High frequency of fusion induced in freely suspended protoplast mixtures by polyethylene glycol and dimethylsulfoxide at high pH

Abstract

Fusion of freely suspended protoplast mixtures (hypocotyl protoplasts of Brassica napus mixed with mesophyll protoplasts of either B. campestris or Nicotiana plumbaginifolia) was induced by a solution containing 10% polyethylene glycol, 10% dimethyl-sulfoxide and 0.1M glycine-NaOH buffer (pH 10.0). The fusion products represented 15 to 17 percent of the surviving cells. More than 50% of the fusion products divided within two days after fusion, indicating that the fusion procedure did not significantly affect the viability of fused cells. The fusion products were not bound to the surface of the fusion vessel, so they could be isolated with a micropipette immediately after fusion.

Studies of protoplast culture and plant regeneration from commercial and rapid-cyclingBrassica species

“…After 4 to 5 weeks of growth approximately 1 g of leaf material was excised and placed in 10ml of W5 solution [17](9g1-1 NaCI, 18.375gl -~ CaC12, 0.373 g1-1 KC1, 0.901 gl -I glucose, pH 5.6) in a 12cm glass Petri dish, and scored on one surface with a six-bladed scalpel. After incubation for 30 min at room temperature the W5 was replaced with 20 ml of an enzyme solution consisting of MS basal medium supplemented with 4gl -t cellulase R10, 2 gl-~ pectolyase Y23 (both from Unwin and Co. Ltd., Welwyn, UK) and 140gl -~ mannitol at pH 5.6.…”

Transformation in Oilseed Rape (Brassica napus L.)

“…Regenerants containing these genes are at present in the course of vernalization and will be examined for increased protection against attack by pathogens. (Menczel and Wolfe 1984) 125 mM CaCl z , 155 mM NaCl, 5 mM KCl, 5 mM glucose, pH 5.6. Overnight culture: Single colonies of A. tumefaciens strains were transferred to 3 ml YEB medium composed of yeast extract 1 gIl, beef extract 5 gIl, peptone 5 gIl, MgS04 x 7H zO 0.05 gil, sucrose 5 gil, pH 7.2.…”

Study of factors affecting the culture of Brassica napus L. and B. juncea Coss. mesophyll protoplasts

“…200 g, 3 min) and the supernatant discarded. The protoplasts were washed by resuspending the pellet in a modified W5 wash solution [15], consisting of 100mM CaCI2.2H20, 124mM NaCI, 4mM KC1, 3mM MES, and 4mM glucose (pH 5.6). Then, the protoplast suspension was repelleted and the supernatant discarded.…”