Development of a fast and user-friendly cryopreservation protocol for sweet potato genetic resources
Plant material
In vitro-grown plantlets of ten different Ipomoea batatas cultivars; Camote Mata Serrano (CAM; Cip-420530); Cinitavo (CIN,Cip-440669); CMR 1112 (CMR; Cip-440145); Espelma (ESP, Cip-421028); Ibarreno (IBA; Cip-400989); Jewel (JEW; Cip-440031); Manchester Hawk (MAN; Cip-400040); Tanzania (TAN; Cip-440166); Tis 87/0029 (TIS; Cip-442764); and Trujillano (TRUJ; Cip-420665) were supplied by CIP (Lima, Peru). These ten cultivars were chosen to represent the diversity of the cultivars present at the sweet potato genebank of CIP as they originate from a broad range of countries over 4 different continents.
Plant multiplication
In vitro plantlets were propagated on “CIP medium” and plain MS medium50). The CIP medium contains half strength MS salts (Duchefa Biochemie, M0221) supplemented with 30 g/L sucrose, 2.8 g/L gelrite, 2 mg/L calciumpanthotenate, 100 mg/L calciumnitrate, 200 mg/L ascorbic acid and 10 mg/L gibberellic acid, pH was set to 6.12 before autoclavation (b.a.). The MS medium contains MS salts and vitamins (Duchefa Biochemie, M0222) supplemented with 25 g/L sucrose and 2.8 g/L gelrite, pH was set to 6.12 b.a.. Nodal fragments from the in vitro plantlets were excised in a sterile laminar flow bench. This was done by removing the leaves and roots of the plantlet, after which 1 cm stem fragments were cut, each containing one axillary meristem in the middle. Three fragments were transferred to each culture tube and grown in a 24 °C growth room on a 16/8 h light/dark regime with the light being provided with 36 W (cool white)/ 840 Lumilux fluorescent lights. The material was subcultured every 5–6 weeks.
Six weeks after initiation, the number of new nodes was counted. This experiment was initially executed on the following 4 cultivars: TIS, IBA, JEW and TAN. Further propagation of all 10 cultivars was done with the medium that proved to be the most productive.
Meristem excision
Two meristem types were excised using a binocular microscope; apical and axillary meristems. The apical meristems were excised by trimming the top leaves until the apical meristem is visible. Then a cut is made in the stem underneath the apical meristem, leaving the apical dome with 2 to 4 leaf primordia (Fig. 7). The axillary meristems were excised by first removing the leaves completely, including the petiole. From this a small cube of 1mm3 was excised containing the axillary meristem on one of the sides (Fig. 7) A movie demonstrating this process is added as supplementary information in the digital version of this paper (see Online Supplementary Resource 1). The exact position of the axillary meristem on the stem was not taken into account in this research, since it was proven that this had no significant impact on the survival rate after cryopreservation39.
Figure 7
Excised apical (left) and axillary meristem (right) from a CIN and ESP cultivar plantlet respectively, displayed on millimetre paper. The black bar represents 1 mm.
Full size image
Preculture
In the “no preculture method”, the excised meristems are transferred on top of a sterile filter paper placed on a MS plate (MS, 30 g/L sucrose and 3 g/L gelrite, pH set to 6.12 b.a.). As soon as sufficient meristems for that specific experiment are excised, they are subjected on the same day to the cryopreservation procedure. In the “preculture method”, the excised meristems are transferred on top of a sterile filter paper placed on a 0.3 M MS plate (MS, 102.7 g/L sucrose; 3 g/L gelrite, pH set to 6.12 b.a.) and kept on this medium in the dark for14 to 16 h before cryopreservation takes place.
The difference between precultured and non-precultured meristems was tested by comparing the post-thaw survival and regeneration rate of 950 of both axillary and apical meristems originating from 3 different cultivars (IBA, CIN, and CMR). These were cryopreserved via the droplet cryopreservation protocol using the following parameters: Loading solution (LS) 20 min; Plant Vitrification Solution (PVS2) 30 min; Recovery Solution (RS) 15 min and 2.22 µM BA regeneration medium.
Droplet cryopreservation
Precultured or non-precultured meristems were transferred to a sterile 30 ml plastic tube containing 15 ml of LS ( MS supplemented with 2 M glycerol and 0.4 M sucrose; pH 5.8), where they remain at room temperature for 20 min. Then the LS was removed from the tube with a sterile plastic boll pipette, taking care not to remove or damage the meristems.
The empty tube was then filled with 15 ml chilled PVS251 with the Murashige- Tucker medium replaced by MS ( MS supplemented with 30% glycerol, 15% ethylene glycol, 15% DMSO and 0.4 M sucrose, pH 5.8) and subsequently placed in an ice bath for 30 min21,23.
Of each sample of 10 meristems, 3 were directly transferred from the PVS2 to the RS (MS supplemented with 1.2 M sucrose, pH 5.8) at room temperature to act as a control. The remaining 7 meristems were transferred with a plastic boll pipet to a sterile aluminium foil strip (4 × 15 mm). From this strip, the remaining PVS2 fluid was removed until only a thin layer of PVS2 surrounds each meristem. Subsequently the aluminium strip was plunged into liquid nitrogen (LN). When the LN surrounding the aluminium strip stopped boiling, the strip containing the meristems was transferred to a 2 mL cryotube filled with liquid nitrogen where the meristems remained for at least 30 min. To warm the meristems, the aluminium strip with meristems was removed from the cryotube with liquid nitrogen and directly plunged in the RS at room temperature.
Both control and cryopreserved meristems were exposed to the RS for 15 min. Following this, they were placed one by one with a plastic boll pipette onto a filter paper placed on a MS plate containing 0.3 M sucrose. The plates were then sealed with parafilm and stored overnight in darkness at a temperature of 24 °C. The next day, the meristems were transferred on to the regeneration medium in an upright position, with the meristematic domes not fully submerged in the medium. The meristems were left in the dark for 7 days, where after they were moved into the light.
After 1 month they were moved from the regeneration medium to new MS plates (Fig. 8).
Figure 8
Step-by-step cryopreservation process of sweet potato.
Full size image
Effect of the age of the in vitro plantlet
The effect of plant age was tested by comparing the post-thaw regeneration rates of 215 meristems, for both apical and axillary each, from 3, 6 and 9 weeks old plantlets from the JEW, IBA, MAN and CMR cultivars, using the following parameters: preculture (0.3 M sucrose); 20 min treatment with LS; 30 min with PVS2; 15 min with RS and regeneration on 0.3 M sucrose medium (1 day) followed by MS with 2.22 µM 6-Benzyladenine (BA) regeneration medium.
Effect of toxicity of the loading solution
For this experiment 24 apical and 72 axillary meristems of the cultivars TIS, TAN, IBA and JEW were subjected to 3 different LS treatment times (20, 180 and 360 min). After the LS treatment the meristems were transferred to a MS plate containing 0.3 M sucrose for one day plate after which they were transferred to an MS plate. Thereafter, the survival and regeneration rates were compared.
Effect of composition of the regeneration media
Three different media were tested: MS (MS supplemented with 25 g/L sucrose and 2.8 g/L gelrite), Hirai* (MS supplemented with 30 g/L sucrose, 1 g/L casein hydrolysate, gibberellic acid 0.5 mg/L and 2 g/L gelrite; which is a slightly altered medium of Hirai and Sakai18, and 2.22 µM BA (MS supplemented with 25 g/L sucrose; 2.8 g/L gelrite and 2.22 µM BA). The pH of the media were set to 6.12 b.a. These media were already previously reported to have been used successfully for cryopreservation of sweet potato meristems. The survival and regeneration rate of these media after cryopreservation were compared for 939 apical and 939 axillary meristems from 4 cultivars (TIS, JEW, IBA and TAN). The cryopreservation parameters were: No preculture; LS 20 min; PVS2 15 min; RS 15 min.
Effect of axillary versus apical meristem
This was tested by comparing the post-thaw regeneration rate of 475 apical and 475 axillary meristems originating from 3 different cultivars (IBA, CIN, and CMR). These were cryopreserved using the following parameters: No preculture; LS 20 min; PVS2 30 min; RS 15 min and 2.22 µM BA regeneration medium.
Post-cryopreservation regrowth
Observations were executed one and two months after cryopreservation using a binocular microscope.
To express the results of the regrowth(survival) and regeneration, 7 categories of post thaw reactions are distinguished. In case of doubt, the lower growth category is taken in order to avoid false positives. The categories are summarized below and a visual representation of a typical meristem in each of the 7 categories is shown in Fig. 5.
Full regeneration (F) are those meristems that have grown multiple leaves, each containing a new meristem in the axil, and that are growing visible roots. These plantlets are able to regenerate into a new plant that can be subcultured and transferred to the soil. A Hyperhydricity (H) score is given to meristems which do form new leaves and meristems, but are growing abnormally. These plantlets have narrow leaves with a thick stem and roots that grow upwards. These are not categorized as regeneration as subculturing these plants will not lead to plantlets that can be transferred to the field. Shoot growth (S) is linked to meristems that produced a limited number of leaves, around 3, and then stop growing. They remain rootless. Tip growth (T) means that the meristem is visibly growing but shows no unfolded leafs. A callus (C) score is given when there is no visible growth other than callus. Black (B) or White (W) is given to meristems that have died either after or before/during cryopreservation. In many cases, callus growth is associated with one of the above categories.
To calculate the post thaw regeneration rate of a plate, the Full regeneration (F) meristems were counted and divided by the total number of meristems on the plate. The survival rate was calculated by counting all meristems with living tissue (F, H, S, T and C).
Statistical analysis
The comparison of the number of nodes after 6 weeks on the 2 propagation media was executed using a one-sided tail, student t-test (homoscedastic) with a P-value More
