Stimulation of Rooting of Citrus Embryoids by Gibberellic Acid and Adenine Sulphate

Embryoids obtained from subcultured ovular callus of the Shamoutiorange (Citrus sinensis) were grown on Murashige and Tuckernutrient medium (BM) in the presence and absence of gibberellicacid (GA₂), adenine sulphate (ADS) and combinations of these.Both GA₂ and ADS stimulated significantly the rooting of smallembryoids with unorganized tissue, and larger embryoids withpartially developed root zones. Only large embryoids with fullydeveloped root zones rooted well on BM but better on BM+GA₂+ADS.Rooting was best on media containing 1 to 5 mg per 1 ADS, andthese two combined. The optimum sucrose concentrations were5 to 6 per cent. Shoot formation on embryoids prior to rootingcompletely inhibited rooting which is otherwise enhanced byGA₂+ADS. Filter-sterilized and autoclaved GA₂, showed similaractivity in rooting.     

Fine Structure of and Embryoid Development from Embryogenic Ovular Callus of 'Shamouti' Orange (Citrus sinensis Osb.)

A morphologically, anatomically and physiologically unique callushas been developed from unfertilized ovules of Shamouti orange.The callus, which is suspected to be of nueellar origin, isnot made up of the normal unorganized parenchymatous tissue,but solely of numerous proembryoids which vary between 0.1 and1.0 mm in diameter. Adventive embryogenesis in this tissue isautonomous and is in fact depressed by the inclusion of growthregulators in the medium, despite having been in culture formore than 2 years. Embryogenesis occurs in single cells on the periphery and withinexisting proembryoids. Cells destined to form new proembryoidsare surrounded by greatly thickened cell walls which lack plasmodesmata.Cell divisions occur within the thickened walls to give riseto globular proembryoids which are freed from encasing thickwalls as these degenerate. Proembryoids may enlarge into spherical pseudobulbils up to4 mm in diameter with an epidermal cell layer but no vascularization.Such structures rarely develop into plantlets but do form furtherproembryoids from surface cells. Alternatively, proembryoidsmay develop into heart-shaped, torpedo, and cotyledonary embryoids,and thence into plantlets with varying degrees of organ fasciation. Since plantlets are derived from single, usually surface cells,this system holds great promise for the production of solidgenetic mutants by irradiation.