Abstract
The microscopic gametophytic phase of the commercial brown alga, Undaria pinnatifida, can be used for several applications, including the production of bioactive compounds, aquaculture and as germplasm bank. Therefore, gametophytes are good candidates for cellular biotechnology techniques, many of which rely on protoplasts (“naked” living plant cells). This study reports on the optimization of protoplast yield from male and female gametophytes of U. pinnatifida using different mixtures of commercial enzymes and chelation pre-treatment. Key conditions for achieving the highest protoplast yield, such as enzyme combinations, chelation pre-treatment, growth, temperature, incubation time, pH and osmolarity, were investigated. Protoplast isolation conditions were modelled by using response surface methodology (RSM) via Box-Behnken design (BBD) and subsequently experimentally verified in its predictability of protoplast production. The enzyme composition with 1% cellulase RS, 2 U mL−1 alginate lyase and 1% driselase with chelation pre-treatment, at 2481–2591 mOsm L−1 H2O and adjusted to pH 5.8–6.1, produced the highest protoplast yields of 3.12 ± 0.51 × 106 protoplasts g−1 fresh weight for male gametophyte and 2.11 ± 0.08 × 106 for female gametophyte when incubated at 20 °C for 4–7 h using cultures at mid or early exponential phase, respectively. These conditions also gave high amounts of protoplasts from other strains of Korea. Our results show the effectiveness of commercial enzymes combined with chelation pre-treatment in protoplast isolation and RSM with BBD is a useful method for rapidly producing the higher yields of protoplasts from brown alga.
Similar content being viewed by others
References
Abràmoff MD, Magalhães PJ, Ram SJ (2004) Image processing with ImageJ. Biophoton Int 11:36–42
Anderson MJ, Whitcomb PJ (2017) RSM simplified: optimizing processes using response surface methods for design of experiments, 2nd edn. Productivity Press, New York
Baweja P, Sahoo D, García-Jimenez P, Robaina RR (2009) Seaweed tissue culture as applied to biotechnology: problems, achievements and prospects. Phycol Res 57:45–58
Benet H, Ar Gall E, Asensi A, Kloareg B (1997) Protoplast regeneration from gametophytes and sporophytes of some species in the order Laminariales (Phaeophyceae). Protoplasma 199:39–48
Bhojwani SS, Razdan MK (1996) Plant tissue culture: theory and practice, a revised edition. Elsevier, Amsterdam
Björk M, Ekman P, Wallin A, Pedersén M (1990) Effects of growth rate and other factors on protoplast yield from four species of Gracilaria (Rhodophyta). Bot Mar 33:433–439
Björk M, Gómez-Pinchetti JL, García-Reina G, Pedersén M (1992) Protoplast isolation from Ulva rigida (Chlorophyta). Br Phycol J 27:401–407
Burns A, Oliveira L, Bisalputra T (1982a) A morphological study of bud initiation in the brown alga Sphacelaria furcigera. New Phytol 92:309–325
Burns A, Oliveira L, Bisalputra T (1982b) A histochemical study of bud initiation in the brown alga Sphacelaria furcigera. New Phytol 92:297–307
Burns AR, Oliveira L, Bisalputra T (1984) A cytochemical study of cell wall differentiation during bud initiation in the brown alga Sphacelaria furcigera. Bot Mar 27:45–54
Burris KP, Dlugosz EM, Collins AG, Stewart CN, Lenaghan SC (2016) Development of a rapid, low-cost protoplast transfection system for switchgrass (Panicum virgatum L.). Plant Cell Rep 35:693–704
Butler DM, Ostgaard K, Boyen C, Evans LV, Jensen A, Kloareg B (1989) Isolation conditions for high yields of protoplasts from Laminaria saccharina and L. digitata (Phaeophyceae). J Exp Bot 40:1237–1246
Choi HG, Kim YS, Lee SJ, Park EJ, Nam KW (2005) Effects of daylength, irradiance and settlement density on the growth and reproduction of Undaria pinnatifida gametophytes. J Appl Phycol 17:423–430
Cocking EC (1960) A method for isolation of plant protoplasts and vacuoles. Nature 187:927–929
Coelho SM, Scornet D, Rousvoal S, Peters N, Dartevelle L, Peters AF, Cock JM (2012) Isolation and regeneration of protoplast from Ectocarpus. Cold Spring Harbor Protoc 2012:361–364
Cronshaw J, Myers A, Preston RD (1958) A chemical and physical investigation of the cell walls of some marine algae. Biochim Biophys Acta 27:89–103
Dawson PL, Martinez-Dawson R (1998) Using response surface analysis to optimize the quality of ultrapasteurized liquid whole egg. Poult Sci 77:468–474
Destombe C, Oppliger V (2011) Male gametophyte fragmentation in Laminaria digitata: a life history strategy to enhance reproductive success. Cah Biol Mar 52:1–9
Dwiranti F, Hiraoka M, Taguchi T, Konishi Y, Tominaga M, Tominaga A (2012) Effects of gametophytes of Ecklonia kurome on the levels of glucose and triacylglycerol in db/db, prediabetic C57BL/6J and IFN-γ KO mice. Int J Biomed Sci 8:64–75
Field A (2009) Discovering statistics using SPSS, 3rd edn. Sage, Los Angeles
Formo K, Aarstad OA, Skjåk-Bræk G, Strand BL (2014) Lyase-catalyzed degradation of alginate in the gelled state: effect of gelling ions and lyase specificity. Carbohydr Polym 110:100–106
Gao J, Zhang Y, Wang H, Qin S (2005) Suspension culture of gametophytes of transgenic kelp in a photobioreactor. Biotechnol Lett 27:1025–1028
Gómez Pinchetti JL, Björk M, Pedersén M, García Reina G (1993) Factors affecting protoplast yield of the carrageenophyte Solieria filiformis (Gigartinales, Rhodophyta). Plant Cell Rep 12:541–545
Gupta V, Kumar M, Kumari P, Reddy CRK, Jha B (2011) Optimization of protoplast yields from the red algae Gracilaria dura (C. Agardh) J. Agardh and G. verrucosa (Huds.) Papenfuss. J Appl Phycol 23:209–218
Huang L, Zhou J, Li X, Peng Q, Lu H, Du Y (2013) Characterization of a new alginate lyase from newly isolated Flavobacterium sp. S20. J Ind Microbiol Biotechnol 40:113–122
Huddy SM, Meyers AE, Vernon EC (2013) Protoplast isolation optimization and regeneration of cell wall in Gracilaria gracilis (Gracilariales, Rhodophyta). J Appl Phycol 25:433–443
Inoue A, Mashino C, Kodama T, Ojima T (2011) Protoplast preparation from Laminaria japonica with recombinant alginate lyase and cellulase. Mar Biotechnol 13:256–263
Kloareg B, Polne-Fuller M, Gibor A (1989) Mass production of viable protoplasts from Macrocystis pyrifera. Plant Sci 62:105–112
Kloareg B, Quatrano RS (1988) Structure of the cell walls of marine algae and ecophysiological functions of the matrix polysaccharides. Oceanogr Mar Biol Annu Rev 26:259–315
Krishnaiah D, Bono A, Sarbatly R, Nithyanandam R, Anisuzzaman SM (2015) Optimisation of spray drying operation conditions of Morinda citrifolia L. fruit extract using response surface methodology. J King Saud Univ Eng Sci 27:26–36
Kuwano K, Kono S, Jo Y-H, Shin J-A, Saga N (2004) Cryopreservation of the gametophytic cells of Laminariales (Phaeophyta) in liquid nitrogen. J Phycol 40:606–610
Lakens D (2013) Calculating and reporting effect sizes to facilitate cumulative science: a practical primer for t-tests and ANOVA. Front Psychol 4:863
Lee W, Marszalek PE, Zeng X, Yang W (2012) Mechanics of polysaccharides. In: Duwez AS, Willet N (eds) Molecular manipulation with atomic force microscopy. CRC Press, Boca Raton, pp 83–128
Matsumura W, Yasuj H, Yamamoto H (2001) Successful sporophyte regeneration from protoplasts of Undaria pinnatifida (Laminariales, Phaeophyceae). Phycologia 40:10–20
Mejjad M, Loiseaux-de-Goër S, Ducreux G (1992) Protoplast isolation, development, and regeneration in different strains of Pilayella littoralis (L.) Kjellm. (Phaeophyceae). Protoplasma 169:42–48
Mori K, Ooi T, Hiraoka M, Oka N, Hamada H, Tamura M, Kusumi T (2004) Fucoxanthin and its metabolites in edible brown algae cultivated in deep seawater. Mar Drugs 2:63–72
Morita T, Kurashima A, Maegawa M (2003) Temperature requirements for the growth and maturation of the gametophytes of Undaria pinnatifida and U. undarioides (Laminariales, Phaeophyceae). Phycol Res 51:154–160
Mussio I, Rusig A-M (2006) Isolation of protoplasts from Fucus serratus and F. vesiculosus (Fucales, Phaeophyceae): factors affecting protoplast yield. J Appl Phycol 18:733–740
Myers RH, Montgomery DC, Anderson-Cook CM (2009) Response surface methodology: process and product optimization using designed experiments, 3rd edn. Wiley, Hoboken
Nakagawa H, Tanaka H, Oba T, Ogura N, Iizuka M (1985) Callus formation from protoplasts of cultured Spinacia oleracea cells. Plant Cell Rep 4:148–150
Park E, Cho M, Ki C-S (2009) Correct use of repeated measures analysis of variance. Korean J Lab Med 29:1–9
R Core Team (2016). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. https://www.R-project.org/. Accessed 9 Nov 2016
Rezazadeh R, Niedz RP (2015) Protoplast isolation and plant regeneration of guava (Psidium guajava L.) using experiments in mixture-amount design. Plant Cell Tissue Organ Cult 122:585–604
Rorrer GL, Modrell J, Zhi C, Yoo H-D, Nagle DN, Gerwick WH (1995) Bioreactor seaweed cell culture for production of bioactive oxylipins. J Appl Phycol 7:187–198
Rorrer GL, Cheney DP (2004) Bioprocess engineering of cell and tissue cultures for marine seaweeds. Aquac Eng 32:11–41
Reddy CRK, Dipakkore S, Kumar GR, Jha B, Cheney DP, Fujita Y (2006) An improved enzyme preparation for rapid mass production of protoplasts as seed stock for aquaculture of macrophytic marine green algae. Aquaculture 260:290–297
Reddy CRK, Gupta MK, Mantri VA, Bhavanath J (2008) Seaweed protoplast: status, biotechnological perspectives and needs. J Appl Phycol 20:619–632
Redmond S, Green L, Yarish C, Kim J, Neefus C (2014) New England seaweed culture handbook-nursery systems. Connecticut Sea Grant CTSG-14-01. Available from: http://seagrant.uconn.edu/publications/aquaculture/handbook.pdf. Accessed Jul 7, 2014
Salmeán AA, Duffieux D, Harholt J, Qin F, Michel G, Czjzek M, Willats WGT, Hervé C (2017) Insoluble (1→3),(1→4)-β-D-glucan is a component of cell walls in brown algae (Phaeophyceae) and is masked by alginates in tissues. Sci Rep 7:2880
Sellke T, Bayarri MJ, Berger JO (2001) Calibration of p values for testing precise null hypotheses. Am Stat 55:62–71
Shahin MM (1972) Relationship between yield of protoplast and growth phase in Saccharomyces. J Bacteriol 110:769–771
SpecialChem—The universal selection source: cosmetics ingredients. Available online: https://cosmetics.specialchem.com/product/i-seppic-ephemer. Accessed on: 6 December 2018
Strauss A, Potrykus I (1980) Callus formation from protoplasts of cell suspension cultures of Rosa ‘Paul’s Scarlet’. Physiol Plant 48:15–20
Sullivan GM, Feinn R (2012) Using effect size-or why the P value is not enough. J Grad Med Educ 4:279–282
Suzuki Y, Kuma K, Kudo I, Matsunaga K (1995) Iron requirement of the brown macro algae Laminaria japonica, Undaria pinnatifida (Phaeophyta) and the crustose coralline alga Lithophyllum yessoense (Rhodophyta), and their competition in the northern Japan Sea. Phycologia 34:201–205
Thayer SS (1985) Protoplasts for compartmentation studies. In: Linskens HF, Jackson JF (eds) Cell components. Springer, Berlin, pp 54–65
Thibault J-F, Rouau X (1990) Studies on enzymic hydrolysis of polysaccharides in sugar beet pulp. Carbohydr Polym 13:1–16
Varvarigos V, Katsaros C, Galatis B (2004) Radial F-actin configurations are involved in polarization during protoplast germination and thallus branching of Macrocystis pyrifera. Phycologia 43:693–702
Wang B, Zhang E, Gu Y, Ning S, Wang Q, Zhou J (2011) Cryopreservation of brown algae gametophytes of Undaria pinnatifida by encapsulation-vitrification. Aquaculture 317:89–93
Wang L, Park Y-J, Jeon Y-J, Ryu BM (2018) Bioactivities of the edible brown seaweed, Undaria pinnatifida: a review. Aquaculture 495:873–880
Wei X, Song X, Dong D, Keyhani NO, Lao L, Zang X, Dong L, Gu Z, Fu D, Liu X, Qiu J, Guan X (2016) Efficient production of Aschersonia placenta protoplasts for transformation using optimization algorithms. Can J Microbiol 62:579–587
Wu S (1988) Isolation and culture of protoplasts from Undaria pinnatifida (Harv.) Suringar. J Ocean Univ China 18:57–65
Wu C, Li D, Liu H, Peng G, Liu J (2004) Mass culture of Undaria gametophyte clones and their use in sporeling culture. Hydrobiologia 512:153–156
Xiaoke H, Xiaolu J, Huashi G (2003) Isolation of protoplast from Undaria pinnatifida by alginate lyase digestion. J Ocean Univ China 2:58–61
Xu Z, Li D, Hu H, Tianwei T (2005) Growth promotion of vegetative gametophytes of Undaria pinnatifida by blue light. Biotechnol Lett 27:1467–1475
Yamanaka R, Akiyama K (1993) Cultivation and utilization of Undaria pinnatifida (wakame) as food. J Appl Physiol 5:249–253
Yetilmezsoy K, Demirel S, Vanderbei RJ (2009) Response surface modeling of Pb (II) removal from aqueous solution by Pistacia vera L.: Box–Behnken experimental design. J Hazard Mater 171:551–562
Zha X, Kloareg B (1996) Gametophyte protoplast culture of an edible alga Undaria pinnatifida. J Anhui Agric Univ 23:602–605 (in Chinese)
Funding
This study was supported by a research grant from Chosun University 2019 to T.O. Cho.
Author information
Authors and Affiliations
Corresponding author
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Avila-Peltroche, J., Won, B.Y. & Cho, T.O. Optimization of protoplast isolation from the gametophytes of brown alga Undaria pinnatifida using response surface methodology. J Appl Phycol 32, 2233–2244 (2020). https://doi.org/10.1007/s10811-020-02095-3
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10811-020-02095-3