Factors affecting cAMP content in jujube pulp callus
Cyclic adenosine monophosphate(cAMP),low content in organisms,functions a second messenger which involves in physiological and biochemical regulation in plants.In addition,cAMP plays an important role in plant growth and development,environmental adaptation,and has certain medicinal values and other practical functions.However rapid propagation of cAMP-rich plant materials through tissue culture method can be used to extract cAMP.Chinese jujube originated from China and its mature fruits contains high level of cAMP compare to other plant.Thus,in order to explore whether it is possible to use the jujube fruit callus to extract cAMP instead of jujube mature fruits,the effects of different jujube genotypes and fruit maturity on the cAMP content in jujube pulp callus,and the regulating effects of culture medium type,the number of sub-cultivation and the actions of the key enzyme adenylate cyclase(AC)on the cAMP content in jujube pulp callus were investigated.A variety of different jujube genotypes and different developmental stages of jujube fruits were selected for callus induction and cultivation after screening the most suitable cultivating conditions.After the callus of different genotypes and different developmental stages were induced,subcultivation and proliferation were carried out.When the callus grew to a well status,the samples were collected to do the cAMP content test.Briefly,the samples were evenly ground with a frozen grinder,and dissolved with pure water.The ratio of material to liquid was 1∶5.Then,it was bathed in water bath at 60 ℃ for 3 h,and centrifuged at the speed of 5 000 rpm for 20 min,and the supernatant was filtrated by proper membrane for the determination of cAMP.In addition,for the ions of the key enzyme AC treatments,three kinds of callus materials with high,medium and low content of cAMP were selected to treat with different concentrations of MgCl2,MnCl2 and CaCl2(20 μmol/L,50 μmol/L,100 μmol/L),respectively.Samples were selected after treatment for 0 h,24 h,48 h and 72 h.The content of cAMP was determined by cAMP ELISA kit.According to the introduction of the kit,OD values of all samples were measured with the enzyme label instrument at 450 nm wavelength,and all samples were tested at least with three repetitions.Finally,linear regression curves were drawn,and the cAMP concentration values of each sample were calculated according to the curve equation.The results showed that the content of cAMP in pulp callus was higher than that in young fruits.The content of cAMP in pulp callus of different genotypes was different,but it was much smaller than that in pulp of different genotypes.The content of cAMP in pulp callus of'Chahuzao'was the highest,significantly higher than that of'Chaoyangyuanzao'and'Wuhe 72',but not significantly different from that of other jujube genotypes.Callus was most easily formed in the pulp of young fruit stage,and it became more and more difficult to induce callus with the increase of fruit maturity.Moreover,the difference of cAMP content in the pulp callus induced by fruits at different developmental stages was not significant,which was much smaller than the difference of cAMP content in the pulp of different developmental stages.Both the young and dot red phase fruits of'Sour jujube 2X'could successfully be induced callus,and there was no significant difference in the cAMP content of the pulp callus between the two stages.Medium type not only affected the callus formation,but also significantly affected the content of cAMP in flesh callus.Moreover,it was found that the cAMP content of the tenth subcultivation was significantly lower than the cAMP content of the pulp callus of the fifth subcultivation.Among them,the difference in cAMP content between the fifth and tenth sucultivating pulp callus was the largest on G7 medium,while the cAMP content of a few genotypes of pulp callus on other mediums remained unchanged.Therefore,with the prolongation of subcultivating times,the overall cAMP content showed a downward trend.Furthermore,adding AC ions(magnesium chloride,manganese chloride and calcium chloride)of different concentrations as well as different treatment periods into the medium has some effect of cAMP content in pulp callus.Treatment with 100 μmol/L manganese chloride for 48 h significantly increased the content of cAMP in the pulp callus of young fruit stage,and treatment with 100 μmol/L calcium chloride for 48 h significantly increased the content of cAMP in the pulp callus of'Xiaozizao'and'Sour jujube 2X'at half-red stage.Treating with 100 μmol/L magnesium chloride for 48 h can significantly increase the cAMP content of callus in the fruit pulp of'Jishanbanzao'in the half red stage.The callus genotypes,fruit maturity,medium type,subcultivating times and AC-coions had certain regulation effects on cAMP content in jujube pulp callus.Thus,when using callus culture to produce cAMP,we should focus on optimizing callus culture environment.This study provided a reference for the production of cAMP through tissue culture and the regulation of cAMP content in jujube fruits.
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