Plant Cell, Tissue and Organ Culture 51: 1–8, 1997.

1

c 1997 Kluwer Academic Publishers. Printed in the Netherlands.

Histology of somatic embryo initiation and organogenesis from rhizome

explants of Musa spp.

K.S. Lee1 , F.J. Zapata-Arias2 , H. Brunner2 & R. Afza2

1
Department of Biology, College of Natural Sciences, Chonbuk National University, Chonju 561-756, Korea;
2
Plant Breeding Unit, FAO/IAEA Agriculture & Biotechnology Laboratory, International Atomic Energy Agency
Laboratories, A-2444 Seibersdorf, Austria ( requests for offprints)

Received 26 February 1996; accepted in revised form 10 September 1997

Key words: banana, somatic embryogenesis, organogenesis

Abstract

The origin of somatic embryos derived from rhizome explants of triploid Musa cv. Grand Nain was the subject of
histological studies during different phases of ontogenetic development. The investigation revealed that the majority
of somatic embryos showed normal root formation and consisted of highly vacuolated cells in the poorly structured
shoot apex. The embryogenic mass and somatic embryos were mostly derived from several morphologically
competent cells. Single cell origin depended on the presence of organogenetically functional vascular cells of
rhizome explants and occurred infrequently. The implications of these findings for genetic improvement of banana
and plantain by in vitro mutation breeding and gene technology are discussed.

Abbreviations: 2,4-D – 2,4-dichlorophenoxyacetic acid; BA – benzyladenine; Dicamba – 3,6-dichloro-2-

methoxybenzoic acid; FAA – formalin-acetic acid-alcohol

Introduction desired traits. Therefore, the integration of biotech-

Bananas and plantains are important staple food crops
for people living in tropical and sub-tropical coun-
tries. Declining yields due to the spread of virulent dis-
eases such as black sigatoka, fusarium wilt and banana
bunchy top virus, has resulted in increased efforts to
genetically improve this crop. However, conventional
breeding of Musa cultivars remains a difficult endeav-
our because of high sterility and polyploidy.
Plant tissue culture techniques can potentially over-
come some of the factors limiting traditional approach-
es to banana and plantain improvement. These tech-
niques enable plants to be regenerated from normal and
genetically modified cells and tissues in an efficient
way under sterile conditions. The genetic improve-
ment of Musa by in vitro mutation breeding or genet-
ic engineering requires the efficient regeneration of
genetically modified single cells into complete plants
which express the desired character(s). This prevents
the formation of chimeras and facilitates screening for
nology into banana and plantain improvement pro-
grammes requires a reliable cell culture protocol.
Cronauer-Mitra and Krikorian (1987) have
described the formation of spherical callus masses
resembling somatic embryos; the regeneration process
from these structures has been characterized as adven-
tive bud formation, i.e. organogenesis. Several authors
have reported somatic embryogenesis and in vitro plant
regeneration in seeded wild species (Cronauer-Mitra
and Krikorian, 1988; Escalant and Teisson, 1988).
Embryogenic calli were derived in these species from
young zygotic embryos, which limits the usefulness
of the procedures only to fertile non-edible Musa. Of
more practical value has been the work of Baner-
jee et al. (1987) and Sannasgala et al. (1990), who
derived embryogenic callus from thin meristematic
cells formed by morphogenetically competent glob-
ular structures in liquid medium that proliferated and
regenerated into plants. Embryogenic banana suspen-
sion cultures were developed from proliferating meris-

ICPC: PIPS No.: 151075 BIO2KAP

ticu2276.tex; 4/12/1997; 9:45; v.7; p.1
2
tems (Dhed‘a et al., 1991), from rhizome tissue and
leaf bases (Novak et al., 1989) from immature zygotic
embryos (Marroquin et al., 1993) and from young male
flowers of banana (Escalant et al., 1994).
The conversion process from somatic embryos into
plants; i.e., synchronized development of the primary
root and formation of shoot apex with leaf primordia is
inefficient. If somatic embryogenesis is to become an
efficient method for genetic improvement, it is essen-
tial to develop protocols that allow efficient plantlet
regeneration. The development of bipolar structures
of somatic embryos on semi-solid cultures has been
demonstrated histologically in Musa spp. (Escalant and
Teisson, 1989; Novak et al., 1989). Dhedı́a et al. (1991)
reported the origin of proembryogenic structures and
the subsequent process of somatic embryogenesis in
suspension cultures. However, a detailed description
of the origin and developmental of somatic embryos
from semi-solid cultures, especially from rhizome tis-
sues, has not been reported yet.
In banana and plantain, where regeneration follows
an embryogenic pathway, somatic embryogenesis may
be limited to specific development stages. Detailed
studies of the embryogenic process, e.g. which particu-
lar cell or tissue develops into embryogenic meristems
or at what stage further development stops, could pro-
vide information for enhancement or manipulation of
the embryogenic response. The objective of the present
study was to assess the effects of different media com-
position on the relative efficiency of embryogenic cul-
ture initiation and to document the origin and develop-
mental stages of banana somatic embryo formation in
Musa spp.
Materials and methods
Plant material
Sliced rhizome tissue from freshly growing shoot tip
cultures of ‘Grand Nain’, a commercially important
banana clone, was used for the induction of somatic
embryogenesis. The methods to culture shoot tips were
based on those developed by Novak et al. (1989).
Culture initiation
Embryogenic calli were induced from cultured rhi-
zome explants on three different media.
– MS (Murashige and Skoog, 1962) or
Table 1. Frequency of embryogenic response on different
media after 8 weeks of culture.
Medium Total no. of Embryogenic response (%)
number explants E EC NEC B
MS 79 6.3 63.3b 13.9 16.5
1/2MS 73 0 97.3a 2.7 0
SH 79 5.0 79.8c 15.2 0
 Embryo forming, (non)embryogenic callus forming explants
or browning explants/ cultured explants x 100
E: embryo formation, EC: embryogenic callus, NEC: non
embryogenic callus; B: browning explant.
a c Means followed by different letter are significantly dif-
ferent at the 0.05 probability level according to LSD test
– 1/2 MS medium with 5 M 2,4-D, 1 M proline,
100 mg l 1 casein hydrolysate, 40 mg l 1 cystein-
HCl, 10 M ascorbic acid, 40 g l 1 sucrose and
1.8 g l 1 gelrite, and
– SH medium (Schenk and Hildebrandt, 1972) with
30 M Dicamba, full-strength B5 vitamins (Gam-
borg et al., 1968), 30 g l 1 sucrose and 1.8 g l 1
gelrite.
All culture media were adjusted to pH 5.8 before auto-
claving (20 min at 120 C and 105 kPa) and the cultures
were incubated at 25 C in the dark for 5–6 weeks.
Embryogenic calli with globular structures devel-
oped from three different media described above were
transferred to hormone-free MS liquid medium for one
week and then to 12 MS liquid medium with 20 M BA
and moved from 24 hours darkness to a 16-h photoperi-
od (mixed cool-white and grolux fluorescent lamps, 50
(mol m 2 s 1 ). Liquid cultures were carried out on
a horizontal gyratory shaker (30 rpm) and subcultures
were performed at 2 week intervals.
Histology
Tissues for histological studies were obtained from
explants cultured on SH medium containing Dicam-
ba. Somatic embryos were vacuum-infiltrated for 20
min with FAA (formalin: acetic acid: ethanol: water
in a ratio of 10:5:50:35 v/v), (Sass, 1958) and fixed
for 7 days at room temperature. Fixed tissues were
washed in running water for 10 min and dehydrated
in a gradual ethanol-toluene series. Sections (10 m)
of paraplast embedded material were obtained using a
sliding microtome (Leitz, 1400); affixed to glass slides
with albumin, stained with hematoxylin, safranin and
fast green, mounted in Canada balsam, and covered
with a cover glass for microscopic observations.
ticu2276.tex; 4/12/1997; 9:45; v.7; p.2

Results
Embryogenic response
The embryogenic response was different depending
on the medium (Table 1) after 8 weeks of culture on
semisolid media (MS, 1/2MS and SH medium). The
frequency of embryo-forming explants on the three
media was 6.3%, 0% and 5%, respectively and the
formation of embryogenic callus from explants 63.3%,
97.3% and 79.8%, respectively. 16.5% of explants with
browning were observed on the MS medium with 2,-
4-D.
The frequency of embryogenic calli and embryos
was higher in SH medium and samples for the histo-
logical studies were taken from this medium (Table 1).
Somatic embryogenesis
After 2 months of culture, embryogenic calli and
embryos developed from vascular tissue in the central
part of the compact and yellow colored rhizomes; non-
embryogenic calli originated from friable and white
to light brown cortex tissue. Somatic embryos that
were regenerated from primary embryogenic cultures
on semisolid media were arrested in development and
failed to germinate on solid induction medium with
Dicamba. These somatic embryos were hyperhydric
and transparent.
Early somatic embryogenesis (Organogenesis)
Sections through rhizome explants revealed two dis-
tinct cell types, a cortex parenchyma in the outer cell
layer and several vascular tissue in the central zone.
The cortex consisted of large cells and the vascular
tissue of small cells (Figure 1A, B) which exhibited a
marked contrast between the tissues.
After 4 to 5 days of culture, vascular cells except
xylem cells had a dense cytoplasm and appeared well
stained. After 8 days of culture, the plane of the first
division in vascular tissues could be periclinal or anti-
clinal (Figure 1C). After 10 days of culture, the second
division of the two-celled proembryo took place in
one of the two cells to form a three-celled proembryo
(Figure 1D) or in both cells simultaneously forming
a four-celled proembryo (Figure 1E). After 12 days
of culture, a five-celled proembryo developed (Fig-
ure 1F). This indicated a single cell origin of somatic
embryos derived from vascular tissue.
3
In most other cases, cell divisions after 8 to 10 days
of culture occurred within groups of adjacent cells of
vascular tissues which developed into cell masses and
globular stages of embryos (Figure 2A, B). Globular
embryos consisted of xylem cells of original rhizome
tissues (Figure 3B). This suggested a multiple cell ori-
gin of somatic embryos from vascular tissues.
Late stage of somatic embryogenesis
Semisolid medium
In longitudinal sections, globular embryos did not con-
sist of suspensor or suspensor-like structures. After 21
to 28 days of culture, globular embryos in different
stages were observed in the upper portion of explants
(Figure 2B,C,D). The late stages of globular embryos
consisted of small, highly cytoplasmic and epidermal
cells (Figure 2D). After 28 days of culture, the plumule
and root pole were distinguished by procambial strands
(Figure 2E). Histological observations after 50 days of
culture showed that the plumule was composed of tiers
of highly elongated vacuolated cells (Figure 2F) and
a functional shoot apex had not developed after two
months of culture (Figure 3D).
Liquid medium
Embryos derived on semisolid medium after 2 months
of culture were transferred to liquid 12 MS medium
supplemented with 20 M BA, 20 g l 1 sucrose and full
strength of B5 vitamin for embryo maturation. After
3 weeks of culture, some embryos developed a green-
colored plumule (p) with a white colored hypocotyl
and a root (r) that consisted of highly vacuolated cells
(Figure 3E) and embryos that developed roots (r) only
(Figure 3F).
A section of matured embryo revealed a cotyle-
donary slit connected with vascular strands from the
hypocotyl to the root (Figure 3G). However, plant
regeneration from these somatic embryos was not
observed.
Discussion
The generation of genetic variation by induced muta-
tions or genetic transformation is a single cell event.
Therefore, treatment of multi-cellular cell initials leads
to the formation of chimeras. Genetically altered and
non-altered cell-lineages compete during the prolifera-
ticu2276.tex; 4/12/1997; 9:45; v.7; p.3
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Figure 1. Somatic embryos of single cell origin from rhizome explants of Musa (AAA) cv. ‘Grand Nain’ cultured on semisolid SH medium
supplemented with 30 M Dicamba; (A) Transverse section of rhizome explants at day 0, showing vascular tissue (v) and cortex cells (c). Bar
= 160 m. (B) Enlarged view of vascular tissues. Arrow indicates xylem (x). Bar = 40 m. (C) Two-celled proembryo development from a
cell of vascular tissue after 8 days of culture. Bar = 50 m. (D) Three-celled proembryo development after 10 days of culture. Arrows indicate
nucleus. Bar = 50 m. (E) Four-celled proembryo development after 10 days of culture. Bar = 50 m. (F) Five-celled proembryo development
after 12 days of culture. Bar = 50 m.

tion and growth of multi-cellular initials and lead to the
formation of mericlinal or sectorial chimeras. These
are not manifested phenotypically and require repeat-
ed subculture to rescue genetically modified homohis-
tont tissue prior to mass-screening and/or selection for
desirable traits. Our histological observations revealed
that the embryogenic mass and somatic embryos devel-
oped in most instances from several morphologically
competent adjacent cells while the occurence of single
cell origin in vascular tissues of rhizome explants of
Musa was less frequent.
These patterns of single and multiple cell ori-
gin during somatic embryogenesis have also been
observed in other species (Tisserat and DeMason,
1980; Button et al., 1974). In general, a unicellular ori-
gin is more likely when embryos are attached by a nar-
row suspensor-like organ (Williams and Maheswaren,
1986). However, our histological examination did not
show a narrow suspensor-like organ. The embryo mor-
phology was similar to the one reported in earlier stud-
ies for Musa accuminata, M. balbisiana and Bluggoe
(Escalant and Teisson, 1989; Dhed’a et al., 1991), but
different from zygotic and somatic embryos of Musa
ornata with a haustorium (Cronauer-Mitra and Kriko-
rian, 1988).

ticu2276.tex; 4/12/1997; 9:45; v.7; p.4

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Figure 2. Somatic embryos of multiple cell origin and development of late stages of somatic embryos; (A) Multiple cells (m) in vascular tissue,
which initiate mitotic activity after 8 days of culture. Bar = 40 m. (B) Somatic embryos (e) developed from multiple cells after 22 days of
culture. Bar = 150 m. (C) Various globular stage somatic embryos developed on the upper part of explants. Bar = 160 m. (D) An enlarged
view of globular stage somatic embryos. Arrow indicates epidermal layer (e). Bar = 50 m. (E) A late stage somatic embryo with procambial
strands (p), which developed after 28 days of culture. Bar = 160 m. (F) Somatic embryo with plumule (p) and root pole (r) developed after 50
days of culture. Arrow indicates highly vacuolated cells (v). Bar = 160 m.

ticu2276.tex; 4/12/1997; 9:45; v.7; p.5

6

Figure 3. Morphogenetic aspects of cultured rhizome explants on semisolid (A,B,C,D) and liquid medium (E, F, G); (A) Section of embryogenic
callus (ec) attached to mother tissue (mt) after 28 days of culture. Bar = 160 m. (B) Globular stage of somatic embryo with xylem of mother
tissue, which developed after 22 days of culture. Arrow indicates xylem cell (x). Bar = 40 m. (C) Various stages of somatic embryos developed
after 8 weeks culture. Arrow indicates plumule (p). Bar = 1.250 m. (D) Longitudinal section of somatic embryo after 2 months, the plumule
part which was composed of vacuolated cells. vp; vacuolated plumule, p; procambial strands, r; root pole. Bar = 160 m. (E) Somatic embryo
showing plumule (p) and root (r) germination after 28 days in liquid culture. Bar = 2,000 m. (F) Somatic embryo with root only (r). Bar =
1,400 m. (G) Longitudinal section through the junction of the hypocotyl with the root. Upper part of somatic embryo developed in liquid
culture, which consisted of highly vacuolated cells (v), the shoot apex was not developed. c; cotyledonary split, p; procambial strands, r; root.
Bar = 160 m.

ticu2276.tex; 4/12/1997; 9:45; v.7; p.6


Regeneration of functional plants from cell cultures
is a major limiting step in the application of biotech-
nology to crop improvement. Somatic embryos grown
on solid media with 2,4-D or Dicamba for 3 months
without any subculture gave rise to a green colored
plumule, but plant regeneration was not observed.
When structures which formed on the 2,4-D- and
Dicamba-supplemented media were transferred to MS
media with or without BA, they failed to develop into
plantlets. Similar structures that never developed into
plants were previously reported in Musa tissue culture
(Cronauer and Krikorian, 1984; Jarret et al., 1985;
Novak et al., 1989).
On the other hand, somatic embryos of good quality
are one of the requirements for high plant regeneration.
Our studies further revealed, that the plumule part of
somatic embryos on semisolid and liquid media did
not germinate. Thus, a failure to establish a functional
shoot meristem may be the cause of conversion failure
in somatic embryos (Nickle and Yeung, 1993; Goeble-
Tourand et al., 1993)
Many different stages of somatic embryos still
remained undeveloped when germination was initiat-
ed. This could explain why the plumule part of embryos
produce callus instead of growing into complete plants.
Filtration by size and shape would permit the selection
of mature embryos, thus increasing the germination
percentage of somatic embryos. The useful applica-
tion of ABA (Verhagen, 1989) and mannitol (Nadel et
al., 1989) for differentiation and further germination
of somatic embryos was demonstrated in some other
species.
Several authors have reported that the conversion
frequency of plant regeneration from embryogenic
banana suspension cultures has remained very low
(Novak et al., 1989; Dhed’a et al., 1991).
Further development of reliable protocols for effi-
cient plant regeneration from single cell derived somat-
ic embryos is therefore imperative for mutation breed-
ing and genetic transformation of Musa spp. This
may be achieved either by manipulating the media or
by selecting different explants where different tissues
are involved in the embryogenic process, using plant
regeneration from protoplast (Panis et al., 1993; Megia
et al., 1993). This would allow the generation of uni-
form genetic variation through avoidance of chimeras
and facilitate early selection of homohistont mutants or
of uniform transformants for desired traits and econo-
mize genetic crop improvement by in vitro techniques.
7
Acknowledgement
We are grateful to Dr J.I. Richards and Dr M.
Maluszynski for critically reading the manuscript and
to Ms Andrea Kodym for her valuable help in editing
this manuscript.
References
Button J, Kochba J & Bornman CH (1974) Fine structure of
and embryoid development from embryogenic ovular callus of
‘Shamouti’ orange (Citrus sinensis Osb. ). J. Exp. Bot. 25: 446–
457
Banerjee N, Schoofs J, Hollevoet S, Dumortier F & De Langhe E
(1987) Aspects of prospects of somatic embryogenesis in Musa,
ABB, cv. Bluggoe. Acta Hort. 212: 727–730
Cronauer-Mitra SS & Krikolian AP (1983) Somatic embryos from
cultured tissues of triploid plantains (Musa ‘ABB’). Plant Cell
Rep. 2: 289–291
Cronauer-Mitra SS & Krikolian AD (1984) Multiplication of Musa
from excised stem tips. Ann. Bot. 53: 321–328
Cronauer-Mitra SS & Krikolian AD (1987) Adventitious shoot pro-
duction from calloid structures of banana. Plant Cell Rep. 6:
443–445
Cronauer-Mitra SS & Krikolian AD (1988) Plant regeneration via
somatic embryogenesis in the seed diploid Musa ornata Roxb.
Plant Cell Rep. 7: 23–25
Dhed’a D, Dumortier Francoise Panis B, Vuylsteke D & De Langhe
E (1991) Plant regeneration in cell suspension cultures of the
banana cv. ‘Bluggoe’ (Musa spp. ABB group). Fruits 46: 125–
135
Escalant JV & Teisson C (1988) Embryogènése somatique chez
Musa sp. Comptes Rendus de l‘Academie des Sciences. Paris
Serie. III, 306: 277–281
Escalant JV & Teisson C (1989) Somatic embryogenesis and plants
from immature zygotic embryos of the species Musa acuminata
and Musa balbisiana. Plant Cell Rep. 7: 665–668
Escalant JV, Teisson C & Cote F (1994) Amplified somatic embryo-
genesis from male flowers of triploid banana and plantain culti-
vars (Musa spp.). In Vitro Cell Dev. Biol. 30: 181–186
Gamborg OL, Miller RA & Ojima K (1968) Nutrient requirements
of suspension cultures of soybean roots cells. Exp. Cell Res. 50:
151–158
Goebel-Tourand I, Mauro MC, Sossountzov L, Miginiac L & Deloire
A (1993) Arrest of somatic embryo development in grapevine:
histological charcterization and the effects of ABA, BAP and
zeatin in stimulating plantlet development. Plant Cell Tiss. Org.
Cult. 33: 91–103
Jarret RL, Fisher JB & Litz RE (1985) Organ formation in Musa
tissue cultures. Plant Physiol. 121: 123–130
Marroquin CG, Paduscheck C, Escalant JV & Teisson C (1993)
Somatic embryogenesis and plant regeneration through cell sus-
pensions in Musa acuminata. In Vitro Cell. Dev. Biol. 29P:
43–46
Murashige T & Skoog F (1962) A revised medium for rapid growth
and bioassays with tobacco tissue cultures. Physiol. Plant. 15:
473–497
Nadel BL, Altman A & Ziv M (1989) Regulation of somatic embryo-
genesis in celery cell suspensions. 1. Promoting effects of man-
ticu2276.tex; 4/12/1997; 9:45; v.7; p.7
8
nitol on somatic embryo development. Plant Cell Tiss. Org. Cult.
18: 181–189
Nickle TC & Yeung EC (1993) Failure to establish a functional
shoot meristem may be a cause of conversion failure in somatic
embryos of Daucus carota (Apiaceae). Am. J. Bot. 80: 1284–
1291
Novak FJ, Afza R, Van Duren M, Perea-Dallos M., Conger BV &
Xiaolang Taang (1989) Somatic embryogenesis and plant regen-
eration in suspension cultures of dessert (AA and AAA) and
cooking (ABB) bananas (Musa spp.). Biotechnology 7: 154–
159
Panis B, Van Wauwe A & Swennen R (1993) Plant regenera-
tion through direct somatic embryogenesis from protoplasts of
banana (Musa spp.) Plant Cell Rep. 12: 403–407
Sannasgala K, Dumortier FM & De Langhe EAL (1990) Somatic
embryogenesis from Musa proliferating shoot tips: Histological
details. In: In vitro Mutation Breeding of Bananas and Plantains
1. IAEA, Vienna (pp. 24–28).
Sass JE (1958) Botanical Microtechnique, 3rd ed. Iowa State College
Press, Iowa
Schenk RU & Hildebrant AC (1972) Medium and techniques for
induction and growth of monocotyledonous and dicotyledonous
plant cell cultures. Can. J. Bot. 50: 199–204
Tisserat B & DeMason DA (1980) A hitological study of develop-
ment of adventive embryos in organ cultures of Phoenix dactylif-
era L. Ann. Bot. 46: 445–472
Verhagen SA & Wann SR (1989) Norway spruce somatic embryo-
genesis high frequency initiation from light cultured mature
embryos. Plant Cell Tiss. Org. Cult. 16: 103–111
Williams EG & Maheswaran G (1986) Somatic embryogenesis: Fac-
tors influencing coordinated behaviour of cells as an embryo-
genic group. Ann. Bot. 57: 443–462
ticu2276.tex; 4/12/1997; 9:45; v.7; p.8