Study The Physiological And Molecular Mechanisms Of IBA And GO Regulating Adventitious Root Formation In Apple Rootstocks

Apple(Malus×domestica Borkh)is one of the most cultivated fruit crops in China.Its area and yield rank first in the world.The development of apple is of great significance to increase farmers’income and adjust industrial structure.M9-T337,an excellent rootstock widely used in commercial orchards,could lead to dwarf tree architecture,early fruiting,and high fruit yield and quality.However,M9-T337 is difficult to root.In this study,the effects of indole-3-butyric acid(IBA)and graphene oxide(GO)on the formation and growth of adventitious roots of M9-T337 in vitro were studied,the effects of different treatment time of IBA,interaction of IBA and ethylene,IBA and GO on rooting rate,number of adventitious roots,length of adventitious roots and expression of related genes of M9-T337 were also studied.The main results were as follows:1.Early signs of callus formation were observed at day 5 in IBA-treated cuttings.However,there were no signs of AR formation in control cuttings before day 7.Anatomical observations showed that IBA induced AR primordium formation at day 3;the AR primordium continued to divide and elongate,and then underwent tissue differentiation,and protruded outward through the cortex and epidermis to form AR at day 7.With the increase of IBA treatment time,the rooting rate and the number of adventitious roots increased.When IBA treatment time was longer than 3 days,the rooting rate could reach 100%,however,long-term IBA treatment significantly inhibited the elongation of adventitious roots.IBA upregulated expression of genes(MdWOX11,MdLBD16,and MdLBD29)involved in AR formation,and induced the formation of root primordia,resulting in a large number of adventitious roots.2.Transcriptome analysis showed that there were 2282 DEGs between IBA group and control group,of which 966 were down-regulated and 1316 were up-regulated at the 12th day,and there were 2592 DEGs between IBA group and control group at the 21st day,of which 1116 were down-regulated,1,476 genes up-regulated.Among the DEGs,plant hormone metabolism and signal transduction genes accounted for a large portion.There were 47 DEGs in the auxin metabolism,transport,and signal transduction pathway.The expression of auxin pathway-related genes(MdYUCCA10B,MdIAA29,MdABCB19)and ethylene synthesis pathway-related genes(MdACO1,MdACS1,MdACS2)in IBA group was higher than that in control group in several periods.For ethylene pathway,there were 13 DEGs in the ethylene synthesis and signal transduction pathways,and 51 DEGs in other hormone synthesis and signal transduction pathway.3.The DEGs related to auxin and ethylene were further verified by RT-qPCR.The relative expression levels of auxin synthesis gene MdYUCCA10 were significantly higher in the IBA treated plants than the control plants at days 8,17,and 21,but significantly lower than control at the day 12.The expression of auxin signal transduction gene MdIAA29 was significantly higher in the IB A treated plants than the control plants at days 17,21,and 27,but significantly lower than control at days 8 and 12.The expression of auxin transport gene MdABCB19 was significantly higher expression in the IBA treated plants than the control plants.The relative expression levels of ethylene synthesis gene MdACO1 was significantly higher in the IB A treated plants than the control plants at days 8,17,21,and 27.The relative expression levels of ethylene synthesis gene MdACO2 was significantly higher in the IBA treated plants than the control plants at days 8,12,and 17,but significantly lower than control at the day 21.The expression of ethylene synthesis gene MdACS1 was significantly higher in the IBA treated plants than the control at days 17,21,and 27,but lower than control plants at days 8 and 12.After IBA 7 days’ treatment,ethylene synthesis inhibitor AgNO3,ethylene precursor ACC and IBA was used to studied adventitious roots.The results showed that AgNO3 could slow down the inhibition of IBA on adventitious roots,while ACC enhanced the inhibition of IBA on adventitious roots,indicating that AgNO3,inhibited ethylene release and ACC promoted ethylene release.The results showed that the inhibition of auxin on adventitious roots elongation was partly mediated by ethylene production.4.Go treatment promoted the rooting of apple rootstock M9-T337,reaching 100%at 20 days,but inhibited the growth of adventitious roots.Compared with control,root length and root fresh weight decreased,but there was no significant difference on the number of adventitious roots.During adventitious root formation period GO alone(GO+7d IBA group)did not inhibit adventitious root,the length of adventitious root was significantly larger than the control group and GO group,and there was no significant difference in rooting rate,indicating that the effect of GO on M9-T337 adventitious root depended on IBA.IBA and GO treatment significantly promoted ethylene production,while AgNO3,an inhibitor of ethylene synthesis,significantly inhibited ethylene release and reduced the inhibition of IBA and GO on adventitious roots to some extent Compared with control group,GO and IBA treatment induced up-regulated expression of rooting-related genes(MdVOX11,MdARRO1),auxin-related genes(MDLAX2,MdLAX3,MdARF19),and ethylene-related genes(MdACO1,MdACS2,MdEIN3,MdERF109).The results indicated that GO depended on IBA to regulate the formation and development of adventitious roots in apple rootstock M9-T337.