Protoplast isolation from Dictyopteris pacifica and Scytosiphon lomentaria, using a simple commercial enzyme preparation

Abstract

Background Protoplasts (i.e., naked plant cells) can be used for in vitro manipulations and genetic improvement in cultivars with economic value. During the last decade, protoplast research in economic brown algae has been scarce, and it is usually hampered by the use of non-commercial enzymes or crude extracts for isolating protoplasts. Dictyopteris pacifica is part of a brown algal genus well known by its wide chemical diversity and biological properties. Scytosiphon lomentaria is an edible brown seaweed with antioxidant, antitumor, and antiviral properties. So far, there are no protoplast isolation protocols using commercial enzymes for these two economic brown algae. In this study, we obtained protoplasts from cultured samples of D. pacifica and S. lomentaria using commercially available enzymes. Additionally, we investigated the effects of Driselase inclusion and Ca-chelation pre-treatment on protoplast yields in order to optimize the conditions for protoplast preparations. Results Protoplasts were isolated from Dictyopteris pacifica and Scytosiphon lomentaria using the commercially available Cellulase Onozuka RS (1%) and Alginate lyase (4 U mL−1), and short incubation time (4 h). Driselase did not show significant effects on protoplast production in both species. Ca-chelation pre-treatment only increased the number of protoplasts in D. pacifica. Under optimal conditions, the protoplast yields from D. pacifica and S. lomentaria were 4.83 ± 2.08 and 74.64 ± 32.49 × 106 protoplasts g−1 fresh weight, respectively. The values obtained for S. lomentaria were 2–3 orders of magnitude higher than previously reported. Conclusions Our results show that high protoplast yields can be obtained from D. pacifica and S. lomentaria using a simple mixture of commercial enzymes (Cellulase RS and Alginate lyase) and short incubation time (4 h). This work also represents the first report of protoplast isolation in D. pacifica. The method proposed here can help to expand protoplast technology in more brown algal species.

Stable overexpression and targeted gene deletion of the causative agent of ash dieback Hymenoscyphus fraxineus

“…The choice of enzymes for protoplasting of filamentous fungi depends on their species and state of growth which has to be tested individually and is therefore a key factor for protoplasting (reviewed in Li et al [ 16 ]). While for some fungi Driselase does not work [ 30 ], it results in the highest number of protoplasts for other fungi [ 31 ]. For the preparation of protoplasts of H. fraxineus , Driselase was also superior in 3 independent experiments in comparison to Lysing Enzymes.…”

Protoplast Preparation for Algal Single-Cell Omics Sequencing

“…Moreover, different algal species need additional enzymes. For instance, green alga Chlorella can be treated with snailase [ 17 ] because it is part of the snail forage; marine algae need alginate lyase [ 15 , 60 ] because their cell wall contains alginate or alginic acid. Similarly, macerase [ 63 ], pronase [ 62 ], and proteinase K [ 62 ] are also needed for some algae.…”

“…Therefore, the disruption of these bonds with chelators such as EDTA or EGTA has proved useful in producing protoplasts [ 54 , 61 ]. EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid) adjusted to appropriate pH (e.g., 6) is mixed with cells, which are rotated gently [ 15 , 61 , 65 ]. Besides the chelator solution, osmotic stabilizers enhance the efficiency of protoplast generation significantly [ 61 ].…”

“…The species of the latter phyla only include a few classes. For example, Rhodophyta only has the classes Bangiophyceae [ 13 , 14 , 15 ], Florideophyceae [ 13 ], and Porphyridiophyceae [ 14 ]. Most studied brown algae are from the same class Phaeophyceae, e.g., Dictyopteris pacifica, Scytosiphon lomentaria [ 15 ], Petalonia fascia [ 9 ], and Undaria pinnatifida [ 16 ].…”

“…For example, Rhodophyta only has the classes Bangiophyceae [ 13 , 14 , 15 ], Florideophyceae [ 13 ], and Porphyridiophyceae [ 14 ]. Most studied brown algae are from the same class Phaeophyceae, e.g., Dictyopteris pacifica, Scytosiphon lomentaria [ 15 ], Petalonia fascia [ 9 ], and Undaria pinnatifida [ 16 ]. In contrast, there are more green algae that have been studied recently, such as Chlorella protothecoides [ 17 ], Ulva prolifera [ 18 ], Haematococcus pluvialis [ 19 ], and Penium margaritaceum [ 20 ].…”