Acta Botanica Sinica Volume 33 Issue 3, Pages .
Transformation of Populus tomentosa by Agrobacterium and Regeneration of Transformed Plantlets

Bu Xue-xian, Lin Zhong-ping and Chen Wei-lun

Abstract:
The establishment of efficient transformation system of Populus tomentosa by Agrobacterium is reported. The strains of Agrobacterium used in experiments were: 1. A. rhizogenes R1000, which harboured the Ri plasmid pRiA4b. 2. A. rhizogenes R1000 (pTVK85), which carried the plasmids pRiA4b and pTVK85 Containing supervirulent region. 3. A. tumefaciens C58C1 (pBZ693), the plasmid pBZ693 containing genes 1 and 2. After being cocultured with the bacteria on media containing 0.5 ppm kinetin for 2 days, explants of P. tomentosa were transferred to MS medium containing 500 ppm cefotaxime. Roots appeared on the explants in a week. The roots induced by A. tume[aciens were morphologically different from those induced by A. rhizogenes. The frequency of the explants transformed by A. rhizogenes R1000 (pTVK85) was nearly up to 60%. Some Ri plasmid transformed roots could spontaneously produce adventitious shoots or calli. By adding appropriate plant growth regulators in the media, we could have all of the root lines transformed produce adventitious shoots which would develop into intact plantlets on a hormone-free medium. Some phenotypical differences were observed among clones of the transformed plantlets. Some clones had short internodes, large number of leaves, reduced apical dominance, rich root systems with a great quantity of branches and root hairs, whereas in other clones aboveground parts of plantlets were morphologically normal and only their root systems were different from those of untransformed plantlets. None of the plantlets transformed by A. rhizogenes had the phenomenon of wrinkle leaves and shapes these leaves were analogous to normal plantlets. It was often observed that roots were regenerated from stems above the medium surfaces. Southern analysis on three clones of the putative transformed plantlets by A. rhizogenes R1000 (pTVKS5) showed that two of them were hybridized positively with the probe covering the TL-DNA region of the plasmid pRiA4b.

Keywords:
Populus tomentosa; Agrobacteriura rhizogenes; A. tumefaciens; Transformation; Plantlet regeneration
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