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

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Abstract

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.

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References

  • Ahlawat YS (1997) Viruses, greening bacterium and viroids associated with citrus (Citrus species) decline in India. J Agric Sci 67:51–57

    Google Scholar 

  • Ahlawat YS, Pant RP (2003) Major virus and virus-like diseases of citrus in India: their diagnosis and management. Ann Rev Plant Pathol 2:447–474

    Google Scholar 

  • Ali S, Mirza B (2006) Micropropagation of rough lemon (Citrus jambhiri lush.): effect of explant type and hormone concentration. Acta Bot Croat 65:137–146

    CAS  Google Scholar 

  • 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 83:777–783

    Article  Google Scholar 

  • Aloni R (1993) The role of cytokinin in organized differentiation of vascular tissues. Aust J Plant Physiol 20:601–608

    CAS  Google Scholar 

  • 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 194:439–442

    Article  CAS  Google Scholar 

  • 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&K

  • 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 26:399–402

    Google Scholar 

  • Biswas KK (2010) Molecular characterization of Citrus tristeza virus isolates from the north eastern Himalayan region of India. Arch Virol 155:959–963

    Article  CAS  PubMed  Google Scholar 

  • 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 84:184–189

    Google Scholar 

  • 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 168:813–821

    Article  CAS  Google Scholar 

  • 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 5:25–31

    Google Scholar 

  • 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 73:8–12

    Article  Google Scholar 

  • Clark MF, Adams AN (1977) Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. J Gen Virol 34:475–483

    Article  CAS  PubMed  Google Scholar 

  • Duncan DB (1955) Multiple range and multiple F test. Biometrics 11:1–42

    Article  Google Scholar 

  • 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 346–380

    Google Scholar 

  • FAOSTAT (2013) Food and Agriculture Organization of the United Nations Rome, Statistical year book, p 169

  • 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 14:1497–1508

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • George EF (1993) Plant propagation by tissue culture. Part 1. The technology (Exegetics Ltd: Edington, Westbury, UK)

  • Ghosh SP (2007) Citrus fruits. ICAR, New Delhi

    Google Scholar 

  • Hazarika TK (2012) Citrus genetic diversity of Northeast India, their distribution ecogeography and ecobiology. Genet Resour Crop Evol 59:1267–1280

    Article  Google Scholar 

  • 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 17:82–83

    Google Scholar 

  • Kose C, Guleryuz M (2006) Effects of auxins and cytokinins on graft union of grapevine (Vitis vinifera) New Zealand. J Crop Hortic Sci 34:145–150

    Article  CAS  Google Scholar 

  • 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 70:396–397

    Google Scholar 

  • Lahoty P, Singh J, Bhatanagar P, Rajpurohit D, Jain SK (2013) Shoot tip grafting in Nagpur mandarin (Citrus reticulata Blanco). Plant Arc 13:173–175

    Google Scholar 

  • 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 35:511–517

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  • 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 7:118–119

    Google Scholar 

  • 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):153–158

    Google Scholar 

  • National Horticulture Board, Horticulture Information Service-2010-2011

  • National Horticulture Board, Indian Horticulture Database-2013

  • 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 59:113–154

    Google Scholar 

  • 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 306–316

    Google Scholar 

  • Navarro L, Roistacher CN, Murashigue T (1975) Improvement of shoot tip grafting in vitro for virus-free citrus. J American Soc Hort Sci 100:471–479

    Google Scholar 

  • 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 27:95–100

    Google Scholar 

  • Parthasarathy VA, Nagaraju V, Rahman SAS (1997) In vitro grafting of Citrus reticulate Blanco. Folia Hort 9:87–90

    Google Scholar 

  • Rahman AAS, Nagaraju V, Parthasarthy VA (1996) Response of embryoids of certain citrus species to two cytokinins. Ann Pl Physiol 10:45–49

    Google Scholar 

  • Ram R, Verma N, Singh AK, Singh L, Hallan V, Zaidi AA (2005) Indexing and production of virus-free chrysanthemums. Biol Plant 49:149–152

    Article  Google Scholar 

  • 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 43:589–594

    Article  Google Scholar 

  • 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 189:208–213

    Article  CAS  Google Scholar 

  • 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 92:85–92

    Article  Google Scholar 

  • 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 9:573–592

    Article  Google Scholar 

  • 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):401–407

    Article  Google Scholar 

  • 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 2:137–143

    Article  Google Scholar 

  • 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 32:171–173

    Google Scholar 

  • Sorce C, Massai R, Picciarelli P, Lorenzi R (2002) Hormonal relationships in xylem sap of grafted and ungrafted Prunus rootstock. Sci Hortic 93:333–342

    Article  CAS  Google Scholar 

  • 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):2051–2056

    CAS  Google Scholar 

  • Thimmappaiah Puthra GT, Anil SR (2002) In vitro grafting of cashew (Anacardium occidentale L.). Sci Hort 92:177–182

    Article  Google Scholar 

  • 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 19–38

    Google Scholar 

  • 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):209–210

    Google Scholar 

  • Weatherhead I (1986) Causes of graft failure in Sitka spruce (Picea sitchensis-Bomg.-Carr.). Ph.D. thesis, University of Reading

Download references

Acknowledgments

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

Funding

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

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Authors and Affiliations

  1. College of Horticulture and Forestry, Central Agriculture University, Pasighat, Arunachal Pradesh, India

    Amit K. Singh

  2. Department of Agricultural Biotechnology, Faculty of Agriculture, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur, Nadia, West Bengal, India

    Amit K. Singh, Ng. T. Meetei, Suprabuddha Kundu, Umme Salma & Nirmal Mandal

  3. College of Post Graduate Studies, Central Agriculture University, Umiam, Shillong, Meghalaya, India

    Ng. T. Meetei

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  1. Amit K. Singh
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Contributions

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.

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Correspondence to Nirmal Mandal.

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Editor: Ewen Mullins

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Singh, A.K., Meetei, N.T., Kundu, S. et al. In vitro micrografting using three diverse indigenous rootstocks for the production of Citrus tristeza virus-free plants of Khasi mandarin. In Vitro Cell.Dev.Biol.-Plant 55, 180–189 (2019). https://doi.org/10.1007/s11627-018-9946-6

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