Interfamilial Somatic Hybridization Between Astragatus Molilotoides Pall. And Zygophyllum Xanthoxylum,Cloning Of PA1 Gene And Transformation Of Alfalfa

The plant regeneration systems were established from protoplasts of Astragalus molilotoides pall. and Zygophyllum xanthoxylum. The conditions of protoplast isolation and culture were optimized. Based on this, somatic hybridization between Astragalus molilotoides pall. and Zygophyllum xanthoxylum was carried out, and somatic hybrid cells were selected by use of physiological complementation of parent protoplasts pretreated with UV radiation and Rhodamine-6G respectively. The hybrid calli were produced. At the same time, a gene of a sulphur-rich and insect-toxic seed albumin—PA1 gene was cloned from Pisum Linn. (cultivar shijiadacaiwan) and transformed into alfalfa thought Agrobacterium tumefaciens method. The transgenic plants were regenerated. The embryoid variations of transgenic plants were examined and PA1 gene expression was characterized. The main results were as follows:1. Plenty of viable protoplasts were isolated from the totally extend young leaves of Astragalus molilotoides pall. after precultured in "dark for 4 days and pretreated in plasmolysal solution for 60 min. Protoplasts were induced to undergo sustained divisions in DPD medium supplemented with 2.0 mg/L 2,4-D, 0.2 mg/L 6-BA, 2%(w/v)sucrose, 0.4 mol/L mannitol and 500 mg/L casein hydrolyasate at a plating density 3×10⁵/ml. The division frequency was about 55.6%. The regenerated calli were cultured on agar solidifed MS medium plus 1.0 mg/L 2,4-D, 1.0 mg/L 6-BA and 0.5 mg/L KT. The plant differentiation frequency was over 96%. The regenerated plants was successfully transplanted in soil.2. The cotyledons and the calli of. Zygophyllum xanthoxylum were used as materials for protoplast preparation, and large amount of protoplasts were isolated from calli. The protoplast yield and viability from calli of Zygophyllum xanthoxylum were higher than those from the cotyledons; 2.4×10⁶/g·FW of protoplast yield and 89% of viability were obtained from light yellow calli after transferred and cultured for 14 days in the dark. The enzyme solution for protoplast isolation was composed of 2% cellulose, 1% hemicellulase and 0.5% pectinase and the enzyme digestion was completed in 13 h. Using liquid thin layer culture method and the proper phytohormone combination, i.e. 2 mg/L 2,4-D and 1.0 mg/L 6-BA, the division frequency of protoplasts was up to 72%.3. Protoplast fusion between Astragalus molilotoides pall. and Zygophyllum xanthoxylum was carried out using modified PEG-high pH, high Ca²⁺ method. The interfamilial somatic hybrid cells were selected based on physiological complementation between both parents which were pretreated respectively with UV radiation and Rhodamine-6G. The system for somatic hybrid cell selection might be extensively useful. Two calli of hybrid were obtained from this selected system and two shoots were produced. Cytology and molecular biology identification confirmed the hybrid nature of two calli.4. A gene of a sulphur-rich and insect-toxic seed albumin—PA1 gene was cloned from Pisum Linn. (cultivar shijiadacaiwan) and the plant expression vector of pCAMBIA1301-PA1 was constructed. The vector was used to transform into alfalfa by Agrobacterium tumefaciens method. The conditions for transformation were optimized. Transgenic regenerated calli and plants were obtained. PCR and Southern blotting identification confirmed that PA1 gene and hygromycin-B phosphotransferase-resistant gene were integrated into the genome of alfalfa, and SDS-PAGE analysis indicated PA1 gene expressed in leaves of transgenic plants.