Evaluation On Rooting Ability Of Cuttings In Different Blueberry Varieties And Research On The Role Of VcWOX Gene In Root Development

The breeding of blueberry seedlings is mainly through tissue culture and cutting propagation.The tissue culture technology has a high propagation coefficient but requires a strict aseptic environment and high cost.Cutting propagation technology is relatively simple,but for blueberries,cutting rooting is more complicated.It has become the“bottleneck”of blueberry cutting propagation.Internal and external factors affect cutting reproduction.Among the internal factors,the differentiation ability of meristems plays a decisive role in the formation of adventitious roots（ARs）.The undifferentiated cells in the meristem are called stem cells,which provide precursor cells for the differentiation of plant tissues and organs.Stem cells can undergo fate transitions in response to auxin and differentiate to form ARs.Many factors are known to participate in the regulation of stem cell differentiation.The WUSCHEL-related homeobox（WOX）transcription factor is unique to plants and participates in the regulation of stem cell differentiation such as apical,root,and stem cells.In this study,we used auxin to induce blueberry branches with different levels of lignification and analyzed the rooting ability to vary blueberry varieties;based on the blueberry genome,we identified WOX transcription factor family members and analyzed their bioinformatics characteristics;using different transcriptome data,and q RT-PCR technology explored the functions of blueberry WOX gene in ARs growth and development,flower bud dormancy release,fruit development,and root acid adaptability,and screened essential genes that regulated root system development;cloned and constructed blueberry WOX gene overexpression vector,genetically transformed to Tobacco（Nicotiana tabacum）,by analyzing the root phenotype of transgenic plants,explored the role of blueberry WOX gene in root development;using paraffin section technology to dissect the root tissue structure of transgenic tobacco,and determined the relative content of various hormones in the root system.Combining the transcriptome data of this plant,preliminary exploration of the mechanism of Vc WOX4 gene suppression on ARs formation.The main results obtained are as follows:1.Through the experiment about the cuttings of blueberry branches with different lignification levels induced by IBA,we found that semi-lignified cuttings were more conducive to the occurrence of blueberry ARs than non-lignified cuttings,and 2.0 mg·L^-1IBA can significantly increase the rooting rate.Thus,we used semi-lignified cuttings of different varieties in blueberries to evaluate the rooting ability,and the result showed that the rooting ability of varying blueberry varieties was different,from largest to smallest:‘Emerald’‘Brightwell’,‘Climax’,‘Star’,‘O’neal’,‘Saphire’,‘Tiffblue’,‘Premier’,‘Woodard’,‘Gardenblue’,‘Powderblue’.Further use of IBA to induce blueberry tissue-cultured cuttings to form ARs,the result showed that the callus increased continuously during the process.Then the rooting ability index gradually increased,indicating that IBA induced blueberry to form ARs mainly through the callus pathway.Further use of paraffin section technology to cross-cut blueberry tissue-cultured seedlings,unlignified and semi-lignified branches,and found that the semi-lignified branch cuttings with the ratio of the number of xylem cells in a single row to the number of wood ray cells on one side of the xylem cells less than 1.5 are better for rooting.2.Through the genome-wide analysis of the blueberry,it was found that 29 WOX genes were obtained by blueberry,which was divided into three clades:WUS branch,middle branch,and ancient branch.Furthermore,bioinformatics analysis of the family revealed that the family has 17 conserved sites and 15 conserved motifs,among which motif 1,motif 2were present in 29 WOX proteins,and motif 3 was present in the WUS branch,indicating that this family was highly conservative.Analysis of the expression patterns of different transcriptome data found that Vc WOX4,Vc WOX5,Vc WOX11,Vc WOX9,Vc WOX13 genes were involved the process of root growth and development.Using q RT-PCR technology,we discovered that Vc WOX4,Vc WOX5,Vc WOX9,Vc WOX11,and Vc WOX13 genes responded auxin and regulated the development of blueberry ARs.The promoter sequences of the Vc WOX4 and Vc WOX13 genes were further cloned.The luciferase activity detection experiment and GUS histochemical analysis showed that the promoters of the Vc WOX4b and Vc WOX13b genes had promoter activity,and the Vc WOX4b gene promoters were constitutively expressed.3.Constructing blueberry Vc WOX4b,Vc WOX5c,Vc WOX9a,Vc WOX11c,and Vc WOX13b gene over-expression vectors,genetically transforming them into tobacco,and obtaining transgenic plants,the root system analysis of transgenic tobacco plants showed that Vc WOX4b,Vc WOX9a,Vc WOX11c,and Vc WOX13b in blueberry promoted the growth of taproots and lateral roots,Vc WOX5c inhibits the growth of taproots and lateral roots;Vc WOX4b inhibits the growth of ARs,and Vc WOX5c,Vc WOX9a,Vc WOX11c,and Vc WOX13b promote the development of ARs.4.Tobacco plants overexpressing the Vc WOX4b gene have malformed roots,with nodules and curled leaves.Analysis of root tissue structure in transgenic tobacco using paraffin section technology found that the xylem area of the transgenic tobacco root system became larger,and the number of cambium layers increased,causing root deformities.Liquid chromatography-mass spectrometry（LC-MS）was used to determine the phytohormone content in tobacco ARs and stem bases with ARs.We found that the ratio of IAA/CKs of the ARs of transgenic plants was significantly lower than that of wild tobacco during the growth of ARs,the IAA/ABA value of the transgenic tobacco gradually decreased in the early cutting period,but the IAA/ABA value of the wild-type tobacco gradually increased,indicating that Vc WOX4b inhibited ARs formation by reducing the IAA/CKs and IAA/ABA values.