CYTOLOGICAL AND MOLECULAR LEVEL OF COCONUT (Cocos nucifera L.) SEEDLINGS RECOVERED FROM CRYOPRESERVATION - Digital Library Universitas Muhammadiyah Purwokerto

  

CYTOLOGICAL AND MOLECULAR LEVEL OF COCONUT (Cocos nucifera L.)

SEEDLINGS RECOVERED FROM CRYOPRESERVATION

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• Sisunandar , Yohannes Samosir , Alain Rival and Steve W. Adkins .

  1) The Muhammadiyah University of Purwokerto, Biology Department, Kampus Dukuhwaluh, Purwokerto, INDONESIA, 53182. Sisunandar@yahoo.com . Ph. +6285869990309, Fax. +62 281637239

  2) Present address : Indonesian Oil Palm Research Institute, Medan, North Sumatera,

  INDONESIA 3) Institut de Recherche pour le Developpement, Montpellier, FRANCE 4) The University of Queensland, Integrated Seed Research Unit, School of Land Crop and

  Food Sciences, Brisbane, AUSTRALIA 4072

  

ABSTRACT

  Coconut (Cocos nucifera L.) plays an important role in the socio-economic life of many millions of people in the tropical regions of the world. However, the genetic diversity of this species is being dramatically decreased and consequently there is a need to undertake coconut germplasm conservation. Cryopreservation is one technique that has been used to conserve genetic materials of many plant species including coconut. A new cryopreservation protocol has been developed and evaluated for its effects upon genetic fidelity. Ygotic embryo from three Indonesian cultivars, Nias Green Dwarf (NGD), Nias Yellow Dwarf (NYD), and Sagerat Orange Dwarf (SOD) were recovered from cryopreservation and germinated into seedlings. The chromosomes isolated from the roots of 5-month old seedlings were examined using N-banding techniques followed by CHIAS 3 analysis to produce idiograms. Cryopreservation did not cause a change in ploidy level and type of chromosome present. Using molecular approaches, microsatellite and global DNA methylation techniques revealed no significant differences between genomic DNA isolated from seedlings that developed from cryopreserved embryos as compared to the genomic those isolated from seedlings that developed from non-cryopreserved embryos. However, these responses were cultivar dependent. The seedlings of NGD that developed from cryopreserved embryos showed significant changes in their chromosome size (the shortening of one arm linked to the lengthening of the other arm), the number of black bands (12 more) and DNA methylation status (1,09 %) as compared to those developed from non-cryopreserved embryos.

  KEYWORDS

  : Coconut, Karyotype, microsatellite, DNA methylation

  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 (see review of 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; Ryynanen and Aronen, 2005). At the cytogenetic level, the karyological information collected on chromosome number indicates that cryopreservation rarely inflicts major ploidy level changes (Hao et al., 2002a; Helliot

  

et al., 2002; Urbanova et al., 2002; Ryynanen and Aronen, 2005; Urbanova et al., 2006). However,

  testing in DNA methylation level revealed that cryopreservation caused significant changes in DNA methylation status (Harding et al., 2000; Hao et al., 2001; Hao et al., 2002a; Hao et al., 2002b; Channuntapipat et al., 2003).

  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 cryopreserved (subsequently known as ‘non-cryopreserved seedlings’).

  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

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  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

  C) 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.

  Cytogenetic analysis

  Actively growing secondary roots (c. 1 cm long) were excised from 20 week-old seedlings at night. The time of isolation (20.00 to 20.30 pm) previously a time identified to be the best for the easy of isolation and karyotype analysis. The excised root tips (c. 2 - 3 mm) were pre-treated in a 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

  C. 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

  C. 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 C 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 C in an incubator before being stained using the N-banding technique Gerlach, 1977 with some slight modifications. Firstly, the slides were soaked in NaH

  2 PO 4 (1 M), pH 4.2 at 88 C 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 (Apisitwanich et al., 2001; Fukui, 2005; Ohmido et al., 2007).

  DNA extraction

  Genomic DNA from first and/or second opened leaf of 10-month old seedlings (three seedlings per cultivar) that developed from cryopreserved and non-cryopreserved embryos was extracted from individual seedlings using a modified Hexadecyltrimethyl-ammoniumbromide (CTAB) method (Hoisington, 1992). The DNA samples were transferred from the University of Queensland, Australia to the Institut de Recherche pour le Developpement (IRD), Montpellier, France, for further analyses. Upon arrival, the DNA concentration of each plant sample was determined using a Fluoroskan Ascent FL fluorometer (Thermo Electron Corp., Waltham, MA, USA). The DNA quality was assessed using an agarose gel (1.2 %) electrophoresis.

  Global DNA methylation analysis

  The DNA methylation analysis was performed using Jaligot et al. (2000) method with a small modification. DNA samples (20 µg) were individually mixed with 10 µL of nuclease P1 (0.5 U

• 1
• 1

  mL ; Sigma N8630) and 35 µL alkaline phospatase (0.0168 U mL ; Sigma P4252), and adjusted to a volume of 200 µL with digestion buffer (30 mM NaCH

  3 COO, 0.1 mM ZnCl 2 , pH 5.3). Then,

  the samples were placed into a water bath (37 ± 1 C) and agitated every hour for a total of 4 hours. The hydrolysis reaction in each tube was stopped by adding 490 µL of absolute ethanol. Following this, the samples were centrifuged (17 000 rpm, 4 ± 1

  C) for 15 minutes. The supernatants were then transferred into new tubes and vacuum-dried for 1 to 2 days. The nucleosides in each tube were then redissolved in 1 mL H

  to high-performance liquid chromatography (HPLC) analysis (Shimadzu Prominence HPLC, Shimadzu Co.,Kyoto, Japan). An isocratic elution protocol with buffer (50 mM KH

  2 PO 4 , 8 %

  methanol (v/v), pH 3.5) was performed using a 250 x 4.6 mm Ultrasphere ODS reverse-phase column (particle diameter 5 µm; Beckman Coulter France S.A.S, Paris, France), with a flow rate of

• 1

  0.8 mm minute and a run time of 23 minutes. The effluence was monitored with a photodiode array detector (Shimadzu Prominence SPD-M20A) at a wavelength of 277 nm for 2’-deoxycytidine

  (dC), and at 285 nm for 5-methyl-2’-deoxycytidine (5mdC). The percentage of 5mdC was calculated according Jaligot et al. (2000) using the formula: 100 × [5mdC] / ([5mdC] + [dC]), where the [5mdC] and [dC] are respective concentrations of the two form of dC.

  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, Minitab (Release 15).

  RESULTS 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 (Figures 3 to 5). 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 (Tables 1 to 3). Only one chromosome was found to be subterminal (chromosome 9 in seedlings that developed from non- cryopreserved embryos of NGD, Table 3). 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.

  In general, length comparisons in the total of short arms, the total of long arms and arm ratio showed no significantly difference between chromosomes isolated from seedlings developed from cryopreserved and non-cryopreserved embryos (Table 1 to 3). 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 (Tables 1). However, only three chromosomes (number 4, 5 and 16) were statistically different. However, SOD chromosomes showed contrasting results to that of NYD (Table 2). 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 (Table 3). 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 (Figures 3 to 5). 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 (Figure 3.C and D). 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 (Figure 4.C and D). 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 (Figure 5.C and D).

  Molecular analysis

  In general, the percentage of global DNA methylation (Figure 6) seen in the genomic DNA isolated from seedlings recovered from cryopreserved embryos (21.50 %) was lower than that seen in genomic DNA isolated from seedlings recovered from non-cryopreserved embryos (21.74 %). However, the difference was only found to be significant in one cultivar. For SOD, the percentage of global DNA methylation was the same, irrespective of the cryopreservation treatment (21.74 %). An opposite result was observed in NYD, in which the percentage of global DNA methylation increased slightly from 21.18 % in the seedlings recovered from cryopreserved embryos, to 21.39 % in the seedlings recovered from non-cryopreserved embryos. However, the differences in the percentage of global DNA methylation in each of these two cultivars (SOD and NYD) were not statistically significant. In contrast, the percentage of global DNA methylation in NGD was significantly different in cryopreserved and non-cryopreserved samples. The percentage of global DNA methylation was significantly lower in the seedlings that developed from cryopreserved embryos (21.23 %) than in the seedlings that developed from non-cryopreserved embryos (22.32 %; Figure 7).

  DISCUSSION 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 (see review of 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.

  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 (Figures 3 to 5) was present. The observed number of chromosomes concurs with all other previous cytological studies undertaken on coconut

  (Nambiar and Swaminathan, 1960; Abraham et al., 1961; Abraham and Mathew, 1963; Ninan and Raveendranath, 1975; Fisher and Tsai, 1978; 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 (Table 1 to 3), 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 (Table 1 and 3). 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 (Table 2). 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 (Figures 3 to 5) 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 (see review of 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 cryopreserved embryos has been reported in previous studies on coconut ( Bajaj, 1984; Chin et al., 1989; Assy-Bah and Engelmann, 1992), but no definitive reasoning for the observations was offered.

  The average global methylation rate (Figure 4) seen in the genomic DNA isolated from seedlings that developed from cryopreserved embryos (21.50 %) and non-cryopreserved embryos (21.74 %) were similar to DNA methylation rates seen in other species. For example, the DNA methylate rate was 25.00 % in tomato (Messeguer et al., 1991), 24.60 % in potato (Messeguer et al., 1991), 27.80 % in tobacco (Nicotiana tabacum L. ;Messeguer et al., 1991), 20.39 % in oil palm (Jaligot et al., 2000), and 25.00 % in ribes (Johnston et al., 2005). This means that the plants recovered from cryopreservation were similar to those recovered from non-cryopreservation.

  In general, no significant changes in the DNA methylation status was induced by the cryopreservation process (Figure 4). However, the DNA methylation was cultivar-dependent. A significant decrease (1.09 %) in the global DNA methylation rate was observed after cryopreservation in NGD, while in the other cultivars (i.e. NYD and SOD), no such differences were noted. The reason for this result for NGD is still not clear. However, it is interesting to note that this cultivar is one that is particularly sensitive to environmental stress (Novarianto et al., 2003).

  The kind of changes in DNA methylation status seen in NGD seedlings that developed from cryopreserved embryos have been reported in other species including mahogany (Harding et al., 2000), paradise apple (Hao et al., 2001), Citrus spp. (Hao et al., 2002b), wild strawberry (Hao et

  

al., 2002a) and almond (Channuntapipat et al., 2003). The DNA methylation changes seen in NGD

  could have occurred in response to dehydration, cryopreservation or tissue culture recovery. The water stress that may occur during dehydration process, for example, has been proved to cause an increase or decrease in DNA methylation status (Lukens and Zhan, 2007). Tissue culture used in recovery step after cryopreservation has also been shown before to cause changes in DNA methylation status (see review Mc Clintock, 1984; Kaeppler and Phillips, 1993; Phillips et al., 1994; Rani and Raina, 2000).

  Interestingly, the lower DNA methylation rates in NGD seedlings that developed from cryopreserved embryos (ca. 1.09 % lower than in non-cryopreserved seedlings) may associate with certain chromosome morphological changes (i.e. a shorter length in one arm of the chromosome and a longer length in another arm). This kind of change may indicate the chromosome breakages occured in NGD seedlings that developed from cryopreserved embryos. It has been reported before that DNA methylation changes may cause changes in chromatin structure (Kaeppler and Phillips, 1993; Phillips et al., 1994; Rani and Raina, 2000), or may lead to a greater rate of chromosome breakage or may change gene expression (Kaeppler and Phillips, 1993; Rani and Raina, 2000). This kind of change in DNA methylation status was not observed in SOD in which no different on their arm ratio of their chromosomes were observed (chromosome length on the both short and long arms).

  N-banding analysis revealed that NGD seedlings that developed from cryopreserved embryos showed the highest addition of black banding compared to NYD and SOD. Interestingly, this banding may correlate to DNA methylation status of the NGD seedlings. The black bands resulted from N-banding analysis has been suggested to be the areas that are very rich with ‘housekeeping’ genes (Holmquist, 1989). Coincidently, DNA methylation status has also been reported to cause some changes in ‘housekeeping’ genes (Rani and Raina, 2000). The increasing number of N- banding region may indicate some changing in the number of housekeeping genes as the response to the changes in DNA methylation status. Certainly, further investigations are required to verify the direct relationship between changes in DNA methylation status and specific chromosome regions affected by this change.

  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.

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