An Efficient and Rapid Protocol for Somatic Shoot Organogenesis from Juvenile Hypocotyl-Derived Callus of Castor Bean cv. Zanzibar Green

Abstract

Aseptic seedlings of different ages derived from surface-sterilized mature seeds were applied as an explant source. Various explants such as 7- and 21-day-old hypocotyl fragments, 42-day-old nodal stem segments, and transverse nodal segments of stem, as well as leaf petioles, were cultured on the agar-solidified Murashige and Skoog (MS) basal medium supplemented with 0.1 mg/L IAA, 5 mg/L AgNO3 and different types and concentrations of cytokinin (1 mg/L zeatin, 0.25 mg/L thidiazuron (TDZ), and 5 mg/L 6-benzylaminopurine (6-BAP)). Consequently, it was found that 7- and 21-day-old hypocotyl fragments, as well as nodal stem segments obtained from adult aseptic seedlings, are characterized by a high explant viability and callus formation capacity with a frequency of 79.7–100%. However, the success of in vitro somatic shoot organogenesis was significantly determined not only by the culture medium composition and explant type but also depending on its age, as well as on the size and explant preparation in cases of hypocotyl and age-matched nodal stem fragments, respectively. Multiple somatic shoot organogenesis (5.7 regenerants per explant) with a frequency of 67.5% was achieved during 3 subcultures of juvenile hypocotyl-derived callus tissue on MS culture medium containing 0.25 mg/L TDZ as cytokinin source. Castor bean regenerants were excised from the callus and successfully rooted on ½ MS basal medium without exogenous auxin (81%). In vitro plantlets with well-developed roots were adapted to ex vitro conditions with a frequency of 90%.