Formation and Growth of Bryopsis hypnoides Lamouroux Regenerated from Its Protoplasts

Abstract

Tissue culture, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and spectra analysis were used for studying the aggregation mechanism of protoplasts from Bryopsis hypnoides Lamouroux and the discrepancy between the protoplast-regenerated plants and the wild type. The aggregation of protoplasts from B. hypnoides was observed in natural seawater and artificial seawater with different pH values, and the location and mechanism of the materials causing the aggregation were also studied. Results showed that the protoplasts could aggregate into some viable spheres in natural seawater and subsequently grow into mature individuals. Aggregation of the protoplasts depended exclusively upon the pH value (6-11), and the protoplasts aggregated best at pH 8-9. Some of the extruded protoplasts were separated into two parts by centrifugation: the pellet (PO) and the supernatant (PL). The PO could aggregate in artificial seawater (pH 8.3) but not in PL. No aggregation was found in PO cultured in natural seawater containing nigericin, which can dissipate the proton gradients across the membrane. These experiments suggest that the aggregation of protoplasts is proton-gradient dependent and the materials causing the aggregation were not in the vacuolar sap, but located on the surface or inside the organelles. Furthermore, the transfer of the materials across the membrane was similar to ∆pH-based translocation (∆pH/TAT) pathway that occurs in the chloroplasts of higher plants and bacteria. Obvious discrepancies in both the total soluble proteins and the ratio of chlorophyll a to chlorophyll b between the regenerated B. hypnoides and the wild type were found, which may be related to the exchange of genetic material during aggregation of the organelles. In the process of development, diatom Amphora coffeaeformis Agardh attached to the protoplast aggregations, retarding their further development, and once they were removed, the aggregations immediately germinated, which showed that diatoms can affect the development of other algae.

The Bryopsis hypnoides Plastid Genome: Multimeric Forms and Complete Nucleotide Sequence

“…The extruded protoplasm without a cell membrane maintains sufficient viability to regenerate into a mature individual [29]. The regenerated individual can grow up to 58 cm, which is three times longer than the wild thalli [30], suggesting that regenerated alga have an advantage over wild individuals in terms of growth. Furthermore, the Bryopsis chloroplast is the largest organelle in the cell [31]and is thought to play an important role in the protoplast regeneration process [30], thus we inferred that its cpDNA may be special regarding the genome size and coding gene compared with other algae.…”

Photosynthetic performance of regenerated protoplasts from disintegrated cells of Bryopsis hypnoides (Chlorophyta)

“…It is noteworthy that the F v /F m ratio remained relatively constant for all the size groups in the initial phase of aggregation (6 h), however Φ PSII exhibited a slight increase during the same period (Fig. 5B); thus, we suppose that during the early stage of protoplasts regeneration, the disorganized cellular material needed to restructure, or to restore its original function, especially there was a de novo synthesis of cell membranes and walls (Kim et al 2001, Ye et al 2005a, this biological process required a large number of carbohydrates, so this energy requirement may lead to the increased activity of PSII.…”

“…Under optimal conditions about 15% of the developing protoplasts were able to regenerate into mature algal thalli (Ye et al 2005a). Our size-distribution assays presented here showed a relationship between size of regenerated sub-protoplasts and their viability.…”

“…Cell membranes and cell walls subsequently formed around the aggregates after 6 and 12 h of wounding. After 1-2 d of cultivation some of them could germinate and develop into fully differentiated Bryopsis (Pak et al 1991, Kim et al 2001, Ye et al 2005a. Based on these studies on the protoplast regeneration of Bryopsis, we focused on the early stage of protoplasts regeneration (before protoplast germination), and chose four representative stages (1, 6, 12, and 24 h after onset of cultivation) to carry out our investigations.…”

“…However, Bryopsis sp., which is a multinucleate cell organism, when cut into several segments that, while only a part of one cell, can each develop into mature individuals. This distinctive regeneration phenomenon has provided an excellent system for the study of protoplast regeneration (Gibor 1965, Tatewaki and Nagata 1970, Kobayashi and Kanaizuka 1985, Menzel 1988, Pak et al 1991, Kim et al 2001, Ye et al 2005a.…”