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Yam (Dioscorea species) has long been cultivated in Ethiopia, where farmers rely on conventional propagation using whole tuber seeds. This leads to a scarcity of clean tuber seeds, which limits production. This study aimed to optimize in vitro propagation and micro-tuberization protocols for the ‘Hatiye’ landrace, which focused on in vitro propagation, including shoot initiation, multiplication, and rooting, and micro-tuberization using MS medium supplemented with different concentrations of plant growth regulators. Experiments were arranged in a completely randomized design with three replications, using nodal segments as explants. For shoot initiation, four levels of α-naphthalene acetic acid (NAA) were combined with four levels of 6-benzylaminopurine (BAP). The highest shoot initiation (99.9%) and shortest initiation period (6.67 days) were obtained with 0.25 mg/L NAA and 1.0 mg/L BAP. During multiplication, seven MS medium treatments with different BAP levels were tested, while rooting was assessed with six NAA concentrations. The highest rooting percentage (99.9%), root number (9.67), and root length (9.5 cm) were recorded at 2 mg/L NAA. For microtuberization, MS medium with two BAP levels (0 and 2 mg/L) combined with five sucrose concentrations was tested under 10/14 light/dark hours. The highest number of microtubers (2.5) and longest microtuber lengths (1.67 mm and 1.23 mm) were achieved with 2 mg/L BAP and 60 g/L sucrose. This study provides a reliable protocol for in vitro propagation and micro-tuberization of the Hatiye yam landrace, addressing the shortage of clean tuber seeds and supporting yam production in Ethiopia.

Multiple-stress is defined as the simultaneous or sequential exposure of plants to multiple abiotic constraints, which triggers regulatory programs that differ fundamentally from single-stress responses. In potato ( L.), drought, salinity, heat, and cold severely impair tuber development, yet the molecular architecture underlying resilience to combined stress remains unclear. We hypothesized that multi-stress conditions activate an integrated regulatory network linking tuber induction with stress-responsive metabolic and redox pathways. RNA-seq profiling of microtuberization under combined osmotic, salinity, heat, and cold stress was performed. Differential expression analysis identified shared differentially expressed genes (DEGs). A subset of upregulated genes was used for protein-protein interaction (PPI) network construction. Comparative regulatory analyses were performed, and selected genes were validated by qPCR. Statistical analyses were conducted to assess differential expression and network enrichment. A total of 2,046 shared DEGs were identified, including 1,212 upregulated and 834 downregulated genes. A PPI network constructed from 1,475 unique upregulated genes revealed 317 highly interconnected components. Network analysis identified the StSP6A-FD tuberigen complex as a central regulatory hub integrating developmental signaling with phenylpropanoid metabolism, oxylipin biosynthesis, and redox regulation. Multiple components were associated with hydrogen sulfide (H₂S) signaling, suggesting redox-gasotransmitter integration. Comparative regulatory analysis revealed conservation of the ERF-NAC-MYB-bZIP transcription factor framework, along with expansion of stress-responsive modules. Collectively, these findings establish a mechanistic framework linking tuber induction with adaptive metabolic remodeling under multi-stress conditions.

Potato (Solanum tuberosum L.), belonging to Solanaceae family, globally ranks as the fourth-largest food (vegetable) crop. In order to sustain the supply of quality propagules, a large-scale in vitro shoot, microtuber and root development protocol was successfully established for potato cv. Badami alu, for the first time. Initially, the tuber sprout explants were inoculated in Murashige and Skoog (MS) basal medium individually with 0.5–1.5 mg/l 6-benzyladenine, kinetin, meta-topolin (mT) and zeatin (Zea) for regeneration. The earliest (~ 3 days) shoot initiation with maximum number (~ 9) and length (81 mm) shoots were recorded in MS medium fortified with 1 mg/l mT after 4 weeks of growth period. The earliest (44 days) microtuber formation with maximum number (4.3) was recorded in MS medium fortified with 1 mg/l Zea after 8 weeks growth stage. On the other hand, the hierarchical clustering heat map, network plot, and principal component analyses illustrated the association among cytokinins (sources and concentrations) and the different growth attributes under study. In vitro-regenerated shootlets were then cultured in MS medium individually with 1–3 mg/l indole-3-acetic acid (IAA) and indole-3-butyric acid. The earliest (~ 7 days) root induction was recorded in MS medium with 1 mg/l IAA, whereas the maximum number (47) with most elongated (28.3 mm) roots were recorded in 2 mg/l IAA after 4 weeks culture period. The micropropagated plantlets were grown in cocopeat (for 1 month) followed by soil (for 2 months) under field conditions (shade net house) that resulted in 95% survival rate. Stability of ploidy level of micropropagated and successively field-grown plantlets with their mother plant was affirmed by flow cytometry, likewise, genetic fidelity was assessed for the same via inter simple sequence repeats, directed amplification of minisatellite-region DNA, start codon targeted polymorphism, and conserved DNA-derived polymorphism markers, and a 100% monomorphism was found, proving that there was no genetic variation among them. Since there is no report of non-conventional mass propagation of this cultivar to date, the present protocol will be immensely helpful for its accelerated large-scale propagation and its genetic improvement.Key messageFirst report on usage of meta-Topolin and zeatin for micropropagation and microtuberization followed by acclimatization and field performance of true-to-type propagules (ensured by flow cytometry and molecular markers) in potato.

This work investigates the interactive effect of six culture media and three photoperiods (darkness, 16h/8h, 8h/16h) on the microtuberization of four potato varieties (Spunta, Désirée, Kondor and Bartina). The objective is to determine the best tuberization under these growing conditions. The measured parameters which are related to the suitability of the tuberization characteristics are: the morphological aspects (shape, position) and the biometric ones (number and diameter of tubers). The obtained results permit to valorize the meristems that constitute the starting explant, and their good organogenetic skills to provide the first generation of micropropagation with healthy vitroplants in a sufficient quantity. Thus, the grown vitroplants on the medium (MS/2+BAP+COU) presented the best values which are related to the percentage of tuberization, the number of microtubercles / vitroplants and weight of tubers. Moreover, the Bartina genotype showed a remarkable superiority over its media and under a 16h/8h photoperiod except for the diameter of the tubers, where this genotype had the best diameter over the medium (MS/2+KIN) under an 8h photoperiod.

Wild Solanum species have contributed many introgressed genes during domestication into current cultivated potatoes, enhancing their biotic and abiotic stress resistance and facilitating global expansion. Abiotic stress negatively impacts potato physiology and productivity. Understanding the molecular mechanisms regulating tuber development may help solve this global problem. We made a transcriptomic analysis of potato microtuberization under darkness, cytokinins, and osmotic stress conditions. A protein–protein interaction (PPI) network analysis identified 404 genes with high confidence. These genes were involved in important processes like oxidative stress, carbon metabolism, sterol biosynthesis, starch and sucrose metabolism, fatty acid biosynthesis, and nucleosome assembly. From this network, we selected nine ancestral genes along with eight additional stress-related genes. We used qPCR to analyze the expression of the selected genes under osmotic, heat–osmotic, cold–osmotic, salt–osmotic, and combined-stress conditions. The principal component analysis (PCA) revealed that 60.61% of the genes analyzed were associated with osmotic, cold–osmotic, and heat–osmotic stress. Seven out of ten introgression/domestication genes showed the highest variance in the analysis. The genes H3.2 and GAPCP1 were involved in osmotic, cold–osmotic, and heat–osmotic stress. Under combined-all stress, TPI and RPL4 were significant, while in salt–osmotic stress conditions, ENO1, HSP70-8, and PER were significant. This indicates the importance of ancestral genes for potato survival during evolution. The targeted manipulation of these genes could improve combined-stress tolerance in potatoes, providing a genetic basis for enhancing crop resilience.

Poly(ADP-ribose) polymerases (PARP) are involved in the regulation of plant growth and development, especially under stress conditions. These enzymes post-translationally modify target proteins by adding ADP-ribose polymers. This study presents the effect of PARP chemical inhibitor 3-methoxybenzamide (3 MB) on in vitro plant growth, microtuber formation, and Agrbacterium-mediated transformation of blue potato (Solanum tuberosum L. subsp. andigenum). The addition of 3 MB (0.2 to 0.6 mM) significantly increased the growth and microtuber formation of in vitro propagated plants. The highest gain in plant fresh weight, root mass, and microtuberization was observed in clonal propagation medium with 0.4 mM 3 MB. Transformation frequency of blue potato internodes increased to 43%, 2.7-fold over the non-treated control (16%), when 0.2 mM 3 MB was included in the culture medium during the whole process. The addition of 3 MB during Agrobacterium transformation did not affect transgene copy number in regenerated plants. Southern blot analyses and histochemical staining for ß-glucuronidase activity confirmed the presence and expression, respectively, of the uidA transgene in plant tissues. From these results, it can be concluded that the inhibition of PARP activity can increase biomass production and transformation rates for genetic improvement of blue potato.

Purple-fleshed potatoes (PFP) are varieties of Solanum tuberosum L., which recently have been recorded to be more and more cultivated and consumed in all European countries, including Romania, as they are promoted for their content in bioactive compounds and benefits to human health. This paper presents a micropropagation protocol study for PFP varieties already traded into the Romanian market, namely Blue Danube (BD), Salad Blue (SB), Violet Negretin (VN), and Violet Queen (VQ). These varieties were tested for in vitro micropropagation also considering asepsis, initiation, callus formation, and microtuberization. To establish the optimum asepsis treatment, a preliminary experiment was performed and, the best results were obtained by using 70% EtOH (1 min) followed by 20% (v/v) Domestos® (20 min). The MS formula (Murashige and Skoog 1962) was tested as the basic culture medium without growth regulators for all tested stages except for callus initiation and its further multiplication stages. The effect of glycine on direct organogenesis and shoot multiplication was evaluated for propagated micro-cuttings. We emphasize that the addition of glycine at a concentration of 15 mg/L to the culture medium induced a better plantlet vigor for all four varieties. Regarding the indirect organogenesis, culture medium supplemented with NAA (5.00 mg/L), GA3 (1.00 mg/L), TDZ (1.00 mg/L) and glycine (15.00 mg/L) induced the best results for shoot cluster regeneration as well as turning of white callus from control to purple callus. Further, the microtuberization was successfully produced when sucrose was supplemented at 8% (w/v) into the culture medium. Among all four tested PFP varieties, SB has proven to give the best results regarding the adaptability for in vitro cultivation.

Ceropegia media is an endemic and endangered plant as its propagation through seeds is unreliable due to low germination, slow growth and seedling decay under natural conditions. Also, tubers of this plant are edible serving as carbohydrate source with medicinal values leading to severe population decline in the natural habitat. To provide a sustainable solution, an efficient in vitro propagation protocol along with phytochemical profiling was developed for C. media. Callus cultures were induced from seedling and wild leaf tissues using the most effective Murashige and Skoog’s (MS) medium with 2,4-dichlorophenoxyacetic acid (2,4-D; 2 µM) and sucrose (3%). Somatic embryos were acquired on MS medium with 1 µM 6-Benzylaminopurine (BAP) and 1 µM 2,4-D. Conversion into plantlets was attained only from tissue culture-derived seedling leaf (TCDSL) explant. Further, in vitro tuberization was achieved from TCDSL callus with BAP and Naphthalene acetic acid (NAA). AgNO3 as an elicitor had a positive effect on both fresh and dry weights of callus. Successful acclimatization (58%) was attained after two months resulting in normal phenotype in pots. Further, metabolite profiles of ten different tissues from wild and in vitro plants were compared. Total 82 compounds comprising alkaloids, fatty acids, fatty acid ester, steroids, terpenes and hydrocarbons were identified. Overall, results suggested enhanced production of selected metabolites with in vitro propagation and AgNO3, alleviating the problem of unavailability of planting materials. Thus, the current study might offer potential ways for the conservation of such RED enlisted species as C. media.Graphic Abstract

Potato microtuber productions through in vitro techniques are ideal propagules for producing high quality seed potatoes. Microtuber development is influenced by several factors, i.e., high content sucrose and cytokinins are among them. To understand a molecular mechanism of microtuberization using osmotic stress and cytokinin signaling will help us to elucidate this process. We demonstrate in this work a rapid and efficient protocol for microtuber development and gene expression analysis. Medium with high content of sucrose and gelrite supplemented with 2iP as cytokinin under darkness condition produced the higher quantity and quality of microtubers. Gene expression analysis of genes involved in the two-component signaling system (StHK1), cytokinin signaling, (StHK3, StHP4, StRR1) homeodomains (WUSCHEL, POTH1, BEL5), auxin signaling, ARF5, carbon metabolism (TPI, TIM), protein synthesis, NAC5 and a morphogenetic regulator of tuberization (POTH15) was performed by qPCR real time. Differential gene expression was observed during microtuber development. Gene regulation of two component and cytokinin signaling is taking place during this developmental process, yielding more microtubers. Further analysis of each component is required to elucidate it.

Potatoes are a cool season crop. Yet, with growth in the world population and rising temperatures, more heat tolerant cultivars are going to be needed. The objectives of this work were to identify heat tolerance in diploid wild Solanum species, as measured by the ability to form microtubers in tissue culture, and to compare four different methods of evaluating heat tolerance. In each of five experiments conducted over time, four nodal cuttings from up to 20 genotypes from each of ten diploid wild potato species were grown in MS medium supplemented with 8% sucrose and cultured at 19 °C (cool) and 25 °C (warm) in the dark. Four methods were used to evaluate heat tolerance: percent microtuberization under warm conditions, difference in percent microtuberization between cool and warm conditions, microtuber yield under warm conditions, and the difference in microtuber yield between cool and warm conditions. Species were ranked for each of these four methods (with rank = 1 being most heat tolerant) and ranks were summed across the four methods to identify the most heat tolerant. There were significant differences among species and the species x temperature interaction was significant for percent microtuberization and microtuber yield. Percent microtuberization in five of the species was similar under cool and warm temperatures; greater under cool temperatures for three species; and, greater under warm temperatures for two species. Microtuber yield in four of the species was similar under cool and warm temperatures; greater under cool temperatures for six species; and, greater under warm temperatures for one species. Rankings of heat tolerance among the species varied by the method used, but S. kurtzianum and S. sogarandinum were the most heat tolerant.