Establishment Of High Efficient Regeneration System Of Lilium And Agrobacterium-mediated Genetic Transformation

Lily is a kind of perennial monocotyledonous bulbar flowers which has high economic and ornamental value, and now has been one of the most important fresh cutting flowers. The rapid development of plant molecule biology has supported a new way to improve character and quality of plant, and also works on lily. Agrobacterium tumefaciens as a carrier is chip and effective which is available for transformation of extrinsic gene. Lily is not sensitive to Agrobacterium tumefaciens because it is a kind of monocotyledons, so we should explore an efficient way to transform extrinsic gene into lily. The experiment uses callus induced by bulb of lilium longiflorum as transformation material and pBI121 (without extrinsic gene) as transformation carrier to establish an acceptor system of Agrobacterium-mediated transformation of lilium longiflorum.1. Establish a high regeneration systemThe bulb of regenerative lily plant and the filament of lily can both be induced a lot of callus and the callus both differentiated deeply. So it is necessary to compare them and choose a proper one. The bulb can reach the highest callus growth level as 90% in the medium for induction and proliferation of callus which plusing 6-BA 0.5mg / L and NAA 0.1 mg / L; The filament can reach the highest callus growth level as 86.3% in the medium for induction and proliferation of callus which plusing 6-BA 1.0mg / L and NAA 0.5 mg / L. So the bulb has higher callus growth level than filament. The bulb can reach the highest callus differentiation level as 93.8% in the medium for callus differentiation which plusing 6-BA 0.5mg / L and 0. 05 mg / L; The filament can reach the highest differentiation growth level as 85.0% in the medium for callus differentiation which plusing 6-BA 1.0mg / L and NAA 0.5 mg / L. So the bulb has higher callus differentiation level than filament.The bulb has both higher callus growth and differentiation level than filament, also it will not be limited by season like filament. So we choose the bulb as the testing material.2. The experiment of the antibiotic sensitivityThe experiment of kannamycin (Km) and cefotasimine (Cef) show that the differentiation of callus is 2.5% in the concentration of Km 125mg/L, so it is the sub-death concentration and also the proper selection concentration for callus of bulb; When the concentration of Km in rooting medium is 125mg/L leaves of lily turn yellow and roots of lily grow no more, so it is the proper selection concentration; When the concentration of Cef is 400mg/L it is efficient but no hurt to explant, so it is the proper concentration for restraining bacterial.3. The factors of transformation.The efficiency of transformation could be increased when NH₄NO₃ is reduced in co-culture medium. It reaches the highest transient expression level when there is no NH₄NO₃ in co-culture medium at all. So we choose the non- NH₄NO₃ MS medium as the co-culture medium.The transformation rate is 61.3% when MS is used for intrusion while the transformation rate is 16.3% when YEB is used for intrusion. So the MS is the proper intrusion solution.It is no sufficient intrusion when the concentration of agrobacterium is too low and the time of intrusion is too little. On the other hand, it is noxious to callus when the concentration of agrobacterium is too high and the time of intrusion is too much. The proper concentration is OD₆₀₀=0.6 and the proper time is 8 min.The tumour can not be induced by agrobacterial if co-culture time less than 16 hours. On the other hand, the cell of lily will be poisoned for co-culture too long time and agrobacterium growing too fast. The proper co- culture time is 3 days.Acetosyringone (AS) is effective for genetic transformation of lilium longiflorum. The transient expression of GUS gene is stable when the concentration of AS is 100-150μmol / L, and the shoots can be obtained.The transient expression of GUS gene is the highest when the concentration of AS is 120μmol / L, and the most shoots can be obtained. But it is noxious to callus when the concentration of AS is too high. When the concentration of AS is more than 200μmol / L, it can not increase the expression of GUS gene.There are three ways to use AS: the first way is pulsing AS in invasion solution; the second way is pulsing AS in co-culture medium; the third way is pulsing AS both in invasion solution and co-culture medium. The highest genetic transformation rate can be got by the third way.