Studies On Histological Localization And Expression Of Genes Related To Hormones In Vivo During Dedifferentiation Of Mature Wheat Embryos

Wheat is one of the most important crops in the world.The utilization of biotechnology in its cultivar improvements,however,is rather retarded comparison with that in other crops.The bottleneck of the problem is the immaturation of wheat tissue culture technique.The immature embryos are current targets in wheat transformation.But acquirement of immature embryos is strictly limited by seasons and consistency of development stages is different to judge which not only increases the labour constraints but also hampers experimental flexibility of wheat genetic transformation heavily.Mature wheat embryos have a lot of advantages in their availability throughout the year and consistency of development stages.Using mature wheat embryos as explants is also not practice at present due to lower regeneration rate in vitro culture.Therefore it is of significance to improve ability of dedifferentiation and regeneration in mature embryo culture and to reveal molecular mechanism at the level of gene expression.The common wheat cultivar Yumai 18(Triticum aestivum L.)in Henan Province were used as experiment material to study the process of mature wheat embryos dedifferentiation from morphology,cell structure to gene expression level.In this study,we confirmed where and when about dedifferentiation of mature embryos using microscopical technology.Dedifferentiation originally arose from the sheath inner cells of the primary radicel.The process of dedifferentiation of this part of cells was formation at 24 h after induction.The four stages were divided apparently according to changes of histological structure in dedifferentiation.The first(0～2 h) was the stage of signal transduction(SST)in which the perception and transduction the stimulation of auxin signal were occurred in cells.The second(2～12 h)was stage of signal response(SSR)in which the cells quickly responded the stimulation and a series of biological changes occurred in the cell wall,cell membrane and cytoplasm, so that the cell changed in morphology.The third(12～24 h)was stage of cell remodification(SCR)in which the ceil structures were remodeled based on the changes occurred in SSR and prepared for cell division and formation of new cell. The fourth(about 24h)was stage of calli formation(SCF).The main missions of this stage were formation of calli.In order to study the molecular mechanism of mature wheat embryos dedifferentiation, we analysised the expression of genes related to auxin,cytokinin,gibberellin,abscisic acid and ethylene in vivo during dedifferentiation of mature wheat embryos at transcriptional level,using a Affymetrix wheat GeneChip.The controlled calli were 0 hours' samples.Those genes whose ratios of 2 h vs 0 h,6 h vs 0 h,12 h vs 0 h,24 h vs 0 h,72 h vs 0 h,were greater than 2 or shorter than 0.5,namely which exhibited a greater than two folds change in gene expression level during dedifferentiation of mature wheat embryos,were meaningful genes.We analysised the expression profiles of these meaningful genes in dedifferentiation of mature wheat embryos.At the results,using the Affymetrix wheat GeneChip including 61127 probe sets, representing 55085 genes,15000 genes were meaningful approximately.In mature embryos,the total number of genes related to those five hormones was 263.The number of genes related to auxin and cytokinin were 114 and 39,respectively,and most of them were up-regulated,so they might play an important role in this dedifferentiation.The mumber of genes related to gibberellin was 31,and the number of up- and down-regulated genes almost was even.The number of genes related to abscisic acid and ethylene were 64 and 15,respectively,and most of them were down, so they could nearly be important.Because PM H~+-ATPase was one of important enzymes in dedifferentiation,the primers were designed by the cDNA sequence of PM H~+-ATPase gene according to Accession Number AY543630 registered in GenBank.So,we assayed the expression of plasma membrane H~+-ATPase gene in dedifferentiation of mature wheat embryos, using the wheat gene chip and Real Time PCR.The result from wheat gene chip was that cross-signaling of 2 and 6 h were stronger;and that from Real Time PCR was that gene copies of 2 and 6 h were more.In conclusion,both the results of plasma membrane H~+-ATPase from the wheat gene chip and Real Time PCR were more consistent,and the plasma membrane H~+-ATPase gene was always up-regulated, which suggest that plasma membrane H~+-ATPase was one of important emzymes and its role was the strangest at 6h in dedifferentiation of mature wheat embryos.