Highly efficient transformation and regeneration of aspen plants through shoot-bud formation in root culture |
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Authors: | Tzvi Tzfira Hagit Ben-Meir Alexander Vainstein Arie Altman |
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Institution: | (1) The Hebrew University of Jerusalem, The Kennedy-Leigh Centre for Horticultural Research and The Otto Warburg Center for Biotechnology in Agriculture, 76-100 Rehovot, Israel |
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Abstract: | The natural capacity of aspen (Populus tremula L.) roots for direct shoot-bud regeneration was harnessed to establish a highly efficient transformation and regeneration procedure that does not require a pre-selection stage on antibiotics. Aspen stem segments were transformed using wildtype Agrobacterium rhizogenes (LBA9402) with the binary p35SGUSINT plasmid carrying the genes coding for -glucuronidase (GUS) and neomycin phosphotransferase II. High levels of transient GUS expression were found in the basal cut surface of 87% of the segments, and 98% of these formed well-developed adventitious roots. Proliferating root cultures were established in liquid culture, and GUS expression was found in 75% of the roots. Shoot-bud regeneration in root cultures was very high: 99% of the roots yielded shoot-buds (4.3 buds per root), of which 91% expressed GUS. Southern blot analysis and polymerase chain reaction confirmed the transgenic nature of the plants expressing GUS. Kanamycin resistance of transformants was tested with respect to callus growth and bud regeneration. Callus from transgenic plants exhibited a high growth rate in the presence of up to 100 g/ l kanamycin, and bud regeneration from transformed roots occurred in the presence of up to 30 g/ l kanamycin. Callus and buds from control (non-transformed) plants failed to proliferate or regenerate, respectively, in the presence of kanamycin at concentrations above 10 g/ l. Ninety-four independent clones from different transformation events were established, of which 52 were phenotypically true-to-type.Abbreviations NAA
-naphthaleneacetic acid
- BA
6-benzylaminopurine
- GUS
-glucuronidase
- NPTII
neomycin phosphotransferase II
- PCR
polymerase chain reaction
- EtOH
ethanol
- CTAB
cetyltrimethylammonium bromide
- SDS
sodium dodecyl sulfate
- NOS
nopaline synthase
- CaMV
cauliflower mosaic virus |
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