CRISPR-Cas9-mediated Gene Editing On Row-type And Caryopsis Traits In Barley

Barley is the fourth largest farming grain crop in the world.As a typical three-purpose crop,it could be used as raw materials for feed and food production or beer brewing.vrs1,an independently inherited recessive gene controlling the row type of the barley(six lateral spikelets)is located on the chromosome 2HL.Its allelic allele,Vrs1,encodes a protein,which inhibits the development of lateral spikelets,leading to the formation of two-rowed barley.A number of studies have demonstrated the close relationship between the row type and the yield.nud,another single recessive gene located on the chromosome 7HL,controls the trait of naked caryopsis,and like the Vrs1 gene,significant impacts quality traits in barley.To produce new barley varieties of high yield and quality,we conducted CRISPR-Cas9-mediated gene editing to modify the Vrs1 and Nud genes for the purpose of transforming two-rowed barley to six-rowed and the hulled caryopsis to naked caryopsis using Agrobacterium tumefaciens-mediated plant transformation for gene delivery.In this paper,we choose the immature embryo callus of Huadamai 5,Huadamai 6,Zhenongda 3 as the acceptors of the vector targeting Vrs1 gene;the immature embryo callus of Huadamai 4 and Huadamai 7 as the acceptors of the vector targeting Nud tgene and the mature embryo callus of Huadamai 5 and Huadamai 6 as the acceptors of the vector targeting Vrs1 gene.Finally,using antibiotic,hygromycin(Hyg)as the selectable marker for plant transformation,we successfully produced some positive transgenic plants from different barley cultivars.In addition,we analyzed the relationship between callus differentiation rate and cultivation time.The main results obtained are as follows:(1)A total of 14 Hyg-resistant plants was obtained by Agrobacterium tumefaciens-mediated transformation of Vrs1 gene into immature embryos.There were no Hyg-resistant plants in Huadamai 5.PCR test of the resistant plants revealed 4 of them positive,including 2 from Zhenongda 3 and 2 from Huadamai 6.(2)When the callus from mature embryos of Huadamai 5 and Huadamai 6 was used for Agrobacterium tumefaciens-mediated transformation to target Vrs1 gene,no Hyg-resistant plants were obtained.(3)Agrobacterium tumefaciens-mediated transformation of the callus from immature embryos with Nud gene resulted in 5 Hyg-resistant plants from Huadamai 7,while there were no Hyg-resistant plants from Huadamai 4.PCR test of the resistant plants revealed positive amplification in 1 Hyg-resistant plant.(4)Upon Agrobacterium tumefaciens infection,immature embryo callus differentiation rates on differentiation media are different in three varieties.For Zhenongda 3 and Huadamai 6,8 weeks of cultivation time resulted in the highest differentiation rate,while the highest differentiation rate was achieved after 12 weeks of cultivation in Huadamai 7.