Somatic Embryogenesis And Plant Regeneration Of Onion

In order to establish high frequency regeneration system of onion in vitro to use for further transgenosis, the seeds of onion (male sterile line 10-8F) was used as experimental explants.The effects of medium types, concentrations of 2,4-D and sucrose in culture medium on callus induction was investigated, the optimal callus induction was found in MS medium supplemented with 0.5 mg-L-1 2,4-D and 40 g·L-1 sucrose, callus induction rate reached to 80.3% after 50 days of culture in vitro.To induce embryogenic callus, effects of the callus in different culture days, medium component, the callus cultured for 45 days was more efficient for induction of embryogenic callus than the callus of other culture days (15,30, and 60 day); the highest embryogenic callus induction rate (88.9%) was found in MS medium (NH4+/NO3-=30/30 mM) supplemented with 2.0 mg·L-1 BA plus 0.25 mg·L-12,4-D, and 50 g·L-1 sucrose under dark. Bioreactor could be used for mass production of embryogenic callus. The proliferation and growth was better in immersion than in ebb&flood bioreactor culture. High fresh (102.2 g) and dry weight (9.5 g) of embryogenic callus as well as growth coefficient (9.2) were observed when 5 g·L-1 inoculums were inoculated into 3 L bioreactor with 2 L working volume and aerated at 0.1 vvm during immersion culture. The kinetic of embryogenetic cell growth was investigated, the yellow and globular particles were observed on the surface of callus after 10 days of culture, these particles increased with increasing culture days, and covered the callus surface after 40 days of culture, the embryogenetic callus were induced. The histomorphology of embryogenic callus and non-embryogenic callus was observed, the cell of embryogenic callus was rounded and small with big nuclear and dense cytoplasm, and intercellular space was little, besides, its cell was stained deeply, fitly arranged, and distributed on the surface of callus. However, the cell of non-embryogenic callus had a big vacuole with small nuclear, was random arranged and its shape was irregular.In order to obtained mature somatic embryos, the embryogenic callus were cultured on MS medium with different concentrations of sucrose and VB1, the result showed that MS medium supplemented with 20 g·L-1 sucrose and 10 mg·L-1 VB1 was optimal for embryo maturation; in addition, uniformed mature somatic embryo was found when the culture medium supplemented with 0.5 mg·L-1 ABA, the germination rate reached to 90.3% and each explant formed 6 mature somatic embryo.For obtaining completed plantlets with roots in vitro, the mature somatic embryos were inoculated into MS medium without plant growth regulators and cultured for 15 days. After then, the plantlets were transplanted into different substances, the high survival rate (80.8%) was found in 1/2 vermiculite plus 1/2 perlite.