In vitro micrografting using three diverse indigenous rootstocks for the production of Citrus tristeza virus-free plants of Khasi mandarin

  • Amit K. Singh
  • Ng. T. Meetei
  • Suprabuddha Kundu
  • Umme Salma
  • Nirmal MandalEmail author
Hairy Root Culture


The present work reports the prospective applicability of three indigenous rootstocks belonging to different species viz. Nemutenga, Tayum and Tasi cultivars of north eastern Himalayan region for producing Citrus tristeza virus (CTV; a viral species of the Closterovirus genus) free quality planting materials of Khasi mandarin (Citrus reticulata Blanco) through micrografting or shoot-tip grafting (STG). The development of disease-free plants through STG is essential as CTV is in vivo transmissible through infected bud sticks. The technique of STG along with the diverse culture parameters like effect of sucrose, plant growth regulators, pre-treatment of scion and stock, effect of scion size after thermotherapy and grafting method was analysed. Among the three indigenous tested rootstocks, Nemutenga was found superior showing maximum STG success of 58.5%. STGs using scion size ranging from 0.3 to 0.5 mm, survival was up to 42.0% and completely virus-free. Pre-treatment of scion and stock with kinetin (1.0 mg L−1) was found more suitable than N6-benzylaminopurine (BAP) and further increased the rate of micrografts. For further growth of micrografts, Murashige and Skoog medium fortified with 0.5 mg L−1 BAP and 0.1 mg L−1 indole-3-acetic acid (IAA) along with 5% sucrose resulted in maximum (56.8%) response. Considering the two techniques applied for STG, cleft grafting was found more suitable than inverted T grafting. Viral assessment with enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase polymerase chain reaction (RT-PCR) revealed negative results for all plants from 0.3 to 0.5 mm scion while 0.7-mm scion-raised micrografts showed 60% negative with RT-PCR for CTV.


Khasi mandarin Indigenous rootstock Citrus tristeza virus Shoot tip grafting ELISA RT-PCR 



Authors acknowledge the laboratory as well as library assistance from BCKV, West Bengal.

Author’s contribution

Amit Kumar Singh, Ng. Tombisana Meetei and Nirmal Mandal conceived the idea, surveyed the literature and designed the experiment. Amit Kumar Singh conducted the experiment and Suprabuddha Kundu as well as Umme Salma assisted for the experiment. All the authors contributed in writing the manuscript and accepted the final version.

Funding information

DBT provided grant (BT/04/NE/2009) under the project Advanced level Institutional Biotech Hub, CHF, CAU, Pasighat.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11627_2018_9946_MOESM1_ESM.docx (13 kb)
ESM 1 (DOCX 13 kb)


  1. Ahlawat YS (1997) Viruses, greening bacterium and viroids associated with citrus (Citrus species) decline in India. J Agric Sci 67:51–57Google Scholar
  2. Ahlawat YS, Pant RP (2003) Major virus and virus-like diseases of citrus in India: their diagnosis and management. Ann Rev Plant Pathol 2:447–474Google Scholar
  3. Ali S, Mirza B (2006) Micropropagation of rough lemon (Citrus jambhiri lush.): effect of explant type and hormone concentration. Acta Bot Croat 65:137–146Google Scholar
  4. Aloni B, Karni L, Deveturero G, Levin Z, Cohen R, Kazir N, Lotan-Pompan M, Edelstein M, Aktas H, Turhan E, Joel DM, Horev C, Kapulnic Y (2008) Physiological and biochemical changes at the rootstock–scion interface in graft combinations between Cucurbita rootstocks and a melon scion. J Hortic Sci Biotechnol 83:777–783CrossRefGoogle Scholar
  5. Aloni R (1993) The role of cytokinin in organized differentiation of vascular tissues. Aust J Plant Physiol 20:601–608Google Scholar
  6. Bangerth F (1994) Response of cytokinin concentration in the xylem exudate of bean (Phaseolus vulgaris L.) plants to decapitation and auxin treatment, and relationship to apical dominance. Planta 194:439–442CrossRefGoogle Scholar
  7. Bhatt KM (2008) In vitro micrografting of apple (Malus domestica Borkh.) cv. Lal Ambri on dwarfing M-9 rootstock. PhD thesis submitted to Sher-e-Kashmir University of Agricultural Sciences and Technology, Shalimar, Srinagar, J&KGoogle Scholar
  8. Bhatt KM, Banday FA, Mir MA, Rather ZA, Hussain G (2013) In vitro grafting in apple (Malus domestica Borkh) cv. LalAmbri. Karnataka J Agric Sci 26:399–402Google Scholar
  9. Biswas KK (2010) Molecular characterization of Citrus tristeza virus isolates from the north eastern Himalayan region of India. Arch Virol 155:959–963CrossRefGoogle Scholar
  10. Biswas KK, Tarafdar A, Sharma SK, Singh JK, Dwivedi S, Biswas K, Jayakumar BK (2014) Current status of citrus tristeza virus incidence and its spatial distribution in citrus growing geographical zones of India. Indian J Agric Sci 84:184–189Google Scholar
  11. Bortolotti C, Murillo L, Fontanet P, Coca M, Segundo BS (2005) Long distance transport of the maize pathogenesis-related PRms protein through the phloem in transgenic tobacco plants. Plant Sci 168:813–821CrossRefGoogle Scholar
  12. Canon C, Mehmet O, Hakan T, Kamil S, Elman I (2006) In vitro micrografting of pistachio (Pistacia vera L.) var. Siirt on wild pistachio rootstocks. J Cell Mol Biol 5:25–31Google Scholar
  13. Chand L, Sharma S, Kajla S (2016) Effect of rootstock and age of seedling on success of in vitro shoot tip grafting in Kinnow mandarin. Indian J Hort 73:8–12CrossRefGoogle Scholar
  14. Clark MF, Adams AN (1977) Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. J Gen Virol 34:475–483CrossRefGoogle Scholar
  15. Duncan DB (1955) Multiple range and multiple F test. Biometrics 11:1–42CrossRefGoogle Scholar
  16. Faccioli G, Marani F (1998) Virus elimination by meristem tip culture and tip micrografting. In: Hadidi A, Khetarpal RF, Koganezawa H (eds) Plant virus diseases control. The American Phytopathological Society, APS Press, St Paul, Minnesota, pp 346–380Google Scholar
  17. FAOSTAT (2013) Food and Agriculture Organization of the United Nations Rome, Statistical year book, p 169Google Scholar
  18. Foster TM, Tony JL, Sarah JE, Robyn HL, John LB, Richard LSF, William JL (2002) A surveillance system regulates selective entry of RNA into the shoot apex. Plant Cell 14:1497–1508CrossRefGoogle Scholar
  19. George EF (1993) Plant propagation by tissue culture. Part 1. The technology (Exegetics Ltd: Edington, Westbury, UK)Google Scholar
  20. Ghosh SP (2007) Citrus fruits. ICAR, New DelhiGoogle Scholar
  21. Hazarika TK (2012) Citrus genetic diversity of Northeast India, their distribution ecogeography and ecobiology. Genet Resour Crop Evol 59:1267–1280CrossRefGoogle Scholar
  22. Hundman SE, Hasegawa PM, Bressan RA (1982) Stimulation of root initiation from cultured rose shoots through the use of reduced concentrations of mineral salts. Hort Sci 17:82–83Google Scholar
  23. Kose C, Guleryuz M (2006) Effects of auxins and cytokinins on graft union of grapevine (Vitis vinifera) New Zealand. J Crop Hortic Sci 34:145–150CrossRefGoogle Scholar
  24. Kumarin V, Shyam S, Kumarin V, Singh S (2000) Shoot tip grafting with growth regulators for virus elimination in Nagpur mandarin (Citrus reticulata). Ind J Agric Sci 70:396–397Google Scholar
  25. Lahoty P, Singh J, Bhatanagar P, Rajpurohit D, Jain SK (2013) Shoot tip grafting in Nagpur mandarin (Citrus reticulata Blanco). Plant Arc 13:173–175Google Scholar
  26. Lbida B, Bennani A, Serrhini MN, Zemzami M (2005) Biological, serological and molecular characterization of three isolates of Citrus tristezaclostero virus introduced into Morocco. EPPO Bull 35:511–517CrossRefGoogle Scholar
  27. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  28. Murashige TW, Bitters R, Nauer EM, Roistacher CN (1972) A technique of shoot apex grafting and its utilization towards recovering virus-free citrus clones. Hort Sci 7:118–119Google Scholar
  29. Mustafa U, Hikmet MS, Bulent P (2005) Comparison of DAS-ELISA and RT-PCR methods for the detection of PrunusNecrotic Ringspot Virus (PNRSV). J Agric Sci 15(2):153–158Google Scholar
  30. National Horticulture Board, Horticulture Information Service-2010-2011Google Scholar
  31. National Horticulture Board, Indian Horticulture Database-2013Google Scholar
  32. Navarro L (1984) Proc. FAO/NORWAY Symp. On plant tissue culture, technology and utilization, micropropagation of root crop, palm, citrus and ornamental plant. Plant Prod Protect Pap 59:113–154Google Scholar
  33. Navarro L, Pina JA, Juárez J, Ballester-Olmos JF, Arregui JM, Ortega C, Navarro A, Duran-Vila N, Guerri J, Moreno P, Cambra M, Zaragoza S (2002) The Citrus variety improvement program in Spain in the period 1975-2001. In: Duran-Vila N, Milne RG, da Graça JV (eds) Proceedings 15th conference international organization Citrus virologists. IOCV, Riverside, pp 306–316Google Scholar
  34. Navarro L, Roistacher CN, Murashigue T (1975) Improvement of shoot tip grafting in vitro for virus-free citrus. J American Soc Hort Sci 100:471–479Google Scholar
  35. Onay A, Pirinc V, Adiyaman F, Isikalen C, Tilkat E, Basaren D (2004) In vivo and in vitro micrografting of pistachio (Pistacia vera L. cv. Siirt). Turkish J Bio 27:95–100Google Scholar
  36. Parthasarathy VA, Nagaraju V, Rahman SAS (1997) In vitro grafting of Citrus reticulate Blanco. Folia Hort 9:87–90Google Scholar
  37. Rahman AAS, Nagaraju V, Parthasarthy VA (1996) Response of embryoids of certain citrus species to two cytokinins. Ann Pl Physiol 10:45–49Google Scholar
  38. Ram R, Verma N, Singh AK, Singh L, Hallan V, Zaidi AA (2005) Indexing and production of virus-free chrysanthemums. Biol Plant 49:149–152CrossRefGoogle Scholar
  39. Ribeiro LM, Peixoto JR, Andrade SRM, Fonseca RS, Vieira LM (2008) Ex vitro micrografting aiming the CABMV virus elimination in passion fruit plant. Pesq Agropec Bras 43:589–594CrossRefGoogle Scholar
  40. Sanabam R, Singh NS, Handique PJ, Devi HS (2015) Disease-free Khasi mandarin (Citrus reticulata Blanco) production using in vitro microshoot tip grafting and its assessment using DAS-ELISA and RT-RCR. Sci Hort 189:208–213CrossRefGoogle Scholar
  41. Sharma S, Balwinder S, Gita R, Zaidi AA, Vipin KH, Avinash KN, Virk GS (2008) In vitro production of Indian citrus ring spot virus (ICRSV) free Kinnow plants employing thermotherapy coupled with shoot tip grafting. Plant Cell Tissue Organ Cult 92:85–92CrossRefGoogle Scholar
  42. Singh AK, Meetei NT, Singh BK, Mandal N (2016) Khasi mandarin: its importance, problems and prospects of cultivation in north-eastern Himalayan region. Int J Agric Environ Biotechnol 9:573–592CrossRefGoogle Scholar
  43. Singh AK, Meetei NT, Singh BK, Mandal N (2017) High incidence of citrus tristeza virus in mandarin (Citrus reticulata) in north-east states of India. VirusDis 28(4):401–407CrossRefGoogle Scholar
  44. Singh B, Sharma S, Rani G, Hallan V, Zaidi AA, Virk GS, Nagpal A (2008) In vitro production of Indian citrus ringspot virus (ICRSV) free Kinnow plants employing thermotherapy coupled with shoot tip grafting. Plant Biotechnol Rep 2:137–143CrossRefGoogle Scholar
  45. Singh SK, Khawale RN, Singh SP (2003) A comparative studies on success of in-vitro shoot tip grafting in Citrus reticulata Blanco on different rootstocks. Haryana J Hort Sci 32:171–173Google Scholar
  46. Sorce C, Massai R, Picciarelli P, Lorenzi R (2002) Hormonal relationships in xylem sap of grafted and ungrafted Prunus rootstock. Sci Hortic 93:333–342CrossRefGoogle Scholar
  47. Steininger C, Kundi M, Aberle SW, Aberle JH, Popow-Kraupp T (2002) Ffectiveness of reverse transcription-PCR, virus isolation, and enzyme-linked Immunosorbent assay for diagnosis of influenza a virus infection in different age groups. J. Clin. Microbiol 40(6):2051–2056Google Scholar
  48. Thimmappaiah Puthra GT, Anil SR (2002) In vitro grafting of cashew (Anacardium occidentale L.). Sci Hort 92:177–182CrossRefGoogle Scholar
  49. Trewavas A (2013) Sensitivity and sensory adoption in growth substance responses. In: Hoad GV, Lenton JR, Jackson MB (eds) Hormone action in plant development — a critical appraisal. ISBN 1483162885. Elsevier, Amsterdam, pp 19–38Google Scholar
  50. Vijayakumari N, Ghosh DK, Das AK, Singh A, Singh S (2006) Elimination of citrus tristeza virus and greening pathogens from exotic germplasm through in vitro shoot tip grafting in citrus. Indian J Agric Sci 76(3):209–210Google Scholar
  51. Weatherhead I (1986) Causes of graft failure in Sitka spruce (Picea sitchensis-Bomg.-Carr.). Ph.D. thesis, University of ReadingGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2018

Authors and Affiliations

  • Amit K. Singh
    • 1
    • 2
  • Ng. T. Meetei
    • 2
    • 3
  • Suprabuddha Kundu
    • 2
  • Umme Salma
    • 2
  • Nirmal Mandal
    • 2
    Email author
  1. 1.College of Horticulture and ForestryCentral Agriculture UniversityPasighatIndia
  2. 2.Department of Agricultural Biotechnology, Faculty of AgricultureBidhan Chandra Krishi ViswavidyalayaNadiaIndia
  3. 3.College of Post Graduate StudiesCentral Agriculture UniversityShillongIndia

Personalised recommendations