Histological study of organogenesis in Cucumis melo L. after genetic transformation: why is it difficult to obtain transgenic plants?

• Published in: Plant cell reports Vol. 30; no. 11; pp. 2001 - 2011
• Main Authors: Chovelon, V., Restier, V., Giovinazzo, N., Dogimont, C., Aarrouf, J.
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer-Verlag 01.11.2011; Springer; Springer Nature B.V; Springer Verlag
• Subjects: Agrobacterium; Agrobacterium - drug effects; Agrobacterium - physiology; Biological and medical sciences; Biomedical and Life Sciences; Biotechnology; Cell Biology; Cell culture; Cotyledons; Cucumis melo; Cucumis melo - anatomy & histology; Cucumis melo - cytology; Cucumis melo - embryology; Cucumis melo - genetics; Culture Media - pharmacology; Explants; Fundamental and applied biological sciences. Psychology; Genotype; Genotypes; Glucuronidase - metabolism; Infection; Life Sciences; Organogenesis; Organogenesis - drug effects; Organogenesis - genetics; Original Paper; Plant Biochemistry; Plant Sciences; Plants, Genetically Modified; Regeneration - drug effects; Regeneration - genetics; Reporter gene; Transformation; Transformation, Genetic - drug effects; Transgenic plants; Vegetal Biology; Genetic transformation; A genes; L; and; Vegetals; Cucurbitaceae; Agrobacterium tumefaciens; Histology; Organogenesis; Dicotyledones; Angiospermae; NptII and uidA genes; Bacteria; Cucumis melo L; Spermatophyta; Transgenic plant; Rhizobiaceae; Cucumis melo
• ISSN: 0721-7714; 1432-203X
• DOI: 10.1007/s00299-011-1108-9

Melon ( Cucumis melo L.) is widely considered as a recalcitrant species for genetic transformation. In this study, we developed different regeneration and transformation protocols and we examined the regeneration process at different steps by histological studies. The highest regeneration rate (1.13 ± 0.02 plants per explant) was obtained using cotyledon explants of the ‘Védrantais’ genotype on Murashige and Skoog (MS) medium supplemented with 0.2 mg/l 6-benzylaminopurine (BAP) and 0.2 mg/l dimethylallylaminopurine (2-iP). Agrobacterium tumefaciens -mediated transformations with the uid A reporter gene were realized on cotyledon explants cultivated in these conditions: 70–90% of explants expressed a transient GUS activity during the early stages of regeneration, however, only few transgenic plants were obtained (1.8–4.5% of stable transformation with the GV2260pBI101 strain). These results revealed a low capacity of melon GUS-positive cells to regenerate transgenic plants. To evaluate the influence of the Agrobacterium infection on plant regeneration, histological analyses were conducted on explants 2, 7, 15, and 28 days after co-culture with the GV2260pBI101 strain. Genetic transformation occurred in epidermal and sub-epidermal cells and reached the meristematic structures expressing a high level of GUS activity during 14 days of culture; but after this period, most of the meristematic structures showed premature cell vacuolization and disorganization. This disruption of the GUS-positive meristematic areas could be responsible of the difficulties encountered to regenerate melon plants after genetic transformation.