Cotton Somatic Embryogenesis And Isolation And Characterization Of Cotton GhAux/IAA4

Cotton is one of the most important economic crops in the world, is also an important industrial raw materials. Research on occurrence and mechanism of the somatic embryo, not only helps to promote the basic research of cotton’s progress, but also has a great significance for the genetic improvement of cotton, In this study, we have wildly screened some upland cotton varieties for their capability to regenerate plants underwent SE. Among these varieties, we optimized the regeneration procedure for Lumian 6, an cultivated variety from North China. Moreover, in order to find crucial genes in cotton SE, different expression transcription factor Aux/IAA was isolated from the RNA-Seq profile of cotton SE. We have further characterize an Ghux/IAA4 gene in this study. The main results were presented as following:1. The regeneability of thirty elite genotypes were compared by using IBA+KT and 2,4-D+KT treatments. The results showed that genotypes namely, "L6" and "Gm933" have predominant ability underwent SE, with their calli loose, grainy and flaxen, and embryogenic-like structure were observed. An efficient somatic embryogenesis system was developed to regenerate plantlets from hypocotyls of upland cotton cultivars Lumian 6(Gossypiumhirsutum L). The cultivar Lumian 6 was cultivated in North China, the same cotton planting region as YZ1, an easy-going genotype somatic embryogenesis. Histological observation demonstrated that the majority of embryogenic calli originated in a familiar way in the two genotypes, it was proved that the cells from primary cambium cells were the original cells for cotton embryogenic calli formation. RT-PCR was used to display the selected genes expression(such as LEC1, PIN1, PIN7, Aux/IAA4, ARF6, SERK1, SERK3, ABA2, PP2CA1) at distinct time-points with Lumian 6 and the comparison YZ1. The results showed that some genes, including PIN1, LEC1, LEC-like, ARF6, PP2CA1 associated with the formation of embryonic callus.2. Based on the sequence of a cDNA fragmentfrom RNA-Seq profile during cotton somatic embryogenesis, we obtained the full length cDNA by rapid amplification of cDNA ends (RACE) and designated as GhAux/IAA4, which contains an ORF of 942 bp.The result of qRT-PCR showed that GhAux/IAA4 transcripts were differentially expressed during cotton SE. Sequence analysis showed that the GhAux/IAA4 protein had four conserved Aux/IAA domain with high similarity to AtAux/IAA4. Overexpression of GhAux/IAA4 in Arabidopsis inhibited taproot length and promoted germination and geotropism. Overexpression of GhAux/IAA4 in cotton resulted in granulate embryogenic callus. GhAux/IAA4 expression was induced by exogenous auxin in wild-type embryogenic callus; However, reduced auxin response was observed in embryogenic callus from overexpression lines.