Cryopreservation for Germplasm Conservation: Progress Report on Indonesian Elite Mutant Coconut “Kopyor”
Proceeding ICGRC 2013
Cryopreservation for Germplasm Conservation: Progress Report on
Indonesian Elite Mutant Coconut “Kopyor”
Sisunandar
Biology Education Department, Muhammadiyah University of Purwokerto, Indonesia 53182
Abstract
Among Indonesian elite materials, the Kopyor mutant is a paramount interest, as it is much sought after by
customers for its soft and sweet flesh, and high prices with more than 10 times higher than the one of a normal fresh
coconut. However, this mutant cannot be conserved by normal means such as field genebanks or seed genebank. An
alternative conservation method would be one that involves a degree of dehydration and then ultra-cold storage
(cryopreservation). However, as yet such protocols developed for Kopyor have been untested and no field-growing
plants have been produced back from these techniques. The cryopreservation of zygotic embryos was achieved
through the following steps viz.: a rapid tissue dehydration step prior to storage and a rapid warming step upon
recovery followed by acclimatization to soil-supported growth. The best protocol was one based on an 8-hour rapid
dehydration step followed by rapid cooling step, rapid warming step and an optimized in vitro culture technique.
Following this protocol almost 20 % of cryopreserved embryo could be returned to normal seedlings growing in
soil. Moreover, 23 % of recovered embryos were viable but unable to produce normal plantlets, mostly showed
stunted shoot. These results indicate that it is possible to store coconut kopyor germplasm on a long-term basis
using cryopreservation approach, even though the production of abnormal seedlings following cryopreservation as
has been seen in many recalcitrant species before remains a major challenge for the cryopreservation protocol. cryopreservation, Embryo culture, Germplasm conservation, Kopyor, Rapid dehydrationKeywords: Introduction
Coconut plays an important role in the socio-economic life of many millions of people in the inter-tropical regions of the world. In Indonesia, coconut is being planted by more than 3 million farmers [1] and production exceeded 19 million metric tons (mt) in 2011 . However, more than 95 % of the coconut farmers are smallholders cultivating less than 0.5 ha. Such growers still live in poverty with an income [2] generated by coconut exploitation accounting for less than US$ 600 per year .
An alternative approach to increase coconut farmers’ income is to plant selected material with higher market value. Among elite Indonesian material, the Kopyor mutant is of paramount interest. Indeed, this type of coconut is very sought after by Indonesian buyers and high prices are driven by very limited production. Each Kopyor nut is sold at a market price of around US$ 2 – 3 per nut, a price which [3] is more than 10 times higher than the one of a normal fresh coconut . There are still limitations and constraints for the large scale plantation of Kopyor coconuts in
Indonesia because the nut has a jelly-like, non-functional endosperm tissue which would not allow the [4] proper germination of the zygotic embryo . Most of Kopyor plantations are originating from mutants seedling which appeared at random amongst non-Kopyor through spontaneous mutation. As a consequence, the percentage of Kopyor nut produced at random is less than 30 %. Biotechnologies now provide an interesting bypass, as it is possible to produce viable Kopyor seedlings through in vitro embryo culture. However, the technique is still need to be developed and it needs some scaling up in order to increase the success rate of seedling production.
Moreover, most of the Kopyor germplasm is found in the gardens of small-holed farmers. As a consequence, the materials may not be easily accessible, can still be affected by pest, diseases, natural disasters or changes in land use. Therefore there is an urgent need to preserve the Indonesian elite mutant of coconut on the long-term basis. Up to now, the only technique available for the long-term storage of coconut without being hampered by various biotic and abiotic factors would be to cryopreserve the embryo. th th Cryopreservation of plant embryo has been used to preserve genetic resources in many plant
4 International Conference on Global Resource Conservation & 10 Indonesian Society for Plant Taxonomy Congress
[5-11]
species including coconut , but has not been applied in Kopyor palm. The present paper reports the [10] progress on the application of a well-developed cryopreservation technique to store Kopyor embryos followed by the recovery of normal seedlings in a soil-supported growth phase.
Materials and Methods Plant Material
Zygotic embryos from coconut (Cocos nucifera L.) were isolated from 11-month old Kopyor fruits which were harvested from the gardens of several farmers in Purbalingga, Central Java, Indonesia. The [12] COGENT methods for embryo isolation from the nut and surface sterilization of explants were applied with a slight modification. The fresh soft endosperm containing the zygotic embryos were isolated using table spoon, then the endosperm were washed several times with tap water and rinsed quickly with ethanol (70 %, v/v). The embryos then were isolated from the soft endosperm in laminar air flow cabinet, then surface sterilized using sodium hypochlorite (2,6 %, v/v) for 15 minutes, followed by rinsed in sterile [9,10,11] water . The embryos were then blotted dry on sterile filter paper before being used for the experiment.
Embryo Rapid Dehydration and Germination
With the aim to optimize the tissue dehydration technique, batches of embryos (10 embryos per treatment) were rapidly dried for 6, 8, and 10 hours under sterile conditions using a physical dehydration [10] [11] apparatus . Five embryos per treatment were used for moisture content determination while another five embryos were used for germination test after drying.
Cryopreservation Protocol and Viability Testing after Cryopreservation
Another ten embryos per treatment were dehydrated for either 6, 8, or 10 hours (as described above), then placed individually into 2 mL cryovials (Techno Plastic Products AG, Trasadingen, Switzerland). The vials were then clipped onto a cryocane holder and then plunged directly into liquid [9] nitrogen for 24 hours . At the time of recovery the vials were thawed in a water bath (40 r 1
C) for 3 minutes, then the embryos were transferred onto a recovery medium (Y3 macro- and micro-nutrients,
- -1 [9,10,11] vitamins, activated charcoal (1 g L ) and sucrose (0.175 M) based on Sisunandar protocol . The whole experiment was repeated three times using embryos from different harvest times. Seedlings were then allowed to grow on the same medium in a growth room (27 ± 1°C) with a 14 hours light / 10 hours [10] dark photoperiod, for a further 3 month period . The seedlings then underwent a 16-week acclimatization period in soil, in a glasshouse before they were ready to plant in the field.
The embryos were scored as being viable when they produced roots, callus, or a shoot, or when they simply enlarged. Dead embryos were scored as those showing none of these re-growth characteristics. The percentage germination was calculated based on the number of embryos that produced shoots, roots or both. The percentage of normal seedlings was calculated based on the number of the embryos that developed normally and produced both a shoot and a root. Scoring these re-growth characteristics was undertaken after 12 weeks of recovery
Results Dehydration process
Changes in moisture content of Kopyor embryos to rapid dehydration for specific duration times took place in two phases (Figure 1). During the first phase (up to 6 h dehyration) the water is lost sharply from 79 % to almost 25 % FW basis). The moisture content is decrease slowly to 20 % during the second phase (up to 10 h dehydration).
Figure 1. Response of embryo moisture content on fresh weight basis after various duration of dehydration using a rapid
dehydration apparatus.Embryo response to dehydration and cryopreservation
The dehydrated embryos then were monitored on their viabilities after each step of the dehydration process using germinating testing methods (Figure 2). During the first 8 hours of dehydration the germination capacity of the dehydrated embryos remain high. The dehydrated embryos for 6 to 8 hours, embryo viability was high (75 %) compared to that of non-dehydrated embryo (95%). However, the percentage of embryo viable decreased sharply to less than 40 % when the embryos were dehydrated for 10 hours. Not all of the viable embryo could properly germinate and produce normal seedlings. Only 42 % of embryo dehydrated for 8 hours could germinate and produce normal seedlings, while almost all of non-dehydrated embryos were found be able to develop into normal seedlings (95 %), then survive on acclimatization process with a high success rate (70 %, Figure 3).
The non-dehydrated embryo could not survive after plunging directly into liquid nitrogen with none of the embryo remained viable after recovery process. However, more than 60 % of embryos dehydrated for 8 hours and store under liquid nitrogen remained viable. The other tested dehydration times (6 and 10 hours) were found were found to generate less viable embryos, only 52 and 22 %, respectively. Moreover, not all of viable embryo could germinate and produce normal seedlings. Indeed, about one third of the viable embryo could germinate (around 20 % of viable embryo was not germinate), and only almost a half of the germinate embryos could develop normal seedlings (20 % of the total number of embryos used in a given treatment), the remaining of the germinate embryos produced abnormal seedlings, either roots only or root with stunted shoots. All of the abnormal seedlings then eventually died at some stage of recovery process (Figure 3).
Figure 2 Re-growth characteristics of rapid dehydrated embryos for specific periods of time ( ) and re-growth characteristics
of cryopreserved embryo ( ) on a recovery medium. Data show the percentage of viable embryo (A), embryo germinating (B) and percentage of embryo producing normal seedling (C). In each bar chart and each data series, treatments with different letters are significantly different at p- value ≤ 0.05.
Figure 3 Re-growth characteristics comparison between 3-month old seedlings produced from non-cryopreserved embryos (A)
compared to the similar seedlings recovered from cryopreserved embryo (B). The abnormal seedlings (20 %) coming from cryopreserved embryos with root and stunted shoot or root only (C). The Kopyor seedlings coming from control embryos after 1 year in screen house ready for field plantation (D), while the seedlings survive from cryopreservation was still in progress for acclimatization.Discussion [13] Up to now, the only technique available for coconut germplasm conservation is ex situ seed garden . However, this technique is costly to maintain and affected by a number of environmental
stresses, pests or diseases. Alternative ex situ conservation such as seed banking is not possible to be applied because coconut has large seed size (600 to 3,000 g each) and recalcitrant for long term storage. The problem then become more difficult in the case of Indonesian elite mutant, Kopyor, because this [14] mutant is a recessive trait and not possible to develop seedling from the Kopyor seed . So, cryopreservation would be the only technique that may be possible to conserve Kopyor embryo for long- term storage.
This is the first report on the cryopreservation of Kopyor embryo that may be possible to be applied for long-term storage. Our experiments showed that Kopyor embryo can be cryopreserved successfully using a rapid dehydration apparatus applied for 8 hours, followed by rapid freezing and rapid thawing. The re-warmed embryo then were recultured using a standart tissue culture protocol to produce a high number of viable embryos from which 20 % of cryopreserved embryos produced normal seedlings (Figure 2). This result means that Kopyor embryos can be treated on the same way with normal embryos [9,10,15] in which can be cryopreserved and gave a high success rate after storage similar to normal embryos .
Two points coming from the present study that will be important for future research on cryopreservation of Kopyor embryo; viz. not all of the viable embryo after recovered from cryopreservation can germinate and not all of the recovered embryo can produce normal seedlings, complete with shoot and root. We observed that almost 20 % of cryopreserved embryo produced root only or root with stunted shoot (Figure 3). The production of abnormal seedling is a common side effect [16] of cryopreservation and has been reported in many recalcitrant species including normal coconut [9,10,11,15]
. The best explanation for the tendency of cryopreserved embryo to produce abnormal seedlings is [16] that the root meristem is more protected by other compact cell than the shoot meristem . So, the cells of shoot apical meristem are possible more damaged during dehydration treatment and ultra low temperature storage. Wesley-Smith et al. (2004) showed some embryo of recalcitrant species failed to produce normal [17] shoot due to excessive freezing injury which is occur during cryopreservation treatments . These observations suggest that research work should be undertaken in the aim of improving the rate of seedling development.
Acknowledgements
This work is supported by The Muhammadiyah University of Purwokerto Research Scheme. I thank to alkhikmah Mr. Rawan for skillful preparation of plant materials and laboratory works.
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