Adventitious shoot regeneration from immature zygotic embryos of Indian Kino tree (Pterocarpus marsupium Roxb.) and genetic integrity analysis of in vitro derived plants using ISSR markers

A simple and highly efficient protocol has been developed for in vitro regeneration of Pterocarpus marsupium using immature zygotic embryo (IZE) as explant. The highest frequency of shoot regeneration (93.8%) was obtained on Murashige and Skoog (MS) medium supplemented with 3.0 mg·L−1 6-benzylaminopurine (BA) and 0.5 mg·L−1 indole-3-acetic acid (IAA), wherein maximum of 17.3 ± 0.9 shoots per explant was induced. When these cultures were sub-cultured on MS medium supplemented with 1.0 mg·L−1 BA, more shoots (27.2 ± 1.1) with an average shoot length of 4.5 cm were observed. The highest rooting (70.8%) and maximum number of roots (3.2 ± 0.3) per shoot were obtained when shoots were dipped in 3.0 mg·L−1 indol-3-butyric acid (IBA) solution for 24 hours and further cultured on half-strength MS medium. Plantlets obtained in vitro were transferred to the field after being hardened with a 74% survival rate. Analysis of regenerated plantlets using inter-simple sequence repeat (ISSR) markers confirmed that there was no genetic variability. All ISSR banding profiles from regenerated plantlets were monomorphic and similar to those of the mother plant. This protocol might be helpful for the mass multiplication and in vitro conservation of P. marsupium.