burger1982

PHYSIOL. PLANT. 56: 324-328. Copenhagen 1982
The isolation, culture and division of protoplasts from citrus
cotyledons
David W. Burger and Wesley P. Hackett
Burger, D. W. and Hackett, W. P. 1982. The isolation, culture and division of
protoplasts from citrus cotyledons. - Physiol. Plant. 56i 324—328.
High yields (2.3 X 10^ to 1.3 X 10' protoplasts/g.f.wt.) of isolated protoplasts were
obtained from cotyledons ot Citrus sinensis (L.) Osb. 'Valencia'. Osmotic potential of
the medium and enzyme concentrations were important in obtaining high viability of
preparations as indicated by FDA fluorescence. Adding malt extract to a
Murashige-Tucker basal medium increased plating efficiencies somewhat, but not the
rate or duration of cell division. However, modifying the NAA and kinetin concen-
tration optimized platiog efficiencies (up to 20%) of protoplasts and also the rate or
duration of cell division. The highest plating efficiency and number of cells per colony
were obtained on a defined medium containing NAA (15 \>M) and kinetin (4.6 (xM).
Coincidence of percentage protoplast viability after 13 days (assessed by FDA
fluorescence) with plating efficiency after 21 days indicates that FDA fluorescence is
an accurate indicator of citrus protoplast viability.
Additional key words - Cellulysin, Macerase, malt extract, plating efficiency, pro-
toplast viability, protoplast yield, Valencia.
D. W. Burger {reprint requests and permanent address), Texas A <& I Univ. Citrus
Center, P.O. Box 2000, Weslaco, TX 78596, USA; W. P. Hackett, Dept. of Environ-
mental Horticulture, Univ. of California, Davis, USA.
Introduction
The successful use of protoplasts to study any problem
depends on the ability to isolate large numbers of viable
protoplasts. The definition of important parameters af-
fecting protoplast yield and viability is the first step to-
ward this goal and is the focus of this paper. Osmoticum
concentration and cell wall digestive enzyme concent-
rations are regarded as important paratneters affecting
protoplast yield and viability (Coltnan and Mawson
1978, Kirby and Cheng 1979). Proper osmotic condi-
tions are required to provide adequate plasmolysis, thus
minimizing hydrolytic enzyme damage to membranes
and reducing membrane damage from over-plasmolysis
or membrane distension (Tribe 1955).
Enzyme concentrations and/or times of exposure to
the enzyme can influence protoplast release as well as
influence the degree of damage to the cell membrane
(Cassells and Badass 1976, Okuno and Furusawa
1977). It is possible that glycoprotein constituents ofthe
cell membrane may be subject to attack if left exposed
to digestive enzymes too long or to high concentrations
of enzymes.
Plating density has been found to be important in
other protoplast systems (Durand 1979, Nagata and
Takebe 1970, Nehls 1978, Power et al. 1976) and malt
extract has been found to increase organogenic events
in citrus tissue cultures (Kochba and Spiegei-Roy
1973). Plating density and malt extract concentration
were varied here in an attempt to inerease the incidence
of cell division.
In the experiments described, factors required for
isolating a large number of viable protoplasts, culture
conditions and techniques necessary to achieve high
plating efficiencies of citrus cotyledon protoplasts were
investigated. Cotyledons were chosen as a tissue source
of protoplasts based on their accessibility and their in-
herent potential for the regeneration of adventitious
organs (D. W. Burger, 1980, Thesis, Univ. of Califor-
Received 26 November, 1981; revised 12 June, 1982
3240031-9317/82/110324-05 $03.00/0 © 1982 Physiologia PlantarumPtiysiot. Plant. 56. t!>82

nia, Davis, USA). These results are important steps in
developing a whole plant-to-protoplast-to-whole plant
system.
Abbreviations — BAP, benzylaminopurine; HDA, fluorescein
diacetate; ME, malt extract; NAA, naphthaleneacetic acid;
MT, Murashige and Tucker.
Materials and methods
Seeds from fruit of Citrus sinensis (L.) Osbeck 'Valen-
cia' were used in all experiments. Mature fruit was har-
vested and stored in a refrigerator at 3—4''C for at least
21 days before seeds were extracted.
Protoplast isolation. The seeds were disinfested under
aseptic conditions by a 1-3 s dip in 95% ethanol fol-
lowed by flaming in an alcohol lamp. The seed coat was
removed and the cotyledons excised, weighed, sliced
into small pieces (less than 2x2 mm), and placed in a
60 X 15 mm plastic petri dish containing a basal
Murashige and Tucker (MT) medium (Murashige and
Tucker 1969) containing mannitol as osmoticum. The
solution was removed after 45-60 min and replaced
with the same medium containing the cell wall hydroly-
tic enzymes Cellulysin and Macerase (Calbiochem). The
petri dish containing the cotyledon pieces and enzyme
solution was rotated at ca. 70 rpm in the dark on a
horizontal shaker. After 30—60 min the enzytne solution
was replaced with identical fresh solution and the tissue
was incubated on the shaker for an additional 120-200
min in the dark. The resulting solution was then passed
through a 37-[xm nylon filter to separate protoplasts
from cell debris and aggregates. The filtrate containing
intact protoplasts was centrifuged at ca. lOOg for five
min. The supernatant was discarded and the pellet was
resuspended in 1-2 ml of MT culture medium. This
washing sequence was repeated twice. The final pro-
toplast suspension was brought to a known volume and
aliquots were taken to measure protoplast yield and
viability. The protoplasts were cultured in hanging
drops or suspended in an equal volume of MT medium
containing molten 1% Taiyo agar (37-^0°C) in the dark
at 25°C.
Yield and viability determinations. Protoplast counts
were made with a hemacytometer. Viability was asses-
sed using fluorescein diacetate (FDA) as a test of mem-
brane integrity and internal diesterase activity
(Widholm 1972). After 2-5 min in 0.01% (w/v) FDA
in culture medium, protoplasts were observed under
ultraviolet light using a Zeiss standard photomicroscope
equipped with epi-fluorescent attachments. The percent
viability was calculated as the number of protoplasts
fluorescing green per total number of intact protoplasts
existing at day-0 x 100.
Determination of optimum enzyme and mattnitol con-
centrations. A 4 X 4 factorial experiment was designed
with Cellulysin at concentrations of 1, 2, 3, and 4% and
Macerase at concentrations of 0.1, 0.3, 0.5, and 1.0%.
Cotyledon segments were exposed to all treatments for
a total of 4 h in 0.6 M mannitol. Yield and viability
measurements were taken each honr.
Eaeh mannitol concentration (0.4 M to 0.65 M)
tested was used for both the isolation and culture of the
protoplasts for 13 days. Enzyme concentrations used lor
isolation were 3% Cellulysin and 0.3% Macerase.
Plating efficiency and optimum plating density. Plating
efficiency, the percentage of isolated protoplasts under-
going division, was estimated after three weeks in cul-
ture. At the time of plating, random fields of protoplasts
in the agar were- marked by etching a circle 1.2 mm in
diameter around the area of interest on the plastic petri
dish. The number of protoplasts in each field was
counted. After three weeks, the number of dividing cells
in each field was determined. The plating efficiency was
calculated as the number of dividing cells (cell colonies)
divided by the number of protoplasts plated x 100. This
method of determination was designated the pre-de-
teimined field method. The isolated protoplasts, 95 ±
5% viable, were cultured at densities ranging from 1 x
10* • ml-' to 1 X 10* • m|-' in MT medium solidified with
0.5% Taiyo agar.
Malt extract, NAA, and kinetin effects on plating effi-
ciency. Protoplasts were cultured in an agar medium
containing various concentrations of malt extract,
naphthaleneacetic acid (NAA), and/or kinetin. The
number of cells per colony was determined by micros-
copic observation of all the colonies.
Results and discussion
Optimum mannitol concentration for yield and viablitj-
Protoplast yield was greatest when 0.6 M mannitol was
used (Fig. 1). Lower mannitol concentrations (0.4 M
and 0.45 M) caused bursting. The results of a viability
•0.4 0.4S5 0.55 C
c0ncentrati{in tM)
Fig. 1. Effect of mannitol concentration on the yield of pro-
toplasts from 'Valencia' cotyledons. Murashige-Tucker basai
medium used in all solutions. Enzyme solution-3% Cellulysin,
0.3% Macerase. Bars represent ± SD from the mean of 5
replicates.
Ptiysiol. POant. 56. 1982325

MannitsI
Q'.4O
0.4 S
O.S0
Oi.SS
ConcBntratiam
M -
H -
M -
M -
0.60 M -
o.esM ~
A
£a
m
D
•
O
g drop culture
Fig. 2. Time course study of viability of 'Valencia' cotyledon
protoplasts isolated and cultured in various mannitol concen-
trations. A Murshige-Tucker basal medium was used in all
solutions. Enzynae solutions contained 3% Cellulysin, 0.3%
Macerase. Bars represent ± SD from the mean of 5' replicates.
time course study tising FDA are shown in Fig. 2. Man-
nitol at 0.6 M gave the highest viability throughout the
time course of the experiment. The decrease in viability
after one day in the 0.4 M mannitol treatment was due
to protoplast bursting. The 0.45 M or 0.5 M mannitol
treatments supported protoplast viability longer than
0.4 M mannitol, but in both treatments protoplasts
eventually lost all viability (Fig. 2). Although bursting
of protoplasts was not observed, the numbers of pro-
toplasts in these treatments dimitiished rapidly just prior
to final loss of viability. This observation suggested a
deterioration of the membrane over time due to osmotic
pressure in the culture medium, insufficient plasmolysis
of the tissue during isolation resulting in enzyme-
induced injury, or to some other unknown factor.
Results in Fig. 2 demonstrate that the viability of
protoplasts measured immediately after isolation may
not necessarily reflect the ability of the protoplasts to
survive in culture over a period of time. It is likely that
varying numbers of protoplasts are injured to some de-
gree due to the duration or composition of the isolation
treatment. Therefore, at the end of an isolation period,
each treatment consists of a population of protoplasts of
variable quality because protoplasts have been exposed
to the digestion conditions for varying lengths of time.
As a result, it is important to follow protoplast viability
over a period of time to obtain a clearer picture of their
response to isolation and culture conditions.
Tab. 1. Viable and total protoplasts (x 10^) released per g fresh
weight from 'Valencia' cotyledons in various concentrations of
Cellulysin and Macerase. Means of 5 replicates separated by
Duncan's New Multiple Range Test, 5% level. Tabulated val-
ues for viable and total were statistically analyzed separately.
Total or viable values followed by the same ietter(s) are not
significantly different.
%
Macerase
0.1
0.3
0.5
1.0
Proto-
plasts
viable
total
viable
total
viable
total
viable
total
1
1.8
1.8
2.9
2.9
1.5
1.5
3.4
5.4
bi
hi
ghi
hi
b
fgh
fgh
%
2
4.7 cde
4.7 fgh
5.3 cd
6.0 efg
3.3 efg
4.2 ghi
3.1 fgh
7.8 defg
Ceilulysin
3
9.4 b
9.4 bcde
13.0 a
14.7 a
4.9 cde
12.6 ab
2.3 ghi
12.0 abc
4
9.2 b
10.5 bed
5.7 c
8.4 cdef
4.3 def
1].] abed
0 i
11.4 abed
enzyme combination gives a graphic illustration of toxic
effects of the enzyme (note the lower right corner of
Tab. 1).
The critical Macerase concentration with regard to
viability is 0.3%. At concentrations of 0.5% and grea-
ter, increasing concentrations of Celiulysin were par-
ticularly detrimental to protoplast viability.
These results do not show the basis of decreased via-
bility frotn exposure to super-critical enzyme concent-
rations. Viability measurements with FDA depend on
membrane integrity and internal diesterase activity. In-
ternal diesterase activity would have to be depressed
indirectly since enzyme molecular size prevents direct
contact with internal constituents. It is most likely that
the digestive enzyme's deleterious effects act primarily
on membrane integrity either by impeding membrane
functions directly or as a result ofthe deleterious effects
of associated impurities. At each enzyme digestion du-
ration for which data were taken (every hour for 4 h),
the same enzyme concentrations (3% Cellulysin and
0.3% Macerase) resulted in the highest yield and via-
bility. Higher concentrations of the two enzymes caused
loss of viability and lower concentrations did not liber-
ate sufficient numbers of protoplasts.
Optimum enz^ime concentrations
Cellulysin at 3% and Macerase at 0.3% was the best
enzyme combination for high yield and viability (Tab.
1). Effects of enzyme concentrations on yield and via-
bility were different. As the concentration of enzymes
increased, so did the yield of protoplasts. However, as
the concentration of either enzyme was increased
(Tab. 1), a point was reached where toxic effects of the
enzyme solution were detected by decreases in viable
protoplasts per gram fresh weight. Comparing the yield
of protoplasts to the yield of viable protoplasts at each
Plating density
The maximum plating efficiency (ca 5%) was obtained
at 1 X 10* protoplasts • mr' (Fig. 3). Vardi et al. (1975)
found that 'Shamouti' orange protoplasts from cell sus-
pension cultures divided and formed colonies best (%
efficiency) at a culture density of ca 1 x 10^ cells • ml~'.
A major difference between the plating efficiencies re-
ported by Vardi et al. (1975) and those reported here is
the method of calculating efficiencies. Vardi and co-
workers calculated plating efficiencies by microscopi-
cally scoring twenty random fields of plated protoplasts
326Physiol. Plant. 56, 1982

10* S«10 10 5»tO 10°
Culture density
Cprotoplasts/ml}
Fig. 3. Effect of culture density on the plating efficiency of
'Valencia' cotyledon protoplasts isolated and cultured in a
Murashige-Tucker basal medium containing 0.6 M mannitol.
Protoplasts were plated in tbe sanae medium containing 0.5%
Taiyo agar. Protoplasts isolated using 3% Celulysin and 0.3%
Macerase. Bars represent ± SD from the mean of not less than
three replicates.
four weeks after plating. This method has a major
drawback in that the total number of protoplasts
counted after four weeks is not an accurate estimate of
the total number of protoplasts plated since some have
surely burst. The burst protoplasts would not be
counted, thus plating efficiency estimates would be
high. However, the random field method may be an
accurate estimate of plating efficiency in systems where
mortality rate is low. The method reported here takes
into account all protoplasts plated since the total
number in a field is counted at the time of plating.
Kao and Michayluk (1975) found that Vicia protop-
lasts developed poorly on defined media at low culture
densities (1-1000 protoplasts • ml"'). The culture den-
sity could be lowered if undefined components such as
coconut water were added to the medium, suggesting
that this low density phenomenon was due to diffusion
of necessary metabolites from the cells to the medium,
depleting the cells to levels too low to survive. These
resuits of Kao and Michayluk and our subsequent re-
sults with malt extract and growth regulators suggest
that optimal plating densities for citrus cotyledon pro-
toplasts may be lower than those reported here if malt
extract or growth regulators at optimal concentrations
are included in the medium.
Malt extract, NAA and kinetin effects on plating efficiency'
Plating efficiency was increased by supplementing a
basal MT medium with 1000 mg- ]"' malt extract (Tab.
Tab. 2. Protoplast plating efficiencies using two methods of
determination and cells per colony as affected by malt extract
additions to a Murashige-Tucker basal medium. Tabulated
values are means of no less than 3 replicates. Mean separation
witbin a column by Duncan's New Multiple Range Test, 5%
level. Plating efficiency values for each method followed by the
same letter are not significantly different.
Malt
(mg/l)
0
100
500
1000
Cells
per
colony
7.1
9.9
8.4
8.8
not significant
Plating efficiency
Random
field method
21.6 b
20.9 b
13.7 b
39.0 a
Pre-determined
field method
• 5.1 b
4.9 b
5.1 b
8.4 a
2). This treattnent increased the number of protoplasts
undergoing division, but did not increase the rate of
division as shown by the "cells per colony" data. Table
2 also shows the difference in two methods of calcula-
ting plating efficiencies. The random field method (as
found in the literattu"e) exaggerated the plating effi-
ciency in citrus preparations. Even though malt extract
slightly increased protoplast plating efficiency and can
increase organogenic events in citrus tissue cultures
(Kochba and Spiegel-Roy 1973), it was eliminated from
subsequent experiments because it is an undefined sub-
stance and therefore complicates the analysis of factors
controlling cell wall reformation and cell division.
Plating efficiencies were significantly increased when
the hormone levels of the MT basal medium were al-
tered (Tab. 3). The protoplasts in the MT basal medium
(Treatment A) had a plating efficiency of 5.1%, which
is consistent with the results of the previous experiment.
These indicate that kinetin is limiting colony formation
in the MT basal medium (Treatment A) since when its
level was raised to 2.3 [iM or 4.6 ^M with the same level
of NAA (30 (iM, Treatments B and C) the plating effi-
ciency increased threefold. When the kieetin:NAA
ratio was increased further (Treatment D), the plating
Tab. 3. Effects of NAA (^lAf) and kinetin (\iM) in a
Murasbige-Tucker basal medium on plating efficiencies of
'Valencia' cotyledon protoplasts. Plating efficiencies were de-
termined by the pre-determined field method. Mean separa-
tion within a column by Duncan's New Multiple Range Test,
5% level. Values in columns followed by tbe same letter are
not significaBtly different.
Treatment
A
B
C
D
E
NAA:Kinetin
30:0.09
30:2.3
30:4.5
15:4.6
2.7:4.6 •
Cells per
colony
7.1 b
5.8 b
5.6 b
15.2 a
2.2 c
Plating
efficiency
5.1 c
14.0 b
13.8 b
19.8 a
2.5 c
22 Pliysiol. Plant. 56, 1982327

efficiency increased to nearly 20 %. This was the highest
plating efficiency observed in any of the treatments.
Results of Treatment E suggested that there was a
threshold level of NAA required, or that the kinetin:
NAA ratio was super-optimal. Treatment D not only
increased numbers of cells undergoing division, it also
increased the rate or duration of division (cells per col-
ony). These results indicate that atixin and cytokinin
levels are very important for initiating and sustaining
division of cells derived from citrus cotyledon proto-
plasts.
Hormone species and levels have been shown to be
important in protoplast development. Power et al.
(1976) found that leaf protoplast plating efficiency in
Petunia was maximized in a Murashige and Skoog
medium containing 11—27 \iM NAA and 1.6—3.2 uM
BAP, very similar to the results reported here. Dudits et
al. (1976) found that carrot protoplasts divided best in a
Kao medium (Kao and Michayluk 1975) containing
1 X iO-^M NAA and 5 x lO'^'M zeatin, an au-
xin: cytokinin ratio of 2:1.
The highest plating efficiency observed here (20%) is
interesting in that it closely coincides with the percen-
tage of the total cotyledon protoplasts that were viable
(fluorescing green) after 13 days (Fig. 2). This fact ar-
gues in favor of using FDA as an accurate indicator of
the viability of citrus protoplasts. It is also of interest in
that the highest plating efficiency and cells per colony
were obtained on a defined medium with quite specific
levels of NAA and kinetin.
Conclusion
Citrus cotyledons liberate adequate numbers of high
quality protoplasts which will reform a celi wall and
divide in defined culture media. Callus cultures derived
from protoplast colonies have yet to be induced to be-
come organogenic. This is the remaining, important
event currently under study, which must occur if this
system is to be useful in following reproductive matura-
tion at the cellular level.
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Edited by C.H.B.
328Physiol. Planl. 56, 19S2