Induction of Polyploidy in Citrus Rootstocks through In Vitro Colchicine Treatment of Seed-Derived Explants

Abstract

Polyploidy, frequently observed in citrus species, aids in achieving better adaptation to environmental stresses. In this context, the current work aims to develop stable tetraploids in citrus rootstock cultivars, viz., Rough lemon, Rangpur lime and Alemow, through in vitro colchicine treatments. Seed-derived explants were obtained by culturing sterile seeds on MS basal media. Seedlings with a size of 5–8 mm (hypocotyl) were exposed to colchicine treatment. After treatment, the surviving seedlings were minigrafted onto six-month-old rootstock for better growth. Colchicine concentrations of 0.1%, 0.2%, 0.3% and control for durations of 16 or 24 h were tested with respect to the induction of polyploidisation. Treatment with 0.1% colchicine for 24 h resulted in high rates of mutation for polyploidisation and showed the highest tetraploid induction percentage (18.3%) in all the rootstock cultivars. High colchicine concentration and long exposure time decreased the survival of the observed seedlings. Flow cytometry and cytological methods were used for confirmation of autotetraploidy in the analysed samples. The surviving seedlings were identified on the basis of morphological and cytological variables, such as leaf area and stomata size, which significantly increased with increasing ploidy level. The proposed method was found to be an effective way to induce the polyploidy in Rangpur lime, Rough lemon and Alemow rootstocks.