Regulating Mechanism Of Hormone In Lotus Petiole Elongation Submerge And Construction Of Nelumbo Nucifera Regeneration System

Lotus is one of traditional flowers in China which has a profound cultural connotation,high ornamental,economic and ecological value.At present,the market demand of lotus(Nelumbo nucifera)is increasing year by year.Lotus can not accommodate to long time submergence which greatly limits the application of lotus.Previously study showed that lotus petiole elongated under flooding stress,and some studies in other plants confirmed that ethylene,gibberellin(GA)and abscisic acid(ABA)might be involved in regulation of this process.To reveal the possible mechanisms involved in of lotus response to submergence,the effect and interaction of ethylene,ABA and GA on lotus petiole elongation induced by flooding stress was explored.Moreover,we cloned a ABA8'-hydroxylase-lv gene NnABA8oxlv,which can regulate ABA metabolism,and analyzed its expression characteristics under submerged stress;For further investigating related gene function in lotus,we constructed the regeneration system(from callus via regeneration seeding to rooting)of lotus successfully,which provides an important carrier to lotus transgene.Callus rate,regenerate rate and rooting rate were improved in our regeneration system.The researches above provide an important technical basis for the next step to carry out the transgenic cultivation of submergence tolerance.The main results are as follows:(1)Using the 'Guangchang white lotus' seedlings(petiole 6 cm in length)as test materials,we observed the growth of lotus seedlings' petiole submerge after 12 h and 24 h submergence treatment.We found that compared with the control,petiole elongated significantly.Treated lotus seedlings with Ethrel,ACC(ethylene precursor),STS(ethylene inhibitor),GA,PAC(GA inhibitor),ABA and Tungstate(ABA inhibitor),ACC and ethylene we found that GA,Tungstate could promote the elongation of petioles at suitable concentrations,but STS,PAC,ABA reduced petiole growth.GA and ethylene caused a decrease in cell length with an increase in the cell number significantly,while ABA decrease petiole cell length and cell number.GA treatment could not reverse the effect of ABA and STS on petiole elongation;ABA was not affected on petiole elongation when Ehtrel was treated.However,PAC could inhibit the effect of Ethrel on petiole elongation;at the same time Tungstate could enhance the effect of GA and Ethrel,the effect of Ethrel on petiole elongation was enhanced when GA was treated.(2)Lotus ABA8'-hydroxylase-1v(NnABA8oxlv)gene were cloned and sequenced.The full-length sequence of this NnABA8oxlv gene encoded a polypeptide of 471 amino acid residues with a predicted molecular mass of 53.6 kDa and a theoretical isoelectric point of 9.51.We test the response of NnABA8oxlv in the tissues of petiole with different submergence time(0 h,6 h,12 h and 24 h)or different treatments(Ethrel,ACC and STS),and results showed that NnABA8oxlv is regulated by ethylene,because seedlings treated with ethylene and its precursor ACC had high NnABA8oxlv transcription level,while STS reversed its expression level.(3)The lotus seeds,ethanol and 3%sodium hypochlorite sterilized,are broken shell by pruning shears,then put complete lotus in medium to developed into aseptic seedling.The study found that using the method of shell breaking after sterilization can avoid the direct damage of disinfection to explants effectively,and pollution rate and mortality were low.Studying on the effect of different parts of explants on callus induction,the results showed that all of the leaf,the stem tip and the petiole could induce callus,and the leaf and the shoot tip had high callus rate,so they are more suitable as material for inducing regeneration.Increasing callus rate of leaf and stem tip by changing amount of explants,data shows that the number of 7 lead to the most prone to leaf callus.And shoot tip explants for the number 5 in the highest rate of callus,callus rate up to 60%.What's more,the number of explants rate had no significant effects on the browning rate.At the same time,differences between callus induction conditions of different varieties were also studied,the results show that the differences between varieties were obvious,the callus of 'Guangchang white lotus' appeared faster and more,while the callus of 'Weishanhu red lotus' and'Heilongjiang red lotus' appeared less in quantity,callus growth rate lower.(4)Studying on the effect of different content of 6-BA medium on the regeneration rate of stem tip and leaf,the results found that the 50 mol/L 6-BA promoted the regeneration of stem tip better,and the different levels of 6-BA had no significant difference in effect on the regeneration rate of leaf.The speed of regeneration of stem tip was obviously faster than the leaf' s under the same 6-BA content,and stem tip had higher regenerate rate than the leaf' s,which proved stem tip are more suitable as materials for regeneration inducing.(5)Studying on the effect of different rooting medium on lotus rooting,results showed that using IB A made the lotus rooting grow well.And low concentration of IB A made the root number increase,higher concentration of IBA increases the rooting rate.