Potato Variety Improvement Using Protoplast Fusion

摘要

Experiments are described that were intended to produce asymmetric somatic hybrids after electrofusion of irradiated gamma kanamycin (kan gamma) transgenic Atlantic protoplasts with Russet Burbank protoplasts and culture of the progeny in the presence of concentrations of kanamycin that are lethal to the Russet Burbank protoplasts was determined to be 65 micrograms/ml. The frequency of protoplast electrofusion increased with increasing concentrations of protoplasts, but the optimal concentration for doublet formation was 480,000 protoplasts/ml. Eight independent putative hybrid p-calli were produced after electrofusion of non-irradiated Russet Burbank with gamma-irradiated (2.5 and 10 krad) transgenic kan gamma Atlantic protoplasts. These p-calli were grown in the presence of 65 micrograms/ml kanamycin, under conditions in which neither parental protoplast type formed p-calli. No regeneration was observed. Three procedural modifications, reported to improve plating efficiency of the protoplast isolation protocol, were tested. Increasing the calcium concentration from 6 to 10 mM in the enzyme digestion and rinsing media had no effect. Performing dark pre-treatment of whole plants instead of placing individual leaves in the dark in flotation medium had a detrimental effect. Culture of plants in PROP medium containing 2, 4, and 8 micrograms/ml silver thiosulfate resulted in smaller plants, rather than more vigorous plants.