Optimization Of Genetic Transformation System For Sweet Sorghum And Pectin Polysaccharides Modification In Tobacco

Sorghum is considered one of the recalcitrant crop for the genetic modulation.Recently,progress in successful transformation has been made for this particular monocot crop through direct DNA delivery method and indirect method via Agrobacterium.But lower transformation rate still proved to be a bottleneck in genetic modification of sorghum.An efficient Agrobacterium transformation system could be attained by optimizing the preliminary assays,comprising of explant source,growth media,antibiotics,Agrobacterium strains and agro-infection response of callus.The selection of competent strains for genetic transformation is also one of the key factors of consideration.The immature embryos from the field source have higher inherent adaptation chances than that of the greenhouse source.A higher concentration of Agrobacterium may damage the explant source.Utilization of antinecrotic treatments and optimized tissue culture timeframe are the adequate strategies to lower down the effect of phenolic compounds.Appropriate selection of culture media vessels at different stages of tissue culture may also assist in a constructive manner.In conclusion,some aspects like culture environment with medium composition,explant sources,and genotypes play an indispensable role in successful Agrobacterium-mediated sorghum transformation system.In the present study we have optimized the regeneration media by utilizing the immature inflorescence rather than immature embryos and mature embryos.We have tested the five local genotypes i.e.JR-105,Ji-2731,Keller,Mn-3025 and Juti'an from field as well as green house sources and compares the callus induction and regeneration frequency after the induction of Agrobacterium transformation.Our results depicted that immature inflorescence produced more callus induction in contrast with immature embryos.Whereas,the production of the phenolics is a serious concern throughout regeneration phase.The above cited factors actually prevails and hinders the genetic transformation in real sense.Furthermore,our results lead us to speculate that sorghum is highly genotype dependent for Agrobacterium transformation whereas,it did not show any transformation in local genotypes.While in case of virus-induced gene silencing,it is a transient based reverse genetic tool used to elucidate the function of unknown genes that might be involved in biological processes.It is the most efficient alternative technique for the functional characterization specifically for the plants which are recalcitrant to stable genetic modification i.e.soghum and legumes.Where as,UDP-D-glucuronate 4-epimerase gene family is a Golgi localized,might have a role in the interconvertion of UDP-D-GlcA and UDP-D-GalA in pectin synthesis.In the current study we have optimized the VIGS system for the functional characterization of NbGAE6 in N.benthamiana tobacco plants.Our results depicted that the downregulation of this gene reduced the amount of GalA in the homogalacturunan which is the major component of the pectin in the primary cell wall.Our biphenyl assay and high performance liquid chromatography results supported each other results that the level of GalA reduced in VIGS plants from 40 % to 51 % as compared to the wild type plants.Furthermore,qRT-PCR also quantifies the downregulation of the NbGAE6 mRNA in VIGS plants as compared to wild type plants.Hence,virus-induced gene silencing provides a rapid silencing of single or multiple genes with a wide host range for the monocot and dicot crops which do not need stable transformants.On the other hand,CRISPR/Cas9 is a second generation genome editing tool and we have succeeded in the mutation of pectin related NtabGAUT4 and lignin related NtabCCoAMT genes with 6.2 % and 9.4 % mutation frequency.