HISTOLOGICAL ANALYSIS OF COCONUT ENDOSPERM
CULTURED IN VITRO
LAZARUS AGUS SUKAMTO*
Research Center for Biology – Indonesian Institute of Sciences
Cibinong, Indonesia
ABSTRACT
The fresh and development of coconut endosperm cultured in vitro were histological analysis.
Solid endosperm of seven month-old postanthesis coconut cultivar “Samoan Dwarf” were picked up
freshly and cultured in vitro on modified Branton & Blake formula. Histological study of fresh
coconut endosperm consisted of parenchyma cells, which were relatively uniform in shape and size,
some nuclei consists of 1 – 5 nucleoli. Three month-old calli in vitro of coconut endosperm in semi
solid media showed its cells were varied in shape and size, characterized by high nucleus to
cytoplasm ratio, high starch, protein, and lipid, underwent many divisions. Seven month-old calli in
vitro of coconut endosperm in liquid media showed embryogenic cells which resembled
proembryos. Fifteen month-old bud-like structure of coconut endosperm in semi solid media
showed a meristematic layer, structure tunica-corpus, cortex-like region, and tracheids of xylem.
These results indicated the bud-like structure is an early stage of shoot bud formation in coconut
endosperm. It is the first report of early stage of shoot bud formation occurring on coconut
endosperm cultured in vitro.
Key words: Histological analysis, coconut endosperm, calli, bud-like structure,
cultured in vitro
*Corresponding author : [email protected]
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INTRODUCTION
Coconut (Cocos nucifera L) is long-lived with a very long juvenile phase (3 - 5 years) and
generally cross-pollinated and very heterozygous (Sukamto 2011a). Studies on the development of
coconut cultured in vitro have been reported for the past sixty years, mostly using embryos
(immature unselected plants). Coconut remains a recalcitrant species that reflected mainly in the
slowness of in vitro morphogenesis and difficulty to monitor the ability of the meristematic cells to
actively divide (Sandoval et al. 2003). Before their differentiations are visually recognizable, many
changes occur at the cellular and tissue levels. Cells could undergo a series of orderly divisions, to
form callus, proembryo, embryo, and bud-like structure. The cell aggregation consists of small,
isodiametric, thin-walled, micro-vacuolated cells, highly basophilic, densely staining nuclei, and
cytoplasm (Thorpe & Murashige 1970; Reinert et al. 1977). They may also contain numerous starch
grains or lipid deposits (Thorpe & Murashige 1970; Ross et al. 1973; Villalobos et al. 1985; Arnold
& Hakman 1988; Kanchanapoom & Tinnongjig 2001; Liu 2013), and produce proembryos that may
develop to become embryos, organs, or vascular tissues (Reinert et al. 1977; Thorpe 1978),
Few studies have been conducted on coconut endosperm culture, the successive results only
produced low callus and failed to develop further morphogenesis (Fisher & Tsai 1978; Bhalla-Sarin
& Bagga 1983; Kumar et al. 1985). Recent improvements have been achieved using endosperm
explants and succeeded to obtain quicker and higher callus formation, also embryo-like structures
(Sukamto 2011b). The calli formed an embryo structure after 21 weeks of culture and grew to
become bud-like structure after 15 month-old of culture that appeared three protuberances on the
top. This study was to know morphogenesis at cellular and tissue levels using histology of fresh
coconut endosperm and cultured in vitro.
MATERIALS AND METHODS
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Plant material
Solid endosperm of seven month-old postanthesis coconut cultivar “Samoan Dwarf” were
picked up freshly from coconut palm trees in Hawaii, USA. The fresh cuts of solid endosperm were
used as plant materials control for histological analysis. The solid endosperms were aseptically
cored with cork borer and scooped with a sterile spoon in laminar air flow. Cylindrical endosperm
shapes with 8 mm diameter and 4 mm thick, used as explants, were grown on various media
treatments. The endosperm formed callus after seven week-old of culture. Three month-old calli in
semi solid culture, seven month-old calli in liquid medium, and 15 month-old bud-like structure (10
mm x 8 mm size) in semi solid medium, were used for histological analysis.
Media culture
Media cultures were a modification of Branton & Blake formula (1986) with addition of 10
mg l-1 putresine, 2.50 g l-1 activated charcoal (AC), 1.70 g l-1 phytagel, 0, 10-6, 10-5, 10-4, 10-3 M 2,4dichlorophenoxyacetic acid (2,4-D) or 4-amino-3,5,6-trichloropicolinic acid (Picloram) combined
with 10-5M 6-benzylaminopurine (BA) at 16 WOC. The pH of the media was adjusted to 5.70
before they were autoclaved. The media were poured into 2.5 x 15 cm test tubes (14 ml) and
autoclaved at 1210C temperature and 1 kg cm-2 pressure for 15 minutes. Media were stored for one
week before using them. Single explant was placed into test tube with the uncut surface upright.
Cultures were incubated at approximately 310C in the dark room.
Histological methods
The fresh of solid endosperm were cut thinly with a sharp and clean razor blade, stained
with or without toluidine blue and covered with a cover glass. The specimens were examined and
photographed with a Zeiss photomicroscope. Plant materials of calli and bud-like structure derived
coconut endosperm in vitro were achieved using the protocol developed previously (Sukamto
2011b).
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Some calli of the three month-old culture in semi solid media, were cut with a sharp and
clean razor blade inside a petri plate containing a small volume of glutaraldehyde fixative. Thin
tissue sections (1 – 2 mm) were placed under vacuum two times for 1 hour each. Tissues were
washed three times with buffer for 10 minutes each and stored in the refrigerator overnight. Then
the tissues were fixed with 2% osmium tetroxide and washed three times with buffer. Specimens
were dehydrated in a graded series of ethanol (10% to 70%) for 15 minutes each. Specimens were
stored overnight in 70% ethanol in the refrigerator in stopper vials. The specimens were dehydrated
with graded series of ethanol (70%, 80%, 90%, 95% and 100%) in 30 minutes steps and transferred
into vials containing 5 ml of 100% ethanol and 1 ml historesin (HR) and mixed by swirling, caped
in vials and stored overnight at room temperature. HR was added daily in increasing volume (3 ml,
3 ml, 4 ml), finally in 2 changes of 5 ml of 100% HR.
Some calli of seven month-old in liquid media, were stained directly to the samples. Cell
suspensions were taken by using pipette onto a glass slide. A few drops of potassium iodide-iodine
(IKI) was added and covered with a cover glass. The specimens were examined and photographed
with a Zeiss photomicroscope.
An embryo structure of the 21 weeks-old culture (Figure 3a) grew to become bud-like
structure after 15 month-old culture (Figure 3b). The bud-like structure was cut longitudinally into
three parts and fixed immediately in a 50 ml mixture of 1% acrolene, 2% glutaraldehyde, 2% para
formaldehyde and 0.05 M sodium cacodylate buffer at pH 7.6 and placed twice under vacuum for 2
days. The fixative was decanted; the tissue was washed three times with buffer (0.05 M cacodylate)
for 30 minutes each, and stored in the refrigerator for several days. The specimens were transferred
to peel-away molds, aluminum pans or beem capsules containing embedding medium. The
containers were closed to exclude oxygen and placed in vacuum overnight. Polymerized blocks
were glued on wood blocks or plastic rods. These were sectioned with a rotary microtome at 5 – 10
µm. The sections were stained with toluidine blue, a combination of toluidine blue and acid
fuchsine, Feulgen-fast green stained DNA and counterstained against light green, or periodic acid
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Schiff (PAS) stained polysaccharides. Acid fuchsine stained most cells components, especially
mitochondria; toluidine blue was used as a counter stain; PAS was used to stain carbohydrates
(Gurr 1965).
RESULTS AND DISCUSSION
Fresh coconut endosperm and calli in semi solid media cultured
The fresh of coconut endosperm from young coconut fruit consist of parenchyma cells,
which relatively uniform in shape and size (Figure 1a). Its nuclei had one to five nucleoli (Figure
1a, 1b). The similar result was reported by Popielarska-Konieczna et al. (2013) in cereal
endosperm. The cells grew to become vary in shape and size in vitro cultured. Calli of the three
month-old culture were stained by acid fuchsin and toluidine blue, showed the tissue on the right
side was darker than those to the left (Figure 1c). The similar result was reported by Fernando et al.
(2003) in coconut plumule cultured in vitro and Popielarska-Konieczna et al. (2013) in cereal
endosperm cultured in vitro. The dark areas of cytoplasm had a lot accumulation of lipid bodies and
dense cytoplasm (Figure 1d). This result was agreed with Ceniza et al. (1992). The majority of lipid
bodies are rod-like shape and the others are droplet/ globular shape, which similar report by Liu
(2013) on ripening of chili pepper fruits. Intercellular spaces of callus tissue were connected with
fibrous and reticular structures. The similar result was reported by Popielarska et al. (2006) in
endosperm-derived callus of kiwifruit. The light area of calli had fewer lipid bodies and more
frequent cell divisions in many planes; some nucleoli appeared long (Figure 1e). Formation of fourcell proembryo structures developed from coconut endosperm culture (Figure 1f), which similar to
proembryo formed by segmenting division of coconut inflorescence culture (Verdeil et al. 1994).
Calli in liquid media
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Seven month-old calli in liquid medium showed proembryo structure developed from
coconut endosperm calli (Figure 2a). The proembryo resembled to embryo-like structure (ELS) in
soybean anther culture (Rodrigues et al. 2005). Proembryos composed of embryogenic cells
characterized by high nucleus to cytoplasm ratio, high starch, and protein reserves (Figure 2b, 2c,
2d). This result resembled to embryo and callus formation of pollen grain culture of coconut (Perera
et al. 2008). Formation of proembryo structures could be without suspensor (Figure 2e) or with
suspensor (Figure 2f). Suspensor of proembryo was important to increase embryo survival on the
development of early globular embryos (Paolicchi et al. 2003).
Bud-like structure in semi solid media
An embryo occurred on callus treated with picloram10-6 M after 21 weeks of culture (Figure
3a). The embryo grew to become bud-like structure after 14 months of culture (Figure 3b).
Histology of bud-like structure showed an apical meristem tip between cotyledonary notch, vascular
strand, and dermal layer (Figure 3c). This structure was primary meristematic state of apical
meristem, tended to organize shoot primordial of coconut embryo development (Guzman et al.
1978), resembled shoot bud proliferating on surface of Pinus eldarica (Wagley et al. 1987), and
revealed a well-structured shoot meristem toward whole coconut plant (Verdeil et al. 1994). There
were tracheids of xylem, inside of vascular strand and lipid droplets in the bud-like structure (Figure
3d). Similar lipid droplets were reported by Kanchanapoom & Tinnongjig (2001) in embryoid cells
of oil palm and Liu (2013) in plastoglobular shape of chili pepper fruits.
Somatic embryogenesis was occurred of coconut endosperm cultured. Many protuberances
were visible in the mantle, one of these resembled early stages of proembryo (Figure 3e). The
similar structure was reported by Dawayati et al. (2012) in embryonic callus of date palm. The
peripheral cells, especially the organized protuberances differed from cells in the adjacent internal
region. The mantle cells were smaller, more densely cytoplasm and had prominent dark nuclei. The
result was similar to cells of leaf and hypocotyl cultures in Torenia fournieri and Anagallis
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arvensis, which destined to become embryos, buds, and shoot apices (Reinert et al. 1977). The
appearance of protuberance has a one layered tunica which covers a large group of irregularlyarranged cells which could be the corpus (Figure 3f). This organization is found in regenerated
shoot bud, which similar to organogenesis in leaf-derived callus of black pepper (Sujatha et al.
2003) and endosperm-derived callus of kiwifruit (Popielarska et al. 2006). This tunica-corpus was
resembled a further organization of bud meristem in Foeniculum vulgare callus (Anzidei et al.
1996).
CONCLUSION
Fresh coconut endosperm consisted of parenchyma cells, where relatively uniform in shape
and size, some nuclei consisted of 1 – 5 nucleoli. Three month-old calli derived from coconut
endosperm in vitro developed cells that varied in shape and size. The calli composed of highly
vacuolated cells and small cells with dense cytoplasm, high nucleus to cytoplasm ratio, high starch,
protein, and lipid. Calli cells underwent many divisions and formed proembryo structures. Seven
month-old calli in liquid medium showed resembled proembryo structures. Fifteen month-old of
bud-like structure in semi solid medium showed meristematic layer with dermal layer, cotyledonary
notch, parenchymatous cells, globular proembryos, tunica-corpus structure, lipid droplets, and
central vascular strand with tracheids of xylem. The histological analysis of bud-like structure
indicated an early stages of shoot bud occurred in coconut endosperm in vitro.
ACKNOWLEDGEMENTS
I wish to thank Dr. D.T. Webb and Dr. Y. Sagawa for advising, Mr. R. Wutzke for giving
material experimentation and Overseas Training Office/ Badan Perencanaan Pembangunan
Nasional, Indonesia for giving fund.
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FIGURES
a
c
b
d
e
f
Figure 1. Fresh endosperm cells showed a). Relatively uniform in shape and size (bar = 4 µm) and
b). Nuclei consists of 4 – 5 nucleoli (bar = 10 µm). Fragmentation of the three month-old
calli consisted of c). Dark and light areas (bar = 10 µm), d). Dark area composed of cells
with dense cytoplasm and a lot of lipid components (bar = 2 µm), e). Light area
composed of highly vacuolated cells composed of many divisions of cells with few lipid
components (l) (bar = 2 µm), f). Formation of proembryos structures with several number
of cells (bar = 20 µm)
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a
b
c
d
a
f
e
a
a
Figure 2. a). Formation of proembryo composed of seven cells (bar = 4 µm), b). Formation of cells
containing starch grains (bar = 4 µm), c). Formation of proembryo composed of seven
month-old calli in vitro in liquid medium (bar = 4 µm), d). Formation of proembryonal
cell complex composed of several proembryo units, showing segmented division (bar = 2
µm), e). Formation of three-celled structures of proembryos in liquid medium (bar = 4
µm), f).
Formation of five-celled structures of proembryo with suspensor in liquid
medium (bar = 2 µm)
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a
b
l
c
e
t
d
f
Figure 3. a). Embryo structure occurred after 21 weeks of cultured (bar = 1 mm), b) Embryo
grew to became bud-like structure (bar = 1 mm) after 14 months of culture, c). Cross
section of the bud-like structure showing cotyledonary notch (s) and vascular strand
(v) toward apical meristem (t) (bar = 30 µm), d). Enlargement of vascular strand
showing parenchymatous cells, tracheids (t) of xylem and lipid droplet (l) (bar = 10
µm), e). Somatic embryogenesis on meristematic area showed globular proembryo (e)
(bar = 10 µm), f). Protoderm layer of tunica (t) and corpus (c) in meristematic area
(bar = 50 µm)
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