Optimizing factors for large-scale production of Arbuscular Mycorrhizal Fungi consortia using root organ cultures
Large-scale production of Arbuscular Mycorrhizal Fungi (AMF) consortia is a crucial stride in harnessing their potential for sustainable agriculture and plant growth enhancement. However, establishing optimal production conditions is challenging due to their obligate nature, variability, lack of standardized protocols, and limited understanding of their specific requirements. Previous attempts to standardize Root Organ Cultures (ROC) for AMF overlooked challenges related to viable inoculum production for field applications. This current investigation reported, for the first time, the optimization of various factors during large-scale production of AMF using ROC. By optimizing factors like gelling agents, media preparation, medium-to-inoculum ratios, incubation conditions, age, harvesting method and drying temperatures, we achieved significant yields of viable propagules. The standardized protocol outlined in this study will greatly influence commercial-scale AMF production. These standardized protocols are poised to contribute to larger-scale AMF production worldwide, with the potential to support sustainable agriculture and ecosystem management.
1. Mycorrhizal symbiosis. Elsevier eBooks. 2008. https://doi.org/10.1016/b978-0-12-370526-6.x5001-6
2. Biermann BJ, Linderman RG. Use of vesicular-arbuscular mycorrhizal roots, intraradical vesicles and extraradical vesicles as inoculum. New Phy. 1983 Sep;95(1):97–105. https://doi.org/10.1111/j.1469-8137.1983.tb03472.x
3. Ghorui M, Chowdhury S, Balu P, Krishnan K, Djearamanec S, Manjunathana J, Preethi N, Ashokkumar KM, Jayanthi M. A review: in vitro cultivation of Arbuscular Mycorrhizal Fungus for commercialization. Oxi Com. 2023;46(3):549–564.
4. Basiru S, Mwanza HP, Hijri M. Analysis of Arbuscular mycorrhizal fungal inoculant benchmarks. Microorganisms. 2020 Dec;9(1):81. https://doi.org/10.3390/microorganisms9010081
5. Adholeya A, Tiwari P, Singh R. Large-Scale inoculum production of arbuscular mycorrhizal fungi on root organs and inoculation strategies. Soil bio. 2005. p. 315–338. https://doi.org/10.1007/3-540-27331-x_17
6. Goh D, Martin JGA, Banchini C, MacLean AM, Stefani F. RocTest: A standardized method to assess the performance of root organ cultures in the propagation of arbuscular mycorrhizal fungi. Fro. in Micro. 2022 Jul 28;13. https://doi.org/10.3389/fmicb.2022.937912
7. Alok A. WO2013098829A1 - Novel mycorrhizae-based biofertilizer compositions and method for mass production and formulations of same - Google Patents. 2011. https://patents.google.com/patent/WO2013098829A1/en
8. Bécard G, Fortin JA. Early events of vesicular–arbuscular mycorrhiza formation on Ri T‐DNA transformed roots. New Phytologist. Wiley-Blackwell; 1988 Feb;108(2):211–218. https://doi.org/10.1111/j.1469-8137.1988.tb03698.x
9. Sandle T PhD. Assessment of Culture Media in Pharmaceutical Microbiology. https://www.americanpharmaceuticalreview.com/Featured-Articles/163589-Assessment-of-Culture-Media-in-Pharmaceutical-Microbiology/
10. Doner LW, Bécard G. Solubilization of gellan gels by chelation of cations. Biotech. Tech.; 1991 Jan;5(1):25–28. https://doi.org/10.1007/bf00152749
11. Sylvia DM, Jarstfer AG. Sheared-Root inocula of Vesicular-Arbuscular mycorrhizal fungi. Applied and Environmental Microbiology. Ame. Soc. for Micro.; 1992 Jan;58(1):229–232. https://doi.org/10.1128/aem.58.1.229-232.1992
12. An Z, Bin G, Hendrix JW. Viability of soilborne spores of glomalean mycorrhizal fungi. Soil Biol. & Biochem.; 1998 Aug;30(8–9):1133–1136. https://doi.org/10.1016/s0038-0717(97)00194-6
13. Phillips J, Hayman DS. Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Trans. of the Bri. Myc. Society; 1970 Aug;55(1):158-IN18. https://doi.org/10.1016/s0007-1536(70)80110-3
14. Cell Culture Contamination | Thermo Fisher Scientific - IE. https://www.thermofisher.com/in/en/home/references/gibco-cell-culture-basics/biological-contamination.html
15. R: The R Project for Statistical Computing. https://www.r-project.org/
16. Ellatif SA, Ali E, Senousy HH, Razik ESA. Production of Arbuscular Mycorrhizal Fungi using In vitro Root Organ Culture and Phenolic Compounds. J. of Pure and App. Micro. 2019 Dec;13(4):1985–94. https://doi.org/10.22207/jpam.13.4.10
17. Das N, Tripathi N, Basu S, Bose C, Maitra S, Khurana S. Progress in the development of gelling agents for improved culturability of microorganisms. Frontiers in Microbiology. 2015 Jul;6. https://doi.org/10.3389/fmicb.2015.00698
18. Jaramillo J, Summers WL. Tomato anther callus production: Solidifing agent and concentration influence induction of callus. J. of the Ame. Soc. for Hor. Sci. 1990 Nov;115(6):1047–50. https://doi.org/10.21273/jashs.115.6.1047
19. McGuffey JC, Leon D, Dhanji EZ, Mishler DM, Barrick JE. Bacterial production of gellan gum as a Do-It-Yourself alternative to Agar. J. of Micro. & Bio. Edu. 2018 Jan;19(2). https://doi.org/10.1128/jmbe.v19i2.1530
20. All you need to know about Gellan Gum. Plant Cell Technology | Your Partner in Plant Tissue Culture. https://www.plantcelltechnology.com/blogall-you-need-to-know-about-gellangum/#:~:text=The%20gellan%20gum%20has%20more,plant%20tissue%20prior%20to%20transplanting
21. Mao R, Tang J, Swanson BG. Water holding capacity and microstructure of gellan gels. Carb. Pol. 2001 Dec;46(4):365–71. https://doi.org/10.1016/s0144-8617(00)00337-4
22. Gupta SD, Ibaraki Y. Plant tissue Culture Engineering. Springer Science & Business Media; 2006.
23. Bhatia NP, Adholeya A, Verma A. Influence of media gelling agents on root biomass and in vitro VA-mycorrhizal symbiosis of carrot with Gigaspora margarita. Biotropia: The Sou. Asi. J. of Tro. Bio. 1997 Jun;0(10). https://doi.org/10.11598/btb.1997.0.10.128
24. Tiwari P, Adholeya A. In vitro co-culture of two AMF isolates Gigaspora margarita and Glomus intraradices on Ri T-DNA transformed roots. Fems Micro. Let. 2002 Jan;206(1):39–43. https://doi.org/10.1111/j.1574-6968.2002.tb10983.x
25. Owen H, Wengerd D, Miller A. Culture medium pH is influenced by basal medium, carbohydrate source, gelling agent, activated charcoal, and medium storage method. Plant Cell Rep. 1991 Dec;10(11). https://doi.org/10.1007/bf00232516
26. Jolicœur M, Williams RD, Chavarie C, Fortin JA, Archambault J. Production of Glomus intraradices propagules, an arbuscular mycorrhizal fungus, in an airlift bioreactor. Biotechnology and Bioengineering. 1999 Apr;63(2):224–32. https://doi.org/10.1002/(sici)1097-0290(19990420)63:2<224::aid-bit11>3.0.co;2-x
27. Wang WK. US6759232B2 - Method of facilitating mass production and sporulation of arbuscular mycorrhizal fungi aseptic in vitro - Google Patents. 2002. https://patents.google.com/patent/US6759232B2/en
28. Fortin JA. US5554530A - Aseptic in vitro endomycorrhizal spore mass production - Google Patents. 1993. https://patents.google.com/patent/US5554530A/en
29. Schuessler A. EP3038456A1 - System and methods for continuous propagation and mass production of arbuscular mycorrhizal fungi in liquid culture - Google Patents. 2013. https://patents.google.com/patent/EP3038456A1/en
30. Siqueira JO, Sylvia DM, Gibson J, Hubbell DH. Spores, germination, and germ tubes of vesicular–arbuscular mycorrhizal fungi. Can. J. of Micro. 1985 Nov;31(11):965–72. https://doi.org/10.1139/m85-183
31. Engelmoer DJP, Behm JE, Kiers ET. Intense competition between arbuscular mycorrhizal mutualists in an in vitro root microbiome negatively affects total fungal abundance. Mol. Eco. 2013 Sep;23(6):1584–93. https://doi.org/10.1111/mec.12451
32. Danesh YR, Goltapeh EM, Alizadeh AHM, M. Modarres Sanavy. Optimizing carrot hairy root production for monoxenic culture of arbuscular mycorrhizal fungi in Iran. J. of Bio. Sci. 2005 Dec. https://doi.org/10.3923/jbs.2006.87.91
33. Costa FA, Haddad LSM, Kasuya MCM, Oton WC, Costa MD, Borges AC. In vitro culture of Gigaspora decipiens and Glomus clarum in transformed roots of carrot: the influence of temperature and pH. Acta Scientiarum-agronomy. 2013 Jul;35(3). https://doi.org/10.4025/actasciagron.v35i3.16581
34. Srinivasan M, Kumar KK, Kumutha K, Marimuthu P. Establishing monoxenic culture of arbuscular mycorrhizal fungus Glomus intraradices through root organ culture. J. of App. and Nat. Sci. 2014 Jun 1;6(1):290–3. https://doi.org/10.31018/jans.v6i1.417
35. D’Souza J. Modified Strullu and Romand (MSR) medium devoid of sucrose promotes higher in vitro germination in Rhizophagus irregularis. J. of Myc. and Plant Path., 43(2), 240–24. 2013. http://irgu.unigoa.ac.in/drs/handle/unigoa/2853
36. Dalpé Y, De Souza FA, Declerck S. Life cycle of glomus species in monoxenic culture. In: Soil biology. 2005. p. 49–71. https://doi.org/10.1007/3-540-27331-x_4
37. Wood T. EP0209627A2 - Method for producing axenic vesicular arbuscular mycorrhizal fungi in association with root organ cultures - Google Patents. 1985. https://patents.google.com/patent/EP0209627A2/en
38. Declerck S, D’Or D, Cranenbrouck S, Boulengé LE. Modelling the sporulation dynamics of arbuscular mycorrhizal fungi in monoxenic culture. Mycorrhiza. 2001 Oct;11(5):225–30. https://doi.org/10.1007/s005720100124
39. St‐Arnaud M, Hamel C, Vimard B, Caron M, Fortin JA. Enhanced hyphal growth and spore production of the arbuscular mycorrhizal fungus Glomus intraradices in an in vitro system in the absence of host roots. Myc. Res. 1996 Mar;100(3):328–32. https://doi.org/10.1016/s0953-7562(96)80164-x
40. Raj BM, Kumar RB, Rao GV, Murthy KSR. An optimised in vitro protocol for mass production of Rhizophagus irregularis spores - for sustainable agriculture. J. of Bus. Res. 2017 Jul;9(4):21–9. https://africaneditors.org/journal/JBR/full-text-pdf/66716-106667
41. Douds DD. Increased spore production by Glomus intraradices in the split-plate monoxenic culture system by repeated harvest, gel replacement, and resupply of glucose to the mycorrhiza. Mycorrhiza. 2002 Aug;12(4):163–7. https://doi.org/10.1007/s00572-002-0174-9
42. Rosikiewicz P, Bonvin JF, Sanders IR. Cost-efficient production of in vitro Rhizophagus irregularis. Mycorrhiza. 2017 Feb;27(5):477–86. https://doi.org/10.1007/s00572-017-0763-2
43. Puri A, Adholeya A. A new system using Solanum tuberosum for the co-cultivation of Glomus intraradices and its potential for mass producing spores of arbuscular mycorrhizal fungi. Symbiosis. 2013 Jan;59(2):87–97. https://doi.org/10.1007/s13199-012-0213-z