Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Comprehensive analysis of in vitro to ex vitro transition of tissue cultured potato plantlets grown with or without sucrose using metabolic profiling technique


This study elucidated the effect of exogenous sucrose on growth parameters and metabolic changes during the in vitro rooting (InVR) and the ex vitro acclimatization (ExVA) stages of potato (Solanum tuberosum L.). During InVR stage, plantlets were cultured on MS medium with 3 % (S+) or without (S−) sucrose, and were then acclimatized under the same ExVA condition. In InVR stage, S+ increased photosynthetic capacity (Amax) and dry matter percentage. Yet, no significant differences in the other growth parameters have been observed. During acclimatization, Amax and respiration were higher in ExVA compared to InVR plants. Most growth parameters were significantly higher in S+ plants. Principal component analysis and hierarchical cluster analysis of 108 metabolites identified by GC–MS clearly demonstrated that in vitro culture had a profound impact on metabolic profile. In vitro S− and S+ plantlets accumulated large quantities of amino acids (specially under S+), photorespiration intermediates, putrescine, tocopherol and organic acids, including oxalic and tartaric acid. However, glycolytic and TCA cycle intermediates were found in lower amount. Under InVR S+ conditions, proline, gamma-aminobutyric acid, sugars and sugar alcohols accumulated in larger amounts. InVR S− plantlets characteristically accumulated large quantity of urea. We suggest that ammonia metabolism was redirected towards urea biosynthesis through urea cycle to sequester nitrogen in condition of low carbon availability. In vitro conditions are causing major disruption in the cellular metabolism, which could produce serious consequences on the capacity of plantlets to adapt to uncontrolled growing conditions and may lead to poor development under these conditions.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7



Murashige and Skoog (1962) medium

Amax :

Light saturated photosynthesis


Culture medium without sugar


Culture medium with sugar


Gas chromatography–mass spectrometry


Tricarboxylic acid cycle


Urea cycle


Gamma-aminobutyric acid


Principal component analysis


3 Phosphoglyceric acid








  1. Alcázar R, Altabella T, Marco F, Bortolotti C, Reymond M, Koncz C, Carrasco P, Tiburcio AF (2010) Polyamines: molecules with regulatory functions in plant abiotic stress tolerance. Planta Med 231(6):1237–1249

  2. Ashraf M, Foolad M (2007) Roles of glycine betaine and proline in improving plant abiotic stress resistance. Environ Exp Bot 59(2):206–216

  3. Badr A, Angers P, Desjardins Y (2011) Metabolic profiling of photoautotrophic and photomixotrophic potato plantlets (Solanum tuberosum) provides new insights into acclimatization. Plant Cell Tissue Org Cult 107(1):13–24

  4. Beharrell B, MacFie J (1991) Consumer attitudes to organic foods. Br Food J 93(2):25–30

  5. Boardman NK (1977) Comparative photosynthesis of sun and shade plants. Annu Rev Plant Physiol 28(1):355–377

  6. Bohnert HJ, Jensen RG (1996) Strategies for engineering water-stress tolerance in plants. Trends Biotechnol 14(3):89–97

  7. Bouché N, Fromm H (2004) GABA in plants: just a metabolite? Trends Plant Sci 9(3):110–115

  8. Bouchereau A, Aziz A, Larher F, Martin-Tanguy J (1999) Polyamines and environmental challenges: recent development. Plant Sci 140:103–125

  9. Carvalho LC, Osório ML, Chaves MM, Amâncio S (2001) Chlorophyll fluorescence as an indicator of photosynthetic functioning of in vitro grapevine and chestnut plantlets under ex vitro acclimatization. Plant Cell Tissue Org Cult 67(3):271–280

  10. Castro A, Young M, Alvarenga A, Alves J (2001) Influence of photoperiod on the accumulation of allantoin in comfrey plants. Rev Bras Fisiol Veg 13:49–54

  11. Cha-um S, Kirdmanee C (2008) Effects of osmotic stress on proline accumulation, photosynthetic abilities and growth of sugarcane plantlets (Saccharum officinarum L.). Pak J Bot 40(6):2541–2552

  12. Choi M-Y, Choi W, Park JH, Lim J, Kwon SW (2010) Determination of coffee origins by integrated metabolomic approach of combining multiple analytical data. Food Chem 121(4):1260–1268

  13. Collakova E, DellaPenna D (2001) Isolation and functional analysis of homogentisate phytyltransferase from synechocystis sp. PCC 6803 and arabidopsis. Plant Physiol 127(3):1113–1124

  14. Cournac L, Dimon B, Carrier P, Lohou A, Chagvardieff P (1991) Growth and photosynthetic characteristics of Solanum tuberosum plantlets cultivated in vitro in different conditions of aeration, sucrose supply, and CO2 enrichment. Plant Physiol 97(1):112–117. doi:10.1104/pp.97.1.112

  15. Desjardins Y, Dubuc J, Badr A (2007) In vitro culture of plants: a stressful activity! Acta Hortic 812:29–50

  16. Doumett S, Lamperi L, Checchini L, Azzarello E, Mugnai S, Mancuso S, Petruzzelli G, Del Bubba M (2008) Heavy metal distribution between contaminated soil and Paulownia tomentosa, in a pilot-scale assisted phytoremediation study: influence of different complexing agents. Chemosphere 72(10):1481–1490

  17. Ehness R, Ecker M, Godt DE, Roitsch T (1997) Glucose and stress independently regulate source and sink metabolism and defense mechanism via signal transduction pathways involving protein phosphorylation. Plant Cell 9:1825–1845

  18. Eskling M, Åkerlund H-E (1998) Changes in the quantities of violaxanthin de-epoxidase, xanthophylls and ascorbate in spinach upon shift from low to high light. Photosynth Res 57(1):41–50

  19. Fait A, Fromm H, Walter D, Galili G, Fernie AR (2008) Highway or byway: the metabolic role of the GABA shunt in plants. Trends Plant Sci 13(1):14–19

  20. Foyer CH, Noctor G (2003) Redox sensing and signalling associated with reactive oxygen in chloroplasts, peroxisomes and mitochondria. Physiol Plant 119(3):355–364

  21. Foyer C, Rowell J, Walker D (1983) Measurement of the ascorbate content of spinach leaf protoplasts and chloroplasts during illumination. Planta 157(3):239–244

  22. Franceschi VR, Nakata PA (2005) Calcium oxalate in plants: formation and function. Annu Rev Plant Biol 56(1):41–71

  23. Gaspar T, Franck T, Bisbis B, Kevers C, Jouve L, Hausman JF, Dommes J (2002) Concepts in plant stress physiology. Application to plant tissue cultures. Plant Growth Regul 37(3):263–285

  24. George E, Hall M, De Klerk G (2007) Plant propagation by tissue culture: 1. The background. Springer, Dordrecht

  25. Gill SS, Tuteja N (2010) Polyamines and abiotic stress tolerance in plants. Plant Signal Behav 5(1):26–33

  26. Grigore M-N, Boscaiu Neagu MT, Vicente Meana Ó (2011) Assessment of the relevance of osmolyte biosynthesis for salt tolerance of halophytes under natural conditions. Eur J Plant Sci Biotech 5:12–19

  27. Grout BWW (1988) Photosynthesis of regenerated plantlets in vitro, and the stress of transplanting. Acta Hortic 230:129–135

  28. Hare PD, Cress WA, Van Staden J (1998) Dissecting the roles of osmolyte accumulation during stress. Plant, Cell Environ 21(6):535–553

  29. Shao H-b, Chu L-y, Shao M-a, Jaleel CA, Hong-mei M (2008) Higher plant antioxidants and redox signaling under environmental stresses. C R Biol 331(6):433–441

  30. Hdider C, Desjardins Y (1994) Effects of sucrose on photosynthesis and phosphoenolpyruvate carboxylase activity of in vitro cultured strawberry plantlets. Plant Cell Tissue Org Cult 36(1):27–33

  31. Hocking P (2001) Organic acids exuded from roots in phosphorus uptake and aluminum tolerance of plants in acid soils. Adv Agron 74:64–99

  32. Jeong ML, Jiang H, Chen H-S, Tsai C-J, Harding SA (2004) Metabolic profiling of the sink-to-source transition in developing leaves of quaking aspen. Plant Physiol 136(2):3364–3375

  33. Jo E-A, Tewari R, Hahn E-J, Paek K-Y (2009) In vitro sucrose concentration affects growth and acclimatization of Alocasia amazonica plantlets. Plant Cell Tissue Org Cult 96(3):307–315

  34. Joyce SM, Cassells AC, Jain SM (2003) Stress and aberrant phenotypes in vitro culture. Plant Cell Tissue Org Cult 74(2):103–121

  35. Kinnersley AM, Turano FJ (2000) Gamma aminobutyric acid (GABA) and plant responses to stress. Crit Rev Plant Sci 19(6):479–509

  36. Kovtun Y, Daie J (1995) End-product control of carbon metabolism in culture-grown sugar beet plants (molecular and physiological evidence on accelerated leaf development and enhanced gene expression). Plant Physiol 108(4):1647–1656

  37. Kozai T, Watanabe K, Jeong B (1995) Stem elongation and growth of Solanum tuberosum L. in vitro in response to photosynthetic photon flux, photoperiod and difference in photoperiod and dark period temperatures. Sci Hortic 64(1–2):1–9

  38. Levy D (1983) Water deficit enhancement of proline and α-amino nitrogen accumulation in potato plants and its association with susceptibility to drought. Physiol Plant 57(1):169–173

  39. Liu J-H, Kitashiba H, Wang J, Ban Y, Moriguchi T (2007) Polyamines and their ability to provide environmental stress tolerance to plants. Plant Biotechnol 24(1):117–126

  40. Mansour MMF (1998) Protection of plasma membrane of onion epidermal cells by glycinebetaine and proline against NaCl stress. Plant Physiol Biochem 36(10):767–772

  41. Martin-Tanguy J (2001) Metabolism and function of polyamines in plants: recent development (new approaches). Plant Growth Regul 34(1):135–148

  42. Morcuende R, Krapp A, Hurry V, Stitt M (1998) Sucrose-feeding leads to increased rates of nitrate assimilation, increased rates of -oxoglutarate synthesis, and increased synthesis of a wide spectrum of amino acids in tobacco leaves. Planta 206(3):394–409

  43. Muller-Moule P, Golan T, Niyogi KK (2004) Ascorbate-deficient mutants of arabidopsis grow in high light despite chronic photooxidative stress. Plant Physiol 134(3):1163–1172

  44. Munné-Bosch S, Alegre L (2002) Interplay between ascorbic acid and lipophilic antioxidant defences in chloroplasts of water-stressed arabidopsis plants. FEBS Lett 524(1–3):145–148

  45. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15(3):473–497

  46. Noctor G (2006) Metabolic signalling in defence and stress: the central roles of soluble redox couples. Plant, Cell Environ 29(3):409–425

  47. Noiraud N, Maurousset L, Lemoine R (2001) Transport of polyols in higher plants. Plant Physiol Biochem 39(9):717–728

  48. Obata T, Fernie AR (2012) The use of metabolomics to dissect plant responses to abiotic stresses. Cell Mol Life Sci 69(19):3225–3243

  49. Pospisilova J, Ticha I, Kadlecek P, Haisel D, Plzakova S (1999) Acclimatization of micropropagated plantlets to ex vitro conditions. Biol Plant 42(4):481–487

  50. Premakumar A, Mercado JA, Quesada MA (2001) Effects of in vitro tissue culture conditions and acclimatization on the content of Rubisco, leaf soluble proteins, photosynthetic pigments, and C/N ratio. J Plant Physiol 158(7):835–840

  51. Reinbothe H, Mothes K (1962) Urea, ureides, and guanidines in plants. Annu Rev Plant Physiol 13(1):129–149

  52. Rodziewicz P, Swarcewicz B, Chmielewska K, Wojakowska A, Stobiecki M (2014) Influence of abiotic stresses on plant proteome and metabolome changes. Acta Physiol Plant 36(1):1–19

  53. Roessner U, Wagner C, Kopka J, Trethewey R, Willmitzer L (2000) Simultaneous analysis of metabolites in potato tuber by gas chromatography–mass spectrometry. Plant J 23(1):131–142

  54. Roessner-Tunali U, Hegemann B, Lytovchenko A, Carrari F, Bruedigam C, Granot D, Fernie AR (2003) Metabolic profiling of transgenic tomato plants overexpressing hexokinase reveals that the influence of hexose phosphorylation diminishes during fruit development. Plant Physiol Biochem 133(1):84–99

  55. Rolland F, Baena-Gonzalez E, Sheen J (2006) Sugar sensing and signaling in plants: conserved and novel mechanisms. Annu Rev Plant Biol 57:675–709

  56. Sakai T, Sakamoto T, Hallaert J, Vandamme EJ (1993) Pectin, pectinase, and protopectinase: production, properties, and applications. In: Saul N, Allen IL (eds) Advances in applied microbiology, vol 39. Academic Press, London, pp 213–294

  57. Shao H, Chu L (2005) Plant molecular biology in China: opportunities and challenges. Plant Molecular Biology Reporter 23(4):345–358

  58. Shelp BJ, Bown AW, McLean MD (1999) Metabolism and functions of gamma-aminobutyric acid. Trends Plant Sci 4(11):446–452

  59. Sima B, Desjardins Y (2001) Sucrose supply enhances phosphoenolpyruvate carboxylase phosphorylation level in in vitro Solanum tuberosum. Plant Cell Tissue Org Cult 67(3):235–242

  60. Sima B, Desjardins Y, Van Quy L (2001) Sucrose enhances phosphoenolpyruvate carboxylase activity of in vitro Solanum tuberosum L. under non-limiting nitrogen conditions. In Vitro Cell Dev Biol Plant 37(4):480–489

  61. Smith AM, Stitt M (2007) Coordination of carbon supply and plant growth. Plant, Cell Environ 30(9):1126–1149

  62. Stepansky A, Leustek T (2006) Histidine biosynthesis in plants. Amino Acids 30(2):127–142

  63. Stitt M, Müller C, Matt P, Gibon Y, Carillo P, Morcuende R, Scheible WR, Krapp A (2002) Steps towards an integrated view of nitrogen metabolism. J Exp Bot 53(370):959–970

  64. Taiz L, Zeiger E (2010) Plant physiology, 5th edn. Sinauer Associates Inc, Sunderland

  65. Tekam ML, Desire TV, Marius-Nicusor G, Maria ZM, Emmanuel Y, Akoa A (2014) Differential responses of growth, chlorophyll content, lipid peroxidation and accumulation of compatible solutes to salt stress in peanut (Arachis hypogaea L.) cultivars. Afr J Biotechnol 13(50):4577–4585

  66. Todd CD, Tipton PA, Blevins DG, Piedras P, Pineda M, Polacco JC (2006) Update on ureide degradation in legumes. J Exp Bot 57(1):5–12

  67. Valero-Aracama C, Wilson S, Kane M, Philman N (2007) Influence of in vitro growth conditions on in vitro and ex vitro photosynthetic rates of easy-and difficult-to-acclimatize sea oats (Uniola paniculata L.) genotypes. In Vitro Cell Dev Biol Plant 43(3):237–246

  68. Van Huylenbroeck JM, Debergh PC (1996) Impact of sugar concentration in vitro on photosynthesis and carbon metabolism during ex vitro acclimatization of Spathiphyllum plantlets. Physiol Plant 96(2):298–304

  69. Venkatesan A, Chellappan KP (1998) Accumulation of proline and glycine betaine in Ipomoea pes-caprae induced by NaCl. Biol Plant 41(2):271–276

  70. Wang P, Kong C, Hu F, Xu X (2007) Allantoin involved in species interactions with rice and other organisms in paddy soil. Plant Soil 296(1):43–51

  71. Witte C-P, Medina-Escobar N (2001) In-gel detection of urease with nitroblue tetrazolium and quantification of the enzyme from different crop plants using the indophenol reaction. Anal Biochem 290(1):102–107

Download references


Thanks to the Egyptian Higher Education and its Missions General Administration for their financial assistance. The authors also wish to thanks NSERC discovery Grant program for their financial support to Yves Desjardins.

Author information

Correspondence to Ashraf Badr.

Electronic supplementary material

Below is the link to the electronic supplementary material.


Changes in metabolites extracted from the leaves of potato (Solanum tuberosum L., cv Norland) grown on 3% and 0% sucrose during in vitro rooting and ex vitro acclimatization stages. The results are mean ± SD (n=15), error bars are not shown where they are smaller than the symbol. a, metabolites 1-36; b, metabolites 37-72 ; c, metabolites 73-107 (TIFF 55130 kb)


Changes in all identified metabolites recovered in methanolic extracts from leaves of potato (Solanum tuberosum L., cv Norland) grown on 3% sucrose, S+ (A, B and C) and 0% sucrose, S- (D, E and F) during in vitro rooting stage (A and D) and ex vitro acclimatization stage (B, C, E and F). Metabolites are shown in order of decreasing normalized peak area in in vitro S+ leaf (A) (TIFF 26721 kb)


PCA loading plot representing the contribution of individual metabolites to principal component clustering of potato leaves plantlet (Solanum tuberosum L., cv Norland) grown on 0% or 3% sucrose in in vitro rooting stage (TIFF 1259 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Badr, A., Angers, P. & Desjardins, Y. Comprehensive analysis of in vitro to ex vitro transition of tissue cultured potato plantlets grown with or without sucrose using metabolic profiling technique. Plant Cell Tiss Organ Cult 122, 491–508 (2015). https://doi.org/10.1007/s11240-015-0786-3

Download citation


  • Potato
  • Micropropagation
  • Stress
  • Gas chromatography–mass spectrometry
  • Principal component analysis
  • Hierarchical cluster analysis
  • Heat map