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

Abstract

The maceration medium comprised a basal nutrient medium (BM) containing an optimum concentration of 3% (w/v) sucrose. Mannitol and sorbitol were inferior osmotica. Addition of potassium dextran sulphate adversely affected maceration. 'Macerozyme' was not as effective as 'Macerase' in the production of single cells. The optimal concentration of 'Macerase' was found to be 2-3% (w/v). Single cells obtained by filtering the macerate were rinsed with BM and cultured in, and on, agar media comprising: BM; BM + 500 mg I-1 malt extract (ME); and BM + 10% (v/v) coconut milk (CM). No growth or organization was observed in cultures where cells were mixed in with warm medium prior to gelling. When spread on the surface of gelled media supplemented with ME and CM, proliferation and organization occurred. Many microscopic globular proembryoids developed within 3 weeks on the supplemented media. Microscopic torpedo-shaped embryoids were frequently observed on BM + CM, rarely on BM + ME, and not at all on unsupplemented BM. The high frequency of microscopic globular proembryoids, and later of macroscopic pseudo bulbils, formed on BM + ME leads us to postulate that pseudobulbils are derived from globular proembryoids in which polarity is not established by the 16 to 32-cell stage. Microscopic torpedo-shaped embryoids probably give rise to macroscopic heart-shaped embryoids which develop into plantlets. The technique reported in this article provides an ideal system for examining embryogenesis per se and for studying the effects of various treatments on embryogenesis and organ differenti ation in vitro. It also affords excellent opportunities for the breeding of solid mutant plants.