Plant Cell Reports
Plant Cell Reports (1991) 1 0 : 1 4 4 - ] 4 7
~D Springer-Verlag 1991
Cryopreservation of immature embryos of
Theobroma cacao
Valerie Creaser Pence Center for Reproduction of Endangered Wildlife, Cincinnati Zoo and Botanical Garden, 3400 Vine Street, Cincinnati, OH 45220, USA Received January 14, ]991/Revised version received March 13, 1991
Communicated by I. K. Vasil
Abstract.
(Grout 1979) of several plant species, using protective techniques such as cryoprotectant solutions and slow freezing. These techniques should also be applicable for the preservation of h y d r a t e d tissues of recalcitrant seeds, although the fact t h a t many recalcitrant seeds, such as cacao, are large is likely to make the uniform application of cryoprotectants and temperature changes problematic. An alternative approach has been to excise embryos or embryo axes from large recalcitrant seeds, desiccate them and subject them to rapid freezing in LN. In some cases, seeds traditionally classified as recalcitrant have embryos which, when isolated, can survive drying and freezing (Grout et al. 1983, Grout 1986). With some exceptions, if seed tissue is capable of surviving the desiccation, it is generally not damaged by LN freezing (Stanwood 1985, Pence 1990). Preliminary experiments with isolated embryo axes of cacao suggested t h a t they cannot survive either h y d r a t e d freezing or desiccation using the procedures available currently (unpublished results). As an alternative, the use of immature cacao embryos as a tissue for cryostorage was explored. Several factors suggest t h a t immature embryos might be more adaptable to manipulation t h a n m a t u r e embryos or mature embryo axes. First, they are smaller t h a n m a t u r e embryos and this should allow more uniform cryoprotection, cooling, and thawing in h y d r a t e d freezing procedures, and more uniform desiccation in dry/freezing procedures, which should improve survival (Withers 1979). I m m a t u r e embryos are also more highly embryogenic t h a n m a t u r e embryos (Pence et al. 1980). They are thus more likely to produce regenerative tissue upon recovery, even if the zygotic embryo is damaged during the freezing procedures. In light of this, the following experiments were undertaken in order to evaluate the ability of p r e m a t u r a t i o n cacao embryos to survive cryostorage. Both h y d r a t e d slow freezing and desiccated fast freezing were tested.
Immature, white zygotic embryos of Theobroma cacao L. (cacao) retained the ability to produce callus and to undergo somatic embryogenesis after slow h y d r a t e d freezing and desiccated fast freezing in liquid nitrogen. The highest rate of somatic embryogenesis occurred in embryos which were precultured on a medium containing 3% sucrose, frozen slowly with cryoprotectants before exposure to liquid nitrogen, and recovered on a medium containing 3 m g / l i t e r NAA. Embryos precultured on media containing sucrose increasing to 21% had a higher rate of survival but were less embryogenic after freezing. These results suggest t h a t immature embryos might be used for l o n g - t e r m germplasm storage of T. cacao germplasm.
Key words:
Cryopreservation, embryogenesis, germplasm preservation, recalcitrant seeds, T h e o b r o m a cacao.
INTRODUCTION T h e o b r o m a cacao (cacao) is an understory tree of the tropical moist forest and is of significant commercial importance for the production of chocolate. Cacao seeds are desiccation-sensitive, or recalcitrant, as well as cold-sensitive, and are thus unsuitable for the dry a n d / o r cold seed storage methods traditionally used for germplasm preservation (King and Roberts 1979). Seed t r a n s p o r t is also difficult because of the short-lived n a t u r e of the seeds. A variety of techniques have been examined for extending the longevity of cacao seeds, including maintaining t h e m in the pod or t r e a t i n g the seeds with fungicides, osmotica, respiration inhibitors, and hormones (Figueiredo 1986). Isolated embryo axes have also been examined for their ability to be maintained in a quiescent state in vitro (Ibafiez 1964, Esan 1977, Mumford and B r e t t 1982). While in some cases storage of several months has been achieved, these methods have thus far not provided a technique for the l o n g - t e r m storage of cacao germplasm. Cryopreservation, or storage in liquid nitrogen (LN), is a technique which promises stable, l o n g - t e r m maintenance of plant germplasm when the plant tissues can survive the freezing process itself. M a n y desiccation- tolerant (orthodox) seeds have been shown to survive LN freezing in the dry state (Stanwood 1985, Pence 1991a). On the other hand, because of their high levels of moisture, freezing of recalcitrant seeds, such as cacao, is generally lethal. Freezing of h y d r a t e d tissues has been accomplished, however, with suspension cultures, protoplasts (Withers 1985), shoot meristems ( K a r t h a 1985), somatic embryos (Withers 1979), pollen and zygotic embryos (Bajaj 1985), and imbibed seeds
MATERIALS AND M E T H O D S Fruit of Theobroma cacao L. for these studies were generously provided by the Hershey Foods Corporation. Pods approximately 5-5.5 c m in diameter were chosen in order to obtain a high percentage of white embryos prior to cotyledon enlargement and filling. Embryos ranged from approximately 4 to 8 rnm in length. Fruit were sprayed with 7 0 % ethanol and the embryos were removed aseptically. Embryos were generally cultured whole, although m a n y were often nicked
during excision. In one experiment, axes and cotyledons were cultured separately. For slow freezing, embryos were pretreated in a liquid cryoprotection medium for 45 minutes at room temperature
145 prior to freezing. For this treatment, embryos were first added to a medium consisting of Murashige and Skoog (1962) (MS) salts and organics and 3% sucrose, pH 5.6. Over a period of 45 minutes an equal volume of MS medium with 1 M sucrose and 2{)% dimethylsulfoxide (DMSO) was added in 3 increments. This gave a final concentration of 0.5 M sucrose and 10% DMSO. For embryos which had been precultured on media containing 21% sucrose, the cryoprotection media were modified so that both solutions contained 21% sucrose. The embryos were then cooled to 4 ~ C for 30 minutes and aseptically transferred from the medium to either aluminum foil in disposable petri dishes or 2 ml cryovials. Slow freezing was accomplished in a controlled freezing apparatus (Le Minicool, Compagnie Fran~aise de Produits Oxygenes) at a rate of [:}.4~ down to -35 ~ or -40~ In some experiments, once this temperature was reached, the embryos were thawed slowly by opening the freezing chamber and allowing it to equilibrate to room temperature at a rate of approximately 0.8~ after which the embryos were transferred to recovery medium. Alternatively, the embryos were transferred to LN, incubated overnight. For thawing the cryovial was placed on the benchtop at ambient temperature for 20 minutes before opening, although in one experiment the tissues were thawed rapidly by immersing the cryovial in a 40~ water bath. Thawed embryos were then placed directly on recovery medium. Control embryos were not precultured but were subjected to the cryoprotectant solution. Some were placed directly on recovery medium, while others were frozen slowly, as for the precultured embryos. For desiccated freezing, drying was accomplished by placing the embryos on sterile filter paper in an open Petri dish under the laminar flow hood overnight. Dried embryos were then placed in a sterile polypropylene 2 ml cryovial and were fast frozen by immersion in LN, incubated and thawed as described above. Embryos were precultured for several days before freezing, either on a single medium or, through successive transfers, on media with increasing concentrations of sucrose. Preculture media consisted of MS medium with 1 g/l casein hydrolysate, 10% deproteinized coconut water, sucrose at 3, 9, 15, or 21%, and 0.8% agar or 0.2% Phytagel, adjusted to pH 5.6. Filter sterilized abscisic acid (ABA) was added, as indicated, after autoclaving at a concentration of 10 uM. Transfers were made every 2 days from 3% to 9%, 15%, and finally 21% sucrose. Controls kept at 3% sucrose were similarly transferred to fresh media on the same schedule. Recovery medium consisted of the preculture medium with 3% sucrose, 1.5 or 3.0 mg/liter naphthaleneaeetic acid (NAA), and 0.05% activated charcoal. Embryos were incubated in a growth chamber at 26~ with a 16:8 hour light:dark cycle. Somatic embryos initiated from frozen zygotic embryos were decotyledonated and placed on half-strength MS medium with 3% sucrose, 2 uM zeatin, and 0.05% charcoal (Duhem et al. 1989) for further growth. Rates of callus and somatic embryogenesis were considered as the number of explants responding of those explants which remained sterile. A white bacterial contamination was occasionally initiated from the explants which appeared to lower their responsiveness. Although initial explant numbers were similar between treatments, only those which appeared not to be contaminated were considered.
RESULTS Preliminary experiments in which newly excised, hydrated immature white embryos were slow-frozen and recovered on a medium with 1.5 mg/liter NAA indicated that such procedures were generally lethal. After freezing, the embryonic axis was brown with the cotyledons pale and lacking turgot. Occasionally, however, these embryos produced callus and, even less frequently, somatic embryos, indicating that the
potential for recovering tissue from the frozen embryos existed. Table 1. The effect of preculture on callus initiation and somatic embryogenesis from immature cacao embryos. Hydrated embryos were frozen slowly to -40~ thawed slowly, and grown on recovery medium with 1.5 mg/liter NAA. Unfrozen controls were cultured after exposure to cryoprotectant solutions without freezing. Total No. Explants No. with No.with (Clean) Callus Embryos
Experiment
Pretreatment
I
None 3% Suc 21% Suc+ABA
18 15 16
1 0 14
0 0 0
II a
None 21% Suc+ABA
33 48
0 18
0 0
III
Unfrozen 3% Suc 3% Suc+ABA 21~ Suc 21% Suc+ABA
18 20 2 19 4
18 0 0 6 0
3 0 0 0 0
IV
Unfrozen None 3% Suc 3% Suc+ABA 21% Suc 21% Suc+ABA
11 11 12 16 14 15
11 0 0 0 1 3
1 0 0 0 0 0
aIn this experiment axes and cotyledons were cultured separately in equal numbers and were counted separately as explants. Table 2. The effect of preculture on callus initiation and somatic embryogenesls from immature white cacao embryos desiccated and fast frozen in liquid nitrogen and recovered on recovery medium with 1.5 rag/liter N.hA. Total No. No. with Explants No. with Somatic (Clean) Callus Embryos
Experiment
Pretreatment
I
21% Suc+ABA (dried, not frozen) 21% Suc+ABA
14
6
0
15
9
1
II
None 21% Suc+ABA
70 52
0 12
0 0
III
3% Suc 3% Suc+ABA 21% Suc 21% Suc+ABA
6 14 11 16
0 0 0 1
0 0 0 0
In a series of four experiments (Table 1), the effectiveness of a preculture period on increasing the survival of embryos through slow freezing was tested. Precultures on 3% sucrose or sucrose increasing to 2196 were compared with and without ABA. Embryos which were not precultured and not frozen expanded somewhat on the recovery medium, became green and pink, and produced callus from the axis or the cotyledons. Somatic embryos were produced from 14% of the nonfrozen embryos, but in these experiments, no somatic embryogenesis occurred from frozen embryos. Callus growth from frozen embryos occurred, however, from those precultured on 21%
146 sucrose media, with and without ABA. In Experiment II, in which axes and cotyledons were cultured separately, of the 18 explants producing callus, 3 of these were axes and 15 were cotyledons. In three of these experiments, embryos were also dried and fast frozen (Table 2). These also produced callus, but only on 21% sucrose medium with ABA. Somatic embryos were initiated from one dry/frozen embryo pretreated on 21~ sucrose plus ABA. In two further experiments, embryos were similarly pretreated, subjected to h y d r a t e d slow freezing, and recovered on a medium containing 3 m g / l i t e r NAA (Table 3). As in the earIier experiments, callus production was greater from embryos precultured on 21~ compared with 3 ~ sucrose. The rate of somatic embryogenesis from unfrozen embryos was also similar to that observed in the previous experiments, but somatic embryogenesis from frozen embryos was more frequent. In the first experiment, frozen zygotic embryos precultured on 3 ~ sucrose with and without ABA had higher rates of somatic embryo production t h a n those observed from frozen embryos on 21~ sucrose with or without ABA. In the second experiment, somatic embryogenesis was observed only from embryos precultured on 21%, but the rate of initiation was similar to that of Experiment I. Frozen precultured embryos became entirely dark brown upon thawing, although many maintained some turgor in the cotyledons. Somatic embryogenesis was observed from the apical meristem area, as well as from mid-hypocotyl tissue. Because of the twisting of the cotyledons, the origin of embryogenic tissue could not be determined in all cases and some may have also originated from the edges of the cotyledons. Somatic embryos originating from f r o z e n / t h a w e d zygotic embryos maintained the potential for developing into plants (Figure 1). Figure 1. Young cacao plant developing from a somatic embryo initiated from a frozen and thawed immature zygotic embryo.
Table 3. Callus and somatic embryo production from hydrated immature cacao embryos pretreated and slow frozen to -35~ transferred to LN overnight, and cultured on recovery medium with 3 mg/liter NAA. Embryos in Experiment II were subjected to rapid thawing.
Pretreatment Experiment I Unfrozen Control Frozen Control, no preculture
Total
No. with Callus
No. with Somatic Embryos
8 11
6 7
1 0
3 ~ Suc 3% Sue+ABA
15 16
6 2
3 6
21~ Suc 21~ Suc+ABA
8 13
4 11
0 1
Summary: All 3 ~ Suc All 21~ Suc
31 21
8 15
9 1
Experiment II Frozen Control, no preculture 3% Suc 21~ Suc
11 36 44
5 2 15
1 0 3
DISCUSSION These results demonstrate that immature cacao embryos can express their embryogenic capacity after freezing in LN. Cacao embryos of this stage are normally embryogenic in the presence of auxin (Pence et al. 1980, A d u - A m p o m a h et al. 1988), and the capacity for embryogenesis decreases as the zygotic embryos mature. Maturation in cacao embryos is characterized by anthocyanin and lipid accumulation and by an increase in f a t t y acid saturation, and these changes can be stimulated in vitro by growing immature embryos on elevated levels of sucrose (Pence et al. 1981a,b). On the other hand, pretreatments with sugar have also been used to prepare tissues ofvarious types for freezing (Withers 1985). Preculture of cacao embryos on elevated sucrose media did improve survival of frozen embryos in all experiments, as evidenced by the production of callus, but because of the effect on maturation in cacao, such treatments might also be expected to decrease the ability of the embryo to undergo somatic embryogenesis. This appears to have been the case, as somatic embryogenesis occurred from zygotic embryos precultured on 21~ sucrose at a very low rate. In the case of zygotic embryos precultured on 3 ~ sucrose, somatic embryogenesis either did not occur, or occurred at a much higher rate than with 21~ embryos. This suggests that the potential for regenerating plants from these embryos is greater than with 21% precultured embryos, but t h a t other factors determine whether this potential is expressed. There was no apparent effect of ABA on the ability to survive hydrated freezing in these experiments. Survival through desiccated fast freezing was observed only in embryos cultured on 21% sucrose plus ABA. Despite the low response, the results presented here demonstrate that callus and somatic embryos can be obtained from pretreated immature cacao embryos after desiccated freezing, as well as after hydrated freezing. Improvements in survival might be made by modifying the ABA pretreatments or by adding other compounds in vitro, as has been reported for somatic embryos (Senaratna et al, 1989). ABA has been implicated in the development of desiccation tolerance during orthodox seed
147 maturation (Koornneef et al. 1989), although developing cacao embryos contain significant levels of ABA as well (Pence 1991b). Media and freezing protocols should also be explored for further optimizing survival and embryogenic frequency, since embryogenesis from frozen hydrated embryos was better with slow freezing to -35~ and a recovery medium with 3 rag/liter NAA, compared with experiments using -40~ and 1.5 mg/liter NAA. Cryostorage of immature embryos of cacao offers a potential technique for long-term germplasm storage, genetically equivalent to a seed bank. Even though the integrity of the original embryo is not maintained through freezing, regenerative cells do survive. These can be used to retrieve the frozen lines through somatic embryogenesis, using techniques which have been reported for germinating cacao somatic embryos (Novak et al. 1986, Adu-Ampomah et al. 1988, Duhem et al. 1989). In order to evaluate the applicability of the procedure, the question of the frequency of genetic change during this process also needs to be addressed. Isozyme markers which have been used to screen cacao germplasm might be useful in this regard (Withers and Williams 1986). A number of other economically important tropical species, such as coconut, mango, and rubber, also have seeds which are desiccation sensitive (King and Roberts 1979). Other, wild species from tropical moist areas, about which little is known, are being threatened by loss of habitat (Raven 1987). Although the exact numbers are unknown, it is likely that many of these possess recalcitrant seeds as well. When immature zygotic embryos of recalcitrant species can be manipulated to undergo somatic embryogenesis, they may be good candidates for attention as tissues for cryopreservation. As a supplement to other conservation and germplasm storage technologies, immature embryo cryostorage offersthe potential for preserving genetic material from both commercially important and endangered wild species which are otherwise recalcitrant to current preservation methods.
ACKNOWLEDGEMENTS The Department of Biological Sciences, University of Cincinnati, is gratefully acknowledged for providing facilities for this research; also Mr. Gordon Patterson, Hershey Foods Corporation, for supplying the fruit for these studies; and Dr. John L. Caruso, in whose laboratory these experiments were conducted.
LITERATURE CITED Adu-Ampomah Y, Novak F J, Afza R, Van Duren M, Perea-Dallos M (1988) Caf~ Cacao Th~! 32:187-200 Bajaj YPS (1985) In: KK Kartha (ed.), Gryopreservation of Plant Cells and Organs. CRC Press, Boca Raton, FL, pp. 227-242 Duhem K, Le Mercier N, Boxus P (1989) Caf~ Cacao Th~ 23: 9-14 Esan EB (1977) In: Proc 5th Int Cacao Res Conf, 1975, Cacao Res Inst Nigeria, Ibadan, pp 116-125 Figueiredo, SFL (1986) Revista Theobroma 16:17-29 Grout BWW (1979) Cryo-Lett 1:71-76 Grout BWW (1986) In: Withers LA, Aldersou PC, eds. Plant Tissue Culture and its Agricultural Applications. Butterworth, London, pp. 303-309. Grout BWW, Shelton K, Pritchard HW (1983) Ann Pot 52: 381-382 Ibafiez ML (1964) Wrop Agric (Trinidad) 41:325-328 Kartha KK (1985) In: KK Kartha (ed.), Cryopreservation of Plant Cells and Organs. C R C Press, Boca Raton, FL, pp. 115-134. King M W , Roberts E H (1979) The Storage of Recalcitrant Seeds--Achievements and PossibleApproaches. International Board for Plant Genetic Resources, Rome
Koornneef M, Hanhart C J, Hilhorst HWM, Karssen CM (1989) Plant Physiol 90:463-469 Mumford PM, Brett AC (1982) Trop Agric 59:306-310 Murashige T, Skoog F (1962) Physiol Plant 15:473-497 Novak F J, Donini B, Owusu G (1986) In: Nuclear Techniques and In Vitro Culture for Plant Improvement. Proc Internat F A O / I A E A Symposium, IAEA, Vienna, pp. 443-449 Pence VC (1990) Cryobiology 27:212-218 Pence VC (1991a) Seed Sci Wech (in press) Pence VC (1991b) Plant Physiol 95 (in press) Pence VC, Hasegawa PM, Janick J (1980) Z Pflanzenphysiol 98:1-14 Pence VC, Hasegawa PM, Janick J (1981a) J Amer Soe Hort Sci 106:381-385 Pence VC, Hasegawa PM, Janick J (1981b) Physiol Plant 53: 378-384 Raven PH (1987) In: Bramwell D, Hamann O, Heywood V, Synge H (eds) Botanic Gardens and World Conservation Strategy. Academic Press Inc, London, pp 19-29 Senaratna T, McKersie BD, Bewley SR. (1989) Plant Science 65:253-259 Stanwood PC (1985) In: KK Kartha (ed.), Cryopreservation of Plant Cells and Organs, CRG Press, Boca Raton, FL, pp. 199-226. Withers LA (1979) Freeze-preservation of somatic embryos and clonal plantlets of carrot (Daucus carota L). Plant Physiol 63:460-467 Withers LA (1985) In: KK Kartha (ed.), Cryopreservation of Plant Cells and Organs. CRC Press, Boca Raton, FL, pp. 243-267 Withers LA, Williams J T (1986) In Vitro Conservation. International Board for Plant Genetic Resources Research Highlights 1984-1985. IBPGR, Rome
Plant Cell Reports (1991) 1 0 : 1 4 4 - ] 4 7
~D Springer-Verlag 1991
Cryopreservation of immature embryos of
Theobroma cacao
Valerie Creaser Pence Center for Reproduction of Endangered Wildlife, Cincinnati Zoo and Botanical Garden, 3400 Vine Street, Cincinnati, OH 45220, USA Received January 14, ]991/Revised version received March 13, 1991
Communicated by I. K. Vasil
Abstract.
(Grout 1979) of several plant species, using protective techniques such as cryoprotectant solutions and slow freezing. These techniques should also be applicable for the preservation of h y d r a t e d tissues of recalcitrant seeds, although the fact t h a t many recalcitrant seeds, such as cacao, are large is likely to make the uniform application of cryoprotectants and temperature changes problematic. An alternative approach has been to excise embryos or embryo axes from large recalcitrant seeds, desiccate them and subject them to rapid freezing in LN. In some cases, seeds traditionally classified as recalcitrant have embryos which, when isolated, can survive drying and freezing (Grout et al. 1983, Grout 1986). With some exceptions, if seed tissue is capable of surviving the desiccation, it is generally not damaged by LN freezing (Stanwood 1985, Pence 1990). Preliminary experiments with isolated embryo axes of cacao suggested t h a t they cannot survive either h y d r a t e d freezing or desiccation using the procedures available currently (unpublished results). As an alternative, the use of immature cacao embryos as a tissue for cryostorage was explored. Several factors suggest t h a t immature embryos might be more adaptable to manipulation t h a n m a t u r e embryos or mature embryo axes. First, they are smaller t h a n m a t u r e embryos and this should allow more uniform cryoprotection, cooling, and thawing in h y d r a t e d freezing procedures, and more uniform desiccation in dry/freezing procedures, which should improve survival (Withers 1979). I m m a t u r e embryos are also more highly embryogenic t h a n m a t u r e embryos (Pence et al. 1980). They are thus more likely to produce regenerative tissue upon recovery, even if the zygotic embryo is damaged during the freezing procedures. In light of this, the following experiments were undertaken in order to evaluate the ability of p r e m a t u r a t i o n cacao embryos to survive cryostorage. Both h y d r a t e d slow freezing and desiccated fast freezing were tested.
Immature, white zygotic embryos of Theobroma cacao L. (cacao) retained the ability to produce callus and to undergo somatic embryogenesis after slow h y d r a t e d freezing and desiccated fast freezing in liquid nitrogen. The highest rate of somatic embryogenesis occurred in embryos which were precultured on a medium containing 3% sucrose, frozen slowly with cryoprotectants before exposure to liquid nitrogen, and recovered on a medium containing 3 m g / l i t e r NAA. Embryos precultured on media containing sucrose increasing to 21% had a higher rate of survival but were less embryogenic after freezing. These results suggest t h a t immature embryos might be used for l o n g - t e r m germplasm storage of T. cacao germplasm.
Key words:
Cryopreservation, embryogenesis, germplasm preservation, recalcitrant seeds, T h e o b r o m a cacao.
INTRODUCTION T h e o b r o m a cacao (cacao) is an understory tree of the tropical moist forest and is of significant commercial importance for the production of chocolate. Cacao seeds are desiccation-sensitive, or recalcitrant, as well as cold-sensitive, and are thus unsuitable for the dry a n d / o r cold seed storage methods traditionally used for germplasm preservation (King and Roberts 1979). Seed t r a n s p o r t is also difficult because of the short-lived n a t u r e of the seeds. A variety of techniques have been examined for extending the longevity of cacao seeds, including maintaining t h e m in the pod or t r e a t i n g the seeds with fungicides, osmotica, respiration inhibitors, and hormones (Figueiredo 1986). Isolated embryo axes have also been examined for their ability to be maintained in a quiescent state in vitro (Ibafiez 1964, Esan 1977, Mumford and B r e t t 1982). While in some cases storage of several months has been achieved, these methods have thus far not provided a technique for the l o n g - t e r m storage of cacao germplasm. Cryopreservation, or storage in liquid nitrogen (LN), is a technique which promises stable, l o n g - t e r m maintenance of plant germplasm when the plant tissues can survive the freezing process itself. M a n y desiccation- tolerant (orthodox) seeds have been shown to survive LN freezing in the dry state (Stanwood 1985, Pence 1991a). On the other hand, because of their high levels of moisture, freezing of recalcitrant seeds, such as cacao, is generally lethal. Freezing of h y d r a t e d tissues has been accomplished, however, with suspension cultures, protoplasts (Withers 1985), shoot meristems ( K a r t h a 1985), somatic embryos (Withers 1979), pollen and zygotic embryos (Bajaj 1985), and imbibed seeds
MATERIALS AND M E T H O D S Fruit of Theobroma cacao L. for these studies were generously provided by the Hershey Foods Corporation. Pods approximately 5-5.5 c m in diameter were chosen in order to obtain a high percentage of white embryos prior to cotyledon enlargement and filling. Embryos ranged from approximately 4 to 8 rnm in length. Fruit were sprayed with 7 0 % ethanol and the embryos were removed aseptically. Embryos were generally cultured whole, although m a n y were often nicked
during excision. In one experiment, axes and cotyledons were cultured separately. For slow freezing, embryos were pretreated in a liquid cryoprotection medium for 45 minutes at room temperature
145 prior to freezing. For this treatment, embryos were first added to a medium consisting of Murashige and Skoog (1962) (MS) salts and organics and 3% sucrose, pH 5.6. Over a period of 45 minutes an equal volume of MS medium with 1 M sucrose and 2{)% dimethylsulfoxide (DMSO) was added in 3 increments. This gave a final concentration of 0.5 M sucrose and 10% DMSO. For embryos which had been precultured on media containing 21% sucrose, the cryoprotection media were modified so that both solutions contained 21% sucrose. The embryos were then cooled to 4 ~ C for 30 minutes and aseptically transferred from the medium to either aluminum foil in disposable petri dishes or 2 ml cryovials. Slow freezing was accomplished in a controlled freezing apparatus (Le Minicool, Compagnie Fran~aise de Produits Oxygenes) at a rate of [:}.4~ down to -35 ~ or -40~ In some experiments, once this temperature was reached, the embryos were thawed slowly by opening the freezing chamber and allowing it to equilibrate to room temperature at a rate of approximately 0.8~ after which the embryos were transferred to recovery medium. Alternatively, the embryos were transferred to LN, incubated overnight. For thawing the cryovial was placed on the benchtop at ambient temperature for 20 minutes before opening, although in one experiment the tissues were thawed rapidly by immersing the cryovial in a 40~ water bath. Thawed embryos were then placed directly on recovery medium. Control embryos were not precultured but were subjected to the cryoprotectant solution. Some were placed directly on recovery medium, while others were frozen slowly, as for the precultured embryos. For desiccated freezing, drying was accomplished by placing the embryos on sterile filter paper in an open Petri dish under the laminar flow hood overnight. Dried embryos were then placed in a sterile polypropylene 2 ml cryovial and were fast frozen by immersion in LN, incubated and thawed as described above. Embryos were precultured for several days before freezing, either on a single medium or, through successive transfers, on media with increasing concentrations of sucrose. Preculture media consisted of MS medium with 1 g/l casein hydrolysate, 10% deproteinized coconut water, sucrose at 3, 9, 15, or 21%, and 0.8% agar or 0.2% Phytagel, adjusted to pH 5.6. Filter sterilized abscisic acid (ABA) was added, as indicated, after autoclaving at a concentration of 10 uM. Transfers were made every 2 days from 3% to 9%, 15%, and finally 21% sucrose. Controls kept at 3% sucrose were similarly transferred to fresh media on the same schedule. Recovery medium consisted of the preculture medium with 3% sucrose, 1.5 or 3.0 mg/liter naphthaleneaeetic acid (NAA), and 0.05% activated charcoal. Embryos were incubated in a growth chamber at 26~ with a 16:8 hour light:dark cycle. Somatic embryos initiated from frozen zygotic embryos were decotyledonated and placed on half-strength MS medium with 3% sucrose, 2 uM zeatin, and 0.05% charcoal (Duhem et al. 1989) for further growth. Rates of callus and somatic embryogenesis were considered as the number of explants responding of those explants which remained sterile. A white bacterial contamination was occasionally initiated from the explants which appeared to lower their responsiveness. Although initial explant numbers were similar between treatments, only those which appeared not to be contaminated were considered.
RESULTS Preliminary experiments in which newly excised, hydrated immature white embryos were slow-frozen and recovered on a medium with 1.5 mg/liter NAA indicated that such procedures were generally lethal. After freezing, the embryonic axis was brown with the cotyledons pale and lacking turgot. Occasionally, however, these embryos produced callus and, even less frequently, somatic embryos, indicating that the
potential for recovering tissue from the frozen embryos existed. Table 1. The effect of preculture on callus initiation and somatic embryogenesis from immature cacao embryos. Hydrated embryos were frozen slowly to -40~ thawed slowly, and grown on recovery medium with 1.5 mg/liter NAA. Unfrozen controls were cultured after exposure to cryoprotectant solutions without freezing. Total No. Explants No. with No.with (Clean) Callus Embryos
Experiment
Pretreatment
I
None 3% Suc 21% Suc+ABA
18 15 16
1 0 14
0 0 0
II a
None 21% Suc+ABA
33 48
0 18
0 0
III
Unfrozen 3% Suc 3% Suc+ABA 21~ Suc 21% Suc+ABA
18 20 2 19 4
18 0 0 6 0
3 0 0 0 0
IV
Unfrozen None 3% Suc 3% Suc+ABA 21% Suc 21% Suc+ABA
11 11 12 16 14 15
11 0 0 0 1 3
1 0 0 0 0 0
aIn this experiment axes and cotyledons were cultured separately in equal numbers and were counted separately as explants. Table 2. The effect of preculture on callus initiation and somatic embryogenesls from immature white cacao embryos desiccated and fast frozen in liquid nitrogen and recovered on recovery medium with 1.5 rag/liter N.hA. Total No. No. with Explants No. with Somatic (Clean) Callus Embryos
Experiment
Pretreatment
I
21% Suc+ABA (dried, not frozen) 21% Suc+ABA
14
6
0
15
9
1
II
None 21% Suc+ABA
70 52
0 12
0 0
III
3% Suc 3% Suc+ABA 21% Suc 21% Suc+ABA
6 14 11 16
0 0 0 1
0 0 0 0
In a series of four experiments (Table 1), the effectiveness of a preculture period on increasing the survival of embryos through slow freezing was tested. Precultures on 3% sucrose or sucrose increasing to 2196 were compared with and without ABA. Embryos which were not precultured and not frozen expanded somewhat on the recovery medium, became green and pink, and produced callus from the axis or the cotyledons. Somatic embryos were produced from 14% of the nonfrozen embryos, but in these experiments, no somatic embryogenesis occurred from frozen embryos. Callus growth from frozen embryos occurred, however, from those precultured on 21%
146 sucrose media, with and without ABA. In Experiment II, in which axes and cotyledons were cultured separately, of the 18 explants producing callus, 3 of these were axes and 15 were cotyledons. In three of these experiments, embryos were also dried and fast frozen (Table 2). These also produced callus, but only on 21% sucrose medium with ABA. Somatic embryos were initiated from one dry/frozen embryo pretreated on 21~ sucrose plus ABA. In two further experiments, embryos were similarly pretreated, subjected to h y d r a t e d slow freezing, and recovered on a medium containing 3 m g / l i t e r NAA (Table 3). As in the earIier experiments, callus production was greater from embryos precultured on 21~ compared with 3 ~ sucrose. The rate of somatic embryogenesis from unfrozen embryos was also similar to that observed in the previous experiments, but somatic embryogenesis from frozen embryos was more frequent. In the first experiment, frozen zygotic embryos precultured on 3 ~ sucrose with and without ABA had higher rates of somatic embryo production t h a n those observed from frozen embryos on 21~ sucrose with or without ABA. In the second experiment, somatic embryogenesis was observed only from embryos precultured on 21%, but the rate of initiation was similar to that of Experiment I. Frozen precultured embryos became entirely dark brown upon thawing, although many maintained some turgor in the cotyledons. Somatic embryogenesis was observed from the apical meristem area, as well as from mid-hypocotyl tissue. Because of the twisting of the cotyledons, the origin of embryogenic tissue could not be determined in all cases and some may have also originated from the edges of the cotyledons. Somatic embryos originating from f r o z e n / t h a w e d zygotic embryos maintained the potential for developing into plants (Figure 1). Figure 1. Young cacao plant developing from a somatic embryo initiated from a frozen and thawed immature zygotic embryo.
Table 3. Callus and somatic embryo production from hydrated immature cacao embryos pretreated and slow frozen to -35~ transferred to LN overnight, and cultured on recovery medium with 3 mg/liter NAA. Embryos in Experiment II were subjected to rapid thawing.
Pretreatment Experiment I Unfrozen Control Frozen Control, no preculture
Total
No. with Callus
No. with Somatic Embryos
8 11
6 7
1 0
3 ~ Suc 3% Sue+ABA
15 16
6 2
3 6
21~ Suc 21~ Suc+ABA
8 13
4 11
0 1
Summary: All 3 ~ Suc All 21~ Suc
31 21
8 15
9 1
Experiment II Frozen Control, no preculture 3% Suc 21~ Suc
11 36 44
5 2 15
1 0 3
DISCUSSION These results demonstrate that immature cacao embryos can express their embryogenic capacity after freezing in LN. Cacao embryos of this stage are normally embryogenic in the presence of auxin (Pence et al. 1980, A d u - A m p o m a h et al. 1988), and the capacity for embryogenesis decreases as the zygotic embryos mature. Maturation in cacao embryos is characterized by anthocyanin and lipid accumulation and by an increase in f a t t y acid saturation, and these changes can be stimulated in vitro by growing immature embryos on elevated levels of sucrose (Pence et al. 1981a,b). On the other hand, pretreatments with sugar have also been used to prepare tissues ofvarious types for freezing (Withers 1985). Preculture of cacao embryos on elevated sucrose media did improve survival of frozen embryos in all experiments, as evidenced by the production of callus, but because of the effect on maturation in cacao, such treatments might also be expected to decrease the ability of the embryo to undergo somatic embryogenesis. This appears to have been the case, as somatic embryogenesis occurred from zygotic embryos precultured on 21~ sucrose at a very low rate. In the case of zygotic embryos precultured on 3 ~ sucrose, somatic embryogenesis either did not occur, or occurred at a much higher rate than with 21~ embryos. This suggests that the potential for regenerating plants from these embryos is greater than with 21% precultured embryos, but t h a t other factors determine whether this potential is expressed. There was no apparent effect of ABA on the ability to survive hydrated freezing in these experiments. Survival through desiccated fast freezing was observed only in embryos cultured on 21% sucrose plus ABA. Despite the low response, the results presented here demonstrate that callus and somatic embryos can be obtained from pretreated immature cacao embryos after desiccated freezing, as well as after hydrated freezing. Improvements in survival might be made by modifying the ABA pretreatments or by adding other compounds in vitro, as has been reported for somatic embryos (Senaratna et al, 1989). ABA has been implicated in the development of desiccation tolerance during orthodox seed
147 maturation (Koornneef et al. 1989), although developing cacao embryos contain significant levels of ABA as well (Pence 1991b). Media and freezing protocols should also be explored for further optimizing survival and embryogenic frequency, since embryogenesis from frozen hydrated embryos was better with slow freezing to -35~ and a recovery medium with 3 rag/liter NAA, compared with experiments using -40~ and 1.5 mg/liter NAA. Cryostorage of immature embryos of cacao offers a potential technique for long-term germplasm storage, genetically equivalent to a seed bank. Even though the integrity of the original embryo is not maintained through freezing, regenerative cells do survive. These can be used to retrieve the frozen lines through somatic embryogenesis, using techniques which have been reported for germinating cacao somatic embryos (Novak et al. 1986, Adu-Ampomah et al. 1988, Duhem et al. 1989). In order to evaluate the applicability of the procedure, the question of the frequency of genetic change during this process also needs to be addressed. Isozyme markers which have been used to screen cacao germplasm might be useful in this regard (Withers and Williams 1986). A number of other economically important tropical species, such as coconut, mango, and rubber, also have seeds which are desiccation sensitive (King and Roberts 1979). Other, wild species from tropical moist areas, about which little is known, are being threatened by loss of habitat (Raven 1987). Although the exact numbers are unknown, it is likely that many of these possess recalcitrant seeds as well. When immature zygotic embryos of recalcitrant species can be manipulated to undergo somatic embryogenesis, they may be good candidates for attention as tissues for cryopreservation. As a supplement to other conservation and germplasm storage technologies, immature embryo cryostorage offersthe potential for preserving genetic material from both commercially important and endangered wild species which are otherwise recalcitrant to current preservation methods.
ACKNOWLEDGEMENTS The Department of Biological Sciences, University of Cincinnati, is gratefully acknowledged for providing facilities for this research; also Mr. Gordon Patterson, Hershey Foods Corporation, for supplying the fruit for these studies; and Dr. John L. Caruso, in whose laboratory these experiments were conducted.
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