Induction Of Hexaploid And Molecular Mechnism Of Its Trait Variation In White Poplarinduction Of Hexaploid And Molecular Mechnism Of Its Trait Variation In White Poplar

Two types of polyploid plant are found.One is higher in vegetative growth than that of its diploids and another is lower in vegetative than that of its diploids.Therefore,studies on molecular basis of growth traits formation in polyploids play an important role in promoting the progress of theories and techniques of plant polyploid breeding.In this study,based on the establishment of a rapid regeneration system,in vitro hexaploid induction of Populus‘Beilin5'was conducted by chromosome doubling via oryzalin using stem as explants.Differential gene expression and the differential mi RNA expression derived from different positions leaves of hexaploids and its counterpart triploids were,then,carried out by transcriptome sequencing?RNA-seq?and mi RNA sequencing.The main results are as follows:?1?Efficient regeneration systems of leaf,petiole and root of Populus‘Beilin5'were established.The results showed that leaf and petiole had better regeneration capacity than that of root.The optimum medium for the regeneration of adventious buds from leaves was MS+0.1mg/L 6-BA+0.15mg/L NAA+0.02mg/L TDZ.The induction rate of shoot was 95%and the mean number of shoot per explant was 7.77.The optimum medium for the regeneration of adventious buds from petioles was MS+0.2mg/L 6-BA+0.05mg/L NAA+0.01mg/L TDZ.The induction rate of shoot was 95%and the mean number of shoot per explant was 8.15.The optimum medium for the regeneration of adventious buds from roots was MS+0.3mg/L 6-BA+0.05mg/L NAA+0.02mg/L TDZ.The induction rate of shoot was 60%and the mean number of shoot per explant was 2.82.?2?Hexaploid plants were successfully obtained by inducing in vitro chromosom doubling of node explants with oryzalin treatment in the‘Beilin5'.The optimum medium,MS+0.1mg/L6-BA+0.11mg/L NAA,for the regeneration of adventious buds from stems was screened.The induction rate of shoot was 93.33%and the mean number of shoot per explant was 7.55.We detected 6 hexaploids from 31 surviving plants arising from oryzalin-treated nodal segments and the hexaploid percentage was19.35%.The optimum treatment of the induction of hexaploidy was stem segments characterized with the length of 5 mm were treated by 5 mg/L oryzalin for 72 h or with the length of 10 mm were treated by 10 mg/L oryzalin for 24 h.?3?Our studies showed that the hexaploid Populus was characterized with slow growing and dwarf.Compared with triploid plants,the leaf cells of hexaploids were larger in size.However,the number of leaf cells,leaf area and height growth were significantly lower than triploids.From the 3^rdto13th leaf,the photosynthetic rate of hexaploid were 78.83%?2.78%higher than corresponding leaf of triploid.But with the increase of leaf age,the photosynthesis of hexaploid was lower 3.33?53.55%than that of triploids.In addition,as leaf age increased,the chlorophyll content and carotenoid content of hexaploid decreased rapidly.They were significantly lower than that of triploids.Scanning electron micrographs revealed that the structure of chloroplasts for the 17th leaf in the triploids organized well.However,the structure of chloroplasts for the 17th leaf in the hexaploids was severely degraded.?4?Differential expression genes?DEGs?involved in plant hormone signal transduction showed regular changes with the development of leaves.The number of DEGs encoding GH3 negatively correlated with auxin signal transduction increased with an increase of leaf age.The number of down-regulated DEGs encoding B-ARR positively correlated with cytomin signal transduction also increased.The number of down-regualted DEGs encoding CTR1 negatively correlated with ethylene signal transduction increased and the number of up-regualted DEGs encoding ERF positively correlated with ethylene signal transduction increased.And the total number of up-regualted DEGs encoding JAR1and MYC2 positively correlated with JA signal transduction increased.Decreased trends in IAA and CTK signal transduction were observed with the increase of leaf age.However,increased trends in ET and JA signal transduction were found.This suggested that changes of plant hormone signal transduction may be one of the important reasons resulting in accelerating leaf senescence and an decrease of growth rate in hexaploids.?5?The results showed that the photosynthesis of hexaploids was significantly less than that of triploids as leaf age increased.Most of DEGs involved in chlorophyll synthesis,carotenoid synthesis,thylakoid reaction and Calvin cycle were up-regulated within the 3^rdand 7^thleaf,and down-regulated within the 17^thleaf.For example,DEGs,such as HEME,CHLH,CHLD and CHLG involved in chlorophyll synthesis.Gene PSY,ZISO,ZDS,LYCB and VDE involved in carotenoids.Gene Psb B,Psa K,Psa F and delta involved thalakoid reaction and FBP,SEBP and PRK involved in the Calvin cycle.?6?Molecular mechanism of slow vegetative growth in hexaploid white poplar was elucidated by the dosage effects of differential expression mi RNA within different aged leaves.With the development of leaves,mi RNA related to vegetative growth displayed dosage effects.The expression level of mi R156a?mi R156g?mi R169aa and mi R530b increaed gradually in hexaploids,which was significant higher than that of triploids.These up-regulated mi RNA caused a relatively stronger of target genes positively correlated to vegetative growth,such as AFH14?AN3?UBP16 and B-ARR.However,the expression of novel135 decreased gradually with leaf aging,which was significant lower than that of triploids.These down-regulated mi RNA caused a slightly weaker negative regulation of target genes negatively correlated to vegetative growth,such as JAR1.As a result,the number of leaf cells and leaf area decreased,and the chloroplast degraded and leaf senescence were accelerated in hexaploid.Thus,the hexaploid white poplar had lower vegetative growth than triploids.