Optimized Protocol for Sorghum Regeneration: Enhancing Embryogenic Callus Formation from Immature Inflorescences

This study aims to develop an efficient and reproducible in vitro protocol for high-frequency embryogenic callus induction and subsequent plant regeneration in multiple sorghum (Sorghum bicolor L. Moench) cultivars, thereby establishing a foundation for genetic transformation, mutation breeding, and other biotechnological applications aimed at enhancing sorghum crop improvement and productivity.

Sorghum (Sorghum bicolor (L.) Moench) is an important cereal crop known for its adaptability to harsh environments and nutritional value. Despite its significance, sorghum remains challenging for in vitro propagation due to difficulties in regenerating callus tissue, especially from monocotyledonous explants. Callus induction and regeneration protocols are crucial for genetic transformation, mutation breeding, and biotechnological applications in sorghum improvement.

To establish an effective in vitro protocol for callus induction and subsequent plant regeneration using different sorghum cultivars, optimizing conditions for highfrequency embryogenic callus formation and plant regeneration.

Six sorghum cultivars (IS 3477, IS 33095, IS 7155, IS 2898, IS 7005, and IS 1202) were selected. Immature inflorescence explants were cultured on a modified Murashige and Skoog's (MS) medium with 3% sucrose, 0.8% agar, and 2.0 mg/l 2,4-D for callus induction. After 14 days, embryogenic and non-embryogenic calli were distinguished. Regeneration media were optimized using embryogenic calli, with 1.5 mg/l 6- benzylaminopurine (BAP) for shoot development and 1 mg/l NAA (1-naphthaleneacetic acid) in a half-strength MS medium for root development.

Two distinct forms of calli were observed: a non-embryogenic light yellow callus and a white, granular embryogenic callus. Embryogenic callus induction frequency varied from 40% to 96% among the cultivars, with IS 3477 and IS 33095 exhibiting the highest frequencies (96% and 88%, respectively), while IS 1202 showed the lowest (40%). Regenerated shoots were successfully developed within 6-18 days and later transferred to a rooting medium, resulting in healthy plantlets. Transplanted plantlets showed normal growth and no morphological abnormalities in the field.

This study provides a reliable protocol for efficient callus induction and plant regeneration in multiple sorghum cultivars. The optimized conditions can be utilized for genetic studies, crop improvement, and biotechnological applications, thus contributing to the advancement of sorghum breeding and biotechnology research.