Bjork M, Pedersen M. 1991. In: Proceedings of COST 48 - Working seminar, "Recent Advances in Seaweed Cellular Biotechnology, Physiology and Intensive Cultivation': University of Las Palmas. Eds. G. Garcia-Reina and M, Pedersen. pp.283-286.

Procedures for Protoplast Isolation from Red Seaweeds.

Demonstration on Gracilaria sordida and Gracilaria tenuistipitata.

Mats Bjork and Marianne Pedersen, Department of Physiological Botany. Uppsala Universiry, Sweden.

This procedure consists of four main steps. The precultivation of the plant material, the pretreatment of the tissue, the incubation of the tissue in an enzyme solution, and finally the purification of the protoplasts. All steps have influence on the final yield and viability of the protoplasts. To prevent contamination all work should be done in a laminar flow hood. Sterilize all tools, andfilter all solutions through a 0,22 jJ.m sterile filter before use. Use standard axenic procedures.

By this method a protoplast yield of 1x1a6 to 1x107 per gram fresh weight has been obtained. These protoplasts were viable and divided up to three times while in culture. (Bjork et al. 1990)

Precultivation of Plant Material:

• Select clean unfertile plants in good condition, if possible from unialgal cultures.

Note: It is important to choose planttissue thaI does TWt contains spores or epiphytes. In this demonstration afemale gametophyte isolaJedjrom a single tetraspore and cultivaJed in isolaJed unialgal culture is used,

• Use only actively growing plants. A growth rate of 15 to 25 % biomass increase per day is good for G, sordida and 25 to 35 % for G. tenuisti pi tata. If no fast growing cultures are available, use clean growing tips without epiphytes.

Note: For Gracilaria tenuistipitata and G. sordida this is very importam, as the growth rate of the plants just prior to the treatments greatly affects the yield of protoplasts. This

283

1.6

1.0

may be due to cell wall composition being dependant upon the plants growth rate .

Yield per IJ'Bmimiliiona)

.... : .... <

/ /

"

' . 10 10 10

Growth rafII ( .. per day)

Protoplast yieldfrom Gracilaria sordida grown at different growth rales (Bjork et al.1990)

Pretreatment:

1- Transfer O,5g of seaweeds into each sterile 5 cm petridish. Add wash

medium. If wild plants are used, rinse them carefully.

Note: The thalli may also be surface sterilized in a diluted Iodine antiseptic such as Betadine or Jodopax.

2- Cut the thalli in millimeter pieces using a clean new razorblade.

Note: Make clean cuts that damage as few cells as possible. The pieces of thallus should be very small to increase the cUI sUrface area as no protoplasts will be released through the "cuticula".

3- Rinse the seaweeds with the wash medium five times. Transfer the pieces to clean petridishes.

Note: This is to get rid of cell sap and debris from broken cells thal may contain substances harmful to the protoplasts.

4- Add preplasmolysis medium for 30 minutes.

Note: This step makes the cells shrink and helps to prevent bursting of the cells when their cell wall are removed. This results in a higher yield and may protect the cells from toxic substances in the media but the high osmolality might also have negative effects on the viability of the cells.

Other pre treatments have been used on other seaweeds to modify the cell walls in a way that will enhance the degradaJion, such as the addition of chelaJors, or tre~nt with proteases.

Enzyme Incubation:

1- Rinse with the wash medium and add 3 mL of the enzyme solution per dish.

Note: The enzyme mixtures usedfor the degradation of the cell walls also have negative e.ffeas on the viability of the cells. Use as low concentration as possiblefor the shortest possible period.

The osmolality of the enzyme solution is also very important Here the osmolality is 1,3 Osmol, which is optimalfor G. sordida. A higher or lower osmolality will decrease the yield of pro top lasts.

2 - Seal the petridish with parafllm and incubate in dark at 25°C, with mild shaking for 2,5 h for G.

284

tenuistipitata and 4 h for G. sordida.

Note: A higher temperature increases the activity of the enzymes but also the activity of unwanted factors in the enzyme solution such as proteases, and may also in itself

be harmful to the protoplasts. The shaking should be mild but enough to

ensure a constant high level of active enzymes close to the degrading cell walls. Also, some mechanical force will break fragments into smaller pieces thereby accelerating cell wall degradation.

Purification of Protoplasts :

1 - Filter enzyme solution with protoplasts through 100J,tm meshfliter

2 - Rinse with the wash solution

3 - Allow to settle for some time with the filter in solution

4 - Filter through 45J,tm mesh filter Note: Rinsing the partly degraded thalli throughly

for afew minutes with the washmedium increases the yield of protoplasts highly.

5 - Put protoplast suspension on top of centrifugation media 1 and 2.

• ProtopIMta wi.

t----t form. band here

6 - Slow centrifugation « 100 x g) for 15 minutes

Note: It is important to use a "swing-out" rotor to prevent protoplasts to attach to the wall of the tube.

7 - Resuspend the band of protoplast in wash medium

8 - Slow centrifugation again for 15 minutes

9 - Resuspend the pellet of protoplasts in wash medium

10- Slow centrifugation again for 15 minutes

11- Resuspend the pellet of protoplast in wash medium/culture medium.

Note: The cenJrijugalion speed is very important. It should be enough to get the protopiasts down fast but when the protopiasts reach the bottom they will be forced together and evenJually the will start to juse. This is normaly not required and one of the reasons is lower cell viability. Thus, the force and time of centriju galion should be minimal.

Culture of the protoplasts:

Protoplasts can be kept in the wash medium for some days if the temperature is maintained at about SoC.

The best results in culture are obtained if a "full" media is used, e.g. MS medium based on seawater with the protoplasts embedded in drops of a high quality agarose. The protoplasts start to regenerate a cell wall in 6-4S h. This is seen by a change in their shape from spherical to elliptical or egg shaped. During this time the culture medium should include 0,2 M mannitol or glucose to stabilize the protoplasts. When cell their walls are regenerated osmolality can be lowered to the level of seawater.

Solutions and Media.

Wash medium: 0,2 M mannitol in seawater, 20 mM Bis-Tris buffer, pH 7. Osmolality 1,1 Osmol

Preplasmolysis medium: O,S M mannitol in seawater, 20 mM Bis-Tris buffer, pH 7. Osmolality 1,7 Osmol.

285

Enzyme Solution: 0,01 % Agarase (Sigma), 2 % Cellulysin (Calbiochem), 0,4 M mannitol in seawater, 20 mM Bis­Tris buffer, pH 5,8. Osmolality 1,3 Osmol.

Centrifugation medium 1: 0,4 M mannitol in seawater, 20 mM Bis-Tris buffer, pH 7.

Centrifugation medium 2: Mix equal parts of percoll (pharmacia) and 1,2 M mannitol

in seawater with 20 mM Bis-Tris buffer, pH 7.

References:

Bjork,M., P. Ekman, A. Wallin, and M. Pedersln. 1990. Effects of growth rate and other

factors on protopiast yieldfromfour species of the red seaweed Gracilaria

(Rhodophyta) Botanica Marina. Vol.33, pp.433-439.

Butler, D., M. K. (Jstgaard, C. Boyen, L.E.Evans, A. Jensen and B. Kloareg. 1989. Isolation Conditions for High Yields of Protoplasts

from Laminaria saccMrina and L.digitata (phaeophyacae).J. Exp. Bot. 40(220): 1237-

1246.

Cheney, D.P., E. Mar, N. Saga and J. van der Meer. 1986. Protoplast isoiaJion and cell

division in the agar producing seaweed Gracilaria (Rhodophyta). J.Phycol 22:238-

243.

Fleck, J., A. Durr, C. Frosch, T. Vernet and L. Birl. 1982. Osmotic-shock "stress proteins" in protopiasts of Nicotiana sylvestris. PlanJ Sci. Lett. 26: 159-165.

Hock, K. and E. Hartman. 1983. Modulation of Fany acid palterns during protoplast isoiaJion. Proc.InJ. Prot. Symp. 6:206-207.

Kaiser, M., G.Kaiser, S. Sconer and S. Neimanis. 1981. Photosynthesis under osmotic stress. PlanJa. 153:430-435.

Seaweed Cellular Biotechnology, Physiology and Intensive Cultivation

Edited by: Guillermo.Garcia Reina & Marianne Pedersen

ISBN 84-604-1766-7 D. L. G. C. 1512-1991

Las Palmas de Gran Canaria-Espana

15 de Noviembre de 1991

No part of the Proceedings may be reproduced or utilized in any form or by any means , without written permission from the authors .

Cover: Morphogenetic callus of Laurencia sp

Seaweed Cellular Biolc~hnology. I'h~'si()logy and Inlensive Cullivation

(;uill,'rnlll j:>1rl:la I(",in~ & MnriallnL' I \:-ri erS('1I

ISBN ~.I.tH).I.17ti6·7 I). I.. (;. (. r ,')1 'l,·19!-Jl

Bjork M, 1991. In: Proceedings of COST 48 - Working seminar, "Recent Advances in Seaweed Cellular Biotechnology, Physiology and Intensive Cultivation': University of Las Palmas. Eds. G. Garcia-Reina and M. Pedersen. pp. 283-286.

Embedding of Protoplasts from Gracilaria tenuistipitata in Agarose

Droplets.

Mats Bjork, Department of Physiological Botany. Uppsala University, Sweden:

Embedding of pro top lasts in different gelling agents has been used in higher plant protoplast research to stabilize the protoplasts and their close environment. The embedding makes the protoplasts easier to handle and increases the viability. In this note a procedure to embed protoplasts from the red seaweed Gracilaria is described.

Procedure:

• Prepare a pure protoplast suspension in culture medium supplied with an osmoticum, eg mannitol to a osmolality of 1,1-1,3 Osmol. (Bjork et al1990)

• Add 1 % agarose of low gelling temperature to culture medium. Heat in microwave oven until agarose is totally dissolved.

• Let agarose medium cool to about 35 °e.

• Mix equal parts of protoplast suspension and agarose medium.

• Rapidly put small drops (about 15 J.'L) on bottom of 5 cm petri dish.

• Let cool further until drops are solid.

• Add culture medium to cover drops.

The protoplasts can now be maintained in the agarose droplets and their development followed in a inverted microscope. After a few days the protoplasts develops a cell wall and the osmotic values of the culture medium can be lowered to the level of seawater.

The major advantage with this method is that it gives possibility to change cultivation medium without stressing the cells. The immediate environment around the protoplasts is protected from fast changes. This is important, e g when the osmolality of the culture medium is lowered after cell wall regeneration.

291

viable cella 100~G-------------------------------------~

10~

1~ ~

0, 1 ~f L.-__ -'----------'-__ ---'-__ '--------'--__ -'----------'-__ --'---__ -'-----'-__ --L--_ o 10 20 30 40 50 60 70 60 90 100 110 120

days Figure 1. Viability of protoplasts in liquid medium (PES) with addition of 0,05 M mannitol and 0,05 M glucose the first 10 days,

viable cell, 100~G_------------------------------------,

o 10~ 0

1~

o

o o

o

o

o

0,1 \II L----'--__ --'---------'-__ --'---__ '--------'-__ --'---------' __ --'---__ -'-----'--'

o 10 20 30 40 SO 60 70 80 '0 100 110 120

days Figure 2. Viability of protoplasts in agarose droplets covered with liquid medium (PES Provasoli 1968) with addition of 0, 05 M mannitol and 0,05 M glucose the first 10 days. Protoplasts regenerated cell walls and started to divide after 2 days, After 110 days 5-10 % of the cells had divided at least one time, afew cells divided up to 3 times.

Ylablo cell. 100\ll~~--~------------~8--------------~

10\11

1\11

0.1 \II L----'---__ -'-------'-__ -'---__ '--------'--__ --'-------'-__ --'---__ -'------'------1

o 10 10 30 40 SO 60 70 80 90 100 110 120

. days Figure 3. Viability of protoplasts in agarose droplets covered with liquid medium "8P" medium (modified according to Glimelius et al1986), excluding honnones and sugars substituted with 0,05 M mannitol and 0,05 M glucose. Cell division was not better than in PES (figure 2).

References:

Bjork,M., P. Ekman, A. Wallin, and M. Pedersin. 1990. Effects of growth rate and other factors on protoplast yieldfromfour species of the red seaweed Gracilaria (Rhodophyta) Botanica Marina. Vol. 33, pp.433-439,

GlimeUus K., Djupsjobacka M. and FeUner-Feldegg. 1986. selection and enrichment of plant protoplast heterokaryones of Brassicaceae. Plant Sci. 45:133-141.

Provasoli, L. 1968. Media and prospects for the cultivation of marine algae. In: (A. Watanabe and A. Hattori, eds.) Cultures and Collections of Algae, (Proc. U.S.-Japan Con!, Hakone) Jpn. Soc. Pl. Physiol. pp. 63-75.

292