Physiological Molecular Mechanism Of Exogenous Spermidine Regulation On Adventitious Roots Formation In Apple Rootstock

Adventitious root induction is a key link in dwarfing apple rootstock breeding.In terms of regulating the occurrence of adventitious roots,relevant studies in apples focus on the five traditional hormones such as auxin,and the research on the induction of adventitious roots by new hormones such as polyamines is relatively weak.In view of this,this study used apple rootstock Malus prunifolia var.ringo tissue cultured seedlings as the test material,treated with exogenous spermidine,studied the regulatory effects of spermidine on the adventitious roots of apple rootstock,analyzed the changes of endogenous hormones,detected changes in the transcriptional expression levels of related genes,screened key genes,and the heterologous expression system of tobacco was used to analyze its function of regulating adventitious roots,which provided theoretical guidance for the efficient breeding of apple rootstock in the apple industry.Below are key research findings:1. After exogenous spermidine treatment,according to the anatomical observations during adventitious root formation,it was found that spermidine treatment promoted the adventitious root formation of apple rootstock'Begonia crassipes'and induced endogenous hormone auxin?IAA?and zeatinriboside?ZR?increased at 1d,3d and 8d,and endogenous hormones abscisic acid?ABA?and gibberellin 3?GA₃?decreased significantly at 3d.Spermidine treatment promoted the relative expression of spermidine?Spd?signaling pathway genes Md SAMDC1,Md PAO and Md SPDS6,increased the expression of auxin pathway genes Md ARF7,Md ARF19,Md IAA3,Md IAA14 and Md IAA23,and promoted the expression of cell cycle gene Md CYCD1;1,Md CYCD3;1 and Md CYCD4;1.2. Clone the ADC genes in different varieties of'Malus prunifolia var.ringo','T337' and'SH6',and find that the coding region length,start codon and stop codon of ADC gene sequence in different varieties are quite different.The results show that the CDS of Mp ADC gene in'Malus prunifolia var.ringo'is 2187 bp in total length,encodes 728 amino acids,has a relative molecular mass of 77.99433 k D,and the theoretical PI value is 5.26.Tobacco lines transformed with Mp ADC gene were treated with auxin,and after 6 days of cultivation,the adventitious roots of tobacco lines overexpressing rooted while the wild type had not been observed.After 11 days of cultivation,the adventitious roots of overexpressed tobacco lines were more than those of wild-type plants.The results showed that overexpression of ADC gene can promote the adventitious roots of plants.3. Select auxin-related auxin response factor ARF22 to clone and construct an overexpression vector to study the effect of Mp ARF22 on spermidine-mediated occurrence of apple adventitious roots.First,the Mp ARF22 gene was cloned and the Mp ARF22 gene overexpression vector was constructed.The results showed that the Mp ARF22 gene CDS was 2682 bp in total length,encoding 893 amino acids,the relative molecular mass was99.09052k D,and the theoretical PI value was 6.25.Treatment with spermidine to transform Mp ARF22 gene into tobacco lines showed that overexpression of ARF22 gene affected spermidine-mediated occurrence of adventitious roots in applesomics area.