Embryo maturity plays an important role for the successful cryopreservation of coconut (Cocos nucifera)

  Embryo maturity plays an important role for the successful cryopreservation of coconut (Cocos nucifera)

  Sisunandar & Hengky Novarianto & Nurhaini Mashud & Yohannes M. S. Samosir & Steve W. Adkins

  Received: 22 January 2014 / Accepted: 1 July 2014 / Published online: 7 August 2014 / Editor: John Forster # The Society for In Vitro Biology 2014

  Abstract Genetic diversity of coconut (Cocos nucifera L.) is being lost due to a combination of pest and disease attack, urban encroachment, natural disasters, as well as introgression with exotic genetic types. Consequently, there is a need to undertake germplasm conservation before further loss occurs. Since coconut has a large, recalcitrant seed (sensitive to des- iccation), it cannot be stored in traditional ways in a seed bank. Cryopreservation of zygotic embryos is now seen as an im- portant storage approach although published techniques are still not reliable. Given the importance of embryo maturity to the success of cryopreservation in other species, the effect of coconut embryo maturity on cryopreservation success was investigated using four cultivars (‘Nias Yellow Dwarf’, ‘Tebing Tinggi Dwarf’, ‘Takome Tall’, and ‘Bali Tall’). After cryopreservation, using a new four-step protocol (rapid desiccation, rapid freezing, rapid thawing, and recovery and acclimatization for 4 mo in the glasshouse), we found that the embryos isolated from an 11-mo-old fruit gave the highest number of normal seedlings (~28%) when compared to coun- terparts excised from younger fruits. In addition, the results showed that fruit could be stored for up to 3 wk prior to embryo isolation before their performance in cryopreservation was compromised. Keywords Embryo maturity . Cryopreservation . Coconut .

  Cocos nucifera

  Introduction Coconut (Cocos nucifera L.) is one of the most important tree crops in the tropics. It is grown by more than 50 million people on 12 million ha of land and in over 90 countries. Crop productivity is continuously being threatened due to a combination of pest and disease attack and natural disasters. As a consequence, the loss of locally well-adapted cultivars has become a major problem, and ex vitro methods to con- serve coconut germplasm have been urgently sought. Traditionally, seed gardens have been used to conserve coco- nut germplasm (Batugal and Jayashree However, more recently, a new approach for the long-term storage of coconut germplasm has been developed using cryopreservation (Sisunandar et al. This new approach uses zygotic embryos as the explant material and has been shown to be efficient in producing a relatively large number of seedlings ready for field planting after cryopreservation. Furthermore, no significant differences in their genetic and epigenetic char- acteristics prior to and after cryopreservation have been ob- served using this approach (Sisunandar et al. ). In its present form, the approach uses embryos isolated from an 11- mo-old fruit; however, the effect of embryo maturity upon the success of the approach has not been determined.

  A number of protocols have been developed for the suc- cessful cryopreservation of plants using zygotic embryos (Pritchard and Prendergast Abdelnour-Esquivel et al.

  Sisunandar ^: Y. M. S. Samosir ^: S. W. Adkins ( *) School of Agriculture and Food Sciences, The University of Queensland, St Lucia, Brisbane, Queensland 4072, Australia e-mail: s.adkins@uq.edu.au H. Novarianto : N. Mashud Indonesian Coconut and Other Palm Research Institute, Mapanget, Manado, North Sulawesi, Indonesia Present Address: Sisunandar Biology Education Department, The University of Muhammadiyah Purwokerto, Kampus Dukuhwaluh, Purwokerto 53182, Indonesia Present Address: Y. M. S. Samosir Bakrie Agriculture Research Institute (BARI), PT Bakrie Sumatera In Vitro Cell.Dev.Biol.—Plant (2014) 50:688–695 DOI 10.1007/s11627-014-9633-1

  

Author's personal copy et al. ). In most of these protocols, the stage of embryo maturity (development) was a factor in determining the degree of success. For orthodox species, fully mature embryos are those that respond best to cryopreservation (Kenmode and Finch- Savage however, for recalcitrant species like coconut, the best embryo maturity stage seems to be species dependent. For example, for coffee (Coffea spp.; Abdelnour-Esquivel et al. ) and jackfruit (Artocarpus heterophyllus Lamk.; Chandel et al.

  Chaudhury and

  Malik the fully mature embryo stage was the best. In earlier studies where embryo maturity was studied in the cryopreservation process of coconut, Chin et al. reported that immature embryos survived better after cryopreservation, while Assy- Bah and Engelmann ) reported fully mature embryos to give the better survival after cryopreservation.

  In determining the best embryo maturity stage for cryo- preservation, it is important to take into account the age of the fruit at harvest and the duration of storage before the embryo is removed. This post-harvest time may include the time taken in transporting the fruit from the field to the laboratory or the time the fruit remains in storage in the laboratory prior to the cryopreservation of the embryo (Berjak et al. ). On many occasions, coconut fruits have to be harvested from remote areas, and the time in transport can be several weeks. On the other hand, some freshly harvested fruits may have to sit in storage for some time before staff can undertake embryo isolation and the cryopreservation step can take place. Thus, this present study evaluates embryo maturity status and post-harvest storage time on the success of the newly developed cryopreservation protocol for coconut (Sisunandar et al.

  Materials and Methods Plant material. The four coconut cultivars used in this study (viz., ‘Nias Yellow Dwarf’ (NYD); ‘Tebing Tinggi Dwarf’ (TTD); ‘Takome Tall’ (TKT); and ‘Bali Tall’ (BAT)) representing two tall and two dwarf cultivars were harvested from the Mapanget Research Seed Garden (The Indonesian Coconut and Other Palm Research Institute, Manado, Indonesia). From each cultivar, four different embryo maturity stages (viz., 8, 9, 10, or 11 mo after pollination) were harvest- ed, based on a leaf counting method. This method assumes that a new coconut leaf and inflorescence is produced every 4 wk (Perera et al. ). Thus, the 8th fruit bunch down from the newly opened inflorescence contains embryos that are 8 mo old. Twelve-mo-old embryos were not used in this present study as such embryos usually start to germinate at

  Embryo characteristics (experiment 1). Six hundred and forty embryos (40 from each of the four cultivars and from each of the four different maturity stages) were aseptically isolated from the fruit using the previously described method of Rillo For the isolation of embryos from very young (8-mo old) fruit, a small metal spoon was used to scoop them from the soft endosperm tissue. All embryos were then surface sterilized using a sodium hypochlorite (2.6%; v/v) solution for 15 min, followed by several rinses in sterile water, and then blotted dry on sterile filter paper. From each of the 40 embryos from each of the four cultivars and four maturity stages, 10 embryos (in two replicates of five) were photographed, and their length and cross-sectional area determined using a free- to-access computer software package Image J 1.37v (Rasband

  The fresh weight of these same embryos was then

  determined using a Precise XT220A analytical balance, and dry weights were determined gravimetrically following oven drying at 103±2°C for 24 h. The remaining 30 embryos (in three replicates of 10) from each batch were germinated as previously described (Rillo ). Briefly, the embryos were placed into a liquid embryo culture medium for 1 mo, then transferred to a solid medium for a further 2 mo, then to a second liquid culture medium for 2 mo. The seedlings after this final stage were then acclimatized and grown in a glass- house (Sisunandar et al. The percentage of viable (embryos showing either root or shoot production), germinat- ing (embryos showing both root and shoot production), and embryos producing seedlings of normal morphology in soil in the glasshouse were determined.

  Maturity and cryopreservation (experiment 2). One day fol- lowing fruit harvest, a further 1,120 embryos (70 from each of the four cultivars and from each of the four different maturity stages) were aseptically isolated using the previously de- scribed method (Rillo ; with modification for the very young embryos) and were allocated to the following treat- ments. Ten embryos (in two replicates of five) were then rapidly dehydrated for 4, then 6, then 8, and finally 10 h in a rapid drying chamber as previously described (Sisunandar et al. ) with their moisture content being determined at each time period. A further 30 embryos (in three replicates of 10) from each batch were dehydrated for 8 h (Sisunandar et al. The remaining 30 embryos (in three replicates of 10 embryos) were dehydrated for 8 h and then plunged into liquid nitrogen where they remained for 24 h (Sisunandar et al. Upon recovery, their viability, germination, and ability to produce seedlings of normal morphology in soil in the glasshouse were determined (Sisunandar et al. ).

  Storage and cryopreservation (experiment 3). An additional

  EMBRYO MATURITY PLAYS A ROLE FOR THE CRYOPRESERVATION OF COCONUT 689

Author's personal copy NYD and stored at 29±3°C and a relative humidity of 79±5% for either 1, 2, or 3 wk. Every week, the embryos from 60 randomly selected fruits were isolated and (in three replicates of 20 embryos) were rapidly dehydrated for 8 h and then plunged into liquid nitrogen where they remained for 24 h (Sisunandar et al. Upon recovery, their viability, ger- mination, and ability to become seedlings of normal morphol- ogy in soil in the glasshouse were determined (Sisunandar et al. ).

  Experimental design and statistical analysis. All experiments were undertaken using a completely randomized design with 5, 20, or 30 embryos per replication, as reported above. An analysis of variance (ANOVA) was undertaken using the Minitab

  

  Figure 1. Representative digital images of ‘Nias Yellow Dwarf’ embryos isolated from four different fruit maturity stages. Top to bottom, taken from 8-, 9-, 10-, or 11-mo-old fruit. 690 SISUNANDAR ET AL.

  ture embryos gave the highest percentage of viable embryos after cryopreservation (~60%), the highest percentage of em- bryos that germinated (~40%), and the highest percentage of normal seedlings (~30%). In contrast, the 8-mo-old, immature embryos gave the lowest percentage of viable embryos after cryopreservation (<10%), the lowest percentage of embryos that germinated (<5%), and the lowest percentage of normal seedlings (<5%). The four cultivars used in this present study did not differ in their response to cryopreservation as was assessed by the recovery of viable embryos and germinating embryos. However, the percentage of embryos producing normal seedlings did vary between cultivars with a low value

   ). The initially 11-mo-old, fully ma-

  In a second batch of embryos that had dehydrated for 8 h and been cryopreserved for 24 h, then recovered for 12 wk, we found that the initial embryo maturity status had a significant impact upon embryo viability, germination, and formation of normal seedlings (Fig.

  dration, water loss followed a simple exponential func- tion and declined from around 80% to around 30%, on a fresh weight basis. The rate of water loss then de- clined and after 8 h reached the optimum level of 20% for cryopreservation (Sisunandar et al. ).

   ). During the first 6 h of dehy-

  Maturity and cryopreservation (experiment 2). Irrespective of maturity status, all embryos of NYD underwent a similar rate of dehydration when placed in the rapid drying chamber (Fig.

  The older embryos not only gave the highest viability and germination percentages but also gave the highest number of normal seedlings (up to 90%) growing in soil. Meanwhile, the seedlings (up to 20%) growing in soil. No differences in the ability to produce normal seedlings from 11-mo-old embryos were noticed between the four cultivars.

  The time-related stage at which the embryos were isolated from the fruit had a significant impact upon their viability (growth observed for either root or shoot) and their ability to germinate (growth observed from both root and shoot). Embryos of 11 mo old were more viable and germinated at a much higher percentage than those that were 8 mo old (Fig.

  ™

  was that the embryos coming from the two tall cultivars (i.e., TKT and BAT) were larger than those of the two dwarf cultivars (i.e., NYD and TTD) when at the same time of maturity.

   ). One minor difference noticed

  Similar patterns of development were observed for their fresh and dry weight gain (Fig.

  

  cross-sectional area) increased rapidly in 8- to 10-mo-old fruits, then to a lesser extent over the next 2 mo (Fig.

   ). Embryo sizes (length and

  Results Embryo characteristics (experiment 1). In visual terms, em- bryos of the four cultivars exhibited a similar pattern of maturation over time (Fig.

  The present studies were undertaken (1) to determine the best time for zygotic embryo isolation from fruit to optimize of the production of normal seedlings after cryopreservation and (2) to determine if a short post-harvest storage period of up to 3 wk will affect the cryopreservation process.

  statistical software package (Minitab Inc., Belmont, CA). Some of the data sets (those violating the ANOVA principles such as homogeneity of variances) were square-root transformed prior to statistical analysis. However, only untransformed data sets are presented here. All experi- ments were repeated with similar results to those reported here.

  

Author's personal copy of 21% being produced for NYD and a high value of 36% being produced for TKT (Fig.

  

  growth in the early period of development but with this decreasing in embryos greater than 9 mo old. This embryo maturity pattern is similar to that seen in other species such as jackfruit (Farant and Walters wheat (Triticum aestivum; Lehner et al. ), and maize (Zea mays; Wen and Song

  11 mo old. All values are expressed as means±SE of 10 embryos. EMBRYO MATURITY PLAYS A ROLE FOR THE CRYOPRESERVATION OF COCONUT 691

  Figure 2. Changes in embryo attributes measured at four maturity stages and for four cultivars: NYD ( ), TTD ( ), TKT ( ), and BAT ( ). a The longitudinal length, b area, c fresh weight, and d embryo dry weight were measured for embryos isolated from fruit at 8, 9, 10, or

  its embryos and the lack of any significant moisture loss

  Such a high level of moisture content within

  declined as embryos approached physiological maturity. The percentage moisture content of embryos found throughout all stages of maturity was about 80% and was similar for all four cultivars (Fig.

  where maturity was rapid in young embryos, but then

   ). There was also a tendency for greater

  Storage and cryopreservation (experiment 3). Post-harvest storage of fruit initially containing 11-mo-old NYD embryos for either 1, 2, or 3 wk caused no significant changes to the embryo moisture content (data not shown). Moreover, these storage treatments did not affect embryo viability (~65%) or their ability to germinate (~35%) and produce normal seed- lings (~25%) after cryopreservation (Fig.

  embryos than the dwarf cultivars, and this has been reported before (Thampan ), with BAT being signif- icantly (P>0.05) bigger in all measured variables than all other cultivars (Fig.

   ). In general terms, the two tall cultivars produced larger

  A similar pattern of embryo development was observed for all of the four cultivars of coconut studied. This similar devel- opment was observed in length and cross-sectional area at- tainment (Fig.

  Engelmann Thus, it was necessary to re-evaluate the effect of embryo maturity on the success of cryopreservation and to do this for a range of coconut cultivars, including both tall and dwarf cultivars.

  Discussion The success of cryopreservation in many species relies upon using tissues of an appropriate physiological age as has been demonstrated for a number of species including coffee (Abdelnour-Esquivel et al. For coconut, both immature and mature embryos have been used in the past for cryopreservation but with different out-

  icant differences in the ability to cryopreserve the embryos were detected following fruit storage for 1, 2, or 3 wk.

   ). Thus, no signif-

  

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692 SISUNANDAR ET AL.

  Figure 3. The re-growth characteristics of embryos taken from fruit of germinating ( ), and embryos producing normal seedlings (■). Bars cultivars, a NYD, b TTD, c TKT, and d BAT embryos at four different below different letters are significantly different at P≤0.05. The bars maturity stages (8, 9, 10, or 11 mo old) and placed onto a recovery represent means±SE of three replications of 10 embryos. medium for 8 wk. The data show the percentage of viable (□),

  species (Engelmann Similar high embryo moisture content has been observed in other recalcitrant species such as landolphia (Landolphia kirkii Dyer; Berjak et al. ). Moisture retention during maturation in recalcitrant species is in contrast to that seen in orthodox species in which the embryo moisture con- tent decreases dramatically as physical maturity approaches, as seen in wheat (Lehner et al. ) and maize (Wen and Song ).

  As might be predicted, the germination capacity of embry- os from all four cultivars increased as they matured. Embryos isolated from 11-mo-old fruit produced the highest germina- tion (~90%) and gave the highest percentage of normal seed- lings (~80%), while those isolated from 8-mo-old fruit pro- duced the lowest germination (~10%) and gave the lowest

  Figure 4. The change in moisture content of embryos taken from fruit of percentage of normal seedlings (~10%; Fig. Similar obser- ), 9 cultivar ‘Nias Yellow Dwarf’ at four different maturity stages (8 (

  vations have been made on other species, such as in areca

  ( ), 10 ( ), or 11 ( ) mo old). Embryos were dehydrated for

  palm (Chrysalidocarpus lutescens; Broschat and Donselman various periods of time (2 to 10 h) using a rapid dehydration apparatus.

   ), pigmy date palm (Phoenix roebelenii), royal palm Figure 5. The viability and germination of embryos taken from fruit of cultivars, a NYD, b TTD, c TKT, and d BAT at four different maturity stages (8, 9, 10, and 11 mo old) that had been dehydrated, cryopreserved, and placed onto a recovery medium for 8 wk. The data show the percentage of viable (□), germinating ( ), and embryos producing normal seedlings (■). Bars below different letters are significantly differ- ent at P≤0.05. The bars are means±SE of three replications of 20 embryos.

  Figure 6. The re-growth attributes of embryos taken from fruit of cultivar ‘Nias Yellow Dwarf’ isolated from 11-mo-old fruit that had been stored for either 0, 1, 2, or 3 wk after harvest. Embryos had been dehydrated for 8 h using a rapid dehydration apparatus, cryopreserved, and placed onto a recovery medium for 8 wk. The data show the percentage of viable (□), germinating (

  ), and embryos producing normal seedlings (■). Bars below different letters are significantly different at P≤0.05. The bars are means± EMBRYO MATURITY PLAYS A ROLE FOR THE CRYOPRESERVATION OF COCONUT 693

  

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  (Roystonea regia; Broschat and Donselman and jackfruit (Chaudhury and Malik ).

  tions used are not identical to those found with long-distance transport to a laboratory, but do indicate that transport times of up to 3-wk duration should not affect the cryopreservation outcomes of the embryos contained within the fruit.

  Farant JM, Walters C (1998) Ultra structural and biophysical changes in developing embryos of Aesculum hippocatanum in relation to ac- 694 SISUNANDAR ET AL.

  Engelmann F (1999) Cryopreservation of coconut germplasm. In: Oropeza C, Verdeil JL, Ashburner GR, Cardena R, Santamaria JM (eds) Current advances in coconut biotechnology. Kluwer Academic Publishers, Dordrecht, pp 289–296

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  Broschat TK, Donselman H (1987) Effect of maturity, storage, presoaking and seed cleaning on germination in three species of palms. J Environ Hort 5:6–9 Chandel KPS, Chudhury R, Radhamani J, Malik SK (1995) Desiccation and freezing sensitivity in recalcitrant seeds of tea, cocoa and jack- fruit. Ann Bot 76:443–450

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  References

  Acknowledgments The project was partly funded by the Australian Agency for International Development (AusAID), the Endeavour Re- search Fellowships Australia 2010, and the Australian Centre for Inter- national Agricultural Research, via project HORT/1998/061.

  Such storage condi-

  When embryos from one cultivar (NYD) and at different stages of maturity were dehydrated, their water loss profiles were similar (Fig.

  typical tropical storage at ambient temperature for up to 3 wk did not influence embryo moisture content (data not shown), capacity after cryopreservation (Fig.

  However, in the present study, storage of fruit in a

  During transport of fruit from the field to the laboratory, or during storage at the laboratory, the embryos contained within the fruit will continue to develop (Berjak et al. espe- cially if this time becomes greater than 1 wk. In recalcitrant species such as coconut, which can germinate soon after harvest (i.e., within 30 to 40 d after harvest; Foale significant delays in post-harvest handling, prior to cryopres- ervation, may result in lower embryo germination or losses in embryo quality. Often, delay is unavoidable as germplasm collection takes place in very remote regions, several days away from the laboratory, and the processing of large numbers of fruit in the laboratory takes time to do. The quality changes that go on inside embryos during this transport and storage period may lead to changes in the tolerance of embryos to dehydration and/or cryopreservation success (Berjak et al.

  normal seedlings. This is a similar result to that reported before (Sisunandar et al.

   ) percentage of

  The four cultivars used in this experiment all had similar responses to cryopreservation when the percentage of viable embryos and the percentage of germinating embryos were taken into account. However, the percentage of embryos that went on to produce normal seedlings did show significant differences between cultivars, NYD giving the lowest (21%) and TKT giving the highest (36%; Fig.

  finding is similar to that of Assy-Bah and Engelmann ( who found that mature coconut embryos gave the best survival percentage after cryopreservation than did immature embryos. Thus, mature embryos, isolated from 11-mo-old fruit, are those recommended for coconut cryopreservation. This is similar to jackfruit where ~30% of the mature embryos survived cryopres- ervation, while no immature embryos survived (Chaudhury and Malik . These results indicate an increase in embryo maturity correlates with an increase in the tolerance of embryos to dehydration and cryopreservation, which is a trend that has been reported for many orthodox species (Hong and Ellis ; Wen and Song ).

   ). This

  Embryos isolated from 11-mo-old fruit gave the highest germination percentage (~40%) and the highest number of normal seedlings (~30%) after cryopreservation (Fig.

  after 8 h of drying. This observation is similar to that of an earlier study (Chaudhury and Malik ) where jackfruit embryos, at different stages of maturity, all dried at a similar rate.

  reaching about 20% moisture content

  

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