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Successful Agrobacterium-Mediated Genetic Transformation of Maize Elite Inbred lines

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Abstract

An efficient transformation system was developed for maize (Zea mays L.) elite inbred lines using Agrobacterium-mediated gene transfer by identifying important factors that affected transformation efficiency. The hypervirulent Agrobacterium tumefaciens strain EHA105 proved to be better than octopine LBA4404 and nopaline GV3101. Improved transformation efficiencies were obtained when immature embryos were inocubated with Agrobacterium suspension cells (A600 = 0.8) for 20 min in the presence of 0.1% (v/v) of a surfactant (Tween20) in the infection medium. Optimized cocultivation was performed in the acidic medium (pH5.4) at 22 °C in the dark for 3 days. Using the optimized system, we obtained 42 morphologically normal, independent transgenic plants in four maize elite inbred lines representing different genetic backgrounds. Most of them (about 85%) are fertile. The transformation frequency (the number of independent, PCR-positive transgenic plants per 100 embryos infected) ranged from 2.35 to 5.26%. Stable integration, expression, and inheritance of the transgenes were confirmed by molecular and genetic analysis. One to three copies of the transgene were integrated into the maize nuclear genome. About 70% of the transgenic plants received a single insertion of the transgenes based on Southern analysis of 10 transformed events. T1 plants were analyzed and transmission of transgenes to the T1 generation in a Mendelian fashion was verified. This system should facilitate the introduction of agronomically important genes into commercial genotypes.

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Abbreviations

BA:

6-benzyladenine

bar:

phosphinothricin acetyltransferase gene

2,4-D:

2,4-dichloro phenoxyactic acid

GUS:

β-glucuronidase

IBA:

indole-3-butyric acid

MES:

2-(N-morpholino) ethanesulfonic acid

PCR:

polymerase chain reaction

PPT:

phosphinothricin

uidA:

β-glucuronidase gene from Escherichia coli

X-gluc:

5-bromo-4-chloro-3-indolyl-β-glucuronide

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  1. Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, The Chinese Academy of Sciences, 200032, Shanghai, P.R. China

    Xueqing Huang & Zhiming Wei

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  1. Xueqing Huang
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  2. Zhiming Wei
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Correspondence to Zhiming Wei.

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Huang, X., Wei, Z. Successful Agrobacterium-Mediated Genetic Transformation of Maize Elite Inbred lines. Plant Cell Tiss Organ Cult 83, 187–200 (2005). https://doi.org/10.1007/s11240-005-5772-8

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