Articles
ASPEN TRANSFORMATION PROCEDURES: ONCOGENIC AGROBACTERIUM RHIZOGENES VERSUS DISARMED AGROBACTERIUM TUMEFACIENS
Article number
447_59
Pages
295 – 300
Language
Abstract
Novel transformation procedures were established for the tree-species model aspen (Populus tremula), using either wild-type Agrobacterium rhizogenes or disarmed A. tumefaciens. The A. rhizogenes-mediated transformation procedure included: (1) induction of transformed roots on stem explants, (2) direct shoot regeneration from roots in liquid culture, (3) rooting of transgenic shoots.
The disarmed A. tumefaciens-mediated transformation procedure included: (1) inoculation of stem explants, (2) regeneration of adventitious shoots from the cut surfaces of the explants, (3) kanamycin selection of transformed shoots, (4) rooting of transgenic shoots.
Both procedures differed from previous ones, which involved regeneration from callus, in that they consist of direct organogenesis of transformed organs.
The efficiency of both procedures was monitored using the uidA (GUS) gene (carried by binary plasmids harboring uidA and nptII genes). Stem explants were highly susceptible to both agrobacteria, as evidenced by transient GUS expression. A. rhizogenes-mediated transformation relied on the natural capacity of aspen roots for direct shoot regeneration in liquid culture, whereas TDZ was required for direct shoot induction on stem explants.
Regeneration percentages from stem segments were very high for both transformed roots and adventitious shoots (using A. rhizogenes or A. tumefaciens, respectively). Over 90% of the regenerating roots were transformed, and about 62% of the adventitious shoots exhibited stable GUS expression following selection on kanamycin.
The formation of transformed roots using A. rhizogenes resulted mainly from expression of the rol genes (donated by the pRi1855 vir-plasmid) in transformed plant cells.
These genes were also found to be responsible for the expression of several unique morphological and physiological traits in transgenic plants, e.g. breaking of apical dominance, extensive root formation and high growth rate.
No morphological abnormalities were observed in the A. tumefaciens-transgenic plants.
The disarmed A. tumefaciens-mediated transformation procedure included: (1) inoculation of stem explants, (2) regeneration of adventitious shoots from the cut surfaces of the explants, (3) kanamycin selection of transformed shoots, (4) rooting of transgenic shoots.
Both procedures differed from previous ones, which involved regeneration from callus, in that they consist of direct organogenesis of transformed organs.
The efficiency of both procedures was monitored using the uidA (GUS) gene (carried by binary plasmids harboring uidA and nptII genes). Stem explants were highly susceptible to both agrobacteria, as evidenced by transient GUS expression. A. rhizogenes-mediated transformation relied on the natural capacity of aspen roots for direct shoot regeneration in liquid culture, whereas TDZ was required for direct shoot induction on stem explants.
Regeneration percentages from stem segments were very high for both transformed roots and adventitious shoots (using A. rhizogenes or A. tumefaciens, respectively). Over 90% of the regenerating roots were transformed, and about 62% of the adventitious shoots exhibited stable GUS expression following selection on kanamycin.
The formation of transformed roots using A. rhizogenes resulted mainly from expression of the rol genes (donated by the pRi1855 vir-plasmid) in transformed plant cells.
These genes were also found to be responsible for the expression of several unique morphological and physiological traits in transgenic plants, e.g. breaking of apical dominance, extensive root formation and high growth rate.
No morphological abnormalities were observed in the A. tumefaciens-transgenic plants.
Authors
T. Tzfira, A. Vainstein, A. Altman, C. Jensen
Keywords
Populus tremula, uidA, GUS, direct regeneration
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