Isolation and Culture of Pollen Tetrad Protoplasts from Solanum tuberosum

Abstract

Pollen protoplasts provide a sexual and haploid system for haploid production, cell fusion and mutation studies used in plant improvement. Due to the multiploidy, heterozygosity, and often self-incompatibility in tetraploid genotypes, haploid potatoes are desirable for breeding schemes via ploidy manipulations. In this study, two tetraploid varieties and two dihaploid lines of potato were used for pollen tetrad protoplast isolation and culture. The meiotic tetrad buds were first pre-treated at 5 °C for 0–12 days, then the tetrads were transferred into enzyme solutions containing different concentrations of snailase (0.5–1.5%), 0.3 M osmolites (sucrose, mannitol, glucose or sorbitol), 1.0% Cellulose, 0.5% Hemicellulase, 0.5% Pectolyase, 0.3% Sucrose, 3 mM 2-(N-Morpholino) ethane sulfonic acid, 1% polyvinyl pyrrolidone, 0.01% casein hydrolysate and K3 medium compositions. Among the four donor materials, tetraploid cv. Gannongshu No. 3 (‘GNS No.3’) showed the greatest protoplast yield (74.6 ± 2.4%). In this variety, most of the tetrad protoplasts regenerated a cell wall and continued cell divisions were observed when they were inoculated in K3 basic medium supplemented with (0.5–1.0) mg/L 2,4-D + (0.1–0.5) mg/L KT + 0.4 mg/L 6-BA +800 mg/L glutamine +100 mg/L serine. ‘GNS No.3’ also showed the greatest first division frequency (21.6 ± 1.5%) and sustained division to form multicellular structures. The study findings suggested that cultured tetrad pollen protoplasts could reverse the gametophytic developmental pattern programmed in vivo to a sporophytic pathway leading to multicellular microspore-derived colonies.