Transgenic Research最新文献

{"title":"Mutation in soybean Lox-2 PLAT/LH2 domain through CRISPR/Cas9 reduces seed lipoxygenase activity: responsible for undesirable flavour.","authors":"Ekta Patel, Piyali Das, Somak Hazra, Manveer Sharma, Gautam Chhabra, Balwinder Singh Gill, Sucheta Sharma, Ajinder Kaur, Deepak Singla, Jagdeep Singh Sandhu","doi":"10.1007/s11248-025-00447-8","DOIUrl":"10.1007/s11248-025-00447-8","url":null,"abstract":"<p><p>Soybean, a protein and oil rich legume is primarily used as livestock feed and to a lesser extent for human consumption due to undesirable flavour in the seeds caused by L-2 isozyme of lipoxygenase. Herein, soybean with reduced isozyme activity was developed through CRISPR/Cas9 targeted mutation in L-2 encoding Lox-2 gene. sgRNA designed from PLAT/LH2 domain in second exon of Lox-2 (Lox-2 E2) was validated by in vitro cleavage assay; inserted in CRISPR/Cas9 binary vector and used for genetic transformation of SL1074 cultivar hypocotyl segments. A total of 12 T₀ putative plants were identified through PCR. Amongst these, four revealed mutation at the target sgRNA site by CEL1 assay and substitution of a base A with G six bp upstream of PAM converting lysine to glutamic acid at 119 position. T₁ and T₂ seeds derived from mutant T0-37 plant showed upto 25.49% reduction in isozyme activity as compared to SL1074. The base substitution was confirmed in T₁ progeny; segregation analysis revealed homozygosity and heritability of mutation in T₂ plants. The interaction between structural models of SL1074, mutant domains and negatively charged substrates revealed strong binding affinity of the substrates with positively charged lysine in SL1074 domain due to formation of two hydrogen bonds. On the contrary, weak binding of the substrates with negatively charged glutamic acid in mutant domain and absence of hydrogen bond explained reduction of isozyme activity in T₂ seeds. The mutant soybean with reduced isozyme activity is an important source for introgressing the trait in plant breeding programs.</p>","PeriodicalId":23258,"journal":{"name":"Transgenic Research","volume":"34 1","pages":"29"},"PeriodicalIF":2.7,"publicationDate":"2025-06-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12158852/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144267291","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Streamlined protoplast transfection system for in-vivo validation and transgene-free genome editing in Banana.","authors":"Hiralben Lakhani, Naveen Kumar, Alka Jangra, Sanjana Negi, Thobhanbhai Dholariya, Siddharth Tiwari","doi":"10.1007/s11248-025-00446-9","DOIUrl":"10.1007/s11248-025-00446-9","url":null,"abstract":"<p><p>The advancement in the CRISPR/Cas system has significantly streamlined genome editing in plants, rendering it simple, reliable, and efficient. However, the development of transgene-free crops is a challenging task for vegetatively propagated plants like banana. In the present study, we established banana protoplasts-based versatile and efficient platform for genome editing to overcome this limitation. Herein, a protocol has been optimized for protoplast isolation by considering leaf and embryogenic cell suspension (ECS) of banana cultivar Grand Naine. Freshly prepared ECS was identified as the best source for protoplast isolation. The protoplast viability and competency were checked by transfection with plasmid and RNP complex. Polyethylene glycol (PEG)-mediated protoplast transfection using pCAMBIA1302 and pJL50TRBO vectors showed GFP expression with 30 and 70% efficiency, respectively, eventually proving the protocol's efficacy. Further, gRNAs targeting banana β-carotene hydroxylase gene are validated by in-vitro cleavage test and subsequently used for RNP complex formation with varied ratios (1:1, 1:2, 1:5, and 1:10) of SpCas9 to gRNA1. Among these, a 1:2 molar ratio proved best to generate indel frequency with 7%. Sequencing analysis of the target amplicon revealed mutations upstream of the PAM region, specifically with gRNA1, among the three in-vitro validated gRNAs. This study evaluated the effectiveness of gRNAs in-vitro and in-vivo, yielding inconsistent results that highlight the need for comprehensive in-vivo validation of their functionality. Conclusively, the optimized protocol for banana transfection has the potential to be harnessed for the generation of transgene-free genetically improved banana.</p>","PeriodicalId":23258,"journal":{"name":"Transgenic Research","volume":"34 1","pages":"28"},"PeriodicalIF":1.9,"publicationDate":"2025-06-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144209615","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"First report of transgenic Agave sisalana 'RLV 19' plants resistant to the herbicide glyphosate-an improved alternative for biofuel production.","authors":"Carolina Rossi de Oliveira, Aline Vitória Corim Marim, Camila Santos Woloche, Paulo Cezar De Lucca, Marcelo Falsarella Carazzolle, Gonçalo Amarante Guimarães Pereira","doi":"10.1007/s11248-025-00445-w","DOIUrl":"10.1007/s11248-025-00445-w","url":null,"abstract":"<p><p>The Agave genus is recognized for its diversity, economic importance, and adaptability to arid and semi-desert climates. Among its species, Agave sisalana stands out for the production of sisal, a resilient natural fiber with various industrial applications and potential to be used as a raw material in biofuel production, due to the accumulation of fermentable sugars in its biomass. Although this species presents significant agronomic potential, challenges in conventional breeding hinder its cultivation. A viable alternative for genetic improvement is the development of transgenic plants that incorporate desirable agronomic traits through the controlled insertion of genes of interest into their genome. The objective of study was to develop a protocol for obtaining transgenic plants of Agave sisalana 'RLV19,' a species widely cultivated in the semi-arid region of Bahia-BA, Brazil, using the cp4-epsps gene aimed at constitutive gene expression. Two transgenic plants of Agave sisalana 'RLV19' were regenerated via organogenesis. PCR analyses and CP4-EPSP protein expression by RT-PCR confirmed the presence and expression of the transgene in these plants. This is the first report of A. sisalana transgenic plants expressing the cp4-epsps gene, and to the best of our knowledge, there have been no prior reports on protocols for the production of transgenic plants of this species.</p>","PeriodicalId":23258,"journal":{"name":"Transgenic Research","volume":"34 1","pages":"27"},"PeriodicalIF":1.9,"publicationDate":"2025-06-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144209614","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"CRISPR mediated gene editing for economically important traits in horticultural crops: progress and prospects.","authors":"Saheb Pal, Ram Krishna, Labdhi Dedhia, Hukum Singh Panwar, Suhas Gorakh Karkute, Nagendra Rai, Rajesh Kumar, Sudhakar Pandey, Achuit Kumar Singh","doi":"10.1007/s11248-025-00444-x","DOIUrl":"10.1007/s11248-025-00444-x","url":null,"abstract":"<p><p>Horticultural crops, with their cost-effectiveness, rich mineral and vitamin content, and high yield potential, have become indispensable worldwide for ensuring food and nutritional security. With the world's population on the rise and climate change becoming more prominent, it is crucial to focus on creating resilient, high-yielding crop varieties that can withstand the changing climate. Genetic improvement of different horticultural crops using conventional tools is both time-consuming and labourious. However, the breeding period can be cut short by adopting modern breeding techniques, including CRISPR/Cas-mediated genome editing. In the present review, we discuss the progress made so far through genome editing to improve several horticultural crops for various traits like stress resistance, morphology, nutritional attributes, quality, shelf life, male sterility, architecture and economic yield. We have also discussed the emerging CRISPR technologies like base editing, epigenome editing, CRE editing, transposon-based editing, prime editing etc., along with their pros and cons and the future prospects. The ethical considerations for commercialization and current regulatory frameworks for gene-edited products have also been discussed.</p>","PeriodicalId":23258,"journal":{"name":"Transgenic Research","volume":"34 1","pages":"26"},"PeriodicalIF":1.9,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144200088","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The high-affinity K⁺ transporter IbHAK5 enhances potassium ion absorption and improves root morphology in sweetpotato (Ipomoea batatas).","authors":"Rong Jin, Peng Zhao, Mengxiao Yan, Ming Liu, Weijuan Fan, Qiangqiang Zhang, Xiaoya Zhu, Jing Wang, Yongchao Yu, Jun Yang, Hongxia Wang, Zhonghou Tang","doi":"10.1007/s11248-025-00437-w","DOIUrl":"10.1007/s11248-025-00437-w","url":null,"abstract":"<p><p>Potassium is a vital element in sweetpotato that plays important roles during its growth and development. In this study, potassium transporter IbHAK5, which is homologous to Arabidopsis HAK5, was cloned and overexpressed in sweetpotato. IbHAK5 encoded a protein of 739 amino acids and localized in the plasma membrane. Two IbHAK5-overexpressing transgenic lines with the highest expression level of IbHAK5 were screened for K⁺-deficiency stress tolerant assay. Compared with wild type sweetpotato plants, transgenic plants grew well with higher chlorophyll content, and maintain great higher K⁺ contents via decreasing more K⁺ effluxes under low potassium ion (- K⁺) stress condition. Additionally, IbHAK5 can help plants improve root morphology and increase endogenous hormone IAA content under both normal condition and - K⁺ stress, which may result in the increased root K⁺ absorption ability. The results indicated that IbHAK5 play an important role in sweetpotato response to - K⁺ stress, as well as support molecular-assisted breeding with the IbHAK5 gene.</p>","PeriodicalId":23258,"journal":{"name":"Transgenic Research","volume":"34 1","pages":"25"},"PeriodicalIF":1.9,"publicationDate":"2025-05-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144120935","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Phenotypic characteristics of interspecific hybrids between wild and cultivated soybean with and without insect-protected biotechnology traits.","authors":"Duška Stojšin, Bill Duncan, Chen Meng","doi":"10.1007/s11248-025-00443-y","DOIUrl":"10.1007/s11248-025-00443-y","url":null,"abstract":"&lt;p&gt;&lt;p&gt;Wild soybean (Glycine soja Sieb. and Zucc.) and cultivated soybean (Glycine max (L) Merr.) can cross-pollinate, albeit at a very low frequency, potentially resulting in an interspecific hybrid with a biotechnology trait inherited from the cultivated soybean parent. As part of environmental risk assessment, it is informative to understand the competitiveness potential of these hybrids in natural habitats. The objective of this research was to evaluate the phenotypic characteristics of the interspecific hybrids and compare them with cultivated and wild soybean. Secondly, the comparisons were conducted between the interspecific hybrids with and without an insect-protected (IP) biotechnology trait. Two wild soybean populations were crossed with cultivated soybean containing either MON 87701 or MON 87751, the IP traits developed to control specific lepidopteran pests. Hybrid plants with and without the IP trait and parental entries were evaluated in growth chamber trials for the plant, pollen and seed characteristics. Compared to the parents, the hybrids had intermediate values for most of the measured characteristics. Compared to the wild soybean parent, hybrids had less twining, shorter plants, fewer seeds and pods, thicker stems, bigger seeds and pollen grains, as well as reduced seed dormancy-all due to domestication genes inherited from cultivated soybean. However, when compared to parents, hybrids had significantly reduced pollen viability (51.8-73.3% vs. &gt; 95%) and number of seeds per pod (1.6-1.7 vs. 2.3) indicating partial reproductive sterility due to chromosome interchange between nonhomologous chromosomes. Seed coat color of the F&lt;sub&gt;3&lt;/sub&gt; hybrids segregated in a manner that seems to involve two major genes (I and T) with some level of inter-locus interaction and/or partial dominance resulting in an observed ratio of 9 green (i&lt;sup&gt;i&lt;/sup&gt;-T-): 3 black (iiT-): 3 brown (-itt): 1 yellow (i&lt;sup&gt;i&lt;/sup&gt;i&lt;sup&gt;i&lt;/sup&gt;tt) seeds. A combination of recessive genes (ii from wild and tt from cultivated soybean) resulted in hybrid seeds with defective, cracked seed coat contributing to reduction in seed dormancy. Generally, hybrids with and without the IP traits had comparable performance indicating that the biotechnology IP trait did not have unintended effects on phenotypic characteristics. However, there were some characteristics that differed. The F&lt;sub&gt;3&lt;/sub&gt; hybrids with MON 87751 had significantly lower seed dormancy (8.1%) compared to hybrids without the transgene (15.1%) likely due to the proximity of the transgene to soybean native domestication-related gene and their co-segregation in hybrid generations. In summary, the results obtained in this research suggest that the fitness, overwintering and general ability of hybrids to compete and persist in nature is reduced when compared to wild soybean. The main contributors to lower survival of hybrids are: (i) domestication genes inherited from the soybean parent, (ii) partial sterility","PeriodicalId":23258,"journal":{"name":"Transgenic Research","volume":"34 1","pages":"24"},"PeriodicalIF":2.7,"publicationDate":"2025-05-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12078375/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144080579","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Knockout of the Agrobacterium ILVC gene generates a valine-isoleucine auxotrophic strain for plant transformation.","authors":"Yanhui Yang, Jun Lu, Tianrui Xue, Zhenli Cao, Mingjie Li, Zhongyi Zhang, Rong Wang, Tongyu Wu","doi":"10.1007/s11248-025-00442-z","DOIUrl":"10.1007/s11248-025-00442-z","url":null,"abstract":"<p><p>The Agrobacterium tumefaciens mediated transformation is the prevailing methodology for plant genetic manipulation; however, A. tumefaciens overgrowth is a common constraint in the process. Exploring auxotrophic A. tumefaciens could reduce overgrowth and enhance plant transformation efficiency. The ILVC gene, which encodes the ketol-acid isomeroreductase, is critical for Valine (Val) and isoleucine (Ile) biosynthesis in some microorganisms. However, its function in A. tumefaciens is unclear. To ascertain the function of ILVC and generate an auxotrophic A. tumefaciens, this study employed an allelic exchange to disrupt the ILVC in A. tumefaciens strain GV3101. This resulted in the loss of ketol-acid isomeroreductase activity and the prevention of Val and Ile biosynthesis, creating a dual-auxotrophic GV3101_∆ILVC. Transient expression assays in Nicotiana benthamiana transformation demonstrated that the GV3101_∆ILVC was capable of T-DNA transfer. Moreover, stable genetic transformation analysis in N. benthamiana indicated that the introduction of GV3101_∆ILVC led to a reduction in overgrowth within infected plant tissues. Additionally, an enhancement in transformation efficiency was observed with the prolongation of the co-cultivation time of the explant-infected strain. This study revealed the function of ILVC and explored a dual-auxotrophic A. tumefaciens for Val and Ile, potentially broadening the utilization of auxotrophic strains in plant genetic transformation.</p>","PeriodicalId":23258,"journal":{"name":"Transgenic Research","volume":"34 1","pages":"23"},"PeriodicalIF":1.9,"publicationDate":"2025-05-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144020042","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of a plant-based vaccine against brucellosis: stable expression of Brucella abortus OMP25 in transgenic tobacco.","authors":"Mansoure Qashqai, Emrah Bertan, Semiha Erisen, Tulin Ozbek, Senay Vural-Korkut","doi":"10.1007/s11248-025-00441-0","DOIUrl":"10.1007/s11248-025-00441-0","url":null,"abstract":"<p><p>Brucellosis, caused by Brucella species, is a global threat to livestock farming, resulting in economic losses and socio-economic challenges, particularly in rural areas. Despite its impact, no licensed human vaccines are available. Animal vaccination remains the most cost-effective control method, but traditional vaccine production is expensive. Edible vaccines, using plants as bioreactors to produce immunogenic antigens, offer a low-cost alternative by eliminating complex purification processes. This study developed a transgenic plant by expressing the Brucella abortus outer membrane protein OMP25 in tobacco plants. OMP25, a conserved transmembrane protein with high immunogenicity, was cloned into a Gateway pDONR vector via a Boundary Pairing reaction and transferred to a binary destination vector via a Left-Right reaction. The destination vector was introduced into Agrobacterium tumefaciens and subsequently used for Agrobacterium-mediated transformation of tobacco plants. Transgenic plants were selected on media containing kanamycin, and the expression of the transgene was verified through the fluorescence of green fluorescent protein. Microcallus formation and shoot development on selective media confirmed kanamycin resistance and the successful integration of the transgene. After phenotypic selection, genomic DNA was extracted from transgenic plants and analyzed by PCR (Polymerase Chain Reaction) using primers specific to the OMP25 gene. Positive PCR results validated the successful integration of the OMP25 gene into the plant genome. Gene expression was further confirmed at the RNA level through real-time quantitative PCR (qRT-PCR) and at the protein level via Western blot analysis. Future studies will evaluate immune responses in animal models. This approach demonstrates the potential for low-cost, effective vaccines to combat brucellosis, addressing critical economic and public health challenges.</p>","PeriodicalId":23258,"journal":{"name":"Transgenic Research","volume":"34 1","pages":"22"},"PeriodicalIF":1.9,"publicationDate":"2025-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12041093/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144050499","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Integrated transcriptomic, transcriptional factors, and protein interaction reveal the regulatory mechanisms of flowering time in rice (Oryza sativa L.).","authors":"Amir Sohail, Liaqat Shah, Syed Mehar Ali Shah, Adil Abbas, Shahzad Ali, Manzoor","doi":"10.1007/s11248-025-00439-8","DOIUrl":"10.1007/s11248-025-00439-8","url":null,"abstract":"<p><p>Appropriate flowering time is important for rice regional adaptation and optimum rice production, but little is known about the omics of heading date in rice. Here, we studied omics including transcriptome, proteome and transcriptional factors to identify regulatory genes related to flowering time. A total of 1402 differentially expressed genes (DEGs, 721 up-regulated and 681 down-regulated) were detected in wild and mutant. These transcripts are classified according to biological processes, cellular components, and molecular functions. Among these differentially expressed genes, many transcription factor genes demonstrated multiple regulatory pathways involved in flowering time. Gene expression analysis showed that Os03g0122600 (OsMADS50), Os08g0105000 (Ehd3), Os06g0275000 (Hd1) were expressed higher and Os06g0199500 (OsHAL3), Os06g0498800 (OsMFT1), Os08g0105000 (Ehd3), Os06g0157700 (Hd3a), and Os02g0731700 (Ghd2), were expressed lower in wild compared to mutant, which are the key genes that regulate the flowering in rice. In addition, Ghd7 interacted with Os10g30860 and Os12g08260 using yeast two-hybrid assay. We identified 28 potential Ghd7 transcriptional regulators using the transcription factor-centered yeast one hybrid (TF-Centered Y1H) assay. Taken together, this study developed a new set of genomic resources to identify and characterize genes, proteins, and motifs associated with flowering time.</p>","PeriodicalId":23258,"journal":{"name":"Transgenic Research","volume":"34 1","pages":"21"},"PeriodicalIF":1.9,"publicationDate":"2025-04-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144041203","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstracts of the 19th Transgenic Technology Meeting (TT2025) : Zurich, Switzerland, April 24-27, 2025.","authors":"","doi":"10.1007/s11248-025-00436-x","DOIUrl":"https://doi.org/10.1007/s11248-025-00436-x","url":null,"abstract":"","PeriodicalId":23258,"journal":{"name":"Transgenic Research","volume":"34 Suppl 1","pages":"19"},"PeriodicalIF":2.7,"publicationDate":"2025-04-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144050077","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}