Articles
PEG FUSION OF CHAMELAUCIUM UNCINATUM PROTOPLASTS
Article number
829_57
Pages
359 – 364
Language
English
Abstract
Somatic hybridisation techniques were used to overcome the crossing barriers of sexual hybridization between parental species in wax flowers breeding program at Western Australia Department of Agriculture and Food.
As part of this program, the effectiveness of chemical fusion using polyethylene glycol (PEG) was investigated.
Parameters that were optimized included: the molecular weight (MW) of PEG, the ratio between PEG solution and the protoplast suspension in the fusion mix, the density of protoplasts, and the duration of fusion.
The experiments were conducted using protoplasts of a white variety of Chamelaucium uncinatum isolated from cell suspension culture or directly from in vitro leaves and yielded more than 80% viable protoplasts.
Results from the trials showed that increasing the MW of PEG caused a reduction of protoplast recovery after elution and washing steps, but increased the percentage of putative hybrids.
The ratio between PEG solution and the protoplast suspension in the fusion mix and the duration of fusion time slightly improved the percent of putative hybrids, but reduced the percent recovery and plating efficiency.
Higher protoplast-density caused an increase in multiple cell fusion.
The optimal fusion conditions to fuse protoplasts at 3×105 cells/ml were to use 3350 MW PEG, with a 1:1 ratio of PEG solution (50%), and the protoplast suspension in the fusion mix over a 20 min period.
As part of this program, the effectiveness of chemical fusion using polyethylene glycol (PEG) was investigated.
Parameters that were optimized included: the molecular weight (MW) of PEG, the ratio between PEG solution and the protoplast suspension in the fusion mix, the density of protoplasts, and the duration of fusion.
The experiments were conducted using protoplasts of a white variety of Chamelaucium uncinatum isolated from cell suspension culture or directly from in vitro leaves and yielded more than 80% viable protoplasts.
Results from the trials showed that increasing the MW of PEG caused a reduction of protoplast recovery after elution and washing steps, but increased the percentage of putative hybrids.
The ratio between PEG solution and the protoplast suspension in the fusion mix and the duration of fusion time slightly improved the percent of putative hybrids, but reduced the percent recovery and plating efficiency.
Higher protoplast-density caused an increase in multiple cell fusion.
The optimal fusion conditions to fuse protoplasts at 3×105 cells/ml were to use 3350 MW PEG, with a 1:1 ratio of PEG solution (50%), and the protoplast suspension in the fusion mix over a 20 min period.
Authors
K. Ratanasanobon , K.A. Seaton
Keywords
Chamelaucium uncinatum, PEG, protoplast fusion
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