Key Gene Mining Of Double Flower Formation And Molecular Design Breeding In Gysophila Paniculata

Gypsophila paniculata L.is a perennial herb of Gypsophila in Caryophyllaceae.It is one of the largest flower matching materials in the world,as well as one of the top ten best-selling cut flowers in the global market.At present,the cultivation of new varieties of G.paniculata mainly depends on traditional breeding methods.Due to the basic sterility of double flower in G.paniculata,it seriously limited the development space of the breeding in G.paniculata.Therefore,understanding the mechanism of double flower formation and establishing the efficient genetic breeding systems will help to break the traditional breeding limitations and provide theoretical basis and technical support for molecular breeding of G.paniculata.The research results are as follows:EMS mutagenesis showed that the best treatment time was 6 h with 150 mmol / L EMS,in which the half lethal rate could be achieved.After EMS treatment,some mutant plants were observed,and one white double flower and two pink double flower mutants were also identified.By referring to the genome information of G.paniculata and the known MADS box gene sequences of other species,multiple sequence alignment and phylogenetic tree were constructed.95 MADS box genes and 62 type II MADS box genes were found in the genome of G.paniculata.The distribution and quantity of ABCE genes in G.paniculata were similar to those in Arabidopsis,and without obviously gene expansion.Compared with wild-type G.paniculata,the expression of class C gene AG in two commercial double flower varieties(’YX1 ’and’ YX4 ’)decreased significantly during flower developmental stages,while the expression of class A,B and E genes increased.The ABCE models were established according to the gene expression of single and double flower.Through gene cloning and sequence alignment analysis,different degrees of mutation were found in AG genes of double flower compared with single flower.Meantime,SNP substitutions(G-C)were identified at mi R172 binding sites of PETa gene in commercial varieties’ YX1 ’and’ YX4 ’,but there was no change that of single flower and EMS mutants.Therefore,our data suggested AG gene mutation and mi R172 mediated AP2/ PET play a joint role in the formation of G.paniculata double flower.In the adventitious bud induction test of G.paniculata,when the adventitious bud induction medium was MS + 2.5 mg / L TDZ + 0.1 mg / L NAA + 30 g / L sucrose + 8 g / L agar,the coefficient of inducing adventitious buds of G.paniculata ’YX4’ was the highest,up to 4.14.On the basis of establishing a good regeneration system,Agrobacterium tumefaciens containing p CAMBIA1301-220.6-Pg F3′5′H vector was used to establish the genetic transformation system of G.paniculata.It was found that G.paniculata had the best effect of inducing bud regeneration under the conditions of pre-culture for 1 day,co-culture for 5 days,Agrobacterium infection for 10 minutes and as concentration of 30 mg / L,and the infection time was the main factor affecting the formation of adventitious buds.As a consequence,281 seedlings were obtained,after the screen,in which 38 seedlings showed positive bands,indicating the positive rate was about 13.8%.However,the flower color of transgenic G.paniculata did not show ang changes but white.It was found that the indigo gene was transiently transformed into the G.paniculata plant by the method of idgs gene transfection.In this study,PDS gene editing vector was also constructed and used to establish the gene editing system in G.paniculata.A total of 34 seedlings were identified,and the T-DNA insertion rate was 50%.Due to the small sample size,the editing of target sites was not detected,which was consistent with the absence of albinism in the leaves of positive plants.On this basis,the AG gene editing vector related to the formation of double flowers was constructed and transferred into wild-type with white flower in G.paniculata.At present,the seedlings are in the stage of recovery culture.