Morphogenesis Of Linum Usitatissimum L. 'Fany' In Vitro And Protoplast Fusion Between L. Usitatissimum 'Shuangya No.5' And Apocynum Venetum L.

The calli of Linum usitatissimum L.'Fany'were emergent with cotyledon and hypocotyl of'Fany'as explants inoculated respectively on different MB media, embryogenic callus obtained only on the medium MB+0.5-4.0 mg·L-1 2,4-D. The 2,4-D concentration needed for embryogenic callus derived from hypocotyl was lower than that from cotyledon. Effect supplemented only with 2,4-D was better than that of other groups in induction of embryoid, the 2,4-D concentration inducing embryoid from cotyledon callus was two-fold higher than that from hypocotyl callus, the frequency of embryogenesis from cotyledon callus was up to 224 g-1 when cultured on MB supplemented with 1.0 mg·L-1 2,4-D, while it reached 265 g-1 when cultured on MB supplemented with 0.5 mg·L-1 2,4-D. The histological observation revealed that there were a large number of normal globular, heart-stage and torpedo-stage embryoids, as well as a few cotyledon-stage embryoids and abnormal embryoids. The adventitious buds of Linum usitatissimum L.'Fany'were induced from calli which were derived from cotyledon and hypocotyl of'Fany'respectively. The optimum differentiation medium was MB+0.5 mg·L-1 6-BA+0.1 mg·L-1 NAA +5% sucrose.The course of adventitious buds production was observed by paraffin section. The optimum conditions on dissociate protoplast, electrofusion and protoplast culture of Linum usitatissimum L.'Shuangya No. 5'and Apocynum venetum Linn. were explored. The two kinds of protoplasts derived from established embryogenic cell suspension lines of'Shuangya No.5'and A. venetum were fused by electrofusion and then cultured. The optimum enzyme combination for isolation of'Shuangya No.5'protoplasts was 0.5% Cellulase Onozuka R-10+1.5% Cellulase Onozuka RS+0.2% Pectolyase Y-23. The proper enzymolysis time was 3.5 h. The protoplast production of flax reached 1.32×106 mL-1 and the survival rate reached 93.6% at the third day after transport. The optimum electrofusion parameters of two kinds of protoplasts were AC voltage 10 v, AC duration seconds 60 s, post fusion AC seconds 6 s, DC voltage 1000 v, pulse length 50μs, number of pulse 3, number of repeats 4. The fusion rate reached 22.8% under the condition.The above work may supply reference and evidence for somatic hybridization breeding between'Fany'and other flax, screening somaclonal variation, relative plant gene engineering and cell engineering research.These results laid the foundation for further study of somatic hybridization breeding between flax and A. Venetum, as well as for other plants.