The association of fraser photinia and its beneficial bacterium (PGB_invit) provided in vitro storage without subculture

  • Irmak Şah
  • Hülya Akdemir
  • Ergun Kaya
  • Özlem AkkayaEmail author
  • Yelda Özden ÇiftçiEmail author
Original Article


Endophytes play crucial roles due to their beneficial influence on plant development, growth, fitness, and diversification. Due to these important capabilities, they have received attention from the scientific community and many papers have been published recently about their beneficial role in in vivo and in vitro plant propagation. However, up to now, there is no research on utilization of these microbial endophytes in prolongation of in vitro storage. Thus, the aim of this study is to assess the influence of fraser photinia associated and putatively endophytic bacterium (Plant Growth Bacteria_ in vitro; PGB_invit) on in vitro storage of its host. When pure strain of the bacterium was inoculated, it enabled the storage of microshoots up to 16 months at 25 °C without requiring periodic subculture while control (unincubated with PGB_invit.) microshoots died after 2 months of storage without subculture as in vitro plant cultures definitely need periodic subcultures (once in every 4–6 weeks) in order to renew media and gaseous atmosphere. Moreover, while the presence of virulence (vir D1), auxin (aux1), and cytokinin (ipt) production genes was confirmed in plasmid DNA of the bacterium, nitrogen fixing gene (nifH) was detected by the PCR analysis using bacterial culture. Overall results demonstrated that with these capabilities PGB_invit could be useful for in vitro conservation of fraser photinia.

Key message

The novelty is the supplementation of in vitro plant growth without either periodic renewal of the media or decreasing the culture temperature by means of a beneficial plant-bacterium interaction.


Aux1 Endophytic Ipt NifH Plant growth promoting bacterium 


Author contributions

YÖÇ and ÖA designed the research project; HA and EK carried out the plant storage analyses; IS and ÖA carried out the molecular analysis; IS, ÖA and YÖÇ wrote the paper.


This research was funded by a grant from TUBITAK (#KBAG 114Z579). A partial support was also obtained from Gebze Technical University (2014-A-09).

Compliance with ethical standards

Conflict of interest

There is no conflict of interest.


  1. Akdemir H, Kaya E, Ozden Y (2010) In vitro proliferation and minimum growth storage of fraser photinia: influences of different medium, sugar combinations and culture vessels. Sci Hortic 126:268–275. CrossRefGoogle Scholar
  2. Akkaya Ö, Gül Şeker M, Özden Çiftçi Y (2019) Plant growth promoting microbiome network. In: Dr SJ (ed) Microbes and the environment: plant-soil-microbe-environment interactions on agricultural production, pollution management and waste recycling towards sustainable development (in press)Google Scholar
  3. Aly AH, Debbab A, Kjer J, Proksch P (2010) Fungal endophytes from higher plants: a prolific source of phytochemicals and other bioactive natural products. Fungal Divers 41:1–16. CrossRefGoogle Scholar
  4. Arshad M, Frankenberger WT (1991) Microbial production of plant hormones. In: Keister DL, Cregan PB (eds) The rhizosphere and plant growth, Kluwer Academic Publishers, Dordrecht, pp 327–334. CrossRefGoogle Scholar
  5. Azevedo JL, Maccheroni Junior W, Pereira JO, Araújo WL (2000) Endophytic microrganisms: a review on insect control and recent advances on tropical plants. Electron J Biotechnol 3:40–65. CrossRefGoogle Scholar
  6. Bashan Y, Holguin G (1997) Azosprillum-plant relationships: environmental and physiological advances (1990–1996). Can J Microbiol 43:103–121. CrossRefGoogle Scholar
  7. Benhamou N, Kloepper JW, Quadth-Hallman A, Tuzun S (1996) Induction of defense-relted ultrastructural modifications in pea root tissues inoculated with endophytic bacteria. Plant Physiol 112:919–929. CrossRefGoogle Scholar
  8. Boddey RM, Urquiaga S, Reis V, Döbereiner J (1991) Biological nitrogen fixation associated with sugar cane. Plant Soil 137:111–117. CrossRefGoogle Scholar
  9. Borque D, Pomerleau Y, Groleau D (1995) High cell density production of polybhydroxybutyrate (PHB) from methanol by Methylobacterium extorquens production of high molecular mass PHB. Appl Microbiol Biotechnol 44:367–376. CrossRefGoogle Scholar
  10. Brader G, Compant S, Mitter B, Trognitz F, Sessitsch A (2014) Metabolic potential of endophytic bacteria. Curr Opin Biotechnol Sci 27:30–37. CrossRefGoogle Scholar
  11. Broothaerts W, Mitchell HJ, Weir B, Kaines S, Smith LMA, Yang W, Mayer JE, Roa-Rodriguez C, Jefferson RA (2005) Gene transfer to plants by diverse species of bacteria. Nature 433:629–633. CrossRefGoogle Scholar
  12. Cabanas CGL, Schiliro E, Valverde-Corredor A, Mercado-Blanco J (2014) The biocontrol endophytic bacterium Pseudomonas fluorescens PICF7 induces systemic defense responses in aerial tissues upon colonization of olive roots. Front Microbiol 5:1–14. Google Scholar
  13. Camilleri C, Jouanin L (1991) The TR_DNA region carrying the auxin synthesis genes of the Agrobacterium rhizogenes agronine-type plasmid pRiA4: nucleotide sequence analysis and introduction into tobacco plants. Mol Plant Microbe Interact 4:155–162. CrossRefGoogle Scholar
  14. Card S, Johnson L, Teasdale S, Caradus J (2016) Deciphering endophyte behavior: the link between endophyte biology and efficacious biological control agents. FEMS Microbiol Ecol 92:1–19. CrossRefGoogle Scholar
  15. Dias ACF, Costa FEC, Andreote FD, Lacava PT, Teixeira MA, Assumpçao LC, Araujo WL, Azevedo JL, Melo IS (2009) Isolation of micropropagated strawberry endophytic bacteria and assessment of their potential for plant growth promotion. World J Microbiol Biotechnol 25:189–195. CrossRefGoogle Scholar
  16. Divakaran M, Babu KN, Peter KV (2006) Conservation of Vanilla species in vitro. Sci Hortic 110:175–180. CrossRefGoogle Scholar
  17. Fahey JW, Dimock MB, Tomasino SF, Taylor JM, Carlson PS (1991) Genetically engineered endophytes as biocontrol agents: a case study in industry. In: Brock TD (ed) Microbial ecology of leaves. Springer, New York, pp 402–411. Google Scholar
  18. George EF, Hall MA, De Klerk GJ (2008) Plant propagation by tissue culture. Springer, Dordrecht. Google Scholar
  19. Gond SK, Bergen MS, Torres MS, White JF (2015) Endophytic Bacillus spp. produce antifungal lipopeptides and induce host defence gene expression in maize. Microbiol Res 172:79–87. CrossRefGoogle Scholar
  20. Gül-Şeker M, Şah I, Kırdök E, Ekinci H, Özden-Çiftçi Y, Akkaya Ö (2017) A hidden plant growth promoting bacterium isolated from in vitro cultures of fraser photinia. Int J Agric Biol 19:1511–1519. Google Scholar
  21. Haas JH, Moore LW, Ream W, Manulis S (1995) Universal PCR primers for detection of phytopathogenic Agrobacterium strains. Appl Environ Microbiol 61:2879–2884Google Scholar
  22. Hallman J, Quadt-Hallmann A, Mahaffee WF, Kloepper JW (1997) Bacterial endophytes in agricultural crops. Can J Microbiol 43:895–914. CrossRefGoogle Scholar
  23. Hardoim PR, van Overbeek LS, van Elsas JD (2008) Properties of bacterial endophytes and their proposed role in plant growth. Trends Microbiol 16:463–471. CrossRefGoogle Scholar
  24. Hardoim PR, van Overbeek LS, Berg G, Pırttila AM, Compant S, Campisano A, Döring M, Sessitsch A (2015) The hidden world within plants: ecological and evolutionary considerations for defining functioning of microbial endophytes. Microbiol Mol Biol Rev 79:293–320. CrossRefGoogle Scholar
  25. Herman EB (2004) Recent advances in plant tissue culture viii. Microbial contaminants in plant tissue cultures: solutions and opportunities 1996–2003. Agritech Consultants, Inc, New YorkGoogle Scholar
  26. Iniguez AL, Dong Y, Triplett EW (2004) Nitrogen fixation in wheat provided by Klebsiella pneumonia 342. Mol Plant Microbe Interact 17:1078–1085. Scholar
  27. Ivanova EG, Doronina NV, Shepelyakovskaya AO, Laman AG, Brovko FA, Trotsenko YuA (2000) Facultative and obligate aerobic methylobacteria synthesize cytokinins. Mikrobiologiya 69:764–769. Google Scholar
  28. Ivanova EG, Doronina NV, Trotsenko YA (2001) Aerobic methylobacteria are capable of synthesizing auxins. Microbiology 70:392–397. CrossRefGoogle Scholar
  29. James EK (2000) Nitrogen fixation in endophytic and associative symbiosis. Field Crops Res 65:197–209. CrossRefGoogle Scholar
  30. Jasim B, Geethu PR, Mathew J, Radhakrishnan EK (2015) Effect of endophytic Bacillus sp. from selected medicinal plants on growth promotion and diosgenin production in Trigonella foenum graecum. Plant Cell Tissue Organ Cult 122:565–572. CrossRefGoogle Scholar
  31. Jimtha JC, Smitha PV, Anisha C, Deepthi T, Meekha G, Radhakrishnan EK, Gayatri GP, Remakanthan A (2014) Isolation of endophytic bacteria from embryogenic suspension culture of banana and assessment of their plant growth promoting properties. Plant Cell Tissue Organ Cult 118:57–66. CrossRefGoogle Scholar
  32. Khosravi-Darani K, Mokhtari Z, Amai T, Tanaka K (2013) Microbial production of poly (hydroxybutyrate) from C1 carbon sources. Appl Microbiol Biotechnol 97:1407–1424. CrossRefGoogle Scholar
  33. Kim S, Lowman S, Hou G, Nowak J, Flinn B, Mei C (2012) Growth promotion and colonization of switchgrass (Panicum virgatum) cv. Alamo by bacterial endophyte Burkholderia phytofirmans strain PsJN. Biotechnol Biofuels 5:37. CrossRefGoogle Scholar
  34. Koenig RL, Morris RO, Polacco JC (2002) tRNA is the source of low-level trans-zeatin production in Methylobacterium spp. J Bacteriol 184:1832–1842. CrossRefGoogle Scholar
  35. Koutsompogeras P, Kyriacou A, Zabetakis I (2007) The formation of 2,5-dimethyl-4-hydroxy-2H-furan-3-one by cell free extracts of Methylobacterium extorquens and strawberry (Fraga-ria ananassa cv. Elsanta). Food Chem 104:1654–1661. CrossRefGoogle Scholar
  36. Kovalchuk I, Lyudvikova Y, Volgina M, Reed BM (2009) Medium, container and genotype all influence in vitro cold storage of apple germplasm. Plant Cell Tissue Org Cult 96:127–136. CrossRefGoogle Scholar
  37. Kulkarni VM, Ganapathi TR, Bapat VA, Rao PS (2004) Establishment of cell-suspension cultures in banana cv. Gr and Naine and evaluation of its sensitivity to gamma-irradiation. Curr Sci 86:902–904Google Scholar
  38. Lambardi M, Sharma KK, Thorpe TA (1993) Optimization of in vitro bud induction and plantlet formation from mature embryos of Aleppo pine (Pinus halepensis Mill.). In Vitro Cell Dev Biol 29:189–199. CrossRefGoogle Scholar
  39. Leifert C, Cassells AC (2001) Microbial hazards in plant tissue and cell cultures. In vitro Cell Dev Biol Plant 37:133–138. CrossRefGoogle Scholar
  40. Leifert C, Morris CE, Waites WM (1994) Ecology of microbial saprophytes and pathogens in tissue culture and field grown plants. CRC Crit Rev Plant Sci 13:139–183. CrossRefGoogle Scholar
  41. Lemoigne M (1926) Produit de d´eshydratation et de polym´erisation de l’acide b-oxybutyrique. Bull Soc Chem Biol 8:770–782Google Scholar
  42. Malboobi MA, Owlia P, Behbahani M, Srakhani E, Moradi S, Yakhchali B (2009) Solubilization of organic and inorganic phosphates by three highly efficient soil bacterial isolates. World J Microbiol Biotechnol 25:1471–1477. CrossRefGoogle Scholar
  43. Malik KA, Bilal R, Mehnaz S, Rasul G, Mirza MS, Ali S (1997) Association of nitrogen-fixing, plant-growth-promoting rhizobacteria (PGPR) with Kallar grass and rice. Plant Soil 194:37–44. CrossRefGoogle Scholar
  44. Marascuilo LA, McSweeney M (1977) Nonparametric and distribution-free method for the social sciences. CA Brooks/Cole Publishing Company, MontereyGoogle Scholar
  45. Mbai FN, Magiri EN, Matiru VN, Ng’ang’a J, Nyambati VCS (2013) Isolation and characterization of bacterial root endophytes with potential to enhance plant growth from Kenyan basmati rice. Am Int J Contemp Res 3:25–40. Google Scholar
  46. Mercado-Blanco J (2015) Life of microbes inside the plant. In: Lugtenberg B (ed) Principles of plant-microbe interactions. Springer, Switzerland, pp 25–32. Google Scholar
  47. Muromtsev GS, Chkanikov DI, Kulaeva ON, Gamburg KZ (1987) Osnovy khimicheskoi regulyatsii rosta I productivnosti rastenii (Basics of chemical regulation of plant growth and productivity). Agropromizdat, MoscowGoogle Scholar
  48. Nair DN, Padmavathy S (2014) Impact of endophytic microorganisms on plants, environment and humans. Sci World J 2014:1–12. CrossRefGoogle Scholar
  49. Negash A, Krens F, Schaart J, Vısser B (2001) In vitro conservation of enset under slow-growth conditions. Plant Cell Tissue Organ Cult 66:107–111. CrossRefGoogle Scholar
  50. Negri V, Tosti N, Standardi A (2000) Slow-growth storage of single node shoots of apple genotypes. Plant Cell Tissue Organ Cult 62:159–162. CrossRefGoogle Scholar
  51. Ozden-Tokatli Y, Akdemir H, Tilkat E, Onay A (2010) Current status and conservation of Pistacia germplasm. Biotechnol Adv 28:130–141. CrossRefGoogle Scholar
  52. Patle PN, Navnage NP, Ramteke PR (2018) Endophytes in plant system: roles in growth promotion, mechanism and their potentiality in achieving agriculture sustainability. Int J Chem Stud 6:270–274Google Scholar
  53. Perez-Rosales E, Alcaraz-Meléndez L, Puente ME, Vázquez-Juárez R, Zenteno-Savín T, Morales-Bojórquez E (2018) Endophytic bacteria isolated from wild jojoba (Simmondsia chinensis L. [Schneider]) roots improve in vitro propagation. Plant Cell Tissue Organ Cult 135:515–522. CrossRefGoogle Scholar
  54. Pham NT, Meier-Dinkel A, Höltken AM, Quambusch M, Mahnkopp F, Winkelmann T (2017) Endophytic bacterial communities in in vitro shoot cultures derived from embryonic tissue of hybrid walnut (Juglans x intermedia). Plant Cell Tissue Organ Cult 130:153–165. CrossRefGoogle Scholar
  55. Pirttilä AM, Laukkanen H, Pospiech H, Myllyla R, Hohtola A (2000) Detection of intracellular bacteria in the buds of Scotch pine (Pinus sylvestris L.) by in situ hybridization. Appl Environ Microbiol 66:3073–3077. CrossRefGoogle Scholar
  56. Quambusch M, Pirttila AM, Tejesvi MV, Winkelmann T, Bartsch M (2014) Endophytic bacteria in plant tissue culture: differences between easy- and difficult-to-propagate Prunus avium genotypes. Tree Physiol 34:524–533. CrossRefGoogle Scholar
  57. Quambusch M, Brümmer J, Haller K, Winkelmann T, Bartsch M (2016) Dynamics of endophytic bacteria in plant in vitro culture—quantification of three bacterial strains in Prunus avium in different plant organs and in vitro culture phases. Plant Cell Tissue Organ Cult 126:305–317. CrossRefGoogle Scholar
  58. Quoirin M, Lepoivre P (1977) Etude de milieux adaptés aux cultures in vitro de Prunus sp. Acta Hortic 78:437–442CrossRefGoogle Scholar
  59. Reinhold-Hurek B, Hurek (1998) Life in grasses: diazotrophic endophytes. Trends Microbiol 139:1–6. Google Scholar
  60. Remy W, Taylor TN, Haas H, Kerp H (1994) Four hundred-million-year-old vesicular arbuscular mycorrhizae. Proc Natl Acad Sci 91:11841–11843. CrossRefGoogle Scholar
  61. Rogowsky PM, Powell BS, Shirasu K, Lin TS, Morel P, Zyprian EM, Steck TR, Kado CI (1990) Molecular characterization of the vir regulon of Agrobacterium tumefaciens: complete nucleotide sequence and gene organization of the 28.63-kbp regulon cloned as a single unit. Plasmid 23:85–106. CrossRefGoogle Scholar
  62. Ryan RP, Germaine K, Franks A, Ryan DJ, Dowling DN (2008) Bacterial endophytes: recent developments and aplications. FEMS Microbiol Lett 278:1–9. CrossRefGoogle Scholar
  63. Santoyo G, Moreno-Hagelsieb G, Orozco-Mosqueda C, Glick BR (2016) Plant growth-promoting bacterial endophytes. Microbiol Res 183:92–99. CrossRefGoogle Scholar
  64. Sarkar D, Naik PS (1998) Factors affecting minimal growth conservation of potato microplants in vitro. Euphytica 102:275–280CrossRefGoogle Scholar
  65. Sarkar D, Kaushik SK, Naik PS (1999) Minimal growth conservation of potato microplants silver thiosulfate reduces ethylene-induced growth abnormalities during prolonged storage in vitro. Plant Cell Rep 18: 897–903CrossRefGoogle Scholar
  66. Schulz B, Boyle C, Draeger S, Römmert AK, Krohn K (2002) Endophytic fungi: a source of novel biologically active secondary metabolites. Mycol Res 106:996–1004. CrossRefGoogle Scholar
  67. Singh M, Kumar A, Singh R, Pandey KD (2017) Endophytic bacteria: a new source of bioactive compounds. 3Biotech 7:1–14. Google Scholar
  68. Thomas P (2004a) In vitro decline in plant cultures: detection of a legion of covert bacteria as the cause for degeneration of long-term micropropagated triploid watermelon cultures. Plant Cell Tissue Org Cult 77:173–179. CrossRefGoogle Scholar
  69. Thomas P (2004b) A three-step screening procedure for detection of covert and endophytic bacteria in plant tissue cultures. Curr Sci 87:67–72Google Scholar
  70. Thomas P (2007) Isolation and identification of five alcohol defying Bacillus spp. covertly associated with in vitro culture of seedless watermelon. Curr Sci 92:983–987Google Scholar
  71. Thomas P, Prabhakara BS, Pitchaimuthu M (2006) Cleansing the long-term micropropagated triploid watermelon cultures from covert bacteria and field testing the plants for clonal fidelity and fertility during the 7–10 year period in vitro. Plant Cell Tissue Org Cult 85:317–329. CrossRefGoogle Scholar
  72. Thomas P, Swarna GK, Roy PK, Patil P (2008) Identification of culturable and originally non-culturable endophytic bacteria isolated from shoot tip cultures of banana cv. Grand Naine. Plant Cell Tissue Organ Cult 93:55–63. CrossRefGoogle Scholar
  73. Triplett EW (1996) Diazotrophic endophytes: progress and prospects for nitrogen fixation in monocots. Plant Soil 186:29–38. CrossRefGoogle Scholar
  74. Vigani G, Rolli E, Marasco R, Dell’Orto M, Michoud G, Soussi A, Raddadi N, Borin S, Sorlini C, Zocchi G, Daffonchio D (2018) Root bacterial endophytes confer drought resistance and enhance expression and activity of a vacuolar H+- pumping pyrophosphatease in pepper plants. Environ Microbiol. Google Scholar
  75. Wang K, Herrera-Estrella A, Van Montagu M (1990) Overexpression of virD1 and virD2 genes in Agrobacterium tumefaciens enhances T-complex formation and plant transformation. J Bacteriol 172:4432–4440. CrossRefGoogle Scholar
  76. Wilson D (1995) Endophyte-the evolution of a term and clarification of its use and definition. Oikos 73:274–276. CrossRefGoogle Scholar
  77. Yuan J, Zhou JY, Li X, Dai CC (2016) The primary mechanism of endophytic fungus Gilmaniella sp. AL12 promotion of plant growth and sesquiterpenoid accumulation in Atractylodes lancea. Plant Cell Tissue Organ Cult 125:571–584. CrossRefGoogle Scholar
  78. Zabetakis I (1997) Enhancement of flavour biosynthesis from strawberry (Fragaria ananassa) callus cultures by Methylobacterium species. Plant Cell Tissue Org Cult 50:179–183. CrossRefGoogle Scholar

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© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Department of Molecular Biology and GeneticsGebze Technical UniversityGebzeTurkey
  2. 2.Department of Molecular Biology and GeneticsMuğla Sıtkı Koçman UniversityMuğlaTurkey

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