Organogenesis from Leaf Tissue of Spondias pinnata (L. f.) Kurz, SEM study and Genetic Fidelity Assessment by ISSR and ScoT

In vitro raised plantlets were obtained from nodal tissue through direct organogenesis and they served as donor plants for the collection of leaf explants. Leaf explants were inoculated on Murashige and Skoog’s medium with different concentrations of 2,4-Dichlorophenoxyacetic acid (2,4-D). Hundred percent callogenesis was observed on medium supplemented with 5 mg l−1 2,4-D. For the multiplication of cells (proliferation), calli were transferred to either basal medium or media containing different types and concentrations of cytokinins. Proliferation was observed maximum on media containing 0.5 mg l−1 BAP or 1.0 mg l−1 BAP. Shoot differentiation from calli took place on media supplemented with BAP in combinations with TDZ or ZN. Initiation of organogenesis was observed in calli within two weeks of their subculture on differentiation medium. Shoot differentiation was maximum, when calli proliferated on medium having 1 mg l−1 BAP were transferred to the medium containing 1 mg l−1 BAP and 0.5 mg l−1 TDZ. Organogenic responses after four weeks of subculture on above differentiation medium were as such: number of shoots per explants (25.33 ± 0.88), number of shoots per calli replicate (3.67 ± 0.33) and maximum shoot length (3.43 ± 0.20 cm). Medium supplemented with 2.5 mg l−1 NAA was most responsive for rooting of shoots (54.16 ± 1.39%). About 62.5% plantlets survived after hardening and 54.17% plantlets got acclimatized. All acclimatized plants were transferred to field condition successfully. Formation of unipolar shoots and their multicellular attachment with callus were observed by scanning electron microscopy. To confirm the genetic fidelity of micropropagated plants, five micropropagated plants derived from different leaf explants and two mother plants (randomly selected from micropropagated plants raised from nodal explants) were subjected to molecular analysis. The genetic fidelity of in vitro regenerated plants was assessed by using SCoT and ISSR molecular markers. 12.5% polymorphism was reported in both studies, which may be due to callus mediated regeneration of shoots.