will be the goal of future research.

Genetic engineering and genome editing techniques in peanut plants

Research has long been associated with human life. In the effort to make a living, many experts who have contributed to the modernization of traditional research methods by conducting various research activities. In this process, professionals, from farmers to senior researchers, have done their part by developing plants that can tolerate or resist to disease. The growing population, climate change and plant disease are having a devastating effect on food security. In particular, it is essential to increase food production by producing high yielding crops of good quality, that may ensure food security. Recently, different gene- editing technologies have been developed. These techniques have been applied in many research fields and their development has provided economic benefits to farmers. Agrobacterium-mediated and biolistic methods are very important techniques for transforming genetic materials in plants. Genome- editing technologies are recent and highly applied in plant research to improve genes associated with yield, disease resistance and drought resistance. For example, Zinc-finger Nucleases (ZFNS), Transcription Activator-like Effector Nucleases (TALEN), and Clustered Regularly Interspaced Short Palindromic Repeats system (CRISPR/ Cas9) methods are now widely applied by researchers and are playing a positive role in increasing production and productivity. Of the gene- editing technology, CRISPR/ Cas9 is widely applied in plant breeding programme as it is easy to use and cost-effective. In this review, we mainly focus on peanut plant, which is an important oil-bearing allotetraploid crop. Therefore, peanut gene editing-technology could increase the oleic acid content in edible peanut oil. Thus, genome editing and gene transformation technologies are extensively explored in this review.

Stress-induced expression of IPT gene in transgenic wheat reduces grain yield penalty under drought

BackgroundThe heterologous expression of isopentenyl transferase (IPT) under the transcriptional control of the senescence-associated receptor-like kinase (SARK) promoter delayed cellular senescence and, through it, increased drought tolerance in plants. To evaluate the effect of pSARK::IPT expression in bread wheat, six independent transgenic events were obtained through the biolistic method and evaluated transgene expression, phenology, grain yield and physiological biomass components in plants grown under both drought and well-irrigating conditions. Experiments were performed at different levels: (i) pots and (ii) microplots inside a biosafety greenhouse, as well as under (iii) field conditions. ResultsTwo transgenic events, called TR1 and TR4, outperformed the wild-type control under drought conditions. Transgenic plants showed higher yield under both greenhouse and field conditions, which was positively correlated to grain number (given by more spikes and grains per spike) than wild type. Interestingly, this yield advantage of the transgenic events was observed under both drought and well-watered conditions. ConclusionsThe results obtained allow us to conclude that the SARK promoter-regulated expression of the IPT gene in bread wheat not only reduced the yield penalty produced by water stress but also led to improved productivity under well-watered conditions.

Production of the sheep pox virus structural protein SPPV117 in tobacco chloroplasts

ObjectiveA chloroplast transgenic approach was assessed in order to produce a structural protein SPPV117 of sheep pox virus in Nicotiana tabacum for the future development of a plant-based subunit vaccine against sheep pox.ResultsTwo DNA constructs containing SPPV117 coding sequence under the control of chloroplast promoter and terminator of psbA gene or rrn promoter and rbcL terminator were designed and inserted into the chloroplast genome by a biolistic method. The transgenic plants were selected via PCR analysis. Northern and Western blot analysis showed expression of the transgene at transcriptional and translational levels, respectively. The recombinant protein accumulated to about 0.3% and 0.9% of total soluble protein in leaves when expressed from psbA and rrn promoter, respectively. Plant-produced SPPV117 protein was purified using metal affinity chromatography and the protein yield was 19.67 ± 1.25 µg g−1 (FW). The serum of a sheep infected with the virus recognised the chloroplast-produced protein indicating that the protein retains its antigenic properties.ConclusionsThese results demonstrate that chloroplasts are a suitable system for the production of a candidate subunit vaccine against sheep pox.

Development of an efficient regeneration system for mature bombarded calli of Moroccan durum wheat varieties

This study examined the effects of various environmental and genetic factors on callus induction and plant regeneration of bombarded calli from mature embryos of durum wheat using the biolistic method. In this study, three Moroccan durum wheat varieties ('Isly', 'Amria', 'Marouane') were cultivated on two induction media (IM1 and IM2) with different nitrogen sources and contents. After that, each variety cultured on both induction media was transferred in to two regeneration media (RM1 and RM2) with different phytohormones, whereas each variety distributed through four combinations of treatments: IM1RM1, IM1RM2, IM2RM1, and IM2RM2. A completely randomized design with five replications per treatment for each genotype was used. Parameters considered in this study were phytohormones, nitrogen source and its content, plant variety, and their interactions. The study found that variety, medium and variety × medium interactions have a statistically significant effect on callus induction and plantlets regeneration. Prior to bombardment, the maximum percentage of callus induction was obtained under IM1. Conversely, the callus survival rate was not affected by the induction media once bombarded for all three varieties. The induction media had a significant effect on all regeneration parameters (p < 0.01). The variety ‘Isly’ showed the best regeneration efficiency after bombardment, with nearly 80% of plantlets regenerated under IM1 and RM2 combination. These media can be used for genetic transformation of durum wheat

Nicotiana tabacum: An Update on PEG-Mediated Plastid Transformation.

Stable plastid transformation in Nicotiana tabacum has been achieved by using two different methods, the biolistic method, using a particle gun, and the polyethylene glycol (PEG)-mediated transformation. PEG-mediated plastid transformation involves the treatment of isolated protoplasts (plant cells without cell wall) with PEG in the presence of DNA. We have previously shown that in Nicotiana tabacum both methods are equally efficient. The PEG-mediated transformation efficiencies range between 20 and 50 plastid transformants per experiment (106 viable treated protoplasts). One advantage of the PEG method is that no expensive equipment such as a particle gun is required. The only crucial points are the handling and the cultivation of protoplasts. Furthermore, markers for the selection of transformed plastids are required. One of the most often used selection markers is the aadA gene which encodes for spectinomycin and streptomycin resistance. Here we describe a simplified and inexpensive protocol for the transformation of plastids in Nicotiana tabacum using an optimized protoplast culture protocol. PEG-mediated plastid transformation has the potential to be developed into a high-throughput, automated pipeline.

Fate of forest tree biotechnology facing climate change

Abstract Woody plants have been cultured in vitro since the 1930s. After that time much progress has been made in the culture of tissues, organs, cells, and protoplasts in tree species. Tree biotechnology has been making strides in clonal propagation by organogenesis and somatic embryogenesis. These regeneration studies have paved the way for gene transfer in forest trees. Transgenics from a number of forest tree species carrying a variety of recombinant genes that code for herbicide tolerance, pest resistance, lignin modification, increased woody bio-mass, and flowering control have been produced by Agrobacterium-mediated and biolistic methods, and some of them are undergoing confined field trials. Although relatively stable transgenic clones have been produced by genetic transformation in trees using organogenesis or somatic embryogenesis, there were also unintended unstable genetic events. In order to overcome the problems of randomness of transgene integration and instability reported in Agrobacterium-mediated or biolistically transformed plants, site-specific transgene insertion strategies involving clustered regularly interspaced short palindromic repeats (CRISPR-Cas9) platform offer prospects for precise genome editing in plants. Nevertheless, it is important to monitor phenotypic and genetic stability of clonal material, not just under greenhouse conditions, but also under natural field conditions. Genetically modified poplars have been commercialized in China, and eucalypts and loblolly pine are expected to be released for commercial deployment in USA. Clonal forestry and transgenic forestry have to cope with rapid global climate changes in the future. Climate change is impacting species distributions and is a significant threat to biodiversity. Therefore, it is important to deploy Strategies that will assist the survival and evolution of forest tree species facing rapid climate change. Assisted migration (managed relocation) and biotechnological approaches offer prospects for adaptation of forest trees to climate change.