Development of an Efficient In Vitro Propagation Protocol for the Elite Bidah Pomegranate (Punica granatum L.) Landrace in Saudi Arabia

Abstract

The Bidah pomegranate (Punica granatum L.) is an elite landrace native to Wadi Bidah in the Al-Baha region of Saudi Arabia. This cultivar is distinguished by its unique taste, high yield, genetic component, and phytochemical profile. In addition, its resilience to harsh environmental conditions aligns closely with national priorities in sustainable agriculture and food security. This study articulated the successful micropropagation of the Bidah pomegranate cultivar using both direct and indirect in vitro approaches. Phenolic exudation was effectively mitigated through sequential subculturing onto a plant growth regulator (PGR)-free medium. Shoot proliferation was highest for Murashige and Skoog (MS) medium supplemented with 3 mg/L benzyladenine (BA), resulting in a 95% shoot induction rate, which was significantly higher than those obtained with the other investigated media. Callus induction and bioreactor-based mass production were maximized on Woody Plant medium (WPM) containing 0.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), yielding significantly higher biomass than MS medium. Rooting of regenerated shoots was successfully achieved on half-strength MS (1/2 MS) medium supplemented with 3 mg/L 1-naphthaleneacetic acid (NAA). The hardening of plantlets was conducted by transferring the rooted shoots into pots containing a soil mixture of vermicompost and perlite. The acclimatization process was performed under controlled greenhouse conditions, where the photosynthetically active radiation (PAR) averaged 4530.5 µmol m−2 s−1, air temperature was maintained at 22 °C, relative humidity (RH) averaged 74.5%, and the pad-wall cooling rate was 2695.56 L. Additionally, the genetic fidelity was investigated using genome size estimation. The in vitro propagation method offers significant advantages over conventional techniques, enabling efficient plant reproduction, genetic conservation, and the obtaining of mass plant material for different uses. Furthermore, genetic enhancement through in vitro selection and the application of CRISPR-Cas9 gene editing technologies can be performed.