| The early abortion of jujube embryo is a serious problem which restricts the process of hybrid breeding of jujube.Although the embryonic development is closely related to the plant endogenous hormone levels,the mechanism of the embryonic development of jujube regulated by the endogenous hormone is not yet clear.Therefore,in order to better reveal the relationship between endogenous hormones and fruit development during the process of jujube embryo abortion,changes of endogenous hormone levels during the development of ‘Lengbaiyu' jujube fruit were studied based on the field investigation and microscopic observation.In addition,high-throughput transcriptome sequencing was also used to analyze the genes related to embryo abortion hormone and its expression characteristics during ‘Lengbaiyu' jujube embryo abortion and fruit development.‘Lengbaiyu' jujube immature embryo culture technology was also studied at the same time.The results of this study will be useful in germplasm innovation,especially crossbreeding in jujube.The main results obtained are as follows:1.The developmental dynamics and the abortion stage of embryos in ‘Lengbaiyu' jujube were clarified.The results showed that the original embryo,small globular embryo,globular embryo and torpedo embryo appeared at the 20 d,30d,40 d and 50 d after flowering,respectively.And the peak of embryos abortion appeared at 40 d after flowering(globular embryo stage).2.The relationship between embryo development and endogenous hormone content during jujube development showed that the GA content in the embryo was significantly lower than that in the pulp,but the IAA content was significantly higher than that in the pulp.In addition,the lower level of GA,higher level of IAA in the embryo,the lower levels of t ABA,c ABA and the high content of IAA/GA were beneficial to the normal development of immature embryos.The decrease of IAA in embryos,the continuous increase of t ABA,the ratio of(GA+IAA)/ABA in embryos and pulp were the important factorsthat leaded to embryo abortion.3.Based on RNA-Seq analysis,1219 differentially expressed genes were screened between normal and abortive embryos at the peak of abortive stage of ‘Lengbaiyu' jujube,among which 16 differential genes were related to the endogenous hormone metabolism pathway.Among the 16 genes,14 genes were down-regulated and 2 genes were up-regulated.The expression of AUX/LAA(auxin-inducible protein gene),SAUR1(auxin early response gene),PYR/PYL(abscisic acid receptor protein gene),ERF1/2(ethylene-responsive transcription factor),GH3(indoleacetamide synthesis))and PR-1(pathogenesis-related protein gene)were down-regulated,and the expression of AHP(histidine phosphotransferase gene)were up-regulated.4.The RT-q PCR results showed that the expression levels of GH3.17(indole acetate synthase gene3.17),AUX22D(Auxin-induced protein)in normal embryos were significantly higher than those in abortive embryos.The expression levels of PLY(abscisic acid receptor),ERF1B(ethylene response transcription factor),EBF1,EBF2(ethylene signaling regulators 1,2)in abortive embryos were significantly higher than those in normal embryos.Therefore,we believed that these up-regulated genes could effectively activate the physiological activities of ABA and ethylene,which in turn promotes the abortion of jujube embryos.In addition,the gene expression level of Aux/IAA18(auxin/indole acetate protein gene),SAUR1(auxin early response gene),GH3.1,GH3.6(indole acetate synthase gene 3.1,3.6)in abortive embryos were also significantly higher than normal embryos.This is the stress response of the gene family in the early response of auxin,which is the feedback of the relative excess auxin in the abortive embryo.It shows that hormone regulatory genes resulting abortion,and embryo abortion causes changes of endogenous hormones in young embryos.5.Hormones are closely related to the development of jujube embryos,and the balance between hormones affects the development of immature embryos.By screening and optimizing the hormone ratio during the jujube embryo culture,the culture of ‘Lengbaiyu' 30 d embryos was finally succeeded.The youngest embryo successfully-rescued was 30 days old.The percentage of immature embryos in20-day-old,30-day-old and 40-day-old could reach to 4.98%,56.82% and 62.31%.The plantlet formed rate of younger embryo in 30-day-old directly was 0.41%.The younger embryos induction ratio was 68.70%.With the culture temperature maintained 24?,the differentiation rate with this formula was the highest,reached to 81.12% at 24 ?.On the basis of endosperm care culture,the immature embryos could bepromoted to grow into embryos along the embryonic growth,and the immature embryos that fail to grow along the embryogenicity were selected from the organogenesis-type callus regeneration pathway to obtain intact plants.The combination of both methods of plant regeneration directly from immature embryos or from callus induction greatly improves the utilization of immature embryos for obtaining sufficient hybrid seedlings for jujube breeding. |
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