Improvement to the D Cryo-plate Protocol Applied to Practical Cryopreservation of In Vitro Grown Potato Shoot Tips

Abstract

We modified the D cryo-plate protocol by paper mounting to the cryo-plate with alginate gel and shoot tips, and cryopreserved shoot tips of 13 potato genotypes (potato genetic resources in Mexico) using the revised and optimized D cryo-plate protocol. There were no significant differences in regrowth of cryopreserved shoot tips by addition of the paper mounting step to the base D cryo-plate protocol, besides a reduction in dropping shoot tips in steps during the whole procedure. Some steps of revised D cryo-plate protocol were optimized or reconfirmed, and the effect of optimizations such as cold-hardening, loading solution treatment, and post-rewarming treatment on the regeneration of shoot tips was studied. This optimized protocol was successfully applied to ‘B-71-240-2’ and 12 additional potato genotypes, resulting in 70.0%–93.3% regrowth with an average of 82.8% and stable storage for 1 year. When introducing new cryopreservation techniques, modification and optimization of the method are required to adjust to each laboratory’s circumstances. This optimized D cryo-plate method will facilitate cryobanking of potato and other plant genetic resources in Mexico for long-term preservation in a genebank.

Introduction

Cryopreservation is increasingly used for long-term storage of plant genetic resources, requiring minimal space and low maintenance (Niino and Valle Arizaga, 2015). In our previous study, V cryo-plate and D cryo-plate methods were proposed as cryopreservation techniques for preservation of potato genetic resources (GRs) (Yamamoto et al., 2015). The D cryo-plate method has been reported for in vitro shoot tips of the mat rush and shoot tips of the persimmon from dormant buds (Matsumoto et al., 2015; Niino et al., 2013). However, this method needs to be modified to be a simple and easy procedure without any shoot tips dropping from the cryo-plate at any step in cryopreservation and regeneration. Also, when a technique is transferred to other country, it is important to adapt the technique to the circumstances of each laboratory, because variations in responses to cryopreservation may arise from differences in operator skills and competence, culture systems, differences in equipment and minor technical details of cryopreservation, the environment of the laboratory room and so on (Keller et al., 2008; Reed et al., 2004). For the implementation of cryostorage, proper cryo-techniques should be determined for each laboratory parameter such as environment, infrastructure, cost, staff skills, and plant material available. The Mexican government recently established a National Genetic Resources Center (CNRG) under the administration of the National Forestry, Crop and Livestock Research Institute (INIFAP) as a key component of its long-term strategy for conservation and sustainable use of genetic resources (Machida-Hirano et al., 2014). A key element of CNRG is the long-term storage of GRs using a cryopreservation technique at super-low temperature. The potato is an essential crop in Mexico and Latin America, which are centers for wild potato diversity. Mexico holds strains with specific resistance to diseases such as late blight, as well as cardinal breeding lines that bear the resistance traits. The national potato program of INIFAP in Mexico maintains approximately 1500 accessions of potato GRs ex situ, and approximately 500 in an in vitro medium-term collection (López-Delgado et al., 1998). Hence, it is imperative to establish a long-term storage method using cryopreservation to provide a steady backup supply of potato GRs at the CNRG in Mexico.

This study investigated modification of the D cryo-plate procedure by paper mounting on an aluminum plate to avoid shoot tips dropping from the cryo-plate and easy handling of cryopreservation and rewarming procedures. We investigated optimization of this revised D cryo-plate procedure using in vitro grown potato shoot tips and adjusting the cryopreservation of CNRG. We subsequently tested the optimized protocol on ‘B-71-240-2’ and 12 additional potato genotypes, and stable storage in liquid nitrogen (LN) for 1 year. As a result, we indicate a new technique for CNRG in Mexico.

Materials and Methods

Plant materials

The potatoes (Solanum tuberosum) used in this study were obtained from the in vitro Collection at Campo Experimental Toluca, INIFAP, Mexico. Experiments to apply the D cryo-plate protocol to potato shoot tips were performed under accession number ‘B-71-240-2’. The optimized procedure was tested on an additional 12 genotypes. Almost all experiments were performed in CNRG in Mexico. Cultured plants were subcultured every 3 months on solid Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) with 3.0% (w/v) sucrose, 0.03% calcium chloride, and 0.3% gellan gum (Yamamoto et al., 2015). Shoots were incubated at 18°C ± 2°C with a 24-h light under a light intensity of 32 μmol·m⁻²·s⁻¹ provided by white fluorescent tubes (standard condition). Apical shoots (about 5 mm) were cut, plated on 20 mL solid MS medium in Petri dishes (90 × 20 mm), and cultured for 2–3 weeks under standard conditions. Shoot tips were dissected from the shoots and precultured overnight on solid MS medium containing 0.3 M sucrose at 24°C. For modification and optimization of the D cryo-plate protocols, we used 2.0–2.5 mm long explants consisting of the apical meristem with two young leaves.

Base D cryo-plate protocol

The aluminum cryo-plates used for the D cryo-plate protocol were 7 × 37 × 0.5 mm, containing 10 oval wells with a length of 2.5 mm, a width of 1.5 mm, and a depth of 0.75 mm (Fig. 1A1). The base D cryo-plate protocol was derived from the protocol developed by Niino et al. (2014) and Yamamoto et al. (2015) as follows:

Fig. 1

Overview of the revised D cryo-plate procedures for cryopreservation of in vitro grown potato shoot tips. (A) 1: Cryo-plate with wells, 2: A sheet of BEMCOT paper; (B) Alginate solution on a cryoplate; (C) Mounting the paper on the cryo-plates; (D) Pouring calcium chloride solution on the paper; (E) Desiccation of shoot tips in a Petri dish with silica gel; (F) Rehydration of cryopreserved shoot tips attached to the paper on the medium. Bars indicate 10 mm.

1. 1.   Droplets (approximately 4 μL) of 2% (w/v) sodium alginate solution with 0.4 M sucrose in MS basal solution were poured into the cryo-plate wells.
2. 2.   Precultured shoot tips were transferred one by one into the wells with a scalpel blade and covered with droplets (approximately 3 μL) of the sodium alginate solution.
3. 3.   The calcium chloride solution (0.1 M calcium chloride in MS basal solution with 0.4 M sucrose) was poured dropwise onto a cryo-plate until it was covered completely. Polymerization was complete after 15 min at room temperature (RT) around 25°C, and the calcium chloride solution was removed by tapping it gently on filter paper in a Petri dish.
4. 4.   The shoot tips on the cryo-plate were treated for 45 min at RT with loading solution (LS) containing 2.0 M glycerol and 1.0 M sucrose in MS basal solution.
5. 5.   Cryo-plates were removed from the LS, tapped gently on filter paper in a Petri dish and dehydrated in a Petri dish with filter paper and 35 g silica gel; shoot tips attached to the cryo-plates were desiccated for 90 min at 24°C (Fig. 1E).
6. 6.   After dehydration, the cryo-plates were plunged directly into LN and transferred into 2 mL cryotubes held on a cryo-cane and filled with LN. The cryo-plates were kept for at least 60 min in LN.
7. 7.   For regeneration, the cryotubes were retrieved from LN. The cryo-plates were immersed and rewarmed in cryotubes containing 2 mL 1 M sucrose solution with MS basal medium in which they were incubated for 15 min at RT. The shoot tips were then removed from alginate gel, plated onto fresh solid MS medium, and cultured under standard conditions at 24°C.

Modification of the base D cryo-plate protocol

One step was added to the base D cryo-plate steps between 2 and 3, because dropping of shoot tips was noted occasionally during cryopreservation treatment and/or rewarming steps, becoming a possible problem in terms of damage or loss of shoot tips. A disinfected sheet of BEMCOT paper (7 × 30 mm) (Fig. 1A2) was used to cover the cryo-plates, taking care to avoid dropping the shoot tips (Fig. 1C). BEMCOT (Clean wipe-P) is a cellulose wiper produced by Ozu Co., Japan. The BEMCOT paper has qualities such as high absorption, easy dehydration, no damage after LN storage, and low impurities contents. After step 2, the sodium alginate solution was poured on the above and below parts of cryo-plate (Fig. 1B). Next dried paper was mounted on an aluminum plate with the sodium alginate solution and shoot tips (Fig. 1C) followed by the calcium chloride treatment on the paper (Fig. 1D). The regeneration was done by step 7. The cryo-plates retrieved from LN were immersed in cryotubes containing 2 mL 1 M sucrose solution with MS basal medium in which they were incubated for 15 min at RT. Then the paper-attached shoot tips were removed from the cryo-plates and plated onto solid MS medium (Fig. 1F). The shoot tips were then removed from alginate gel and plated onto fresh solid MS medium. Effect of BEMCOT paper mounting was tested on the regeneration of cryopreserved shoot tips.

Optimization of the revised D cryo-plate protocol

Various steps of the revised D cryo-plate protocol were optimized or reconfirmed, and the effect of these optimizations on the regeneration of shoot tips was studied. Optimizations were as follows:

1. 1.   Effect of cold hardening and preculture: after apical culture for 2 weeks, the shoots were cold hardened at 5°C for 1 week under an 8-light photoperiod with intensity of 16 μmol·m⁻²·s⁻¹ (Hirai and Sakai, 1999). Cold-hardened shoot tips were then precultured overnight on solid MS medium containing 0.3 M sucrose at 24°C.
2. 2.   Comparison of LS sucrose concentrations: sucrose concentrations in LS were 0.6, 0.8, 1.0, 1.2, and 1.4 M.
3. 3.   Effect of duration of exposure to LS and duration of dehydration in Petri dish with silica gel: durations of exposure to LS were 30, 45, 60, and 90 min. Dehydration periods in a Petri dish with silica gel were 30, 60, 90, and 120 min at 24°C.
4. 4.   Effect of three initial light conditions after rewarming: standard light conditions (32 μmol·m⁻²·s⁻¹), 3 days dim condition (16 μmol·m⁻²·s⁻¹), and 3 days dark condition.
5. 5.   Effect of rehydration condition and removal of alginate gel: after rewarming in 1.0 M sucrose solution for 15 min, shoot tips with alginate gel were plated on the medium for 0, 1, and 16 h, and then shoot tips were removed from the alginate gel.

For large-scale cryostorage, the optimized D cryo-plate procedures with and without the cold hardening were assessed on ‘B-71-240-2’ and 12 additional potato genotypes. For safety long-term storage, the 1 month, 6 months, and 1 year storage in LN were tested using these procedures.

Regeneration assessment and statistical analyses

Post-LN regrowth was evaluated after 4 weeks of culture under standard conditions at 24°C by counting the number of shoots that developed into normal shoots. Three replicates for each of the 10 samples were tested in each experimental treatment. Statistical analysis was performed using Tukey’s test or one way ANOVA through the program on the web site (http://www.gen-info.osaka-u.ac.jp/MEPHAS/tukey.html) to compare the means and determine significant differences (P < 0.05) (Yamamoto et al., 2015).

Results and Discussion

Modification of the D cryo-plate protocol by paper mounting on cryo-plate

When using the D cryo-plate method, it is important to adhere the shoot tips firmly to the cryo-plates throughout the whole procedure for efficient performance. Niino et al. (2014) indicated the need to avoid some shoot tips detaching themselves from the cryo-plates during manipulations. The addition of 1 M glycerol and 0.4 M sucrose to the sodium alginate gel prevented the dropping of mat rush buds from the cryo-plates (Niino et al., 2014). However, this was not enough to avoid dropping the shoot tips from the cryo-plate perfectly, especially just after taking it out from the LN for rewarming and LS treatment. The effect of mounting the BEMCOT paper on the aluminum plate on the regrowth of cryopreserved shoot tips was evaluated (Table 1). There were no significant differences in regrowth of cryopreserved shoot tips between paper mounting and no paper mounting, regardless of the dehydration time by silica gel. We observed no negative effect on regrowth of cryopreserved potato shoot tips after rewarming for paper mounting. The paper mounting increased the performance of each step, preventing detachment of explants during cryopreservation manipulations, because shoot tips were firmly fastened between the paper and cryo-plate with the alginate gel. We also observed no dropping of shoot tips during regeneration steps. After rewarming, the shoot tips attached to the paper by the alginate gel were easily transferred to the medium by removing the paper. The paper-attached shoot tips can be used for transferring to the medium for rehydration (Fig. 1F). The modification step of paper mounting may allow the use of D and V cryo-plate procedures for other plant species, larger materials, and longer treatments using an osmoprotective solution. In this study, we adapted the paper mounting procedure (revised D cryo-plate protocol) in all other experiments.

Table 1

Effect the BEMCOT paper mounting on the aluminium plate with the sodium alginate solution and shoot tips on regrowth of cryopreserved shoot tips of potato accession ‘B-71-240-2’ using the D cryo-plate protocol.

Optimization of the revised D cryo-plate protocol

Cold hardening and preculture

Cold hardening and preculture are effective and necessary for high levels of regrowth (Yamamoto et al., 2011). The dimethyl sulfoxide (DMSO) droplet method of potato cryopreservation is now being used for cold accumulation of shoots under an 8-h photoperiod at 21°C/8°C day/night temperature for 7 days (Kaczmarczyk et al., 2008). Using the droplet vitrification method, cold hardening of potato germplasms positively affected regrowth after cryopreservation, especially in varieties with low regrowth ability (Folgado et al., 2014; Panta et al., 2014). These results indicate cold hardening and preculture of shoot tips are necessary to achieve high regrowth after cryopreservation. When the shoot tips were precultured, regrowth after cryopreservation was high (80% and 90%) regardless of cold hardening. In contrast, the shoot tips without preculture demonstrated a low regrowth rate, regardless of cold hardening (Table 2). In potato shoot tips that were cold hardened at 5°C for 1 week and precultured overnight on solid MS medium containing 0.3 M sucrose at 24°C, regrowth after cryopreservation was highest (90%). Cold hardening following preculture is effective and necessary to obtain high regrowth in many potato genotypes. However, if potato shoots are cultured under high intensity light (> 100 μmol·m⁻²·s⁻¹), elimination of the cold acclimatization step may be possible (Kim et al., 2006; Yamamoto et al., 2015).

Table 2

Effect of cold hardening and preculture on regrowth of cryopreserved shoot tips of potato accession ‘B-71-240-2’ using the revised D cryo-plate protocol.

LS treatment and dehydration duration

LS treatment and dehydration in a Petri dish with silica gel are crucial to obtain vitrification of shoot tips and high regrowth using the D cryo-plate protocol (Engelmann et al., 2008; Niino et al., 2013). High regrowth of cryopreserved shoot tips was obtained after 90 min silica gel dehydration reaching 73.3%–86.7% regardless of the LS treatment duration (Table 3). The highest regrowth was obtained after 45 min of LS treatment and 90 min dehydration. Differences in sucrose concentration (0.6, 0.8, 1.0, 1.2, and 1.4 M) resulted in no significant difference in regrowth (Table 4). To standardize the dehydration step, the use of silica gel must be considered as the dehydration rate can fluctuate between laboratories due to differences in RT, relative humidity (RH), and laminar air flow speed. Dehydration in the D cryo-plate procedure could be performed with silica gel if an air conditioner is not installed in the transfer room (Niino et al., 2013). There were no differences in regrowth after rewarming of cryopreserved mat rush buds between those dehydrated by laminar air flow and by silica gel (Niino et al., 2014). We adopted silica gel as a dehydration step in the CNRG laboratory due to difficulty in controlling RT and RH, resulting high regrowth. The optimal dehydration time for the potato shoot tips on cryo-plates by silica gel was 1.5–2.0 h, while that of mat rush buds on cryo-plates by silica gel was 2.0–2.5 h (Niino et al., 2014). This difference may be related to the volume of the shoot tips.

Table 3

Effect of LS treatment and dehydration durations on regrowth (% ± SE) of cryopreserved shoot tips of potato accession ‘B-71-240-2’ using the revised D cryo-plate protocol.

Table 4

Effect of sucrose concentration in LS on regrowth of cryopreserved shoot tips of potato accession ‘B-71-240-2’ using the revised D cryo-plate protocol.

Post-warming regrowth

To achieve high recovery after LN exposure, an efficient tissue culture procedure and post-rewarming handling system are necessary (Rafique et al., 2015). When cryopreserved shoot tips were placed in dim light or dark for 3 days, their regrowth was significantly higher than that of shoot tips that were kept in standard light conditions (Table 5). Plating the shoot tips on medium for 1 h or overnight before removal of the gels was more effective in achieving higher regrowth than without plating. Dehydrated shoot tips encapsulated in alginate gel were rehydrated and swollen on the medium for more than 1 h, resulting in high regrowth due to the ease of handling during gel removal, preventing damage to the shoot tips (Table 6). Removal of alginate gel from shoot tips was important to obtain high regrowth as seen by comparison to shoot tips encapsulated in alginate gel (Table 6). Post-rewarming handling systems are vital to achieve high regrowth after LN. In the cryopreservation of in vitro sugarcane shoot tips using V cryo-plates, regrowth was improved by removing the alginate gel, keeping the cultures in the dark for 7 days after LN and using an optimal medium with growth regulators (Rafique et al., 2015). For the cryopreservation of in vitro potato shoot tips by droplet vitrification, post-cultures are kept in the dark for a week on medium containing progressively decreasing sucrose concentrations, from 0.3 M to 0.07 M (Panta et al., 2014). In addition, cryopreserved potato shoot tips are post-cultured on semisolid MS at 24°C under low light intensity for 7 days (Kim et al., 2006).

Table 5

Effect of light conditions during the first 3 days after rewarming on regrowth of cryopreserved shoot tips of potato accession ‘B-71-240-2’ using the revised D cryo-plate protocol.

Table 6

Effect of the rehydration method and alginate gel removal on regrowth of cryopreserved shoot tips of potato accession ‘B-71-240-2’ using the revised D cryo-plate protocol.

Optimized procedure for cryopreservation

Based on the above results, we developed an optimized procedure as follows for efficient application of the D cryo-plate protocol to Mexican potato GRs.

Cold-hardened shoot tips were excised to 2.0–2.5 mm in length, precultured for 16 h at 24°C on MS with 0.3 M sucrose, attached on the cryo-plates by alginate gel with paper, treated with 2.0 M glycerol and 1.0 M sucrose solution for 45 min at 24°C, and dehydrated in a Petri dish with 35 g silica gel at 24°C for 90 min before direct immersion in LN. For regeneration, the cryo-plates were immersed in 2 mL cryotubes containing 2 mL 1 M sucrose solution with MS basal medium and incubated for 15 min at RT. The BEMCOT paper with cryopreserved shoot tips attached was removed from the cryo-plate, and plated on the medium for 1 h. Shoot tips were then removed from the alginate gel, plated on fresh solid MS medium, and cultured for 3 days under dim light and then under standard light conditions.

This optimized procedure was applied to shoot tips of ‘B-71-240-2’ and 12 additional potato genotypes (Table 7). Regrowth on D cryo-plates was high for all genotypes, ranging from 70.0% to 93.3%, with an average of 82.8%. However, regrowth of some genotypes without cold hardening was lower than those with cold hardening. Shoot tips resumed growth within 5 days after plating and developed normal shoots without initial callus formation, growing into complete normal plantlets with roots (Fig. 2). There were no significant differences in regrowth after LN storage ranging from 1 h to 1 year (Data not shown).

Table 7

Regrowth of shoot tips of 13 potato genotypes cryopreserved using the optimized D cryo-plate protocol.

Fig. 2

Regenerated potato shoots of accession ‘B-71-240-2’ by the optimized D cryo-plate method. A, B, C, D, E, and F refer to regenerated shoots from cryopreserved shoot tips at 3 days, 3 weeks, 2 months, 3 months, 5 months, and 6 months after plating, respectively. Bar indicates 10 mm.

The optimized D cryo-plate procedure developed here for CNRG produced high regrowth in 13 potato genotypes that were maintained by the slow growth method in INIFAP. This procedure appears to be an efficient and practical method for the cryopreservation of potato shoot tips and will facilitate the implementation of cryobanking of potato GRs in Mexico. This technique, including paper mounting to a cryo-plate, was the first trial and could be useful to avoid losing cryopreserved shoot tips due to simple handling error. Also, this will make it possible to use larger materials and materials requiring longer treatment with solutions for D and V cryo-plate protocols in other plant species.

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