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In vitro Growth Responses and Plant Regeneration from Cryopreserved Meristems of Cassava (Manihot esculenta Crantz)
In vitro Growth Responses and Plant Regeneration from Cryopreserved Meristems of Cassava (Manihot esculenta Crantz)*) K. K.
KARTHA,
N. 1. LEUNG and 1. A. MROGINSKI**)
Prairie Regional Laboratory, National Research Council of Canada, Saskatoon, Saskatchewan, Canada S7N OW9 Received April 23, 1982 . Accepted May 6,1982
Summary A droplet-freezing method has been developed for the cryopreservation of cassava, Manihot esculenta Crantz, meristems. The meristems treated with a 15 % DMSO + 3 % sucrose solution for 15 min were frozen over an 18/Lm aluminium foil in 2-3/LI droplets of the cryoprotectants in plastic petri dishes at a cooling rate of 0.5 °CI min to various sub-zero temperatures (-20; -25; -30 and -40°C) and stored in liquid nitrogen (-196°C). The meristems retrieved from liquid nitrogen storage, upon thawing and return to in vitro culture on plant regeneration medium, exhibited various morphogenetic responses such as differentiation of callus and leaves and whole plantlets. The plantlets were successfully grown in pots.
Key words: Manihot esculenta, cryopreservation, dropletJreezing method, dimethylsulfoxide (DMSO), plant regeneration in vitro, germplasm preservation.
Introduction Cassava (Manihot esculenta Crantz), an annual root crop, is extensively grown as a source of human food in the humid tropics of the world. It is propagated vegetatively through the use of stem cuttings. Techniques to regenerate plants from shoot apical meristems of cassava cultured in vitro have been developed in this laboratory (Kartha et al., 1974) and have been applied to the production of mosaic disease-free plants (Kartha and Gamborg, 1975). The meristem culture technique has been successfully extended to short-term preservation of cassava collections (Roca, 1980), but alternative approaches have to be sought to warrant germplasm preservation for any extended periods of time. In recent years, cryogenic techniques have been developed for preservation of meristems of a few species, such as carnation (Seibert, 1976; Uemura and Sakai, 1980), potato (Grout and Henshaw, 1978; Towill, 1981), pea (Kartha et al., 1979), strawberry (Sakai *) NRCC No. 20411. **) Visiting Scientist, Facultad de Ciencias Agrarias, Instituto de Bot:inica del Nordeste, Casilla de Correos 209, Corrientes (3400) Argentina. Abbreviations: DMSO, dimethylsulfoxide; BA, benzyladenine; NAA, naphthaleneacetic acid; GA3, grbberellic acid.
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et al., 1978; Kartha et al., 1980) and vegetative buds of hardy fruit trees (Sakai and Nishiyama, 1978) (for a review see Kartha, 1981). It would be desirable to similarly cryopreserve Icassava rneristeIlIls for long periods of time. The need for preservation is pressing especially due to the vegetative mode of propagation of this crop. Bajaj (1977) claim~d to have succeeded in regenerating plantlets from the cryopreserved meristems of cassava. Three years of research at this laboratory aimed at this direction with this crop failed until the development of a new technique, i.e. the dropletfreezing technique. This paper presents the details of the new technique as well as the various morphogenetic responses of cassava meristems subsequent to their exposure to sub-zero temperatures and cryopreservation.
Materials and Methods Dormant stakes of cassava (Manihot escutenta Crantz) cultivars, MVEN-218, CM 321-188, CM 489-1 and CM 1684 were obtained from CIAT (Centro Internacional de Agricultura Tropical). The stakes were cut ir sections with 3-4 nodes each, the upper cut ends sealed with paraffin, and grown in a growth room at 26°C, 16 h photoperiods of 7500 Ix intensity and 70 % relative humidity. Fdr cryopresetvation experiments buds of sprouted cuttings as well as subsequently develqped secondary buds were used. After removing the outer whorls of visible leaves, buds were surface sterilized in 70 % ethanol for 60.sec and rinsed thoroughly in 3 changes of sterile distilled water. Meristems measuring 0.4-0.5 mm in length and containing a pair of leaf primordia and some subjacent tissue were isolated under a stereo-mi~roscope and transferred to 10 ml of hormone-free liquid MS (O-MS) medium (Murashige ana Skoog, 1962) in a centrifuge tube. An equal volume of a 30 % solution (v/v) of dimethylsulfli>xide (DMSO) in O-MS medium containing 3 % sucrose was gradually added to this solution in 10 instalments over a period of 30 min until a final concentration of 15 % was reached. The meristems were allowed to equilibrate in this «freezing solution» for' 15 min. The free~ing of meristems was performed in 9 cm plastic petri dishes in droplets. An 18/Lm gauge sterile aluminium foil was placed inside each petri dish and 20-30 DMSO-treated meristems were evenly distributed over the foil along with 2-3 /LI of the freezing solution for each meristem (droplet-freezing method, Fig. 1). The petri dishes were then covered with their lids and transferred to the freezing chamber of a programmable Cryo-Med 1000 Biological Freezing System. A small pertoration was made in the lid of one of the dishes. Through it the temperature probe of the freezing machine was introduced into one of the droplets for monitoring the cObling rates. Based on detailed investigations on the effect of various cooling rates on the survival of meristems, a rate of 0.5 °C/min was chosen and the meristems were frozen at this'rate to terminal freezing temperatures of -20, -25, -30 and -40°C. With one batch of meristems, the freezing was terminated at each indicated temperature. With other batches the dishes containing the meristems were completely immersed in liquid nitrogen for 1 h. The aluminium foils containing the frozen meristems from both groups were removed from the petri dishes and thawed for 10 min by immersion in O-MS medium held at 37°C. The meristems were then removed and cultured on cassava plant regeneration medium (MS + 0.5 ~ BA + I/LM NAA + 0.1 /LM GA3 and 0.8 % Bacto Difco agar) at 26°C, 16h photoperiods at 4000lx as previously reported (Kartha et al., 1974; Kartha and Gamborg, 1975) in order to assess the viability.
Results and Discussion All 4 cassava cultivars behaved almost identical with regard to their survival or loss of viability subsequent to exposure to various sub-zero temperatures (referred to as
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Table 1: Effect of various terminal freezing temperatures on the survival and morphogenetic responses of cassava meristems a). Terminal Freezing Temperature (0C)
Percentage Survival Callus or Leaves Plantlets
-20 -25 -30 -40
30.0 12.7 3.3
100 61 1.8
o
a) The meristems were treated with 15 % DMSO + 3 % sucrose for 15 min and frozen at 0.5°C/min to indicated terminal freezing temperatures. The results represent averages of 5 experiments.
terminal freezing temperatures). The viability of meristems treated with 15 % DMSO + 3 % sucrose for 15 min and frozen in 2-3 p.l of freezing solution over an aluminium foil (droplet-freezing method) at a cooling rate of 0.5 °el min to various terminal freezing temperatures depended upon the temperature to which they were frozen (Table 1). The meristems frozen to only -20 o e upon thawing regenerated plantlets at extremely high frequency (100 %) (Fig. 2 a). Since the freezing solution contained 15 % DMSO and 3 % sucrose and such a high concentration of the cryoprotectant, especially DMSO, depresses the freezing point considerably, the result was failure of total phase change or incomplete freezing of the freezing solution and
Fig. 1: The droplet-freezing method: distribution of cassava meristems in 2-3/LI of cryoprotectant solution over an 18/Lm aluminium foil in 9 cm plastic petri dishes, prior to freezing. Fig. 2: Morphogenetic responses of cassava meristems frozen at 0.5 °C/min to various sub-zero temperatures using 15 % DMSO + 3 % sucrose as cryoprotectants. Figures «a» and «b» represent plant regeneration from meristems frozen to -20°C (a) and -25 °C (b). Most of the meristems frozen to -30 °C (c) and -40 °C (d) differentiated either into callus or callus and leaves. Pictures taken after 3 weeks of incubation of frozenthawed meristems on the plant regeneration medium.
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the meristems. At -25 °e the freezing process was complete since the droplets comprising the freezing solution had transformed into ice embedding the frozen meristems. Over 90 % of the meristems retrieved and recultured on plant regeneration medium survived; over 60 % of them differentiated into plantlets, the rest into callus and leaves (Fig. 2 b). The meristems which grew into callus and leaves failed to develop shoots. It is quite probable that here only the leaf primordia survived. Similar observations have been made with some of the cryopreserved pea meristems (Haskins and Kartha, 1980). The mechanism by which the cells of primordial leaves withstand exposure to sub-freezing temperatures remains to be elucidated. Meristems retrieved from much lower temperature, -30 and -40 o e, showed further loss in viability. Meristems developed callus and leaves rather than plantlets (Fig. 2 c, d). In subsequent experiments, the meristems after freezing to various terminal freezing temperatures were immersed in liquid nitrogen (-196°C) (Table 2,3,4). Since after retrieval and reculture considerable variation was observed in the morphogenetic responses of meristems, the results from a number of experiments are separately tabulated instead of presenting the mean values which may not be a true representation of any given treatment. Table 2 represents the morphogenetic Table 2: Survival and morphogenetic responses of cassava meristems frozen at 0.5 °C/min -25°C and stored in liquid nitrogen for 1 h. Expt. No.") Survival b) (%) 1 2 3 4 5 6 7
12/15 13/31 8/12 10/31 5/22 6/38 19/37
(80) (42) (67) (32) (23) (16) (51)
to
Number of meristems forming: Callus Callus + Leaves Plantlets 9 4 8
10 0 0 16
0 5 0 0
3 4 0
0
4
1
6 0
0 3
.) The meristems were treated with 15 % DMSO + 3 % sucrose for 15 min. D) Numerator represents the number of meristems showing signs of indicated responses; denominator is the total number of meristems frozen, percentage survival is in parentheses.
responses of meristems frozen to -25°e followed by 1 h storage in liquid nitrogen. Initially, the frozen-thawed meristems turned white in color within 24 h of transfer to plant regeneration medium. Gradual greening was observed within 5-7 days in meristems which were alive. Depending upon the degree of survival these meristems differentiated either into plantlets or callus and leaves within 15 days (Fig. 3, 4). No sign of growth was observed in meristems frozen without cryoprotectant treatment (control) even after prolonged incubation. The variation noticed in the survival of meristems was in the range of 16 to 80 %. The trend of the results indicated that most of the cryopreserved meristems regrew either into callus or callus and leaves thereby indicating partial survival of meristem tissue. However, a few meristems regenerated Z. Pjlanzenphysiol. Ed. 107. S. 133-140. 1982.
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Fig. 3-4: Differentiation of callus and leaves and plantlets from cassava meristems frozen by droplet-freezing method at 0.5 °CImin to -25°C followed by 1 h storage in liquid nitrogen. Fig. 5: Plantlets shown in Fig. 4 grown in pots. Fig. 6: Callus, root formation and localized greening of 5 % DMSO-treated meristems frozen by direct immersion in liquid nitrogen (rapid freezing). Plants could not be regenerated from this callus.
into plantlets which were subsequently transplanted to pots (Fig. 5). The heterozygosity of cassava might explain the variation in the morphogenetic responses as well as the resistance or susceptibility of the cells to freezing stress. The meristems which were frozen to -30°C and stored in liquid nitrogen for 1 h showed a survival rate of 14-86% (Table3). Although it appeared that there was not too much difference in survival between the meristems frozen to -25 and -30°C prior to storage in liquid nitrogen, the number of plantlets regenerated was greater in the former while callus and leaf differentiation was more pronounced in the latter. Comparing the data presented in Table 1 and Table 3, solely on the basis of total survival, a lower rate of survival was obtained if the freezing was terminated at -30°C as opposed to those which had undergone liquid nitrogen storage. This discrepancy in the rate of viability in these 2 cases is hard to explain except to attribute it to the differences in the duration of thawing. Meristems which were frozen to -40°C followed by storage in liquid nitrogen for 1 h exhibited the lowest survival (16-44 %) and plant regeneration {Table 4). Here again, the rate of survival was different for meristems frozen to only -40°C as opposed to those which were then stored in liquid nitrogen. On the other Z. Pjlanzenphysiol. Bd. 107. S. 133-140. 1982.
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Table 3: Survival and morphogenetic responses of cassava meristems frozen at 0.5 DC/min to -30°C and stored in liquid nitrogen for 1 h. Expt. No. a)
Survivalb) (%)
Number of meristems forming: Callus Callus + Leaves Plantlets
1 2 3 4 5 6 7
18/21 11121 6/17 4/28 9/34 8/45 9/27
2 4 0 4 1 0 0
(86) (52) (35) (14) (26) (18) (33)
16 6 6 0 6 8 8
0 1 0 0 2 0 1
.) Meristems were treated with 15 % DMSO + 3 % for 15 min. D) Numerator represents the number of meristems showing indicated responses; denominator is the total number of meristems frozen, percentage survival is in parentheses. Table 4: Survival and morphogenetic responses of cassava meristems frozen at a cooling rate of 0.5 DC/min to -40°C followed by 1 h storage in liquid nitrogen. Expt.No. a) Survivalb) (%) 1 2 3 4 5
7/28 6/38 7/16 4/23 6/34
(25) (16) (44) (17) (18)
Number of meristems forming: Callus Callus + Leaves Plantlets 0 5 0 4 0
7 0 7 0 5
0 1 0 0 1
a) Meristems were treated with 15 % DMSO + 3 % for 15 min. b) Numerator represents the number of meristems showing indicated responses; denomina-
tor is the total number of meristems frozen, percentage survival is in parentheses.
hand maximum survival of cryopreserved pea and strawberry meristems was obtained when the meristems were frozen to -40 DC prior to storage in liquid nitrogen (Kartha et al., 1979, 1980). Low temperature response of tropical crops such as cassava is thought to be different from those of temperate crops and this may be attributable to the observed differences. Investigations on other freezing techniques, such as slow freezing in cryogenic ampoules as reported for pea and strawberry meristems (Kartha et al., 1979, 1980) at cooling rates ranging from 0.25 to 10 DC/min, step-wise freezing, rapid freezing in liquid nitrogen and liquid helium, were also carried out. A number of cryoprotectants such as DMSO, glycerol, methanol, ethylene glycol, polyethylene glycol (M.W. 4000 and 6000), polyvinylpyrrolidone, pyridine-n-oxide, N-N-dimethylformamide, ethanolamine, decenylsuccinic acid, proline, sucrose, glucose, lactose, mannitol, raffinose, individually or in combination at various concentrations were tested for each freezing technique. Similarly the effect of preculturing the meristems in nutrient medium with and without added cryoprotectants was also examined. Cassava meristems failed to survive when frozen slowly in cryogenic ampoules in 1 ml freez-
z. Pjlanzenphysiol. Ed. 107. S. 133-140. 1982.
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139
ing solution of various cryoprotectants either alone or in combination followed by liquid nitrogen storage. Similarly rapid freezing of the cryoprotectant-treated meristems by direct immersion in liquid nitrogen or liquid helium was also unsuccessful. In a few isolated instances,S % DMSO-treated meristems when frozen rapidly in liquid nitrogen grew into only callus. The callus thus recovered turned green on plant regeneration medium, produced few roots but did not differentiate into either leaves or shoots (Fig. 6). Bajaj (1977) claimed to have obtained 13 % plantlets and 8 % callus and roots from cassava meristems frozen rapidly by immersion in liquid nitrogen using 10 % glycerol and 5 % sucrose as cryoprotectants. However, we were unable to reproduce his results with all the 4 cultivars tested. We are not certain if the particular genotype utilized in his study (M-4) might account for the observed contradiction. The droplet-freezing technique reported here has resulted in achieving some success in the cryopreservation of cassava meristems. The advantage of droplet-freezing on aluminium foil lies in the fact that the metal has an efficient thermal conductivity resulting in homogenous cooling of sample by uniform dispersion of temperature. The droplet-freezing performed without using aluminium foil, although successful, resulted in reduced survival (data not presented here). Moreover, since a very small amount of the freezing solution (2-3 {tl) was used for each meristem, the freezing appeared to be more uniform thus facilitating the various constituent cells of the explants to freeze at a uniform rate. Since lower concentrations of DMSO were found to be ineffective, a 15 % concentration had to be chosen. This high concentration alone could account for the loss of viability in some of the meristems and the observed alterations in the morphogenetic responses. However the final concentration of DMSO taken up by the cells is expected to be much lower for it is prone to be diluted with the cell sap. At present, most of the world collections of cassava germplasm are located in international agricultural research centres such as CIA T in Colombia and IITA (International Institute of Tropical Agriculture) in Nigeria. In CIAT alone over 2200 collections are maintained every year in the field. This practice in addition to being labor intensive and expensive also invites problems such as possibilities of a sudden major disease outbreak, pest infestation, environmental perturbation and human error, resulting in loss of valuable collections of genetic material. The technique and the results reported here form the basis for detailed investigation into the applied aspects of preserving cassava germplasm by cryogenic methods. With further improvement in the technique and the plant regeneration rate, a strategy could be envisaged for the preservation of cassava germplasm in a disease-free and genetically stable condition and would facilitate international exchange of valuable genetic material. Acknowledgements Part of this research was supported by a financial grant from the International Development Research Centre, Ottawa, Canada, under a contract research project on cryopreservation of cas-
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sava meristems. The authors are very grateful to Dr. J. c. Toro and W. M. Roca, CIAT, Colombia, for the supply of cassava material and also to Mr. A. Lutzko for the preparation of illustrations.
References BAJAJ, Y. P. S.: Clonal multiplication and cryopreservation of cassava through tissue culture. Crop Improv. 4, 198-204 (1977). GROUT, B. W. W. and G. G. HENSHAW: Freeze-preservation of potato shoot-tip cultures. Ann. Bot.42,1227-1229(1978). HASKINS, R. H. and K. K. KARTHA: Freeze preservation of pea meristems: Cell Survival. Can. J. Bot. 58, 833-840 (1980). KARmA, K. K.: Meristem culture and cryopreservation, methods and applications. In: T. A. THORPE (Ed.): Plant tissue culture, methods and applications in agriculture, 181-211. Academic Press, New York, 1981. KARTHA, K. K. and o. L. GAMBORG: Elimination of cassava mosaic disease by meristem culture. Phytopathology 65,826-828 (1975). KARmA, K. K., O. L. GAMBORG, F.CONSTABEL, andJ. P. SHYLUK: Regeneration of cassava plants from apical meristems. Plant Sci. Lett. 2,107-113 (1974). KARTHA, K. K., N. L. LEUNG, and O. L. GAMBORG: Freeze-preservation of pea meristems in liquid nitrogen and subsequent plant regeneration. Plant Sci. Lett. 15, 7-15 (1979). KARTHA, K. K., N. L. LEUNG, and K. PAHL: Cryopreservation of strawberry meristems and mass propagation of plantlets. J. Amer. Soc. Hort. Sci. 105, 481-484 (1980). MURASHIGE, T. and F. SKOOG: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473-497 (1962). ROCA, W. M.: Genetic resources unit: Plant tissue culture section. CIAT Annual Report (1980). SAKAI, A. and Y. NISHIYAMA: Cryopreservation of winter-vegetative buds of hardy fruit trees in liquid nitrogen. Hort.Science 13, 225-227 (1978). SAKAI, A., M. YAMAKAWA, D. SAKATA, T. HARADA, and T. YAKUWA: Development of a whole plant from an excised strawberry runner apex frozen to -196°C. Low Temp. Sci. Ser. Bull. 36, 31-38 (1978). SEIBERT, M.: Shoot initiation from carnation shoot apices frozen to -196°C. Science 191, 1178-1179 (1976). TOWILL, L. E.: Solanum etuberosum: A model system for studying the cryobiology of shoot-tips in the tuber bearing Solanum species. Plant Sci. Lett. 20, 315-324 (1981). UEMURA, A. and A. SAKAI: Survival of carnation (Dianthus caryophyllus L.) shoot apices frozen to the temperature of liquid nitrogen. Plant and Cell Physiol. 21, 85-94 (1980).
Z Pjlanzenphysiol. Ed. 107. S. 133-140. 1982.
KARTHA,
N. 1. LEUNG and 1. A. MROGINSKI**)
Prairie Regional Laboratory, National Research Council of Canada, Saskatoon, Saskatchewan, Canada S7N OW9 Received April 23, 1982 . Accepted May 6,1982
Summary A droplet-freezing method has been developed for the cryopreservation of cassava, Manihot esculenta Crantz, meristems. The meristems treated with a 15 % DMSO + 3 % sucrose solution for 15 min were frozen over an 18/Lm aluminium foil in 2-3/LI droplets of the cryoprotectants in plastic petri dishes at a cooling rate of 0.5 °CI min to various sub-zero temperatures (-20; -25; -30 and -40°C) and stored in liquid nitrogen (-196°C). The meristems retrieved from liquid nitrogen storage, upon thawing and return to in vitro culture on plant regeneration medium, exhibited various morphogenetic responses such as differentiation of callus and leaves and whole plantlets. The plantlets were successfully grown in pots.
Key words: Manihot esculenta, cryopreservation, dropletJreezing method, dimethylsulfoxide (DMSO), plant regeneration in vitro, germplasm preservation.
Introduction Cassava (Manihot esculenta Crantz), an annual root crop, is extensively grown as a source of human food in the humid tropics of the world. It is propagated vegetatively through the use of stem cuttings. Techniques to regenerate plants from shoot apical meristems of cassava cultured in vitro have been developed in this laboratory (Kartha et al., 1974) and have been applied to the production of mosaic disease-free plants (Kartha and Gamborg, 1975). The meristem culture technique has been successfully extended to short-term preservation of cassava collections (Roca, 1980), but alternative approaches have to be sought to warrant germplasm preservation for any extended periods of time. In recent years, cryogenic techniques have been developed for preservation of meristems of a few species, such as carnation (Seibert, 1976; Uemura and Sakai, 1980), potato (Grout and Henshaw, 1978; Towill, 1981), pea (Kartha et al., 1979), strawberry (Sakai *) NRCC No. 20411. **) Visiting Scientist, Facultad de Ciencias Agrarias, Instituto de Bot:inica del Nordeste, Casilla de Correos 209, Corrientes (3400) Argentina. Abbreviations: DMSO, dimethylsulfoxide; BA, benzyladenine; NAA, naphthaleneacetic acid; GA3, grbberellic acid.
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K. K. KARTHA, N. L. LEUNG and L. A. MROGINSKI
et al., 1978; Kartha et al., 1980) and vegetative buds of hardy fruit trees (Sakai and Nishiyama, 1978) (for a review see Kartha, 1981). It would be desirable to similarly cryopreserve Icassava rneristeIlIls for long periods of time. The need for preservation is pressing especially due to the vegetative mode of propagation of this crop. Bajaj (1977) claim~d to have succeeded in regenerating plantlets from the cryopreserved meristems of cassava. Three years of research at this laboratory aimed at this direction with this crop failed until the development of a new technique, i.e. the dropletfreezing technique. This paper presents the details of the new technique as well as the various morphogenetic responses of cassava meristems subsequent to their exposure to sub-zero temperatures and cryopreservation.
Materials and Methods Dormant stakes of cassava (Manihot escutenta Crantz) cultivars, MVEN-218, CM 321-188, CM 489-1 and CM 1684 were obtained from CIAT (Centro Internacional de Agricultura Tropical). The stakes were cut ir sections with 3-4 nodes each, the upper cut ends sealed with paraffin, and grown in a growth room at 26°C, 16 h photoperiods of 7500 Ix intensity and 70 % relative humidity. Fdr cryopresetvation experiments buds of sprouted cuttings as well as subsequently develqped secondary buds were used. After removing the outer whorls of visible leaves, buds were surface sterilized in 70 % ethanol for 60.sec and rinsed thoroughly in 3 changes of sterile distilled water. Meristems measuring 0.4-0.5 mm in length and containing a pair of leaf primordia and some subjacent tissue were isolated under a stereo-mi~roscope and transferred to 10 ml of hormone-free liquid MS (O-MS) medium (Murashige ana Skoog, 1962) in a centrifuge tube. An equal volume of a 30 % solution (v/v) of dimethylsulfli>xide (DMSO) in O-MS medium containing 3 % sucrose was gradually added to this solution in 10 instalments over a period of 30 min until a final concentration of 15 % was reached. The meristems were allowed to equilibrate in this «freezing solution» for' 15 min. The free~ing of meristems was performed in 9 cm plastic petri dishes in droplets. An 18/Lm gauge sterile aluminium foil was placed inside each petri dish and 20-30 DMSO-treated meristems were evenly distributed over the foil along with 2-3 /LI of the freezing solution for each meristem (droplet-freezing method, Fig. 1). The petri dishes were then covered with their lids and transferred to the freezing chamber of a programmable Cryo-Med 1000 Biological Freezing System. A small pertoration was made in the lid of one of the dishes. Through it the temperature probe of the freezing machine was introduced into one of the droplets for monitoring the cObling rates. Based on detailed investigations on the effect of various cooling rates on the survival of meristems, a rate of 0.5 °C/min was chosen and the meristems were frozen at this'rate to terminal freezing temperatures of -20, -25, -30 and -40°C. With one batch of meristems, the freezing was terminated at each indicated temperature. With other batches the dishes containing the meristems were completely immersed in liquid nitrogen for 1 h. The aluminium foils containing the frozen meristems from both groups were removed from the petri dishes and thawed for 10 min by immersion in O-MS medium held at 37°C. The meristems were then removed and cultured on cassava plant regeneration medium (MS + 0.5 ~ BA + I/LM NAA + 0.1 /LM GA3 and 0.8 % Bacto Difco agar) at 26°C, 16h photoperiods at 4000lx as previously reported (Kartha et al., 1974; Kartha and Gamborg, 1975) in order to assess the viability.
Results and Discussion All 4 cassava cultivars behaved almost identical with regard to their survival or loss of viability subsequent to exposure to various sub-zero temperatures (referred to as
Z. PjlanzenphysioL Ed. 107. S. 133-140. 1982.
Cryopreservation of cassava meristems
135
Table 1: Effect of various terminal freezing temperatures on the survival and morphogenetic responses of cassava meristems a). Terminal Freezing Temperature (0C)
Percentage Survival Callus or Leaves Plantlets
-20 -25 -30 -40
30.0 12.7 3.3
100 61 1.8
o
a) The meristems were treated with 15 % DMSO + 3 % sucrose for 15 min and frozen at 0.5°C/min to indicated terminal freezing temperatures. The results represent averages of 5 experiments.
terminal freezing temperatures). The viability of meristems treated with 15 % DMSO + 3 % sucrose for 15 min and frozen in 2-3 p.l of freezing solution over an aluminium foil (droplet-freezing method) at a cooling rate of 0.5 °el min to various terminal freezing temperatures depended upon the temperature to which they were frozen (Table 1). The meristems frozen to only -20 o e upon thawing regenerated plantlets at extremely high frequency (100 %) (Fig. 2 a). Since the freezing solution contained 15 % DMSO and 3 % sucrose and such a high concentration of the cryoprotectant, especially DMSO, depresses the freezing point considerably, the result was failure of total phase change or incomplete freezing of the freezing solution and
Fig. 1: The droplet-freezing method: distribution of cassava meristems in 2-3/LI of cryoprotectant solution over an 18/Lm aluminium foil in 9 cm plastic petri dishes, prior to freezing. Fig. 2: Morphogenetic responses of cassava meristems frozen at 0.5 °C/min to various sub-zero temperatures using 15 % DMSO + 3 % sucrose as cryoprotectants. Figures «a» and «b» represent plant regeneration from meristems frozen to -20°C (a) and -25 °C (b). Most of the meristems frozen to -30 °C (c) and -40 °C (d) differentiated either into callus or callus and leaves. Pictures taken after 3 weeks of incubation of frozenthawed meristems on the plant regeneration medium.
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K. K. KARTHA, N. L. LEUNG and L. A. MROGINSKI
the meristems. At -25 °e the freezing process was complete since the droplets comprising the freezing solution had transformed into ice embedding the frozen meristems. Over 90 % of the meristems retrieved and recultured on plant regeneration medium survived; over 60 % of them differentiated into plantlets, the rest into callus and leaves (Fig. 2 b). The meristems which grew into callus and leaves failed to develop shoots. It is quite probable that here only the leaf primordia survived. Similar observations have been made with some of the cryopreserved pea meristems (Haskins and Kartha, 1980). The mechanism by which the cells of primordial leaves withstand exposure to sub-freezing temperatures remains to be elucidated. Meristems retrieved from much lower temperature, -30 and -40 o e, showed further loss in viability. Meristems developed callus and leaves rather than plantlets (Fig. 2 c, d). In subsequent experiments, the meristems after freezing to various terminal freezing temperatures were immersed in liquid nitrogen (-196°C) (Table 2,3,4). Since after retrieval and reculture considerable variation was observed in the morphogenetic responses of meristems, the results from a number of experiments are separately tabulated instead of presenting the mean values which may not be a true representation of any given treatment. Table 2 represents the morphogenetic Table 2: Survival and morphogenetic responses of cassava meristems frozen at 0.5 °C/min -25°C and stored in liquid nitrogen for 1 h. Expt. No.") Survival b) (%) 1 2 3 4 5 6 7
12/15 13/31 8/12 10/31 5/22 6/38 19/37
(80) (42) (67) (32) (23) (16) (51)
to
Number of meristems forming: Callus Callus + Leaves Plantlets 9 4 8
10 0 0 16
0 5 0 0
3 4 0
0
4
1
6 0
0 3
.) The meristems were treated with 15 % DMSO + 3 % sucrose for 15 min. D) Numerator represents the number of meristems showing signs of indicated responses; denominator is the total number of meristems frozen, percentage survival is in parentheses.
responses of meristems frozen to -25°e followed by 1 h storage in liquid nitrogen. Initially, the frozen-thawed meristems turned white in color within 24 h of transfer to plant regeneration medium. Gradual greening was observed within 5-7 days in meristems which were alive. Depending upon the degree of survival these meristems differentiated either into plantlets or callus and leaves within 15 days (Fig. 3, 4). No sign of growth was observed in meristems frozen without cryoprotectant treatment (control) even after prolonged incubation. The variation noticed in the survival of meristems was in the range of 16 to 80 %. The trend of the results indicated that most of the cryopreserved meristems regrew either into callus or callus and leaves thereby indicating partial survival of meristem tissue. However, a few meristems regenerated Z. Pjlanzenphysiol. Ed. 107. S. 133-140. 1982.
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Fig. 3-4: Differentiation of callus and leaves and plantlets from cassava meristems frozen by droplet-freezing method at 0.5 °CImin to -25°C followed by 1 h storage in liquid nitrogen. Fig. 5: Plantlets shown in Fig. 4 grown in pots. Fig. 6: Callus, root formation and localized greening of 5 % DMSO-treated meristems frozen by direct immersion in liquid nitrogen (rapid freezing). Plants could not be regenerated from this callus.
into plantlets which were subsequently transplanted to pots (Fig. 5). The heterozygosity of cassava might explain the variation in the morphogenetic responses as well as the resistance or susceptibility of the cells to freezing stress. The meristems which were frozen to -30°C and stored in liquid nitrogen for 1 h showed a survival rate of 14-86% (Table3). Although it appeared that there was not too much difference in survival between the meristems frozen to -25 and -30°C prior to storage in liquid nitrogen, the number of plantlets regenerated was greater in the former while callus and leaf differentiation was more pronounced in the latter. Comparing the data presented in Table 1 and Table 3, solely on the basis of total survival, a lower rate of survival was obtained if the freezing was terminated at -30°C as opposed to those which had undergone liquid nitrogen storage. This discrepancy in the rate of viability in these 2 cases is hard to explain except to attribute it to the differences in the duration of thawing. Meristems which were frozen to -40°C followed by storage in liquid nitrogen for 1 h exhibited the lowest survival (16-44 %) and plant regeneration {Table 4). Here again, the rate of survival was different for meristems frozen to only -40°C as opposed to those which were then stored in liquid nitrogen. On the other Z. Pjlanzenphysiol. Bd. 107. S. 133-140. 1982.
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Table 3: Survival and morphogenetic responses of cassava meristems frozen at 0.5 DC/min to -30°C and stored in liquid nitrogen for 1 h. Expt. No. a)
Survivalb) (%)
Number of meristems forming: Callus Callus + Leaves Plantlets
1 2 3 4 5 6 7
18/21 11121 6/17 4/28 9/34 8/45 9/27
2 4 0 4 1 0 0
(86) (52) (35) (14) (26) (18) (33)
16 6 6 0 6 8 8
0 1 0 0 2 0 1
.) Meristems were treated with 15 % DMSO + 3 % for 15 min. D) Numerator represents the number of meristems showing indicated responses; denominator is the total number of meristems frozen, percentage survival is in parentheses. Table 4: Survival and morphogenetic responses of cassava meristems frozen at a cooling rate of 0.5 DC/min to -40°C followed by 1 h storage in liquid nitrogen. Expt.No. a) Survivalb) (%) 1 2 3 4 5
7/28 6/38 7/16 4/23 6/34
(25) (16) (44) (17) (18)
Number of meristems forming: Callus Callus + Leaves Plantlets 0 5 0 4 0
7 0 7 0 5
0 1 0 0 1
a) Meristems were treated with 15 % DMSO + 3 % for 15 min. b) Numerator represents the number of meristems showing indicated responses; denomina-
tor is the total number of meristems frozen, percentage survival is in parentheses.
hand maximum survival of cryopreserved pea and strawberry meristems was obtained when the meristems were frozen to -40 DC prior to storage in liquid nitrogen (Kartha et al., 1979, 1980). Low temperature response of tropical crops such as cassava is thought to be different from those of temperate crops and this may be attributable to the observed differences. Investigations on other freezing techniques, such as slow freezing in cryogenic ampoules as reported for pea and strawberry meristems (Kartha et al., 1979, 1980) at cooling rates ranging from 0.25 to 10 DC/min, step-wise freezing, rapid freezing in liquid nitrogen and liquid helium, were also carried out. A number of cryoprotectants such as DMSO, glycerol, methanol, ethylene glycol, polyethylene glycol (M.W. 4000 and 6000), polyvinylpyrrolidone, pyridine-n-oxide, N-N-dimethylformamide, ethanolamine, decenylsuccinic acid, proline, sucrose, glucose, lactose, mannitol, raffinose, individually or in combination at various concentrations were tested for each freezing technique. Similarly the effect of preculturing the meristems in nutrient medium with and without added cryoprotectants was also examined. Cassava meristems failed to survive when frozen slowly in cryogenic ampoules in 1 ml freez-
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ing solution of various cryoprotectants either alone or in combination followed by liquid nitrogen storage. Similarly rapid freezing of the cryoprotectant-treated meristems by direct immersion in liquid nitrogen or liquid helium was also unsuccessful. In a few isolated instances,S % DMSO-treated meristems when frozen rapidly in liquid nitrogen grew into only callus. The callus thus recovered turned green on plant regeneration medium, produced few roots but did not differentiate into either leaves or shoots (Fig. 6). Bajaj (1977) claimed to have obtained 13 % plantlets and 8 % callus and roots from cassava meristems frozen rapidly by immersion in liquid nitrogen using 10 % glycerol and 5 % sucrose as cryoprotectants. However, we were unable to reproduce his results with all the 4 cultivars tested. We are not certain if the particular genotype utilized in his study (M-4) might account for the observed contradiction. The droplet-freezing technique reported here has resulted in achieving some success in the cryopreservation of cassava meristems. The advantage of droplet-freezing on aluminium foil lies in the fact that the metal has an efficient thermal conductivity resulting in homogenous cooling of sample by uniform dispersion of temperature. The droplet-freezing performed without using aluminium foil, although successful, resulted in reduced survival (data not presented here). Moreover, since a very small amount of the freezing solution (2-3 {tl) was used for each meristem, the freezing appeared to be more uniform thus facilitating the various constituent cells of the explants to freeze at a uniform rate. Since lower concentrations of DMSO were found to be ineffective, a 15 % concentration had to be chosen. This high concentration alone could account for the loss of viability in some of the meristems and the observed alterations in the morphogenetic responses. However the final concentration of DMSO taken up by the cells is expected to be much lower for it is prone to be diluted with the cell sap. At present, most of the world collections of cassava germplasm are located in international agricultural research centres such as CIA T in Colombia and IITA (International Institute of Tropical Agriculture) in Nigeria. In CIAT alone over 2200 collections are maintained every year in the field. This practice in addition to being labor intensive and expensive also invites problems such as possibilities of a sudden major disease outbreak, pest infestation, environmental perturbation and human error, resulting in loss of valuable collections of genetic material. The technique and the results reported here form the basis for detailed investigation into the applied aspects of preserving cassava germplasm by cryogenic methods. With further improvement in the technique and the plant regeneration rate, a strategy could be envisaged for the preservation of cassava germplasm in a disease-free and genetically stable condition and would facilitate international exchange of valuable genetic material. Acknowledgements Part of this research was supported by a financial grant from the International Development Research Centre, Ottawa, Canada, under a contract research project on cryopreservation of cas-
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sava meristems. The authors are very grateful to Dr. J. c. Toro and W. M. Roca, CIAT, Colombia, for the supply of cassava material and also to Mr. A. Lutzko for the preparation of illustrations.
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