Construction Of Mutant Library And Genetic Transformation Of Multiple Bud Dumps In Setaria Viridis

Setaria viridis is a tufted and self-pollinated, C4 summer annual; reproducing by seed;diploid species, 2n=36. S. viridis belongs to Poaceae and is a member of the Panicoideae clade. Itis a true diploid with a relatively small genome of 510 Mb. Its short stature, simple growthrequirements, and rapid life cycle will greatly facilitate genetic studies of the C4 grasses.Importantly, S. viridis uses an NADP-malic enzyme subtype C4 photosynthetic system to fixcarbon and therefore is a potentially powerful model system for dissecting C4 photosynthesis.Here, I summarize my work about S. viridis as a genetic system, including construction ofmutant library and the genetic transformation of multiple bud dumps.In this study, seeds of S. viridis was induced with 0.1％, 0.2％, 0.3％, 0.4％EMS for 24hand 150 Gy, 200 Gy, 300 Gy60Coγ-rays. There are 12,000 seeds of EMS-induced M1 and13,000 seeds of M1 radiated with60Coγ-rays. Biological characters and agronomic traits wereinvestigated in M2 generation. For 6,700 M2 seedings, total of 206 mutants of morphologicaltraits has been created with the mutant-induced frequency of 3.07％after sereening in 6,000 M1and transfer to the field. There are 158 EMS-induced mutant with the mutant-induced frequencyof 2.6％. 48 mutant was sceened in 700 M2 radiated with60Coγ-rays and the mutationfrequency was 3.07%. Mutation involves in plant height, stem, leaf, culm and tilling, panicle andspikelet and vein. Among them, leaf trait mutants were most abundant with frequeney of 1.03%,followed by vein density, dwarf and spikelet mutants with frequeney of 0.55%, 0.43% and 0.41%.Leaf mutant involves in leaf sheap and color, busy leaf. Spike mutants inculde short spike mutant,curve spike mutant, spike stems grow, a stem and two spike mutant and so on. Vein mutantinvolves in more major veins, the branch of major vein in the middle, side veins and so on. And75 mutants with decreased vein density have been found with the frequeney of 1.1%. We havecheck microstructure of the mutant with paraffin sections. But there was not the increasingnumber of M cells between veins in those mutants. Because of big size cell, the vein spacebecome wide.The transformation methods for S. viridis is not yet mature. Regeneration of plants from callus induced from seed is rate limiting. In this experiment, I have constructed regenerationsystem from multiple bud clumps, and multiple bud clumps was been used for genetictransformation. Firstly, it was cultureing multiple bud clumps. The bud tips were derived frometiolated shoot tips of stweile seeding. And we plated the shoot tips onto induction mediumwhich was modified MS medium containing 2.0 mg/L 6-BA and 0.5 mg/L 2,4-D. Then multiplebud clumps were obtained on proliferation medium containing 2.0 mg/L 6-BA. The single budand bud clumps derived from the multiple bud clumps were prepared for biolisticstransformation with plasmid. Then transfer bud clumps to medium with 4.0 mg/L 6-BA for 7days in order to recovery growth. The resistant bud clumps were gained by screening on theselection medium. It was 4 times and every time was two weeks. Survival buds were transferredto rooting medium to induce root. Rooted plantlets were transferred to the soil and transgenicplants were obtained. PCR assay results of the transgenic plants showed that 8 were PCRpositive in T0of 80 lines. Now we have succeed in regeneration of plants, but transgenic plantsare being identification and analysis.