Activated charcoal–alginate beads (“Black Pearls”) in an alternative citrus micrografting approach: a preliminary morphological and molecular assessment

Abstract

Micrografting is an important technique for citrus propagation; however, maintaining stable contact between the scion and rootstock and limiting damage at the graft union remain important challenges. In this study, activated charcoal–alginate beads, termed “Black Pearls,” were developed as an alternative support material for micrografting Citrus deliciosa Ten. “Bodrum” (CD) scions onto C. aurantium L. (CA) and C. limonia L. (CL) rootstocks. Two scion-preparation approaches, involving either removal or retention of the scion leaves, were evaluated. Based on scion viability, new leaf formation, shoot development, and root development, approximately 93% of the evaluated plants maintained viability and continued vegetative growth during in vitro culture. The two approaches produced different morphological development patterns, particularly in root growth, while the synthetic gel beads remained around the graft union and gradually decreased in volume. In the second phase, ungrafted C. deliciosa “Bodrum” plants and C. deliciosa scions micrografted onto C. aurantium and C. limonia rootstocks were exposed to 25, 50, 100, and 200 mM NaCl for 15 days. Preliminary RT-qPCR analysis showed that HKT1 and NHX1 expression patterns varied according to salt concentration and rootstock combination. In ungrafted C. deliciosa, HKT1 expression increased mainly at intermediate salt concentrations, whereas NHX1 expression progressively decreased with increasing salinity. In grafted plants, HKT1 expression generally remained closer to the control level in the CD/CL combination than in CD/CA, while NHX1 expression increased under all salt treatments in CD/CL. Because a single pooled biological sample represented each treatment, these molecular findings are interpreted as descriptive preliminary trends rather than statistically validated biological differences. Overall, the Black Pearls system represents an alternative citrus micrografting approach, the feasibility of which was demonstrated under laboratory conditions.