Plant Cell Rep (2002) 21:226–232
DOI 10.1007/s00299-002-0516-2
CELL BIOLOGY AND MORPHOGENESIS
K.E. Danso · B.V. Ford-Lloyd
Induction of high-frequency somatic embryos
in cassava for cryopreservation
Received: 25 January 2002 / Revised: 1 August 2002 / Accepted: 10 August 2002 / Published online: 17 September 2002
© Springer-Verlag 2002
Abstract Methods for inducing high-frequency somatic
embryos in cassava on cotyledons and 33 clonal acces-
sions by the addition of supplementary copper sulphate
to the induction medium were investigated. The addition
of copper sulphate enhanced primary embryo induction
and significantly increased secondary embryo produc-
tion. All accessions from Latin America (CIAT) were
embryogenically competent on medium supplemented
with 8 mg l–1 2,4-dichlorophenoxyacetic acid (2,4-D)
plus 1 µM copper sulphate as were 15 of the 18 acces-
sions from Africa. The percentage of calli producing
somatic embryos ranged from 7.5% in M. Bra 12 to
100% in M. Col. 1505, while the number of embryos
produced per callus ranged from 0.3 in M. Bra 383 to
13.5 in TEK. The frequency of embryo production was
dependent on the concentration of copper sulphate. The
number of primary embryos produced per callus was
also comparatively higher in the medium supplemented
with copper sulphate than in the controls. The optimal
concentration of copper sulphate for number of embryos
produced in most accessions was 5 µM, and at this con-
centration the number of embryos produced was double
that of the controls. Copper sulphate also reduced the
maturation time of somatic embryos to 25 days from
embryo initiation. High levels of 2,4-D were detrimental
to embryo production. Similarly, fragmented embryos
incubated in the dark produced more embryos tan those
incubated under light conditions. On the basis of these
results, the use of cassava somatic embryo micropropa-
gules for germplasm conservation and synthetic seed
development seems to be a strong possibility.
Keywords Copper sulphate · Embryogenic calli ·
Somatic embryo · Dark incubation
Communicated by M.R. Davey
K.E. Danso · B.V. Ford-Lloyd (✉)
School of Biosciences, University of Birmingham, Edgbaston,
Birmingham, B15 2TT, UK
e-mail: B.Ford-Lloyd@bham.ac.uk
Introduction
Somatic embryogenesis (SE) as an efficient and repro-
ducible regeneration system for the transformation of
cassava (Manihot esculenta Crantz) has proven success-
ful in some cultivars (Puonti-Kaerlas 1997; Raemakers
et al. 1997). Particle bombardment of embryogenic sus-
pensions of accessions TMS 60444, M. Col 1505 and
Bonoua allowed the regeneration of transformed cassava
(Taylor et al. 2001). However, the application of SE is
not restricted to its transformation capacity alone but can
also be used for the rapid multiplication of healthy and
disease-free planting materials of cassava. According to
Raemakers (1993) the multiplication rate in cyclic SE in
cassava is comparatively higher than micropropagation
via nodal cuttings. In addition, somatic embryos can be
used for long-term germplasm conservation via cryo-
preservation and synthetic seed development (Zhou et al.
2000). Synthetic seeds promise to be a valuable alterna-
tive to stem cuttings of cassava, which are associated
with the transmission of viruses and are bulky and highly
perishable. However, the conservation of germplasm us-
ing somatic embryos is rare. This can be attributed to the
low rates of primary embryo induction, the highly geno-
typic dependent nature of the whole embryogenic system
as well as the low rates of plant recovery from somatic
embryos. The embryogenic system must be improved
to remove these drawbacks if somatic embryos or em-
bryogenic calli are to be used for cryopreservation or
synthetic seed development. Several methods, including
the pretreatment of donor explants with 2,4-dichloro-
phenoxyacetic acid (2,4-D) (Raemakers et al. 1997), the
addition of paclobutrazol to the culture medium (Li et al.
1995) or the addition of activated charcoal (Matthews
et al. 1993), have been used to improve the embryogenic
system in cassava.
In recent years, the effects of supplementary micronu-
trients such as Ag+ Co2+, Ni2+, Zn2+and Cu2+ on plant
regeneration via SE have received considerable attention
(Sahrawat and Chand 1999). In rice (Sahrawat and
Chand 1999) and barley (Castillo et al. 1998), the addi-

tion of copper sulphate to the induction medium in-
creased plant regeneration from immature embryos and
embryogenic calli, respectively. In this paper we report
on screening cassava accessions for their embryogenic
competence on a medium containing 2,4-D and supple-
mentary copper sulphate. The effects of copper sulphate
on primary and secondary embryo production as well as
light/dark effects on secondary embryo production are
also reported.
Materials and methods
Plant materials
Seeds from ten individual open-pollinated plants of cassava
(Manihot esculenta Crantz) accession SM-2075-1 as well as 15
clonal accessions were received from the Centro International de
Agricultura Tropical (CIAT) (courtesy of Dr. Fregene). The
remaining clonal accessions were obtained from the International
Institute of Tropical Agriculture (IITA, Nigeria, 13 accessions)
and the Biotechnology and Nuclear Agriculture Research Institute
(BNARI, Ghana, five accessions). Clonal accessions were main-
tained through a 5-week serial subculture on medium containing
MS (Murashige and Skoog 1962) basal salts and vitamins supple-
mented with 30 g l–1 sucrose, 0.05 mg l–1 6-benzylaminopurine
(BAP), 0.01 mg l–1 α-naphthaleneacetic acid (NAA), 2 µM CuSO4
solidified with 7 g l–1 agar. The cultures were incubated at a tem-
perature of 24–25°C under a 16/8-h (light/dark) photoperiod with
light provided by white fluorescent tubes at an intensity of
40 µmol m–2 s–1.
Induction of somatic embryos from cotyledons
Seeds were soaked in 350-ml honey jars containing 250 ml of tap
water overnight to rehydrate. They were then surface-sterilized by
immersion in 70% ethanol for 1 min, followed by immersion in
5% commercial bleach (active ingredient: sodium hypochlorite)
containing two drops of Tween 20 for 6 min and finally rinsed
with three changes of sterilized distilled water. Cotyledons were
excised and cultured on MS basal medium supplemented with
20 g l–1 sucrose, 1 µM CuSO4 and 0–16 mg l–1 2,4-D in 8.5-cm-
diameter petri dishes containing 25 ml of the induction medium.
The medium was adjusted to pH 5.7 prior to the addition of 7 g l–1
agar and autoclaving at 120°C. The growth regulator 2,4-D was
filter-sterilized before it was added to the medium. Five cotyle-
dons were placed in one petri dish and incubated in the dark
(step 1). After 14 days in culture, calli with or without globular
embryos were transferred to the same medium supplemented with
0.1 mg l–1 BAP but without 2,4-D and incubated in light for the
maturation of embryos (step 2).
Screening for embryogenic competence
in clonal cassava accessions
Excised young leaf lobes (1–3 mm) from 33 cassava clonal acces-
sions were cultured on MS basal medium supplemented with
8 mg l–1 2,4-D plus 1 µM copper sulphate (step 1) as described
above. After 14 days of incubation in the dark, calli with or with-
out embryos were transferred to the maturation medium (step 2),
which consisted of MS supplemented with 0.1 mg l–1 BAP,
100 mg l–1 myo-inositol, 1 µM CuSO4, 30 g l–1 sucrose and
1 mg l–1 thiamine HCl, 1.5 mg l–1 pyridoxine HCl, 1.5 mg l–1
nicotinic acid, 2 mg l–1 glycine [the latter four referred to as
cassava vitamins (Mathews et al. 1993)]. The number of leaf lobes
that formed calli and subsequent embryos were recorded, while
the number of somatic embryos that matured was counted under
the microscope after 14 days on maturation medium. Somatic
227
embryos were defined as having matured when distinct hypocotyls
and cotyledons were visible. Additionally, the effect of 4 mg l–1 or
16 mg l–1 2,4-D on somatic embryo production was compared
with the effect of 8 mg l–1 using accessions TMS 60444,
TME 2019, M. Col 1505, M. Col 1468 and M. Per 183.
Effect of copper sulphate on the embryogenic competence
of clonal accessions
The effect of copper sulphate on the embryogenic competence of
clonal accessions was tested on selected accessions based on the
results of the screening experiment. Young leaf lobes were cul-
tured on MS basal medium supplemented with 20 g l–1 sucrose,
8 mg l–1 2,4-D and 0, 1, 2 or 5 µM CuSO4. The cultures were incu-
bated as described above. The number of leaf lobes that formed
calli and subsequent somatic embryos was recorded. In addition,
the number of somatic embryos produced per callus was also
counted under the light microscope 14 days after the latter was
transferred to the maturation medium.
Effect of copper sulphate on cyclic embryogenesis
Aliquots (100 mg) of isolated matured somatic embryos from
each of TME 2019, TME 279, TME 9 and TME 60444 were frag-
mented and cultured on induction medium supplemented with
8 mg l–1 2,4-D and 0, 1, 2 or 5 µM copper sulphate. The frag-
mented embryos were distributed over four petri dishes, each dish
containing 25 ml of induction medium, and incubated in the dark.
After 14 days, embryogenic clumps were transferred to maturation
medium supplemented with 1 µM CuSO4 and incubated in the
light. The rates of emergence of matured somatic embryos as well
as the number of embryos produced per embryogenic clump were
recorded.
Effect of light/dark on secondary embryo production
Aliquots (100 mg) of mature somatic embryos isolated from coty-
ledons of SM1-2075-1 and SM2-2075-1 were fragmented and re-
cultured on induction medium supplemented with 8 mg l–1 2,4-D
for the production of secondary somatic embryos. The cultured
fragments were incubated under either light or dark conditions.
After 10 days of culture, calli were transferred to maturation medi-
um (S2). The number of mature embryos derived from fragments
incubated under either the light or dark conditions was counted
after 10 days.
Results
Response of cotyledon explants to SE
Cotyledons from seeds cultured on hormone-free medium
did not form callus or somatic embryos. However, the
embryonic axes attached to the cotyledons developed into
complete plants with a prominent taproot and shoots after
14 days of culture in the dark. Cotyledons cultured on
medium with 2,4-D were swollen within 7 days and
developed callus on the adaxial surfaces. Upon transfer of
the calli to maturation medium there were varied morpho-
genic responses: within 10 days, foliose structures, so-
matic embryos or roots had developed. Calli derived from
culture on medium with 4 mg l–l 2,4-D predominantly
formed roots with no somatic embryos, while calli that
developed in the presence of 8 mg l–l 2,4-D formed
somatic embryos.
228
Table 1 Cassava accessions of different geographical origins tested for their embryogenic competence

Accessions from Africa Accessions from Latin America

Accessions Geographical origin Classificationa Accessions Geographical origin Classificationa

TME 1808 Republic of Benin Moderate M. Col 2215 Columbia Very high
TME 1801 Republic of Benin High M. Col 22 Columbia Very high
TME 1939 Republic of Benin Very high CM 523-7 Columbia Moderate
TME 2019 Republic of Benin Moderate CM 3306-19 Columbia High
TME 2037 Republic of Benin Low CM 3555-6 Columbia Moderate
TME 279 Nigeria Very high CM 4365-3 Columbia Moderate
I 4 (2) 1425 Nigeria Low CM 6740-7 Columbia Moderate
TME 60444 Nigeria High M. Col 1468 Brazil Moderate
TME 2 Nigeria Non-embryogenic M. Bra 12 Brazil Low
TME 9 Nigeria High M. Bra 191 Brazil Low
I 30572 Nigeria Moderate M. Bra 383 Brazil Low
TME 3 Nigeria Low M. Col 1505 Venezuela Very high
TME 24 Nigeria High M. Ven 77 Venezuela Very high
Afisiafi (AFI) Nigeria Non-embryogenic M. Per 183 Peru High
Abasa fitaa (ABA) Nigeria Low NGA-2 Nigeria Moderate
Santom (SM) Ghana Very high
Tek bankye (TEK) Ghana Low
Biafra (BF) Ghana Non-embryogenic
a Very high: 80–100%; high: 60–80%; moderate: 20–60%; low: less than 20%

Somatic embryogenetic response of young leaf lobes

of clonal accessions

Young leaf lobes (1–3 mm) from 33 accessions of cassava

Fig. 1 Embryogenic calli from cotyledon of SM1-2075-1 line 1
with somatic embryos. c Callus, se somatic embryos
However, cotyledons from SM3-2075-1, SM4-2075-1
and SM5-2075-1 did not develop somatic embryos on
MS basal medium supplemented with 8 mg l–l 2,4-D
even after prolonged culture on maturation medium.
Cotyledons from SM1-2075-1 and SM2-2075-1 devel-
oped somatic embryos (Fig. 1), with more than 50
embryos per cotyledon. Only one cotyledon from
SM9-2075-1 developed somatic embryos in the presence
of 16 mg l-l 2,4-D, and the number of embryos produced
was comparatively lower (fewer than ten).
from BNARI (Ghana), IITA (Nigeria) and CIAT (Colum-
bia) were screened for their embryogenic competence.
These accessions represent breeding lines, improved culti-
vars and landraces of cassava of different geographical
origins (Table 1).
Leaf-lobe explants formed callus within 7 days of cul-
ture on induction medium. The calli were soft, friable or
compact and yellowish-cream, cream or brown in colour.
Globular or torpedo-shaped embryos were visible on
the yellowish-cream or cream embryogenic calli within
14 days of culture, while brown calli seldom developed
embryos. Upon transfer to maturation medium, the globu-
lar and torpedo-shaped somatic embryos matured within
10 days. In some accessions, embryogenic calli that did not
develop somatic embryos developed roots. The percentage
of leaf lobes that developed somatic embryos is shown in
Fig. 2a, b. Of the 18 accessions from Africa, 15 showed
embryogenic competence. The percentage of leaf lobes
that produced somatic embryos varied depending on the
accession and was not influenced by the geographical
origin of the accession (Fig. 2a, b). Of the 18 accessions
from Africa, only three did not produce somatic embryos.
Among the accessions responding to embryogenesis,
TME 2037 showed the least embryogenic competence
(8.3%), while TME 279 showed the highest (92.4%).
Seven accessions – TME 1801, TME 1939, TME 279,
TME 60444, TME 3, TME 24 and Santom (SM) – had
more than 60% of their leaf lobes producing somatic em-
bryos, while the remaining accessions had less than 40%.
In contrast, all 15 accessions of Latin American ori-
gin developed somatic embryos. However, the percent-

Fig. 2a, b The response of leaf lobe-derived calli to somatic em-
bryogenesis on a medium with added copper sulphate. a Acces-
sions from Africa, b accessions from Latin America. Numbers
above bars represent the mean number of somatic embryos pro-
duced
age of leaf lobes that developed somatic embryos varied
depending on the accessions (Table 1). The M. Col series
from both Venezuela and Columbia showed very high
embryogenic competence, while the CM series from
Columbia and M. BrA series from Brazil showed moder-
ate and low embryogenic competence, respectively
(Table 1, Fig. 2b). M. BrA 12 had the lowest percentage
of leaf lobes (7.7%) developing somatic embryos, while
in M. Col 1505 all of the leaf lobes developed somatic
embryos. The mean number of somatic embryos pro-
duced per responding callus of African origin ranged
from 1.0 to 13.5, while in the Latin American accessions
it was 1.1–9.5 (Table 1). Based on these results, clonal
accessions were classified as being very highly compe-
tent (80–100% of embryogenic calli developing somatic
embryos), high (60–80%), moderate (20–60%), low (less
than 20%) or non-embryogenic (no somatic embryos
produced) (Table 1).
Effect of 2,4-D concentration on somatic embryo
production from leaf lobes
We tested the effect of different 2,4-D concentrations
(4, 8 or 16 mg l-l) on selected accessions. In contrast to
cotyledon explants, young leaf lobes cultured in the pres-
ence of 4 mg l-l 2,4-D formed calli, which subsequently
developed somatic embryos upon transfer to maturation
229
Fig. 3 The effect of 2,4-D on the frequency of embryo production
(a) and the mean number of embryos produced per callus (b)
medium. However, in all of the accessions tested fewer
calli formed somatic embryos on medium with 4 mg l–1
2,4-D than on medium with 8 mg l–1 2,4-D, except for
CM 523-7 (Fig. 3). In only two accessions (M. BrA 191
and M. Per 183) was the production of somatic embryos
better in the presence of 16 mg l–-1 2,4-D than in the
presence of 8 mg l–l2,4-D (Fig. 3). Where 16 mg l–1 was
the optimum concentration of 2,4-D, the percentage of
calli forming somatic embryos increased with increasing
2,4-D concentration. Among all of the accessions tested,
M. Col 1468 had the least embryogenic competence.
Percentage embryogenic calli that formed somatic
embryos ranged from 12.5 on 4 mg l–1 2,4-D to 42.4 on
8 mg l–1 2,4-D. The number of embryos produced per
callus in all of the accessions tested showed a different
trend (Fig. 4). It was low on calli derived from culture in
the presence of 16 mg l–1, except in M. Per 183. The
optimal concentration for embryo production was either
4 mg l–1 or 8 mg l–l depending on the accession.
Effect of copper sulphate on somatic embryogenesis
Based on the results of the screening experiment using
young leaf lobes, clonal accessions were classified as
very high, high, moderate, low and non-embryogenically
competent with respect to embryogenic competence
(Table 1). We selected accessions from each classifica-
tion group to test for the effect of different concentra-
tions of copper sulphate on embryogenic competence,
with an emphasis on accessions that had been classified
as having a low embryogenic competence. Embryos in-
230
Table 2 The effect of copper
sulphate on secondary embryo Concentrationa of CuSO4 (µM) TME 2019 TME 279 TME 9 TME 60444
production
0.0 23.75 52.50 53.75 45.25
1.0 27.50 58.75 67.00 66.00
2.0 40.25 55.50 73.25 68.75
a Critical
5.0 15.25 42.75 55.00 100.75
value (P=0.05): 4.20

Table 3 Effect of light/dark incubation of fragmented embryo explants on the number of embryos produced at secondary somatic
embryogenesis

Genotypea Incubation Number of Number of Total no. Mean no.

treatment calli clumps calli with of somatic of somatic
cultured somatic embryos embryos per
embryos isolated callus

SM1-2075-1 (SE line 1) Dark 35 34 210 6.0±0.6

Light 25 23 60 2.4±0.5
SM1-2075-1(SE line 2) Dark 20 18 119 5.9±1.1
Light 20 16 74 3.7±0.7
a SM
1, Seeds from one particular plant; SE, somatic embryo; line, embryos produced from seed

However, the stimulatory effect of copper sulphate

varied from one accession to the other. The optimal con-
centration of copper sulphate for the induction of
somatic embryos was either 2 µM or 5 µM depending
on the accession. In TME 2019, Abasa Fitaa (ABA) and
Biafra (BF) the optimal concentration was 2 µM; at this
concentration, 33.3%, 60% and 90%, respectively, devel-
oped somatic embryos. In contrast, for M. Per 183,
M. Col 1505, Santom (SM) and Afisiafi (AFI) the opti-
mal concentration of copper sulphate was 5 µM. Acces-
sion TME 2 had been classified as being non-embryo-
genic, but in the presence of 2 µM copper sulphate 3.7%
of the calli developed somatic embryos.
Similarly, the mean number of embryos produced was
also comparatively higher in the treated calli than in the
controls in all of the accessions (Fig. 4). In Biafra and
M.Col 1505, the optimal concentration was 2 µM, while
in the remaining accessions tested, 5 µM was optimal.
The highest number of embryos produced per callus (12)
was obtained in M. Col 1505.

The effects of copper sulphate

Fig. 4 The effect of copper sulphate on the frequency of embryo
production (a) and the mean number of embryos produced per
callus (b)
duced on media supplemented with copper sulphate had
well-developed green cotyledons within 14 days, indicat-
ing early maturity, compared to the controls where most
of the embryos were in the late-torpedo stage or early-
cotyledonary stages. The number of embryogenic calli
that subsequently developed somatic embryos was also
comparatively higher on the copper sulphate-supple-
mented medium (Fig. 4).
on secondary embryo production
Fragmented somatic embryos cultured on maturation
medium containing supplementary copper sulphate pro-
duced embryos within 7 days of transfer to maturation
medium. The effect of copper sulphate on the production
of secondary somatic embryos is shown in Table 2. The
presence of supplementary copper sulphate in the induc-
tion medium significantly (P=0.05) increased somatic
embryo production over the controls, except at the high-
est concentration of copper sulphate where there was on-
ly a significant increase in the number of embryos in
TME 60444. In three of the accessions there were no
significant differences between 1 µM and 2 µM copper

sulphate, indicating that the optimal concentration of
copper sulphate was highly dependent on the genotype.
In TME 2019 and TME 9, 2 µM was optimal. There
was a significant (P=0.05) positive interaction between
copper sulphate and the accessions when a two-way
analysis of variance was performed.
The effect of light/dark incubation on the production
of secondary somatic embryogenesis
Primary embryos from SM1-2075-1 and SM2-2075-1
were used to study the effect of light/dark incubation
on the number of embryos produced. Somatic embryos
produced under a dark/light incubation regime developed
mature somatic embryos (within 7 days) earlier than
those cultured under continuous light. Fragmented
primary embryos of SM1-2075-1 incubated in the dark
followed by light incubation at the maturation stage
produced more embryos than explants incubated under
continuous light at the induction and maturation stages
(Table 3).
Discussion
In cassava, cotyledons and young leaf lobes are the two
main explants used for the induction of primary somatic
embryos, with cotyledons being more embryogenically
competent than leaf lobes. We observed a high frequency
of embryo production when we cultured these explants on
a medium supplemented with 8 mg l–1 2,4-D plus 1 µM
copper sulphate. However, due to high heterozygosity
and poor seed production in some cultivars, leaf-lobe ex-
plants are frequently used for somatic embryo production.
Of the 33 clonal cassava accessions we tested for embry-
ogenic competence, 30 produced somatic embryos. An
increased concentration of copper sulphate in the medium
increased the percentage of leaf-lobe explants that devel-
oped somatic embryos and also doubled the number of
embryos produced per callus relative to the controls. The
high response could be due to the addition of supplemen-
tary copper sulphate to the induction medium. Using the
same protocol without supplementary copper sulphate,
Szabados et al. (1987) reported that 60–70% of leaf lobes
of cassava accession M. Col 1505 formed somatic embry-
os. Mathews et al. (1993) incorporated copper sulphate
into the induction medium but did not add BAP to the
maturation medium, and 85% of the leaf lobes developed
embryos. In our study, 100% of the leaf lobes of
M. Col 1505 developed somatic embryos. Raemakers
(1993) did not obtain any somatic embryos from
M. Ven 77 on a medium supplemented with 8 mg l–1
2,4-D, while in our study 80% of the cultured explants of
M. Ven 77 produced somatic embryos when cultured with
copper sulphate.
Copper sulphate is known to improve plant regenera-
tion from immature embryos in rice (Sahrawat and
Chand 1999) and embryogenic calli in barley (Castillo et
231
al. 1998). While the exact function of copper sulphate in
plant regeneration has not been determined, it is known
that copper sulphate is a component or activator of many
enzymes involved in electron transport, photosynthesis
and protein and carbohydrate metabolism. The stimula-
tory effect of copper sulphate could be important by
playing a key role in morphogenesis at an optimum con-
centration (Sahrawat and Chand 1999). It is also known
that copper sulphate at concentrations of 1.0–25.0 µM
inhibits ethylene, a modulator of plant growth in a
hydroponic rice nutrient solution (Lidon et al. 1995).
Copper sulphate may therefore enhance somatic embryo
production through its inhibitory effect on ethylene.
El Meskaoui and Temblay (2001) demonstrated that
limiting ethylene biosynthesis by the addition of inhibi-
tors to the maturation medium of low-capacity embryo-
genic lines of black spruce promoted somatic embryo
production.
The incubation of fragmented embryos in darkness
followed by a transfer to light produced more somatic
embryos within a relatively short time than did incuba-
tion under continuous light. Some species, such as
poplar and Podophyllum, have an absolute requirement
for dark during embryogenesis (Merkle et al. 1995).
However, cassava fragmented embryos require an initial
dark incubation period before the transfer to maturation
medium for embryo maturation.
Successful transformation procedures in cassava via
particle bombardment (Schöpke 1996; Raemakers et al.
1996; Taylor et al. 2001) or Agrobacterium mediation
(Gonzalez et al. 1998; Schreuder et al. 2001) rely on SE
for both gene insertion and recovery of transgenic plants.
Thus, it is essential that cassava accessions, independent
of their geographical background, are made embryogen-
ically competent to enable transformation procedures to
address agronomic problems such as susceptibility to
cassava mosaic virus diseases. Of the 30 responding
accessions, there were no differences in embryogenicity,
indicating a strong possibility for genetic transformation
independent of their origin. Our results compare with
those of Taylor et al. (2001) who produced friable em-
bryogenic callus of a similar nature in 14 cultivars of dif-
ferent geographical origin; eight of these have success-
fully been regenerated into plants, and transgenic plants
have been obtained from three.
The application of SE in micropropagation and germ-
plasm storage requires an efficient system of embryo pro-
duction. In our study we have shown that the addition of
copper sulphate enhanced early maturation of the somatic
embryos; somatic embryos matured within 25 days com-
pared to the 35 days reported by Matthews et al. (1993).
We have also shown that incubation of fragmented em-
bryos in the dark produced more somatic embryos within
a relatively short time than did incubation under continu-
ous light. Furthermore, we have demonstrated that an in-
creased concentration of copper sulphate in the induction
medium increased embryogenicity in all accessions as
well as doubled the number of embryos produced. Thus,
a system for the rapid generation of high-frequency so-
232
matic embryos independent of accessions (genotype) has
been obtained, making somatic embryos alternative mi-
cropropagules for the long-term conservation of plant ge-
netic resources and, more importantly, a potential means
of propagation on an agricultural scale. Somatic embryo-
genesis has the potential to accelerate the introduction of
improved cultivars into commercial production, since
they can be encapsulated and handled as seeds. However,
further investigations on the frequency of conversion
of somatic embryos into plants and an optimization of
cryogenic protocols are needed to make somatic embryos
of cassava more useful as ideal micropropagules for both
the mass multiplication and long-term conservation of
cassava.
Acknowledgements The authors wish to thank The Common-
wealth Scholarship Commission, UK for their financial support to
Mr. Kenneth Ellis Danso. We also thank Mrs. Ng at IITA and
CIAT for sending us in vitro plantlets of cassava for this investiga-
tion.
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