Recovery and Indexing of Avocado Plants (Persea Americana) from Embryogenic Nucellar Cultures of an Avocado Sunblotch Viroid-Infected Tree

Avocado (Persea americana Mill.) plants were recovered from somatic embryos derived from the nucellus of an avocado sunblotch viroid (ASBVd)-infected tree (Vero Beach SE2). Embryogenic cultures were induced on semi solid medium consisting of B5 major salts, MS minor salts and organics, 45 g sucrose and 0.41 micromolar picloram, and were maintained as suspension cultures in MS3:1N medium (MS containing 12 mg l-1 NH4NO3, 30.3 mg l-1 KNO3 , 0.41 micromolar picloram and (in mg l-1) thiamine HCl, (0.4), myo-inositol (100), and sucrose (45,000). Somatic embryos developed on semi solid MS medium without growth regulators. Plants were recovered from mature somatic embryos on semi solid MS supplemented with 4.44 micromolar BA and 2.89 micromolar GA3, and thereafter were maintained on semi solid MS basal medium. RT-PCR indexing of embryogenic cultures, somatic embryos and leaf tissue of regenerants demonstrated that all developmental stages were ASBVd-positive. Variation in the viroid increased with developmental stage. Five ASBVd variants were isolated from embryogenic cultures, five ASBVd variants were isolated from somatic embryos and 33 ASBVd variants were isolated from leaves of regenerated plants. Nucellar culture cannot be considered a strategy for elimination of ASBVd from infected elite avocado material, and appears to stimulate variation in the viroid.