Modification of plant pre-cultivation alters regeneration competence in recalcitrant model Andean potato

Modification of plant pre-cultivation alters regeneration competence in recalcitrant model Andean potato | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Modification of plant pre-cultivation alters regeneration competence in recalcitrant model Andean potato Jan Konečný, Helena Lipavská, Petra Mašková This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9573528/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Solanum tuberosum ssp. andigena line 7540, a key model for tuberization, exhibits a recalcitrance to Agrobacterium -mediated transformation. This study aimed to improve leaf regeneration efficiency by modifying the source plant's pre-cultivation. We compared plants cultivated in vitro under standard heterotrophic conditions (non-permeable lids, 2% sucrose) and under mixotrophic and photoautotrophic regimes (air-permeable lids, 2% or 0% sucrose). The use of air-permeable lids significantly increased biomass and leaf size. Moreover, we found a link between plant pre-cultivation and leaf physiological state. In photoautotrophic plants, regeneration competence was strongly favoured in leaf explants from upper half of source plants, while heterotrophic control exhibited an opposite behaviour. Mixotrophic variant preserved regeneration competence along the shoot axis. In conclusion, the competence for regeneration varies with tissue physiological status and thus can be shifted via low-input adjustment of the source plant pre-cultivation. This approach might prove beneficial also for regeneration of other difficult-to-master crops. Agrobacterium-mediated transformation air-permeable lid de novo regeneration in vitro tissue culture Solanum tuberosum ssp. andigena line 7540 trophic status Figures Figure 1 Figure 2 Introduction In potato research, Solanum tuberosum ssp. andigena , line 7540 serves as an indispensable model for tuberization signaling studies, which can guide potato breeding strategies in general (Dutt et al. 2017 ; Hannapel et al. 2017 ; Kondhare et al. 2020 ). However, line 7540 is a genotype recalcitrant to Agrobacterium -mediated transformation, a challenge faced by various potato genotypes (Dale and Hampson 1995 ; Park et al. 2023 ; Trujillo et al. 2001 ). The genetic improvement of many crops is frequently bottlenecked by low regeneration efficiencies. Standard approaches to overcome such recalcitrance typically rely mostly on time-consuming optimization of growth regulators treatment, a strategy that is costly and genotype-dependent (Dale and Hampson 1995 ; Heeres et al. 2002 ; Park et al. 2023 ; Sheerman and Bevan 1988 ; Wenzler et al. 1989 ). Alternatively, revisiting primary reports behind broad reviews (e.g. Vinterhalter et al. 2008 ) can reveal critical, yet often overlooked, constraints. To this end, the protocol established by Banerjee et al. ( 2006 ) explicitly identifies explant size as a decisive factor, stating that large leaf explants (0.5-1 cm²) are critical for high regeneration frequencies. Also Park et al. ( 2023 ) observed different regeneration potential in cultivars that obviously differed in leaf size although the authors did not systematically evaluate this correlation. Recent studies demonstrate that plant morphology and physiology can be dramatically altered by modifications of in vitro cultivation towards nature-like conditions via increase of air permeability and/or photoautotrophy (Ševčíková et al. 2019 ; Vale et al. 2025 ; Vollmer et al. 2024 ). Thus, we propose an alternative strategy: manipulating the physiological status of the source plant itself by shifting plants from heterotrophic (sugar supply and tight non-permeable lid) to mixo- or even photoautotrophic (low or no sugar supply, air-permeable lid) conditions. Based on Banerjee et al. ( 2006 ), we hypothesized that the increased leaf size resulting from advanced plant cultivation would enlarge the pool of suitable-sized leaves and thus improve overall regeneration (and hence transformation) efficiency. Material and methods For experiments, we used in vitro grown Solanum tuberosum ssp. andigena , line 7540 (CGN 17637, Centre of Genetic Resources, Wageningen, NL). Plants were vegetatively propagated under in vitro conditions from apical segments (three per flask) in 150-mL Erlenmeyer flasks (Kavalierglass, CZ) on MS medium (Sigma-Aldrich, USA; (Murashige and Skoog 1962 ) supplemented with 2% (w/v) sucrose under a 16 h photoperiod at 21°C with irradiance 100 µmol·m − 2 ·s − 1 (daylight fluorescent tubes; Osram, Winterthur, Switzerland) and subcultivated every four weeks. For plant pre-cultivation, we compared three different regimes: heterotrophic control (MS medium, 2% (w/v) sucrose, non-permeable lid; 2% NP), a mixotrophic regime in vitro (MS medium, 2% (w/v) sucrose, air-permeable lid; 2% P), and a photoautotrophic regime (MS medium, 0% sucrose, air-permeable lid; 0% P). The lid was prepared from a transparent polypropylene foil without (air non-permeable variant; NP) or with a hole (27.25 mm 2 ) covered by one layer of a breathable tape (Leucopore, Duchefa) (air-permeable variants; P). Other cultivation conditions were identical with plant in vitro maintenance. Selected growth parameters were evaluated after 3 weeks of cultivation and the experiment was repeated two and three times, for NP versus P lid variant and 0% versus 2% P variant, respectively. The total fresh biomass of both, shoots and roots as well as the leaf number was determined for each variant. Leaf area measurements were done using ImageJ software ( www.imagej.nih.gov Rasband, W.S., ImageJ, US National Institutes of Health, Bethesda, MA) and leaves sorted into three groups: ≤ 0,5cm 2 , 0,5 − 1 cm 2 and ≥ 1 cm 2 . The leaves of source plants (larger than 0,5 cm 2 ) with different pre-cultivation history were used for the regeneration competence experiment (repeated twice): heterotrophic regime following Banerjee et al. ( 2006 ) (25°C, GA-7 Magenta vessels (Sigma-Aldrich, USA), 90 mL MS medium with 2% sucrose non-permeable lid; 25°C 2% NP), newly tested regimes - mixotrophic (21°C, Erlenmeyer flasks, 25 ml MS with 2% sucrose, air-permeable lid; 21°C 2% P) and photoautotrophic (21°C, Erlenmeyer flasks, 25 ml MS with 0% sucrose, air-permeable lid; 21°C 0% P). Transformation of leaves was performed using Agrobacterium tumefaciens strain C58C1 carrying empty vector pGreen0029 (kanamycin resistance, nptII ), otherwise strictly following the transformation and regeneration protocol by Banerjee et al. ( 2006 ) - see Supplementary Material S1 for brief procedure description and media composition. Six weeks after the transfer on shoot inducing media, the number of regenerating explants (visible shoot buds present) was counted. Data distributions were presented either as box plots or bar charts with error bars that represent standard deviations. For statistical analysis, NCSS 9 statistical software (NCSS, LLC. Kaysville, Utah, USA) was employed, ANOVA-One-Way Analysis of Variance using Kruskal-Wallis Z-test. Statistical significance was determined at P ≤ 0.001, P ≤ 0.01, P ≤ 0.05, and P ≤ 0.1 levels marked by ***, **, *, and (*), respectively. Results and discussion Prior to the specific experiments, we performed a preliminary screening to optimize the in vitro growth of Solanum tuberosum ssp. andigena , line 7540. We tested various cultivation parameters, including vessel type, medium volume, MS salt strength, sucrose concentration, and temperature. The protocol by Banerjee et al. ( 2006 ) prescribes higher temperature (up to 27°C). Based on our long-term good experience with potato growth at lower temperatures, we compared cultivation at 21 and 25°C also for andigena and found 21°C superior for the plant growth and proportion of leaves larger than 0,5 cm 2 (61 ± 3% (21°C) vs 32 ± 3% (25°C); p < 0.001). Nevertheless, most plants grown at 25°C remained healthy, validating the suitability of the 20–25°C range generally observed for potato (Hussey and Stacey 1981 ; Vinterhalter et al. 2008 and references therein). Published data on modern potato repeatedly showed that the modification of cultivation regime by increased gas exchange significantly altered overall plant habitus (Hussey and Stacey 1981 ; Chanemougasoundharam et al. 2004 ; Ševčíková et al. 2019 )(. Thus, we proceeded with comparison of the morphology of andigena plants grown at 21°C under hetero-, mixo- and photoautotrophic conditions using non-permeable (NP) and air-permeable (P) lids. Total fresh biomass of both, shoots and roots, consistently higher under P treatment (Fig. 1 a). The P variant also tended to have a higher average number of leaves compared to their NP counterparts (Fig. 1 b). Importantly, there was a marked increase in leaf size of P variant resulting in formation of more suitable-sized leaves (> 0.5 cm²) in P variant than in NP one that favoured production of smaller leaves (< 0.5 cm²) (Fig. 1 c). Furthermore, handling comfort, important factor for frequent subcultivations during transformation and subsequent regeneration process, was also improved as P lid-grown tissues were mechanically more resilient compared to the fragile, easily damageable tissues of NP variant. It confirms that improved gas exchange stimulates formation of nature-like leaves better withstanding mechanical constraints. To determine whether exogenous sucrose might be beneficial even under improved ventilation, we compared the mixotrophic regime (2% sucrose, air-permeable; 2% P) with the photoautotrophic regime (0% sucrose, air-permeable; 0% P) under the same cultivation conditions. We observed no significant differences between the 2% and 0% sucrose P variants regarding the fresh biomass (Fig. 1 d), average number of leaves (Fig. 1 e) or leaf size distribution (Fig. 1 f). This suggests that under adequate irradiation and lid air-permeability, the plants are fully capable of sustaining growth through photosynthesis alone. The only significant deviation was observed in the root-to-shoot ratio, which was lower in the photoautotrophic variant (0.39 ± 0.03 (0% P) vs 0.57 ± 0.06 (2% P); p < 0.018), reflecting the dominant way of acquiring C source: photosynthesis or sugar uptake from the medium. Noteworthy, the leaves of mixotrophic plants exhibited a dark green colour (Figure S1 ), which indicates strengthened anthocyanin production, a common response to enhanced sugar availability (reviewed e.g. by Steyn et al. 2002 ). Thus, photoauto- or mixotrophic cultivation of potato plants could help to meet the condition proposed by Banerjee et al. ( 2006 ) for achieving good results, i.e., using healthy leaves with a larger leaf area as starting material for the transformation process. To verify this assumption, we tested regeneration capacity of the newly established cultivation setupversus the original one established by Banerjee et al. ( 2006 ). After six weeks on shoot inducing media, the number of regenerating explants (visible shoot buds present) was compared. Surprisingly, despite the marked differences in the leaf area distribution, we observed the overall regeneration efficiency being comparable across variants (Fig. 2 a). This result clearly indicates that the leaf size alone is not a proxy for regeneration competence. Instead, we found a surprising correlation between the pre-cultivation regime, the leaf insertion on source plants and regeneration efficiency. In autotrophic plants (0% P), the regeneration competence was position-dependent: while the explants arisen from the upper part of the source plantexhibited the highest regeneration rates (Fig. 2 b), this potential was lost in the explants from lower part (Fig. 2 c). In contrast, the standard heterotrophic control (2% NP) showed a reverse pattern, with the lowest efficiency in the leaves of the upper part. The differences in regeneration capacity of leaves of a given insertion in both arrangements (0% P versus 2% P) were statistically significant. Interestingly, the mixotrophic variant (2% P) consistently occupied an intermediate position, preserving regeneration competence along the entire shoot axis (Fig. 2 c). Together, these results underscore the importance of the physiological state of leaves, which reflects the C and energy status. However, this characteristic depends on a combination of the influence of the cultivation regime (0% P, 2% NP, 2% P) and the position of the leaf on the stem. Establishing this optimal metabolic status is crucial, as the subsequent de novo shoot organogenesis relies heavily on the explant's capacity to properly uptake and transduce morphogenic auxin and cytokinin signals (Raspor et al. 2021 ). Previous studies showed a correlation between tissue/organ ageing and lowering of the regeneration potential in potato (Mohapatra and Batra 2017 ; Tavazza et al. 1989 ) as well as in other recalcitrant species ranging from legumes to conifers (Bidabadi and Jain 2020 ; Mahalakshmi et al. 2006 ; Sainger et al. 2015 ). Our data refine this view by demonstrating that this correlation is environmentally plastic. Under photoautotrophic conditions, photosynthesis promoted more rapid leaf development resulting in faster production of competent leaves. Conversely, heterotrophic conditions inverted this trend, favouring the lower leaves near the exogenous sugar. The advantage of both is combined in the 2% P variant where all leaves exhibit comparable regeneration competence. Ultimately, this proves that the window of competence is not immutable. By employing simple, low-input interventions—such as changing pre-cultivation regime of source plants by enabling air-permeability and/or adjusting sucrose supply—researchers can shift this physiological window to align with their specific, often genotype-dependent, needs. To overcome genotype-dependent recalcitrance, researchers typically focus on optimizing the in vitro regeneration phase itself. This often involves extensive adjustments to phytohormones and incubation parameters (Bakhsh 2020 ; Chauhan et al. 2025 ; Kaur et al. 2020 ; Kaur et al. 2022 ; Molla et al. 2022 ; Patel et al. 2025 ), or employing modifications such as substituting carbon sources (Hamama et al. 2019 ) or utilizing machine learning to balance macronutrients (Mahdavi et al. 2025 ). While these post-excision optimizations are undeniably valuable, our findings demonstrate that the physiological baseline of the source plant is equally critical. Whether aiming for the rapid multiplication in autotrophic systems or the axis-wide leaf competence of mixotrophic cultures, regeneration can be effectively fine-tuned through clever management of the source plant environment —a strategy often overlooked even in comprehensive reviews (Vinterhalter et al. 2008 ). In conclusion, our results highlight that the optimal regeneration competence of leaf explants is not a fixed temporal marker but a variable dependent on the pre-cultivation and we propose modifications of pre-cultivation regime as a possible tool to improve regeneration efficiency also suitable for other recalcitrant species. Declarations Author contribution statement All authors contributed to the study conception and design. J. K. performed cultivations and data analyses and wrote the first manuscript version. All authors contributed to editing and finalization of the manuscript. Compliance with Ethical Standards The authors have no competing interests to declare. Funding acknowledgement statement Supported by the by project no. 1522218 of the Grant Agency of Charles University, Czech Republic and by the project TowArds Next GENeration Crops [CZ.02.01.01/00/22_008/0004581] from the ERDF Programme Johannes Amos Comenius under the Ministry of Education, Youth and Sports of the Czech Republic. Author Contribution All authors contributed to the study conception and design. J. K. performed cultivations and data analyses and wrote the first manuscript version. All authors contributed to editing and finalization of the manuscript. Acknowledgement The Agrobacterium tumefaciens strain C58C1 and the empty vector pGreen0029 were kindly provided by Dr. Lukáš Fischer, Charles University. Data Availability The raw datasets to Figures are freely available in the Zenodo repository (https://doi.org/ 10.5281/zenodo.19846193). References Bakhsh A. (2020). 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9573528","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":638460941,"identity":"cac8e810-4f9e-4443-9ab2-c8a3dfc3708f","order_by":0,"name":"Jan Konečný","email":"","orcid":"","institution":"Charles University","correspondingAuthor":false,"prefix":"","firstName":"Jan","middleName":"","lastName":"Konečný","suffix":""},{"id":638460942,"identity":"ca1884ee-7faa-405a-bfe2-34a67d09f89f","order_by":1,"name":"Helena Lipavská","email":"","orcid":"","institution":"Charles University","correspondingAuthor":false,"prefix":"","firstName":"Helena","middleName":"","lastName":"Lipavská","suffix":""},{"id":638460943,"identity":"d0de5bc5-22fc-42b9-a959-a71c54d035e2","order_by":2,"name":"Petra Mašková","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABD0lEQVRIiWNgGAWjYFADHhBRgcbHDpiRlZxBKCVSC2MbXqUQYM7ef+zDD4Zt8vI9h49J/Jx3R95eIvcBw8+2OzIM7McfYNNi2XOYeWYPw23DDWfb0iR7tz0z7JFIN2DsbXvGw8CTkIBNi8GNZGagW24zbuDnMTbg3XaYsUcijYGZse0wD4MEwwGsWu4/Zmb8w3Dbfn4/j7Hh3zmH7ZG0MDZgt4WZmRloS2LD2R7Dx7wNhxORtDBj0wH0S7Ixs4zB7eQNZ44lPpY5dji558wzhoM95w7zsPGk4Qixg48Z31Tctp3fk3zg4Juaw7bt7WmMD36UHbbnxxFiBkgkAoC9zYbVDkzFo2AUjIJRMAowAQC7OVqCbBH2lQAAAABJRU5ErkJggg==","orcid":"","institution":"Charles University","correspondingAuthor":true,"prefix":"","firstName":"Petra","middleName":"","lastName":"Mašková","suffix":""}],"badges":[],"createdAt":"2026-04-30 07:27:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9573528/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9573528/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109172066,"identity":"758b7feb-8b9f-4ceb-8259-6ff65784f3c1","added_by":"auto","created_at":"2026-05-13 09:02:21","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":79639,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of sucrose availability and lid permeability on biomass production, number and size of leaves. \u003c/strong\u003ePlants were cultivated in Erlenmeyer flasks containing 25 mL of MS medium supplemented with either [0%] or [2%] sucrose, using air-permeable [P] or non-permeable [NP] lids. \u003cstrong\u003e(a, d) \u003c/strong\u003eAverage shoot and root fresh weight per plant. \u003cstrong\u003e(b, e) \u003c/strong\u003eTotal number of leaves per plant. \u003cstrong\u003e(c, f) \u003c/strong\u003eRelative proportion of leaves categorized by leaf area (see legend). Data are presented as means ± SE (a, c, d and f) or as boxplots (b, e) (n = 15–16 for a, b, c ; n = 16–19 for d, e, f). Statistical significance was determined by ANOVA, Kruskal-Wallis Z-test: ns - non-significant, (*) P ≤ 0.1, * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9573528/v1/e0fe52107bc94f2db9bb2b52.jpg"},{"id":109172067,"identity":"9e87f798-ce34-4354-a1a2-22d3dcde86c6","added_by":"auto","created_at":"2026-05-13 09:02:22","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":63537,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eDe novo \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eshoot bud regeneration as affected by the pre-cultivation regime and leaf position on source plant. \u003c/strong\u003eRegeneration frequency of explants irrespective of the leaf insertion on the source plant (\u003cstrong\u003ea\u003c/strong\u003e), leaf explants arisen from the upper (\u003cstrong\u003eb\u003c/strong\u003e) and lower part (\u003cstrong\u003ec\u003c/strong\u003e) of the source plant. Source plants were maintained under three regimes: \u003cstrong\u003e2% 25 °C NP\u003c/strong\u003e: 25 °C, Magenta vessels (90 mL MS, 2% sucrose, non-permeable lid); \u003cstrong\u003e2% 21 °C P\u003c/strong\u003e: 21 °C, Erlenmeyer flasks (25 mL MS, 2% sucrose, permeable lid); \u003cstrong\u003e0% 21 °C P\u003c/strong\u003e: 21 °C, Erlenmeyer flasks (25 mL MS, 0% sucrose, permeable lid). Data present frequency of leaf explants forming shoot buds per plate (\u003cem\u003en\u003c/em\u003e = 4–8). Asterisks indicate statistical significance determined by ANOVA, Kruskal-Wallis Z-test: ns - non-significant, * \u003cem\u003eP\u003c/em\u003e ≤ 0.05.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9573528/v1/52e7a2b7d8aa57635cd2206b.jpg"},{"id":109172158,"identity":"48792285-3510-41fd-b7c4-df6537187f1e","added_by":"auto","created_at":"2026-05-13 09:03:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":352027,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9573528/v1/6dc33f65-1996-46d3-a030-066e514ca270.pdf"},{"id":109172089,"identity":"e53b273c-36a1-4d43-880d-65b46936b116","added_by":"auto","created_at":"2026-05-13 09:02:32","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1673853,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterialS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-9573528/v1/276c23a3f5b773d99c8a768c.docx"},{"id":109172091,"identity":"692d3ef7-c200-4068-bae1-a9e153c667a2","added_by":"auto","created_at":"2026-05-13 09:02:34","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6716119,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-9573528/v1/5b687a566d9e73b9d5512381.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Modification of plant pre-cultivation alters regeneration competence in recalcitrant model Andean potato","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn potato research, \u003cem\u003eSolanum tuberosum\u003c/em\u003e ssp. \u003cem\u003eandigena\u003c/em\u003e, line 7540 serves as an indispensable model for tuberization signaling studies, which can guide potato breeding strategies in general (Dutt et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Hannapel et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Kondhare et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, line 7540 is a genotype recalcitrant to \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation, a challenge faced by various potato genotypes (Dale and Hampson \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Park et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Trujillo et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). The genetic improvement of many crops is frequently bottlenecked by low regeneration efficiencies. Standard approaches to overcome such recalcitrance typically rely mostly on time-consuming optimization of growth regulators treatment, a strategy that is costly and genotype-dependent (Dale and Hampson \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Heeres et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Park et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Sheerman and Bevan \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Wenzler et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). Alternatively, revisiting primary reports behind broad reviews (e.g. Vinterhalter et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) can reveal critical, yet often overlooked, constraints. To this end, the protocol established by Banerjee et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) explicitly identifies explant size as a decisive factor, stating that large leaf explants (0.5-1 cm\u0026sup2;) are critical for high regeneration frequencies. Also Park et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) observed different regeneration potential in cultivars that obviously differed in leaf size although the authors did not systematically evaluate this correlation. Recent studies demonstrate that plant morphology and physiology can be dramatically altered by modifications of \u003cem\u003ein vitro\u003c/em\u003e cultivation towards nature-like conditions via increase of air permeability and/or photoautotrophy (Ševč\u0026iacute;kov\u0026aacute; et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Vale et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Vollmer et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Thus, we propose an alternative strategy: manipulating the physiological status of the source plant itself by shifting plants from heterotrophic (sugar supply and tight non-permeable lid) to mixo- or even photoautotrophic (low or no sugar supply, air-permeable lid) conditions. Based on Banerjee et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), we hypothesized that the increased leaf size resulting from advanced plant cultivation would enlarge the pool of suitable-sized leaves and thus improve overall regeneration (and hence transformation) efficiency.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cp\u003eFor experiments, we used \u003cem\u003ein vitro\u003c/em\u003e grown \u003cem\u003eSolanum tuberosum\u003c/em\u003e ssp. \u003cem\u003eandigena\u003c/em\u003e, line 7540 (CGN 17637, Centre of Genetic Resources, Wageningen, NL). Plants were vegetatively propagated under \u003cem\u003ein vitro\u003c/em\u003e conditions from apical segments (three per flask) in 150-mL Erlenmeyer flasks (Kavalierglass, CZ) on MS medium (Sigma-Aldrich, USA; (Murashige and Skoog \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1962\u003c/span\u003e) supplemented with 2% (w/v) sucrose under a 16 h photoperiod at 21\u0026deg;C with irradiance 100 \u0026micro;mol\u0026middot;m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u0026middot;s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (daylight fluorescent tubes; Osram, Winterthur, Switzerland) and subcultivated every four weeks. For plant pre-cultivation, we compared three different regimes: heterotrophic control (MS medium, 2% (w/v) sucrose, non-permeable lid; 2% NP), a mixotrophic regime \u003cem\u003ein vitro\u003c/em\u003e (MS medium, 2% (w/v) sucrose, air-permeable lid; 2% P), and a photoautotrophic regime (MS medium, 0% sucrose, air-permeable lid; 0% P). The lid was prepared from a transparent polypropylene foil without (air non-permeable variant; NP) or with a hole (27.25 mm\u003csup\u003e2\u003c/sup\u003e) covered by one layer of a breathable tape (Leucopore, Duchefa) (air-permeable variants; P). Other cultivation conditions were identical with plant \u003cem\u003ein vitro\u003c/em\u003e maintenance. Selected growth parameters were evaluated after 3 weeks of cultivation and the experiment was repeated two and three times, for NP versus P lid variant and 0% versus 2% P variant, respectively. The total fresh biomass of both, shoots and roots as well as the leaf number was determined for each variant. Leaf area measurements were done using ImageJ software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.imagej.nih.gov\u003c/span\u003e\u003cspan address=\"http://www.imagej.nih.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e Rasband, W.S., ImageJ, US National Institutes of Health, Bethesda, MA) and leaves sorted into three groups: \u0026le; 0,5cm\u003csup\u003e2\u003c/sup\u003e, 0,5\u0026thinsp;\u0026minus;\u0026thinsp;1 cm\u003csup\u003e2\u003c/sup\u003e and \u0026ge;\u0026thinsp;1 cm\u003csup\u003e2\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe leaves of source plants (larger than 0,5 cm\u003csup\u003e2\u003c/sup\u003e) with different pre-cultivation history were used for the regeneration competence experiment (repeated twice): heterotrophic regime following Banerjee et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) (25\u0026deg;C, GA-7 Magenta vessels (Sigma-Aldrich, USA), 90 mL MS medium with 2% sucrose non-permeable lid; 25\u0026deg;C 2% NP), newly tested regimes - mixotrophic (21\u0026deg;C, Erlenmeyer flasks, 25 ml MS with 2% sucrose, air-permeable lid; 21\u0026deg;C 2% P) and photoautotrophic (21\u0026deg;C, Erlenmeyer flasks, 25 ml MS with 0% sucrose, air-permeable lid; 21\u0026deg;C 0% P). Transformation of leaves was performed using \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain C58C1 carrying empty vector pGreen0029 (kanamycin resistance, \u003cem\u003enptII\u003c/em\u003e), otherwise strictly following the transformation and regeneration protocol by Banerjee et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) - see Supplementary Material S1 for brief procedure description and media composition. Six weeks after the transfer on shoot inducing media, the number of regenerating explants (visible shoot buds present) was counted.\u003c/p\u003e \u003cp\u003eData distributions were presented either as box plots or bar charts with error bars that represent standard deviations. For statistical analysis, NCSS 9 statistical software (NCSS, LLC. Kaysville, Utah, USA) was employed, ANOVA-One-Way Analysis of Variance using Kruskal-Wallis Z-test. Statistical significance was determined at P\u0026thinsp;\u0026le;\u0026thinsp;0.001, P\u0026thinsp;\u0026le;\u0026thinsp;0.01, P\u0026thinsp;\u0026le;\u0026thinsp;0.05, and P\u0026thinsp;\u0026le;\u0026thinsp;0.1 levels marked by ***, **, *, and (*), respectively.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cp\u003ePrior to the specific experiments, we performed a preliminary screening to optimize the \u003cem\u003ein vitro\u003c/em\u003e growth of \u003cem\u003eSolanum tuberosum\u003c/em\u003e ssp. \u003cem\u003eandigena\u003c/em\u003e, line 7540. We tested various cultivation parameters, including vessel type, medium volume, MS salt strength, sucrose concentration, and temperature. The protocol by Banerjee et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) prescribes higher temperature (up to 27\u0026deg;C). Based on our long-term good experience with potato growth at lower temperatures, we compared cultivation at 21 and 25\u0026deg;C also for andigena and found 21\u0026deg;C superior for the plant growth and proportion of leaves larger than 0,5 cm\u003csup\u003e2\u003c/sup\u003e (61\u0026thinsp;\u0026plusmn;\u0026thinsp;3% (21\u0026deg;C) vs 32\u0026thinsp;\u0026plusmn;\u0026thinsp;3% (25\u0026deg;C); \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Nevertheless, most plants grown at 25\u0026deg;C remained healthy, validating the suitability of the 20\u0026ndash;25\u0026deg;C range generally observed for potato (Hussey and Stacey \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1981\u003c/span\u003e; Vinterhalter et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2008\u003c/span\u003e and references therein).\u003c/p\u003e \u003cp\u003ePublished data on modern potato repeatedly showed that the modification of cultivation regime by increased gas exchange significantly altered overall plant habitus (Hussey and Stacey \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1981\u003c/span\u003e; Chanemougasoundharam et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Ševč\u0026iacute;kov\u0026aacute; et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e)(. Thus, we proceeded with comparison of the morphology of andigena plants grown at 21\u0026deg;C under hetero-, mixo- and photoautotrophic conditions using non-permeable (NP) and air-permeable (P) lids. Total fresh biomass of both, shoots and roots, consistently higher under P treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). The P variant also tended to have a higher average number of leaves compared to their NP counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Importantly, there was a marked increase in leaf size of P variant resulting in formation of more suitable-sized leaves (\u0026gt;\u0026thinsp;0.5 cm\u0026sup2;) in P variant than in NP one that favoured production of smaller leaves (\u0026lt;\u0026thinsp;0.5 cm\u0026sup2;) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Furthermore, handling comfort, important factor for frequent subcultivations during transformation and subsequent regeneration process, was also improved as P lid-grown tissues were mechanically more resilient compared to the fragile, easily damageable tissues of NP variant. It confirms that improved gas exchange stimulates formation of nature-like leaves better withstanding mechanical constraints.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine whether exogenous sucrose might be beneficial even under improved ventilation, we compared the mixotrophic regime (2% sucrose, air-permeable; 2% P) with the photoautotrophic regime (0% sucrose, air-permeable; 0% P) under the same cultivation conditions. We observed no significant differences between the 2% and 0% sucrose P variants regarding the fresh biomass (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed), average number of leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee) or leaf size distribution (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). This suggests that under adequate irradiation and lid air-permeability, the plants are fully capable of sustaining growth through photosynthesis alone. The only significant deviation was observed in the root-to-shoot ratio, which was lower in the photoautotrophic variant (0.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 (0% P) vs 0.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 (2% P); \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.018), reflecting the dominant way of acquiring C source: photosynthesis or sugar uptake from the medium. Noteworthy, the leaves of mixotrophic plants exhibited a dark green colour (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), which indicates strengthened anthocyanin production, a common response to enhanced sugar availability (reviewed e.g. by Steyn et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Thus, photoauto- or mixotrophic cultivation of potato plants could help to meet the condition proposed by Banerjee et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) for achieving good results, i.e., using healthy leaves with a larger leaf area as starting material for the transformation process.\u003c/p\u003e \u003cp\u003eTo verify this assumption, we tested regeneration capacity of the newly established cultivation setupversus the original one established by Banerjee et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). After six weeks on shoot inducing media, the number of regenerating explants (visible shoot buds present) was compared. Surprisingly, despite the marked differences in the leaf area distribution, we observed the overall regeneration efficiency being comparable across variants (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). This result clearly indicates that the leaf size alone is not a proxy for regeneration competence. Instead, we found a surprising correlation between the pre-cultivation regime, the leaf insertion on source plants and regeneration efficiency. In autotrophic plants (0% P), the regeneration competence was position-dependent: while the explants arisen from the upper part of the source plantexhibited the highest regeneration rates (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), this potential was lost in the explants from lower part (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). In contrast, the standard heterotrophic control (2% NP) showed a reverse pattern, with the lowest efficiency in the leaves of the upper part. The differences in regeneration capacity of leaves of a given insertion in both arrangements (0% P versus 2% P) were statistically significant. Interestingly, the mixotrophic variant (2% P) consistently occupied an intermediate position, preserving regeneration competence along the entire shoot axis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Together, these results underscore the importance of the physiological state of leaves, which reflects the C and energy status. However, this characteristic depends on a combination of the influence of the cultivation regime (0% P, 2% NP, 2% P) and the position of the leaf on the stem. Establishing this optimal metabolic status is crucial, as the subsequent \u003cem\u003ede novo\u003c/em\u003e shoot organogenesis relies heavily on the explant's capacity to properly uptake and transduce morphogenic auxin and cytokinin signals (Raspor et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Previous studies showed a correlation between tissue/organ ageing and lowering of the regeneration potential in potato (Mohapatra and Batra \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Tavazza et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1989\u003c/span\u003e) as well as in other recalcitrant species ranging from legumes to conifers (Bidabadi and Jain \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Mahalakshmi et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Sainger et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Our data refine this view by demonstrating that this correlation is environmentally plastic. Under photoautotrophic conditions, photosynthesis promoted more rapid leaf development resulting in faster production of competent leaves. Conversely, heterotrophic conditions inverted this trend, favouring the lower leaves near the exogenous sugar. The advantage of both is combined in the 2% P variant where all leaves exhibit comparable regeneration competence. Ultimately, this proves that the window of competence is not immutable. By employing simple, low-input interventions\u0026mdash;such as changing pre-cultivation regime of source plants by enabling air-permeability and/or adjusting sucrose supply\u0026mdash;researchers can shift this physiological window to align with their specific, often genotype-dependent, needs. To overcome genotype-dependent recalcitrance, researchers typically focus on optimizing the\u003cem\u003ein vitro\u003c/em\u003e regeneration phase itself. This often involves extensive adjustments to phytohormones and incubation parameters (Bakhsh \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Chauhan et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Kaur et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Kaur et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Molla et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Patel et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), or employing modifications such as substituting carbon sources (Hamama et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) or utilizing machine learning to balance macronutrients (Mahdavi et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). While these post-excision optimizations are undeniably valuable, our findings demonstrate that the physiological baseline of the source plant is equally critical. Whether aiming for the rapid multiplication in autotrophic systems or the axis-wide leaf competence of mixotrophic cultures, regeneration can be effectively fine-tuned through clever management of the source plant environment \u0026mdash;a strategy often overlooked even in comprehensive reviews (Vinterhalter et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). In conclusion, our results highlight that the optimal regeneration competence of leaf explants is not a fixed temporal marker but a variable dependent on the pre-cultivation and we propose modifications of pre-cultivation regime as a possible tool to improve regeneration efficiency also suitable for other recalcitrant species.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eAuthor contribution statement\u003c/h2\u003e \u003cp\u003eAll authors contributed to the study conception and design. J. K. performed cultivations and data analyses and wrote the first manuscript version. All authors contributed to editing and finalization of the manuscript.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompliance with Ethical Standards\u003c/h2\u003e \u003cp\u003eThe authors have no competing interests to declare.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eacknowledgement statement\u003c/p\u003e \u003cp\u003eSupported by the by project no. 1522218 of the Grant Agency of Charles University, Czech Republic and by the project TowArds Next GENeration Crops [CZ.02.01.01/00/22_008/0004581] from the ERDF Programme Johannes Amos Comenius under the Ministry of Education, Youth and Sports of the Czech Republic.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors contributed to the study conception and design. J. K. performed cultivations and data analyses and wrote the first manuscript version. All authors contributed to editing and finalization of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe Agrobacterium tumefaciens strain C58C1 and the empty vector pGreen0029 were kindly provided by Dr. Luk\u0026aacute;š Fischer, Charles University.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe raw datasets to Figures are freely available in the Zenodo repository (https://doi.org/ 10.5281/zenodo.19846193).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBakhsh A. (2020). Development of Efficient, Reproducible and Stable Agrobacterium-Mediated Genetic Transformation of Five Potato Cultivars. Food Technol Biotechnol 58 (1):57-63. doi:10.17113/ftb.58.01.20.6187\u003c/li\u003e\n\u003cli\u003eBanerjee A.K., S. Prat, D.J. Hannapel. (2006). 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Ninković (2008) Protocols for Agrobacterium-mediated Transformation of Potato. In: Benkeblia N, Tennant P (eds) Potato I. Fruit, Vegetable and Cereal Science and Biotechnology, vol 2 (Special Issue 1). Global Science Books, pp 1-15\u003c/li\u003e\n\u003cli\u003eVollmer R., J. Espirilla, A. Espinoza, R. Villagaray, M. Castro, S. Pineda, J.C. Sanchez, A.F.S. Mello, V.C.R. Azevedo. (2024). Effect of Gas Exchange Rate, Vessel Type, Planting Density, and Genotype on Growth, Photosynthetic Activity, and Ion Uptake of In Vitro Potato Plants. Plants-Basel 13 (19). doi:10.3390/plants13192830\u003c/li\u003e\n\u003cli\u003eWenzler H., G. Mignery, G. May, W. Park. (1989). A rapid and efficient transformation method for the production of large numbers of transgenic potato plants. Plant Sci 63 (1):79-85. doi:10.1016/0168-9452(89)90103-9\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Agrobacterium-mediated transformation, air-permeable lid, de novo regeneration, in vitro tissue culture, Solanum tuberosum ssp. andigena line 7540, trophic status","lastPublishedDoi":"10.21203/rs.3.rs-9573528/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9573528/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eSolanum tuberosum\u003c/em\u003e ssp. \u003cem\u003eandigena\u003c/em\u003e line 7540, a key model for tuberization, exhibits a recalcitrance to \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation. This study aimed to improve leaf regeneration efficiency by modifying the source plant's pre-cultivation. We compared plants cultivated \u003cem\u003ein vitro\u003c/em\u003e under standard heterotrophic conditions (non-permeable lids, 2% sucrose) and under mixotrophic and photoautotrophic regimes (air-permeable lids, 2% or 0% sucrose). The use of air-permeable lids significantly increased biomass and leaf size. Moreover, we found a link between plant pre-cultivation and leaf physiological state. In photoautotrophic plants, regeneration competence was strongly favoured in leaf explants from upper half of source plants, while heterotrophic control exhibited an opposite behaviour. Mixotrophic variant preserved regeneration competence along the shoot axis. In conclusion, the competence for regeneration varies with tissue physiological status and thus can be shifted via low-input adjustment of the source plant pre-cultivation. This approach might prove beneficial also for regeneration of other difficult-to-master crops.\u003c/p\u003e","manuscriptTitle":"Modification of plant pre-cultivation alters regeneration competence in recalcitrant model Andean potato","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-13 08:59:49","doi":"10.21203/rs.3.rs-9573528/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"42e3e086-088a-4a8b-868b-58fa43f80f66","owner":[],"postedDate":"May 13th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"255409861096917392300408005355134697750","date":"2026-05-15T15:24:54+00:00","index":21,"fulltext":""},{"type":"reviewerAgreed","content":"112460109302836131877461227666719504827","date":"2026-05-14T08:36:35+00:00","index":20,"fulltext":""},{"type":"reviewersInvited","content":"11","date":"2026-05-05T03:16:45+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-05-01T04:46:56+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-05-01T04:46:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"American Journal of Potato Research","date":"2026-04-30T07:15:16+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-13T08:59:49+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-13 08:59:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9573528","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9573528","identity":"rs-9573528","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}