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Tissue culturing of marine algae

Micropropagule production

Method

  • Minimum explant size, epiphyte-free main axes of four forms were cut into 3, and 5 mm segments.
  • Sterile procedures and growth was monitored over a 4 week period.
  • The most effective antibiotic soak for producing axenic explants was 30 min in polymixin B sulfate (300 mg 100 ml- ') which reduced contamination rates (< 25 %).
  • To insure axenic culture, 1 ml of the stock antibiotic of polymixin B was added to each litre of ESS enriched seawater.
  • Transferred to 3ml wells of Falcon Multiwell Tissue Culture Plates with 2 ml of sterilized seawater enriched with ESS (without PGRs)
  • The multiwell plates were incubated at 25 C, 25 µmol photons m – 2 s-1 and 12:12 h light:dark photoperiod..
  • Factorial combinations (N=3; 0, 1, 5, 10mgl- 1) of auxins (PAA = phenylacetic acid; IBA = indol-3-butryic acid; IAA = indole-3-acetic acid; NAA = -naphthalenacetic acid) and cytokinins (2iP = N6-[-2-isopentenylladenine; BAP = 6-benzylaminopurine; Z = zeatin; K = kinetin) were tested over a 4-week period.

  • Weekly cleaning to remove epiphytes was required for the first 3 to 6 weeks.
  • Cleaning procedures included rinsing under tap or distilled water, brushing with a soft toothbrush under running water, or using an ultrasonic bath (1 megacycles- ') with distilled water for 30 s.

Callus production

  • After arrival, the branches were trimmed to 15 cm placed in Fernbach flasks (up to four 15 cm branches) with 1800 ml 34 ppt salinity autoclaved seawater and left overnight in a growth chamber (25 C, 10 to 12 h photoperiod, 25mol photon m - 2 s- 1).

  • The next day, the branches were gently damp dried with paper towels and rinsed in distilled water.

  • If bacterial contamination was evident (turbid media), the branches were rinsed for 30 s in an antibiotic solution (15 mg erythromycin, 10 mg gentamicin, 30 mg neomycin, 35 mg polymixin B 1- 1) and transferred without rinsing to either Fernbach flasks (15 cm branches, Fig. 1) or 300 ml storage dishes (5 cm branches, Fig. 2). Enriched seawater media were tested using 5cm branches in 300 ml storage dishes.

  • Media included

- A modified form of ESS (Saga, 1986) which contained plant growth regulators (PGR's) and an antibiotic mixture (see Azanza-Corrales & Dawes, 1989 for modifications)

- f/2 medium

- f1 mother liquid.

- SWMD1

- Aquil - artificial seawater

Aeration/mixing

  • Epiphyte-free main axes of were cut into 0.5 cm sections using sterile procedures and the explants
  • Soaked in polymyxin B sulfate (3000 units 1-') for up to 3 h, based on the results from micropropagule production.
  • The segments were shaken gently every 3 to 5 min while in the antibiotic solution.
  • Substrates used were 3%, and 8% agar all dissolved in seawater and ESS enriched.
  • The media was also supplemented with 1 mg 1- NAA (or 0.1and 1 mg 1- of PAA ) and 1 mg 1- 2iP (or BAP) in addition to the base levels of those plant growth regulators in the ESS media (Azanza-Corrales & Dawes, 1989).
  • The segments were placed on the agar substrate so that one of the cut surfaces was exposed to the air.

Mixing is necessary to prevent sedimentation of the algae, to ensure that all cells of the population are equally exposed to the light and nutrients, to avoid thermal stratification (e.g. in outdoor cultures) and to improve gas exchange between the culture medium and the air. The latter is of primary importance as the air contains the carbon source for photosynthesis in the form of carbon dioxide. For very dense cultures, the CO2 originating from the air (containing 0.03% CO2) bubbled through the culture is limiting the algal growth and pure carbon dioxide may be supplemented to the air supply (e.g. at a rate of 1% of the volume of air). CO2 addition furthermore buffers the water against pH changes as a result of the CO2/HCO3- balance. Depending on the scale of the culture system, mixing is achieved by stirring daily by hand (test tubes, Erlenmeyer), aerating (bags, tanks), or using paddle wheels and jet pumps (ponds). However, it should be noted that not all algal species can tolerate vigorous mixing.

Transportation

Light intensity

Requirements vary greatly with the culture depth and the density of the algal culture. At higher depths and cell concentrations the light intensity must be increased to penetrate through the culture (e.g. 1,000 lux is suitable for Erlenmeyer flasks, 5,000-10,000 is required for larger volumes). Light may be natural or supplied by fluorescent tubes. Too high light intensity (e.g. direct sun light, small container close to artificial light) may result in photo-inhibition. Also, overheating due to both natural and artificial illumination should be avoided. Fluorescent tubes emitting either in the blue or the red light spectrum should be preferred as these are the most active portions of the light spectrum for photosynthesis. The duration of artificial illumination should be minimum 18 h of light per day.

  • The plants should be cleaned of epiphytes and debris and rinsed in seawater prior to transportation.
  • packing material – clear filtered (Whatman # 2) seawater

Radiation

Salinity

Optimum growth around 60µmol photon m - 2 s- 1.

Temperature

Species

Open ocean away from land the salt content (or salinity) of the seawater tends to range between 30 and 35 ppt (parts per thousand).

Marine phytoplankton are extremely tolerant to changes in salinity. Most species grow best at a salinity that is slightly lower than that of their native habitat.

Caulerpa- The salinity should not be lower than 30 ppt.

G. edulis - Salinity of 20 to 24 ppt appears to be optimal for growth.

Others - growth effect was better in 25–30.

  • 20–26°C were suitable temperatures for the growth.
  • Higher than 35°C are lethal for a number of species.
  • 16°C will slow down growth.

pH

  • For most cultured algal species is between 7 and 9, with the optimum range being 8.2-8.7.
  • In the case of high-density algal culture, the addition of carbon dioxide allows to correct for increased pH, which may reach limiting values of up to pH 9 during algal growth (with highest growth rates (1.5% d- 1) around 7.5 to 8.0)
  • At lower pH levels the plants showed tip discoloration and dieback suggesting a limitation in CO2 availability

Red Algae : Gracilaria edulis -Vegetative apical cuttings of 3 mm , thallus, spores

Chondrophycus ceylanicus, Gelidiella acerosa, Gracilaria corticata

Harvest branches up to 30 c.m long and3 cm diameter

Green Algae : Caulerpa racemosa - apical branches (reproduces vegetatively by fragmentation. When pieces of the plant get broken off they develop into new plants. Small pieces of tissue only a few millimeters across are capable of doing this)

Chaetomorpha crassa

Brown Algae : Chnoospora minima – medullary tissue, thallus (50mm), clavate structure (55µm)

Sargassum cassifolium

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