Cryopreservation and storage of embryogenic callus cultures of several Citrus species and cultivars

Abstract

Nucellus-derived embryogenic callus cultures of Salustiana sweet orange were subjected to cryoconservation assays. Cryoprotection with 10%(vol/vol) dimethylsulfoxide, freezing by slow cooling and thawing by fast warming was suitable to recover viable growing cultures and whole plants through embryogenesis. Evaluation of liquid phase R ₁ and solid phase R ₂ cooling rates using a programmable freezing unit indicated that 100% of embryogenic cultures survived when frozen using a range of cooling rates (R ₁ not above 0.5°C min^–1 and R ₂ not above 1°C min ¹) and thawed by fast warming. Storage up to 2 years in liquid nitrogen did not affect the growth of the cryopreserved cultures and the recovery of whole plants. Cultures of four cultivars of sweet orange (C. sinensis Osb.), three cultivars of grapefruit (C. paradisi Macf.), and one cultivar each of lemon [C. limon (L.) Burm. f.], Cleopatra mandarin (C. reshni Hort. ex Tan.), sour orange (C. aurantium L.) and Mexican lime [C. aurantifolia (Christm.) Swing.] have been successfully cryopreserved. Problems using a viability assessment using fluorescein diacetate staining are discussed.