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An Efficient Somatic Embryogenesis Protocol for Citrus jambhiri and Assessment of Clonal Fidelity of Plantlets Using RAPD Markers

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Abstract

Citrus jambhiri Lush. (rough lemon) is considered a major rootstock for a number of Citrus species. Different explants were used for somatic embryogenesis: leaf, nodal, and root segments excised from in vitro seedlings, stigmas, styles, and ovaries from flower buds, nucellar tissues and cotyledons from seeds, and juice vesicles from fruits. The explants were cultured on MS medium supplemented with different concentrations of 6-benzylaminopurine (BAP), kinetin (KN), malt extract (ME), and 2,4-dichlorophenoxyacetic acid (2,4-D) for callus induction. All explants formed callus except styles and juice vesicles. The most responsive explants were leaf and nodal segments cultured in the presence of 2,4-D, and ovary and nucellar tissues cultured in a medium with BA 4 mg/l. Callus obtained from all explants was transferred to MS medium supplemented with different concentrations and combinations of 2,4-D, BA, KN, ME, and ABA (abscisic acid) for somatic embryo induction. Only calli from ovaries and nucellar tissue were competent to form somatic embryos, mainly when they were transferred to a induction medium supplemented with BA 4 mg/l and ME 500 mg/l (89.33 and 83.50 %, respectively). The somatic embryos were shifted to MS medium containing different concentrations of sucrose with or without activated charcoal (AC) or polyethylene glycol (PEG) for somatic embryo germination. Maximum somatic embryo germination (98.85 %) was noticed from somatic embryos from ovary calli on MS medium consisting of 8 % sucrose. By increasing and decreasing the concentration of sucrose from 8 %, a corresponding decrease in somatic embryo germination was observed. The addition of AC and PEG to somatic embryo germination medium reduced the percentage of germination of somatic embryos. RAPD analysis was employed to assess the genetic fidelity of plantlets. The plantlets regenerated through somatic embryogenesis from nucellar tissues showed no variation in their RAPD profile but the plantlets raised through somatic embryogenesis from ovaries showed variation in around 20–30 % plantlets.

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  1. Savita

    Present address: Deen Dayal Upadhayaya College, University of Delhi, Karampura, India

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  1. Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar, 143005, India

    Savita, G. S. Virk & Avinash Nagpal

  2. Department of Biotechnology, Guru Nanak Dev University, Amritsar, 143005, India

    Pratap Kumar Pati

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Savita, Pati, P.K., Virk, G.S. et al. An Efficient Somatic Embryogenesis Protocol for Citrus jambhiri and Assessment of Clonal Fidelity of Plantlets Using RAPD Markers. J Plant Growth Regul 34, 309–319 (2015). https://doi.org/10.1007/s00344-014-9465-6

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