Establishment of cell suspension culture protocol for a seagrass (Halodule pinifolia): Growth kinetics and histomorphological characterization

• Published in: Aquatic botany Vol. 117; pp. 33 - 40
• Main Authors: Subhashini, Pon, Raja, S., Thangaradjou, T.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01.07.2014; Elsevier
• Subjects: Animal and plant ecology; Animal, plant and microbial ecology; Autoecology; Biological and medical sciences; Cell suspension culture; Fundamental and applied biological sciences. Psychology; Growth kinetics; Halodule pinifolia; Histomorphology; Marine; Plants and fungi; Scanning electron microscopy; Seagrass; Scanning electron microscopy; Seagrass; Histomorphology; Cell suspension culture; Growth kinetics; Cell culture; Monocotyledones; Growth; Zannichelliaceae; Characterization; Suspension culture; Angiospermae; Spermatophyta; Kinetics; Sea grass; Aquatic plant
• ISSN: 0304-3770; 1879-1522
• DOI: 10.1016/j.aquabot.2014.04.005

•Cell suspension culture protocol for seagrass was established for the first time.•Growth kinetics of the suspension culture was studied and standardized.•Granular to milky form of cell suspension was achieved consistently.•This protocol ensures continuous and reliable supply of seagrass cells. Seagrasses are known for their secondary metabolites of great commercial value and this study provides the first report on the establishment of cell suspension culture for a seagrass. Basal medium supplemented with 3% sucrose, 2,4-dichlorophenoxy acetic acid (2,4-D – 2.0mgl−1) and N6-benzylaminopurine (BAP – 1.0mgl−1) was used for callus induction, and same medium with 2,4-D (2.5mgl−1) was used for establishing cell suspension cultures of Halodule pinifolia. A growth curve was observed with a lag phase until day 12, a log phase from day 12 to day 24, and a stationary phase from day 24 to day 36 with an average absolute cell growth rate of the suspension culture of 0.425biomassd−1 and a doubling time (td) of 51h. The growth curve was derived with a Michaelis constant, Km (half the time required to attain the maximum cell cycle frequency) of 1.7597h−1 and a Vmax (maximum cell cycle frequency) of 0.0016h−1, and the relationship between the cell growth and substrate hydrolysis was significant (r2=0.9206). The histomorphological characterization of the established cell culture revealed different stages of the suspension of cells from a granular form of the suspension to a fine milky form. Consistent reproducibility of the growth pattern was observed in the suspension culture over one year so that this technique promises to be a step forward to the in vitro production of potential natural products from seagrass, since it is more convenient for large scale production in bioreactors.