Dehydration improves cryopreservation of coconut (Cocos nucifera L.)

  Dehydration improves cryopreservation of coconut (Cocos nucifera L.) q

  [12,31,38] specific tissues or organs that could be regenerated into

  

Cryobiology

  Contents lists available at ScienceDirect

  

Cryobiology 61 (2010) 289–296

  E-mail address: Sisunandar@ump.ac.id (Sisunandar). ₁ Present address: Indonesian Oil Palm Research Institute, Jl Brigjen Katamso 51, Medan, North Sumatera, Indonesia.

  Corresponding author. Present address: The Muhammadiyah University of Purwokerto, Biology Education Department, Kampus Dukuhwaluh, Purwokerto 53182, Indonesia. Fax: +61 62281637239.

  ⇑

  doi: 10.1016/j.cryobiol.2010.09.007 ^q This work was supported by the Australian Agency for International Develop- ment (AusAID), the Australian Centre for International Agricultural Research (ACIAR), the Graduate School Research Travel Grants (GSRTG) scheme from the University of Queensland and Endeavour Research Fellowships Australia 2010.

  plants and retain them under protected conditions. However, to be able to apply cryopreservation techniques for germplasm conser- vation of coconut on a routine basis, the technique should not only be able to store germplasm for many years, but also ensure that all regenerated plants produced from preserved material are true-to- type. Recently, genetic and epigenetic assessment of seedlings recovered from cryopreserved coconut material have shown that 0011-2240/$ - see front matter ! 2010 Elsevier Inc. All rights reserved.

  tain and are subjected to a number of environmental stresses, pests and diseases. An alternative approach would be to cryopreserve

  Sisunandar a ,

  ex situ seed gardens [5] . However, these gardens are costly to main-

  Up to now, coconut germplasm conservation has been based on

  ! 2010 Elsevier Inc. All rights reserved. Introduction Coconut (Cocos nucifera L.) is a major tropical crop, grown on ca. 10.7 million ha and producing ca. 55 million t (Mt) of dehusked fruit per annum [18] . However, this palm is susceptible to various abiotic factors and its development is hampered by a number of production constraints. Present plantations, which consist of unim- proved cultivars that are now over 50 years old, are declining in productivity due to several devastating diseases (e.g. lethal yellow- ing, cadang-cadang) or pests (e.g. Oryctes and Brontispa); they are subsequently being removed and the land is being used for other purposes. A direct consequence of this reduction in traditional coconut-growing land is the loss of locally-adapted germplasm. Therefore there is an urgent need to preserve agro-biodiversity on the long term. Preserving living collections is of a paramount interest in genetic improvement programs that are aimed at devel- oping diseases-resistant and abiotic stress-tolerant cultivars for replanting in traditional coconut growing areas.

  Cryopreservation of coconut can be used as a strategy to back up the establishment of living collections which are expensive to maintain and are under constant threat from biotic and abiotic factors. Unfortu- nately, cryopreservation protocols still need to be developed that are capable of producing a sizeable num- ber of field-grown plants. Therefore, we report on the development of an improved cryopreservation protocol which can be used on a wide range of coconut cultivars. The cryopreservation of zygotic embryos and their recovery to soil-growing plants was achieved through the application of four optimised steps viz.: (i) rapid dehydration; (ii) rapid cooling; (iii) rapid warming and recovery in vitro and (iv) acclimatisation and soil-supported growth. The thermal properties of water within the embryos were monitored using dif- ferential scanning calorimetry (DSC) in order to ensure that the freezable component was kept to a mini- mum. The feasibility of the protocol was assessed using the Malayan Yellow Dwarf (MYD) cultivar in Australia and then tested on a range of cultivars which were freshly harvested and studied in Indonesia. The most efficient protocol was one based on an 8-h rapid dehydration step followed by rapid cooling step. Best recovery percentages were obtained when a rapid warming step and an optimised in vitro culture step were used. Following this protocol, 20% (when cryopreserved 12 days after harvesting) and 40% (when cryopreserved at the time of harvest) of all MYD embryos cryopreserved could be returned to normal seed- lings growing in soil. DSC showed that this protocol induced a drop in embryo fresh weight to 19% and sig- nificantly reduced the amount of water remaining that could produce ice crystals (0.1%). Of the 20 cultivars tested, 16 were found to produce between 10% and 40% normal seedlings while four cultivars generated between 0% and 10% normal seedlings after cryopreservation. This new protocol is applicable to a wide range of coconut cultivars and is useful for the routine cryopreservation of coconut genetic resources.

  The University of Queensland, Integrated Seed Research Unit, School of Land, Crop and Food Sciences, Brisbane, Queensland 4072, Australia _b The University of Queensland, Centre for Nutrition and Food Sciences, Brisbane, Queensland 4072, Australia _c Cirad BIOS, UMR DIAPC, Palm Development Group, IRD, BP5045, F-34394 Montpellier Cedex 5, France a r t i c l e i n f o Article history: Received 14 June 2010 Accepted 29 September 2010 Available online 16 October 2010 Keywords: Dehydration Differential scanning calorimetry Germplasm conservation Embryo culture Recalcitrant seeds a b s t r a c t

  , Steve W. Adkins a _a

  1 , Alain Rival c

  , Yohannes M.S. Samosir a ,

  ⇑ , Peter A. Sopade b

  j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / y c r y o cryopreservation can maintain fidelity in the regenerated plants

  [33] . In the past several research projects have attempted to under-

  D H =

  D H = 28.5 J g ^!1

  ), mercury (T

  m = !38.9 "C, D

  H = 11.4 J g ^!1

  ), tin (T

  m

  = 231.9 "C,

  60.6 J g ^!1 ), zinc (T m = 419.5 "C,

  Thermal analysis

  D H = 108.0 J g ^!1

  ), and sapphire (heat capacity). After thermal analysis, each aluminium pan was punctured and dried at 103 "C for 24 h and weighed to measure the final tissue

  DW. The differences between FW and DW of samples corre- sponded to the sample moisture content, as previously described. The thermograms were analysed using the DSC Universal Analy- sis™ 2000 version 3.9A software package, in order to determine the glass transition temperature (T

  g

  ), change in heat capacity dur- ing the glass transition (

  D C p ), and the melting temperature (T m ) of

  the frozen water formed during cooling. The proportion of frozen water (or melted water) in samples (freezable water) was calcu- lated from the enthalpy of water and the total water content of the sample [32] . To understand the changes measured in the DSC and their trends, typical thermograms were overlaid with offsets

  A thermal analysis of the water present in the dehydrated (2, 4, 6, 8, or 10 h) and non-dehydrated embryos, and during the rewarming process, was undertaken using a TA 2920 DSC unit (TA instruments, Newcastle, Delaware, USA) connected to a liquid nitrogen cooling system. The meristematic tissues of embryos were exposed by cut- ting away the side portions of the embryo. Tissues (three embryos per treatment: 10 to 20 mg) were then placed into an aluminium pan and hermetically sealed. The pans were held at 25 "C for 5 min, cooled to –140 "C at 10 "C per minute and held at this temper- ature for 5 min prior to warming up to 40 "C at 10 "C per minute. An empty hermetically sealed pan was used as a reference pan. The dif- ferential scanning calorimeter was routinely calibrated [34] for tem- perature and enthalpy using indium (T m = 156.6 "C,

  Batches of embryos (10 per treatment) imported from the Phil- ippines were dehydrated in the rapid dehydration apparatus. Dehydration was applied for different periods of time (2, 4, 6, 8, 10 or 12 h). The final moisture content of the embryos was deter- mined on a fresh weight (FW) basis. The embryo dry weight (DW) was measured after further drying at 103 "C for 24 h.

  take coconut cryopreservation although, to our knowledge, no field-grown plants have been produced from cryopreserved tissues

  We aimed to develop a rapid and reliable cryopreservation pro- tocol for coconut. Our specific goals were to (i) design and assess a rapid dehydration step applied prior to cryopreservation followed by the recovery of normal seedlings in a soil-supported growth phase and to (ii) provide an efficient protocol that was applicable to a wide range of coconut cultivars.

  [1,2,4,14,22,21,24] .

  For many plant species, one of the most important variables contributing to the success of cryopreservation is the moisture content of the tissues prior to cooling [6] . For orthodox species, tis- sues can be dehydrated and cryopreserved at low moisture levels (3–5% of tissue fresh weight) without damage [3,40] . However, re- calcitrant species do not survive dehydration below 30–50% FW

  [3,6] . Coconut has been considered as a recalcitrant species by sev- eral authors [3,16] .

  For many species, physical dehydration is the preferred method for tissue drying prior to cryopreservation, particularly for plants which are considered as recalcitrant. The most widely applied physical dehydration technique uses a steady flow of sterile air over the tissue [2,14] . However, a more efficient approach consists of drying tissues in a closed system over silica gel. This protocol en- ables a higher uniformity of the drying process [32] and may also promote vitrification of intracellular solutes as previously demon- strated in the embryos of Aesculus hyppocastanum L. [19] . With the installation of a fan into the desiccator, the silica gel approach can be used to more rapidly dehydrate tissues to specific moisture lev- els. This approach has been used to dehydrate jackfruit (Artocarpus

  heterophyllus Lamk.) embryonic axes from 3.5 to ca. 0.2 g g ^!1

  DW in just 90 min [39] . Such a rapid dehydration step was required to maximise the survival of jackfruit tissues after cryopreservation. The reasons for this improved response following rapid dehydra- tion still remain unclear, although it may relate to the lower cell damage caused by rapid dehydration. The silica gel dehydration technique has been previously applied to coconut embryos prior to cryopreservation [22] , but not as part of a rapid dehydration approach.

  To cater for the special needs of rapid dehydration, the thermal characteristics of the water present in dehydrating tissues are of- ten monitored. Differential scanning calorimetry (DSC) is used in order to assess the effects of dehydration upon its ability to modify the glass transition temperature. Such observations are helpful in predicting which physical dehydration approach will be the most suitable one for a given species.

  Materials and methods

  Moisture content

  Plant material

  During the first stage of the study, zygotic embryos from coco- nut ( C. nucifera L.) cultivar Malayan Yellow Dwarf (MYD) were used. Embryos were imported from the Philippine Coconut Authority, Albay Research Centre in the Philippines and analysed at the University of Queensland (UQ), Australia. In this stage, 10 to 12 surface-sterilized embryos per culture tube (polycarbonate; 3 cm diameter " 10 cm height) containing 10 mL of a solidified embryo culture medium [30] were packed into a styrofoam box and sent to Australia using an international courier. In total, about 12 days were needed to transport the embryos from the field in the Philippines to the laboratory at UQ. Upon arrival, em- bryos were selected for their health and uniformity in size and solution for 3 min) prior to being washed in three rinses of sterile deionised water.

  Rapid dehydration apparatus

  A physical dehydration apparatus, modified from an apparatus previously designed by Wesley-Smith et al. [39] , was used to dry embryos rapidly and under sterile conditions ( Fig. 1 ). The modified apparatus consisted of a glass jar (21 cm height " 11 cm diameter) equipped with a stainless steel mesh (1 mm

  2

  ) platform and an autoclavable fan (12 volt, 500 mA; Sunbeam Pty. Ltd., NSW, Austra- lia; air flow directed upwards) placed below the platform and housed in a polycarbonate tube (17 cm length " 4 cm diameter). Activated silica gel (680 g; 8–10 mesh, Biolab Pty. Ltd., Clayton, Victoria, Australia) was placed into the lower portion of the glass jar in two separate zones (150 g in the bottom and 530 g in the mid section of the jar) separated by further layers of stainless steel mesh. The separation of silica gel into two zones ensured a good airflow around the tissues to be dried. The electrical connections for the fan were glued onto the jar cap and the cap was screwed on tightly and sealed with a layer of waterproof tape. The unit was autoclaved (121 "C, 1.2 kg cm ^!2 for 20 min) then dried

  (65 ± 1 "C for 24 h) before being used in the following experiments in a tissue culture room (25 ± 1 "C).

  290 Sisunandar et al. / Cryobiology 61 (2010) 289–296

  Cryopreservation protocol

  Eighteen to 20 embryos per treatment were dehydrated for either 2, 4, 6, 8, or 10 h (as described above), then transferred indi- vidually into 2 mL cryovials (Techno Plastic Products AG, Trasadin- gen, Switzerland). The vials were then clipped onto a cryocane holder, placed into a cryosleeve (both from Nalgene, Rochester, New York, USA) and then plunged directly into liquid nitrogen for 24 h. In order to recover the cryopreserved embryos, the vials were removed, warmed in a water bath (40 ± 1 "C for 3 min) and the em- bryos transferred onto a recovery medium (Y3 macro- and micro- nutrients [15] vitamins [30] activated charcoal (1 g L ^!1 ) and sucrose (0.175 M). The whole experiment was repeated three times using embryos from batches that were imported at different times.

  Viability testing and seedling establishment

  The viability of embryos was determined before and after dehy- dration and following dehydration and cryopreservation using a germination test. To do this, embryos were transferred into a liquid culture medium (see above) and incubated in the dark (27 ± 1 "C) for 3–8 weeks. Soon after the embryos started to germinate, they were subcultured onto a fresh medium of the same composition but gelled with agar (7 g L ^!1 ). Seedlings were then allowed to grow on the same medium in a growth room (27 ± 1 "C) with a 14 h light/10 h dark photoperiod, for a further 3 month period. The seedlings then underwent a 16-week acclimatisation period in soil, in a glasshouse before they were ready to plant in the field.

  An embryo was scored as viable when either roots or shoots, or a callus developed, or when the embryo enlarged. A germinated embryo was determined to be one that either produced shoots, roots, or both. A normal seedling was determined to be one that produced both a shoot and a root system. Scoring of these re- growth characteristics was undertaken after 8 weeks on the em-

  Application of the protocol to a range of cultivars

  During the second stage of the study, the newly developed cryo- preservation approach was applied to a wide range of coconut germplasm originating from the Indonesian Coconut and Other Palm Research Institute (ICOPRI) in Manado, Indonesia. Eleven- month old fruit from 20 cultivars (10 from Dwarf and 10 from Tall types) were harvested at Mapanget Research Garden. They were: Bali Yellow Dwarf (BAYD), Jombang Green Dwarf (JGD), Malayan Yellow Dwarf (MYD), Nias Green Dwarf (NGD), Nias Yellow Dwarf (NYD), Raja Brown Dwarf (RBD), Sagerat Orange Dwarf (SOD), Salak Green Dwarf (SKD), Tebing Tinggi Dwarf (TTD), Waingapu Red Dwarf (WRD), Bali Tall (BAT), Ilo-Ilo Tall (IIT), Jepara Tall (JPT), Kinabuhutan Tall (KNT), Lubuk Pakam Tall (LPT), Mapanget Tall number 99 (MPT), Palu Tall (PUT), Sawarna Tall (SAT), Takome Tall (TKT) and Tenga Tall (TGT). After dehusking and splitting the nut, endosperm cylinders (containing the embryos) were removed from the endosperm using a sterile cork borer, then washed under run- ning tap water, then rinsed with ethanol (95%, v/v). In a laminar airflow cabinet, the embryos were isolated from the endosperm cylinders and placed into a sterile glass beaker (250 mL), surface- sterilized with a sodium hypochlorite solution (2.6%, v/v; 15 min), and rinsed in sterile water. The embryos were then blot- ted on sterile filter paper before being cryopreserved, then rescued using the previously developed protocols.

  Results

  The process of rapid dehydration

  Moisture loss from embryos during the dehydration process took place in two phases ( Fig. 2 ). During the first 6 h, the moisture content decreased sharply from 80% FW down to 25% FW, with a

  

Fig. 1. Original apparatus designed for rapid dehydration: (A) the rapid dehydration apparatus and (B) details of the air flow created by the fan; (C) embryos under

dehydration treatment.

  Sisunandar et al. / Cryobiology 61 (2010) 289–296 291

• 1

  (0.72 J g ^!1 "

  Fig. 2. Changes in moisture content of embryos on fresh weight (FW) basis after various durations of dehydration time using a rapid dehydration process. Each data point is the mean of 10 embryos ± SE.

  The response to cryopreservation following rapid dehydration was partly cultivar-dependent. Indeed, the percentage of viable embryos recovered after cryopreservation ranged from 12% (SKD) to 89% (SOD) compared to 68% to 98% for embryos not cryopre- served ( Fig. 6 ). Not all of the viable embryos cultured after cryo- preservation germinated ( Fig. 6 ); germination percentages ranged from 0% (WRD) to 70% (IIT). The percentage of embryos that pro- duced normal seedlings after cryopreservation was also found to vary between cultivars, ranging from 0% (WRD) to 40% (MYD). Some degree of variation in seedling production was also observed in the embryos not cryopreserved, ranging from 38% (JGD) to 89% (TKT). Based on the percentage of embryos that produced normal

  Cultivar response to cryopreservation

  20% of the total number of embryos used in a given treatment) developed into normal seedlings which could be subsequently established in soil ( Fig. 5 ). The remaining abnormal seedlings pro- duced either roots or roots with a stunted shoot and all of them eventually died at some stage in the recovery process ( Fig. 5 ).

  The transfer of 8 h dehydrated embryos (19% FW moisture con- of viable embryos (64%) upon their recovery. The other tested dehydration times (viz. 4, 6 and 10 h) were found to generate 20%, 52% and 23% viable embryos, respectively ( Fig. 3 ). Only em- bryos which were dehydrated for 6 or 8 h gave any germination after cryopreservation, with the highest germination (43%) ob- tained for 8 h of dehydration. However, not all germinated em- bryos generated normal seedlings. Indeed, about half of them (i.e.

  Cryopreservation using rapid dehydration

  14% FW after 10 h of dehydration. Moreover, the frozen water of both dehydrated and non-dehydrated embryos melted at temper- atures lower than the melting temperature of pure water (0 "C). This may occur because of possible effects of solutes due to their ability to depress melting point of frozen water. However, the pres- ence of solutes is not expected to substantially change the enthal- py, and 65% of the total water present in the non-dehydrated embryos was estimated to be freezable water and this decreased to almost zero (0.02%) following 10 h of dehydration.

  C ^!1 ). In addition, it was observed ( Table 1 ) that while melting of the frozen water was effectively independent of the duration of dehydration, the enthalpy of melting was dependent upon the moisture content of the embryos. The non-dehydrated embryos (79% FW) gave the highest enthalpy (ca. 260 J g ^!1 ) which decreased to ca. 0.33 J g ^!1 when the moisture content decreased to

  " C ^!1 ) to embryos that were dehydrated for 8 h

  As stated above, embryo viability was monitored after each step of the dehydration process ( Fig. 3 ). For the first 8 h (to a moisture content of 19% FW), embryo viability was high (70%) and not sig- nificantly different to that of non-dehydrated embryos (control). However, embryo viability decreased sharply (to about 37%) when dehydrated for 10 h (to a moisture content of 14% FW) or more.

  (heat capacity change), were also moisture con- tent dependent with about 35-fold increase from non-dehydrated embryos (0.02 J g

  D C p

  value increased as the moisture content of the embryos de- creased ( Table 1 ). Also the changes in the endothermic reaction as revealed by

  g

  the T

  Fig. 4 ). Generally,

  The DSC thermograms obtained from the dehydrated and cryo- preserved embryos revealed two thermal transitions (the glass transition and that of the melting of freezable water) when the fro- zen embryos were heated from !140 to 40 "C (

  The germination capacity of the embryos, when estimated after 8 h of dehydration (ca. 19% FW), was high (61%) with only 9% of the viable embryos not germinating. However, when dehydration was applied for 10 h (14% FW), the percentage of germinating embryos decreased sharply to ca. 23%. The same downward trend was also observed during the production of normal seedlings produced by this treatment. Indeed, after 8 h of dehydration, 43% of embryos developed into normal seedlings, while this value decreased to 13% when embryos were dehydrated for 10 h. All normal seedlings produced from any treatment were found to grow and develop into normal soil-grown plants without further loss.

  Fig. 3. The re-growth characteristics of embryos following rapid dehydration for specific periods of time (h) and cryopreservation ( ). These characteristics were recorded after 12 weeks of recovery. Data show the percentage of viable embryos (A), embryos germinating (B) and embryos producing normal seedlings (C). In each bar chart and each data series, treatments that are ascribed with different letters are significantly different at p-value 6 0.05. The bars are means of three replications of 18 to 20 embryos ± SE. 292 Sisunandar et al. / Cryobiology 61 (2010) 289–296 divided into three groups: (a) those difficult to recover after cryo- preservation and generating less than 10% normal seedlings (four dwarf cultivars; WRD, SKD, BAYD and JGD); (b) those moderately difficult to recover after cryopreservation and producing between 10% and 30% normal seedlings (three dwarf, NYD, RBD and TTD, and eight tall cultivars, JPT, KNT, SAT, TGT, IIT, BIT, MPT and LPT) and (c) those easy to recover after cryopreservation and producing between 30% and 40% normal seedlings (PUT, TKT, NGD, SOD and MYD). Discussion

  A cryopreservation protocol using rapid dehydration

  This is the first report on the generation of viable plantlets from cryopreserved coconut material which were amenable to field planting. Our experiments showed that coconut embryos can be cryopreserved successfully using a rapid dehydration technique applied for 8 h only and a rapid freezing approach ( Fig. 3 ). Embryos treated in this way could be rewarmed rapidly from liquid nitrogen a high number of viable embryos (64%) from which a significant number (20% on average) generated normal, soil-established plants. The percentage of conversion of cryopreserved embryos into normal seedlings (20%) was found to be similar to that re- ported for other recalcitrant species such as cork-oak (Quercus sub-

  er L.) and evergreen oak (Quercus ilex L. [20] . However, it was

  higher than that reported for cacao (Theobroma cacao L., [13] ) and cape ash (Ekebergia capensis Sparrm. [28] ), for which no nor- mal seedlings could be generated. This percentage was also higher than that reported for peach palm (Bactris gasipaes Kunth. [35] ), in which less than 12% of the cryopreserved embryos developed into normal seedlings.

  This rapid dehydration approach can decrease the embryo moisture content to 19% of their normal moisture content in just 8 h, without reducing their viability (retained at 70%) and germina- tion capacity (43% producing normal seedlings; Fig. 3 ). These re- sults support the view held by other authors [6,7,25,39] that a rapid dehydration step is necessary for preparing tissues from re- calcitrant plant species for cryopreservation.

  The rapid dehydration technique employed a newly designed

  Fig. 4. Typical thermograms for the coconut embryos, showing T g and T _m . Embryos were dehydrated for 4 ( ), 6 ( ), 8 ( ) or 10 h ( ) and were compared to non-dehydrated embryo ( ). The curves have been shifted down in order to avoid overlap.

  Table 1

Physical characteristics of MYD embryos obtained from differential scanning calorimetry analysis during the warming process after the cooling of embryos that had been

dehydrated for different periods of time (0–10 h). Each data set is the mean of three samples with two replications in each treatment ± SE. In each row, values that are followed

with different letter are significantly different (P < 0.05).

  Physical characteristics Duration of dehydration (hours)

  4

  6

  8

  10 Water content (% FW) 79.01 ± 0.70 a 43.86 ± 1.50 b 27.07 ± 2.07 c 19.37 ± 0.06 d 14.01 ± 1.65 e Melting Onset temperature, T _m,onset, ("C) !10.20 ± 0.14 a !24.02 ± 1.31 b !18.65 ± 8.31 ab !20.03 ± 0.11 ab !9.81 ± 0.54 a Peak temperature, T _m,peak , ("C) !0.28 ± 1.12 a !8.84 ± 0.19 b !5.91 ± 4.52 ab !7.32 ± 0.49 ab !1.92 ± 0.53 ab Melt water (J g ^!1 ) 260.75 ± 0.25 a 86.13 ± 0.59 b 70.37 ± 3.86 c 1.64 ± 0.06 d 0.33 ± 0.01 d

Frozen water (% of total) 65.87 ± 0.01 a 11.96 ± 0.01 b 6.03 ± 0.01 c 0.10 ± 0.02 d 0.01 ± 0.01 d

Glass transition Onset temperature, T _onset ("C) !122.93 ± 0.82 a !90.69 ± 0.86 b !89.33 ± 0.40 b !57.90 ± 1.53 c !53.05 ± 0.27 d Glass transition temperature, T g ("C) !123.00 ± 0.97 a !86.42 ± 2.21 b !84.14 ± 0.82 b !48.34 ± 0.63 c !44.76 ± 0.51 c End temperature, T _end ("C) !121.98 ± 0.31 a !83.02 ± 0.62 b !80.92 ± 0.30 b !41.31 ± 0.86 c !34.26 ± 0.68 d Change in heat capacity, DCp (J g ^{!1 "} C ^!1 ) 0.02 ± 0.01 a 0.10 ± 0.01 b 0.10 ± 0.01 b 0.72 ± 0.02 c 0.72 ± 0.04 c

  Sisunandar et al. / Cryobiology 61 (2010) 289–296 293 dehydrate embryonic axes of jackfruit [39] , has the added advan- tage of being autoclavable and subsequently useable under sterile conditions. Thus, the dehydrated embryos developed in this study did not have to be re-surface-sterilized, and in so doing avoiding a step that may have lead to tissue damage.

  Thermal analysis showed that the glass transition temperature of coconut embryos, when reheated following cryopreservation, was higher (closer to zero) in embryos that had been dehydrated for the longest time ( Table 1 and Fig. 4 ). This is consistent with re- sults from other authors who have shown water to be a plasticiser and to reduce the T g of tissues [34] . Similar results have been re- ported for bull rush (Typha latifolia L.) pollen [11] and black currant shoot tips [32] . Interestingly, the T g of embryos that were rapidly dehydrated (!48 "C) was much higher than that provided by liquid nitrogen at –196 "C. It has been demonstrated that the storage of living materials needs to be below the T

  g

  temperature if viability is to be maintained for many years [10,11,36,38] . Thus, the present results do clearly support the idea that the storage of zygotic em- bryos in liquid nitrogen will be able to preserve coconut embryos for significant periods of time.

  Applicability of the protocol to a range of cultivars

  When dehydrated embryos taken from 20 different cultivars were subjected to cryopreservation, the percentage of normal, soil-established seedlings recovered varied from 0% to 40% ( Fig. 6 ). The performance of the cultivars fell into three classes (viz. difficult; moderately difficult; and moderately easy) based on their ease to produce plants after cryopreservation. For the ‘dif- need to be refined, while for the other two classes the developed protocol is thought to be suitable. A similar cultivar-dependent re- sponse to cryopreservation has been already described in sweet potato [8] , cassava [17] , Japanese persimmon [23] and banana

  [26,27] .

  Several factors may be responsible for this cultivar-dependent response and include the geographic origin and their sensitivity to drought. For Japanese persimmon, cultivars originating from temperate regions responded better to cryopreservation than those from subtropical origin [23] . In banana, drought-tolerant cultivars showed a better shoot growth after cryopreservation than drought-sensitive cultivars [26] . There are differences in drought tolerance [29] amongst the various coconut cultivars used. For example, the four cultivars which generated the lowest number of normal seedlings following cryopreservation are those that have previously been described as drought sensitive [29] .

  An interesting observation can be made for MYD material when the two parts of the present study are compared; in the first part the embryos used had been subjected to a 12-day importation time while in the second part embryos used were studied just after har- vest. The latter embryos gave rise to a much high percentage of normal seedlings ready for field-planting following cryopreserva- tion (40%; Fig. 6 ) while the former only gave rise to only 20% nor- mal seedlings ( Fig. 3 ). Based on these observations, it appears that the use of high quality, freshly isolated embryos is essential to achieve successful cryopreservation of coconut.

  Apart from the 20% soil-established coconut seedlings, we pro- duced a further 23% of recovered embryos which were viable, although unable to produce normal plantlets (they lacked roots

  

Fig. 5. Various steps in the recovery process of coconut embryos after cryopreservation. (A) 8-week recovered seedlings on the recovery medium showing normal growth;

(B) 20-week recovered seedlings that are ready for acclimatisation; (C) 16-week acclimatised seedlings (D) 8-week embryos cultivated on recovery medium showing

abnormal growth; (E) abnormal seedlings showing poor root growth with or without stunted shoot growth after 16 weeks recovery. 294 Sisunandar et al. / Cryobiology 61 (2010) 289–296

  Sisunandar et al. / Cryobiology 61 (2010) 289–296 295

Fig. 6. The re-growth characteristic of cryopreserved (h) and non-cryopreserved embryos ( ) isolated from 20 coconut cultivars. The cryopreserved embryos had been

dehydrated using a rapid dehydration step for 8 h, cryopreserved and then recovered for 8 weeks. The data show the percentage of viable embryos (A), embryos germinating

(B) and embryos producing normal seedlings (C). The bars are means of three replications of 18 to 20 embryos ± SE. The cultivars could be categorised into three classes;

difficult, moderately difficult or moderately easy to cryopreserve.

  [4] H.D.D. Bandupriya, S.C. Fernando, J.L. Verdeil, B. Malaurie, Effect of absisic acid

  following cryopreservation has been seen in many species before

  on survival and recovery of cryopreserved plumule explants of coconut (Cocos

  and the conversion of these seedlings into normal ones remains a nucifera L.), Cocos 18 (2007) 58–66. major challenge for many cryopreservation protocols [28,35] .

  [5] P. Batugal, K. Jayashree, COGENT’s multi-site international coconut genebank, in: P. Batugal, V. Ramanatha Rao, J. Oliver, (Eds.), Coconut Genetic Resources, International Plant Genetic Resources Institute-Regional Office for Asia, the

  Acknowledgments Pacific and Oceania (IPGRI-APO), Serdang, Selangor DE, Malaysia, 2005, pp.

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  tional Agricultural Research (ACIAR), the Graduate School

  developmental status, desiccation sensitivity and state of water in axes of Research Travel Grants (GSRTG) scheme from the University of Landolphia kirkii Dyer, Planta 186 (1992) 249–261. [8] M.H. Bhatti, T. Percival, C.D.M. Davey, G.G. Henshaw, D. Blakesley, Queensland and Endeavour Research Fellowships Australia 2010.

  Cryopreservation of embryogenic tissue of a range of genotypes of sweet

  We thank Dr Hengky Novarianto and his staff at the Indonesian

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