Role of the host cell cycle in the Agrobacterium-mediated genetic transformation of Petunia: evidence of an S-phase control mechanism for T-DNA transfer

Chimeric β-glucuronidase (GUS) gene expression in an efficient Agrobacterium-mediated transformation system utilising mesophyll cells of Petunia hybrida synchronized with cell cycle phase-specific inhibitors (mimosine and colchicine) was used to show the absolute requirement of S-phase for transfer...

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Bibliographic Details
Published in:Planta 1997, Vol.201 (2), p.160-172
Main Authors: Villemont, E, Dubois, F, Sangwan, R.S, Vasseur, G, Bourgeois, Y, Sangwan-Norreel, B.S. (Universite de Picardie Jules Verne, Amiens (France). Faculte des Sciences, Lab. Androgenese et Biotechnologie)
Format: Article
Language:English
Subjects:
ADN
AGROBACTERIUM
Agronomy. Soil science and plant productions
Biological and medical sciences
Biotechnology
Cell cycle
Cell division
Cell growth
CELLS
CELLULE
CELULAS
Cultured cells
DNA
EXPRESION GENICA
EXPRESSION DES GENES
FEUILLE
Flow Cytometrie
Fundamental and applied biological sciences. Psychology
GENE EXPRESSION
Genetic engineering
Genetic engineering applications
Genetic technics
GENETIC TRANSFORMATION
Genetics and breeding of economic plants
genetischer Kontrollmechanismus
HOJAS
HOSTS
HOTE
HUESPEDES
LEAVES
MESOFILO
MESOPHYLL
Mesophyll cells
MESOPHYLLE
Methods. Procedures. Technologies
NOYAU CELLULAIRE
NUCLEO
NUCLEUS
PETUNIA
Petunia hybrida
Phytohormon
Plant breeding: fundamental aspects and methodology
Plant cells
PLANT GROWTH SUBSTANCES
PLANTAS TRANSGENICAS
PLANTE TRANSGENIQUE
Plants
SUBSTANCE DE CROISSANCE VEGETALE
SUSTANCIAS DE CRECIMIENTO VEGETAL
TRANSFORMACION GENETICA
TRANSFORMATION GENETIQUE
Transgenic animals and transgenic plants
TRANSGENIC PLANTS
Zellkern
Zellzyklus
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Summary:Chimeric β-glucuronidase (GUS) gene expression in an efficient Agrobacterium-mediated transformation system utilising mesophyll cells of Petunia hybrida synchronized with cell cycle phase-specific inhibitors (mimosine and colchicine) was used to show the absolute requirement of S-phase for transfer and/or integration of the transferred DNA (T-DNA). Flow-cytometric analysis of nuclear DNA content and immunohistological detection of bromodeoxyuridine (BrdUrd) incorporation showed that, prior to phytohormone treatment, most (98%) mesophyll cells were at G0—G1-phase (quiescent phase) and no cell division was occurring. After 48 h and 72 h of phytohormone treatment, there was a rapid increase in S—G2—M-phase populations (> 75%) and a concomitant decrease (down to 24%) in G0—G1-phase cells. Assays of GUS showed that maximum transformation (> 95% of explants) also occurred after this period. Our data showed that mimosine and colchicine blocked the mesophyll cells at late G1-phase and M-phase, respectively. No transformation (= GUS expression) was observed in phytohormone-treated cells inhibited in late G1 by mimosine. However, after removal of mimosine, 82% of the explants were transformed, indicating the non-toxic and reversible effect of the inhibitor. On the other hand, a relatively high transformation frequency (65% of explants) was observed after blocking the cell cycle at M-phase with colchicine. However, only transient, but no stable, gene expression (= kanamycin-resistant callus formation) was observed in colchicine-treated M-phase-arrested cells. Similarly, endoreduplication of nuclear DNA, which occurred during the 48 h of phytohormone treatment in some mesophyll cells and cells located along the minor veins in the leaf explants, resulted in transient GUS expression only. These observations indicate a direct correlation between endoreduplication and transient GUS gene expression. Obviously, for stable GUS gene expression, cell division and proliferation are required, indicating that both DNA duplication (S-phase) and cell division (M-phase) are strongly related to stable transformation. We propose that the present system should facilitate further dissection of the process of T-DNA integration in the host genome and therefore should aid in developing new strategies for transformation of recalcitrant plants.
ISSN:0032-0935
1432-2048
DOI:10.1007/BF01007700