GENERATION OF LARGE NUMBERS OF INDEPENDENTLY TRANSFORMED FERTILE BARLEY PLANTS
GENERATION OF LARGE NUMBERS OF INDEPENDENTLY TRANSFORMED FERTILE BARLEY PLANTS
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DOI：
10.1104/pp.104.1.37
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发表时间：
1994-01-01
期刊：
影响因子：
7.4
通讯作者：
LEMAUX, PG
中科院分区：
文献类型：
--
作者：
WAN, YC;LEMAUX, PG
描述了一个快速，高效，可再生的系统，以产生大量的独立转化，自交，转基因大麦（Hordeum vulgare L.）植株。用含有bar和uidA的质粒单独或与含有大麦黄矮病毒外壳蛋白（BYDVcp）基因的质粒联合轰击未成熟的合子胚、幼小的愈伤组织和小孢子衍生的胚。共有91个独立的抗双磷愈伤组织表达了bar的产物-功能性膦-丙氨酸乙酰转移酶。通过DNA杂交证实了棒材的整合性。两个连锁基因（bar和uidA）和两个非连锁基因（bar和BYDVcp基因）的共转化频率分别为84%和85%。36个转化的愈伤组织在含双磷培养基上再生出500多株绿色、可育的转基因植株；白化植株仅从41个品系再生。对25个株系（每系3株）的T-0植株进行DNA杂交分析，结果均含棒材。引入基因（bar、uidA和BYDVcp基因）的整合模式与亲本愈伤组织的整合模式相同。通过DNA杂交分析，证实了基因在5个家族中传递给T-1后代。未成熟的T-1胚在含双磷培养基上的萌发试验对于选择积极表达bar的个体是有用的，尽管这不是一个很好的指标。bar基因在某些后代植株中的表达表现为对除草剂面食的抗性。T-1植株在未成熟胚轰击后大约7个月生长在土壤中。
A rapid, efficient, and reproducible system to generate large numbers of independently transformed, self-fertile, transgenic barley (Hordeum vulgare L.) plants is described. Immature zygotic embryos, young callus, and microspore-derived embryos were bombarded with a plasmid containing bar and uidA either alone or in combination with another plasmid containing a barley yellow dwarf virus coat protein (BYDVcp) gene. A total of 91 independent bialaphos-resistant callus lines expressed functional phosphinothricin acetyltransferase, the product of bar. Integration of bar was confirmed by DNA hybridization in the 67 lines analyzed. Cotransformation frequencies of 84 and 85% were determined for the two linked genes (bar and uidA) and for two unlinked genes (bar and the BYDVcp gene), respectively. More than 500 green, fertile, transgenic plants were regenerated from 36 transformed callus lines on bialaphos-containing medium; albino plants only were regenerated from 41 lines. T-0 plants in 25 lines (three plants per line) were analyzed by DNA hybridization, and all contained bar. Most contained the same integration patterns for the introduced genes (bar, uidA, and the BYDVcp gene) as their parental callus lines. Transmission of the genes to T-1 progeny was confirmed in the five families analyzed by DNA hybridization. A germination test of immature T-1 embryos on bialaphos-containing medium was useful for selecting individuals that were actively expressing bar, although this was not a good indicator of the presence or absence of bar. Expression of bar in some progeny plants was indicated by resistance to the herbicide pasta. The T-1 plants were in soil approximately 7 months after bombardment of the immature embryo.