Efficient Transformation Protocol Research Articles

Generation of tools for expression and purification of the phage-encoded Type I restriction enzyme inhibitor, Ocr.

DNA manipulation is an essential tool in molecular microbiology research that is dependent on the ability of bacteria to take up and preserve foreign DNA by horizontal gene transfer. This process can be significantly impaired by the activity of bacterial restriction modification systems; bacterial operons comprising paired enzymatic activities that protectively methylate host DNA, while cleaving incoming unmodified foreign DNA. Ocr is a phage-encoded protein that inhibits Type I restriction modification systems, the addition of which significantly improves bacterial transformation efficiency. We recently established an improved and highly efficient transformation protocol for the important human pathogen group A Streptococcus using commercially available recombinant Ocr protein, manufacture of which has since been discontinued. In order to ensure the continued availability of Ocr protein within the research community, we have generated tools and methods for in-house Ocr production and validated the activity of the purified recombinant protein.

Agrobacterium-Mediated Transformation of the Dwarf Soybean MiniMax.

This study aims to establish an Agrobacterium-mediated transformation system for use with the 'MiniMax'soybean cultivar. MiniMax is a mutant soybean whose growth cycle is around 90 days, half that of most other soybean varieties, making it an optimal model cultivar to test genes of interest before investing in modification of elite lines. We describe an efficient protocol for Agrobacterium-mediated transformation using MiniMax seeds. It uses a modified 'half seed' regeneration protocol for transgenic soybean production, utilizing the rapid generation MiniMax variety to obtain T1 seeds in approximately 145 days. Addition of phloroglucinol (PG) to the regeneration protocol was key to obtaining high-efficiency rooting of the regenerated shoots. Transfer to soil was accomplished using an organic soil amendment containing nutrients and mycorrhiza for plants to thrive in the greenhouse. This combination of genotype and stimulants provides a transformation protocol to genetically engineer MiniMax seeds with a transgenic lab-to-greenhouse production efficiency of 4.0%. This is the first report of MiniMax soybean whole plant transformation and heritable T1 transmission. This protocol provides an ideal resource for enhancing the genetic transformation of any soybean cultivar.

Development of an Efficient and Reproducible Embryonic Axis-targeted Tissue Culture-based Transformation Protocol for Pigeonpea (Cajanus cajan)

Background: Tissue culture-based transformation approach poses significant challenges for genetic modification of recalcitrant pigeonpea due to poor regeneration efficiency. In this study, we present a comprehensive high-frequency regeneration and transformation protocol using embryonic axesas explants. Methods: Optimization of transformation parameters and culture conditions for different stages of adventitious shoot regeneration, including shoot induction, elongation and rooting, significantly enhanced transformation efficiency. Agrobacterium-mediated transformation was carried out using embryonic axes explants with pCAMBIA 2301 vector. Culturing of explants on MS basal media supplemented with BAP 2.5 in Preculture, BAP 2.5 in Selection Medium I, IAA 0.5, GA 0.5 and Zeatin 1 in Selection Medium II resulted in formation of numerous multiple shoots from primordia-forming calli at axillary bud regions. Result: Shooting efficiency was 82% and 77%, for cultivars, Pusa 992 and Asha respectively. Rooting efficiency was nearly 100% for both when cultured on half-strength MScontaining 15 g/l sucrose and 0.5 mg/l IAA. Regenerated plants exhibited a high level ofsurvival of 90% upon transplantation into pots. Successful gene integration and expression were confirmed through GUS histochemical and molecular analyses, with approximately 57% and 65% transformation efficiencies for cultivars Pusa 992 and Asha, respectively. This study presents a robust and reproducible tissue culture-based transformation system for pigeonpea, applicable across diverse genotypes, offering new possibilities for genetic improvement in this crucial legume crop.

Quantum Communication Complexity of Linear Regression

Quantum computers may achieve speedups over their classical counterparts for solving linear algebra problems. However, in some cases—such as for low-rank matrices—dequantized algorithms demonstrate that there cannot be an exponential quantum speedup. In this work, we show that quantum computers have provable polynomial and exponential speedups in terms of communication complexity for some fundamental linear algebra problems if there is no restriction on the rank. We mainly focus on solving linear regression and Hamiltonian simulation. In the quantum case, the task is to prepare the quantum state of the result. To allow for a fair comparison, in the classical case, the task is to sample from the result. We investigate these two problems in two-party and multiparty models, propose near-optimal quantum protocols, and prove quantum/classical lower bounds. In this process, we propose an efficient quantum protocol for quantum singular value transformation, which is a powerful technique for designing quantum algorithms. We feel this will be helpful in developing efficient quantum protocols for many other problems.

In planta transformation in wheat: an improved protocol to develop wheat transformants.

Lack of efficient transformation protocol continues to be a major bottleneck for successful genome editing or transgenic development in wheat. An in planta transformation method was developed in Indian bread wheat in earlier study (Vasil et al. in Nat Biotechnol 10:667-674, 1992) which was labour-intensive and time-consuming. In the present study, in planta transformation method was improved to make it simple, efficient, less labour-intensive and time-saving. PCR-based screening for generated transformants at T0 stage was introduced in this method. Shoot apical meristem of two days old wheat seedling was inoculated with the routine active culture of Agrobacterium tumefaciens harboring plasmid pCAMBIA1300-Ubi-GFP having gene GFP under the control of Zea mays ubiquitin promoter. PCR analysis at T0 stage confirmed 27 plants to be transgene positive. These 27 plants were only taken to the next generation (T1) and the rest were discarded. At T1 generation 6 plants were analyzed to be PCR positive. Out of them, 4 plants were confirmed to have stable integration of transgene (GFP). Fluorescent microscopy at T1 stage confirmed the 4 Southern hybridization positive plants to be expressing reporter gene GFP. Screening at T0 stage, reduced the load of plants to be taken to T1 generation and their screening thereof at T1 with no overall loss in transformation efficiency. We successfully transformed wheat genotype HD2894 with 3.33% transformation efficiency using a simple, effective method which was less labour-intensive and less time-consuming. This method may be utilized to develop wheat transgenic as well as genome edited lines for desirable traits.

Establishment of protoplasts isolation and transient transformation system for kiwifruit

Protoplast isolation and transient transformation have proven to be valuable tools for studying gene function in plants. However, these techniques are rarely utilized in most woody plants due to the lack of an efficient protoplast isolation system. In this study, we successfully established a protocol for isolating and purifying protoplasts from kiwifruit callus. First, loose kiwifruit callus was induced using young true leaves as explants on MS medium containing 0.5 mg·L−1 zeatin and 0.5 mg·L−1 2,4-dichlorophenoxyacetic acid (2,4-D), followed by propagation on medium supplemented with 1.0 mg·L−1 2,4-D + 0.5 mg·L−1 6-benzylaminopurine + 0.5 mg·L−1 1-naphthlcetic acid. Subsequently, the callus subcultured at 15 d was digested with an enzyme solution consisting of 2.0 % Cellulase R-10, 0.5 % Macerozyme R-10, and 0.7 M mannitol for 7 h in the dark to obtain protoplasts at a yield of approximately 2.8 × 106 protoplasts·g−1FW with viability reaching up to 95.2 %. Furthermore, we optimized the conditions for transient transformation in kiwifruit protoplasts, achieving an approximately 43.5 % transformation efficiency using a concentration of PEG4000 at 40 %. By employing this method, we successfully verified the subcellular localization of AcMYB6l-GFP fusion protein. In conclusion, we have successfully established an efficient protocol for protoplast isolation and transient transformation in kiwifruit, laying a foundation for future research on gene function and molecular breeding in Actinidia.

Injection-based hairy root induction and plant regeneration techniques in Brassicaceae

BackgroundHairy roots constitute a valuable tissue culture system for species that are difficult to propagate through conventional seed-based methods. Moreover, the generation of transgenic plants derived from hairy roots can be facilitated by employing carefully designed hormone-containing media.ResultsWe initiated hairy root formation in the rare crucifer species Asperuginoides axillaris via an injection-based protocol using the Agrobacterium strain C58C1 harboring a hairy root-inducing (Ri) plasmid and successfully regenerated plants from established hairy root lines. Our study confirms the genetic stability of both hairy roots and their derived regenerants and highlights their utility as a permanent source of mitotic chromosomes for cytogenetic investigations. Additionally, we have developed an effective embryo rescue protocol to circumvent seed dormancy issues in A. axillaris seeds. By using inflorescence primary stems of Arabidopsis thaliana and Cardamine hirsuta as starting material, we also established hairy root lines that were subsequently used for regeneration studies.ConclusionWe developed efficient hairy root transformation and regeneration protocols for various crucifers, namely A. axillaris, A. thaliana, and C. hirsuta. Hairy roots and derived regenerants can serve as a continuous source of plant material for molecular and cytogenetic analyses.

Diversity of transgene integration and gene-editing events in wheat (Triticum aestivum L.) transgenic plants generated using Agrobacterium-mediated transformation.

Improvement in agronomic traits in crops through gene editing (GE) relies on efficient transformation protocols for delivering the CRISPR/Cas9-coded transgenes. Recently, a few embryogenesis-related genes have been described, the co-delivery of which significantly increases the transformation efficiency with reduced genotype-dependency. Here, we characterized the transgenic and GE events in wheat (cv. Fielder) when transformed with GROWTH-REGULATING FACTOR 4 (GRF4) and its cofactor GRF-INTERACTING FACTOR 1 (GIF1) chimeric gene. Transformation efficiency in our experiments ranged from 22% to 68%, and the editing events were faithfully propagated into the following generation. Both low- and high-copy-number integration events were recovered in the T0 population with various levels of integrity of the left and right T-DNA borders. We also generated a population of wheat plants with 10 different gRNAs targeting 30 loci in the genome. A comparison of the epigenetic profiles at the target sites and editing efficiency revealed a significant positive correlation between chromatin accessibility and mutagenesis rate. Overall, the preliminary screening of transgene quality and GE events in the T0 population of plants regenerated through the co-delivery of GRF-GIF can allow for the propagation of the best candidates for further phenotypic analysis.

CRISPR/Cas as a Genome-Editing Technique in Fruit Tree Breeding

CRISPR (short for “Clustered Regularly Interspaced Short Palindromic Repeats”) is a technology that research scientists use to selectively modify the DNA of living organisms. CRISPR was adapted for use in the laboratory from the naturally occurring genome-editing systems found in bacteria. In this work, we reviewed the methods used to introduce CRISPR/Cas-mediated genome editing into fruit species, as well as the impacts of the application of this technology to activate and knock out target genes in different fruit tree species, including on tree development, yield, fruit quality, and tolerance to biotic and abiotic stresses. The application of this gene-editing technology could allow the development of new generations of fruit crops with improved traits by targeting different genetic segments or even could facilitate the introduction of traits into elite cultivars without changing other traits. However, currently, the scarcity of efficient regeneration and transformation protocols in some species, the fact that many of those procedures are genotype-dependent, and the convenience of segregating the transgenic parts of the CRISPR system represent the main handicaps limiting the potential of genetic editing techniques for fruit trees. Finally, the latest news on the legislation and regulations about the use of plants modified using CRISPR/Cas systems has been also discussed.

A Versatile Protocol for Efficient Transformation and Regeneration in Mega Indica Rice Cultivar MTU1010: Optimization through Hormonal Variables.

Rice is one of the apex food crops in terms of meeting the daily calorific and dietary requirement of the majority of the world population. However, rice productivity is severely limited by various biotic and abiotic attributes, causing a severe threat to global food security. In the use of functional genomics and genome editing for the generation of trait-enhanced genotypes, it is necessary to have an efficient genetic transformation and regeneration protocol. The recalcitrant nature and paucity of efficient and versatile genetic transformation and regeneration protocols for indica cultivars remains a constraint. In the present study, we have optimized a tissue culture method for MTU1010, a mega indica rice variety. We conducted a combinatorial analysis of different plant growth regulators on embryogenic callus induction efficiency, and it was observed that MSB5 medium supplemented with 2.5 mg/L 2-4D and 0.25 mg/L 6-BAP results in maximum embryogenic callus induction, i.e., 92%. The regeneration efficiency of a transformed callus can be enhanced by up to 50% with the supplementation of 1 mg/L kinetin alongside 2.5 mg/L BAP and 0.5 mg/L NAA in the shooting medium. Furthermore, our results unveiled that the pre-activation of Agrobacterium culture for 30 min with 150 µM acetosyringone significantly increased the transformation efficiency of calli. Additionally, descaling the salt concentration to half strength in resuspension and co-cultivation increased the efficiency of transformation up to 33%. Thus, the protocol developed in this study will be instrumental for the genome editing and genetic engineering of indica rice cultivars for functional genomics studies and crop improvement.

Engineering the Maize Root Microbiome: A Rapid MoClo Toolkit and Identification of Potential Bacterial Chassis for Studying Plant-Microbe Interactions.

Sustainably enhancing crop production is a global necessity to meet the escalating demand for staple crops while sustainably managing their associated carbon/nitrogen inputs. Leveraging plant-associated microbiomes is a promising avenue for addressing this demand. However, studying these communities and engineering them for sustainable enhancement of crop production have remained a challenge due to limited genetic tools and methods. In this work, we detail the development of the Maize Root Microbiome ToolKit (MRMTK), a rapid Modular Cloning (MoClo) toolkit that only takes 2.5 h to generate desired constructs (5400 potential plasmids) that replicate and express heterologous genes in Enterobacter ludwigii strain AA4 (Elu), Pseudomonas putida strain AA7 (Ppu), Herbaspirillum robiniae strain AA6 (Hro), Stenotrophomonas maltophilia strain AA1 (Sma), and Brucella pituitosa strain AA2 (Bpi), which comprise a model maize root synthetic community (SynCom). In addition to these genetic tools, we describe a highly efficient transformation protocol (107-109 transformants/μg of DNA) 1 for each of these strains. Utilizing this highly efficient transformation protocol, we identified endogenous Expression Sequences (ES; promoter and ribosomal binding sites) for each strain via genomic promoter trapping. Overall, MRMTK is a scalable and adaptable platform that expands the genetic engineering toolbox while providing a standardized, high-efficiency transformation method across a diverse group of root commensals. These results unlock the ability to elucidate and engineer plant-microbe interactions promoting plant growth for each of the 5 bacterial strains in this study.

Methylome-dependent transformation of emm1 group A streptococci.

Genetic intractability presents a fundamental barrier to the manipulation of bacteria, hindering advancements in microbiological research. Group A Streptococcus (GAS), a lethal human pathogen currently associated with an unprecedented surge of infections worldwide, exhibits poor genetic tractability attributed to the activity of a conserved type 1 restriction modification system (RMS). RMS detect and cleave specific target sequences in foreign DNA that are protected in host DNA by sequence-specific methylation. Overcoming this "restriction barrier" thus presents a major technical challenge. Here, we demonstrate for the first time that different RMS variants expressed by GAS give rise to genotype-specific and methylome-dependent variation in transformation efficiency. Furthermore, we show that the magnitude of impact of methylation on transformation efficiency elicited by RMS variant TRDAG, encoded by all sequenced strains of the dominant and upsurge-associated emm1 genotype, is 100-fold greater than for all other TRD tested and is responsible for the poor transformation efficiency associated with this lineage. In dissecting the underlying mechanism, we developed an improved GAS transformation protocol, whereby the restriction barrier is overcome by the addition of the phage anti-restriction protein Ocr. This protocol is highly effective for TRDAG strains including clinical isolates representing all emm1 lineages and will expedite critical research interrogating the genetics of emm1 GAS, negating the need to work in an RMS-negative background. These findings provide a striking example of the impact of RMS target sequence variation on bacterial transformation and the importance of defining lineage-specific mechanisms of genetic recalcitrance. IMPORTANCE Understanding the mechanisms by which bacterial pathogens are able to cause disease is essential to enable the targeted development of novel therapeutics. A key experimental approach to facilitate this research is the generation of bacterial mutants, through either specific gene deletions or sequence manipulation. This process relies on the ability to transform bacteria with exogenous DNA designed to generate the desired sequence changes. Bacteria have naturally developed protective mechanisms to detect and destroy invading DNA, and these systems severely impede the genetic manipulation of many important pathogens, including the lethal human pathogen group A Streptococcus (GAS). Many GAS lineages exist, of which emm1 is dominant among clinical isolates. Based on new experimental evidence, we identify the mechanism by which transformation is impaired in the emm1 lineage and establish an improved and highly efficient transformation protocol to expedite the generation of mutants.

Optimization of in vitro and ex vitro Agrobacterium rhizogenes-mediated hairy root transformation of soybean for visual screening of transformants using RUBY.

In vitro and ex vitro Agrobacterium rhizogenes-mediated hairy root transformation (HRT) assays are key components of the plant biotechnology and functional genomics toolkit. In this report, both in vitro and ex vitro HRT were optimized in soybean using the RUBY reporter. Different parameters including A. rhizogenes strain, optical density of the bacterial cell culture (OD600), co-cultivation media, soybean genotype, explant age, and acetosyringone addition and concentration were evaluated. Overall, the in vitro assay was more efficient than the ex vitro assay in terms of the percentage of induction of hairy roots and transformed roots (expressing RUBY). Nonetheless, the ex vitro technique was deemed faster and a less complicated approach. The highest transformation of RUBY was observed on 7-d-old cotyledons of cv. Bert inoculated for 30 minutes with the R1000 resuspended in ¼ B5 medium to OD600 (0.3) and 150 µM of acetosyringone. The parameters of this assay also led to the highest percentage of RUBY through two-step ex vitro hairy root transformation. Finally, using machine learning-based modeling, optimal protocols for both assays were further defined. This study establishes efficient and reliable hairy root transformation protocols applicable for functional studies in soybean.

Agrobacterium-mediated Genetic Transformation of two Peanut (Arachis hypogaea L.) Varieties using Salinity Tolerant PDH45 Gene

An efficient Agrobacterium-mediated genetic transformation protocol has been developed in the present investigation to integrate salinity tolerant gene pea DNA helicase (PDH45) in two peanut varieties, Dhaka-1 and BARI Chinabadam-8. Decapitated half-embryo explants of the above-mentioned varieties were also used to develop a transformation compatible in vitro regeneration system. MS medium supplemented with 5.0 mg/l BAP and 0.5 mg/l Kn showed maximum response for shoot induction in both varieties. Half strength of MS medium supplemented with 0.2 mg/l IBA showed better response towards root induction for both varieties. Genetic transformation experiments were performed using Agrobacterium strain LBA4404 containing pCAMBIA1301-PDH45 conferring reporter gus gene, hygromycin resistance hpt gene, and salinity tolerant gene pdh45. The transformation variables were optimized, OD600 1.4 with 45 minutes of incubation period, and three days of co-culture showed the best performance. The transformed shoots were selected using 30 mg/l hygromycin. The overall transformation frequency was very low, where 0.10% shoots of Dhaka-1 and 0.07% shoots of BARI Chinabadam-8 survived the selection pressure. The stable integration of the transgene in the transformed shoots was confirmed through PCR analysis. Plant Tissue Cult. & Biotech. 33(1): 37-46, 2023 (June)

Melatonin strongly enhances the Agrobacterium- mediated transformation of carnation in nitrogen-depleted media

With the rising demand for new cultivars of carnation, efficient transformation protocols are needed to enable the bioengineering of new traits. Here, we established a novel and efficient Agrobacterium-mediated transformation system using callus as the target explant for four commercial carnation cultivars. Leaf-derived calli of all cultivars were inoculated with Agrobacterium tumefaciens strain LBA4404 containing the plasmid pCAMBIA 2301 harboring genes for β-glucuronidase (uidA) and neomycin phosphotransferase (nptII). Polymerase chain reaction (PCR) and histochemical assays confirmed the presence of uidA and β-glucuronidase (GUS), respectively in transgenic shoots. The effect on transformation efficiency of medium composition and the presence of antioxidants during inoculation and co-cultivation was investigated. The transformation efficiency was increased in Murashige and Skoog (MS) medium lacking KNO3 and NH4NO3, and also in MS medium lacking macro and micro elements and Fe to 5% and 3.1% respectively, compared to 0.6% in full-strength medium. Transformation efficiency was increased dramatically to 24.4% across all carnation cultivars by the addition of 2 mg/l melatonin to nitrogen-depleted MS medium. Shoot regeneration was also doubled in this treatment. The establishment of this efficient and reliable transformation protocol can advance the development of novel carnation cultivars through molecular breeding approaches.

An efficient transformation method for genome editing of elite bread wheat cultivars.

An efficient genetic transformation protocol is necessary to edit genes for trait improvement directly in elite bread wheat cultivars. We used a protein fusion between a wheat growth-regulating factor 4 (GRF4) and its interacting factor (GIF1) to develop a reproducible genetic transformation and regeneration protocol, which we then used to successfully transform elite bread wheat cultivars Baj, Kachu, Morocco, Reedling, RL6077, and Sujata in addition to the experimental cultivar Fielder. Immature embryos were transformed with the vector using particle bombardment method. Transformation frequency increased nearly 60-fold with the GRF4-GIF1-containing vectors as compared to the control vector and ranged from ~5% in the cultivar Kachu to 13% in the cultivar RL6077. We then edited two genes that confer resistance against leaf rust and powdery mildew directly in the aforementioned elite cultivars. A wheat promoter, TaU3 or TaU6, to drive the expression of guide RNA was effective in gene editing whereas the OsU3 promoter failed to generate any edits. Editing efficiency was nearly perfect with the wheat promoters. Our protocol has made it possible to edit genes directly in elite wheat cultivars and would be useful for gene editing in other wheat varieties, which have been recalcitrant to transformation thus far.

CRISPR/Cas genome editing in tomato improvement: Advances and applications.

The narrow genetic base of tomato poses serious challenges in breeding. Hence, with the advent of clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein9 (CRISPR/Cas9) genome editing, fast and efficient breeding has become possible in tomato breeding. Many traits have been edited and functionally characterized using CRISPR/Cas9 in tomato such as plant architecture and flower characters (e.g. leaf, stem, flower, male sterility, fruit, parthenocarpy), fruit ripening, quality and nutrition (e.g., lycopene, carotenoid, GABA, TSS, anthocyanin, shelf-life), disease resistance (e.g. TYLCV, powdery mildew, late blight), abiotic stress tolerance (e.g. heat, drought, salinity), C-N metabolism, and herbicide resistance. CRISPR/Cas9 has been proven in introgression of de novo domestication of elite traits from wild relatives to the cultivated tomato and vice versa. Innovations in CRISPR/Cas allow the use of online tools for single guide RNA design and multiplexing, cloning (e.g. Golden Gate cloning, GoldenBraid, and BioBrick technology), robust CRISPR/Cas constructs, efficient transformation protocols such as Agrobacterium, and DNA-free protoplast method for Cas9-gRNAs ribonucleoproteins (RNPs) complex, Cas9 variants like PAM-free Cas12a, and Cas9-NG/XNG-Cas9, homologous recombination (HR)-based gene knock-in (HKI) by geminivirus replicon, and base/prime editing (Target-AID technology). This mini-review highlights the current research advances in CRISPR/Cas for fast and efficient breeding of tomato.

An efficient Agrobacterium-mediated transformation and regeneration protocol for aromatic black rice, Oryza sativa L. (cv. Chakhao Poireiton and Chakhao Amubi)

An efficient Agrobacterium-mediated transformation and regeneration protocol for aromatic black rice, Oryza sativa L. (cv. Chakhao Poireiton and Chakhao Amubi)

Optimization of Regeneration and Agrobacterium-Mediated Transformation Protocols for Bi and Multilocular Varieties of Brassica rapa

The regeneration of the high-yielding multilocular types has not been attempted, although successful regeneration and transformation in brassica have been done. Here, we report efficient regeneration and transformation protocols for two B. rapa genotypes; UAF11 and Toria. The B. rapa cv UAF11 is a multilocular, non-shattering, and high-yielding genotype, while Toria is the bilocular type. For UAF11 8 shoots and for Toria 7 shoots, explants were observed on MS supplemented with 3 mg/L BAP + 0.4 mg/L NAA + 0.01 mg/L GA3 + 5 mg/L AgNO3 + 0.75 mg/L Potassium Iodide (KI), MS salt supplemented with 1 mg/L IBA and 0.37 mg/L KI produced an equal number of roots (3) in UAF11 and Toria. For the establishment of transformation protocols, Agrobacterium-mediated floral dip transformation was attempted using different induction media, infection time, and flower stages. The induction medium III yielded a maximum of 7.2% transformants on half-opened flowers and 5.2% transformants on fully opened flowers in UAF11 and Toria, respectively, with 15 min of inoculation. This study would provide the basis for the improvement of tissue culture and transformation protocols in multilocular and bilocular Brassica genotypes.

An efficient soybean transformation protocol for use with elite lines

An efficient soybean transformation protocol for use with elite lines