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2. Introduction
• Protoplasts can be isolated from plant tissues or cultured cells
by enzymatic digestion to remove the cell walls.
• The enzymes for this purpose are preparations which are
commercially available.
• The success of protoplast isolation depends especially on the
condition of the tissue and the combination of enzymes being
used.
• Methods and procedures for protoplast isolation from plant
tissues have long been known.
• Recent advances in the isolation, culture, and regeneration of
plants from protoplasts of a wide diversity of species have
been reported.
3. Objectives and Goals :
• To present the technique for isolation of protoplasts at high
yield from stem cortex tissues of Brassica napus (canola) and
an intergeneric hybrid of tomato.
• To present general procedures for the isolation of protoplasts
from cell suspension cultures.
• To provide procedures for protoplast culture, and to observe
cell wall reformation and cell division in such cultures.
4. Equipment and Supplies :
• Autoclave or steam sterilizer (pressure cooker)
• Laminar air flow hood, Canadian Cabinets or equivalent
• Clinical centrifuge, IEC Centra 4B or equivalent, Fisher Scientific
• Gyratory or orbital shaker
• Conical or round bottom tubes, 50 ml glass or plastic
• Inverted microscope, Zeiss 1M or equivalent
• Filtration filter units, Millipore 0.2 f.Lm membranes
• Incubator or growth room, with temperature, light, and humidity controls
• Fine forceps and sterile scalpels
• Erlenmeyer flasks, 125 ml
• Funnels, 55 mm diameter
• Sterile wide-mouth pipettes, glass or plastic
• Pasteur pipettes with medium bore size (Note. Cut standard Pasteur
pipettes to obtain a bore diameter of about 3 mm) .
5. jh
• Hemacytometer
• Sterile petri dishes, 100 X 15mm, 60 X 15mm
• Nylon mesh filters, 64 and 44 f.Lm pore sizes
• Calcium hypochlorite
• Agarose, SeaKem LE or equivalent purity, FMC BioProducts or Sigma Co -
Gelrite or Phytagel, Sigma Co - Macerozyme R-10, Yakult Honsha Co Ltd,
Tokyo, Japan
• Cellulase R-lO, Yakult Honsha Co Ltd, Tokyo, Japan
• Pectolyase, Sigma Co (effective pectinase, not used in the protocols
6. Procedures:
• Enzyme Stock Solutions : Each is prepared by
dissolving in 0.4 M mannitol (72.8 gil) at pH 5.8,
stored frozen at -20 DC:
• Macerozyme R-10, 10% w/v, O.lg in 10ml.
• Cellulase R-10, 10% w/v, 0.1 gin 10ml.
• When needed, the enzyme stocks are diluted to the
desired concentration in 0.4 M mannitol and
sterilized using 0.22 !-tm filters.
• Donor Plant Materials : Procedures are described for
the isolation and culture of stem cortical tissue
protoplasts of Brassica species (Klimaszewska and
Keller 1987) and a Lycopersicon intergeneric hybrid.
7. h
• Growth of Donor Plants of Brassica napus (Canola, Oilseed
Rape) : Seeds of cv. Westar are obtained from the Agriculture
Canada Research Station, Saskatoon, SK, Canada.
• Protoplasts are produced in good yields from stem cortex
tissues of this genotype. The protoplast isolation, culture, and
plant regeneration procedures can possibly be adapted to
other cultivars and varieties of B. napus.
• Plants are grown in 150-cm diameter fiber pots in a
soil:sand:peat (2: 1: 1) mixture under a 16/8 h day/night
photoperiod, 360 ftmol m- 2 S-I, at 20/ 15°C. Plants are
watered daily and receive weekly fertilizer with a 20:20:20 N:
P: K liquid nutrient solution at 1 gIl.
• Good quality, vigorous, healthy plants are essential for the
success of protoplast culture to achieve sustained cell division
and plant regeneration.
8. Preparation of Explants :
• Excise three to four terminal internodes from B. napus cv. Westar
plants at the early flowering stage (three to five flower buds open
per plant), or from the intergeneric hybrid of tomato.
• Surface sterilize in 7% w/v of saturated calcium hypochlorite
solution for 10 min and rinse the internodes in sterile distilled water
3 X for 5 min each.
• A sterile plastic petri dish, 100 X 15 mm, is opened and used as a
sterile tray to cut the tissues from the stem sections.
• Peel off epidermal and subepidermal cell layers from the stems
using sterile thin scalpel blades and fine jeweller's forceps. A good
explant is 3-6 mm long, depending upon the plant species.
• The epidermal segments are precultured in the dark at 4°C
overnight in lOIS ml of liquid MS medium in 100 X 15mm petri
dishes.
9. Protocols :
• Enzymatic Digestion to Release Protoplasts :
• The pretreated epidermal segments are transferred
to 100 X 15-mm petri dishes containing 10-15 ml of
filter sterilized enzyme solution consisting of 1%
(w/v) Macerozyme R-lO and 1% Cellulase R-lO in 0.4
M mannitol, pH 5.8.
• Seal the petri dishes with parafilm and incubate for
16 h at 28°C in the dark on a slow gyratory shaker.
10. Protoplast Filtration and Washing (Purification) :
• . Gently transfer the protoplasts, enzyme solution, and
undigested tissues with a sterile, medium-bore Pasteur
pipette onto a sterile 64 or 44 ~m pore-size filter inside a
sterile funnel.
• Collect the filtrate in a sterile 50 ml centrifuge tube, cover
with sterile tin foil or autoclaved metal closure, and centrifuge
at 500 g for 5-10 min.
• Remove the enzyme solution carefully with a sterile Pasteur
pipette.
• Add 12 ml of 0.4 M sucrose to the pellet, and gently disperse
and resuspend the protoplasts in the solution. Use gentle with
drawing and delivery with the pipette.
11. r
• When protoplasts are completely dispersed, add carefully
with a Pasteur pipette, by layering over the sucrose solution: 3
ml of a solution of 0.4 M sorbitol, 10 mM CaCI2 • 2HP, 5 mM
H MES, pH 5.8, and centrifuge at 250 g for 6min.
• To wash the protoplasts, use a Pasteur pipette to collect the
protoplasts carefully at the interphase of the two solutions,
and transfer to 4 ml of salt solution containing 0.2% w/v CaCl2
and 2.5% KCI, pH 6.0. Gently suspend and centrifuge at low
speed to collect the protoplasts. Remove the washing solution
carefully with a sterile Pasteur pipette.
12. j
• Resuspend the protoplasts in the centrifuge tubes to a density
of 1 X 105 per ml in L Medium (Klimaszewska and Keller 1987)
for Brassica and in SCM1 Medium (Tan et al. 1987) for tomato.
To determine the density, a hemacytometer and inverted
microscope are used to make a count of the protoplasts.
• Count the protoplasts in the hemacytometer. Count 64 fields,
each being 0.25mm X 0.25 mm (see Fig. 1). Calculate the
average number of protoplasts per field and per ml. Because
each field is 0.25 mm X 0.25 mm X 0.1 mm (depth), or 6.25 X
10-3 mm each field is 6.25 X 10-6 ml.
13. Protoplast Culture :
• The protoplasts may be cultured in one of three ways:
• as thin liquid layers of 1-1.5 ml per 60 X 15 mm sterile
petri dish;
• as micro droplets of 10-25 III each, spotted in the petri
dishes
• as thin liquid layers of 1.5 ml over agarose underlayers.
• Agarose underlayers consist of 2 ml of the respective culture
medium solidified with 0.4% (w/v) agarose, in the bottom of
60 X 15 mm petri dishes. L Medium is the resuspension and
culture medium for Brassica protoplasts while the SCM-l
Medium is the resuspension and culture medium for hybrid
tomato protoplasts.
