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

Abstract

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 uidA 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.

Regeneration and Agrobacterium-mediated transformation of multiple lily cultivars

“…In many species, e.g. in melon (Chovelon et al 2011), a great difference was found between transient expression efficiencies and stable integration frequency. Also in other crops optimal conditions for transient and stable transformation are not always similar (De Bondt et al 1994;Altpeter et al 1996;Sandhu and Gosal 2009).…”

Histología de la regeneración por organogénesis en Paulownia tomentosa (Paulowniaceae)

“…The development of a breeding programme associated with the biotechnological tools depends on the establishment of an efficient in vitro plant regeneration and transformation system (Chovelon et al, 2011). However, the application of such biotechnological approaches has not been possible for P. tomentosa, due to a lack of success in transformation.…”

“…Attempts in our laboratory to transform Paulownia tissues by kanamycin selection have produced transgenic calluses, but have failed to produce regenerated transgenic plants. The major prerequisite for genetic transformation through Agrobacterium tumefaciens is the availability of a reliable plant regeneration system and a suitable method of transformation (Chateau, Sangwan, & Sanhgwan-Norreel, 2000;Chovelon, Restier, Giovinazzo, Dogimont, & Aarrouf, 2011). An understanding of the regeneration process and the identification of the optimal target tissue is essential for the success of genetic engineering techniques.…”

Plant regeneration and Agrobacterium-mediated transformation of Achyranthes bidentata using cotton EREBP gene

“…However, the petiole of Populus sp had strong differentiation potential (Jafari et al 1995). On the basis of totipotency theory, the explants from different parts of plant can differentiate and develop, however the differentiation potential appears to be variable because cell structures, physiological status and levels of endogenous hormones in all kinds of organs also are various (Chovelon et al 2011). Therefore, the explants should be selected to establish the system of plant regeneration and genetic transformation.…”