Isolation of protoplasts from tissue fragments of Philippine cultivars of Kappaphycus alvarezii (Solieriaceae, Rhodophyta)

Abstract

Protoplasts were isolated from tissue fragments (<1 mm²) of three Philippine cultivars of Kappaphycus alvarezii: the giant cultivar, cultivar L and Bohol wild type, by enzymatic dissolution of cell walls. Yields of viable protoplasts from young and old thalli (apical, middle, basal segments) were compared at various temperatures, duration of treatment and pH using eight combinations of commercial enzymes (abalone acetone powder and cellulase), and prepared extracts from fresh viscera of abalone (Haliotis asinina) and a terrestrial garden snail. Isolated protoplasts were grown in various culture media, temperatures, photoperiods and irradiance values to determine the conditions that favor germination and growth.

Protoplast yields in tissues treated with commercial enzymes and the garden snail extract were lower than those obtained in tissues treated with fresh abalone extracts. Generally, the number of viable protoplasts increased with duration of enzyme treatment at 25 °C with a maximum yield of 8.2 × 10³ g⁻¹ tissue at 48 h. Yields were consistently higher in all cultivars at pH 6.1. The yields were also high from the middle segments of the giant cultivar (3.7 × 10³ g⁻¹ tissue) and Bohol wild type (4.5 × 10³ g⁻¹ tissue) treated with fresh abalone extract, and from basal segments of cultivar L and tissues treated with garden snail extract. The germination rate of protoplasts was highest (39.8%) at 25 °C and 20 μmol photon m⁻² s⁻¹, using a 12:12 light dark photoperiod. The filament was 3.7 mm long by Day 5. These findings are relevant to developing cultures from protoplasts for genetic or strain improvement of K. alvarezii cultivars.