Protoplast regeneration and fusion in Cucumis: melon × cucumber

Abstract

The aim of this investigation was to develop a protocol to be used in asymmetric protoplast fusions with breeding material of melon and cucumber. Efficient methods for plant regeneration from unfused protoplasts of commercial melon lines were established and are reported. Ploidy levels of explant material and plants, regenerated from protoplasts were analyzed. Electrofusion was carried out between melon protoplasts and irradiated and non-irradiated donor protoplasts of cucumber. Although initial plating efficiencies of heterokaryons were high, development stopped after a few divisions. In control experiments, shoots were regenerated at high frequencies. In only two fusion experiments, development continued to the callus stage, but further development was not observed. When analyzed with PCR using arbitrary primers, the majority of these calli DNA were identical to the melon DNA. However, a few of the examined calli, although being mainly homologous to melon, were observed to have new bands corresponding to bands specific for cucumber. Due to sexual incompatibility, successful hybridization between cucumber and melon was never obtained by sexual crosses. We suggest that the failure to regenerate plants in our fused material could be explained partially by an analogous somatic incompatibility reaction. Abbreviations: BA -benzyl adenine; 2,4-D -2,4-dichlorophenoxyacetic acid; FDA -fluorescein diacetate; FITC fluorescein isothiocyanate; IAA -indoleacetic acid; MS -Murashige & Skoog; NAA -naphtalene acetic acid; PEG -polyethylene glycol; PE -plating efficiency; RAPD -random amplified polymorphic DNA

Melon Biotechnology

“…Since then, there have been few reports of successful regeneration of melon plants from protoplasts (Bokelmann et al, 1990;Li et al. 1990;Debeaujon and Branchard, 1992;Tabeiet aI., 1992;Jarl et aL, 1995). Tabei et al (1992) found that several subcultures of the protoplast-derived shoots in the presence of BAP and GAl allowed the recovery of nonnal plants at high frequency.…”

“…Almost the same conditions have been used for fusion of C. melo protoplasts with a Cuculnis wild species (C. metuliferus and Cucurbita lnartinezii; Roig et al t 1986c). Attempts of symmetrical protoplast fusion between C. melD and C. sativus (Jarl et al, 1995) led to the recovery of a few calli, but no plants. However, molecular analysis by peR of the calli indicated that some of them incorporated a limited amount of the genetic material from cucumber.…”

Biotechnological methods utilized in Cucumis research - A review

“…In Cucumis species, somatic hybridization by protoplast fusion was described between C. sativus and C. melo (electrofusion: Jarl et al 1995), C. metuliferus and C. melo (electrofusion: Debeaujon, Branchard 1990; chemical fusion: Roig et al 1986), C. melo and C. anguria (electrofusion: Dabauza et al 1998), and C. melo and C. myriocarpus (electrofusion: Bordas et al 1998).…”

Transformation efficiencies and progeny analysis after varying different parameters of direct gene transfer of Nicotiana tabacum protoplasts

“…Doses used for nuclear fragmentation and inhibition are, however, usually much higher (e.g. Bates 1990, Jarl et al 1995. One cannot exclude that the stability also of the introduced DNA will be affected on a longer term.…”