biologia plantarum

International journal on Plant Life established by Bohumil Němec in 1959

Biologia plantarum 42:361-369, 1999 | DOI: 10.1023/A:1002457000944

Hygromycin B - An Alternative in Flax Transformant Selection

S. Rakouský1, E. Tejklová2, I. Wiesner1, D. Wiesnerová1, T. Kocábek1, M. Ondřej1
1 Institute of Plant Molecular Biology, Academy of Sciences of the Czech Republic, České Budějovice, Czech Republic
2 AGRITEC Ltd., ¦umperk, Czech Republic

The in vitro regeneration of three flax (Linum usitatissimum L.) breeding lines (cv. Jitka, cv. Areco and NLN 245) and selection of transgenic plants were studied. A. tumefaciens derived binary vector GV3101 (pPM90RK)(pPCVRN4) bearing tetramer of 35S promoter enhancer was used in transformation experiments. Following 3 weeks of cultivation on shoot inducing Murashige and Skoog agar medium containing BAP (0.1 µM) and NAA (0.005 µM) from 82.6 % to 98 % of hypocotyl segments formed shoots. While ticarcillin (500 mg dm-3) used to eliminate Agrobacterium following the transformation decreased the organogenic response by about 10 % only, the addition of 20 mg dm-3 hygromycin to ticarcillin efficiently suppressed the regeneration of untransformed control plants. To look up for genomic mutations caused by T-DNA insertion from Agrobacterium transformation or originated from somaclonal variation over 500 regenerated plants have been cloned, transferred into soil and evaluated especially for their morphological characteristics. Up to now among plants of cv. Areco-background at least 8 genotypes showed changes either in flower or filament and stigma colour and one clone of plants with pollen sterility was identified. Among fifty four plant clones evaluated in 7 clones the presence of transgene specific sequence hpt was detected and simultaneously Agrobacterium contamination of tissues was firmly excluded.

Keywords: Agrobacterium tumefaciens; mutant induction; nested PCR; plant transformation; T-DNA
Subjects: Agrobacterium tumefaciens, transformation; flax, transgenic plants; genomic mutation, T-DNA; hygromycin B, Agrobacterium transformation; in vitro culture, regeneration; Linum usitatissimum; mutant induction, flax; nested polymerase chain reaction, PCR; T-DNA, genomic mutation; transgenic plants, flax

Published: November 1, 1999  Show citation

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Rakouský, S., Tejklová, E., Wiesner, I., Wiesnerová, D., Kocábek, T., & Ondřej, M. (1999). Hygromycin B - An Alternative in Flax Transformant Selection. Biologia plantarum, 42(3), 361-369. doi: 10.1023/A:1002457000944
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