Optimization Of Somatic Embryogenesis System And Detection Of Molecular Research In Xin Hai 16

Gossypium barbadense plays an important role in economic production because of its high quality fiber quality.The combining application of genetically modified biotechnology and traditional breeding provides an effective way to improve the quality of Gossypium barbadense production and improve stress resistance.Somatic embryogenesis is a key link in Agrobacterium tumefaciens mediated transgenic technology.However,somatic embryogenesis in Gossypium barbadense is heavily dependent on genotype,which greatly reduces the efficiency of Agrobacterium genetic transformation and limits the process of Gossypium barbadense breeding.In order to establish an efficient and stable marine Gossypium barbadense somatic embryogenesis system,this study is firstly to optimize the embryogenic callus induction system,Based on the established Gossypium barbadense ’Xinhai 16’ somatic embryogenesis system,MSB is supplemented with 0.1 mg/L 2,4-D and 0.1 mg/L.KT was used as the basic medium,and the effects of different parts of explants,light and medium components on callus were studied by single factor randomized experiment.The modified phenol fuchsin staining method was used to record the embryogenic callus induction process.The morphology of non-embryonic callus cells appeared,followed by the observation of early somatic embryogenesis by paraffin sectioning on 0 d,20 d embryogenic callus(EC,ECG)and 40 d globular embryo(GE).The tissue morphology and cell changes were used as materials for studying the molecular mechanism of somatic embryogenesis in the Gossypium barbadense.The molecular mechanism of the transformation process from embryogenic callus to somatic embryogenesis was studied by using transcriptome sequencing technology(RNA-seq).The main results are as follows:(1)The explants were more likely to differentiate into the callus from the upper green shoots of the hypocotyls;the best treatment for callus induction was MS1 with 1.2 g/L Gelrite or Phytagel under 2000 lux.It can significantly improve the physical and chemical state of callus,reduce the appearance of hoarfrost structure,Increasing the rate of callus differentiation to 30-31%;the callus with a round cell shape and a large nucleus located in the center of the cell and having a loose graininess can further form embryogenic callus and differentiate into somatic embryos.(2)The transcriptome data analysis showed that after the two periods of gene pairwise comparison(EC-vs-ECG,ECG-vs-GE),4223 and 1016 DEGs were obtained respectively.During the embryogenic callus development of the Gossypium barbadense,the transcripts showed temporal changes.Set analysis found that DEGs are mainly involved in protein dimers,transcriptional regulation,plant hormones,enzyme catalytic activity and other pathways.A large number of transcription factors and hormone signal transduction related genes were differentially expressed during the embryonic process of Gossypium barbadense embryogenic callus differentiation.Selecting differentially expressed genes with 8 different functions to verify transcriptome data by qRT-PCR,the results showed that the RNA-Seq data showed the same expression as the qRT-PCR results,and the transcriptome data were accurate and reliable.