Mahideen Afridi

The hairy root induction system has been widely applied in studying gene expression and function in plant species due to its rapidity and efficiency. The hairy root system is an efficient tool for evaluating the activities of CRISPR/Cas systems. Cotton hairy roots were primarily induced through cotton tissue culture under aseptic conditions and by injecting cotton stem tips under non-aseptic conditions. However, both methods are lab-intensive and time-consuming. In this study, an efficient cotton hairy root induction procedure was established via infecting cotton hypocotyls with Agrobacterium rhizogenes under non-sterile conditions. Cotton seedlings with expanded cotyledons were decapitated with a slanted cut, and the residual hypocotyl (maintained 1 cm apical portion) was inoculated with A. rhizogenes. Over 90% of the infected explants from all three tested varieties could produce hairy roots after 8 days of inoculation. The effictiveness of the method was tested by overexpressing two reporter genes (eGFP and GUS). The transformation efficiency of the GUS and eGFP were ranged from50–68.18% and 40.9–68.18%. In addition, the editing efficiency of target sites in different CRISPR/Cas systems were also tested in hairy root. This method provided technical support for screening suitable target sites for cotton gene editing.

Background: Non-synchronized pods shattering in the Brassicaceae family bring upon huge yield losses around the world. The shattering process was validated to be controlled by eight different genes in the model plant Arabidopsis thaliana, including SHATTERPROOF1, SHATTERPROOF2, FRUITFULL, INDEHISCENT, ALCATRAZ, NAC, REPLUMLESS and POLYGlACTOURANAZE. To obtain gene family & examine their expression patterns into fresh & mature silique, then completed genome wide identification, characterization, and expression analysis of shattering genes in B. napus and B. juncea.Results: Complete genome analysis of B. napus and B. juncea revealed 32 shattering genes, which were identified and categorize based on protein motif structure, exon-intron organization and phylogeny. The phylogenetic study revealed that these shattering genes contain little duplications that were determined with a distinct chromosome number. Motifs of 32 shattering proteins were also observed where motifs 6 were found to be more conserved. A single motif was observed for other genes like BrnS7, BrnS8, BrjS23 and BrjS26. Comparative genomics for synteny analysis was performed that validated a conserved pattern of blocks among these cultivars. RT-PCR based expressions profile showed higher expression of shattering genes in B. juncea as compared to B. napus. FUL gene was expressed more in the mature silique. ALC gene was not expressed in the fresh silique of B. napus but highly expressed in the mature silique. Conclusion: This study authenticates that shattering genes exist in the local cultivars of Brassica. ALC exhibited strong expression in both the mature and fresh silique of B. juncea. Our results showed that shattering genes expression occurred more in B. juncea as compared to B. napus. It also contributes to the screening of more candidate gene for further investigation and characterization.