| In order to obtain the synchronous artificial embryo of Pinellia ternata,the leaves of Pinellia ternata were used as explants and different plant growth regulators and nutrients were added for induction.The conditions suitable for the induction of loose callus were screened and the loose callus was used for suspension culture.Finally,the key factors affecting the synchronization of artificial embryo were identified.The main research results are as follows:?1?The effects of different carbon sources?Sucrose,Maltose,Glucose?,nitrogen sources?Peptones,Beef extract,Acidic hydrolyzed casein?,6-benzylamine?6-BA?,2,4-dichlorophenoxyacetic acid?2,4-D?on loose callus induction in Pinellia ternata leaves were studied.The results showed that 2,4-D had asignificant effect on the induction of loose callus,and sucrose had a small effect on callus induction.The fresh weight of callus increased first and then decreased with the increase of 6-BA concentration;30 g·L-1sucrose treatment group callus fresh weight maximum;callus treated with 0.4 g·L-1 acid hydrolyzed casein protein had loose texture.Based on the analysis of fresh weight of callus and the degree of callus porosity,the appropriate conditions for callus induction were:MS+1.0 mg·L-1 2,4-D+1.5 mg·L-1 6-BA+20 g·L-1 sucrose+0.4 g·L-1 CH.?2?Using Pinellia ternata loose callus as test material,study different carbon sources?Sucrose,Maltose,Glucose?,nitrogen sources?Peptone,Meat extract,Acid hydrolyzed casein?,and liquid contents?30 m L,40 m L,50 m L?,the inoculation amount?2 g,3 g,4 g?and the effect of different types andconcentrations of plant growth regulators on the proliferation of Pinellia ternata suspension culture cells.The results showed that sucrose was a suitable carbon source and CH was a suitable nitrogen source.When the volume of the solution was 50 m L,the callus was fresh and the cell sedimentation volume was thelargest.When the inoculation amount was 3g,the cell viability was strongest and low.Cell proliferation was better under the concentration of 2,4-D.When 2,4-D 0.5 mg·L-1,6-BA 1.5 mg·L-1,NAA 1.5 mg·L-1,the cell proliferation was the largest and the most vigorous.Based on the combination of cell viability,proliferation factor and cell sedimentation volume,the suitable culture conditions for cell proliferation of Pinellia ternata suspension culture are:MS+0.5 mg·L-1 2,4-D+1.5 mg·L-1 6-BA+1.5 mg·L-1 NAA+30 g·L-1 sucrose+0.4 g·L-1 CH,the inoculation volume is 3 g,and the liquid volume is 50 m L.?3?In this work,1.0 mg·L-1 2,4-D+0.5 mg·L-1 6-BA+30 g·L-1 sucrose+0.3 g·L-1 CH was added as the basic medium for suspension culture.The effects of temperature,caffeine,and sucrose concentration on the synchronization of Pinellia ternata suspension culture cells were investigated.Interestingly,thesynchronization effect was the best and then cell division index was 13.86%when the temperature was set at 8?for 12 h and then recovered for 36 h.Meanwhile,the effect was the better and the cell divisionindex was 9.42%when the cell was treated with 2.00 mmol·L-1 caffeine for 2 h followed by recovering for48 h.And the effect was moderate and the cell division index was 5.33%when the cell was treated with 40g·L-1 sucrose for 5 d and then recovered for 4 d.?4?Using Pinellia ternata suspension microcells as test materials,MS+30 g·L-1 sucrose+0.3 g·L-1 CH as the basic medium,to investigate the effects of different 6-BA and IBA concentrations and inoculation amount on the synchronous expansion of suspension microcells in Pinellia ternata.The results showed that the most significant effect was 6-BA,and the synchronization effect was the best when 0.5 mg·L-1 IBA+0.5mg·L-1 6-BA+inoculation amount was 3 g.?5?The effect of different 6-BA,IBA,and sucrose concentrations on the synchronous differentiation of Pinellia ternata suspension microcells was studied using the expanded microcells as test materials and MS+0.3 g·L-1 CH as the basic medium.The results showed that 0.5 mg·L-1 6-BA+0.1 mg·L-1 IBA+10 g·L-1sucrose had the highest simultaneous differentiation rate.The results of this paper lay a good foundation for further study on the induction and proliferation of loose callus in Pinellia ternata tissue culture,especially the regulation of seed embryo synchronization,and provided theoretical guidance for solving the problem of artificial seed embryo synchronization in Pinellia ternata,which has theoretical significance for the industrialization of artificial seeds in Pinellia ternata. |
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