Establishment Of The Rapid Propagation System Of Platanus × Acerifolia ’Huanong Fufeng’ And Application Of PaAGL15 Gene In Germplasm

Platanus × acerifolia is widely used in urban greening because of its excellent characteristics such as beautiful tree shape and thick crown shade.However,the problem of falling fruit flying in spring every year not only pollutes the urban environment,but also endangers people’s health.Theoretically,genetic engineering breeding can solve this problem completely,but the efficient and stable genetic transformation system of Platanus × acerifolia remains to be explored.In this study,the substalk bud of ’Huanong Fufeng’,an improved strain obtained by radiation mutagenesis,was used as the experimental material to establish a rapid propagation system,which laid a foundation for the popularization and application of late flowering and little fruit Platanus × acerifolia and the establishment and optimization of genetic transformation system of Platanus × acerifolia.At the same time,two AGL15 homologous genes with differential expression between wild type and late flowering and little fruit specific germplasm of Platanus × acerifolia were screened from the transcriptome database to explore their functions,which provided theoretical guidance for further improvement and expansion of gene resources of Platanus ×acerifolia germplasm.The main research results are as follows:1.The substalk bud of ’Huanong Fufeng’ of Platanus × acerifolia was used as experimental material to explore the effects of different disinfectants and combinations of disinfection duration,different combinations of plant growth regulators and concentrations,different basic medium and different transplanting medium on the proliferation,seedling strengthening,rooting and transplanting of’Huanong Fufeng’ tissue culture seedlings,so as to establish a rapid propagation system of ’Huanong Fufeng’.The process is as follows: The substalk buds collected from August to November were disinfected with 75% alcohol 30 s+1% Na Cl O 18 min and inoculated on germination medium MS+0.3 mg/L 6-BA+0.05 mg/L NAA for germination.The contamination rate and germination rate were 12.78% and 91.88%,respectively.About 0.7 cm of normally germinated buds were cultured in the proliferation medium MS+0.5 mg/L 6-BA+0.05 mg/L NAA,and the proliferation coefficient was 4.30±0.37.After 30 days of treatment,the average height of the seedlings reached 2.113±0.22 cm.The propagating shoots in good condition of about0.7 cm were transferred to the strong seedling medium MS+0.3 mg/L 6-BA+0.05mg/L NAA.The rooting rate was up to 91.2% after 40 days in WPM medium.The rooting seedlings were transplanted into a mixture of substrate soil: vermiculite:perlite =3:1:1,and then transplanted into the field when the plants grew to a certain size and the roots were well developed.2.Two AGL15 homologous genes with differential expression characteristics were screened and cloned by referring to the specific germplasm and wild-type transcriptome databases of Platanus × acerifolia,and named as PaAGL15-1 and PaAGL15-2 by amino acid sequence alignment and phylogenetic tree analysis.The results of annual expression pattern analysis showed that the expression levels of PaAGL15-1 and PaAGL15-2 were higher in two flower development stages and lower in the rest stages.The expression of PaAGL15-1 was mainly expressed in cotyledon and PaAGL15-2 was mainly expressed in hypocotyl of the 2 seedlings at the true leaf stage,and the expression of PaAGL15-2 was highest in the stems of the 6seedlings at the true leaf stage.The main expression sites of PaAGL15-1 in mature Platanus × acerifolia were root and cone.However,PaAGL15-2 was highly expressed in cones.The analysis of circadian rhythm showed that PaAGL15-1 and PaAGL15-2 showed obvious rhythmicity under both long and short day treatments.Subcellular localization of PaAGL15-1 and PaAGL15-2 protein was observed in the nucleus.3.Overexpression vectors of PaAGL15-1 and PaAGL15-2 were constructed for allogenic transformation of Arabidopsis thaliana and Nicotiana benthamiana.The transgenic plants of Arabidopsis thaliana and Nicotiana benthamiana showed significant late flowering,plant dwarfization and smaller leaf size compared with the control.Among them,the leaf shape of PaAGL15-1 Nicotiana benthamiana was slender,and all tissues and organs showed a tendency to become smaller,especially in the leaves,calyx and fruit pod.The leaves of strong phenotype plants also showed chlorophyll deposition and serious curls,and the corolla could not open and then withered and faded.However,the leaf shape of the overexpressed PaAGL15-2 strains was rounded,the leaf edges of the strong phenotypic strains were smooth and uprolled,the calyx was significantly enlarged,and the abnormal flower development led to abnormal flowering and setting.The interaction between PaAGL15-1 and PaAGL15-2 and At FT promoter of Arabidopsis thaliana was demonstrated by yeast single hybridization.The results indicated that PaAGL15-1 and PaAGL15-2 may regulate vegetative and reproductive growth of plants,and may regulate flowering in transgenic Arabidopsis thaliana through direct interaction with At FT.