Assessment of genetic stability in somatic embryo derived plantlets of Pterocarpus marsupium Roxb. using inter-simple sequence repeat analysis
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An efficient plantlet regeneration protocol using immature zygotic embryos (IZEs) via somatic embryogenesis has been developed in Pterocarpus marsupium Roxb. The regenerated plantlets were evaluated for their genetic stability. IZEs were incubated on Murashige and Skoog (MS) media augmented with 1.07–16.11 μM naphthalene acetic acid (NAA) or 0.90–13.97 μM 2,4-dichlorophenoxyacetic acid. The optimum callus induction (96.6%) was observed on MS medium augmented with 5.37 μM NAA. Induction of somatic embryos (SEs) was observed after sub-culture of calli on medium with decreased concentrations of NAA (0.54–5.37 μM), either alone or 2.69 μM NAA in combination with 2.22–8.90 μM benzyladenine (BA) or 2.32–9.30 μM Kinetin. Maximum number (33.4 ± 0.85) of SEs occurred on MS medium augmented with 2.69 μM NAA + 4.40 μM BA + 3% sucrose. Highest percentage (67.3 ± 0.37) of SEs matured and developed into cotyledonary stage by subsequent subculture on the same medium. SE formation and maturation decreased when sucrose concentrations were higher than 3%. Seventy percent of mature somatic embryos developed into plantlets on half strength MS medium augmented with 5.80 µM gibberellic acid. The various stages of development during somatic embryogenesis include globular, heart, torpedo and mature stages as revealed by the stereomicroscopic and histological studies of explants. Plantlets derived from SEs were successfully acclimatized in the greenhouse with a survival rate of 78%. Among the survived plantlets, 9 plantlets were randomly selected for inter-simple sequence repeat (ISSR) analysis. Of the 13 primers used, 8 produced reproducible and monomorphic bands. ISSR analysis revealed a homogenous amplification profile for all regenerated plantlets analyzed validating the genetic stability of somatic embryo derived plantlets.
KeywordsIndian Kino tree Immature zygotic embryos Somatic embryogenesis Plantlet conversion Genetic stability
Murashige and Skoog
Plant growth regulators
Immature zygotic embryo
Inter-simple sequence repeats
T. Radhika is thankful to Jawaharlal Nehru Memorial Fund, New Delhi, India for financial support (Ref: SU-A/180/2011-12/404). The authors gratefully acknowledge Prof. A. Sadanandam, Department of Biotechnology, Kakatiya University, Warangal, Telangana, India for providing ISSR primers.
The authors have made the following declarations regarding their contributions. CT, RSN: Conceived and designed the experiments. RT: Performed the experiments. RT, PM: Analyzed the data. CT, RSN: Contributed reagents/materials. RT: Wrote the paper. CT, RSN, PM: Reviewed the manuscript. All the authors have read and approved the final manuscript.
Compliance with ethical standards
Conflict of interest
None of the authors of the submitted manuscript has declared any conflict of interest.
The protocol is useful for mass multiplication, genetic engineering, synthetic seed preparation and cryopreservation of P. marsupium plants.
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