Genetic Engineering Research Research Articles

Comparative analysis of chloroplast genome of Lonicera japonica cv. Damaohua

Abstract Lonicera japonica is a well-known medicinal plant, and the Damaohua cultivar is one of the oldest known honeysuckle cultivars in China. The 155,151 bp chloroplast genome of this cultivar was obtained through Illumina sequencing. The genome includes a pair of inverted repeats (IRa and IRb; 23,789 bp each), a large single-copy region (88,924 bp), and a small single-copy (SSC) region (18,649 bp). In total, 127 unique genes were identified: 80 protein-coding, 39 tRNA, and 8 rRNA genes. Only ycf3 contained two introns. Eighty-nine large repetitive sequences and 54 simple sequence repeats were detected. Fifty potential RNA editing sites were predicted. Adaptive evolution analysis revealed that infA, matK, petB, petD, rbcL, rpl16, rpl2, rps3, ycf1, and ycf2 were positively selected, possibly reflecting the specific environmental adaptations of this cultivar. Sequence alignment and analysis revealed several candidate fragments for Lonicera species identification, such as the intergenic regions rpoB-petN, rbcL-accD, and psaA-ycf3. The IR region boundary and phylogenetic analysis revealed that the L. japonica cv. Damaohua chloroplast genome was most closely related to the L. japonica genome, but there were five distinct differences between the two. There are four sites with high variability between L. japonica and L. japonica cv. Damaohua with nucleotide variability (Pi) greater than 0.002, including rps2-rpoC2, atpB-rbcL, ycf1, and ycf1-trnN GUU. The differences between L. japonica and L. japonica cv. Damaohua were further confirmed by the single nucleotide polymorphism sites between these two species. Therefore, this study revealed that the chloroplast genome can serve as a universal super barcode for plant identification, which can identify differences and help distinguish Lonicera japonica from related species. An understanding of Lonicera japonica cv. Damaohua chloroplast genomics and a comparative analysis of Lonicera species will provide a scientific basis for breeding, species identification, systematic evolution analysis, and chloroplast genetic engineering research on medicinal honeysuckle plants.

Identification and functional characterization of bidirectional gene pairs and their intergenic regions in cotton

BackgroundIn research to improve the quality of transgenic crops, it is often necessary to introduce multiple functionally related genes into recipient plants simultaneously to improve crop genetic traits effectively. Compared with unidirectional promoters, bidirectional promoters simultaneously regulate the expression of multiple genes and improve the efficiency of biotechnology. Therefore, in this study, bidirectional gene pairs were systematically analyzed in Gossypium hirsutum TM-1, and the structure, function and evolutionary relationships of the bidirectional genes were analyzed. The endogenous bidirectional promoters of cotton were mined, and their specific regulatory elements and biological functions were explored to provide useful promoter resources and a theoretical basis for cultivating new cotton germplasms with excellent fiber quality.ResultsUsing an improved search model, a total of 1,383 bidirectional transcript pairs were identified in the Gossypium hirsutum TM-1 genome, and their gene structure and functional annotations were systematically analyzed. Thirty bidirectional intergenic sequences were randomly screened for promoter activity analysis via a transient expression system, and 25 intergenic sequences were found to have bidirectional promoter activity. Comparative analysis of the bidirectional gene profiles of the four cotton subspecies revealed that these subspecies presented abundant bidirectional gene pairs with high homology and that the bidirectional genes in the cotton subspecies were more similar in terms of their molecular functions, cellular components and biological processes. In addition, parallel analysis of bidirectional genes in dicotyledons and monocotyledons revealed that abundant bidirectional gene pairs exist in different species. Although the total number of orthologous bidirectional genes was similar, there was a significant difference in the number of orthologous bidirectional gene pairs between dicotyledons and monocotyledons. This evolutionary analysis of the function and structure of homologous bidirectional gene pairs in different varieties and different subspecies of the same species revealed potential pathways by which these gene pairs originated, which may be necessary for the evolution of a new species.ConclusionIn this study, many bidirectional gene pairs in Gossypium hirsutum TM-1 were identified using computer programming, and systematic analysis was conducted to explore their functions and evolutionary relationships. In addition, the promoter activity of the bidirectional intergenic sequences was verified. The combination of computer programming screening, experimental validation and other methods is expected to provide preferred bidirectional promoters for transgenic breeding work via multigene cotransformation methods, and this information is valuable for genetic engineering research and applications.

Application of Developmental Regulators for Enhancing Plant Regeneration and Genetic Transformation.

Establishing plant regeneration systems and efficient genetic transformation techniques plays a crucial role in plant functional genomics research and the development of new crop varieties. The inefficient methods of transformation and regeneration of recalcitrant species and the genetic dependence of the transformation process remain major obstacles. With the advancement of plant meristematic tissues and somatic embryogenesis research, several key regulatory genes, collectively known as developmental regulators, have been identified. In the field of plant genetic transformation, the application of developmental regulators has recently garnered significant interest. These regulators play important roles in plant growth and development, and when applied in plant genetic transformation, they can effectively enhance the induction and regeneration capabilities of plant meristematic tissues, thus providing important opportunities for improving genetic transformation efficiency. This review focuses on the introduction of several commonly used developmental regulators. By gaining an in-depth understanding of and applying these developmental regulators, it is possible to further enhance the efficiency and success rate of plant genetic transformation, providing strong support for plant breeding and genetic engineering research.

The complete Chloroplast genome of Stachys geobombycis and comparative analysis with related Stachys species

Herb genomics, at the forefront of traditional Chinese medicine research, combines genomics with traditional practices, facilitating the scientific validation of ancient remedies. This integration enhances public understanding of traditional Chinese medicine’s efficacy and broadens its scope in modern healthcare. Stachys species encompass annual or perennial herbs or small shrubs, exhibiting simple petiolate or sessile leaves. Despite their wide-ranging applications across various fields, molecular data have been lacking, hindering the precise identification and taxonomic elucidation of Stachys species. To address this gap, we assembled the complete chloroplast (CP) genome of Stachys geobombycis and conducted reannotation and comparative analysis of seven additional species within the Stachys genus. The findings demonstrate that the CP genomes of these species exhibit quadripartite structures, with lengths ranging from 14,523 to 150,599 bp. Overall, the genome structure remains relatively conserved, hosting 131 annotated genes, including 87 protein coding genes, 36 tRNA genes, and 8 rRNA genes. Additionally, 78 to 98 SSRs and long repeat sequences were detected , and notably, 6 highly variable regions were identified as potential molecular markers in the CP genome through sequence alignment. Phylogenetic analysis based on Bayesian inference and maximum likelihood methods strongly supported the phylogenetic position of the genus Stachys as a member of Stachydeae tribe. Overall, this comprehensive bioinformatics study of Stachys CP genomes lays the groundwork for phylogenetic classification, plant identification, genetic engineering, evolutionary studies, and breeding research concerning medicinal plants within the Stachys genus.

Genome-Wide Computational Analysis of Dirrigent Proteins in Solanum Tuberosum

Dirrigent proteins are required for the synthesis of lignans, a distinct and widely distributed class of plant-derived secondary metabolites with promising pharmacologic characteristics and a potential role in plant defense. However, no detailed data on the Dirrigent family of Solanum tuberosum is known. Comparatively, 33 DIR sequences were explored in Solanum tuberosum in this research study. There are no introns in the majority of StDIR genes. A single Dirigent domain is found in all StDIR proteins. The abundance of amino acids, sequence similarity analysis, phylogenetic analysis, genomic location, gene structure, conserved domain analysis, evolutionary analysis, and gene promoter assessment are all studied. This study lays a foundation for potential plant genetic engineering and crop improvement research by providing an in-depth and thorough explanation of the detailed molecular mechanism and structural characterization of StDIR proteins in the genome of Solanum tuberosum. This work will provide useful data for enabling the proper selection of dirrigent proteins in higher plants and will support future studies on the DIR gene family.

Improvement of Culture Conditions and Plant Growth Regulators for In Vitro Callus Induction and Plant Regeneration in Paeonia lactiflora Pall.

Owing to its high ornamental, medicinal and horticultural values, herbaceous peony (Paeonia lactiflora Pall.) has been widely used as a landscaping and economical plant around the world. However, the lack of an efficient and stable regeneration system in P. lactiflora restricts its rapid propagation and large-scale production. By testing the key factors affecting callus formation, proliferation, adventitious bud induction and rooting, here, we developed an in vitro system for callus induction and regeneration in P. lactiflora. Our results show that callus formation was affected by explant types, culture environment, basal medium and plant growth regulators. Using cotyledons as explants, we established good conditions for P. lactiflora callus induction and callus proliferation. We effectively obtained adventitious buds differentiated from callus in Murashige and Skoog (MS) medium containing kinetin (KT) and thidiazuron (TDZ). Adventitious bud growth can be further promoted by adding gibberellin 3 (GA3), 1-naphthaleneacetic acid (NAA) and 6-benzyleaminopurine (6-BA) into the MS medium. A high percentage of rooting can be achieved by adding indolebutyric acid (IBA) and activated carbon (AC) to ½ MS medium. Overall, our system promotes callus induction and adventitious bud regeneration for P. lactiflora through improved culture conditions and plant growth regulators in the culture media, and lays a foundation for subsequent genetic engineering research.

Long-term culture induces Bax-dependent apoptosis in rat preimplantation embryos.

Although rat preimplantation embryos are necessary for producing genetically modified rats, their in vitro culture remains a challenge. Rat zygotes can develop from the one-cell stage to the blastocyst stage in vitro; however, long-term culture reduces their developmental competence via an unknown mechanism. In this study, we examined how in vitro conditions affect rat preimplantation embryos, which may explain this reduced competence. Comprehensive gene expression analysis showed that genes related to apoptosis and energy metabolism were differentially expressed in rat embryos cultured long-term in vitro compared with those developed in vivo. Furthermore, we found that the expression of Bak1 and Bax, which are responsible for mitochondrial outer membrane permeabilization, were more upregulated in embryos cultured in vitro than those developed in vivo. Similarly, apoptosis-dependent DNA fragmentation was also exacerbated in in vitro culture conditions. Finally, gene disruption using CRISPR/Cas9 showed that Bax, but not Bak1, was responsible for these effects. These findings suggest that long-term in vitro culture induces Bax-dependent apoptosis through the mitochondrial pathway and may provide clues to improve the long-term culture of rat preimplantation embryos for genetic engineering research.

Cyanobacterial Biofuel: A Platform for Green Energy

Cyanobacteria have great potential as a platform for biofuel production because of their fast growth, ability to fix CO<sub>2 </sub>gas, and genetic tractability. They also preserve the sustainability of an ecosystem without harming the environment. High-performance biofuels made from cyanobacteria can be utilized as a base for the production of green energy. Although a lot of studies have been conducted where plants and crops are used as the source of energy, cyanobacteria have been reported to have a more efficient photosynthetic process strongly responsible for increased production with limited land input along with affordable cost. The production of cyanobacteria-based biofuels can be accelerated through genetic engineering or genomics research, which may help to meet the global demand for these fuels on a large scale. Cyanobacterial strains that have undergone genetic modifications have been developed as part of a green recovery approach to transform membrane lipids into fatty acids to produce cheap and eco-friendly green energy. Cyanobacteria also produce different biofuels such as butanol, ethanol and isoprene. The four different generations of biofuel production to meet the energy requirement have been discussed in this review. This review presents a comprehensive strategy for the commercial viability of green energy production utilizing cyanobacteria to achieve a price for biofuels that can compete with the present or future market.

Functional characterization of microbes and their association with unwanted substance for wastewater treatment processes

Nowadays, microorganisms can be used to eliminate a variety of pollutants such as toxic metal ions from wastewater. These emergences of harmful elements in wastewater, high-priced cultivation of microbes and technical hitches in industrial scale production appeared as main challenges for thriving coupling of microbes with wastewater. These microbes serve as potential sorbents by following suitable adsorption mechanisms. There are some photobioreactors have been also mentioned in this review which is based on microbial biofilm and emerged as an alternative technology to predictable photosynthetic systems for treatment of wastewater based on biomass production at low cost. Bioremediation using different microbes showed contrast results to remove heavy metals from wastewater. Microorganism such as Nostoc sp., Aspergillus versicolor, Aspergillus lentulus and Aspergillus niger remediate 99.6, 99.89, 99.7 and 98 % of Pb, Cr, Cu and Ni, respectively. In this review, mechanistic approaches and distinct pathways of the microbes for removal of various inorganic and organic compounds from wastewater have been methodically discussed. We have also discussed some major commercial production challenges such as techno-economic feasibility genetic engineering research and biorefinery approach. Overall the review discussed the microbial biodiversity in wastewater and their role in remediation of wastewater and their ability to be a potent candidate headed for sustainable industrial wastewater treatment applications through different approaches such as phytoremediation and bioremediation. This article provides valuable insights into multiple aspects of environmental biotechnology, including photobioreactors, metal uptake capacity of microorganisms, heavy metal contamination and its effects and bioremediation using molecular approaches and wastewater treatment through phytoremediation. Moreover, it contributes to our understanding of these topics and can help in the development of sustainable solutions for environmental remediation and pollution control in wastewater though microorganisms.

Genome-Wide Tissue-Specific Genes Identification for Novel Tissue-Specific Promoters Discovery in Soybean.

Promoters play a crucial role in controlling the spatial and temporal expression of genes at transcriptional levels in the process of higher plant growth and development. The spatial, efficient, and correct regulation of exogenous genes expression, as desired, is the key point in plant genetic engineering research. Constitutive promoters widely used in plant genetic transformation are limited because, sometimes, they may cause potential negative effects. This issue can be solved, to a certain extent, by using tissue-specific promoters. Compared with constitutive promoters, a few tissue-specific promoters have been isolated and applied. In this study, based on the transcriptome data, a total of 288 tissue-specific genes were collected, expressed in seven tissues, including the leaves, stems, flowers, pods, seeds, roots, and nodules of soybean (Glycine max). KEGG pathway enrichment analysis was carried out, and 52 metabolites were annotated. A total of 12 tissue-specific genes were selected via the transcription expression level and validated through real-time quantitative PCR, of which 10 genes showed tissue-specific expression. The 3-kb 5' upstream regions of ten genes were obtained as putative promoters. Further analysis showed that all the 10 promoters contained many tissue-specific cis-elements. These results demonstrate that high-throughput transcriptional data can be used as effective tools, providing a guide for high-throughput novel tissue-specific promoter discovery.

System network analysis of Rosmarinus officinalis transcriptome and metabolome-Key genes in biosynthesis of secondary metabolites.

Medicinal plants contain valuable compounds that have attracted worldwide interest for their use in the production of natural drugs. The presence of compounds such as rosmarinic acid, carnosic acid, and carnosol in Rosmarinus officinalis has made it a plant with unique therapeutic effects. The identification and regulation of the biosynthetic pathways and genes will enable the large-scale production of these compounds. Hence, we studied the correlation between the genes involved in biosynthesis of the secondary metabolites in R. officinalis using proteomics and metabolomics data by WGCNA. We identified three modules as having the highest potential for the metabolite engineering. Moreover, the hub genes highly connected to particular modules, TFs, PKs, and transporters were identified. The TFs of MYB, C3H, HB, and C2H2 were the most likely candidates associated with the target metabolic pathways. The results indicated that the hub genes including Copalyl diphosphate synthase (CDS), Phenylalanine ammonia lyase (PAL), Cineole synthase (CIN), Rosmarinic acid synthase (RAS), Tyrosine aminotransferase (TAT), Cinnamate 4-hydroxylase (C4H), and MYB58 are responsible for biosynthesis of important secondary metabolites. Thus, we confirmed these results using qRT-PCR after treating R. officinalis seedlings with methyl jasmonate. These candidate genes may be employed for genetic and metabolic engineering research to increase R. officinalis metabolite production.

URA3 as a Selectable Marker for Disruption and Functional Assessment of PacC Gene in the Entomopathogenic Fungus Isaria javanica.

An effective selection marker is necessary for genetic engineering and functional genomics research in the post-genomic era. Isaria javanica is an important entomopathogenic fungus with a broad host range and prospective biocontrol potentials. Given that no antibiotic marker is available currently in this fungus, developing an effective selection marker is necessary. In this study, by applying overlap PCR and split-marker deletion strategy, combining PEG-mediated protoplasm transformation method, the uridine auxotrophy gene (ura3) in the I. javanica genome was knocked out. Then, using this transformation system, the pH response transcription factor gene (IjpacC) was disrupted successfully. Loss of IjpacC gene results in an obvious decrease in conidial production, but little impact on mycelial growth. The virulence of the ΔIjpacC mutant on caterpillars is similar to that of the wild-type strain. RT-qPCR detection shows that expression level of an acidic-expressed S53 gene (IF1G_06234) in ΔIjpacC mutant is more significantly upregulated than in the wild-type strain during the fungal infection on caterpillars. Our results indicate that a markerless transformation system based upon complementation of uridine auxotrophy is successfully developed in I. javanica, which is useful for exploring gene function and for genetic engineering to enhance biological control potential of the fungus.

Human retina – A history of biological research and the development of genetic engineering

AbstractThe development of genetic engineering (also called genetic modification) started in the 40‐ties of the 19th c. After the Second World War the rapid process that used laboratory‐based technologies to conduct researches on cells, tissues, alter the DNA makeup of an organism and virus become the basis of modern paradigm of medical sciences. This was the culminating point of the process, which is sometimes called “the war against diseases”. One of the most successful moments was the creation of vaccines by combining genetic engineering research with research on viruses. It was a time of scientific optimism. For the first time in human history people could get the knowledge of aetiology of diseases, could fight them and protect themselves against infection. Its seems that the development of medical sciences could serve mankind, therefore there should be no limits as to its development. This optimism was not hindered by Nazists medical experimentations performed during the Second World War. However, The main ethical questions about the limits of medical science and human biological material research were left unanswered. The development of genetic engineering gave rise to questions on legalization of abortion, euthanasia, and transplantology. From 60‐ties to 90‐ties of the 20th century, large‐scale research began on diploid cells of fetal origin. All these events have called for discussion in the bioethics community and the question of the limits to which medical sciences can be pushed.Key word: history of genetic engineering, cell biology, history of human biological material research.

Construction of a novel plasmid for an industrial yeast Candida glycerinogenes by dual-autonomously replicating sequence strategy

Due to the lack of available episomal plasmid, the improvement of many industrial strains, especially exogenous gene expression, is severely restricted. The failure of autonomous replication or low copy number of episomal plasmids is the main reason for the failure of many episomal plasmids construction. In this paper, Candida glycerinogenes, an industrial strain lacking episomal plasmids, was employed as the topic. A series of GFP-based plasmids containing autonomously replicating sequence (ARS) from different strain sources were constructed and analyzed for performance, and it was found that only the panARS from Kluyveromyces lactis compared with other nine low capacity ARSs proved to have the best performance and could be used to construct episomal plasmid. Further, the dual-ARS strategy was used to optimize the episomal plasmid, and the results indicated that only the dual-ARS plasmid+PPARS2 with double different ARSs, not the dual-ARS plasmid+panARS with double same ARSs, showed an improvement in all properties, with an increase in transformation efficiency of about 36% and a synchronous trend of fluorescence intensity and copy number, both by about 40%. In addition, constructed episomal plasmids were used to express the exogenous gene CrGES, and the fact that geraniol was found proved the versatility of the plasmids. The successful construction of episomal plasmids will also substantially facilitate genetic engineering research and industrial use of C.glycerinogenes in the future, as well as providing a feasible approach to create episomal plasmids for industrial strains.

Gene-independent therapeutic interventions to maintain and restore light sensitivity in degenerating photoreceptors.

Neurodegenerative retinal diseases are a prime cause of blindness in industrialized countries. In many cases, there are no therapeutic treatments, although they are essential to improve patients' quality of life. A set of disease-causing genes, which primarily affect photoreceptors, has already been identified and is of major interest for developing gene therapies. Nevertheless, depending on the nature and the state of the disease, gene-independent strategies are needed. Various strategies to halt disease progression or maintain function of the retina are under research. These therapeutic interventions include neuroprotection, direct reprogramming of affected photoreceptors, the application of non-coding RNAs, the generation of artificial photoreceptors by optogenetics and cell replacement strategies. During recent years, major breakthroughs have been made such as the first optogenetic application to a blind patient whose visual function partially recovered by targeting retinal ganglion cells. Also, RPE cell transplantation therapies are under clinical investigation and show great promise to improve visual function in blind patients. These cells are generated from human stem cells. Similar therapies for replacing photoreceptors are extensively tested in pre-clinical models. This marks just the start of promising new cures taking advantage of developments in the areas of genetic engineering, optogenetics, and stem-cell research. In this review, we present the recent therapeutic advances of gene-independent approaches that are currently under clinical evaluation. Our main focus is on photoreceptors as these sensory cells are highly vulnerable to degenerative diseases, and are crucial for light detection.

Characterization of the gene expression profile response to drought stress in Haloxylon using PacBio single-molecule real-time and Illumina sequencing.

Haloxylon ammodendron and Haloxylon persicum are important drought-tolerant plants in northwest China. The whole-genome sequencing of H. ammodendron and H. persicum grown in their natural environment is incomplete, and their transcriptional regulatory network in response to drought environment remains unclear. To reveal the transcriptional responses of H. ammodendron and H. persicum to an arid environment, we performed single-molecule real-time (SMRT) and Illumina RNA sequencing. In total, 20,246,576 and 908,053 subreads and 435,938 and 210,334 circular consensus sequencing (CCS) reads were identified by SMRT sequencing of H. ammodendron and H. persicum, and 15,238 and 10,135 unigenes, respectively, were successfully obtained. In addition, 9,794 and 7,330 simple sequence repeats (SSRs) and 838 and 71 long non-coding RNAs were identified. In an arid environment, the growth of H. ammodendron was restricted; plant height decreased significantly; basal and branch diameters became thinner and hydrogen peroxide (H2O2) content and peroxidase (POD) activity were increased. Under dry and wet conditions, 11,803 and 15,217 differentially expressed genes (DEGs) were identified in H. ammodendron and H. persicum, respectively. There were 319 and 415 DEGs in the signal transduction pathways related to drought stress signal perception and transmission, including the Ca2+ signal pathway, the ABA signal pathway, and the MAPK signal cascade. In addition, 217 transcription factors (TFs) and 398 TFs of H. ammodendron and H. persicum were differentially expressed, including FAR1, MYB, and AP2/ERF. Bioinformatic analysis showed that under drought stress, the expression patterns of genes related to active oxygen [reactive oxygen species (ROS)] scavenging, functional proteins, lignin biosynthesis, and glucose metabolism pathways were altered. Thisis the first full-length transcriptome report concerning the responses of H. ammodendron and H. persicum to drought stress. The results provide a foundation for further study of the adaptation to drought stress. The full-length transcriptome can be used in genetic engineering research.

Efficient assembly of long DNA fragments and multiple genes with improved nickase-based cloning and Cre/loxP recombination.

SummaryFunctional genomics, synthetic biology and metabolic engineering require efficient tools to deliver long DNA fragments or multiple gene constructs. Although numerous DNA assembly methods exist, most are complicated, time‐consuming and expensive. Here, we developed a simple and flexible strategy, unique nucleotide sequence‐guided nicking endonuclease (UNiE)‐mediated DNA assembly (UNiEDA), for efficient cloning of long DNAs and multigene stacking. In this system, a set of unique 15‐nt 3′ single‐strand overhangs were designed and produced by nicking endonucleases (nickases) in vectors and insert sequences. We introduced UNiEDA into our modified Cre/loxP recombination‐mediated TransGene Stacking II (TGSII) system to generate an improved multigene stacking system we call TGSII‐UNiE. Using TGSII‐UNiE, we achieved efficient cloning of long DNA fragments of different sizes and assembly of multiple gene cassettes. Finally, we engineered and validated the biosynthesis of betanin in wild tobacco (Nicotiana benthamiana) leaves and transgenic rice (Oryza sativa) using multigene stacking constructs based on TGSII‐UNiE. In conclusion, UNiEDA is an efficient, convenient and low‐cost method for DNA cloning and multigene stacking, and the TGSII‐UNiE system has important application prospects for plant functional genomics, genetic engineering and synthetic biology research.

Kombinasi BAP dan NAA untuk Media Perbanyakan Nanas Varietas Smooth Cayenne, Toboali in Vitro

Toboali pineapple variety is a smooth cayenne variety because it contains high antioxidants in the form of vitamins A and C. In addition, it is a source of citric and malic acids, substances that can be used to enhance the taste of the fruit. Pineapple is a plant that is propagated with vegetative organs such as suckers, slips, and crowns. However, conventional propagation has a low propagation rate. Tissue culture is an in vitro technique used in the rapid propagation of plants and in large quantities to meet the needs of cultivation and research. The selection of regeneration media is very influential on the development of plants in tissue culture. This study aimed to obtain the optimal media combination with BAP and NAA in the regeneration of Smooth Cayenne pineapple (Toboali) in vitro for mass propagation of seedlings and genetic engineering research. This study used 6 levels of BAP, namely 0, 1, 2, 3, 4, 5 mg.L<sup>-1</sup> and 4 levels of NAA, namely 0, 1, 2, 3 mg.L<sup>-1</sup>. The treatment used was a combination of BAP and NAA as many as 24 treatments with 3 replications. The best pineapple regeneration medium for mass plant propagation in this study was BAP 3 mg.L<sup>-1</sup> without NAA media which produced the highest shoots of 17.33 shoots per explant at 12 week after planting. The best pineapple regeneration medium for genetic engineering research was a combination BAP 3 mg.L<sup>-1</sup> and NAA 2 mg.L<sup>-1</sup> media which produced 18.33 shoots per explant from embryogenic callus.

Constitutive expression of a pea apyrase, psNTP9, increases seed yield in field-grown soybean

To address the demand for food by a rapidly growing human population, agricultural scientists have carried out both plant breeding and genetic engineering research. Previously, we reported that the constitutive expression of a pea apyrase (Nucleoside triphosphate, diphosphohydrolase) gene, psNTP9, under the control of the CaMV35S promoter, resulted in soybean plants with an expanded root system architecture, enhanced drought resistance and increased seed yield when they are grown in greenhouses under controlled conditions. Here, we report that psNTP9-expressing soybean lines also show significantly enhanced seed yields when grown in multiple different field conditions at multiple field sites, including when the gene is introgressed into elite germplasm. The transgenic lines have higher leaf chlorophyll and soluble protein contents and decreased stomatal density and cuticle permeability, traits that increase water use efficiency and likely contribute to the increased seed yields of field-grown plants. These altered properties are explained, in part, by genome-wide gene expression changes induced by the transgene.

Characterization of the Gene Expression Profile Response to Drought Stress in Populus ussuriensis Using PacBio SMRT and Illumina Sequencing.

In this study, we characterized the gene expression profile in the roots of Populus ussuriensis at 0, 6, 12, 24, 48 and 120 h after the start of polyethylene glycol (PEG)-induced drought stress using PacBio single-molecule real-time sequencing (SMRT-seq) and Illumina RNA sequencing. Compared to the control, 2244 differentially expressed genes (DEGs) were identified, and many of these DEGs were associated with the signal transduction, antioxidant system, ion accumulation and drought-inducing proteins. Changes in certain physiological and biochemical indexes, such as antioxidant activity and the contents of Ca2+, proline, and total soluble sugars, were further confirmed in P. ussuriensis roots. Furthermore, most of the differentially expressed transcription factors were members of the AP2/ERF, C2H2, MYB, NAC, C2C2 and WRKY families. Additionally, based on PacBio SMRT-seq results, 5955 long non-coding RNAs and 700 alternative splicing events were identified. Our results provide a global view of the gene expression profile that contributes to drought resistance in P. ussuriensis and meaningful information for genetic engineering research in the future.