In vitro production of Indian citrus ringspot virus-free plants of Kinnow mandarin (Citrus nobilis Lour × C. deliciosa Tenora) by nucellar embryo culture.
Indian citrus ringspot virus (ICRSV)-free plants of Kinnow mandarin (Citrus nobilis × C. deliciosa) were produced from a virus-infected plant using nucellar embryo culture. The parent kinnow plant was tested by indirect ELISA and RT-PCR before using its explants. An amplified product of 539 bp (partial cp gene) was obtained by RT-PCR in ICRSV-infected plants. The nucellar embryos obtained from seeds collected from immature fruits of the infected plant were cultured on Murashige and Skoog's (MS) basal medium supplemented with various concentrations of 2,4-D and malt extract (ME) alone or in various combinations. Maximum embryogenic callus induction (33.33%) was observed on MS medium supplemented with 2,4-D (9.02 µM) in combination with malt extract (400 mg l-1). Transfer of embryogenic calluses to MS medium containing different concentrations of malt extract alone or in combination with ABA resulted in somatic embryogenesis with a maximum of 56.94% cultures in MS medium supplemented with malt extract (500 mg l-1) and ABA (7.56 µM). Cotyledonary shaped embryos when transfered to different strengths of MS medium supplemented with NAA (10.74 µM) developed into complete plantlets in maximum of 72.22% cultures on 1/2 MS medium. The plantlets were successfully acclimatized, transferred to screen house and indexed for ICRSV employing indirect ELISA and RT-PCR and all were found negative of virus. A distinct feature of this study is the induction of somatic embryogenesis from nucellar embryos to produce virus-free plants.