Transformation of sweetgum via microprojectile bombardment of nodule cultures |
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Authors: | Myoung K Kim Harry E Sommer Jeffrey F D Dean Scott A Merkle |
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Institution: | (1) Daniel B. Warnell School of Forest Resources, University of Georgia, 30602 Athens, Georgia;(2) Present address: Institute of Biological Chemistry, Washington State University, 99164-6340 Pullman, Washington |
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Abstract: | Summary A sweetgum (Liquidambar styraciflua) nodule culture system was developed and integrated with genetic transformation by microprojectile bombardment. Nodule cultures
were established from seedling hypocotyls and proliferated in liquid medium containing 0.1 mg (0.45 μM) thidiazuron (TDZ) per 1 and 0.01 mg (0.045 μM) 2,4-dichlorophenoxyacetic acid (2,4-D) per 1. Shoots differentiated from the nodules in liquid media containing (per 1)
1 mg (4.4 μM) benzyladenine (BA), 0.5 mg (2.2 μM) BA, and 0.01 mg (0.054 μM) naphthaleneacetic acid (NAA), or 0.5 mg BA, 0.01 mg NAA, and 0.05 mg (0.23 μM) TDZ under the light. Differentiating shoots required 4 wk of dark treatment for further development on semisolid medium
containing 1 mg BA per 1. Elongated shoots were harvested and the basal ends were soaked in a solution containing 10 mg (49.2
μM) indole-3-butyric acid (IBA) per 1 before being planted in potting mix for ex vitro rooting. Roots formed and leaves expanded
in 2 wk. Sweetgum nodules were stably transformed by microprojectile bombardment with a 7.4-kb plasmid, pTRA 140, harboring
CaMV 35S-HPH and CaMV 35S-GUS. Evidence that nodules growing in the presence of hygromycin B were stably transformed was provided
by polymerase chain reaction analysis and β-glucuronidase activity. Sweetgum shoots differentiated in liquid medium in the
presence of hygromycin B. Shoots transferred to solid medium lacking hygromycin B elongated and displayed β-glucuronidase
activity in their expanding leaves and stems. Southern analysis confirmed the presence of the GUS gene in nodules and shoots.
Transgenic shoots initiated roots and showed leaf expansion 2 wk after being planted in potting mix. |
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Keywords: | gene transfer Liquidambar styraciflua biolistics adventitious shoots |
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