Plant material
Raspberry ‘Stiora’ (Fig. 1a) was used to optimize droplet-vitrification protocol for cryopreservation and to assess genetic stability and field performance in cryo-derived regenerants. Thirty additional raspberry genotypes were subsequently used for testing the optimized cryopreservation protocol (Table 2). In vitro stock shoots of all raspberry genotypes used in the present study were cultured on shoot maintenance medium (SMM) containing 4.4 g/L Murashige and Skoog (1962, MS), 30 g/L sucrose, 0.5 mg/L 6-benzylaminopurine (BAP), 0.1 mg/L indole-3-butyric acid (IBA), 36 mg/L ethylenediamine di-2-hydroxyphenyl acetate ferric (Fe-EDDHA), and 6 g/L agar. The pH was adjusted to 5.7 before autoclaving at 121 ℃ for 20 min. The cultures were maintained at 22 ± 2 ℃ under a 16-h photoperiod with a light intensity of 50 μmol s-1 m-2) provided by cool-white fluorescent tubes). Subculturing was performed every 4 weeks.
Optimization of droplet-vitrification protocol
Size of shoot tips: Three different sizes of shoot tips were used for optimizing droplet-vitrification protocol. They are 0.5 mm in length with 1-2 leaf primordia (LPs), 1 mm in length with 3-4 LPs (Fig. 1b) and 2 mm in length with 5-6 LPs. Shoot tips were excised from 4-weeks old stock shoots (Fig. 1a) and step-wise precultured with 0.25 M sucrose for 1 day, 0.5 M sucrose for 1 day, and 0.75 M sucrose for 1 day. The shoot tips were then transferred to a loading solution containing 2 M glycerol and 0.4 M sucrose for 20 min. After loading, the shoot tips were exposed to plant vitrification solution 2 (PVS2, Sakai et al. 1960) at room temperature (RT) for 20 min. PVS2 was composed of 4.4 g/L MS, 15% (w/v) ethylene glycol, 15% (w/v) dimethylsulfoxide (DMSO), 30% (w/v) glycerol and 0.4 M sucrose (pH 5.8). Following vitrification, the shoot tips were transferred into PVS2 droplets (15 µl for each) on aluminium foil (about 3 × 0.8 cm, Fig. 1c) and directly immersed in LN. After LN treatment for 2 min, aluminium foil strips were removed out from LN and rapidly transferred into an unloading solution for 20 min. Unloading solution contained 0.75 M sucrose in MS medium. The cryopreserved shoot tips were post-cultured on 0.75 M sucrose medium in the dark for 3 days, and then transferred on SMM medium in the light condition for recovery. Subculturing performed every 4 weeks.
Preculture: Shoot tips (Fig. 1b), measuring approximately 1 mm, were subjected to different sucrose concentrations (0.25 M, 0.5 M, and 0.75 M) for a duration of 3 days. Each treatment was identified with a letter label (A-G) representing the combination of sucrose concentration and preculture duration (Table S1). All subsequent procedures, including loading, vitrification, unloading, and post-culture, followed the same protocol as previously described for optimizing shoot tip size.
PVS2 vitrification: Shoot tips (1 mm in length, Fig. 1b) were exposed to PVS2 at RT for varying durations of 0, 5, 10, 20, 40, and 60 min to determine the optimal vitrification time. Other procedures including preculture, loading, unloading, and post-culture were the same as described above for optimizing size of shoot tips.
Post-culture: Following cryopreservation, the shoot tips were post-cultured on 0.75 M sucrose medium in darkness for durations of 0, 1, 3, and 7 days, respectively, and then transferred to the light condition for recovery. Other procedures including preculture, loading, vitrification, and unloading were the same as described above for optimizing size of shoot tips.
Testing the optimized droplet-vitrification protocol to other 30 raspberry genotypes: Additional thirty raspberry genotypes (Table 1) from in vitro collection in Norway were used to test wide-spectrum ability of the optimized droplet-vitrification protocol developed in the present study.
Assessments of genetic integrity in cryo-derived regenerants
After three months of post-culture, cryo-derived regenerants (Fig. 1f) were collected and used for assessments of genetic integrity by ISSR, SNPs and InDels. In vitro cultured shoots were used as control.
Total DNA was extracted using the DNeasy plant Mini Kit (Qiagen GmbH, Hilden, Germany) with minor modifications (home-made lysis buffer have been applied to replace buffer AP1 in kit), following the manufacturer’s instructions. The lysis buffer contains 6 mM Tris-HCl (pH 8.0), 12 mM EDTA (pH 8.0), 17.4 g NaCL, 6 g Sodium dodecyl sulfate (SDS), and water to a total volume of 100 ml.
ISSR was performed as described by Zhang et al. (2015). Ten primers were selected from 35 candidates based on the number of amplifications. Each polymerase chain reaction (PCR) reaction contained 2.5 μL of 10 × PCR buffer, 0.75 μL of MgCl2 (50 mM), 0.5 μL of dNTP Mix (10 mM), 0.5 μL of primer (10 μM), 1.25 μL KB Extender, 0.1 μL Platinum™ Taq DNA Polymerase (Invitrogen, USA), and 1 μL of template DNA (50 ng/μL). PCR conditions included an initial denaturation at 94°C for 2 min, followed by 40 cycles of denaturation at 94°C for 30 s, annealing at 30°C for 45 s, and extension at 72°C for 2 min, with a final extension at 72°C for 7 min. Each resulting PCR product (10 µL) was subjected to electrophoresis in a 1.2% agarose gel previously stained with SYBR safe DNA stain (Invitrogen, ThermoFisher Scientific, USA), and their sizes were estimated using a 100 bp DNA ladder (New England BioLabs Inc, Ipswich, UK).
Paired-end sequencing was conducted on the DNBseq platforms (BGI TECH SOLUTIONS (HONGKONG) CO., LIMITED) with an average depth of 15-fold genome coverage. Cleaned reads were mapped against the reference genome of Raspberry ‘Anitra’ (Davik et al. 2022). The MarkDuplicates (Picard, https://sourceforge.net/projects/Picard/,version 2.25.2) was then applied to remove the potential PCR duplicates. GATK software v4.1.0.0 (McKenna et al. 2010) was used to perform SNP (single nucleotide polymorphisms) calling. Raw vcf files were filtered using the GATK standard filter method with the following parameters: “QD < 2.0 || mq < 40.0 || FS > 60.0 || MQRankSum < -12.5 || ReadPosRankSum < -8.0” for SNP and “QD < 2.0 || FS > 200.0 || ReadPosRankSum < -20.0” for InDels. To assess the SNP rates in cryopreserved and in vitro cultures compared to the Raspberry ‘Anitra’ genome, a sliding-window approach (2 m windows sliding in 1 m steps) was applied.
Assessments of field performance in cryo-derived plants grown in greenhouse
Cryo-derived and in vitro (control) shoots (>2 cm in length) were rooted (Fig. 1g) and grown in greenhouse at 24 ± 2℃ with a 16-h photoperiod of light intensity of 200 μmol⋅m−2⋅s−1. Root and shoot growth were measured after 9 weeks of growth. Shoot growth was measured in plant height, number of fully opened leaves, leaf area, fresh weight, and dry weight. Leaf area was measured from the top three fully opened leaves, using WinFOLIATM, EPSON, WinRHIZO Pro2003a, Regent Instruments Inc., Quebec, Canada). Root growth was measured in root total length, root average diameter, root surface area, and root volume by WinRHIZOTM (EPSON 1680, WinRHIZO 2003d, Regent Instruments Inc., Quebec, Canada).
Experimental design and data statistical analysis
In all experiments, the optimal shoot tip size, preculture, PVS2 treatment time, and post culture were determined. Each treatment includes ten shoot tips, and three replicates were conducted in two independent experiments. The optimized droplet-vitrification protocol was assessed across different raspberry genotypes with thirty shoot tips of each genotype utilized. Survival and regrowth rate were defined as the ratio between survival and regrowth number/total number of treatment shoot tips, and recorded after 7 days and 4 weeks of post-culture.
Genetic stability was evaluated using ISSR analysis on twenty cryopreserved samples and ten in vitro cultures (control) derived from in vitro plants. In addition, InDels and SNPs analysis were performed on five cryopreserved plants and five in vitro cultured (control) samples, with three replicates for each experiment. Furthermore, field performance was evaluated by comparing twenty cryopreserved plants with twenty in vitro (control) plants in greenhouse setting. Statistical analysis of significant differences among treatments was performed using Student’s t-test (P < 0.05).