Plant Regeneration Research Articles

Peptide REF1 is a local wound signal promoting plant regeneration

Plants frequently encounter wounding and have evolved an extraordinary regenerative capacity to heal the wounds. However, the wound signal that triggers regenerative responses has not been identified. Here, through characterization of a tomato mutant defective in both wound-induced defense and regeneration, we demonstrate that in tomato, a plant elicitor peptide (Pep), REGENERATION FACTOR1 (REF1), acts as a systemin-independent local wound signal that primarily regulates local defense responses and regenerative responses in response to wounding. We further identified PEPR1/2 ORTHOLOG RECEPTOR-LIKE KINASE1 (PORK1) as the receptor perceiving REF1 signal for plant regeneration. REF1-PORK1-mediated signaling promotes regeneration via activating WOUND-INDUCED DEDIFFERENTIATION 1 (WIND1), a master regulator of wound-induced cellular reprogramming in plants. Thus, REF1-PORK1 signaling represents a conserved phytocytokine pathway to initiate, amplify, and stabilize a signaling cascade that orchestrates wound-triggered organ regeneration. Application of REF1 provides a simple method to boost the regeneration and transformation efficiency of recalcitrant crops.

Regeneration niche of Cortaderia selloana in an invaded region: Flower predation, environmental stress, and transgenerational effects

Background and aimsBiological invasions are causing massive environmental, social, and economic impacts worldwide. Understanding the ecological factors that affect the regeneration of invasive plants is critical to develop action plans based on empirical biological evidence. In this study, we investigate ecological limits in the regeneration of Cortaderia selloana, an invasive species with a strong impact in temperate regions. MethodsWe sampled 22 sites invaded by C. selloana along an environmental gradient in northern Spain to evaluate the influence of flower predation, environmental stress, and transgenerational effects on the performance of C. selloana offspring. Key resultsWe found that over half of the sampled flowers per individual contained larvae of Spanolepis selloanae, a recently discovered predator of C. selloana. This finding suggests that the presence of this natural enemy extends over a larger area than previously thought, which might drastically reduce seed production. A common-garden experiment conducted with over 5,200 seeds revealed that seed germination is significantly enhanced with increasing temperatures (with an optimum temperature of 22/12 °C day/night temperatures), and that seedling survival and growth are highest under waterlogged soil conditions. Our experiments also suggest that the minimum temperature for germination of C. selloana is 10 °C, and that seedlings can survive up to one month without water. These findings can improve management programs by delineating which areas are at high risk of invasion and which ones are unsuitable for the regeneration of C. selloana. We also found that seed mass and maternal plant size are positively correlated with seed germination and early seedling growth. Progeny from dry maternal environments performed better under continuous drought conditions and the opposite was found under wet conditions. ConclusionsOverall, our results suggest that flower predators, environmental stress, and transgenerational effects have complex and profound effects on early demographic stages of C. selloana, influencing seed production, seed germination, seedling growth, and seedling survival.

Germination niche of co-occurring threatened native and alien species: a case study in Lindernia procumbens and L. dubia

Plant invasions have a negative impact on biodiversity and lead to species loss. Although studies on traits promoting invasiveness have been largely developed, comparative studies on germination traits of closely related native versus alien invasive species, which also take into account threatened species, are lacking. We analysed the germination requirements of two congeneric Lindernia species inhabiting paddy field in Northern Italy. Germination tests were conducted exposing seeds of both species across a broad range of temperature and conditions (i.e., cold-wet stratification, dark condition, alternate temperature, gibberellic acid). The invasive L. dubia showed higher and faster germination, occurring over a broader range of temperatures compared to the native threatened L. procumbens, along with a lighter non-deep physiological dormancy. These results show a competitive advantage of the alien species already in the early stages of plant regeneration and provide a comprehensive overview of the germination requirements, necessary for the development of future conservation and management plans.

Contribution of early succession regeneration plants to heavy metal remediation in abandoned Artisanal small-scale mining sites in Ghana

Artisanal Small-Scale mining (ASM) is a major land use activity in Ghana. Despite its tremendous contribution to livelihoods, ASM is largely unregulated in the country, illegally operated, leaving most mined out sites unrehabilitated. In this study, we explored the extent to which early succession naturally regenerating plants on two abandoned illegal artisanal small scale mined-out sites (Chamfi and Alluvial types) remediate heavy metals. The study was undertaken in three districts of the Western Region of Ghana. Plots were randomly established in the alluvial, chamfi and reference (control) sites. Within each plot, samples of soil and naturally regenerating plants were collected for determination of bioaccumulation potential, translocation and contamination factor of cadmium (Cd), arsenic (As), lead (Pb), and mercury (Hg) using the Atomic Absorption Spectrophotometer. The results revealed that the concentration of As, Cd, Hg, Pb from soil samples, as well as roots and shoots of the plants collected from the two mined sites were significantly higher than the concentrations in the samples from the control plots. Our results show that heavy metal removal is more significant in plants at the alluvial mined soils than those at the chamfi sites. C. pubescens, C. odorata, and M. pudica were observed to be better cadmium translocators than the other vegetations sampled. Furthermore, plants at the alluvial sites recorded higher Bioaccumulation Factor (BF) in the roots than those at the chamfi sites for lead and arsenic. The study implies that natural regeneration of early successional species can promote contaminant removal on abandoned artisanal mine sites. They however vary in their capacity to accumulate different metals on abandoned soils. Within the context of illegal ASM prevalence, our results suggest that efforts should be made to encourage and minimize disturbances to the growth of early succession plant species as short-term alternative measures in abandoned artisanal mined sites, where reclamation is often neglected.

Effect of plant-soil system on the restoration of community stability after wildfire in the northeast margin of Qinghai-Tibet plateau

Wildfires, as an environmental filter, are pivotal ecological disturbances that reshape plant communities and soil dynamics, playing a crucial role in regulating biogeographic patterns and ecosystem services. In this study, we aim to explore the effects of wildfires on forest ecosystems, specifically focusing on the plant-soil feedback mechanisms within the northeastern margin of the Qinghai-Tibet Plateau (QTP). Utilizing Partial Least Squares Path Modeling (PLS-PM), we investigated the interrelationships among soil physicochemical properties, enzyme activities, species diversity, and community stability at varying post-fire recovery stages (5, 15, and 23 years). Results indicated that in the early recovery stages, rapid changes in soil properties such as decreased pH (p < 0.001) and increased nutrient availability facilitate the emergence of early successional species with high resource utilization traits. As the ecosystem evolved toward a climax community, the soil and vegetation exhibit increased stability. Furthermore, soil enzyme activities displayed dynamic patterns that corresponded with changes in soil nutrient content, directly influencing the regeneration and diversity of plant communities. Importantly, our study documented a transition in the influence of soil properties on community stability from direct positive effects in initial recovery phases to negative impacts in later stages, while indirect benefits accrue through increased species diversity and enzyme activity. Vegetation composition and structure changed dynamically with recovery time during community succession. Plant nutrient absorption and accumulation affected nutrient dynamics in the soil, influencing plant regeneration, distribution, and diversity. Our results underscore the complex interactions between soil and vegetation that drive the recovery dynamics post-wildfire, highlighting the resilience of forest ecosystems to fire disturbances. This study contributes to the understanding of post-fire recovery processes and offers valuable insights for the management and restoration of fire-affected forest ecosystems.

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.

Effects of subtle variation in forest canopy openness on cache pilferage and its implications for forest regeneration.

Scatter-hoarding rodents play important roles in plant regeneration and species coexistence in many forest ecosystems. Cache pilferage, the behavior of rodents seeking or relocating seeds cached by other individuals, is ubiquitous during the scatter-hoarding process. The effects of canopy openness on cache pilferage have received considerable attention, most of which have focused on the comparison between full canopy cover and completely open areas, such as forest gaps. However, little attention has been given to whether the subtle variation in forest canopy openness affects cache pilferage, although subtle variation in light environments exists in many forests, especially tropical and subtropical forests, where the overall canopy is large and the forest window is relatively small. Here, we directly tested these questions by simulating 400 artificial caches, each containing one seed from four selected tree species, in a subtropical forest in southwestern China. The overall canopy openness of the forest was relatively small (with a mean value of 11.1%), but subtle spatial variation still existed (ranging from 5.7% to 19.5%). Overall, caches with lower canopy openness were more likely to be pilfered and removed faster, although not all species showed the same pattern. Our study highlights that subtle variation in forest canopy openness, even in a closed primary forest, has significant effects on cache pilferage by rodents, which may influence the following seed germination and forest regeneration processes. Additionally, seedling species composition may further be altered because the canopy effects on cache pilferage are species-specific.

Elimination of cherry virus A from Prunus domestica ‘Besztercei Bt. 2’ using in vitro techniques

Cherry virus A, a capillovirus, can infect different Prunus species and be present as a latent infection in orchards. CVA infection was detected in a Hungarian stock collection of Prunus domestica ‘Besztercei Bt. 2’. In our study, different in vitro virus elimination techniques (thermotherapy combined with shoot tip culture and chemotherapy alone or combined with thermotherapy) were used and compared for their efficiency in eliminating CVA from ‘Besztercei Bt. 2’ plum cultivar. Thermotherapy was carried out at 38/36°C (day/night) in a heat chamber followed by the excision of 1–2 mm long shoot tips for plant regeneration. As a chemotherapy agent, ribavirin or zidovudine was added to the multiplication medium at two different concentrations with or without two weeks of thermotherapy pretreatment. The plum shoots tolerated 14–18 days of heat treatment, 64% of the plants regenerated from shoot tips, and 75% of the tested regenerated plants were confirmed by RT‒PCR to be CVA-free. Ribavirin and zidovudine did not negatively affect the survival of the plum shoots at any applied concentrations. Zidovudine alone was not able to efficiently eradicate virus from the treated plantlets, but when zidovudine was combined with heat treatment, the number of CVA-positive plants decreased to 60%. In contrast, ribavirin alone was very efficient at eliminating CVA from all the tested plants when used at a concentration of 25 mg L− 1. To our knowledge, this is the first report of eliminating CVA from Prunus sp. via in vitro techniques.

Optimization process for de-novo organogenesis and regeneration of transgenic papaya plants using leaf tissue as explants

Optimization process for de-novo organogenesis and regeneration of transgenic papaya plants using leaf tissue as explants

Life cycle assessment to evaluate the integral water cycle in industrial supply: A real case study

Wastewater recycling technologies are developed in areas where the necessity of water resources cannot be satisfied by natural sources. Nevertheless, nowadays trends and European Union Plans show an increasing interest on using these technologies to reduce environmental impacts. This manuscript aims to address the question of the real environmental results of using these technologies and the differences between each specific case using the Life Cycle Assessment (LCA) methodology. A real case study is analyzed to answer this question: the integral water cycle of a northern of Spain, comparing a traditional water supply system (system I), and an alternative wastewater regeneration plant (system II). System II presents a higher impact for all categories (between 1.2 and 37 times higher), except for land use, where it is reduced by 53 %. These results show a larger impact produced by the alternative system due to higher energy and chemical product consumption. Energy consumption is the main factor causing the highest impact in most of the impact categories for both studied systems, including the one associated to the water resource consumption. It accounts for at least 50 % of the total impact for each system in 7 of the 16 evaluated impact categories. In terms of climate change, energy consumption is not particularly significant in system I, but it is for system II, where it represents around 50 % of that impact. In the categories where the impact is not determined by energy consumption, chemical product consumption and waste and discharge treatment are the most relevant factors. In this sense, this paper highlights the importance of analysing each case specifically and underscores the usefulness of using LCA methodology as a tool to improve decision-making in resource management, with water resources emerging as a crucial focal point.

Establishment of plant regeneration protocol through callus induction of sugarcane (Saccharum officinarum l.) var. China using leaf-sheath

Establishment of a plant regeneration system through direct organogenesis or via callus is also a prerequisite to further in vitro genetic manipulation of the cultivar through somaclonal variation, in vitro mutagenesis and genetic transformation. Production of sufficient numbers of plants of a unique genotype is possible using in vitro culture system. In this study, the effect of various concentrations and combinations of plant growth regulators for in vitro regeneration of sugarcane via callus was described using leaf-sheath as explants. Thus, a reproducible protocol for induction of callus and regeneration into plantlets were developed in sugarcane var. china using leaf-sheath as explants. Potential callus induction was observed on MS medium supplemented with 4.5 mg/l 2,4-D, in which 80% explants induced callus after culture initiation of 90 days. Shoot formation from the callus was optimum on MS + 1.0 mg/l GA3 + 0.5 mg/l Kin., in which 70% of the cultured calli formed shoots. The average number of shoot formed per culture callus was 27.0 ± 2.50 and the average shoot length of 6.5 ± 0.50 cm were achieved in this medium after culture of 60 days. Regenerated shoots rooted well when they were transferred into half strength of MS + 2.50 mg/l NAA, in which 90% shoots rooted within 30 days of culture. The average number of root produced per shoot was 18.0 ± 2.25 and the average root length of 7.50 ± 1.20 cm were observed in this medium. Ninety percent of the in vitro raised plantlets were survived in the natural environment.

Different phosphorus preferences among arbuscular and ectomycorrhizal trees in a subtropical forest

Phosphorus (P) is one of the most limiting elements for efficient plant growth and regeneration, coexisting plants are found to be able to partition soil P components through different types of mycorrhizal symbiosis. However, previous evidences were mostly based on artificial addition of different forms of P chemicals, there is still a lack of observational and experimental verifications on how tree species respond to soil P heterogeneity in natural soils. Herein, we conducted a field survey to investigate how spatial distribution of tree species with different types of mycorrhizal associations are related with soil P compositions. We found that arbuscular mycorrhizal (AM) trees tend to distribute in soils rich in inorganic P, while ectomycorrhizal (ECM) trees distribute in habitats with low inorganic P. We further established a shade-house experiment to evaluate how P availability of field soils affects seedling performance of AM and ECM trees. Biomass of AM seedlings was highest when grew in soil with the medium level of inorganic P, while ECM seedlings grown best in soil with the lowest inorganic P level. Seedling survival and mycorrhizal colonisation rates decreased, while specific root length and specific area of seedling roots increased, with increasing inorganic P concentrations. Using field soil with natural composition of P-forms and spatial heterogeneity, our findings confirmed the hypothesis that AM trees prefer inorganic P while ECM trees are able to utilise organic P in a more realistic setting, which regulated spatial distributions and promoted coexistence of subtropical tree species at local scale.

Exploring somatic embryogenesis in Ajuga multiflora Bunge: Profiling lipophilic metabolites via HPLC, GC-FID, and GCMS– analysis

The present study aims to assess the effect of different concentrations of auxins and combinations of plant growth regulators (PGRs), specifically auxin and cytokinin, on somatic embryogenesis in Ajuga multiflora. We also aim to compare the lipophilic metabolite profiles, such as carotenoids (CAs), fatty acids (FAs), phytosterols (PSs), and tocopherols (TPs), in globular somatic embryos (SEs), germinated SEs, and shoots of SEs derived plantlets. This assessment is carried out utilizing high performance liquid chromatography, gas chromatography-flame-ionization detection, and gas chromatography–mass spectrometry techniques. The results reveal that the application of 2,4-dichlorophenoxyacetic acid (2,4-D) had the most significant impact on somatic embryogenesis, inducing both SE initiation (34.4%) and the number of SEs (3.5) per explant. Among the explants used, leaf explants were found to be the most effective for SE induction (SEI). Additionally, the study explores the combined effect of 2,4-D and cytokinins (N6-benzyladenine, N6-BA, or 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea, TDZ) on somatic embryogenesis, highlighting optimal conditions for SEI percentages and the number of SEs per explant. The maximum SE induction rate (100%) and the number of SEs (45.9) per leaf explant were obtained on an SEI medium with 30 µM of 2,4-D and 2.3 µM of TDZ. The maturation and conversion of SEs were also examined, emphasizing the role of GA3 in achieving the highest conversion percentage (94.0 ± 2.7 at 0.8 µM GA3). Furthermore, the study details the biochemical composition during various stages of somatic embryogenesis, including CAs, TPs, PSs, and FAs. Globular SEs displayed comparatively lower concentrations of CAs than their germinated counterparts and shoots from SE-derived plantlets, while the total PSs content remained relatively consistent across all stages. Conversely, fatty acids tended to accumulate more in globular SEs, with the exception of α-Linolenic acid. These findings provide valuable insights into the optimization of somatic embryogenesis protocols in A. multiflora, with implications for plant tissue culture, regeneration and lipophilic metabolites production.

Somatic embryogenesis and plant regeneration from suspension cultures of Sapindus trifoliatus

Somatic embryogenesis and plant regeneration from suspension cultures of Sapindus trifoliatus

Genome editing of a recalcitrant wine grape genotype by lipofectamine-mediated delivery of CRISPR/Cas9 ribonucleoproteins to protoplasts.

The main bottleneck in the application of biotechnological breeding methods to woody species is due to the in vitro regeneration recalcitrance shown by several genotypes. On the other side, woody species, especially grapevine (Vitis vinifera L.), use most of the pesticides and other expensive inputs in agriculture, making the development of efficient approaches of genetic improvement absolutely urgent. Genome editing is an extremely promising technique particularly for wine grape genotypes, as it allows to modify the desired gene in a single step, preserving all the quality traits selected and appreciated in elite varieties. A genome editing and regeneration protocol for the production of transgene-free grapevine plants, exploiting the lipofectamine-mediated direct delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) to target the phytoene desaturase gene, is reported. We focused on Nebbiolo (V. vinifera), an extremely in vitro recalcitrant wine genotypeused to produce outstanding wines, such as Barolo and Barbaresco. The use of the PEG-mediated editing method available in literature and employed for highly embryogenic grapevine genotypes did not allow the proper embryo development in the recalcitrant Nebbiolo. Lipofectamines, on the contrary, did not have a negative impact on protoplast viability and plant regeneration, leading to the obtainment of fully developed edited plants after about 5 months from the transfection. Our work represents one of the first examples of lipofectamine use for delivering editing reagents in plant protoplasts. The important result achieved for the wine grape genotype breeding could be extended to other important wine grape varieties and recalcitrant woody species.

Comparative transcriptome and functional analyses provide insights into the key factors regulating shoot regeneration in highbush blueberry.

Establishing an efficient plant regeneration system is a crucial prerequisite for genetic engineering technology in plants. However, the regeneration rate exhibits considerable variability among genotypes, and the key factors underlying shoot regeneration capacity remain largely elusive. Blueberry leaf explants cultured on a medium rich in cytokinins exhibit direct shoot organogenesis without prominent callus formation, which holds promise for expediting genetic transformation while minimizing somatic mutations during culture. The objective of this study is to unravel the molecular and genetic determinants that govern cultivar-specific shoot regeneration potential in highbush blueberry (Vaccinium corymbosum L.). We conducted comparative transcriptome analysis using two highbush blueberry genotypes: 'Blue Muffin' ('BM') displaying a high regeneration rate (>80%) and 'O'Neal' ('ON') exhibiting a low regeneration rate (<10%). The findings revealed differential expression of numerous auxin-related genes; notably, 'BM' exhibited higher expression of auxin signaling genes compared to 'ON'. Among blueberry orthologs of transcription factors involved in meristem formation in Arabidopsis, expression of VcENHANCER OF SHOOT REGENERATION (VcESR), VcWUSCHEL (VcWUS), and VcCUP-SHAPED COTYLEDON 2.1 were significantly higher in 'BM' relative to 'ON'. Exogenous application of auxin promoted regeneration, as well as VcESR and VcWUS expression, whereas inhibition of auxin biosynthesis yielded the opposite effects. Overexpression of VcESR in 'BM' promoted shoot regeneration under phytohormone-free conditions by activating the expression of cytokinin- and auxin-related genes. These findings provide new insights into the molecular mechanisms underlying blueberry regeneration and have practical implications for enhancing plant regeneration and transformation techniques.

Development of indirect somatic embryogenesis and plant regeneration system with immature embryos of the cultivated traditional Chinese medicinal herb Polygonatum cyrtonema

The dried rhizome of Polygonatum cyrtonema represents a highly valued traditional Chinese medicine. With a growing demand, the imperative arises for the establishment of an efficient tissue culture system for large-scale propagation. Here, we successfully developed a tissue culture system capable of yielding P. cyrtonema seedlings through somatic embryogenesis using immature embryos as explants within a 120-day timeframe. An optimum callus induction rate was achieved through a combination of 1.0 mg/L 6-BA and 0.5 mg/L 2,4-D in MS medium, resulting in the formation of numerous highly active callus. Subsequent transfer of primary callus to a medium containing 1.0 mg/L 6-BA and 1.0 mg/L 2,4-D in MS medium for 30 days yielded induction rates of 97.78%, 89.62%, 73.33%, and 66.67% for globular, heart, torpedo, and cotyledonary embryos, respectively. The highest rate of somatic embryo normal germination (73.33%) was observed in the MS+6-BA 2.0 mg/L+NAA 1.0 mg/L medium. Subsequent transfer of germinated somatic embryos to MS+IAA 0.5 mg/L+IBA 1.0 mg/L medium for 20 days resulted in robust tissue culture seedlings, with a survival rate of 93.33% when transplanted into a seedling substrate consisting of soil: perlite (3:1, v/v). This study not only marks the inaugural instance of somatic embryogenesis in Polygonatum species but also paves a foundation for the advent of breeding traditional Chinese medicinal herb P. cyrtonema via recent advanced biotechnological strategies.

A Walk on the Wild Side: Genome Editing of Tuber-Bearing Solanum bulbocastanum.

Solanum bulbocastanum is a wild diploid tuber-bearing plant. We here demonstrate transgene-free genome editing of S. bulbocastanum protoplasts and regeneration of gene-edited plants. We use ribonucleoproteins, consisting of Cas9 and sgRNA, assembled in vitro, to target a gene belonging to the nitrate and peptide transporter family. Four different sgRNAs were designed and we observed efficiency in gene-editing in the protoplast pool between 8.5% and 12.4%. Twenty-one plants were re-generated from microcalli developed from individual protoplasts. In three of the plants we found that the target gene had been edited. Two of the edited plants had deletion mutations introduced into both alleles, whereas one only had a mutation in one of the alleles. Our work demonstrates that protocols for the transformation of Solanum tuberosum can be optimized to be applied to a wild Solanum species.

N6-benzyladenine (BAP)-Based Seed Preconditioning Enhances the Shoot Regeneration of Seedling-Derived Explants for Subsequent Indirect Gene Transfer in Soybeans (Glycine max [L.] Merrill.)

This study evaluated the effects of N6-benzyladenine (BAP) seed preconditioning and seedling-derived explants on in vitro plant regeneration potential in soybeans (Glycine max [L.] Merrill.). The findings showed that seed preconditioning with 2.55 mg/L BAP prior to germination significantly influenced seedling establishment and the development of shoots, shoot elongation, and rooting on MS media supplemented with BAP and TDZ, compared to the negative (MS-NC) and positive (MS-NP) controls. The results also showed significant differences based on the genotypes, with Dundee recording 91.0% germination over a minimum of 5 days, compared to 74.2% with Peking, followed by 87.5% and 80.0% overall shoot induction frequency in these genotypes, respectively. Regenerated shoots were successfully elongated on MS medium supplemented with 0.5 mg/L BAP plus 0.6 mg/L GA3 and rooted on hormone-free medium, for 3‒4 weeks, and then hardened in the acclimatization growth room under elevated light levels. Overall, this study revealed that BAP preconditioning of seeds enhances the frequency of bud initiation and shoot proliferation, mostly in whole-seedling and cotyledonary node explants subcultured on MS-E and MS-A media supplemented with BAP in combination with TDZ.

Autonomous differentiation of transgenic cells requiring no external hormone application: the endogenous gene expression and phytohormone behaviors.

The ectopic overexpression of developmental regulator (DR) genes has been reported to improve the transformation in recalcitrant plant species because of the promotion of cellular differentiation during cell culture processes. In other words, the external plant growth regulator (PGR) application during the tissue and cell culture process is still required in cases utilizing DR genes for plant regeneration. Here, the effect of Arabidopsis BABY BOOM (BBM) and WUSCHEL (WUS) on the differentiation of tobacco transgenic cells was examined. We found that the SRDX fusion to WUS, when co-expressed with the BBM-VP16 fusion gene, significantly influenced the induction of autonomous differentiation under PGR-free culture conditions, with similar effects in some other plant species. Furthermore, to understand the endogenous background underlying cell differentiation toward regeneration, phytohormone and RNA-seq analyses were performed using tobacco leaf explants in which transgenic cells were autonomously differentiating. The levels of active auxins, cytokinins, abscisic acid, and inactive gibberellins increased as cell differentiation proceeded toward organogenesis. Gene Ontology terms related to phytohormones and organogenesis were identified as differentially expressed genes, in addition to those related to polysaccharide and nitrate metabolism. The qRT-PCR four selected genes as DEGs supported the RNA-seq data. This differentiation induction system and the reported phytohormone and transcript profiles provide a foundation for the development of PGR-free tissue cultures of various plant species, facilitating future biotechnological breeding.