A More Accessible, Time-Saving, and Efficient Method for In Vitro Plant Regeneration from Potato Protoplasts

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%.

Invited Mini-Review Research Topic: Utilization of Protoplasts to Facilitate Gene Editing in Plants: Schemes for In Vitro Shoot Regeneration From Tissues and Protoplasts of Potato and Rapeseed: Implications of Bioengineering Such as Gene Editing of Broad-Leaved Plants

“…Here, we present and comment on a successful regenerating protocol from gene-edited potato protoplasts to shoots (Figure 3), mainly based on our method published by Nicolia et al (2015) and with recent adjustments as described in Moon et al, 2021. 1. Maximum yield of protoplasts has been achieved using leaflets from 4-week-old in vitro material by cutting both sides from the midrib (using leaf strips with midrib), thus reducing wounding to a minimum (Moon et al, 2021). Finely cut leaf slices are incubated in medium C containing 1% (w/ v) cellulase and 0.5% (w/v) Macerozyme for 18 h in complete darkness (Moon et al, 2021).…”

“…Here, we present and comment on a successful regenerating protocol from gene-edited potato protoplasts to shoots ( Figure 3 ), mainly based on our method published by Nicolia et al (2015) and with recent adjustments as described in Moon et al, 2021 . 1.…”

“…Maximum yield of protoplasts has been achieved using leaflets from 4-week-old in vitro material by cutting both sides from the midrib (using leaf strips with midrib), thus reducing wounding to a minimum ( Moon et al, 2021 ). Finely cut leaf slices are incubated in medium C containing 1% (w/v) cellulase and 0.5% (w/v) Macerozyme for 18 h in complete darkness ( Moon et al, 2021 ). Note: Gentle shaking at 40 rpm in the darkness can increase the protoplast yield substantially.…”

“… 3. The solution containing released protoplasts is gently aspirated with a pipette and sieved through the filters, the remaining protoplasts are washed from the filters using 30 ml of wash solution (i.e., 3x more washing buffer than the amount normally used increased the yield ( Moon et al, 2021 ). 4.…”

“…In general, the regeneration capacity of explants from juvenile plants is much greater than mature plants due to the ability of responsiveness to plant growth regulators (PGR) ( Bidabadi and Jain, 2020 ). Therefore, the young leaves are mostly used for the protoplast extraction of potatoes ( Moon et al, 2021 ). Moreover, the plant lines that have been maintained for an extended period in vitro tend to confer a reduced vitality as they acclimatize to in vitro conditions.…”