High Frequency Regeneration Of Protoplasts From Litchi Transgenic Embryogenic Calli

In this experiment, the conditions of subculture and maintenance of 11 cell lines of trangenic resistant embryogenic calli (TREC) with htp and gus {uid A) genes via particle bombardment, were first optimized and the GUS histochemical assays of TREC were also detected in Litchi chinensis Soon. cv. 'Xiafanzhi'; and then the TREC cell lines Q811, Q81.535, Q9221, Q9223 and Q1016 were selceted as materials for the isolation and hgih-frequency culture of protoplasts, and embryogenic calli derived from protoplasts were further used as materials for inducing somatic embryogenesis and plant regeneration. The main results showed as follows:1. Optimization of the conditions of subculture and maintenance of TREC. Trangenic embryogenic calli grew well and expressed GUS steadily through alternative subculture of the two media for 2 years, and the suitable meida for alternative subculture were MS medium supplemented with 1.0 mg·L^-1 2,4 —D and MS medium supplemented with 1.0 mg·L^-12,4—D and 50 mg ·L^-1 Hygromycin B.2. The GUS histochemical assays of litch TRECs. The GUS histochemical assays of litchi TRECs during long-term subculture indicated that TREC cell lines Q811 and Q81.535 were steadily expressed GUS; TREC cell lines Q9221 and Q9223 were chimerically expressed GUS; TREC cell lines Q1016 Q6L61 Q6L161 Q42-1 Q42—2 N31 and N21 were steadily unexpressed GUS.3. Optimization of the conditions of protoplast isolation from litchi TREC. The optimized procedures were as follows: litchi transgenic embryogenic calli after culture for 16-18 d were transferred into the CPW-13M enzymatic solution containting 0.8% Cellulose R-10, 0.4% Macrease R-10 continuously shaking at 30— 40 r · min^-1 for 10—11 h at (25±2)℃ in darkness for innoculation; and the yield of 1.82× 10⁷ protoplasts·g^-1 and viability of 97.5% were obtianed under the optimized procedures in the resistant cell line Q811.4. Establishment of high-frequency regeneration system of protoplast from litchi TREC. Litchi TREC protoplasts regenerated at the frequcey of division of 18.78%, colonies formation (culture for 20 d) of 22.30% and colonies formation (culture for...