Effect Of Red Light On Somatic Embryogenesis And Its Application In Cotton Tissue Culture

In China,cotton is not only one of the important cash crops,but also the world?s second largest raw material of textile fiber and edible oil.With the continuous development of cotton genetic engineering and transgenic breeding,transgenic breeding has become the most efficient way of cotton breeding.Somatic embryogenesis,as the main way of cotton regeneration system,has the advantages of good genetic stability and few chimeras.The establishment of somatic embryogenesis(SE)system is the basis of efficient cotton genetic transformation technology.In recent years,cotton regeneration systems of different genotypes have been established successively,and the molecular mechanism of cotton somatic embryogenesis system has been studied more and more.Among them,auxin and light play an important role in the regulation of cell fate in somatic embryogenesis,but the crosstalk network of auxin and light is not well understood in SE.In this study,the embryonic callus of Arabidopsis thaliana was positively regulated by the low flux of red light by using the somatic embryogenesis system of Arabidopsis thaliana.The optimal light combination was identified through observation of different light treatments,and the relationship between red light and auxin was analyzed by DR5:GUS:GFP and pPIN1:PIN1:GFP,and then applied to cotton tissue culture.The following experimental results were obtained:1.Low flux red light affects the formation of embryogenic callus in Arabidopsis thaliana embryosThe Arabidopsis SE system was used to evaluate the effect of red light at different stages of somatic embryogenesis,and different combinations of lights were used during the PSE induction and EC induction stages.The EC differentiation rate at 5 d on ECIM was calculated following the different treatments.The PSE was firstly induced under white light and then cultured in ECIM under dark light and red light conditions.Compared with the dark light,the EC induction efficiency of the red light was significantly increased.2.Red light induces embryogenic callus formation by promoting auxin accumulation and distribution during embryogenic callus inductionThe system that primary somatic embryo induction was induced under white light,embryogenic callus induction under dark and red light respectively,which was used for further study.To investigate the function of red light in auxin accumulation and distribution,the A.thaliana line containing DR5:GUS:GFP was used to monitor auxin distribution.The results indicated that GFP and GUS expression were more obvious and more widely distributed in the embryonic callus induction stage 2-5 days after treatment with red light than in the dark.This indicates that red light promotes the formation of embryonic callus by promoting the synthesis and distribution of auxin.3.Application of low-flux red light in somatic embryogenesis of cottonIn order to define the low flux red light on the influence of cotton somatic embryo differentiation,will the above methods in translation to cotton somatic embryogenesis system research,we found that,compared with controls(16 h light / 8 h dark),red(16 h dark red / 8 h)on the cotton callus induction of somatic embryogenesis phase will greatly enhance the efficiency of the differentiation of embryonic callus.This indicated that red light could be applied in cotton tissue culture to optimize somatic embryogenesis system and assist cotton genetic transformation system.The use of DR5:GFP transgenic cotton material demonstrated that red light accelerates the establishment of embryogenic callus by promoting the accumulation of auxin.And red light has a significant improvement in the callus differentiation of CCIR 12.This indicates that red light can be applied to cotton tissue culture to optimize somatic embryogenesis system to assist cotton genetic transformation.In summary,low flux red light can significantly increase the differentiation of cotton embryogenic callus and improve the induction of CCRI 12 callus,and is closely related to the accumulation and distribution of auxin.It broke the bottleneck of low efficiency of differentiation from cotton callus to embryogenic callus,and laid the foundation for the application of cotton varieties breeding.