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

• Published in: Planta Vol. 201; no. 2; pp. 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: Journal Article
• Language: English
• Published: Berlin Springer-Verlag 1997; Springer
• 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; β-Glucuronidase; Petunia hybrida; Stability; Genetic transformation; Enzyme; Agrobacterium tumefaciens; Cell division; Transforming DNA; Gene expression; Dicotyledones; Glycosidases; Angiospermae; Cell cycle; Bacteria; Hydrolases; Flower crop; Genetic engineering; Spermatophyta; Developmental stage; Rhizobiaceae; Solanaceae; O-Glycosidases
• ISSN: 0032-0935; 1432-2048
• DOI: 10.1007/BF01007700

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.