Finger millet (Eleusine coracana) is a nutritionally important and climate-resilient cereal cultivated in rainfed regions of India and Eastern Africa, yet its genetic improvement has been limited by the lack of efficient and reproducible transformation systems. In this study, we developed a rapid and efficient Agrobacterium tumefaciens-mediated transformation and regeneration system using shoot apical meristem (SAM) explants, enabling direct, callus-free shoot organogenesis. Optimal regeneration and shoot elongation were achieved on Murashige and Skoog (MS) medium supplemented with 3.5mg L⁻1 6-benzylaminopurine (BAP), 1.5mg L⁻1 kinetin, 0.1mg L⁻1 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.2mg L⁻1 gibberellic acid (GA₃). Genotype-dependent responses were observed, with PR-202 requiring 2mg L⁻1 AgNO3 to reduce phenolic browning, whereas VL-376 regenerated efficiently without AgNO3. Transformation efficiencies of 30-32% were achieved in PR-202 and VL-376, respectively, by optimising infection and co-cultivation conditions, including reduced MS salt strength and pre-incubation of Agrobacterium. Molecular analyses, including PCR and Southern blot hybridisation, confirmed stable T-DNA integration in independent lines, while segregation analysis of T₁ progenies demonstrated Mendelian inheritance of the transgene. In addition, CRISPR/Cas9 constructs targeting EcCKX2 were successfully introduced via Agrobacterium, demonstrating the suitability of this system for genome engineering applications. Overall, this optimised SAM-based protocol provides a rapid (45-50days), efficient, and reproducible platform for stable genetic transformation in finger millet and establishes a strong foundation for transgenic research and future genome editing studies in this underutilized crop.

Leucine aminopeptidases (LAPs) are multifunctional enzymes with roles in both defence and development. In plants, they are reported to be induced by wound-inflicting Lepidopteran insects and regulate wound response pathways leading to an effective defence response. Infestation by Hemipteran mustard aphid, Lipaphis erysimi (L.) Kaltenbach has been reported to induce wound response as well as a wound-responsive Arabidopsis thaliana Lap1 homologue (RI01; GenBank Accession: JK034053) in Rorippa indica (L.) Hiern. This is interesting as Hemipteran insects like aphids are assumed to inflict minimal wounding. In the present study, starting with the RI01 sequence information, we isolated the full length (1566bp) sequence of a novel R. indica Lap (RiLap) gene, performed in silico analyses and developed transgenic R. indica plants with suppressed RiLAP activity by expressing a 565bp antisense fragment of RiLap cDNA. We found that the isolated RiLAP is an acidic LAP of M17 family and suppressing it causes a significant increase in aphid herbivory but reduction in total chlorophyll content and possibly photosynthetic capacity in aphid infested transgenic plants of the T1 generation. These findings though preliminary suggest that RiLap could have a role in deterring aphids by acting as a regulatory protein simultaneously balancing defence response and photosynthetic capacity or plant growth. Noting the dearth of research in this area, this pilot study will be useful for designing future in depth analyses in understanding the role of Laps in defence response against Hemipteran insects. The study has implications in the development of sustainable pest management avenues.

A new line of cedar pollen allergen-accumulating transgenic rice, which accumulated the 7Crp peptide comprised of 7 concatenated major T cell epitopes of cedar pollen allergens, was developed; it improved on the shortcomings of an original transgenic line. The new line has one copy of inserted T-DNA and a substantial expression of the 7Crp peptide, and was named Os7Crp2. Regulatory approval is needed before commercializing this cedar pollen peptide rice as a genetically modified food product in Japan. Therefore Os7Crp2 was evaluated for the criteria required for food safety assessments, under the guidance of Standards for the Safety Assessment of Genetically Modified Foods (Seed Plants) in Japan. No statistically significant differences were found between Os7Crp2 and the control (parental) Dongtokoi cultivar in physicochemical properties, expressed traits, or concentrations of key components, except for the insertion of the T-DNA and expression of the recombinant protein derived from the transgenes. These results suggest that Os7Crp2 is compositionally equivalent to non-transgenic rice.

Molecular breeding and gene function studies in plants require high transformation efficiency. Agrobacterium-mediated transformation has contributed significantly to molecular research in many plants, but is inefficient and inconsistent in rice that do not host Agrobacterium. Transformation efficiency in rice remains low. Therefore, this study aimed to establish a simple and efficient transformation method for rice using Agrobacterium. Two foreign genes (CISP1-GFP and CISP2-GFP) and Agrobacterium strain (EHA105) was used in the experiments. Then, Agrobacterium infection of rice seeds that had absorbed water and germinated under reduced pressure infiltration conditions showed that an average of 14% of the seeds formed after growth (12% with CISP1-GFP and 16% with CISP2-GFP) carried the foreign gene, and it was also confirmed by PCR, Western blot, GFP fluorescence and TAIL-PCR. Since this method does not involve callus formation or re-differentiation of rice plants, no special equipment or complicated operations are required, and transformants can be obtained in only three months. Therefore, this method is expected to simplify rice genetic manipulation and promote molecular breeding of rice.

Chia (Salvia hispanica L.), a nutritionally valuable crop, is the richest source of α-linolenic acid, a key omega-3 fatty acid. Despite its nutritional benefits, a stable genetic transformation method for chia is not available. This study presents a sonication-assisted Agrobacterium-mediated transformation protocol optimized for high-efficiency transformation of chia seedlings. Key parameters including bacterial cell density, acetosyringone concentration, sonication duration, vacuum infiltration, and infection time were optimized. Results demonstrated that an optical density at 600nm (OD600) of 0.5, acetosyringone concentration of 100µM, 20min of sonication, 10min of vacuum infiltration, and 60min of infection significantly enhanced transformation efficiency and GUS expression. This optimized protocol was validated through Polymerase Chain Reaction and β-glucuronidase (GUS) assay in transformed plants. Our findings establish a robust and reliable transformation protocol, paving the way for future genetic engineering efforts aimed at enhancing the nutritional and agronomic traits of chia.

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.

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.

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.

Food and nutritional security are the top priorities in Indian agriculture. Exponential population growth coupled with climate change effects has become a serious challenge for sustainable agriculture. Genome editing has revolutionized the agricultural sector because of its ability to create precise, stable and predictable modifications in the genome and therefore, offers great opportunities for crop improvement in India. However, for harvesting the real benefits of this technology in agriculture sector, there is a strong need of creating awareness among the end users and development of suitable policies for regularization of genome edited products. Many regulatory agencies around the world have been modernizing their regulatory approaches to be more risk proportionate and to reflect a more science-based approach. In this article, recent research initiatives and developments undertaken by different Indian institutes/organizations for the genetic improvement of agricultural and horticultural crops via genome editing technologies are summarized. Furthermore, to benefit from this potential technology in our country, regulatory policies must be clear, science-based and proportionate. Therefore, in the present review, the regulatory policies related to the genome editing of crop products in India are discussed in detail. This review will sensitize researchers and stakeholders to the application of genome editing techniques in crop improvement and various biosafety committees involved in the development and regulation of genome edited crops.