Genetic Transformation Of MiR396 Gene Into Barley Callus By Agrobacterium Tumefaciens Infection

MiRNAs are about 21 nucleotide, non-coding small RNAs that play an important role in plant growth, development and disease resistance by participating in post-transcriptional regulation. It has been proved that miR396 involves in cell division,leave-characters formation, floral organ variation and drought tolerance etc.In order to effectively utilize the excellent genetic resource, we over-expressed the miR396 from rice using Agrobacterium-mediated transformation, excepting getting barley lines with new good traits, especially with strong drought tolerance. In this study, the immature embryos of 6 barley varieties and mature embryos of 3 varieties were used as the transferred acceptors, hptⅡ as the selective marker, we successfully obtained some transformmed barley plants with Os-miR396. Meanwhile, we analyzed and optimized the factors influencing transferred efficiency. The mean results were as follows:(1) Genotype is a major factor affecting the immature and mature callus-induced rate as usual;(2) when callus are induced only use mature embryo scutellum, there’s no need to add ABA to inhibit budding as it will suppress the callus growing;(3) Different immature embryo callus has different sensitivity of hygromycin when differentiation in the same selection pressure, 25mg/L hygromycin may be too high to Misato Gloden, Huadamai 4 and Huadamai 7 as affecting its growth and reproduction;(4) Timentin concentration influences its antibacterial effect, it should be increased when the infection OD600 rise in mature callus transformation;(5) We obtained 17 hygromycin-resistant barley lines in Agrobacterium-mediated transformation of immature embryo callus, of which 5 lines showed positive of gene miR396 in PCR analysis, including one line of Amagi Nigo, two lines of Supi 1 and two lines of Zhenongda 3;(6) In the mature callus transformation mediated by Agrobacterium, we got 13 regenerated plants, PCR analysis revealed that 4 lines were positive of miR396,both of them were all from variety Amagi Nigo.