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Improvement of the Doplet-vitrification Method for the Cryopreservation of Cultivated Potato Shoot Tips
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The germplasm of vegetatively propagated crops, such as potato, is usually conserved either in the field or by in vitro maintenance. In vitro preservation is not only time- consuming and labor-intensive, but it may not even ensure good genetic stability of micropropagated plantlets (Harding, 1991). Therefore, cryopreservation of shoot tips in liquid nitrogen is the preferred method for long-term storage of potato germplasm. Studies on the cryopreservation of potato shoot tips began in the late 1970s, and since then, various methods have been developed using slow and ultra-rapid freezing or other modified freezing procedures (Bajai, 1977; Grout and Henshaw, 1978; Towill, 1981, 1984; Benson et al. 1989; Schäfer-Menuhr et al. 1997). Potato shoot tips have been cryopreserved by vitrification (Sarkar and Naik, 1998), encapsulation-vitrification (Hirai and Sakai, 1999), encapsulation-dehydration (Bouafia et al. 1996), and a special vitrification method using aluminum foil as a carrier for cryoprotectant droplets, called the ‘droplet method’ (Kartha et al. 1982; Schäfer-Menuhr et al. 1997). Of these methods, vitrification and droplet-freezing have been implemented on a large scale (CIP, Peru; DSMZ, Germany). Our previous studies have shown that the droplet-vitrification procedure, a combination of the droplet-freezing and vitrification protocols, is a very efficient cryopreservation method (Kim et al. 2006; Yoon JW et al. 2006).
A direct comparison of cryopreservation methods reported in the literature is problematic because the results have been obtained in different locales with different plant sources. The applicability of a cryopreservation protocol to diverse genotypes is also an important consideration to researchers in charge of cryobanks to carry out long-term and large- scale germplasm conservation using cryotechnology. The different survival levels of different potato species observed after freezing could be due to the vigor of the plants, or it could depend on the genotype (Engelmann, 1997). Therefore, the optimization of a protocol for various species is a prerequisite for implementing cryopreservation of potato germplasms in genebanks. Thus, an analysis of the factors causing differences in survival and regeneration percentages among diverse species after freezing is needed.
In this study, to improve the droplet-vitrification cryopreservation method for potato shoot tips, we tested three types of dehydration solution with four cultivated potato germplasms. We selected the most efficient dehydration solution and determined the optimum application time for cryopreservation. We also compared the conventional post- culture medium with an improved one to select the most efficient recovery medium.
MATERIALS AND METHODS
In vitro-grown plantlets of four cultivated potatoes (Solanum tuberosum L.), ‘Dejima’, ‘Alpha’, ‘Washeshiro’, and ‘Kitaakari’ were used. These were obtained as in vitro cultures from the Highland Agriculture Research Center, a branch institute of the National Institute of Crop Science, RDA. The accessions had been maintained at the Highland Agriculture Research Center as tubers in the field and as in vitro plants. For in vitro propagation, nodal segments from the in vitro cultures, consisting of a piece of stem, were transferred to Murashige and Skoog (MS) basal medium (Murashige and Skoog, 1962) containing 30 g･L-1 sucrose and 2.2 g･L-1 phytagel (Sigma-Aldrich, USA) without growth regulator, and then incubated at 23±1℃ under a photoperiod of 16 h light/8 h dark. After 6 weeks, axillary shoot tips were isolated by dissection from the upper to middle part of the propagated plants, and the plants were subcultured.
Cryopreservation procedure and rewarming
The droplet-vitrification procedure was used for freezing shoot tips. Isolated shoot tips were pre-cultured in liquid MS medium containing 0.3 M sucrose for 7 h, after which the shoot tips were further pre-cultured in liquid MS medium containing 0.7 M sucrose for 17 h under the same conditions. No loading treatment was performed. The pre- cultured shoot tips were dehydrated in PVS2, modified PVS2, and PVS3 vitrification solution (Table 1) for 20, 30, and 90 min, respectively. Five drops of dehydration solution were placed on an aluminum foil strip (8 × 20 mm) using micropipette. One shoot tip was put in each of the five dehydration solution drops and then the foil strip was immediately plunged into liquid nitrogen. After a few minutes in the liquid nitrogen, two foil strips were transferred to a single 2 mL cryovial that had been previously filled with liquid nitrogen and stored in a liquid nitrogen tank for more than 1 day. For warming, foil strips were taken out of the cryovials and immediately plunged into a pre-heated (40℃) unloading solution of 0.8 M sucrose, and after 30 s, another 5 mL of room temperature unloading solution was added. The shoot tips were then incubated at room temperature for 30 min to facilitate unloading.
Survival and regeneration assessment
For assessment of viability, the shoot tips were post- cultured on three kinds of MS medium (Table 2) at 23±1℃ under dim light for 7 days and then transferred to standard culture conditions. Survival rates were evaluated 14 days after cryopreservation by counting the number of shoot tips that were green and swollen (>3 mm). Regeneration rates were estimated at 5 to 6 weeks after cryopreservation by counting the number of shoot tips that were differentiated. The survival and regeneration percentages were averaged for four cultivated potatoes.
Table 1 Compositions of the three vitrification solutions used in this study.
Table 2 Comparison of the key steps in the conventional versus the improved protocol, which uses the droplet-vitrification method for the cryopreservation of potato shoot tips.
Table 3 Effects of various vitrification solutions and their application times on the survival and regeneration rates of control (-LN) and cryopreserved (+LN) potato shoot tips.
Data analysis and statistical procedures
The results were obtained as average percentages with standard deviations. Each experiment consisted of three replicates per treatment, and each cryovial held 10 samples. The results were analyzed by ANOVA, and the means were separated using Duncan’s multiple-range test (p<0.05).
RESULTS AND DISCUSSION
Effects of vitrification solution and application time on recovery
No survival was observed in dehydrated and rewarmed shoot tips cooled to -196℃ in the absence of pre-culture (data not shown), suggesting that pre-culture is an essential step for successful cryopreservation of potato shoot tips. In our previous study (Kim et al. 2006), we found no difference in the survival and regeneration percentages between treatments, with or without loading. Therefore, no loading treatment was performed in this study. Rather, we compared three types of vitrification solutions and their application times to choose the most appropriate dehydration method.
Vitrification solutions contain cryoprotectants that have been formulated for their ability to vitrify during cooling. PVS2, the vitrification solution most commonly employed for cryopreservation of plant species, has a total molarity of 7.8 M and is highly toxic (Sakai et al. 1990). As shown in Table 3, application of modified PVS2 for 30 min yielded higher survival (88.3%) and regeneration rates (80.9%) of cryopreserved explants than the similar application of PVS2 or PVS3. In our previous study (Kim et al. 2006), treatment with PVS2 for 20 min produced the best survival and regeneration rates, whereas in the present study, treatment with PVS2 produced the lowest recovery of potato shoot tips. There are many factors affecting recovery in vitro, and inconsistent survival and regeneration rates after cryopreservation are often related to the genotypes and nutritive condition of the germplasms (Matsumoto et al. 1994). In this experiment, we evaluated both survival and regeneration rates of shoot tip explants after rewarming from preservation in liquid nitrogen. The important criterion for evaluation of cryopreservation methods, however, is the ability to regenerate entire plants from the cryopreserved shoot tips.
Droplet-vitrification is a modification of the vitrification method. Vitrification is the phase transition of water from liquid directly into a non-crystalline amorphous phase by extreme elevation in viscosity during cooling (Fashy et al. 1984). In the vitrification method, the cells of the shoot tips are sufficiently dehydrated using a highly concentrated vitrification solution before direct transfer to liquid nitrogen (Sakai, 1995). Cell dehydration in vitrification-based procedures is performed prior to freezing by exposing the samples to concentrated cryoprotective media or air desiccation followed by rapid cooling (Engelmann, 1997). During the cooling and warming steps, rapid heat transfer is crucial to avoid freeze injury. In our previous study (Kim et al. 2006), the droplet-vitrification protocol, which entails a combination of rapid cooling by plunging aluminum foil strips on which explants have been placed in droplets of cryoprotectants into liquid nitrogen and rapid warming by dipping the cryopreserved foil strips in pre-heated (40℃) 0.8 M sucrose for 30 s produced the highest regeneration ratio. Thus, the current study was undertaken to optimize an effective chemical-induced vitrification procedure for cryopreservation of potato shoot tips.
Direct exposure of pre-cultured shoot tips to concentrated vitrification solution was detrimental to the viability of vitrified shoot tips. Presumably, this harmful effect was due to excessive osmotic stress or chemical toxicity from the PVS2 (Matsumoto et al. 1994). Therefore, exposure to modified PVS2, which has lowered concentrations of toxic agents like DMSO for 30 min produced a substantially greater recovery of potato shoot tips than did the other treatments (Table 3).
Effects of regeneration medium on recovery
After rewarming from cryopreservation, significantly more shoot tips were regenerated on the improved post-cryo medium, SIM1 (Table 4), than on the conventional post- culture medium or SIM2. Solid regeneration medium is usually used for regrowth of cryopreserved plant explants (Bouafia et al. 1996), but some studies have suggested that liquid medium is better (Hirai and Sakai, 1999). In this study, we used solid medium for the first 5 weeks after rewarming, and then we transferred the regenerated shoots to liquid multiplication medium containing 0.1 mg･L-1 GA3, followed by shaking.
Sarkar and Naik (1998) reported that vitrified and cryopreserved shoot tips were significantly recovered on semi- solid MS medium containing 0.2 M sucrose, 5.8 uM GA3, 1.0 uM BA, and 6 g･L-1 noble agar. In comparison, in our study, we used a combination of 0.1 mg･L-1 GA3 and 0.1 mg･L-1 kinetin in place of the 5.8 uM GA3 and 1.0 uM BA, and we used 0.09 M sucrose in place of 0.2 M sucrose as a carbon source. Another study group reported that excised dome meristems cultivated on MS medium containing 0.1 mg･L-1 NAA, 0.5 mg･L-1 kinetin, and 2.25 mg･L-1 phytagel for 21 days regenerated plantlets (Mahfouze et al. 2010).
As shown in Table 4, SIM1 containing 0.1 mg･L-1 GA3 and 0.1 mg･L-1 kinetin produced the highest survival and regeneration rates (93.1% and 80.9%, respectively) after cryopreservation. This suggests that SIM 1 is more efficient for the direct regeneration of potato shoot tips than the conventional post-culture medium or SIM2. Fig.1 compares the potato shoot tips regenerated on the conventional post- culture medium with those regenerated on improved SIM1; the regenerated plantlets on SIM1 were more plentiful and more vigorous. An additional benefit was the lowered expense and easier handling of SIM1, compared with the conventional medium due to the absence of zeatin. In the conventional protocol, we had to sterilize zeatin by filtering instead of autoclaving because of its vulnerability to high temperature, and zeatin is also expensive.
In this study, we compared different combinations of phytohormones for shoot tip culture, and we selected the most appropriate post-cryo medium. However, according to Sarkar and Naik (1998), post-cryo culturing of vitrified shoot tips on a high-sucrose (0.2 M) medium for the first week after cryoculturing permitted a favorable osmotic adjustment that allowed the cryopreserved shoot tips to better recovery from the trauma of cryo-shock. Additionally, they found that diffuse light was beneficial to shoot tip survival and direct shoot formation, in agreement with Henshaw et al. (1985), who reported that cryopreserved potato shoot tips required a low light level during the initial post-thaw culture phase.
Direct regeneration from shoot tips minimizes the possibility of genetic variation, but longer culture periods and elevated sucrose concentrations can cause genetic variation. Thus, there is some risk of genetic or somaclonal variation in plants regenerated through adventitious shoot development or vitrification (Benson et al. 1996). Since 2011, we have applied our improved protocol to 50 accessions of in vitro potato cultures. To confirm the genetic and phenotypic stability of cryopreserved potato germplasms, we are planning to compare the molecular and agronomic traits of regenerated plants derived from conventional micropropagation methods and cryopreservation.
Table 4 Effects of various post-culture media on the survival and regeneration rates of control (-LN) and cryopreserved (+LN) potatoshoot tips.
This study was carried out with the support of “Research Program for Agricultural Science & Technology Development (Project No. PJ007386)”, National Academy of Agricultural Science, Rural Development Administration, Republic of Korea.
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