Evaluation Of Red-Leaf Chinese Tallow Trees,Establishing Its Propagation Technique And Uncovering Regulators Involved In The Formation Of Red-Leaf

Triadica sebifera is a deciduous tree of the Euphorbiaceae family,with a wide distribution,high adaptability,rapid growth,high seed oil content,and economic,medicinal and horticultural value.Because of its beautiful tree shape,colourful autumn foliage,white seed hanging from the branches in winter and significant seasonal variation,T.sebifera has good prospects for use in the garden.Based on the germplasm resources of T.sebifera of red-leaf,we established the asexual propagation system and the Agrobacterium rhizogenes-mediated genetic transformation system,and uncovered regulators involved in red-leaf formation in T.sebifera.The following results have been obtained:（1）After 3 years of phenological observation,the phenology of T.sebifera growing in the Wuhan area has been summarised.The germination begins in mid-March,with a leaf budding period of 7 days and a leaf growth period of 25-30 days.Its leaves start to discolour at the beginning of October,with a leaf colouring period of 12-25 days.In mid-November,it begins to defoliate,with a defoliation period of 6-15 days and complete defoliation in mid to late November.（2）By investigating Luotian County and Wuhan city in the Dabie Mountains,106varieties of T.sebifera were collected.By comparing the phenological period,leaf colour,leaf discoloration rate and health level of these varieties,two excellent varieties were selected:X3 and X8.The characteristics of X3 excellent variety:bright red autumn leaves,broad leaves,95%of leaf discoloration rate,stable leaf color and 20 days of the leaf colouring period.The characteristics of X8 excellent variety:dark red autumn leaves,small and flat leaf shape,95%of the leaf discoloration rate,stable leaf color,22 days of the leaf colouring period.（3）The tissue culture system of T.sebifera:（1）Explant disinfection method:Using the young stems of the chamber as explants,the survival rate after sterilisation with 0.2%Na Cl O for 5 minutes and 0.1%Hg Cl₂for 8 minutes was 81.25±1.33%.Using the seed embryos as explants,the survival rate after sterilisation with 0.1%Hg Cl₂for 10 minutes was 77.78±2.78%.（2）Induced culture of the leaf callus:For scratched leaves cultured on WPM medium supplemented with 1.0 mg/L 6-BA and 0.4 mg/L NAA,the average callus induction rate was 82.22±2.00%.（3）Induction culture of adventitious buds:The average callus differentiation rate obtained on WPM medium supplemented with 1.0mg/L 6-BA,0.2 mg/L IBA and 0.1 mg/L NAA was 93.75±7.98%,and the average number of differentiated buds was 3.38±0.45.（4）Induced culture of adventitious roots:Adventitious buds cultivated on 1/2 MS medium supplemented with 0.5 mg/L IBA had90.59±0.15%rooting,with a average number of roots of 6.12±0.58 and a average longest root of 4.13±0.25 cm.（4）Softwood cutting propagation system of T.sebifera:The effect of different IBA treatment time on cutting of T.sebifera showed that the IBA treatment time had no significant effect on rooting of cuttings,but it had a certain promoting effect.The effect of cuttings with different levels of lignification showed that the lower the lignification of cuttings of T.sebifera,the higher the rooting rate of cutting,the shorter the rooting time.In a word,it is a feasible softwood cutting method of T.sebifera that the softwoods of 1-5months were inserted in a river sand matrix containing 0.5 mg/L IBA and sprayed daily with 0.5 mg/L IBA solution in later days,with an average rooting rate of about 50%to70%.（5）Agrobacterium rhizogenes-mediated genetic transformation system of T.sebifera:（1）75 mg/L was chosen as the screening concentration for Kan resistance.（2）The OD₆₀₀values of the agrobacterium solution ranged from 0.8 to 1.2.（3）Agrobacterium was activated for 1 to 2 hours in WPM liquid medium containing 100μM AS.（4）The infection treatment method we adopted was to vacuum the leaves in the bacterial liquid for 15 minutes,and then shake them for 15 minutes.（5）The explants were grown in dark on WPM medium containing 100μM AS for 3 days.（6）The explants were cultured in hairy roots in WPM medium supplemented with 0.1 mg/L 6-BA,0.2 mg/L NAA,and75.0 mg/L Kan.（7）The petiole and leaves of 20 days are suitable for the genetic transformation plants of T.sebifera,and the hairy root positive rate can reach about 20%.（6）Uncovering regulators involved in the formation of red-leaf in T.sebifera:The anthocyanin species in T.sebifera leaf was Cyanidin-3-O-glucoside（Cy3G）,analysed by UHPLC-MS and HPLC.And the content of Cy3G in its red-leaf was 139.26±1.35ng/mg,1160 times higher than in green-leaf.This suggests that Cy3G is closely related to the red-leaf formation of T.sebifera.In addition,seven regulatory genes were selected by RNA-Seq sequencing of the red-leaf and green-leaf,which were six genes of the MYB family and one gene of the b HLH family and named RW1 to RW7.It was confirmed by q RT-PCR that the relative expression levels of the seven genes were significantly greater in the red-leaf compared to the green-leaf of T.sebifera.Therefore,it is speculated that these seven genes are involved in the regulation of red-leaf formation in T.sebifera.To uncovering these seven regulators,the full-length sequence of RW1,RW3 and RW5 genes was successfully cloned and the over expression vector was constructed.Using the Agrobacterium rhizogenes-mediated genetic transformation system,we obtained positive transgenic hairy roots of RW5.However,the positive hairy roots did not show red,so it is speculated that this gene is not a regulator of red-leaf formation in T.sebifera.In summary,by phenological observation and investigating and collecting germplasm resources of T.sebifera,this study selected two excellent varieties with stable characteristics and high ornamental value,and established the tissue culture system,softwood cutting propagation system and agrobacterium rhizogenes-mediated genetic transformation system of T.sebifera.At the same time,the major anthocyanidin species in the T.sebifera leaves was Cy3G,and seven regulators related to the red-leaf formation of T.sebifera were excavated,and the positive transgenic hairy roots of T.sebifera were obtained.We hope to provide a valuable reference basis for the advancement and application of red-leaf T.sebifera.