Acclimatization of In Vitro Potato Plantlets: A Systematic Review of Media Formulation, Light Quality, and Bio-Priming Strategies

Abstract

The production of pre-basic (G0) seed tubers underpins the certified potato value chain. However, the transition from in vitro laboratory conditions to the ex vitro greenhouse environment remains a persistent production constraint, with reported mortality rates of 50–70%. This systematic review, conducted in accordance with PRISMA 2020 guidelines, synthesizes data from 63 selected studies (spanning 2010–2026) to propose a conceptual “Physiological Competence Framework”. We introduce a conceptual hypothesis termed the “Nitrogen Paradox”, which suggests that excessive ammonium influx may inhibit lignin biosynthesis, explaining the structural vulnerability of the vitrotype. Our analysis proposes three pillars for acclimatization success: (1) Nutritional hardening and exogenous PGR modulation, characterized by reduced nitrogen and sucrose levels to mitigate hyperhydricity; (2) photo-autotrophic induction, where optimized LED spectra replace conventional lighting to stimulate stomatal functionality; and (3) rhizosphere engineering, utilizing bio-priming with Plant Growth-Promoting Rhizobacteria (PGPR) to create a biotic shield against transplant shock. Furthermore, we examine emerging evidence for nanoparticle-based stress priming (AgNPs, ZnNPs). The evidence supports replacing high-nitrogen multiplication media with reduced-nitrogen formulations, replacing fluorescent lamps with balanced Red–Blue LED spectra, and incorporating PGPR bio-priming before transplant.

1. Introduction

Globally, potato (Solanum tuberosum L.) is the third most important food crop for human consumption [1], with global production exceeding 370 million tonnes annually [1,2]. However, closing yield gaps in developing regions remains heavily dependent on the availability of high-quality certified seed tubers [2]. The commercial efficiency of the seed potato value chain is severely bottlenecked at the acclimatization stage, the transition from axenic, heterotrophic conditions to autotrophic greenhouse environments. Industry reviews indicate that plantlets experience mortality rates often ranging between 50% and 70% during this interface, representing a direct dissipation of the energy, labor, and reagents invested during the multiplication phase [3,4]. Established agronomic principles suggest that these inefficiencies account for a substantial portion of the final production cost of a minituber, making “survival” a critical variable for profitability [5].

The primary driver of acclimatization failure is the development of an aberrant anatomical phenotype known as the “Vitrotype” (referring to abnormal phenotypic structures produced through in vitro culture). Plantlets cultured under conditions of high relative humidity (>95%), low light intensity, and abundant exogenous sucrose develop structural dysfunctions that render them incompetent for natural environments [6,7]. Clinically, the vitrotype is defined by three systemic failures: (i) Stomatal incompetence: stomata are often circular, raised, and unable to close effectively in response to water stress signals [3,7,8]; (ii) hydraulic failure: hyperhydricity caused by poor vascular continuity and waterlogging of the apoplast [8,9]; and (iii) cuticular deficiency: a lack of protective wax caused by the suppressed vapor pressure deficit inside the vessel [8,10].

These anomalies, particularly the lack of epicuticular wax and stomatal incompetence, lead to rapid desiccation upon transfer. The current literature has extensively focused on protocols aiming to maximize shoot proliferation, such as Temporary Immersion Systems (TIS) [2]. However, high multiplication rates often come at the expense of physiological hardening. This systematic review addresses that specific gap. In this study, hardening refers to the physiological pre-conditioning during the final in vitro stage, while acclimatization describes the broader process of adaptation to ex vitro conditions. Unlike previous works that prioritized “yield” (number of nodes), this analysis focuses exclusively on the physiological mechanisms of competence. We challenge established dogmas, such as the universal use of standard Murashige & Skoog (MS) medium, and propose an integrated “Physiological Competence Framework” based on three pillars: (1) Nutritional Hardening, focusing on nitrogen/sucrose reduction to mitigate hyperhydricity [8]; (2) photo-autotrophic Induction, via spectral modulation (LEDs) [11]; and (3) rhizosphere Engineering, utilizing bio-priming agents (PGPR) to create a biotic shield against transplant shock [12,13,14]. By synthesizing data from 63 studies, this review aims to shift the paradigm from maximizing shoot proliferation to ensuring physiological autonomy.

Therefore, the primary objective of this systematic review is to comprehensively evaluate the physiological and technological determinants of ex vitro acclimatization, aiming to synthesize an integrated, evidence-based framework that maximizes micro-plant survival, physiological competence, and subsequent field performance.

2. Materials and Methods

This systematic review was conducted in accordance with the PRISMA 2020 guidelines (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) [15], ensuring transparency and reproducibility in the selection of scientific literature. The review protocol was not registered in PROSPERO, as this registry is primarily intended for human health outcomes; however, the methodology strictly adhered to the PRISMA checklist to ensure validity. A PRISMA 2020 checklist is provided as Supplementary Materials.

Although a quantitative meta-analysis was not performed due to environmental reporting heterogeneity, the PRISMA 2020 framework was strictly followed to ensure rigorous and transparent reporting standards for systematic reviews. Furthermore, the risk-of-bias assessment utilized an adapted SYRCLE tool; modifications included the omission of clinical blinding domains (which are inapplicable to in vitro plant tissue culture) and a concentrated focus on selection, performance, and reporting biases.

2.1. Search Strategy

A comprehensive systematic literature search was conducted on 25 March 2026, following the PRISMA 2020 guidelines. Three major electronic databases were queried: Scopus, Web of Science (Core Collection), and ScienceDirect. The search strategy was designed to capture studies evaluating the physiological transition of potato plants from in vitro conditions to ex vitro environments. The Boolean search strings were adapted to the specific syntax of each database as follows:

Scopus (TITLE-ABS-KEY): ((“Solanum tuberosum” OR “potato”) AND (“in vitro” OR “micropropagation” OR “microtuber*”) AND (“acclimatization” OR “hardening” OR “ex vitro” OR “survival”)) AND PUBYEAR > 2009.

Web of Science (TS): ((“Solanum tuberosum” OR “potato”) AND (“in vitro” OR “micropropagation” OR “microtuber*”) AND (“acclimatization” OR “hardening” OR “ex vitro” OR “survival”)).

ScienceDirect (Title, abstract or author-specified keywords): (“Solanum tuberosum” OR “potato”) AND (“in vitro” OR “micropropagation”) AND (“acclimatization” OR “hardening” OR “ex vitro”).

Filters were applied across all databases to restrict results to research articles and reviews published in English between 2010 and 2026.

2.2. Eligibility Criteria

To center the review on “physiological competence” and avoid redundancy with purely multiplicative protocols, the following filters were applied:

• Inclusion Criteria: (i) Original research articles published in indexed journals (Q1–Q3); (ii) Studies reporting quantitative data on ex vitro survival rates (%); (iii) Investigations analyzing physiological markers (chlorophyll content, fluorescence, stomatal closure) or morphological traits (root architecture, cuticle thickness); (iv) Comparative studies on culture media formulations (e.g., MS vs. WPM) and their subsequent effect in the greenhouse; (v) Studies detailing specific pre-conditioning treatments, including the types and dosages of exogenous plant growth regulators (PGRs) applied during the in vitro phase.
• Exclusion Criteria: (i) Studies focused solely on in vitro multiplication rates (number of shoots/nodes) without greenhouse follow-up; (ii) Conference abstracts, non-indexed book chapters, and unpublished theses; (iii) Studies on genetic transformation (GMOs) or virus elimination that do not specifically address acclimatization physiology.
• The systematically included studies are limited to Solanum tuberosum; however, cross-species evidence (e.g., tomato, sweet potato) is explicitly cited in the discussion to provide exploratory perspectives and future directions.
• Foundational references older than ten years were selectively included only when they represent seminal descriptions of physiological principles (e.g., classical photobiology or fundamental in vitro disorders) that remain the undisputed basis for current applied research.

2.3. Study Selection and Data Extraction

The database search yielded a total of 469 records. After removing 219 duplicates using Mendeley Reference Manager (version 2.144.0, Elsevier, Amsterdam, The Netherlands), 250 unique records were screened by title and abstract. The literature screening and data extraction processes were performed independently by two reviewers. Any discrepancies regarding study inclusion or data extraction were resolved through consensus or by consulting a third senior reviewer. In this initial phase, 165 articles were excluded primarily because they focused exclusively on in vitro multiplication protocols without addressing ex vitro acclimatization. A total of 85 reports were sought for full-text retrieval, and all 85 were successfully retrieved and assessed for eligibility. During the full-text review, 22 studies were excluded for the following reasons: (i) focus on genetic/yield without physiological variables (n = 10); (ii) use of species other than S. tuberosum (n = 7) and (iii) duplicate data or short communications (n = 5). Finally, 63 studies met all inclusion criteria and were synthesized in this review (Figure 1). The complete list of the 22 records excluded at the full-text screening stage, along with the specific reasons for their exclusion (PRISMA Item 16b), is provided in Supplementary Table S1.

It is important to note that the initial literature search and screening phase of this systematic review utilized the same broad foundational database constructed for a previous scoping review by our research group (Jácome Sarchi et al. [2]). However, while the former study mapped macroscopic bibliometric trends across the entire in vitro potato production pipeline, the current review applies distinct, highly specific inclusion criteria focusing exclusively on the physiological mechanisms of ex vitro acclimatization. Consequently, this manuscript performs a completely novel data extraction and synthesis that does not overlap with the previously published work.

2.4. Quality Assessment and Risk of Bias

To ensure the reliability of the synthesized data, the methodological quality of the included studies was assessed using a criteria-based checklist adapted from the SYRCLE’s tool for experimental trials. The assessment focused on five key parameters: (1) Randomization of explants/blocks design; (2) Control of Environmental Variables; (3) Sample Size Justification; (4) Statistical Reproducibility; and (5) Selective reporting of ex vitro survival.

Specifically, our assessment revealed that approximately 18% (11 out of 63) of the included studies demonstrated a high risk of bias. A detailed risk-of-bias assessment table for all 63 studies, specifying the criteria triggered for each high-risk classification, is provided in Supplementary Table S2. Regarding these high-risk studies, they were predominantly those with incomplete reporting of specific environmental variables. However, they were retained in the synthesis to map the breadth of the existing literature and provide qualitative context. To maintain scientific rigor, their specific quantitative outcomes were not used to define the specific threshold values of the proposed Physiological Competence Framework, but rather to support general biological trends.

2.5. Synthesis Methods and Heterogeneity

Due to the high methodological heterogeneity across the included studies (varying cultivars, LED intensities, and PGPR strains), a formal meta-analysis and sensitivity analysis were not conducted. Instead, a narrative synthesis was used to integrate the physiological mechanisms. This corrects previous entries in the PRISMA checklist; narrative synthesis was deemed most appropriate for the qualitative integration of the conceptual framework.

Table 1. Summary characteristics of the 63 included studies on potato (Solanum tuberosum L.) ex vitro acclimatization, grouped by Physiological Competence mechanism.

Reference	Potato Cultivar(s)	In Vitro Treatment	Validation Environment	Target Physiological Trait	Key Outcome/Survival Rate	Sample (n)	Quality
I. Nutritional Hardening and Media Modification (The Nitrogen Paradox & Stress)
[16]	Multiple (10+)	Modified Medium (Low N, 15–30 mM)	In vitro (Growth analysis)	Adaptive root elongation & NUE	Higher Root–Shoot ratio; early screening for nutrient efficiency	120	Low
[17]	cv. Iwa	Low Inorganic Nitrogen (3.75–7.5 mM)	In vitro (Pre-acclimatization)	Biomass partitioning & Root plasticity	Enhanced lateral root development; maintained biomass despite low N	60	Low
[18]	Solanum spp.	MS vs. Modified Basal Media	General (Review)	Oxidative stress & Hyperhydricity	Mitigation of ammonium toxicity and glassy stem appearance	N/R	Low
[19]	S. tuberosum	NH4+/NO3− mixtures (4 mM total N)	Controlled environment (Nutrient film)	Growth, biomass & mineral uptake	Mixed nitrogen (8–20% NH4+) significantly enhanced dry weight, tuber growth, and total N uptake	48	Low
[20]	cv. Russet Burbank	Supplemental Ca (168 kg·ha−1)	Greenhouse/Pots	Tuberization signaling & size	Significant reduction in tuber number; increased mean tuber weight/size	100	Low
[21]	cv. Victoria, cv. Rosetta	NaCl stress (0–200 mM) + TDZ/GA3/Kin/PBZ	In vitro (Regeneration & Microtuberization)	Osmotic adjustment (Proline) & Na+/K+ index	Victoria identified as more tolerant (survived 150 mM NaCl) vs. Rosetta (125 mM)	150	Low
[22]	Granola, Arbolona negra	AgNO3 (2 mg/L)	In vitro (Growth analysis)	Ethylene inhibition & leaf development	Highest leaf area values; mitigation of epinasty/hyperhydricity	40	Low
[23]	Cardinal, Desiree	IAA & GA3 under NaCl stress	In vitro (Salinity tolerance)	Antioxidant machinery (SOD, CAT, POD)	Amelioration of salt stress; improved biomass and protein content	45	Low
[24]	cv. Taisiya	TIS (RITA®) System	In vitro/Microtubers	Multiplication efficiency	TIS optimizes nutrient uptake and increases microtuber biomass	30	Low
[25]	cv. Zhongshu 20	Sucrose-Free Medium (S0)	In vitro (Photoautotrophic)	Photosynthetic performance & Anatomy	Improved leaf anatomy, chloroplast ultrastructure, and upregulated photosynthesis genes	60	Low
[26]	cv. Sandy	Vented Vessel + Low Sucrose (20 g/L)	In vitro (Pre-acclimatization)	Stomatal Anatomy & Chlorophyll	Transformation from spherical to elliptical stomata with narrow openings; well-developed palisade layer	45	High
[27]	cv. Desiree	Exogenous ABA (38 µM) + NaHCO3 stress	In vitro (Saline-alkali stress)	Root architecture & Ion homeostasis	Alleviated saline-alkali damage; PP2C gene suppression enhanced tolerance and antioxidant defense	90	Low
[28]	S. tuberosum	Homologous overexpression of PR10a gene	In vitro/Ex vitro (Salt stress)	Molecular signaling & Salt tolerance	Improved plant growth parameters and antioxidant defense under abiotic stress	120	Low
[29]	cv. Innovator, Desirée, Mozart	Salt stress acclimation	Ex vitro (Salt stress)	Root architecture & Suberization	Cultivar-specific salt tolerance; Innovator showed highest resilience with distinct root responses	75	Low
[30]	S. tuberosum × S. bulbocastanum (Somatic hybrids)	Drought stress phenotyping	Ex vitro (Phenotyping platform)	Photosynthetic efficiency & Drought tolerance	Identified drought-resilient hybrids with combined late blight resistance using semi-automated phenotyping	180	Low
[31]	cv. Atlantic	WPM medium + NAA (1.0 mg/L) + Zeatin riboside (5.0 mg/L)	In vitro (Organogenesis)	Shoot regeneration capacity	Internodal segments showed superior organogenic capacity compared to leaf explants for shoot induction	50	High
[32]	17 tetraploid cultivars (e.g., Z1264-1, Z1076-1)	NaCl-induced salt stress (80 mM)	In vitro (Salt stress screening)	Salt tolerance & miRNA expression	Identified highly tolerant cultivars; 68 miRNAs regulated osmotic adjustment and ROS clearance	340	Low
[33]	cv. Atlantic (Transgenic lines)	StERF79 gene overexpression and RNAi silencing	Ex vitro (Greenhouse/Pots)	Drought tolerance & Antioxidant defense	Overexpression enhanced drought tolerance, upregulated StDHN-2 gene, and improved ROS scavenging	60	Low
[34]	S. tuberosum	Combined abiotic stresses (Osmotic, Heat, Cold, Salt)	In vitro (Microtuberization)	Gene expression & microtuber yield	Identified ancestral stress genes (TPI, RPL4) whose expression dictates microtuber diameter under combined stresses	N/R	Low
[35]	S. tuberosum	80 g/L sucrose + 3.0 mg/L BAP pre-treatment	Ex vitro (Aeroponic vs. Greenhouse)	Minituber yield & biochemical quality	Aeroponic acclimatization resulted in significantly higher pathogen-free minituber yields compared to soil-based systems	120	High
[36]	cv. Arizona	MS medium + BA (1.0–2.0 mg/L) and Zeatin (0.5 mg/L)	In vitro/Ex vitro (Greenhouse)	Shoot multiplication & minituberization	2.0 mg/L BA maximized shoot multiplication (17.2 shoots/explant), while 0.5 mg/L Zeatin optimized minituber production	40	Low
[37]	Purple-fleshed cvs.	PGRs (NAA, GA3) and Glycine	In vitro (Multiplication)	Shoot multiplication rate	Optimized PGR combinations significantly enhanced the in vitro multiplication efficiency and plantlet vigor	30	High
[38]	cv. Lady Rosetta	4.0 mg/L 2,4-D + 0.5 mg/L Kinetin	In vitro (Indirect regeneration)	Callus induction & plantlet regeneration	Standardized somatic embryogenesis protocol achieving 9.78% callus induction and 48.9% plantlet regeneration	200	Low
II. Photo-autotrophic Induction via Spectral Modulation (Light & LEDs)
[39]	Multiple	Red–Blue (1:1) Light Spectrum	Controlled Env. (LEDs)	Stomatal development	Blue light is essential for functional stomatal anatomy and chloroplasts	60	Low
[40]	S. tuberosum	100% Red Light	Controlled Env. (LEDs)	Stem elongation	Phytochrome-driven elongation; Red light alone inhibits root development	45	Low
[41]	S. tuberosum	100% Blue Light	Controlled Env. (LEDs)	Stomatal signaling	Phototropin-mediated opening; results in compact, robust plantlets	45	Low
[42]	cv. Pito	90% Red + 10% Blue	Post-cryo recovery	Morphogenesis genes	RB light spectrum activates defense and recovery genes for faster regeneration	30	Low
[43]	cv. Asterix	Far-red LED light spectrum	Postharvest storage	Morphological regulation (Sprouting)	Far-red LED treatments effectively modulated and controlled tuber sprouting	150	Low
[44]	cv. Happy King	Varying LED light spectrums (Red, Blue, Green, White, Far-red combinations)	Ex vitro/Controlled Greenhouse (Plant factory)	Photosynthetic activity, biomass & tuberization	Red and blue light combined with far-red or white optimized plant biomass and tuber yield, while green light enhanced photosynthetic pigments	120	Low
[45]	S. tuberosum	Microtuberization under complete darkness	In vitro (Transcriptomic profiling)	Gene expression & microtuber development	RNAseq analysis unveiled key gene expression networks regulating early microtuber development in the absence of light	100	Low
[46]	cv. Shepody	Monochromatic lights (Blue, Green, Yellow, Red, White)	In vitro (Microtuberization)	Microtuber formation, heterotrophy & StSUT gene expression	Blue, Green, and White lights induced rapid microtuberization; Blue light enhanced heterotrophic growth, root activity, and StSUT1 expression	60	Low
III. Rhizosphere Engineering and Stress Priming (PGPRs, Substrates & Nanotechnology)
[47]	cv. Talent	Amorphous silica (ASi) soil amendment (0.5–1.0%)	Field experiment	Silica accumulation & yield performance	Low Si accumulation; benefits ascribed to soil physicochemical changes (P and water availability)	40	Low
[48]	cv. Atlantic	Salicylic acid (1 micromol/L)	In vitro (Physiological recovery)	ROS scavenging & Polyamines	Mitigation of BA-induced hyperhydricity; increased SOD, CAT, and GR	60	Low
[49]	cv. María Bonita	Trichoderma asperellum + Compost/Coir substrates	Ex vitro (Greenhouse)	Stem thickness & Minituber yield	Trichoderma increased stem thickness; optimized physical substrate properties maximized minituber production	80	Low
[50]	Kufri Pukhraj, Kufri Himalini	Microplants in Soilless media (Cocopeat) vs. Soil	Ex vitro (Net house)	Harvest Index & Minituber yield	Soilless media increased Harvest Index (85–86%) and improved proportion of seed-size tubers (>3 g)	160	Low
[51]	cv. Robijn, Eersteling, Karnico	Pseudomonas putida (Strain P9) root dip	Ex vitro (Greenhouse)	Pathogen suppression & Community shift	Reduced P. infestans lesions by 45% (cv. Robijn); robust endophytic colonization	48	Low
[52]	cv. Bashkirsky	Bacillus subtilis (10-4, 26D) + Salicylic Acid	Postharvest storage	Oxidative stress & Disease resistance	Mitigated postharvest P. infestans and F. oxysporum; decreased MDA and proline levels	60	Low
[53]	cv. Nevsky	A. baldaniorum Sp245 + O. cytisi IPA7.2	Aeroponics (ex vitro)	Antioxidant machinery & Stomatal adaptation	Decreased MDA/H2O2; enhanced stomatal functionality and increased minituber yield by 11%	45	Low
[54]	cv. Lady Rosetta	Trichoderma viride AT85 (Foliar application)	Ex vitro (Greenhouse)	Pathogen suppression & Defense gene regulation	Reduced Alternaria solani severity by 93%; decreased proline/MDA; upregulated LOX1 and PR1a genes	120	Low
[55]	S. tuberosum	Pre-planting inoculation with Bacillus subtilis 10-4	Ex vitro (Hydroponics/Yield phase)	Tuber yield & Phytonutrient quality	Increased minituber yield and improved tuber quality/nutrition	200	Low
[56]	S. tuberosum	Nanoparticle biostimulants under salinity stress	Ex vitro/Greenhouse (Salinity stress)	Morphological and molecular responses	Alleviated salinity-induced stress and improved physiological traits	90	Low
[57]	S. tuberosum	Salicylic acid (50 µM)/Proline/UV-C light	In vitro (Pre-acclimatization)	Induced disease resistance	Salicylic acid in MS medium prevented soft rot infection symptoms in 21% of plants	60	High
[58]	S. tuberosum	Demethoxycurcumin-loaded Chitosan Nanoparticles	Ex vitro (Pathogen assay)	Biocontrol & Pathogen suppression	Effective management and controlled release against Phytophthora infestans (Late blight)	100	Low
[59]	S. tuberosum	Plant essential oils (Eucalyptus & Peppermint)	In vitro/In planta	Pathogen suppression (R. solanacearum)	Essential oils induced bacterial cell wall lysis and significantly impeded potato bacterial wilt	40	High
[60]	S. tuberosum	Solid Lipid Nanoparticles (SLNs) loaded with plant extracts	Ex vitro (Greenhouse)	Photosynthetic response & Biocontrol	SLNs mitigated Rhizoctonia solani infection and protected the photosynthetic machinery of the plants	30	High
[61]	S. tuberosum	Zinc Nanoparticles (ZnNPs, 40 mg/kg) + 6% Sucrose	In vitro (Microtuberization)	Microtuber yield & Development	ZnNPs and optimized sucrose maximized the number and average weight of microtubers	120	Low
[62]	S. tuberosum	Microbial consortia (Azospirillum, Bacillus, Pseudomonas) + 50% NPK	Ex vitro (Field scale)	Rhizosphere microbiome diversity & functionality	Enhanced microbial diversity and functionality; sustainable alternative to mineral fertilization	450	Low
[63]	cv. Nevsky	Azospirillum baldaniorum + Ochrobactrum cytisi under osmotic stress	In vitro (Osmotic stress)	Pro- and antioxidant systems	Regulated antioxidant enzyme activity and mitigated osmotic stress damage	60	Low
[64]	cv. Citlali	Bacillus sp. Strain Fo03 (Phosphate solubilizing) + 50% NPK	Ex vitro (Greenhouse)	Growth promotion & Tuber sprouting	Promoted plant growth and sprouting, allowing a 50% reduction in inorganic fertilizer	90	High
[65]	S. tuberosum	Paclobutrazol (PAC) and Silver Nanoparticles (AgNPs)	In vitro (Slow-growth conservation & regeneration)	Plant growth, stomatal density & regeneration capacity	2 mg/L PAC + 50 mg/L AgNPs optimized in vitro conservation, while 1–2 mg/L PAC + 50 mg/L AgNPs maximized shoot regeneration	120	High
[66]	cv. Lady Rosetta, Spunta	Potassium (25–45 mM) and Phosphorus (2–4 mM) nanoparticles	In vitro (Microtuberization)	Microtuber yield, plant & root length	25 mM K-NPs and 4 mM P-NPs maximized the number of microtubers and enhanced root length for both cultivars	80	Low
[67]	cv. Agria	Proline nanocomposite coated with chitosan + Moderate salinity (50 mM NaCl)	In vitro (Microtuberization under salinity stress)	Microtuber yield & developmental stimulation	120 mg/L proline nanocomposite under moderate salinity significantly increased the number and yield of microtubers	90	Low
[68]	cv. Nevsky	Polymer-stabilized Silver Nanoparticles (AgNPs, 0.1–9.0 g/ha) foliar application	Ex vitro/Field scale (Pathogen challenge)	Immune priming, oxidative stress (POX/CAT) & disease resistance	AgNPs primed the immune system, increased peroxidase activity, and suppressed P. infestans and A. solani by >60% while maintaining yield	300	Low
[69]	cv. Fianna	Biotic elicitors (Activane, Micobiol, Stemicol)	In vitro (Organogenesis)	Shoot, root & callus development	Moderate levels of biotic elicitors enhanced in vitro development, serving as a sustainable alternative to synthetic regulators	60	High
[70]	S. tuberosum	Bio-agents (P. fluorescens, B. subtilis, T. viride) + 0.5% glycerol	Ex vitro (Acclimatization)	Plantlet survival & growth promotion	Consortia of bio-agents with glycerol significantly enhanced plantlet survival (73.33%) and plant height during hardening	75	Low
[71]	S. tuberosum	1% Chitosan nanoparticles loaded with 5 ppm NPK	Ex vitro (Greenhouse)	Mass yield & nutrient absorption	Foliar application increased mass yield by 37% and significantly improved NPK absorption	100	Low
[72]	cv. Agria	Nanosheet Graphene Oxide (NGO, 25–75 mg/L)	In vitro (Micropropagation & microtuberization)	Microtuber yield, plantlet growth & proliferation	NGO enhanced microtuberization without growth regulators; 25 mg/L optimized length and diameter, while 75 mg/L maximized microtuber weight	60	Low
[73]	cv. Kuroda, cv. Cardin	PGPR Bio-priming (Azospirillum, Bacillus, Pseudomonas)	Ex vitro (Greenhouse/Pots)	Plantlet survival, root architecture & shoot growth	PGPR consortia significantly mitigated transplant shock, enhanced lateral root development, and increased ex vitro survival and final tuber yield	150	Low
[74]	cv. Solara	Endophytic bacteria inoculation (Bacillus & Paenibacillus)	In vitro (Plantlet inoculation)	Plant growth (biomass, stalk & root length)	Selected endophytes successfully established in vitro and significantly improved stalk length, root number, and total biomass	45	Low
[75]	S. tuberosum	PGPR screening from rhizosphere and root tissues	In vitro (Biochemical screening)	PGPR functional traits (IAA, ACC deaminase)	Identified high-potential inoculants (e.g., Serratia sp.) capable of producing IAA, ACC deaminase, and siderophores for sustainable biofertilization	300	Low
[76]	cv. Canchay × Ccompis	Bacillus halotolerans and Streptomyces decoyicus	In vitro/Ex vitro (Greenhouse)	Biocontrol (R. solani) & plant biomass	S. decoyicus strongly inhibited pathogens and enhanced root biomass; B. halotolerans significantly increased tuber yield and reduced stem canker	120	Low
[77]	S. tuberosum	PGPR inoculation (Bacillus spp.) under drought stress	Ex vitro (Greenhouse/Field)	Drought tolerance & antioxidant machinery	PGPR significantly improved shoot/root biomass, relative water content, and ex vitro survival by upregulating SOD and CAT under stress	100	Low
[78]	S. tuberosum	Evaluation of acclimatization substrates	Ex vitro (Warm tropical agroecosystems)	Acclimatization & plantlet adaptation	Optimized growing media provided essential aeration and moisture, ensuring successful adaptation to heat-stress environments	48	High

synthesizes the characteristics and main outcomes of the systematically included studies. Due to the density of the extracted data, studies applying similar standard protocols have been methodologically grouped, while highly specific interventions are detailed individually. Readers are advised to note that the reported trends highlight genotype-specific physiological responses rather than universal absolute values. To facilitate the interpretation of this dense literature, the data reveal three dominant global trends in potato acclimatization: (1) a shift from full-strength MS media toward reduced nitrogen and sucrose concentrations to combat hyperhydricity; (2) the integration of specific LED spectra to functionalize stomata; and (3) an increasing reliance on microbial bio-priming (PGPR) to enhance ex vitro survival.

3. Results

The systematic search and subsequent eligibility screening resulted in the inclusion of 63 qualitative and quantitative studies detailing the physiological transition of S. tuberosum from in vitro to ex vitro environments (Table 1). Due to the high methodological heterogeneity in cultivars and applied treatments, the results are synthesized narratively, categorizing the evidence into three primary domains of physiological competence: nutritional hardening, photo-autotrophic induction, and stress priming.

3.1. Phase I: Nutritional Hardening and the Nitrogen Paradox

The meta-synthesis of the selected studies reveals that the traditional focus on high multiplication rates often compromises the structural integrity of the plantlet. While standard Murashige & Skoog (MS) medium remains the most cited basal formulation [18], our analysis identifies its chemical composition, specifically the high inorganic nitrogen concentration, as a primary driver of the aberrant vitrotype.

3.1.1. The Nitrogen Paradox: Ammonium Toxicity and Hyperhydricity

Standard MS medium provides a high total nitrogen content (60 mM), with a significant proportion as ammonium (NH₄⁺: 20.6 mM). While Cao and Tibbitts [19] demonstrated the broad effects of ammonium/nitrate ratios on potato growth parameters, the specific metabolic consequences during in vitro culture require deeper mechanistic integration. This proposed hypothesis, termed the Nitrogen Paradox, suggests that excessive NH₄⁺ influx creates a metabolic competition for carbon skeletons (Box 1). Assimilation via the GS/GOGAT pathway [18] consumes organic acids and energy (ATP/NADPH) that would otherwise be channeled into the shikimate and phenylpropanoid pathways—the primary routes for lignin precursor synthesis. Drawing from inferential data, this metabolic diversion is hypothesized to downregulate lignin biosynthesis. However, it is important to note that no study included in this review directly measured Phenylalanine Ammonia-Lyase (PAL) activity in relation to nitrogen form in Solanum tuberosum. Consequently, this framework serves as a speculative, deductive model (Figure 2). While high NH₄⁺ accelerates vertical growth, it simultaneously inhibits the structural lignification necessary for ex vitro mechanical support, resulting in the physiological syndrome known as hyperhydricity [8,9].

Box 1. Testable Predictions for the Nitrogen Paradox Hypothesis.

Prediction 1: In vitro potato plantlets cultured with a low NH₄⁺:NO₃⁻ ratio will exhibit significantly higher PAL enzyme activity and increased lignin content in stem tissues compared to those on standard MS medium.

Prediction 2: Isotopic labeling (¹⁵N) will show a preferential redirection of carbon skeletons toward amino acid synthesis over phenolic compounds under high ammonium stress.

Prediction 3: Supplemental application of silicon or calcium will partially alleviate the structural vulnerability induced by high nitrogen by promoting alternative cell wall fortification mechanisms.

3.1.2. Nutritional Mitigation Strategies and Stress Priming

To counteract this metabolic drain, the reviewed literature supports a “Nutritional Hardening” strategy. Protocols substituting standard MS with Woody Plant Medium (WPM) [31], which contains 25% less total nitrogen and a significantly reduced NH₄⁺:NO₃⁻ ratio [19], or reducing MS macrosalts to 50% impose a mild, beneficial stress. This nutritional restriction signals the plantlet to shift metabolic resources from rapid vertical elongation to cell wall thickening and root proliferation. Comparative studies demonstrate that potato plantlets cultured on low-nitrogen media exhibit higher dry matter content and an optimized root-to-shoot ratio, significantly enhancing their morphological readiness for ex vitro acclimatization compared to full-strength MS controls [16,17].

As previously detailed in our consolidated framework (Table 1), specific physiological competence can be engineered through various basal salt modifiers and stress-signaling agents. For instance, correcting the NH₄⁺/NO₃⁻ imbalance directly mitigates oxidative stress and prevents the “glassy stem” phenotype [18,19]. Additionally, supplementing the media with structural elements such as Silicon (Si) [47] or Calcium (CaCl₂) [20] strengthens cell wall pectin linkages and increases cuticular resistance to transpiration shock.

Furthermore, controlled abiotic priming using moderate NaCl induced osmotic stress [21] or chemical elicitors like Silver Nitrate (AgNO₃) [22] and Salicylic Acid (SA) [48] actively upregulates the plantlet’s ROS scavenging mechanisms (SOD, CAT) and prevents ethylene-induced leaf epinasty. Finally, PGR priming with IAA and GA₃ [23] and the physical optimization of gas exchange via Temporary Immersion Systems (TIS) [24] maintain the membrane stability index under stress, maximizing both the multiplication coefficient and subsequent survival rates.

3.1.3. PGR Residual Effects (The “Carry-Over” Risk)

While nutritional hardening forms the structural basis of the plantlet, PGR balance is equally critical. Our review identified that protocols utilizing potent cytokinins, such as Thidiazuron (TDZ) or high concentrations of Benzyladenine (BA), often result in negative “carry-over effects” during the acclimatization phase. Although these regulators maximize shoot proliferation rates and microtuber induction in vitro [36,37], studies confirm that their high stability and residual activity can lead to severe morpho-physiological disorders, including fasciation and hyperhydricity, which persist even after transplanting to the greenhouse [3,8]. Therefore, ensuring a PGR-free “elongation step” or utilizing strictly optimized, low-concentration plant growth regulator [37] is recommended to detoxify the tissue and restore vascular functionality before ex vitro transfer.

3.2. Phase II: Photo-Autotrophic Induction via Spectral Modulation

Light quality is the most influential environmental factor regulating the transition from heterotrophy to autotrophy. While traditional fluorescent lamps (CFL) provide diffuse, broad-spectrum light, they often fail to stimulate the functional stomatal apparatus required for ex vitro survival.

3.2.1. The Failure of Fluorescent Lighting

CFL systems typically emit a high proportion of green/yellow wavelengths and insufficient flux in the photosynthetically active radiation (PAR) region (400–700 nm). Under these conditions, the synthesized evidence indicates that potato plantlets develop a “shade avoidance syndrome,” characterized by weak, etiolated stems and permanently open stomata that lack the capacity to respond to vapor pressure deficit (VPD) changes upon transplanting [11,41].

3.2.2. Spectral Quality, Gas Exchange, and Morphological Plasticity

To overcome the physiological limitations of fluorescent lighting, the application of Light-Emitting Diodes (LEDs) allows for the precise modulation of morphological plasticity. As schematized in Figure 3, monochromatic red light (660 nm) triggers a significant phytochrome-mediated response, promoting rapid stem elongation but severely inhibiting root development and mechanical resistance, which can reduce overall tuber numbers [39,40]. Conversely, monochromatic blue light (450 nm) activates phototropins (phot1 and phot2) and cryptochromes, which are the primary photoreceptors governing stomatal functionality and the induction of a compact, robust, “hardened” phenotype [11,39].

For successful acclimatization, an integrated approach is required. The meta-synthesis confirms that a balanced Red–Blue (R:B) spectrum integrates PGR and photosynthetic signaling. This balanced spectrum optimizes the root-to-shoot ratio, increases grana stacking and starch accumulation in chloroplasts, and ensures that the stomatal apparatus is physiologically competent before the transition to the greenhouse environment [11,39,40]. Furthermore, specific spectral interventions, such as a 90% Red to 10% Blue ratio, have been shown to upregulate defense genes crucial for recovery following cryopreservation stress [42], while far-red treatments effectively regulate morphological control and tuber sprouting [43].

Beyond spectral quality, the physical environment of the culture vessel also dictates physiological competence. Eliminating exogenous carbon (sucrose-free medium) forces autotrophy, inducing active Rubisco expression and higher photosynthetic rates [25]. Coupling this with vented vessels facilitates proper gas exchange, driving the transformation of aberrant, spherical in vitro stomata into functional, elliptical stomata capable of regulating water loss [26]. It is imperative to integrate the foundational principles of photoautotrophic micropropagation into modern acclimatization frameworks. Pioneering work by Kozai demonstrated that utilizing sucrose-free media combined with CO₂ enrichment and forced ventilation significantly enhances the photosynthetic competence of plantlets prior to transplanting [79].

3.3. Phase III: Rhizosphere Engineering and the Biotic Shield

The final barrier to acclimatization is the functional competence of the root system. In vitro roots are often devoid of root hairs and possess weak vascular connections to the stem, limiting water uptake upon transplanting. Furthermore, the sterile nature of the substrate leaves the plantlet vulnerable to immediate pathogen attack.

3.3.1. PGR Pulse Treatments and Substrate Optimization

Root induction is strictly regulated by endogenous auxin gradients, which can be effectively modulated by exogenous PGR pulse treatments (e.g., synthetic auxins like IBA or NAA). While continuous exposure to these exogenous PGRs in the medium can lead to callus formation, a non-functional undifferentiated tissue, our review supports the efficacy of optimized in vitro treatments, such as PGR pulses, to optimize multiplication and vigor [37]. A short-term exposure is sufficient to trigger rhizogenesis without blocking vascular continuity, promoting the development of true lateral roots capable of nutrient uptake.

Equally critical is the physical matrix receiving the plantlet. The physicochemical properties of the growing media dictate root architecture and moisture retention [78]. Transitioning from agar to optimized substrates with high air-filled porosity (e.g., specific peat–perlite ratios or cocopeat) prevents root asphyxia, maximizes bacterial adherence, and significantly improves the cost–benefit ratio and harvest index compared to traditional soil alone [49,50].

3.3.2. Bio-Priming: Installing a Biotic Shield

To mitigate transplant shock, recent protocols advocate for rhizosphere engineering. The inoculation of roots with Plant Growth-Promoting Rhizobacteria (PGPR), such as Bacillus subtilis or Pseudomonas putida, establishes a robust endophytic colonization and plant growth regulator modulation that improves water status and drought resistance [51,52]. Similarly, the use of antagonistic fungi like Trichoderma viride creates a true Biotic Shield against soil-borne pathogens [54].

These beneficial microorganisms colonize the rhizosphere, trigger Induced Systemic Resistance (ISR) and regulate defense gene expression. Treated plantlets exhibit higher antioxidant enzyme activity (SOD, CAT), lower lipid peroxidation (reduced MDA), and a measurable reduction in mortality caused by pathogens like Rhizoctonia solani and Alternaria solani compared to non-inoculated controls [52,54,76]. Furthermore, advanced high-tech acclimatization systems, such as aeroponics, have successfully integrated Azospirillum strains to expand root surface area and boost antioxidant machinery [53], ultimately stabilizing the rhizosphere microbiome against environmental fluctuations [62].

3.3.3. Chemical Priming and Emerging Frontiers: Nanotechnology

Beyond biologicals, chemical priming using exogenous Abscisic Acid (ABA) effectively regulates antioxidant pathways and mitigates saline-alkaline stress during acclimatization [27]. An emerging field identified in this review is the application of nanotechnology. Silver nanoparticles (AgNPs) have demonstrated dual efficacy: acting as potent elicitors to prime the plant’s immune system against phytopathogens while simultaneously optimizing in vitro conservation and regeneration capacity [65,68]. In parallel with microbial inoculation, emerging nano-elicitors have demonstrated significant efficacy. Specifically, the application of Zinc nanoparticles (ZnNPs or ZnO-NPs) has emerged as a powerful tool to reinforce the ex vitro transition. Evidence indicates that ZnO-NPs actively stimulate microtuber development and enhance plantlet vigor by improving chlorophyll content and stimulating the antioxidant enzymatic system. This provides a robust biochemical defense against the oxidative stress typical of the first weeks of acclimatization, significantly reducing mortality rates [61]. Similarly, fostering Silica (Si) accumulation within the plant tissues reinforces cell wall mechanical strength and improves yield performance, offering a promising acclimatization strategy [47].

3.3.4. Integration into the Physiological Competence Protocol

The transition from heterotrophic in vitro growth to autotrophic ex vitro survival requires a multi-faceted approach. We integrate these findings into a Physiological Competence Protocol (Figure 4), which operates through three integrated pillars. First, Nutritional Hardening reduces the osmotic and metabolic stress caused by excessive NH₄⁺ [18,19]. Crucially, Phase I also demands strict exogenous PGR management (e.g., utilizing PGR-free media or significantly reducing cytokinins) to prevent negative morpho-physiological carry-over effects and restore the plantlet’s endogenous hormonal balance. Second, Photo-autotrophic Induction via LED technology ensures that the photosynthetic machinery is primed for immediate activity [39,44]. Finally, Rhizosphere Engineering using PGPRs, optimized substrates, and elicitors provides the biological shield necessary to minimize transplant shock and maximize G0 seed production [50,73]. This comprehensive integration represents a shift from traditional empirical hardening to a precision-based physiological priming strategy.

4. Discussion

Synthesizing the systematic data, a clear divergence emerges between traditional “multiplication protocols” (aimed at numbers) and modern “acclimatization protocols” (aimed at survival). The significant mortality rates reported in older literature are largely preventable by shifting the focus from yield to physiological competence.

The nitrogen paradox hypothesis proposed in this review suggests a metabolic trade-off that goes beyond simple ammonium toxicity. We argue that under high NH₄⁺ conditions, the plant prioritizes the GS/GOGAT cycle to prevent ammonium accumulation, creating a massive drain on ATP and carbon skeletons. This ‘metabolic emergency’ likely de-prioritizes the phenylpropanoid pathway, specifically reducing the activity of Phenylalanine Ammonia-Lyase (PAL). Consequently, the lack of lignin precursors during the critical window of cell wall differentiation leads to the structural ‘glassiness’ of the vitrotype, explaining why simple nutritional adjustments are often insufficient without an integrated physiological approach.

However, it is crucial to explicitly state the limitations of the proposed Physiological Competence Protocol. This framework integrates data across a wide range of Solanum tuberosum cultivars, varying baseline acclimatization conditions, and diverse experimental setups (e.g., differing LED intensities and substrate compositions). Given the high genotypic plasticity inherent to potato varieties, the general applicability of these findings should not be implied as an absolute standard. The specific thresholds for nitrogen reduction or spectral ratios may not be directly comparable across all commercial environments without prior pilot-scale calibration. Therefore, this protocol represents a flexible conceptual guide rather than a rigid, universally applicable recipe.

4.1. Summary of Evidence

The evidence synthesized from 63 studies indicates that ex vitro acclimatization in potato is a multi-factorial process where physiological competence is built through cumulative pre-conditioning. While standard protocols often prioritize rapid multiplication, this review demonstrates that survival rates exceeding 90% are consistently associated with a strategic shift toward photoautotrophic traits, reduced ammonium-induced stress, and the establishment of a “Biotic Shield” through microbial priming. The integration of these three pillars—nutritional, spectral, and biological—constitutes the most effective path to overcoming the transplant shock in Solanum tuberosum.

4.2. Genotype Dependence and Microbiome Specificity

One of the major challenges in acclimatization is the genotype-specific response to ex vitro conditions. The literature emphasizes that there is no ‘one-size-fits-all’ solution. For instance, evidence indicates that while some cultivars respond well to generic PGPR inoculation, others exhibit highly differential responses and require taxonomically diverse bacterial consortia to trigger the Induced Systemic Resistance (ISR) mechanism [73]. Similarly, combining microbial inoculation with reduced inorganic fertilization has been shown to enhance rhizosphere diversity, yet the degree of colonization and functionality varies significantly between potato varieties [62]. This suggests that future protocols must be tailored to the “genetic architecture” of the cultivar, particularly for somatic hybrids, which require specific phenotyping platforms to demonstrate differential drought tolerance strategies [30].

4.3. The Biotic Shield: Beyond Simple Inoculation

The sterile plantlet requires a “microbiome transplant” to survive the septic environment of the greenhouse. It is not just about adding bacteria, but about creating a bioactive shield. Furthermore, expanding the ‘Biotic Shield’ concept, the combined application of mycorrhizal inoculation (e.g., Rhizophagus irregularis) [80] and silicon supplementation [81] has been shown to fortify cell walls and improve osmotic adjustment, representing a vital strategy for mitigating transplant shock.

Antioxidant Regulation: Bio-priming agents like Bacillus subtilis and Pseudomonas do not merely solubilize nutrients; they actively regulate the pro- and antioxidant systems of the plantlet. During transplant shock, these endophytic interactions significantly reduce lipid peroxidation and oxidative stress, thereby increasing the adaptation potential of the microclones [51,63].

Pathogen Suppression: The acclimatization phase is the most vulnerable window for infection. Innovative approaches using Trichoderma viride have proven effective as sustainable biocontrol agents against Early Blight (Alternaria solani) [54], while specific plant essential oils demonstrate effective antibacterial activity against Ralstonia solanacearum [59]. Furthermore, green-synthesized chitosan and silver nanoparticles represent a modern, circular economy approach to combat devastating phytopathogens like Phytophthora infestans in the substrate [58,68].

4.4. Stress Alleviation Mechanisms

Finally, the gap between the heterotrophic (in vitro) and autotrophic (soil) phase is an acute stress event. Novel elicitors and molecular insights are reshaping how we manage this transition.

Salinity and Drought: Recent investigations indicate that potato cultivars utilize distinct physiological mechanisms for salt stress acclimation, reinforcing that stress management strategies must be genotype specific [29]. At the molecular level, homologous overexpression of the PR10a gene significantly enhances salt stress tolerance and antioxidant defense [28].

Nanotechnology as a Buffer: Expanding the nanomaterial spectrum, recent studies highlight the role of Zinc nanoparticles (ZnNPs) [61] and Graphene Oxide nanosheets [72] as powerful tools to stimulate microtuber development and mitigate abiotic stress. Furthermore, controlled-release chitosan nanofertilizers [71] and surface-functionalized Silver nanoparticles (AgNPs) [65,68] act as novel elicitors that enhance the antioxidant machinery and boost regeneration capacity. At the transcriptomic level, abiotic stresses and physical treatments upregulate specific stress-responsive transcription factors and miRNAs, providing a molecular basis for the observed resilience [32,33]. Nevertheless, the translation of these nano-biological tools from the laboratory to commercial scale must strictly adhere to regulatory frameworks regarding potential environmental nanotoxicity, requiring comprehensive risk assessments and soil-microbiome impact evaluations before widespread field application.

Reconciling Contradictions: A critical analysis of the synthesized evidence reveals significant contradictions, particularly regarding genotype-specific responses to these treatments. For instance, while most studies report a universal benefit of PGPR in enhancing root architecture and biomass [53,74], other results indicate that the efficacy of bio-priming is highly modulated by the host’s endogenous plant growth regulator levels and microbiome specificity. Similarly, the nitrogen paradox appears to be influenced by light intensity; under high photosynthetic photon flux density (PPFD), plantlets can occasionally overcome the metabolic drain of high ammonium, explaining divergent results across laboratories. These discrepancies highlight that acclimatization protocols cannot be rigid but must be calibrated to the specific physiological thresholds of each potato cultivar.

4.5. Protocol and Study Limitations

A critical limitation of the proposed physiological competence protocol is the high degree of experimental variability across the reviewed literature. The synthesized studies encompass a wide range of cultivars, light regimes, and acclimatization substrates. Consequently, this protocol cannot be interpreted as a rigid, universally applicable formula. There is no “one-size-fits-all” approach for potato micropropagation. Rather, this framework represents a highly adaptable baseline. The efficacy of specific interventions, particularly bio-priming responses and precise spectral requirements, is highly genotype-dependent, dictating that these strategies must be empirically calibrated for the specific physiological thresholds of each potato cultivar.

Furthermore, despite the systematic approach, this review presents inherent methodological limitations. First, the high heterogeneity in environmental reporting across the included studies (e.g., lack of precise vapor pressure deficit (VPD) or specific light quality data in older publications) prevented a formal quantitative meta-analysis. Second, while physiological competence is well-documented in laboratory settings, there is a scarcity of long-term field data tracing the performance of these hardened plantlets through subsequent seed generations (G1–G4). Finally, the economic projections regarding the physiological competence protocol are conceptual and require site-specific validation to account for varying energy, nanomaterial, and infrastructure costs.

4.6. Relationship to Previous Reviews

This systematic review fills a critical mechanistic gap left by previous literature. Unlike the scoping review by Jácome Sarchi et al. [2], which mapped general bibliometric trends of the entire in vitro pipeline, this study provides a deep physiological analysis exclusively of the ex vitro transition. Compared to the production-oriented work of Buckseth et al. [4], which focuses on aeroponic multiplication efficiencies, our analysis prioritizes the plantlet’s internal metabolic readiness. Furthermore, while Hazarika [3] identified broad morpho-physiological disorders, we specifically link these anomalies to the GS/GOGAT-phenylpropanoid competition framework. Finally, this work integrates the foundational photoautotrophic principles described by Kozai [79] into a consolidated protocol, emphasizing that the “vitrotype” is a reversible state rather than a permanent defect.

4.7. Future Research Directions

Rather than generalized calls for further investigation, this review identifies three specific, testable questions to advance the field:

• To what extent does the NH₄⁺:NO₃⁻ ratio during the final in vitro stage determine the activity of Phenylalanine Ammonia-Lyase (PAL) and total lignin content in potato stems?
• Can specific PGPR-derived metabolites, such as ACC-deaminase, effectively substitute synthetic plant growth regulators (PGRs) to induce ex vitro mechanical robustness?
• What is the optimal Red–Blue LED ratio that maximizes stomatal conductance while minimizing hyperhydricity across diverse potato genotypes?

5. Conclusions

The transition of Solanum tuberosum from in vitro culture to ex vitro environments remains a critical bottleneck in pre-basic seed potato production. This systematic review demonstrates that successful acclimatization requires a paradigm shift from traditional multiplication-focused protocols toward pre-conditioning strategies that build physiological competence. Based on the synthesized evidence, the most effective acclimatization protocol integrates three core components: (1) nutritional hardening via reduced nitrogen/ammonium media to mitigate hyperhydricity and prevent lignin downregulation [16,18]; (2) photoautotrophic induction using balanced Red–Blue LED spectra [11,39] to stimulate functional stomatal development; and (3) rhizosphere engineering through PGPR bio-priming [51,73] to fortify the plantlet against early transplant shock and pathogenic pressure. The integration of these pillars is consolidated in the Physiological Competence Protocol (Figure 4).

When implemented in synergy, these interventions correlate with improved structural robustness and high ex vitro survival rates, frequently exceeding 85–90% across various cultivars. However, this review identifies a significant knowledge gap: the vast majority of the current literature focuses exclusively on short-term survival metrics within the greenhouse environment (G0 phase). The primary limitation in the field is the absence of multi-center, long-term field validations that track how these specific pre-conditioning treatments influence agronomic performance and tuber quality across subsequent seed generations (G1–G4). Future research must prioritize standardized field trials to validate the long-term economic and agronomic viability of these physiologically integrated protocols.