Regulation Of Endogenous Phytohormone To Improve Regeneration And Transformation Efficiency Of Citrus

Citrus is commercially grown over world,where crops are exposed to a variety of abiotic and biotic stresses that often negatively affect fruit quality and productivity.Conventional breeding methods have demonstrated limitations with respect to citrus improvement due to some of the biological characteristics of woody plants such as nucellar polyembryony,high heterozygosity,long juvenile period,and autoincompatibility.The development of transgenic plant technologies tools has made it possible to overcome of these problems,and have been recognized as a powerful tool to do molecular breeding.In this study,the epicotyl internode stem of ‘Carrizo' citrange and ‘Eureka' lemon were used as explants to study the relationship between shoots forming and auxin polar transport,and the key role of auxin in citrus regeneration and genetic transformation,meanwhile,improve the regeneration efficiency and transformation efficiency of the explants by regulating the concentration of endogenous and exogenous auxin;I n order to increase genetic transformation efficiency of juvenile citrus by KN1 and IPT gene,and the effect of KN1 and IPT overexpression on the growth and development of citrus plants,the epicotyl internode stem of ‘Pineapple' orange,‘Hamlin' orange,‘Sucarri' orange,‘Valencia' orange,‘Carrizo' citrange and ‘Eureka' lemon were used as explants to do transformation with KN1 and IPT gene.In addition,internodal stem of mature ‘Pineapple' orange,‘Valencia' orange and ‘Washington' navel orange were used as explants to study that use KN1 and IPT gene to increase transformation efficiency,and use auxin polar transport inhibitor NPA and methylation inhibitor to enhance regeneration efficiency.The main results were as follows: 1.The function of auxin in citrus Regeneration and Transformation Efficiency and Increase Citrus Regeneration and Transformation Efficiency via Regulating Auxin DistributionThe results showed that the polar transport of endogenous auxin resulted in the higher concentration of auxin in the basal end of the explant,which inhibited the regeneration of adventitious buds.While NPA enhance the growth of adventitious buds by r educing the inhibition of adventitious shoots regeneration by a uxin,and NPA itself enhanced the function of cytokinin to improve the regeneration efficiency of adventitious buds.The results showed that NPA can improve the regeneration rate of ‘Carrizo' citrange and ‘Eurake' lemon 2 fold respectively,the highest average number of shoots per explant is 6.6,and each lemon explant can regenerate up to 1.2 a dventitious shoot.In addition,auxin could significantly increase the number of transformed cells on the explants,add 1 mg/L IAA in co-culture medium,and add 20 mg/L NPA in the selection medium,the conversion efficiency was 3 fold higher than control,transformation efficiency up to 82.2%.Our results provide some theoretical basis for future plant regeneration and transgenic research.2.KN1 gene overexpression drastically improves genetic transformation efficiencies of citrus cultivarsThe effects of a maize knotted1(KN1)gene under the control of the cauliflower mosaic virus 35 S promoter on genetic transformation efficiencies of six citrus genotypes were tested.Our results demonstrate that expression of the KN1 gene enhances transformation efficiencies from 3 to 15 f old compared to a control vector,3 t o 11 fold relative to the highest transformation efficiencies reported for these citrus genotypes.Stable incorporations of T-DNA into the citrus genome have been confirmed with both histochemical staining of GUS activity and molecular analyses.The majority of KN1 over-expressing transgenic plants grow and develop normally.With all six genotypes of citrus tested including ‘Eureka' lemon,a cultivar difficult to be transformed,our results demonstrate that the KN1 gene provide an effective molecular tool to enhance genetic transformation efficiencies of various citrus varieties.High transformation efficiency of citrus is of great importance for cultivar development via transgenic and genome editing technologies.3.The study of enhancing regeneration and genetic transformation efficiency of mature citrusThe effect of KN1 and IPT gene on t he transformation efficiency of adult citrus material was studied,and the application of auxin polar transport inhibitor NPA and methylation inhibitor 5-Aza(5-Azacytidine)were studied to improve regeneration efficiency.The results showed that KN1 and IPT could significantly increase the transformation efficiency of mature citrus by 1.6 to 3.4 fold,which indicated that regulation of endogenous cytokinin was an effective way to improve the transformation efficiency of adult material.The regeneration efficiency increased by 2 t o 5 fold after explants treated by methylation inhibitor 5-Aza.In addition,NPA can significantly improve the regeneration efficiency of juvenile citrus,but there is no increasing regeneration efficiency significantly when the adult citrus explants treated by NPA.