Disease-free khasi mandarin (Citrus reticulata Blanco) production using in vitro microshoot tip grafting and its assessment using DAS-ELISA and RT-PCR

Citrus plantation using nucellar seedlings suffers from various limitations. Biotechnological tools of microshoot-tip-grafting (STG) for disease elimination and finding compatible rootstock for budding Khasi mandarin need to be implemented so as to rejuvenate the declining citrus industry of North-East India. Considering this fact, study of micrografting using three rough lemon varieties (Champra Maunthabi, Kachai Lemon and Meitei Champra) and different parameters of culture like concentration of sucrose and benzyl aminopurine (BAP), scion size and photoperiod was analysed. Depending on the genotypes used, the success rate of STG ranged from 30.15 to 42.69%. A sucrose concentration of 6% gave the optimum performance with 51.54% success on micrografting, with observable decrease in success rate on either increase (35.43% success at 9%) or decrease (33.44% success at 3%) in Murashige and Tucker (MT) liquid media supplemented with BAP 0.5mg/l. Photoperiod also has significant influence, as 51.08% success could be achieved in dark incubation for 2 weeks after micrografting, compared to no observable response at continuous light exposure (16h photoperiod). Size of scion also has significant influence on STG as smaller scion <0.5mm size gave a low performance of 7.24% success, compared to a higher success rate of 45.09% at optimal scion size of 0.5–0.65mm. Different concentrations of BAP in liquid media (MT with 6% sucrose) gave variable responses – 1mg/l of BAP gave scion vitrification and poor response while a stable growth of scion and higher micrograft success was obtained at 0.5mg/l BAP. Disease elimination from the micrografted plants using this technique of STG was confirmed using DAS-ELISA and RT-PCR techniques. Samples tested negative for citrus tristeza virus (ctv) with DAS-ELISA were further analysed with RT-PCR using ctv-specific primer pairs CTV P25F/CTV P25R and PM50/PM 51 for confirmation of the absence of viral pathogen. Providing a high humidity and ambient temperatures during acclimatization, 72.35% survival of the micrografted plants was achieved.