THE SURVIVAL AND THE GENETIC FIDELITY OF COCONUT EMBRYOS AFTER CRYOPRESERVATION
Sisunandar1,2 (*)
, Yohannes Samosir1,3, Alain Rival4 and Steve W. Adkins1.
1) The University of Queensland, Integrated Seed Research Unit, School of Land Crop and Food Sciences, Brisbane, AUSTRALIA 4072
2) Present address : The University of Muhammadiyah Purwokerto, Biology Education Department, Kampus Dukuhwaluh, Purwokerto, INDONESIA, 53182.
Sisunandar@yahoo.com. Ph. +6285869990309, Fax. +62 281637239
3) Present address : Indonesian Oil Palm Research Institute, Medan, North Sumatera, INDONESIA
4) Institut de Recherche pour le Developpement, Montpellier, FRANCE
ABSTRACT
INTRODUCTION
Coconut plays an important role in the socio-economic life of many millions of people in the
tropical regions of the world. However, this species is suffering from a number of important pests
and diseases and its coastal habitat is highly susceptible to certain kinds of natural disaster. In
addition, many old coconut plantations are now being removed in some countries to make way for
the planting of potentially higher valuable crops. All of these factors are resulting in the loss of
traditional, locally adapted coconut germplasm and consequently there is a need to undertake
coconut germplasm conservation.
Cryopreservation is one technique that has been used to conserve genetic material of many
plant species including coconut (Bajaj 1984; Chin et al. 1989; Assy-Bah and Engelmann 1992;
Hornung et al. 2001; Malaurie et al. 2004; N'Nan et al. 2008). However, as yet the technique
applied to coconut is inconsistent and the success of recovery is highly variable between cultivars.
Thus, a new technique for coconut cryopreservation utilizing a desiccation step has been proposed
(Sisunandar et al., 2005) and is being evaluated.
Apart from the developmental aspects of such treatments before and after cryopreservation,
the uniformity testing of established plants is a crucial step for the success of the cryopreservation
system. The degree of genetic fidelity found in cryopreserved tissue and recovered plants have been
studied in a range of plant species and using a range of fidelity tests. The kind of fidelity test used
includes those that study the recovered plant phenotype, cytogenetic, and aspects of their
biochemistry or DNA structure (Harding 2004).
The results from such tests are contradictory with some reports showing no significant
differences between plants derived from cryopreservation as compare to non-cryopreserved tissues,
while other reports indicate significant differences. At the phenotypic level, several previous cases,
the growth rate of the cryopreserved plants was less than that observed for non-cryopreserved plants
(Moukadiri et al. 1999; Harding and Staines 2001). However, in many other cases, no such
differences were observed (Ahuja et al. 2002; Wilkinson et al. 2003). At the cytogenetic level, the
karyological information collected on chromosome number indicates that cryopreservation rarely
inflicts major ploidy level changes (Hao et al. 2002b; Urbanova et al. 2006).
In coconut, genetic fidelity testing of the plants coming from cryopreservation has not been
reported. The present paper brings together observations on phenotypic, cytological and molecular
levels, applied to seedlings coming from embryos that had been cryopreserved (from here-on,
known as ‘cyopreserved seedlings’) and also seedlings coming from embryos that had not been
MATERIALS AND METHODS
Plant material, cryopreservation and embryo culture methods
Embryos of coconut cv. Nias Yellow Dwarf (NYD), Nias Green Dwarf (NGD) and Sagerat
Orange Dwarf (SOD) were isolated from 11 month old fruits which were harvested from the field of
Mapanget Coconut Genebank, Manado, Indonesia. The methods used to isolate and surface
sterilize were those developed by COGENT (Rillo 2004) with some minor modification. Briefly,
this involved the isolation of the solid endosperm cylinders containing the embryos from fruits in
the field and their transportation back to the laboratory in a glass jar filled with coconut water. In
the laboratory, the cylinders were washed several times with tap water and quickly rinsed with
ethanol (95 %, v/v). Then the embryos were isolated from the endosperm cylinders under sterile
condition in a laminar air flow cabinet followed by surface sterilization using a sodium hypochlorite
solution (2.6 %, v/v) applied for 15 min, then several rinses in sterile water. The embryos were then
blotted dry on sterile filter papers before being physically dehydrated in a rapid drying chamber for
8 hours ((Sisunandar et al. 2005)), prior to being individually placed into 2 mL cryovial (TPP®, Trasadingen, Switzerland) which were then immersed into liquid nitrogen for 48 hours. After the
vials were removed and thawed in a water bath (40 ± 1 0C) for 3 minutes, then the embryos were
cultured into a liquid embryo culture medium (Rillo, 2004) for 4 weeks. After germination had
taken place, the seedlings were cultured on a solid medium (Rillo, 2004) for 8 weeks, followed by a
second liquid medium for the weeks following this until the seedling were ready to be acclimatised
and eventually established in soil approximately 8 weeks later.
Phenotypic analysis
After 16 weeks of acclimatisation in the glasshouse, all seedlings were removed from the
potting compost, cleaned carefully with running tap water, and blotted dry with paper towels. Other
observations were made on their shoot length, the number of opened leaves present, the number of
primary roots present, and the total length of the primary roots system. The seedling FWs were then
determined as were their DWs following drying in an oven (70 ± 2 0C) for 3 weeks, or until a constant weight had been achieved.
Cytogenetic analysis
Actively growing secondary roots (c. 1 cm long) were excised from 20 week-old seedlings at
dark condition with 8-hydroxyquinoline (2 mM) for 2 hours on a rotary shaker (100 rpm) at room
temperature, followed by dipping in a fixative solution (absolute ethanol and glacial acetic acid, 3 :
1, v/v) for 72 hours at 5 ± 1 0C. After washing the root tips with a series of ethanol solutions of
decreasing concentration (70; 50; 30 and 15 % each for 3 min), they were twice washed in aquadest
(10 min each wash), then the meristematic tissue (c. 1 mm long) were removed from the root caps
and other dead cell layer using a sharp scalpel blade before being hydrolysed in hydrochloric acid
(2.5 M) for 15 min and at 45 0C. They were then washed in NaEDTA (10 mM, 10 min) and
digested with an enzymes mixture of cellulase (2 %, Onozuka R-10, Yakult Honsa Co Ltd.) and
macerozyme (1 %, R-10, Yakult Honsa Co Ltd.) in NaEDTA (10 mM) at pH 4.0, 37 0C for 6 hours.
The root tips were washed again in aquadest and placed on a slide with a drop of the fixative
solution, then tapped with the tip of a fine forcep to create small, almost invisible cell massed. The
slides were then air dried for 24 hours at room temperature and stored at 5 0C in an incubator before
being stained using the N-banding technique (Gerlach 1977) with some slight modifications.
Firstly, the slides were soaked in NaH2PO4 (1 M), pH 4.2 at 88 0C for 20 min. Then, after washing
with aquadest and air drying, the slides were stained with giemsa (4 %) for 10 minutes. They were
washed again with aquadest and once more air dried at room temperature. Finally, the chromosomes
were viewed and photographed under an Olympus BX 51 microscope under 1,000 time
magnification with an Olympus DP 70 camera on. The photographic files were then stored in 2040
x 1536 pixel format for banding pattern analysis.
The chromosome short arm (p) and long arm (q) were measured using free access computer
software ImageJ 1.37v (Rasband 2006). Chromosomes were classified on the basis of arm ratio (r =
q/p) using Levan’s nomenclature ((Levan et al. 1964)). The construction of idiograms are based on
a computer software analysis, CHIAS3 ((Kato et al. 2004)) of six metaphase plates. The software
manual as described by Kato et al. (2004) was followed to produce idiograms as has been used to
produce idiograms of several species (Ohmido et al. 2007; Fukui 2005)).
Statistical analysis
All data sets were statistically analysed for variance using ANOVA, and the statistical comparisons
in each all data set between seedlings recovered from cryopreserved embryos and seedlings
recovered from non cryopreserved embryos (Chapter 8 and 9) were undertaken using Student’s
t-test at a 0.05 significance level. The analyses were performed using the statistical software package,
RESULTS
Phenotypic analysis
After 16 weeks of acclimatisation in the glasshouse, the growth rate of the seedlings coming
from non-cryopreserved was little more slowly than those from cryopreserved embryos. However,
only fresh and dry weights of NGD seedlings and length of shoot and primary root of NYD were
different significanly, while other growth characteristics and other cultivars showed no significant
different between cryopreserved and non-crypreserved seedlings. The FW of the seedlings that
grew from non-cryopreserved embryos ranged from 14.3 g (or 2.2 g DW) in SOD, to 44.9 g (or 8.1
g DW) in NGD. Meanwhile the FW of seedlings from cryopreserved embryos ranged from 6.9 g (or
1.0 g DW) in NYD, to 21.5 g (or 3.4 g DW) in NYD. The shoot lengths for the seedlings from
non-cryopreserved embryos varied from 25.3 in SOD to 37.7 cm in NGD, while in the non-cryopreserved
seedlings it varied from 18.0 cm in NYD to 30.3 cm in NGD. The number of expanded leaves
produced was ca. three to five leaves in the seedlings coming from non-cryopreserved embryos but
ca. two to four leaves in the seedlings coming from cryopreserved embryos. The total length of the
primary root in seedlings coming from the non-cryopreserved seedlings varied from 23.8 cm in
SOD to 47.6 cm in NGD, while in the seedlings coming from cryopreserved embryos ranged from
15.7 cm in NYD to 29.5 cm in NGD. The number of primary roots produced also varied from two
to four both in the seedlings that developed from non-cryopreserved and cryopreserved embryos.
Overall, the results obtained from these morphological character analyses showed that seedlings
from both cryopreserved and non-cryopreserved embryos were undergoing a normal development
pattern with healthy shoots and roots.
Cytogenetic analysis
Karyotype analysis of the seedlings that developed from cryopreserved and non-cryopreserved
embryos showed that all were diploid with a chromosome number of 2n = 32. The chromosomes
from the two seedling types had a similar centromeric index and relative length, and based on their
centromeric position, were all median and submedian types. Only one chromosome was found to be
subterminal (chromosome 9 in seedlings that developed from non-cryopreserved embryos of NGD.
In general, the chromosomes of the three cultivars were all small to medium in length. They ranged
from 1.80 to 5.47 µm in seedlings that developed from non-cryopreserved embryos, and from 1.96
to 6.40 µm in seedlings that developed from cryopreserved embryos.
cryopreserved and non-cryopreserved embryos. Similarly, the comparison on the total length, the
difference in length between arms, the centromeric index and the relative length also also showed
no significant difference (data not shown). However, these responses were cultivar-dependent. For
NYD, five chromosomes (number 1, 4, 5, 6 and 16), with of the seedlings that developed from
cryopreserved embryos were being longer in the short arms, and shorter in the long arms than their
non-cryopreserved counterparts. One chromosome (7) showed an opposite trend, being shorter in
the short arm. However, only three chromosomes (number 4, 5 and 16) were statistically different.
However, SOD chromosomes showed contrasting results to that of NYD. Nine out of the 16
chromosome sets (1, 2, 3, 4, 6, 9, 10, 11 and 12) showed overall length increases in both their short
and long arms following cryopreservation, with only two chromosomes (14 and 16) exhibiting no
statistically detectable length changes. However, these changes did not affect on their arm ratio of
all 16 chromosomes. For NGD, six chromosomes from seedlings that developed from
cryopreserved embryos (numbers 3, 4, 5, 7, 9 and 16) were longer in their long arms and shorter in
their short arms than were the same chromosomes isolated from non-cryopreserved samples. Two
further chromosomes (numbers 2 and 6) were shorter in their long arms and longer in their short
arms. However, of all the chromosomes that differed between cryopreserved and non-cryopreserved
samples, only three chromosomes (number 3, 7 and 9) were exhibited differences that were
statistically significant both on their long arm and arm ratio.
The chromosome idiograms of seedlings that grew from cryopreserved embryos exhibited a
greater number of black bands than did the idiograms of seedlings that grew from
non-cryopreserved embryos. For NYD, eight more black bands were observed on chromosomes isolated
from seedlings that developed from cryopreserved embryos than was observed on chromosomes
from seedlings that developed non-cryopreserved embryos. The additional black bands mostly
appeared on the short arm (numbers 11, 12, 14 and 15), while two chromosomes (number 5 and 6)
had additional black bands on their long arms. Chromosome 16 showed additional black bands on
both its short and long arms. For SOD, the additional 10 black bands were observed on the long
arms (numbers 2, 3, 4 and 12) or on the short arms (numbers 8, 9 and 10), while one chromosome
(number 1) showed additional black bands on both arms. For NGD, The additional 12 black bands
were observed mostly on the long arms (i.e. in chromosomes numbers 1, 2, 3, 5, 6, 7, 13 and 16),
with the extra bands occurring on the short arm of only one chromosome (number 12). Finally, one
chromosome (number 4) showed 3 more black bands on each arm in samples that were
cryopreserved as compared to non-cryopreserved samples.
When plant tissues are stored at low temperatures, changes can occur in their cell cytoplasm.
Such changes may include the accumulations of certain compounds and ice crystals, and changes in
their cell membrane structure ((Berjak and Pammenter 2004)). As a result, cells may die or the
genetic fidelity of regenerated plants from such treated cells may be adversely affected. With
appropriate cryopreservation pre-treatments, such as tissue desiccation, some of this
freezing-induced cell and tissue death can be prevented (see review of Engelmann, 2004). However, when
not applied properly, the desiccation pre-treatment steps themselves can also lead to cell and tissue
death. Seed tissues of recalcitrant species such as coconut are particularly sensitive to drying. Tissue
desiccation has been shown to not only cause the loss of structural integrity of certain cells, cellular
metabolism failure, production of free radicals, and to damage the protective antioxidant system of
the cell (see review of Pammenter and Berjak, 1999; Berjak and Pammenter, 2001), but also to
affect genetic fidelity. In addition to the steps of desiccation and freezing, tissue thawing and
recovery steps can also cause cell death and genetic fidelity changes (see review of Harding, 2004).
Thus, seedlings recovered from explants that have been cryopreserved following a dehydration
pre-treatment step and then recovered using a tissue culture step, may lose genetic fidelity due to one or
all of these processes.
Phenotypic analysis
No morphological abnormalities were observed in any of the seedlings that survived
acclimatisation, and this was true for those that came from cryopreserved and non-cryopreserved
embryos . The normal development of seedlings recovered following embryo cryopreservation has
also been reported in other species such as Grecian fir (Abies cephalonica Loudon; (Aronen et al.
1999), apel paradise (Hao et al. 2001) yam ((Ahuja et al. 2002)), aerial yam (Dixit et al. 2003), and
silver birch (Ryynanen and Aronen 2005).
Although the seedlings developed from cryopreserved embryos had normal morphology, these
seedlings generally had much slower growth rates than those from non-cryopreserved embryos.
This has been seen before in other species (Moukadiri et al. 1999). It is believed that the differences
seen were not due to genetic change rather due to slow re-growth of the seedlings following
cryopreservation. This slower growth of the plants coming from cryopreserved embryos may only
be observable during early stage of growth. This kind of slow early growth in plants coming from
cryopreservation has been reported in other species, such as in rice (Moukadiri et al. 1999) and
become negligible after some time in the field. For example, in Saccharum species, cryopreserved
plants showed lower growth than non-cryopreserved plants during the first 6 months of a field trial,
yet after a further 6 and 9 months, the cryopreservation treatments were no longer distinguishable
(Martinez-Montero et al. 2002). We were unable to test this in the present study due to constraints
of the Australian Quarantine Service Permit which did not allow us to grow plant to such advanced
stages in soil.
Karyotypic Analysis
For coconut, this study represents the first karyotype analysis that has been undertaken on
seedlings recovered from cryopreservation. The results obtained show that the recovered seedlings
had the same ploidy level as those that were not subjected to cryopreservation. This finding is
similar to that seen in a number of other species (Hao et al. 2002a; Helliot et al. 2002; Urbanova et
al. 2002; Urbanova et al. 2006) where cryopreservation did not lead to changes in ploidy level.
Seedlings of all three cultivars that developed from cryopreserved or non-cryopreserved embryos
were diploid with a chromosome number of 32 was present. The observed number of chromosomes
concurs with all other previous cytological studies undertaken on coconut (Nambiar and
Swaminathan 1960; Abraham and Mathew 1963; Abraham et al. 1961; Da Vide et al. 1996).
The length comparison between chromosomes isolated from seedlings that grew from
cryopreserved and non-cryopreserved embryos was that the total of short arms, the total of long
arms and the sum of total length also showed the same response, as well as the comparison on the
arm ratio, the difference in length between arms, the centromeric index and the relative length were
not significantly different. However, these responses were cultivar-dependent. Chromosomes of
NYD and NGD showed significant changes on their arm lengths especially the shortening of one
arm linked to lengthening of the other arm. These changes caused three chromosomes of NYD and
NGD showed significant differences on their arm ratio. In contrast, nine chromosomes of SOD out
of 16 chromosomes showed overall increases in both their short and long arm, but no one of the
chromosomes showed significant change on their arm ratio. The reason for these changes is unclear;
however, these cultivars may be particularly sensitive to drying or respond differently to recovery in
tissue culture.
The N-banding technique demonstrated that chromosomes that were isolated from seedlings
produced from cryopreserved embryos had a greater amount of banding than those chromosomes
isolated from seedling developed from non cryopreserved embryos with NGD showed the highest
increased in black banding (12 black bands) when compared to NYD and SOD (8 and 10 more
black bands, respectively). The fact that more black bands were observed on chromosomes isolated
from seedlings that developed from cryopreserved embryos may indicate that chromosomal changes
were induced by the cryopreservation treatment. As the location of N-banding on the chromosomes
may indicate an area that is particularly sensitive to denaturation of chromosomal protein
(Holmquist 1989), dehydration may be the reason for the change in N-banding patterns. The
increased number of N-banding regions may also indicate some changes in the number of
‘housekeeping’ genes (Holmquist 1989), genes that are responsible for the maintenance of the basal
cellular function (Eisenberg and Levanon 2003). If this is the case, it may explain the reason why
the embryos recovered from cryopreservation exhibited a slower growth rate than
non-cryopreserved embryos. The slower growth of non-cryopreserved embryos has been reported in previous
studies on coconut (Bajaj 1984; Chin et al. 1989) but no definitive reasoning for the observations
was offered.
The results obtained in this report revealed that it is strongly recommeded that all plants that
developed from cryopreserved materials should be monitored for potential genetic changes using
both chomosomal and molecular analyses. The reason is that no single method alone can be used to
assess genetic changes that may occur in the genome (Harding, 2004). In general, the phenotype
analysis showed no morphological abnormalities were observed, as well as the chromosome
analysis resulted from this study revealed that no changes on ploidy level and type of chromosome
present in recovered plants. The molecular analyses using DNA methylation also revealed that no
variations were observed on the seedlings recovered from cryopreservation. However, this response
is cultivar dependent. In the case of NGD, the genetic changes were observed in chromosomal and
molecular level. Thus, given the results of this study, it is strongly recommended that the genetic
fidelity of coconut materials be scrutinised during and after cryopreservation. This is particularly
important for coconut as it is a long-lived species where the effect of genetic change may not be
immediately obvious in the morphology of young plants, but could be expressed later in mature
trees.
