Thidiazuron Role in In vitro Flowering of Murraya paniculata and Fortunella hindsii via Embryogenic Protoplast and Hypocotyl Segments Cultures

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Background: Procedures to regenerate protoplast into plants of citrus relatives is important to establish the genetic improvement for sweet oranges by using somatic hybridization and transgenic. Protoplasts are a good material to genetically manipulate and to become gene transfer. This study aims to characterize the regeneration sequence of Fortunella hindsii and Murraya paniculata protoplasts via somatic embryogenesis into flowering plants in vitro. This study also describes the sequence of flowering in vitro originated from hypocotyl segments of plantlets of two species. Methods: Protoplast sources that were isolated from callus of Fortunella hindsii and Murraya paniculata. Embryogenic callus was put in Erlenmeyer and mixed together with 0.4% macerozyme, 0.2% cellulose and 0.1% driselase and pH was adjusted to 6.0. Two drops of purified protoplasts were cultured with media supplemented with 2.0% glucose and 0.0, 0.001, 0.01, 0.1 or 1.0. 10.0 mg/l thidiazuron (TDZ) and thicken with 0.9% gelrite. Result: The media supplemented with 0.001 mg/l TDZ yielded the maximum plating efficiency, while 0.001 mg/l TDZ produced the highest percentage of shoot formation, approximately 80%. After being cultured on the same TDZ concentration for 14 days, the protoplasts that survived developed cell walls. Fortunella hindsii and Murraya paniculata underwent somatic embryogenesis to grow into plantlets. Thidiazuron has demonstrated efficacy in forming flower buds that grow normally. Murraya paniculata and Fortunella hindsii shoots that emerged from hypocotyl segmments flowered in vitro on half-strength MT media containing 0.001 to 0.01 mg/l TDZ and 2-3% sucrose after two months of culture and they eventually went on to flower.