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標題: Trypsin Inhibitor 轉移於花椰菜之研究
Studies on the Trypsin Inhibitor gene transfer to cauliflower (Brassica oleracea var. botrytis)
作者: 丁樑泉
Ding, Lang Chang
關鍵字: transgenic plant;植物基因轉移;cauliflower;Agrobacterium tumefaciens;binary vector;Trypsin Inhibitors;biotechnology;花椰菜;農桿菌;雙載體;抗蟲效應;生物技術
出版社: 農業生物科技學研究所
自台灣栽培種甘薯的Trypsin Inhibitors基因轉入花椰菜中,觀察轉移植
程中瞭解:花椰菜下胚軸的分化能力較強,若將下胚軸分化以2.4-D 前處
理3 天,並置於含銀離子29.4mM的分化培養基中,下胚軸分化成不定芽的
能力均可達到95% 以上,其中以雪姬的分化能力較強。在以農桿菌媒介轉
液稀釋100 倍及共培養72小時的轉移效率最高。感染後轉移植物的篩選時
第3 週加入30mg/l的G418,直至抗G418的再生苗出現為止。在分生檢測方
面先以TI assay來測定抗G418的再生苗TI表現的程度。選取表現量較高的
再生苗,以PCR 及南方氏核酸雜交分析,證明TI基因已移入再生苗中,再

The aim of this research project is to establish a gene
transfer system for the Taiwan cauliflower cultivars.
Agrobacterium mediated transformation was used to introduce a
trypsin inhibitor (TI) gene into cauliflower hypocotyl explants.
This target gene was isolated from a strain of Taiwan cultivated
sweet potato. Our regeneration studies indicated that, among
various tested explant types, the 3-day-old seedling hypocotyl
segments had the highest regeneration capacity. Five-mm long
hypocotyl explant were first pretreatedwith 1 mg/l 2,4-D for
three days. They were then transferred onto a silver ion
(29.4mM) containing shoot formation medium. More than 95% of
the explantsregenerated adventitious shoots using this
protocol. The following optimumtransformation conditions were
determined. The 2,4-D pretreated explants wereinoculated with an
1:100 dilution of an 18-h Agrobacterium culture harboring TI and
NPTII gene containing plasmid. Co-cultivation was carried out at
20-25 ℃ for 72hs. G418 selection was initiated one-week after
co-cultivation at a concentration of 15 mg/l. G418 concentration
was doubled three-weeks afterward.Confirmation of the transgenic
ststus of the resultant G418 resistant R0 plantswas carried out
with Southern and Western hybridization, PCR analysis assay.
From 18 tested plants, 14 expressed positive reactions to all
the above four molecular assays. All the transgenic plants
demonstrated resistance to the chewing insects Plulella
xyloshella and Spodophera litura to which the nontransformed
control plants were vulnerable.
Appears in Collections:生物科技學研究所

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