The genetic fidelity study of different callus-derived plantlets of Ficus religiosa L. using ISSR markers

Abstract

Ficus religiosa contributes huge significance in medicinal and ornamental properties. Recently, some obstacles have arisen for their in vivo propagation. Therefore, the present investigation was undertaken to produce genetically stable plants from callus of this species using shoot tip explants. Here, morphologically different calluses were formed based on several plant growth regulator treatments on Murashige and Skoog (MS) medium. Maximum callus (98.89%) was achieved with 2 mg L⁻¹ α-naphthaleneacetic acid (NAA) and 0.5 mg L⁻¹ both for 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BAP) in combination. The green compact callus exhibited superior result of organogenesis (50%) whereas light brown fragile callus recorded the lowest results (16.17%). The highest organogenesis response was 94.44% with green compact callus cultured on MS medium containing 0.5 mg L⁻¹ BAP and 0.1 mg L⁻¹ NAA. The maximum number of buds per shoot and longest shoot length were 3.72 and 4.51 cm, respectively, following incubation on MS medium with a combination of 1 mg L⁻¹ BAP and 0.5 mg L⁻¹ NAA. The regenerated microshoots were further multiplied by subculturing, and the second subculture generated the highest buds per shoot and longest shoot length of 5.36 and 5.22 cm, respectively, within 1 mg L⁻¹ BAP and 0.5 mg L⁻¹ NAA containing MS medium. The superior level of root response (96.92%), number of roots per shoot (4.44), and highest root length (4.02 cm) were gained with 0.5 mg L⁻¹ NAA combined with 1 mg L⁻¹ indole-3-butyric acid (IBA), 1 mg L⁻¹ IBA, and 0.5 mg L⁻¹ NAA, respectively, on ½ strength MS medium. Among the several types of potting mixture tested for primary hardening, the soil:organic manure (1:1) exhibited the best result (91.67%) for survival of plantlets after a month. It increased to 92.2% after 2 mo of secondary hardening to a field environment containing vermiculite-soil mixture (2:1). ISSR fingerprinting revealed that all bands between mother and hardened plants exhibited to be monomorphic, indicating no dissimilarity was detected in banding profiles of hardened plants with their donor mother plant. It demonstrated the absence of any variation among the callus-derived plantlets with their respective mother donor plant. The present study describes an effective protocol for the mass multiplication and conservation of this species through different textures of callus.