Functional Identification Of Apple Baby Boom In Genetic Transformation And Somatic Embryogenesis

The low efficiency of genetic transformation in Apple(Malus × Domestica),as a perenni al woody plant,has limited the research of apple gene function and molecular mechanism as well as the development of molecular breeding.Regeneration,a key step in genetic transform ation,has been less studied in apples with respect to its key genes and mechanisms.BBM(Ba by Boom)belongs to AP2/ERF transcription factor family,which is located in arabidopsis see ds and embryos,and can promote adventitious shoot regeneration in Arabidopsis and rape,an d improve the efficiency of genetic transformation by promoting adventitious shoot regenerati on in maize.Somatic embryos can develop directly into complete plants,so the study of soma tic embryo regeneration is of great importance to woody plants.Overexpression of BBM induc es spontaneous formation of somatic embryos in Arabidopsis and oilseed rape.In apples,the f unction of BBM related to adventitious shoot regeneration is not well studied and there is a lac k of studies on the function of somatic embryo regeneration.In this study,the homologous sequence MdBBM(MD04G1247700)with the highest sim ilarity to the amino acid sequence of Arabidopsis thaliana BBM was found in the apple databa se.Bioinformatics analysis was conducted on apple and agrobacterium-mediated genetic trans formation was used to obtain transgenic material overexpressing apple BBM.Studies were ca rried out on its functions in genetic transformation efficiency,adventitious bud regeneration,g rowth and development,and somatic embryogenesis.The specific research results are as follo ws:1 MdBBM was most closely related to peach,European sweet cherry,almond and plum by phylogenetic tree analysis.In addition,MdBBM contains specific motif bbm-1 and conser ved domain AP2 by multiple sequence alignment,suggesting the conserved function of Md B BM.Comparison of the predicted tertiary structure of Arabidopsis(Arabidopsis thaliala)BB M revealed that the tertiary structure of MdBBM is more complex,suggesting that in apple,MdBBM may have evolved more complex functions.2 Analysis of the promoter cis-acting elements revealed that the MdBBM gene promoter contains elements related to light response,seed-specific regulation,and Me JA response.Prot ein interaction network prediction revealed that MdBBM had potential interaction with merist em regulation-related proteins WUSCHEL and SCARECROW.3 The q RT-PCR analysis revealed that the expression level of MdBBM was higher in the ovary,root and seed organs,all of which indicated that MdBBM might play an important role in the regeneration process.4 In order to further explore the function of MdBBM,the empty expression vector p RI101-AN and overexpression vector p RI101-MdBBM were simultaneously transformed into appl e leaves in vitro.It was found that the regeneration efficiency of p RI101-MdBBM was 16 time s higher than that of p RI101-AN.These results indicated that MdBBM overexpression could p romote the efficiency of genetic transformation in apple.5 During the continuous growth of the transgenic tissue culture seedlings,it was found th at the height of overexpressed MdBBM apple tissue culture seedlings was 1.6 times as high as that of the control,and the number of reproductive seedlings was more than 2 times as high as that of the control,indicating that overexpressed MdBBM could promote the internode elonga tion and improve the regeneration efficiency of apple tissue culture seedlings.Ectopic express ion of MdBBM promotes flowering in Arabidopsis thaliana,suggesting that overexpression of MdBBM has a pleiotropic effect on apple growth and development.6 The effect of MdBBM expression on regeneration was further studied.There was no sig nificant difference in the shoot regeneration rate between the overexpressed MdBBM leaves a nd the control leaves,while the root regeneration rate of the overexpressed MdBBM leaves wa s 3.75 times higher than that of the control leaves,and only the overexpressed MdBBM leaves regenerated somatic embryos,suggesting that the overexpression of MdBBM promoted advent itious root generation and induced somatic embryogenesis.In the process of inducing somatic embryos containing 2,4-D,overexpression of MdBBM promoted the formation of embryogen ic callus,increased the number of somatic embryos and accelerated the process of somatic em bryo formation.Overexpression of MdBBM was found to promote somatic embryogenesis by q RT-PCR analysis by increasing the expression level of Md LEC2,a gene related to embryoni c development.In summary,we mainly found that overexpression of MdBBM has functions in promotin g genetic transformation,adventitious root regeneration and somatic cell embryogenesis regen eration.At the same time,we found that overexpression of MdBBM has a multi-effective effec t on growth and development,which lays the foundation for an in-depth study of the molecula r mechanisms regulating apple regeneration,and lays the theoretical foundation for improving apple transformation efficiency with the help of MdBBM and establishing an efficient genetic transformation system for apple by molecular means.