Plant Science Letters, 17 (1980) 459--465 459

© Elsevier/North-Holland Scientific Publishers Ltd.

CASSAVA MESOPHYLL PROTOPLASTS: ISOLATION, PROLIFERATION,

AND SHOOT FORMATION

E.A. S H A H I N and J.F. S H E P A R D

Department of Plant Pathology, Kansas State University, Manhattan, KS 66506
(U.S.A.)
(Received October 19th, 1979)
(Revision received November 30th, 1979)
(Accepted December 3rd, 1979)

SUMMARY

Isolated mesophyll protoplasts of cassava (Manihot esculenta Crantz.)

were induced to regenerate cell walls, divide and form calli via the potato
protoplast system previously developed in this laboratory. Shoot formation
was occasionally observed in cultivar Mexico No. 35 under the specific con-
ditions described herein.

INTRODUCTION

The opportunities that protoplasts offer for the improvement of important

crop species have been extensively reviewed by many authors. However, plant
regeneration from callus and/or plant protoplasts has been described only in
a few major crops. Cassava, for instance, is a vital crop in many developing
countries as a food source for both humans and animals [ 1 ]. The establish-
m e n t of a protoplast regeneration system in cassava, though of unexplored
potential, could be of significant value in genetic modification studies aimed
at improving this important crop plant. To date, cassava plants have only
been regenerated from meristem culture [2,3], and stem callus [4], but not
from protoplast cultures [5,6]. This paper describes procedures for the iso-
lation, proliferation, and shoot formation of mesophyll cell protoplasts of
cassava utilizing the basic potato protoplast system developed by Shepard [ 7 ].

MATERIALS AND METHODS

Manihot esculenta Crantz cultivars Mexico No. 35 and CMC 76 were sup-
plied by Dr. Peter Carlson, Michigan State University, through the Centro
International de Agricultura Tropical (CIAT) in Cali, Colombia, and by the
Instituto Colombiano Agropecuario (ICA) in Colombia, respectively. Three-
node stem sections of the cultivars were planted in plastic pots of 20 cm diam.
460

and 12 cm depth which were filled with vermiculite. Plants were maintained
in an environmentally controlled growth chamber (Lab-Line Instruments,
Inc. Melrose Park, Illinois) at a constant temperature of 28°C and a relative
humidity of 90%. The plants were given a 12 h photoperiod of 3000 lux
white fluorescent light {Type F20T12/CW), and were kept well moistened
with deionized water. Pots were fertilized weekly with the mineral salts solu-
tion of Murashige and Skoog [8] containing two-fold amounts of CaC12 and
KH2PO4.
For protoplant isolation, well expanded leaves (7--9 cm) in length were
collected from preconditioned plants and floated on a distilled water solu-
tion containing 1 mM CaCl:, 1 mM NH4NO3, 1 ppm naphthaleneacetic acid
(NAA) and 5 ppm benzylaminopurine (BAP) [7]. After 48 h incubation in
the dark, the leaves were surface sterilized in 70% ethanol for 30 s, 3 min in
10% Chlorox and then rinsed three times in sterile distilled water. Leaves
were then lightly brushed, and soaked overnight at 4°C in a dilute solution
of mineral salts [9]. Mesophyll protoplasts were prepared in an enzyme solu-
tion (0.5% macerozyme R 10, 1.0% cellulase R 10, Kinki Yakult Ltd., Japan,
in 0.3 M sucrose at pH 5.7) incubated at 25°C (50--70 rev./min) for 5 h.
Liberated protoplasts were collected in Babcock bottles [9] after centrifu-
gation (International model HN-S) at 800 g for 20 min.
Floated protoplasts were removed from the surface of centrifuged prepa-
rations with a sterile Pasteur pipet and then placed in a 0.3 M sucrose solu-
tion containing one-half the concentration of mineral salts of CL-medium
(Table I), and again centrifuged at 800 g for 20 min. Floating protoplasts
were then suspended at a concentration of 5 × 10S/ml in the same solution
for 1 h before plating. The protoplasts were then plated in the CL-media
along with R-medium (Table I) in 100 mm plastic quadrant (X-plate, Falcon,
Oxnard, CA) petri-dish [7], then the plates were sealed with parafllm and
incubated at 24°C under 4000 lux continuous fluorescent light (Type
F20T12. CW). Three weeks after protoplast plating, medium in the CL-
compartment of the quadrant plates was removed with a small spatula or
pipet and 0.5--1.0 ml were evenly distributed over the surface of petri-dishes
containing 20 ml of medium-C (Table II). Within two weeks, after the calli
became green in color and 0.1--1.0 mm in diameter, they were transferred
individually to another fresh medium-C for 14 days and then to medium-D
(Table II) or other shoot medium at a light intensity of 4000 lux and a day
length of 16 h.

RESULTS AND DISCUSSION

Upon isolation, protoplasts were spherical with a mean diameter of 38/~m

and with peripherally aligned chloroplasts (Fig. la). The average yield of
mesophyll protoplasts was 5.6 × 106/g fresh wt of leaves and with a high
apparent viability. The presence of specific sugar alcohols (D-mannitol, m y o -
inositol, sorbitol, and xylitol) along with sucrose, as the osmotic stabilizer, in
 461

TABLE I

COMPOSITION OF CELL L A Y E R (CL) AND R E S E R V O I R (R) MEDIA

Constituent CL-Medium (mg/l) R-Medium (rag/l)

Major salts
KNO 3 7600 1900
CaCl~- 2H20 1760 440
MgSO 4 o 7H20 1480 370
KH2PO , 680 170

Iron and minor elements

Na 2 • EDTA 18.5 18.5
F e S O , - 7H~O 13.9 13.9
H3BO 3 3.1 3.1
MnC12 • 4H20 9.9 9.9
ZnSO 4 o 7H20 4.6 4.6
KI 0.42 0.42
Na2MoO 4 • 2H~O 0.13 0.13
CuSO 4 • 5H20 0.013 0.013
C O S O , . 7H20 0.015 0.015

Organic addenda
Thiamine • HCI 0.5 0.5
Glycine 2.0 2.0
Nicotinic acid 5.0 5.0
Pyridoxine o HC1 0.5 0.5
Folic acid 0.5 0.5
Biotin 0.05 0.05
Casein Hydrolysate 50 100

Osmoticum
Sucrose 0.200 M 0.05
myo-Inositol 0.025 M
D-Mannitol 0.025 M 0.25
Sorbitol 0.025 M
Xylitol 0.025 M

Other
1-Napthaleneacetic acid 1.0 1.0
6-Benzylaminopurine 0.5 0 .5
Agara 0.2%b 0.4%c
pH 5.6 5.6

aWeight: volume. Medium components filter sterilized then mixed with molten agar.
b Sigma Type VII Agarose.
cDifco purified agar, washed.
462

TABLE II

COMPOSITION OF CULTURE MEDIA C, D, A N D E

Constituent Medium-C (mg/l) M e d i u m - D (mg/l) M e d i u m - E (mg/1)

NH,CI 107 267.5 267,5

KNO 3 1900 1900 1900
CaCl 2 • 2H20 440 440 440
M g S O , - 7H20 370 370 370
KH~PO, 170 170 340
Na~ ° E D T A 18.5 18.5 18.5
F e S O 4 • 7H20 13.9 13.9 13.9
H,BO 3 3.1 3.1 3.1
MnC12 ° 4H20 9.9 9.9 9.9
Z n S O 4 , 7H20 4.6 4.6 4.6
KI 0.42 0.42 0.42
Na2MoO 4 • 2H20 3.13 0.13 0.13
CuSO 4 , 5H20 0.013 0.013 0.013
COSO 4 • 7H20 0.015 0.015 0.015

myo-Inositol 1 O0 100 100

T h i a m i n e , HC1 0.5 O.5 1.0
Glycine 2 2 2
N i c o t i n i c acid 5 5 5
P y r i d o x i n e • HCI 0.5 0.5 0.5
Folic acid 0.5 0.5 0.5
Biotin 0.05 0.05 0.05
Casein H y d r o l y s a t e 100 I00
A d e n i n e sulfate 40 80 40

1 - N a p h t h a l e n e a c e t i c acid 0.1 -- 0.05

3 - I n d o l e a c e t i c acid 0.1
6-Benzylaminopurine 0.5
Zeatin 0.5
Gibberellic acid a -- 0.2

D-Mannitol 0.3 M 0.2 M 0.1 M

Sucrose b 0.25% 0.25% 1%
MES c 5 mM 5 mM 5 mM
A gard 1% 1% 0.5%
pile 5.6 5.6 5.6

aGibberellic acid added after autoclaving.

bWeight: volume.
c2-N-Morpholino-ethane sulfonic acid.
dWeight : volume, Difco Purified Agar.
epH after autoclaving.
 463

Fig. 1. (a) Freshly isolated cassava mesophyll protoplasts. Bar = 25 # m ; (b) First division
of cell regenerated from a single protoplast. Bar = 50 ~ m ; (c) Photograph of quadrant
petri plates three weeks after cell layers were plated with mesophyll protoplasts; (d) Callus
from protoplast grown on solidified medium. Bar = 5 ram; (e) Shoot formation.
464

CL-medium, was indispensable for protoplast division. Under these condi-

tions, and over a wide range of protoplast concentrations, more than 90% of
the plated protoplasts formed a cell wall and increased in size. Cell division,
however, when tested in a protoplast dilution series, was restricted to a range
of 1.0--3.0 × 105 protoplast/ml of CL-media. At this population density,
60--75% of viable cells divided between the 7th and 12th day of culture and
formed colonies (Fig. lb). Colonies were macroscopically visible in the agar
after 3 weeks of culture (Fig. lc). When transferred to medium-C, small calli
grew rapidly and a green mass was obtained within two weeks. The calli were
then transferred individually to the same medium and incubated at 8000 lux
with 16 h photoperiod at 27°C. Best results were obtained on medium-C that
contained 0.5 mg/1 BAP, 0.1 mg/1 NAA, 0.25% sucrose and 2.46% mannitol.
Those calli were green in the center and white or pale yellow in color at their
margins (Fig. ld). In an attempt to regenerate shoots, calli were placed on
media that had been proved successful in plant regeneration from meristem
culture [2,3], D-medium [7] and MS-medium [8]. Different hormonal
regimes and light conditions were tried with the media used. Shoot forma-
tion occurred when calli were placed on MS-medium supplemented with
0.045 mg/1 BAP, 0.25 mg/1 IAA and 0.014 mg/1 GA3 after passage for 8 days
through SH-medium [10] with 10 rag/1 2,4-D and 1 pM kinetin. This last
procedure allowed for rapid increase in callus size. Shoot formation was
observed sporadically among calli of cultivar Mexico No. 35 (Fig. le). For
root initiation, the shoots were transferred to E-medium (Table II) contain-
ing 0.2 mg/1 GA and 0.05 rag/1 NAA, and placed in 12 h daylength and
2500 lux.
Despite this progress, our knowledge of morphogenesis in vitro is still frag-
mentary, and we have not yet fully defined the conditions for efficient shoot
formation. However, efforts to this end are continuing.

ACKNOWLEDGEMENTS

Support for this research was provided by Grant GA COH 7910 from the
Rockefeller Foundation. Kansas Agricultural Station Contribution No.
80-124~J.

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