Abstract
Both obtaining high-yielding, viable protoplasts and following reliable regeneration protocols are prerequisites for the continuous expansion and development of newly emerging systems involving protoplast utilization. This study determines an efficient process from protoplast isolation to shoot regeneration in vitro. The maximum yield of protoplast extraction, which was 6.36 ± 0.51 × 106 protoplasts/g fresh weight (FW), was approximately 3.7 times higher than that previously reported for potato protoplasts. To obtain data, wounded leaves were used by partially cutting both sides of the midrib, and isolated protoplasts were purified by the sucrose cushion method, with a sucrose concentration of 20%. We confirmed a significant effect on the extraction efficiency by measuring enzymolysis during a 6 h period, with three times more washing buffer than the amount normally used. Protoplasts fixed in alginate lenses with appropriate space were successfully recovered and developed into microcalli 2 weeks after culture. In addition, to induce high efficiency regeneration from protoplasts, calli in which greening occurred for 6 weeks were induced to develop shoots in regeneration medium solidified by Gelrite, and they presented a high regeneration efficiency of 86.24 ± 11.76%.
Highlights
Since 1975 [1], potato protoplast cultures have been studied by researchers worldwide to establish efficient and reproducible methods for shoot regeneration
The results reveal that material preparation, enzymolysis conditions, alginate fixation, and medium and culture periods for callus greening affect regeneration efficiency, and fine tuning these factors can lead to time savings and easy plant regeneration
The efficiency of protoplast extraction was 4.45 ± 1.23 × 105 protoplasts/g fresh weight (FW), and this result was the lowest compared to the results of the other two methods (Table 1)
Summary
Since 1975 [1], potato protoplast cultures have been studied by researchers worldwide to establish efficient and reproducible methods for shoot regeneration. Varietal improvements, such as non-browning, heat tolerance, increased resistance, improved starch content and yield, and increased nutrient and antioxidant contents in potato, have been achieved through somaclonal variation [6,7,8,9,10]. Another significant field in which protoplasts are used is the development of novel genotypes via somatic fusions. Protoplasts of Lycopersicon pennellii Corr., a wild relative of tomato, were electrofused with those of a dihaploid potato clone
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