TL;DR
A successful method for indirect somatic embryogenesis and regeneration from leaf explants of Salvia sclarea is demonstrated and will be highly valuable for the effective use of emerging biotechnological tools.
A reproducible protocol for somatic embryogenesis was developed from mature stem and leaf explants of Gmelina arborea Roxb., an economically important forestry species. Explants were cultured on Murashige and Skoog (MS) medium supplemented with varying concentrations (0.1–5.0 mg L⁻¹) of naphthaleneacetic acid (NAA) or 2,4-Dichlorophenoxyacetic acid (2,4- D), each combined with 1.0 mg L⁻¹ benzylaminopurine (BAP). No embryogenic response was observed on hormone-free medium. Auxin-supplemented media successfully induced globular somatic embryos within 35–40 days. The highest embryogenic response was obtained with 4.0 mg L⁻¹ 2,4-D + 1.0 mg L⁻¹ BAP (50.77% in stem; 46.76% in leaf explants), followed by 4.0 mg L⁻¹ NAA + 1.0 mg L⁻¹ BAP. Stem explants showed higher embryogenic competence than leaf explants. Higher auxin concentrations (>4.0 mg L⁻¹) resulted in tissue browning and reduced embryo formation. Maturation of globular embryos was achieved on MS medium containing 0.5 mg L⁻¹ NAA and kinetin (0.5– 3.0 mg L⁻¹), facilitating the sequential development of heart, torpedo and cotyledonary evolutionary stages within 25 days. The results were statistically significant (p ≤ 0.05), demonstrating the reproducibility and reliability of the protocol. This study provides an efficient somatic embryogenesis system from mature explants of G. arborea, addressing a key limitation in woody plant tissue culture and enabling large-scale clonal propagation and forestry biotechnology applications.
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.
Somatic embryogenesis (SE) underpins plant regeneration, genetic transformation and virus-free seedling propagation, yet efficient SE systems using root explants remain lacking in Allium fistulosum. Here, we establish and characterize two novel high-efficiency SE pathways mediated by frog egg-like bodies (FELBs) and rhizoid tubers (RTBs) in A. fistulosum, and clarify the regulatory effects of NAA, 2,4-D and TDZ on morphogenesis and plant regeneration. Screening of multiple explants demonstrated that only root segments and stem tips could form translucent mucilaginous callus, while single 2,4-D treatment failed to induce callus at all tested concentrations. Under dark conditions, 10 mg/L NAA maximized callus induction (94.233%) and FELB formation (87.833%) in root explants with significant dose-dependent effects. Phytocytohistological analysis confirmed that FELBs/RTBs develop via three unique stages (pro-embryo, globular embryo, heart/torpedo transitional embryo), distinct from the five canonical stages of typical somatic embryos. For RTB pathway, 5.0 mg/L NAA induced the maximum rhizoid number (54.867 per explant), and subsequent treatment with 20.0 mg/L TDZ under high light achieved optimal RTB production (51.333 per explant). Stage-specific BAP application yielded 100% FELB germination and over 90% RTB plantlet regeneration, with both regenerated seedlings exhibiting > 90% acclimatization survival rates. This study first elucidates the ontogeny of FELB and RTB in monocot A. fistulosum. This is the first study characterizing FELB/RTB developmental stages and ontogeny in a monocot, showing striking homology to eudicotyledons, confirming that FELB- and RTB-mediated somatic embryogenesis can also be achieved in monocots. These optimized systems provide robust technical support for high-frequency regeneration, genetic modification and virus-free seedling production of Allium crops.
Somatic embryogenesis (SE) represents a central regeneration system for the implementation of new breeding technologies in grapevine ( Vitis spp.). However, its practical use remains constrained by genotype dependence, prolonged developmental timelines, and variability associated with explant type and culture conditions. This study evaluated SE and embryo-to-plant conversion across fifteen recalcitrant European genotypes and hybrids from Italy, Spain, Portugal, and Hungary using anthers and ovaries cultured on three induction media, followed by embryo germination. Ovaries generally exhibited higher responsiveness to SE than anthers, the PIV medium showed the broadest effectiveness, although the genotype was the primary determinant of the SE process. Noteworthy, differences in embryogenesis competence did also emerge among clones of Nebbiolo cultivar highlighting intra-cultivar variability effects. By extending the duration of callus culture beyond the conventional evaluation windows and using a two-media induction protocol, the embryogenesis success increased in several genotypes, suggesting that premature callus elimination may underestimate SE potential. Regeneration experiments confirmed that the embryo-to-plant transition was the second critical step of the process. For instance, Kadarka was a cultivar with low aptitude to SE and high regeneration efficiency, whereas other genotypes displayed poor embryo germination. Overall, all fifteen recalcitrant grapevine genotypes produced SE although plant regeneration was not achieved in Graciano and Fernão Pires. The results support a two-bottleneck-based model of grapevine regeneration controlled by genotype. Inefficiencies in the embryo-to-plant conversion constitute a highly limiting factor for downstream plant breeding applications, underscoring the need for genotype-specific optimization strategies able to address both embryogenic induction and regeneration efficiency.
Tomato (Solanum lycopersicum L.) is one of the most economically important horticultural crops in China, and Xinjiang is the main region in China for processing tomato production. The establishment of a robust in vitro regeneration system is a fundamental prerequisite for genetic improvement and cultivar innovation in this crop. Among the factors influencing regeneration efficiency, exogenously applied plant growth regulators (PGRs) are the most important because they regulate endogenous phytohormone homeostasis and signaling pathways. In this study, true leaf explants of the processing tomato Ligeer 87-5 were used to optimize combinations of 6-benzyladenine (6-BA) and indole-3-acetic acid (IAA) and to establish an efficient in vitro regeneration system. Full strength MS medium supplemented with 2 mg/L 6-BA and 0.2 mg/L IAA was optimal for callus induction and organogenic competence, resulting in a callus induction rate of 97.00%, an embryogenic cell incidence of 63.88%, and an adventitious bud formation rate of 21.33% after 20 days; the budding rate further increased to 63.00% after 30 days. For rapid shoot induction, a medium composed of MS + 1 mg/L 6-BA + 0.2 mg/L IAA yielded the highest adventitious bud formation rate, reaching 89.00% after 10 days. Rooting was most effective on MS medium supplemented with 0.2 mg/L IAA, achieving a 100% rooting rate after 21 days, an average root length of 11.76 cm, a 76.67% acclimatization rate after 28 days, and a 100% survival rate after hardening and transplantation. Collectively, the stage-specific application of different media substantially improved true-leaf regeneration efficiency in processing tomato plants, providing a robust platform for future genetic transformation and molecular breeding.
ABSTRACT Background: Euterpe edulis Martius, a symbolic species of the Atlantic Forest in Brazil, popularly known as the Juçara palm tree, does not produce axillary or basal shoots, which prevents its vegetative propagation through conventional techniques. Plant tissue culture techniques have been proposed for the clonal propagation of superior genotypes of this species. The present study aimed to evaluate the effect of 2,4-dichlorophenoxyacetic acid (2,4-D) on the induction of embryogenic calluses from mature zygotic embryos of E. edulis. Mature zygotic embryos were inoculated in culture media supplemented with different concentrations (20, 40, 80, 160, 320, 480, and 600 mg L⁻¹) of 2,4-D, while the control treatment contained no 2,4-D. The experiments were conducted in a completely randomized design. Results: After 70 days in the induction medium, three morphogenic responses were observed: seedlings, abnormal seedlings, and calluses. Increasing concentrations of 2,4-D reduced the formation of normal seedlings while enhancing callogenesis up to concentrations between 160 and 279 mg L⁻¹. At higher concentrations, embryogenic calluses or oxidized explants predominated. Morphoanatomical evaluations confirmed the embryogenic identity of the calluses, which consisted of a meristematic center surrounded by a parenchymatic zone. Neutral polysaccharides and starch grains were mainly observed in the parenchymatic zone, which was not directly involved in somatic embryo regeneration. Conclusion: This study demonstrates the formation of embryogenic calluses from in vitro cultures of zygotic embryos of Euterpe edulis. These findings contribute to the development of a complete regeneration protocol and may support the clonal propagation and conservation of this important Atlantic Forest palm species.
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