In vitro micrografting using three diverse indigenous rootstocks for the production of Citrus tristeza virus-free plants of Khasi mandarin
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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.
KeywordsKhasi 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.
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.
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.
- Ahlawat YS (1997) Viruses, greening bacterium and viroids associated with citrus (Citrus species) decline in India. J Agric Sci 67:51–57Google Scholar
- Ahlawat YS, Pant RP (2003) Major virus and virus-like diseases of citrus in India: their diagnosis and management. Ann Rev Plant Pathol 2:447–474Google Scholar
- Ali S, Mirza B (2006) Micropropagation of rough lemon (Citrus jambhiri lush.): effect of explant type and hormone concentration. Acta Bot Croat 65:137–146Google 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 83:777–783CrossRefGoogle 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&KGoogle Scholar
- 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
- 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
- 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
- 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
- FAOSTAT (2013) Food and Agriculture Organization of the United Nations Rome, Statistical year book, p 169Google Scholar
- George EF (1993) Plant propagation by tissue culture. Part 1. The technology (Exegetics Ltd: Edington, Westbury, UK)Google Scholar
- Ghosh SP (2007) Citrus fruits. ICAR, New DelhiGoogle 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 17:82–83Google 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 70:396–397Google Scholar
- Lahoty P, Singh J, Bhatanagar P, Rajpurohit D, Jain SK (2013) Shoot tip grafting in Nagpur mandarin (Citrus reticulata Blanco). Plant Arc 13:173–175Google 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 7:118–119Google 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):153–158Google Scholar
- National Horticulture Board, Horticulture Information Service-2010-2011Google Scholar
- National Horticulture Board, Indian Horticulture Database-2013Google Scholar
- 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
- 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
- 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
- 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
- Parthasarathy VA, Nagaraju V, Rahman SAS (1997) In vitro grafting of Citrus reticulate Blanco. Folia Hort 9:87–90Google Scholar
- Rahman AAS, Nagaraju V, Parthasarthy VA (1996) Response of embryoids of certain citrus species to two cytokinins. Ann Pl Physiol 10:45–49Google 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 32:171–173Google 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):2051–2056Google 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 19–38Google 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):209–210Google Scholar
- Weatherhead I (1986) Causes of graft failure in Sitka spruce (Picea sitchensis-Bomg.-Carr.). Ph.D. thesis, University of ReadingGoogle Scholar