THE SURVIVAL AND THE GENETIC FIDELITY OF COCONUT EMBRYOS AFTER CRYOPRESERVATION

  

THE SURVIVAL AND THE GENETIC FIDELITY OF COCONUT EMBRYOS AFTER

CRYOPRESERVATION

1,2 ( ) 1,3

  4

  1

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

  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

  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 by diseases, natural disasters and through land cleaning for the planting of the so called ‘more valuable’ crops. Coconut seed cannot be conserved by normal means as it is big and sensitive to drying (recalcitrant). Cryopreservation is an alternative conservation approach but as yet, this method is still poorly developed for coconut. Thus, there is a need to develop a new, more reliable cryopreservation protocol for this crop. A new cryopreservation protocol, based on a rapid tissue desiccation step was developed to produce a high plant recovery rate after cryopreservation. Embryos from four coconut cultivars (Malayan Yellow Dwarf - MYD, Nias Yellow Dwarf - NYD, Nias Green Dwarf and Sagerat Orange Dwarf) were rapidly desiccated for 8

• 1

  hours (water content decreased from 80 % fresh weight or 4.14 g g dry weight to 19 % fresh

• 1

  weight or 0.24 g g dry weight). This dehydration pre-treatment prevented ice crystal formed in the embryo cells during freezing and a differential scanning calorimetry analysis approach indicated that the glass transition temperature had been raised from ca. –123 to –48

  C. Upon recovery from liquid nitrogen, 24 hours later, the percentages of embryos that produced normal seedlings was high and varied from 21 % in NYD to 40 % in MYD. Most of the seedlings produced after cryopreservation could be field planted after a further 4 months growth in the glasshouse. In general, genetic fidelity testing undertaken on these plants showed that no morphological abnormalities could be detected. In addition, karyotype analysis also showed no ploidy level or gross chromosomal abnormalities to have taken place. While molecular analysis, using microsatellite and global DNA methylation techniques, revealed no significant differences in the genomic DNA isolated from seedlings that developed from cryopreserved or non-cryopreserved embryos. KEYWORDS: Cocos nucifera L., Cryopreservation, Genetic fidelity, Karyotype, 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 (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 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

  ®

  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.

  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

  C) 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 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 (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, Minitab (Release 15).

  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 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.

  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. 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.

  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 ((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 potato (Harding and Staines 2001). Indeed, in some species, although growth differences between cryopreserved and non-cryopreserved plants can be seen in the glasshouse, the differences may 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.

  N-Banding Analysis

  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 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.

  Bibliography

  Abraham A, Mathew PM (1963) Cytology of coconut endosperm. Annals of Botany 27 (107):505 - 513