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《ACTA BOTANICA BOREALI-OCCIDENTALIA SINICA》 1996-03
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STUDIES ON A NEW GENE TRANSFORMATION METHOD:VACUUM INFILTRATION TRANSFORMATION

Gong Zhenghui(Department of Horticulturen, Northwester Agricultural University, Yangling Shaanxi 712100 ) Milner Joel Joseph(Department of Botany,University of Glasgow,U. K. )He Yuke(Shanghai Institute of Plant Physiolology,Academia Sinica,Shanghai 200032  
A new gene transformation method,vacuum infiltration transformation was studiedby the GV3101 strain of Agrobacterium tumefaciens carrying the construction plasmidpJo530:: Bari- 1 GVI in Arabidopsis thaliana Hegn.,ecotype Landsberg erecta. Manytransgenic plants were obtained by this simple,convenient, speedy,reliable and nontissue cultural method. The suitable conditions of vacuum infiltration method was as follows. The robust plants were cultured in the greenhouse. Emerging bolts were clippedoff to encourage growth of multiple secondary bolts. The infiltration would be done fourto eight days after clipping. The agrobacterium carrying pJo530:: Bari-1 GVI wasgrown,centrifuged and then resuspended in infiltration medium (1/2 × MS, 1 × B5 vitamins, 5. 0% sucrose,0. 04 μmol/L Benzylamino purine) for a final OD600 approximate 0.8. The agrobacterium(in infiltration medium) was added to a dish and the plants(pot,soil and all)were inverted into it,and then to place them into bell jar of vacuum pump ofwhich sauction capacity was 1. 7 m2/h. The infiltrated plants would be removed to greenhouse for continuous culture at continuous light until the time of vacuum infiltrationlasted for 2 min/30 s (Drawing a vacuum for 2 minutes, then releasing vacuum veryrapidly, and closing the pump valve to draw a vacuum for another 30 seconds). Whensiliques on plants were very dry,the seed were harvested and were selected on selectionplates (1/2 × MS, 0. 8% agar, 1× B5 vitamins and 30 μg/ml hygromycin). The frequence of transformants was as high as 0. 71% by the analysis of PCR and ELISA.
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