Apical callus formation and plant regeneration controlled by plant growth regulators on axenic culture of the red alga Gracilariopsis tenuifrons (Gracilariales, Rhodophyta)

Abstract

SUMMARY Axenic cultures of Gracilariopsis tenuifrons (Bird et Oliveira) Fredericq et Hommersand (Gracilariales, Rhodophyta) were established in ASP12-NTA solid medium (0.4% agar and 1.0% sucrose) supplemented with plant growth regulators to evaluate the effects on apical callus formation and plant regeneration. Indole-3-acetic acid (IAA), 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BA) were added individually or in combinations (IAA : BA) over a range of concentrations from 0.5 to 5 mg L−1. Growth of apical and intercalary segments was stimulated by high concentrations of 2,4-D (5 mg L−1) and a high IAA to BA ratio (IAA : BA = 5:1 mg L−1) respectively. Apical calluses were originated from divisions of apical and cortical cells located at apical regions of thallus segments and lateral branches. Low concentration of IAA (0.5 mg L−1) or a high IAA to BA ratio (IAA : BA = 5:1 mg L−1) were the optimal treatments for inducing apical callus formation in apical segments, while high concentration of IAA (5 mg L−1) stimulated the highest callus induction rate in intercalary segments. Conversely, equal parts IAA and BA (IAA : BA = 1:1 mg L−1) and low concentration of 2,4-D (0.5 mg L−1) stimulated growth of apical calluses from apical and intercalary segments, respectively. Two processes of regeneration were observed: direct regeneration (upright axis originated from cells of proximal region of intercalary segments) and indirect regeneration (adventitious plantlet originated from cells of apical calluses). Direct regeneration was promoted significantly by treatment with a low IAA to BA ratio (IAA : BA= 1:5 mg L−1), and treatments with IAA (0.5 mgL−1) or 2,4-D (0.5 or 5 mg L−1) significantly stimulated the elongation of upright axis. Plant growth regulators are essential to inducing indirect regeneration, and a high concentration of IAA (5 mg L−1) and BA (5 mg L−1) were the optimal treatments for inducing the regeneration of plantlets from apical calluses in apical and intercalary segments, respectively. Regenerating plantlets grew into plants morphologically similar to those formed from germinating spores, and became fertile after 6 weeks. The results suggest that auxins and cytokinins are involved in developmental regulatory processes in G. tenuifrons. The regeneration process from calluses in species of Gracilariales was observed for the first time in the present study. The culture system described for G. tenuifrons could be useful for micropropagation and for biotechnological applications in agarophytic algae.