Research On The Morphological Changes And Its Mechanism Of Pyrus Betulaefolia Bunge Tissue Culture Seedlings During Subculture

Pyrus betulaefolia Bunge has strong adaptability and high grafting affinity with pear trees.It is the main rootstock in pear producing areas in north China.At present,the main way of Pyrus betulaefolia propagation is seed propagation.Because of the variation of progeny,it is easy to cause the uneven seedlings.The weak rooting ability of the adult trees vegetative propagation has become an important factor restricting the promotion of superior rootstock plants.It is of great significance to establish a rapid and efficient propagation system for the improvement of rooting ability by means of regeneration.In this study,the multiple shoots from multiple rejuvenation generations of Pyrus betulaefolia were used as materials to select suitable subculture medium and rooting medium,and to studied the morphology,physiological indexes and related gene expression of adventitious root development during the rooting process of rejuvenation multiple shoots.On this basis,the reproductive efficiency,leaf morphology and endogenous hormone content of multiple shoots were compared among different subculture passages.Furthermore,the transcriptome data were used to compare the gene expression before and after rejuvenescence of Pyrus betulifolia.,and to focuse on the molecular basis of Pyrus betulifolia rejuvenescence.The main research results are as follows:1.By comparing and analyzing the growth indexes of Pyrus betulifolia smultiple shoots,medium with1.0 mg·L^-1 6-BA was more suitable as a subculture medium for Pyrus betulifolia.Exogenous auxins can promote the occurrence of adventitious roots of smultiple shoots,and the concentrations of 0.5 and 1.0mg· L^-1 are more suitable for the induction of adventitious roots under the treatment of same kind of auxins.The callus induced by NAA was large,soft and easy to fall off,which was not conducive to the survival of later transplanting.The rooting rate of IBA was significantly higher than that of NAA and IAA,and the rooting index was the best when the concentration of IBA was 0.5 mg· L^-1.2.The results of the observation on the tissue morphology during the rooting process showed that the root primordium occurred at the junction between phloem and vascular cambium after 4 days of treatment with IBA,callus was induced 8 days later,and adventitious roots broke through the epidermis and grew outward after 12 days.In the process of adventitious bud rooting,the activity of SOD and PPO increased rapidly in 0 4 d,decreased in 4 12 d and increased slowly in 12 16 d.The activity of IAAO decreased first and then increased in general and the activity of each period was significantly lower than that of CK.The activity of POD showed a trend of "increasing→decreasing→increasing→decreasing",and the two peaks appeared at 4 d and 12 d,respectively.The relative expression of ARRO-1 was significantly higher than that of control group at 4 d and 16 d after rooting induction.The relative expression levels of auxin early response genes ARF9,ARF17 and ARF19 were significantly higher than those of the control group at8 16 d after rooting induction.3.Through continuous subculture,the rooting ability of Pyrus betulifolia smultiple shoots can be significantly improved.The rooting rate of Pyrus betulifolia smultiple shoots increased from 0 to 66.70 %,and the reproduction coefficient increased from 2.13 in the first subculture to 4.20 in the 10 th subculture.The leaves showed a lobes at 3rd subculture and then the degree of lobes gradually deepened;During the continuous subculture,leaf area and vein number of multiple shoots were decreased significantly,and leaf perimeter and leaf index showed a trend of decreasing firstly and then increasing.The endogenous IAA content of multiple shoots reached 46.39 ng·g^-1 at 6th subculture,which was significantly higher than that of 0th subculture;With the increase of subculture passages,the endogenous ZR showed a trend of increasing firstly and then decreasing,and the endogenous GA₃ content did not change significantly,while the endogenous ABA content gradually decreased in the leaves;The values of IAA/ABA and IAA/ZR in the leaves increased with the number of subculture.The ABA content and IAA/ZR of multiple shoots leaves showed significant negative correlation and significant positive correlation with rooting rate.The number of leaf lobes and IAA/ABA showed a significant positive correlation with rooting rate.4.The leaves of different subculture passages multiple shoots of Pyrus betulifolia and the leaves of seedling were used as the materials,and the transcriptome sequencing data was used to analyze the expression of related genes before and after the Pyrus betulifolia rejuvenation.The number of up-regulated genes and down-regulated genes in the seedling leaves were higher than those in the leaves of the rejuvenative multiple shoots,compared to the leaves of the 0th-subculture multiple shoots.Further analysis was performed on 1865 differential expression genes.The GO functional classification showed that these differential genes were mainly focused on metabolic processes and catalytic activities.KEGG enrichment analysis shows that these common differential genes are mainly involved in biological pathways such as phenylpropionic acid biological metabolites,plant hormone signaling,endoplasmic reticulum protein processing,and plant pathogen interaction.Among them included IAA synthesis genes ALDH2B7;IAA signaling genes AUX / IAA,SAUR,GH3;ABA metabolism related genes,PYL and PP2 C.Real-time fluorescence quantitative PCR（qRT-PCR）was used to analyze the expression of the 6 differentially expressed genes in the transcriptome throughout the subculture process,which proved the accuracy of the RNA-Seq results.Combined with the difference in endogenous hormone content before and after rejuvenation,it is speculated that rejuvenation can regulate the expression of hormone-related genes to change the content and distribution of endogenous hormones in plants,and further enable plants to acquire juvenile characters such as good rooting ability.