Multiple shoot induction from embryonic axes of mature seeds supports a novel regeneration platform for biolistic DNA delivery in Theobroma cacao L.

Seedling-derived split internode and leaf explants as efficient alternatives for callus induction in tropical maize

The development of effective callogenesis and regeneration protocols remains a major challenge for tropical maize genotypes, which are often recalcitrant to in vitro culture techniques. This study aimed to evaluate the callogenic potential of seedling-derived split internodes and leaf explants in four local maize varieties. Explants were cultured on five auxin-cytokinin induction media. Callus induction percentage and relative fresh weight growth rate (RFWGR) were measured. Data were analyzed using two-way ANOVA followed by Duncan’s Multiple Range Test (α = 0.05). Callus induction and proliferation were strongly genotype-dependent. Split internodes consistently produced higher RFWGR than leaf explants. Media containing 2-2.5 mg/L 2,4-D combined with BAP or kinetin (M1-M3) yielded the highest responses, particularly in variety CHABA. Seedling-derived explants represent efficient alternatives to immature embryos for tropical maize callogenesis. The identified genotype-explant-medium combinations provide a basis for developing regeneration and transformation protocols adapted to tropical germplasm. This study does not involve human participants or clinical trials.

Development of plant regeneration and stable Agrobacterium tumefaciens–mediated transformation methods for Solanum retroflexum Dunal

Solanum retroflexum is cultivated for both its fruit and leaves in Africa, China, India, and Indonesia. However, it is still considered an underutilized crop due to limited genetic and genomic resources to support improvement. To enable gene function studies for future improvement by new breeding technologies, this study established efficient plant regeneration and Agrobacterium tumefaciens –mediated transformation approaches. Cotyledon and hypocotyl explants were used to test plant regeneration on a Murashige and Skoog salts-based medium supplemented with 0.5 mg L −1 zeatin. Both explant types showed 100% regeneration; however, cotyledons produced a greater number of whole plants than hypocotyls. For transformation experiments, cotyledon explants were infected with A. tumefaciens AGL1 carrying the pJL33 binary vector containing the Green Fluorescent Protein ( GFP ) reporter and the Neomycin Phosphotransferase-II ( nptII ) selectable marker genes. Following cocultivation, firstly, kanamycin at various concentrations (75.0 to 400.0 mg L −1 ) was evaluated to determine the optimal concentration for maximizing transgenic line recovery. However, a significant number of escapes even at 400.0 mg L −1 kanamycin were observed. Because the nptII gene also confers resistance to G418, in this study’s subsequent experiments the current authors tested the effectiveness of a range of G418 concentrations (25.0 to 150.0 mg L −1 ). Fluorescence microscopy and PCR analysis showed that 25.0 mg L −1 G418 resulted in the highest transformation efficiency at 88% and the fewest escapes. Ploidy levels of regenerated, transgenic, and wild-type plants were assessed using flow cytometry, confirming stable ploidy. The optimized selection medium facilitated an efficient and stable transformation system that will be utilized to support crop improvement efforts of S. retroflexum .

Establishment of a High-Efficiency In Vitro Regeneration and Agrobacterium-Mediated Genetic Transformation System for Ajania achilleoides (Turcz.) Poljakov

Ajania achilleoides, a wild species with strong environmental adaptability, is a valuable germplasm resource for genetic improvement and functional gene studies. However, efficient in vitro regeneration and genetic transformation systems for this species remain undeveloped, limiting functional gene characterization and genetic improvement of this valuable germplasm resource. In this study, leaf explants were used to establish an efficient regeneration system by evaluating different combinations of plant growth regulators and to develop an Agrobacterium tumefaciens-mediated genetic transformation system for A. achilleoides. MS medium supplemented with 2.0 mg/L 6-benzyladenine (6-BA) and 1.0 mg/L α-naphthaleneacetic acid (NAA) achieved the highest adventitious shoot regeneration rate (99%), while 1/2 MS medium containing 0.5 mg/L indole-3-butyric acid (IBA) was optimal for rooting, resulting in a 100% rooting rate. Critical hygromycin concentrations for shoot regeneration and rooting selection were determined to be 25 mg/L and 10 mg/L, respectively. For transformation, the best results were obtained with a 3-day preculture, bacterial suspension at OD600 = 0.2, 30 min infection, and 2-day co-cultivation. PCR and sequencing confirmed the successful integration of the target genes into the genome, yielding three positive transgenic lines with a molecular confirmation rate of 30%. This study establishes a stable and efficient regeneration and transformation system for A. achilleoides, providing a platform for functional gene analysis and molecular breeding of wild chrysanthemum species.

Direct regeneration pathway for the mass propagation of commercial banana cv. Ney Poovan (AB) using immature male flower bud explants

This study developed an efficient, variety-specific in vitro regeneration protocol for the commercially important yet recalcitrant banana cultivar Ney poovan (AB). The investigation aimed to optimise a direct organogenesis system using immature male floral hands as explants and to validate the genetic fidelity and field performance of regenerated plants. Immature male flower buds proved to be a highly effective explant source, offering reduced contamination risk compared to conventional shoot-tip cultures commonly employed for large-scale production of disease-free planting material. Among the growth regulator treatments evaluated, BAP (8 mg L-1) combined with Thidiazuron (TDZ) (0.4 mg L-1) induced the earliest greening response in cv. Ney poovan, with explants exhibiting greening within 23.40 days. The TDZ (0.6 mg L-1) alone was found to be optimal for meristematic clump formation. Maximum shoot proliferation (10.80 shoots per explant) was achieved on medium supplemented with 6-Benzylaminopurine (BAP) (5 mg L-1) and indole-3-acetic acid (IAA) (1 mg L-1). Root induction was significantly enhanced by the combined application of indole-3-butyric acid (IBA) and α-naphthaleneacetic acid (NAA) (1 mg L-1 each), resulting in rapid rooting within 5.20 days. Field evaluation of regenerated plants demonstrated normal vegetative growth, flowering and yield performance. The regenerated plants produced bunch weights comparable to conventionally propagated suckers, recording an average bunch weight of 14.52 kg. Genetic fidelity assessment using inter simple sequence repeats (ISSR) markers revealed very low polymorphism (6.64 %), confirming the high genetic stability and true-to-type nature of the regenerants. The protocol developed in this study provides a reliable, scalable and commercially viable approach for mass propagation of banana cv. Ney poovan banana, facilitating the production of genetically uniform, high-quality planting material for sustainable banana cultivation.

Efficient in-vitro regeneration and transformation for CRISPR/Cas9-mediated genome editing of phytoene desaturase (PDS) gene in pea (Pisum sativum L.)

The present study addresses optimization of in-vitro regeneration via direct organogenesis and Agrobacterium-mediated genetic transformation, enabling efficient multiplex CRISPR/Cas9-based genome editing of the phytoene desaturase (PsPDS) gene in pea. Pea (Pisum sativum L.) is an important legume crop valued for food, plant-based protein, vegetable, and green manure. Although genome editing offers a precise and rapid strategy for crop improvement, its application in pea remains challenging due to inherent recalcitrance to in-vitro regeneration and genotype-dependent transformation. The regeneration and Agrobacterium-mediated transformation systems were optimized, and the dicotyledonary node (DCN) was identified as the preferred explant for multiplex CRISPR/Cas9-based genome editing in pea. Among three explant types (embryonic axis, DCN and nodal segment), DCN showed the highest regeneration efficiency, producing 100% shoot bud induction and 39.70 shoots per explant on MS medium augmented with 6-benzylaminopurine (BAP; 6.00 mg/L) and kinetin (1.00 mg/L). Shoot elongation and rooting efficiencies were improved using GA3 (1.00 mg/L), BAP (1.00 mg/L), IAA (0.10 mg/L), and NAA (0.5 mg/L), respectively. Manipulating explant type, Agrobacterium optical density, vacuum infiltration, acetosyringone concentration, infection time, and co-cultivation duration improved the transient transformation efficiency. We noted efficiency from 23.33% to 90.00% in DCN and from 6.66% to 93.33% in embryonic axis explants across 10 pea cultivars. Stable transformed lines generated from the DCN of cultivar Kashi Samridhi were confirmed by GUS staining and PCR. The optimized regeneration and transformation system facilitated targeted editing of phytoene desaturase (PsPDS) in pea, achieving ICE-estimated mutation frequencies of upto 97% in independent lines. The study provides a robust platform for functional genomics and accelerates the deployment of genome-editing technologies for pea improvement.

Efficient somatic embryogenesis and plant regeneration in Salvia sclarea L.: Assessment of genetic fidelity and stage-specific variation in terpene profiles