Fusion of plant protoplasts: a study using auxotrophic mutants of Nicotiana plumbaginifolia, Viviani

Abstract

Protoplast fusion studies between various auxotrophic mutants of Nicotiana plumbaginifolia were performed to optimize conditions for PEG-mediated fusion and to identify factors influencing the plant protoplast fusion process. Numerous parameters in the isolation, culture, and fusion of protoplasts were tested, and established fusion protocols were compared. Fusion rates, calculated on the basis of colony growth on selection medium (genetic complementation), ranged from 10(-4) to 10(-2). Conditions that allow rapid and reproducible fusions at the highest rates were established. Particular emphasis was given to fusion of mesophyll-derived protoplasts, for which the ability to regenerate fertile plants from fusion products was shown to be particularly high. Preliminary experiments using electric-field mediated fusion suggest that electrofusion may offer significant advantages over the traditional chemical fusion.

Hybrid genes in the analysis of transformation conditions

“…The following basic solutions were used: PEG 6R, PEG 6000 at 24 or 40% in 0.4 M mannitol, 15 mM MgC12 and 0.1% MES, pH 5 . 6 -7 (23), PEG CMS (14) containing PEG 1500, 4000 or 6000 (Merck) at 40% in 0.4 M mannitol and 0.1 M Ca(NO3) 2 • 4H20, pH 7 -9 (filter sterilized, stored at -2 0 °C).…”

Protoplast fusion technology

“…Asymmetric hybridization coupled with efficient in vitro selection systems for disease and stress tolerances may prove to be very powerful genetic manipulation techniques. This test-tube protoplast fusion technique, together with the modifications described elsewhere (4,7,13,14,20), shouM allow researchers to choose and adapt a simple procedure for plant gene transfer.…”

“…PEG may be used in a variety of ways in addition to the test-tube technique described in this report and elsewhere {4). Some of these include the cover slip or petri dish method (13,20), or at the interface of a glucose and sucrose solution (14). Solutions of 15 to 45% (wt/vol) of PEG (MW 1 540 to 6 000) are commonly used in treatment times of 10 to 40 min.…”

“…Solutions of 15 to 45% (wt/vol) of PEG (MW 1 540 to 6 000) are commonly used in treatment times of 10 to 40 min. Modifications to the elution of PEG with culture medium include the use of high calcium solutions an buffers at various pHs (7,20).…”

Asymmetric protoplast fusion between sweet potato and its relatives, and plant regeneration

“…Protoplasts from I. tri$da and I. lacunosa were washed once and re-suspended in W5 solution (Negrutiu et al, 1986), followed by X-ray irradiance in the range of 0 to 20 krad at the rate of 87.6 rad min-'. Protoplasts from the sweet potato cultivars Shirosatsuma and Kanto 101 were treated with IOA-solution ranging from 0.0 to 20.0 mM for 15 min at 4 "C in the dark.…”