High Efficiency Induction Of Artificial Spike Buds From Switchgrass And P_SAG12-IPT Gene Genetic Transformation

Switchgrass(Panicum virgatum L.)is a perennial warm season high yield forage grass,which is originated from North American. It has a wide adaptability to barren soil, droughtand salinity conditions. It is the ideal plant for soil erosion and desertification control.Inrecent years, because of its biological yield and high rate of ethanol production,switchgrasshas become an essential bio-energy plants.Nowadays, the use of agriculture biotechnology toincrease the biological production of switchgrass, to lower lignin content and increase the rateof ethanol production has become the current research focuses.In modern crop genetics and breeding, beside the conventional breeding, biotechnologybreeding, such as cell engineering and genetic engineering, plays more and more importantrole in breeding.However, plant tissue culture was an essential part in the cell engineering,genetic engineering research and application. So, study of plant tissue culture was theprerequisite and guarantee of use of modern biotechnology. According to the current situationof the development of the technology of transgenic plants, it is the foundation and prerequisitefor the establishment to research and create a more efficient system of plant tissue culturegenetic transformation system.So in this research, firstly with inflorescence of switchgrass as explants, establish thehighly efficient regeneration system of switchgrass artificial spike buds, and finally providethe receptor materials for switchgrass biological engineering. And at the same time, theAgribacterium-mediated transformation method was used to transform gene to matureembryogenic callus of switchgrass, And in the hope of providing technology and methods forAgrobacterium-mediated transformation of monocotyledons. The main results were asfollows:1. With inflorescence of three genotypes of xiji1, xiji2, xiji3as materials, using singlefactor design consider factors including genotype, panicle length, phytohormone, the length ofspike buds in the initial cutting period and in mass cultivating period, the carbon source, pH,spike buds cutting way and root induction medium, to optimize the regeneration system of switchgrass artificial spike buds, The results showed that the optimal regeneration system was:genotype xiji1, panicle length1.5cm,3.0mg/L6-BA+0.1mg/L2,4-D as phytohormone, thelength of spike buds in the initial cutting period and in mass cultivating period was1.5cm,maltose as carbon source, pH5.8, vertical spike buds cutting, and1/2MS as root inductionmedium. The proliferation efficiency of switchgrass artificial spike buds might reach up to4167.00%.2. The highly efficient regeneration system of switchgrass artificial spike buds wereoptimized by quadratic regression orthogonal rotary tests.The quadratic orthogonal regressionmodel was spike buds proliferation efficiency of switchgrass(Y) to seven factors of paniclelength (X₁), phytohormone(X₂), the length of spike buds in the initial cutting period(X₃),processing times(X₄), Size of ear buds block(X₅), in mass cultivating period(X₆), pH(X₇) andestablished as Y=4020.37+38.89X₁+252.78X₂+247.22X₃+941.67X₄+47.22X₅-322.22X₆-433.33X₇-566.67X₁X₂-12.50X₁X₃-25X₁X₄+88.89X₁X₅-70.84X₁X₆+714.28X₁X₇+175.0X₂X₃+20.83X₂X₄-145.83X₂X₅-41.67X₂X₆-212.5X₂X₇+74.07X₃X₄+351.85X₃X₅-533.34X₃X₆-597.84X₃X₇+752.38X₄X₅-677.78X₄X₆-1200X₄X₇-488.9X₅X₆+76.2X₅X₇+538.16X₆X₇-3814.29X₁²-3640.18X₂²-3632.08X₃²-3745.45X₄²-33850.73X₅²-33595.65X₆²-3134.07X₇²ã€‚It was concludedfrom the model that the optimal regeneration system were: panicle length1.5cm,3.0mg/L6-BA+0.1mg/L2,4-D as phytohormone, the length of spike buds in the initial cutting periodand in mass cultivating period was1.5cm,5times of processing, size of spike buds block2.0cm and pH6.0. The conclusion was as similar as the results of single factor design.3.With the embryogenic callus from mature embryos as acceptor, usingAgrobacterium-mediated transformation methods to transform PSAG12-IPT gene to switchgrass. Afterscreening, differentiation, transplanting,318transgenic plants were obtained. Through PCRidentification, nineteen plants were positive, and with the transformated efficiency of0.223%.