Enzymic Maceration of Citrus Callus and the Regeneration of Plants from Single Cells

The maceration medium comprised a basal nutrient medium (BM)containing an optimum concentration of 3% (w/v) sucrose. Mannitoland sorbitol were inferior osmotica. Addition of potassium dextransulphate adversely affected maceration. ‘Macerozyme’was not as effective as ‘Macerase’ in the productionof single cells. The optimal concentration of ‘Macerase’was found to be 2–3% (w/v). Single cells obtained by filtering the macerate were rinsedwith BM and cultured in, and on, agar media comprising: BM;BM + 500 mg 1^–1 malt extract (ME); and BM + 10% (v/v)coconut milk (CM). No growth or organization was observed incultures where cells were mixed in with warm medium prior togelling. When spread on the surface of gelled media supplementedwith ME and CM, proliferation and organization occurred. Manymicroscopic globular proembryoids developed within 3 weeks onthe supplemented media. Microscopic torpedo-shaped embryoidswere frequently observed on BM + CM, rarely on BM + ME, andnot at all on unsupplemented BM. The high frequency of microscopic globular proembryoids, andlater of macroscopic pseudo-bulbils, formed on BM + ME leadsus to postulate that pseudobulbils are derived from globularproembryoids in which polarity is not established by the 16to 32-cell stage. Microscopic torpedo-shaped embryoids probablygive rise to macroscopic heart-shaped embryoids which developinto plantlets. The technique reported in this article provides an ideal systemfor examining embryogenesis per se and for studying the effectsof various treatments on embryogenesis and organ differentiationin vitro. It also affords excellent opportunities for the breedingof solid mutant plants.