Optimization of an in vitro Regeneration Protocol for Rough Lemon Rootstock (Citrus jambhiri L.) via Direct Organogenesis

Abstract

Standardization of a reproducible protocol for in vitro rough lemon rootstock mass propagation was conducted at Tigray Biotechnology Center Plc., Plant Tissue Culture Laboratory, Mekelle, Ethiopia in 2015/2016 cropping season. Rough lemon is the frequently used rootstock both in the world and Ethiopia citrus fruit production, particularly in the Tigray region due to its superior performance over other rootstocks. However, seedlings produced through conventional ways are not recommended to be used in orchards due to variability problems caused by its polyembrony nature. To overcome such variations, in vitro regeneration of rough lemon rootstocks was performed using nodal segments and shoot tips as explant types. The explants were inoculated on MS medium supplemented with 5% scurose and 250 mg/L streptomycin followed to surface sterilization. The most effective and reproducible auxin (NAA), cytokinin (BA) and gebrillenllic acid (GA3) for in vitro shoot and root induction in rough lemon rootstocks were determined. Almost all IBA and BA treatments resulted in almost 100% shoot induction except for at 0.0 and 0.1 mg/L IBA and at 1.5 and 2.0 BA mg/L. Nodal segments induced a higher percentage of explant response with longer shoots in a shorter period of time than shoot tips, which produced more shoots and leaves than nodal segments. The effect different BA and IBA concentrations on various parameters of proliferation were studied. Full strength medium produced more regenerated shoots and leaves per shoot than half-strength MS medium. In addition, longer shoots formed with 0.1 mg/L GA3 than culture medium without this plant growth regulator. Root length decreased with higher concentration of NAA and the longest root (2.5 ± 0.22 cm) was found in the 1.0 mg/L NAA and followed by (1.95 ± 0.22 cm) at 0.5 mg/L of NAA. The rooted plants were successfully established in the greenhouse on the substrate called coco-peat and sand, and their survival rate was found to be 98%. These results suggest that standardization of these factors can help in development of a commercially viable tissue culture system for rough lemon. Moreover, it signifies the need of plant variety based in vitro protocol development and optimization across citrus species.