Generation Of Transgenic Plants Research Articles

Verification of the peanut vitamin C synthesis-related gene AhPMM and its role in stress resistance

Vitamin C plays an important role in plant antioxidation, photosynthesis, growth and development, and metabolism. In this study, a gene AhPMM, which is involved in vitamin C synthesis and responds significantly to low temperature, NaCl, polyethylene glycol (PEG) and abscisic acid (ABA) treatments, was cloned from peanut. An AhPMM overexpression vector was constructed, and transferred to a peanut variety Junanxiaohong using the pollen tube injection method. PCR test on the T3 generation transgenic peanut plants showed a transgenics positive rate of 42.3%. HPLC was used to determine the content of reducing vitamin C (AsA) and total vitamin C in the leaves of transgenic plants. The results showed that the content of AsA in some lines increased significantly, up to 1.90 times higher than that of the control, and the total vitamin content increased by up to 1.63 times compared to that of the control. NaCl and ABA tolerance tests were carried out on transgenic seeds. The results showed that the salt tolerance of transgenic seeds was significantly enhanced and the sensitivity to ABA was weakened compared to that of the non-transgenic control. Moreover, the salt tolerance of the transgenic plants was also significantly enhanced compared to that of the non-transgenic control. The above results showed that AhPMM gene not only increased the vitamin C content of peanut, but also increased the salt tolerance of transgenic peanut seeds and plants. This study may provide a genetic source for the molecular breeding of peanut for enhanced salt tolerance.

Cotton plants overexpressing the Bacillus thuringiensis Cry23Aa and Cry37Aa binary-like toxins exhibit high resistance to the cotton boll weevil (Anthonomus grandis)

The cotton boll weevil (CBW, Anthonomus grandis) stands as one of the most significant threats to cotton crops (Gossypium hirsutum). Despite substantial efforts, the development of a commercially viable transgenic cotton event for effective open-field control of CBW has remained elusive. This study describes a detailed characterization of the insecticidal toxins Cry23Aa and Cry37Aa against CBW. Our findings reveal that CBW larvae fed on artificial diets supplemented exclusively with Cry23Aa decreased larval survival by roughly by 69%, while supplementation with Cry37Aa alone displayed no statistical difference compared to the control. However, the combined provision of both toxins in the artificial diet led to mortality rates approaching 100% among CBW larvae (LC50 equal to 0.26 PPM). Additionally, we engineered transgenic cotton plants by introducing cry23Aa and cry37Aa genes under control of the flower bud-specific pGhFS4 and pGhFS1 promoters, respectively. Seven transgenic cotton events expressing high levels of Cry23Aa and Cry37Aa toxins in flower buds were selected for greenhouse bioassays, and the mortality rate of CBW larvae feeding on their T0 and T1 generations ranged from 75% to 100%. Our in silico analyses unveiled that Cry23Aa displays all the hallmark characteristics of β-pore-forming toxins (β-PFTs) that bind to sugar moieties in glycoproteins. Intriguingly, we also discovered a distinctive zinc-binding site within Cry23Aa, which appears to be involved in protein-protein interactions. Finally, we discuss the major structural features of Cry23Aa that likely play a role in the toxin’s mechanism of action. In view of the low LC50 for CBW larvae and the significant accumulation of these toxins in the flower buds of both T0 and T1 plants, we anticipate that through successive generations of these transgenic lines, cotton plants engineered to overexpress cry23Aa and cry37Aa hold promise for effectively managing CBW infestations in cotton crops.

High Efficiency In vitro Whole Plant Regeneration via Desiccated Callus in Oryza sativa cv. MTU1010

For most people across the planet, rice is also considered as most beneficial crops in terms of fulfilling every day's energy and nutritional needs. Worldwide food safety is seriously threatened since severe limitations on rice output caused by both abiotic and biotic variables. Even though rice features much more developed regeneration mechanism than other agricultural crops, majority of desi cultivars continue to stay as impervious to genetic alterations and regeneration. Therefore, among preliminary steps in the transgenic plant generation necessitates to improve tissue culture procedures to produce viable plants via embryogenic calli mediated regeneration. In this study, we reporting that enhanced rate of plant regeneration was observed from mature seed-derived embryogenic calli of the indica rice cultivar MTU1010 following partially desiccation procedure. We studied the effects of several plant growth regulators on the efficiency of inducing embryogenic callus and regeneration of whole plantlets. The MS-CIM medium with 2.0 mg-1 2,4, D showed the highest callus induction percentage. Maximum rate of shoot regeneration (92%) was observed from 48hr partially dehydrated calli, when enriched with high cytokinin (2 mg/l kinetin) and lower auxin (0.5 mg/l NAA) concentrations. Calli that had been moderately dehydrated showed significantly greater regeneration rates than undehydrated calli. Upon being transferred onto a rooting medium, the well-rooted plantlets were shifted to shaded glass house for hardening. Hence, this approach is rather simple to apply and can be employed in genetic modification studies alongside other biotechnological endeavours.

Streamlined Agrobacterium rhizogenes-mediated hairy root transformation for efficient CRISPR/Cas9-based gene editing evaluation in diverse Citrullus varieties

The CRISPR/Cas9 genome-editing system serves as a pivotal tool for enhancing crop genetics. Within this system, single guide RNAs (sgRNAs) are instrumental in the precise cleavage of DNA double strands. However, the efficiency of gene editing varies among sgRNAs, emphasizing the need to meticulously select target sites, especially in the context of Citrullus lanatus, a species notorious for its challenging and inefficient generation of transgenic plants through stable transformation. This study employed an Agrobacterium rhizogenes-mediated hairy root method to assess effective target sites for gene editing of ClCIPK17 across various Citrullus species. Hairy roots were successfully induced in different plant tissues at diverse growth stages of Citrullus lanatus, Citrullus mucosospermus, and Citrullus amarus. Employing a vector with two sgRNAs (sgRNA1 and sgRNA5) positioned within conserved regions of exon 1 and exon 5 of ClCIPK17 in the CRISPR/Cas9 system, targeted mutations were detected in 90.9% of accessions across the four Citrullus species. Notably, 73.94% of all examined hairy roots exhibited mutations at the sgRNA1 site, while the sgRNA5 site showed no mutations. Among the 31 different mutation types identified at the sgRNA1 site, base deletion was the most prevalent. Using the sgRNA1 site of ClCIPK17, stable transgenic watermelon buds were obtained from explants, and the targeted mutations of the sgRNA1 site were confirmed. These findings underscore the viability of the hairy root transformation system in assessing the editing efficiency of sgRNA targets in diverse Citrullus species.

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.

Hairy Root Transformation and Regeneration in Arabidopsis thaliana and Brassica napus.

Hairy root transformation represents a versatile tool for plant biotechnology in various species. Infection by an Agrobacterium strain carrying a Root-inducing (Ri) plasmid induces the formation of hairy roots at the wounding site after the transfer of T-DNA from the Ri plasmid into the plant genome. The protocol describes in detail the procedure of the injection-based hairy root induction in Brassica napus DH12075 and Arabidopsis thaliana Col-0. The hairy roots may be used to analyze a transgene of interest or processed for the generation of transgenic plants. Regeneration medium containing cytokinin 6-benzylaminopurine (5 mg/L) and auxin 1-naphthaleneacetic acid (8 mg/L) successfully elicits shoot formation in both species. The protocol covers the genotyping and selection of regenerants and T1 plants to obtain plants carrying a transgene of interest and free of T-DNA from the Ri plasmid. An alternative process leading to the formation of a composite plant is also depicted. In this case, hairy roots are kept on the shoot (instead of the natural roots), which enables the study of a transgene in hairy root cultures in the context of the whole plant.

Single-cell transcriptome sequencing of plant leaf expressing anti-HER2 VHH–FcK cancer therapeutic protein

The transgenic plant is a promising strategy for the production of highly valuable biotherapeutic proteins such as recombinant vaccines and antibodies. To achieve an efficient level of protein production, codon sequences and expression cassette elements need to be optimized. However, the systematical expression of recombinant proteins in plant biomass can generally be controlled for the production of therapeutic proteins after the generation of transgenic plants. Without understanding the transgene expression patterns in plant tissue, it is difficult to enhance further production levels. In this study, single-cell RNA-sequencing (scRNA-seq) analysis of transgenic tobacco (Nicotiana tabacum) leaf, expressing an immunotherapeutic llama antibody against breast cancer, anti-HER2 VHH–Fc, was conducted to obtain data on the expression pattern of tissue-specific cells. These high-quality scRNA-seq data enabled the identification of gene expression patterns by cell types, which can be applied to select the best cell types or tissues for the high production of these recombinant antibodies. These data provide a foundation to elucidate the mechanisms that regulate the biosynthesis of recombinant proteins in N. tabacum.

Construction of the CRISPR/Cas9 module and genetic transformation of aromatic rice cv. Mentik Wangi for developing bacterial leaf blight resistance

Abstract. Rifhani NF, Apriana A, Sisharmini A, Santoso TJ, Trijatmiko KR, Slamet-Loedin IH, Yunus A. 2023. Construction of the CRISPR/Cas9 module and genetic transformation of aromatic rice cv. Mentik Wangi for developing bacterial leaf blight resistance. Biodiversitas 24: 3258-3268.Rice cv. Mentik Wangi is a local aromatic rice that is susceptible to pests and diseases, such as bacterial leaf blight (BLB) caused by Xanthomonas oryzae(Xoo). This bacteria can cause damage to plants thereby reducing crop yields. This study aimed to obtain the CRISPR/Cas9 module construct as well as introducing the construct into rice cv. Mentik Wangi for developing BLB resistance. The manufacture of the CRISPR/Cas9 module was carried out using the Golden Gate method, and the introduction of that construct into rice cv. Mentik Wangi was carried out using Agrobacterium tumefaciens. Constructing a CRISPR/Cas9 module with multiple gRNA of OsSWEET11 and OsSWEET14 genes has succeeded, 129 putative transformant lines of T0 generation produced with the regeneration and transformation efficiency values ??are respectively 9.4% and 9.8%. The results showed that 36 lines were positive for the hpt gene, indicating that the CRISPR/Cas9-gRNAOsSWEET module construct was successfully entered into rice cv. Mentik Wangi. Further analysis is needed to identify mutagenesis in the target gene region of TI generation transgenic plants as well as phenotypic tests for BLB resistance.

A holistic and sustainable approach linked to drought tolerance of Mediterranean crops.

The rapid increase in average temperatures and the progressive reduction in rainfalls caused by climate change is reducing crop yields worldwide, particularly in regions with hot and semi-arid climates such as the Mediterranean area. In natural conditions, plants respond to environmental drought stress with diverse morphological, physiological, and biochemical adaptations in an attempt to escape, avoid, or tolerate drought stress. Among these adaptations to stress, the accumulation of abscisic acid (ABA) is of pivotal importance. Many biotechnological approaches to improve stress tolerance by increasing the exogenous or endogenous content of ABA have proved to be effective. In most cases the resultant drought tolerance is associated with low productivity incompatible with the requirements of modern agriculture. The on-going climate crisis has provoked the search for strategies to increase crop yield under warmer conditions. Several biotechnological strategies, such as the genetic improvement of crops or the generation of transgenic plants for genes involved in drought tolerance, have been attempted with unsatisfactory results suggesting the need for new approaches. Among these, the genetic modification of transcription factors or regulators of signaling cascades provide a promising alternative. To reconcile drought tolerance with crop yield, we propose mutagenesis of genes controlling key signaling components downstream of ABA accumulation in local landraces to modulate responses. We also discuss the advantages of tackling this challenge with a holistic approach involving different knowledge and perspectives, and the problem of distributing the selected lines at subsidized prices to guarantee their use by small family farms.

RNA-seq analysis of synchronized developing pollen isolated from a single anther.

Pollen development, from unicellular microspores to anthesis, is a complex process involving the coordinated specification, differentiation and functions of different cell types. Key to understanding this development is identifying the genes expressed at precise stages of development. However, transcriptomic studies on pollen prior to anthesis are complicated by the inaccessible nature of pollen developing in the anther and the resistant pollen wall. To assist with understanding gene expression during pollen development we have developed a protocol to perform RNA-Seq on pollen isolated from a single anther (SA RNA-Seq). The protocol involves removing pollen from a single anther for analysis and viewing the remaining pollen to determine the developmental stage. The isolated pollen is chemically lysed and mRNA isolated from the lysate using an oligo-dT column before library preparation. Here, we report on the development and testing of our method and the generation of a transcriptome for three stages of pollen development from Arabidopsis (Arabidopsis thaliana) and two stages from male kiwifruit (Actinidia chinensis). This protocol enables the transcriptome of precise developmental stages of pollen to be analyzed, and uses a small number of plants, potentially facilitating studies that require a range of treatments or the analysis of the first generation of transgenic plants.

Specific suppression of long terminal repeat retrotransposon mobilization in plants.

The tissue culture passage necessary for the generation of transgenic plants induces genome instability. This instability predominantly involves the uncontrolled mobilization of LTR retrotransposons (LTR-TEs), which are the most abundant class of mobile genetic elements in plant genomes. Here, we demonstrate that in conditions inductive for high LTR-TE mobilization, like abiotic stress in Arabidopsis (Arabidopsis thaliana) and callus culture in rice (Oryza sativa), application of the reverse transcriptase (RT) inhibitor known as Tenofovir substantially affects LTR-TE RT activity without interfering with plant development. We observed that Tenofovir reduces extrachromosomal DNA accumulation and prevents new genomic integrations of the active LTR-TE ONSEN in heat-stressed Arabidopsis seedlings, and transposons of O. sativa 17 and 19 (Tos17 and Tos19) in rice calli. In addition, Tenofovir allows the recovery of plants free from new LTR-TE insertions. We propose the use of Tenofovir as a tool for studies of LTR-TE transposition and for limiting genetic instabilities of plants derived from tissue culture.

Cre-mediated autoexcision of selectable marker genes in soybean, cotton, canola and maize transgenic plants.

Efficient selectable marker gene autoexcision in transgenic plants of soybean, cotton, canola, and maize is achieved by effective Cre recombinase expression. Selectable marker genes are often required for efficient generation of transgenic plants in plant transformation but are not desired once the transgenic events are obtained. We have developed Cre/loxP autoexcision systems to remove selectable marker genes in soybean, cotton, canola and maize. We tested a set of vectors with diverse promoters and identified promising promoters to drive cre expression for each of the four crops. We evaluated both the efficiency of generating primary transgenic events with low transgene copy numbers, and the frequency of marker-free progeny in the next generation. The best performing vectors gave no obvious decrease in the transformation frequency in each crop and generated homozygous marker-free progeny in the next generation. We found that effective expression of Cre recombinase for marker gene autoexcision can be species dependent. Among the vectors tested, the best autoexcision frequency (41%) in soybean transformation came from using the soybean RSP1 promoter for cre expression. The cre gene expressed by soybean RSP1 promoter with an Arabidopsis AtpE intron delivered the best autoexcision frequency (69%) in cotton transformation. The cre gene expressed by the embryo-specific eUSP88 promoter from Vicia faba conferred the best marker excision frequency (32%) in canola transformation. Finally, the cre gene expressed by the rice CDC45-1 promoter resulted in 44% autoexcision in maize transformation. The Cre/loxP recombinase system enables the generation of selectable marker-free transgenic plants for commercial product development in four agriculturally important crops and provides further improvement opportunities for more specific and better marker excision efficiency.

Functional Analysis of Genes GlaDFR1 and GlaDFR2 Encoding Dihydroflavonol 4-Reductase (DFR) in Gentiana lutea L. Var. Aurantiaca (M. Laínz) M. Laínz.

Anthocyanins are important pigments for flower color, determining the ornamental and economic values of horticultural plants. As a key enzyme in the biosynthesis of anthocyanidins, dihydroflavonol 4-reductase (DFR) catalyzes the reduction of dihydroflavonols to generate the precursors for anthocyanidins (i.e., leucoanthocyanidins) and anthocyanins. To investigate the functions of DFRs in plants, we cloned the GlaDFR1 and GlaDFR2 genes from the petals of Gentiana lutea var. aurantiaca and transformed both genes into Nicotiana tabacum by Agrobacterium-mediated leaf disc method. We further investigated the molecular and phenotypic characteristics of T1 generation transgenic tobacco plants selected based on the hygromycin resistance and verified by both PCR and semiquantitative real-time PCR analyses. The phenotypic segregation was observed in the flower color of the transgenic tobacco plants, showing petals darker than those in the wild-type (WT) plants. Results of high-performance liquid chromatography (HPLC) analysis showed that the contents of gentiocyanin derivatives were decreased in the petals of transgenic plants in comparison to those of WT plants. Ours results revealed the molecular functions of GlaDFR1 and GlaDFR2 in the formation of coloration, providing solid theoretical foundation and candidate genes for further genetic improvement in flower color of plants.

The Use of Fluorescent Protein Fusions to Monitor the Unfolded Protein Response and Protein Foldase-Substrate Interactions in Plant Protoplasts.

Endoplasmic reticulum (ER) stress and the resulting unfolded protein response (UPR) are critical stress response pathways in eukaryotes. To study these types of interactions in plants, a wide range of methods have been used, including generation of transgenic plants, subcellular immunolocalization of protein foldases, and co-immunoprecipitation (co-IP) assays. Although these more time-consuming methods have been successfully implemented, there is a need for a versatile and rapid in vivo system to investigate ER stress and UPR. Here, we describe a transient expression system that uses plant protoplasts to define in vivo subcellular localizations and protein-protein interactions of protein foldases and their substrates fused to fluorescent protein reporters. This accurate and robust assay utilizes a variety of analyses, such as subcellular localization, FLIM-FRET, co-IP, mutagenesis, and RT-PCR in the genetically amenable Arabidopsis model system. We demonstrate the methodology by using the representative protein foldase, protein disulfide isomerase-9 (PDI9), as well as subcellular markers, secretory proteins, and dithiothreitol (DTT)-mediated induction of the UPR as monitored by RT-PCR. Together, these methods yield reliable high output results for investigating subcellular localization and protein-protein interactions in plants to decipher the UPR pathways.

Metabolic flux analysis of the non-transitory starch tradeoff for lipid production in mature tobacco leaves

The metabolic plasticity of tobacco leaves has been demonstrated via the generation of transgenic plants that can accumulate over 30% dry weight as triacylglycerols. In investigating the changes in carbon partitioning in these high lipid-producing (HLP) leaves, foliar lipids accumulated stepwise over development. Interestingly, non-transient starch was observed to accumulate with plant age in WT but not HLP leaves, with a drop in foliar starch concurrent with an increase in lipid content. The metabolic carbon tradeoff between starch and lipid was studied using 13CO2-labeling experiments and isotopically nonstationary metabolic flux analysis, not previously applied to the mature leaves of a crop. Fatty acid synthesis was investigated through assessment of acyl-acyl carrier proteins using a recently derived quantification method that was extended to accommodate isotopic labeling. Analysis of labeling patterns and flux modeling indicated the continued production of unlabeled starch, sucrose cycling, and a significant contribution of NADP-malic enzyme to plastidic pyruvate production for the production of lipids in HLP leaves, with the latter verified by enzyme activity assays. The results suggest an inherent capacity for a developmentally regulated carbon sink in tobacco leaves and may in part explain the uniquely successful leaf lipid engineering efforts in this crop.

Small molecules mediate cellular reprogramming across two kingdoms.

The fertilized egg is the single totipotent cell from which multicellular organisms arise through the processes of cell division and differentiation. While animals typically lose their capacity to redifferentiate cells that are already fully differentiated, plant cells are thought to remain totipotent (Su et al., 2020). Every gardener knows well that plants can regenerate a full array of plant tissues from already differentiated organs. This also seems to be true for single plant cells such as protoplasts, which, under proper in vitro culture conditions, served as the initial source for generation of transgenic plants (Skoog and Miller, 1957; Birnbaum and Sánchez Alvarado, 2008). However, the mechanisms behind the totipotency of plant cells remain elusive, with the exception of the knowledge that the developmental fate of regenerating tissues can be directed by the ratio of two plant hormones, auxin and cytokinin (Skoog and Miller, 1957).

Rapeseed Transformation with aroA Bacterial Gene Containing P101S Mutation Confers Glyphosate Resistance.

Field weed infestations can cause serious problems and require regular and planned programs to control them. Glyphosate is a broad-spectrum herbicide that inactivates the 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) enzyme and causes plant death. It has been reported that the mutation of proline 101 to serine in EPSPS is one of the effective mutations to reduce the affinity of glyphosate to EPSPS enzyme. In this study, we investigated the effect of the bacterial P101S mutant aromatic acid (aroA) gene on glyphosate resistance in transgenic rapeseeds. For this purpose, the mutant gene was synthesized and cloned into the pUC18 and pBI121 vectors. The gene was transferred to rapeseed by the Agrobacterium-mediated method. In this experiment, three generations of transgenic plants (T0, T1, and T2) were studied under in vitro and in vivo conditions. After the treatment of 75 putative transgenic plants with 10mM glyphosate in T0 generation, resistant plants were identified and their seeds were harvested. In the T1 generation, out of 200 cultivated plants, 141 showed resistance. After the plants were treated with herbicides and resistance was determined, the seeds were harvested when they mature. In the T2 generation, most plants (162 plants of 200) were resistant to glyphosate. Therefore, the inheritance of resistance followed Mendel's first law, which is a sign of the monogenic character of resistance. Purification and increasing the percentage of resistant plants will be carried out in the next generations. It is concluded that P101S mutation guarantees glyphosate resistance of rapeseed and is recommended to study it in other plants.

The ACC deaminase-producing plant growth-promoting bacteria: Influences of bacterial strains and ACC deaminase activities in plant tolerance to abiotic stress.

Global climate change results in frequent occurrences and/or long durations of abiotic stress. Field grown plants are affected by abiotic stress, and they modulate ethylene in response to abiotic stress exposure and use it as a signaling molecule in stress tolerance mechanisms. However, frequent occurrences and/or long durations of stress conditions can cause plants to induce ethylene levels higher than their thresholds, resulting in a reduction of plant growth and crop productivity. The use of plant growth-promoting bacteria (PGPB) that produce 1-aminocyclopropane-1-carboxylate (ACC) deaminase has increased in various plant species to ameliorate the deleterious effects of stress-induced ethylene and promote plant growth despite abiotic stress conditions. Unfortunately, there are restrictions that limit the use of ACC deaminase-producing PGPB to protect plants from abiotic stresses. This review describes how abiotic stress induces ethylene and how stress-induced ethylene adversely affects plant growth. In addition, this review emphasizes the importance of the compatibility of PGPB strains and specific host plants and ACC deaminase activities in the reduction of stress ethylene and the promotion of plant growth, based on the research published in the last 10 years. Moreover, due to the restrictions in PGPB use, this review highlights the potential generation of transgenic plants expressing the AcdS gene that encodes the ACC deaminase enzyme as a substitute for PGPB in the future to support and uplift agricultural sustainability and food security globally.

Functional characterization of late embryogenesis abundant genes and promoters in pearl millet (Pennisetum glaucum L.) for abiotic stress tolerance.

Late embryogenesis abundant (LEA) genes display distinct functions in response to abiotic stresses in plants. In pearl millet (Pennisetum glaucum L.), a total of 21 PgLEA genes were identified and classified into six groups including LEA1, LEA2, LEA3, LEA5, LEA7, and dehydrins (DHN). Open reading frames (ORFs) of PgLEAs range from 291 bp (PgLEA1-1) to 945 bp (PgLEA2-11) and distributed randomly among the seven chromosomes. Phylogenetic analysis revealed that all PgLEA proteins are closely related to sorghum LEA proteins. The PgLEAs were found to be expressed differentially under high progressive vapor pressure deficit (VPD), PgLEA7 was significantly expressed under high VPD and was selected for functional validation. In silico analysis of the PgLEA promoter regions revealed abiotic stress-specific cis-acting elements such as ABRE, CCAAT, MYBS, and LTRE. Based on the type of motifs, PgLEAPC promoter (758 bp), its deletion 1 (PgLpd1, 349 bp) and deletion 2 (PgLpd2, 125 bp) were cloned into the plant expression vector pMDC164 having the promoter-less uidA gene. All the three plant expression vectors were introduced into tobacco through Agrobacterium tumefaciens-mediated transformation to obtain T1 and T2 generations of transgenic plants. Based on expression of the uidA gene, tissue-specific expression was observed in mature stems, roots and seedlings of PgLEAPC and PgLpd1 carrying transgenics only. While the transgenic PgLEAPC plants displayed significantly higher uidA expression in the stem and root tissues under salt, drought, heat, and cold stresses, very low or no expression was observed in PgLpd1 and PgLpd2 transgenics under the tested stress conditions. The results of this study indicate that the complete promoter of PgLEAPC plays a role in developing abiotic stress tolerance in plants.

Agrobacterium-Mediated Transformation of Setaria viridis, a Model System for Cereals and Bioenergy Crops.

Setaria viridis is an emerging model system for the genetic and molecular characterization of cereals and bioenergy crops. Here, we describe a detailed procedure for genetic transformation of the S. viridis accession line ME034V-1. This method utilizes callus generated from mature seeds for infection with Agrobacterium tumefaciens strain AGL1 to regenerate hygromycin-resistant stable transgenic plants. It takes approximately 7 weeks to generate callus from mature seeds, 11-17 weeks from infection to the regeneration of transgenic lines, and an additional 3-4 weeks for plant growth in the greenhouse for seed collection. The protocol as presented consistently results in transformation frequency of approximately 25% for the generation of transgenic plants, with fewer escapes and higher survivability in soil for optimal seed collection. © 2021 Donald Danforth Plant Science Center. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of S. viridis (Accession ME034V-1) callus from mature seeds Basic Protocol 2: Agrobacterium-mediated transformation of callus to generate transgenic plants Basic Protocol 3: Plantlet transplantation in soil, plant growth in greenhouse, and seed collection Support Protocol: Preparation of Agrobacterium culture for infection.