Introduction

To ensure safe conservation of the vegetatively propagated crops in genebanks, it is recommended to duplicate this material in the field, in vitro and/or with cryo-collections (Reed et al. 2004). According to published reports, Rubus clonal accessions are mostly conserved in field collections (EU GEN RES 2017). Field and in vitro collections are the sources used to establish cryobanks. The cryo-collections of Rubus germplasm are not numerous and contain a comparatively small number of accessions cryopreserved using several methods. Table 1 summarizes the data on the numbers of red raspberry and blackberry accessions preserved employing different cryopreservation methods in the established cryogenic storage worldwide, and in the research centers in different countries where raspberry cryopreservation has just started.

Table 1. Rubus shoot tips collections in cryopreservation worldwide
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The largest Rubus germplasm cryo-collection is maintained at the National Laboratory for Genetic Resources Preservation (NLGRP) in Ft Collins, CO (USA). It includes over 209 Rubus accessions (from 39 species) cryopreserved between 1996 and 2013, using the methods of slow cooling (National Council for Geocosmic Research (NCGR) 1996–2000), vitrification (NCGR 2000–2005), and droplet vitrification (NLGRP 2005–2013) (Maria Jenderek personal communication). Currently, the method of droplet vitrification (DV) developed by Panis et al. (2005) has broad applications for cryopreservation of a broad range of plant species (Panis et al. 2016). This DV method is relatively quick and simple and ensures good regeneration results after rewarming.

In addition to the studies aimed at developing new and modifying existing methods to raise efficiency of cryopreservation, one of the important research directions is the study of genetic stability of the regenerants obtained after rewarming (Castillo et al. 2010; Kaczmarczyk et al. 2011; Wang et al. 2014, 2017). Various DNA markers are widely used to monitor genetic stability and these studies have been done on Rubus cryo-regenerants (Castillo et al. 2010). In general, no differences in the recovered and control plants were found.

This present paper presents results of cryopreservation research performed at the N.I. Vavilov Institute of Plant Genetic Resources (VIR) on the Rubus in vitro collection held at VIR, which consists of 177 accessions. This collection contains 87 released red raspberry cultivars, from which 27 originated abroad and 60 were bred in Russia between 1920 and 2000 by various research organizations located in different ecogeographic regions of the Russian Federation. Sixty-five of the 87 accessions of red raspberry cultivars were transferred to in vitro cultures from individual plants of the field-grown accessions that were previously genotyped by simple sequence repeats (SSR markers) (Lamoureux et al. 2011), and 48 had known biochemical composition of the berries (Lefèvre et al. 2011). The combined chemo-thermotherapy was successfully applied for eradication of Raspberry Bushy Dwarf Virus (RBDV) in the in vitro plants (Antonova et al. 2015). Accessions from the in vitro collection of red raspberry cultivars are being indexed for the presence of bacterial contaminants once a year according to the method of Reed et al. 2004. Pathogen-free samples from the in vitro collection were used as a basis for cryopreservation.

The goal of the study was to analyze the post-cryogenic recovery of red raspberry cultivars using modifications of the droplet vitrification method. The results obtained from cryo-regeneration studies were compared with the published data on red raspberry cultivar cryopreserved by the droplet vitrification method (Nukari et al. 2011; Condello et al. 2011). In addition, the first results of genetic uniformity study of the recovered plants obtained after rewarming performed using inter-simple sequence repeat (ISSR) and start codon targeted (SCoT) markers are presented.

Materials and methods

Plant materials

Red raspberry cultivars of different origins from the N.I. Vavilov All-Russian Institute of Plant Genetic Resources (VIR) in vitro collection were used for cryopreservation experiments (Table 2). These cultivars were transferred into in vitro culture in 2010. All microplants of each cultivar were represented by the same clone because they originated from an individual field-grown plant (Lamoureux et al. 2011).

Table 2. List of red raspberry (Rubus. idaeus L.) cultivars used in cryopreservation in present study with the information on their parentages and origins
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Establishment of in vitro shoot cultures and tissue culture conditions

Chemical reagents were obtained from Sigma-Aldrich®, St. Louis, MO, with catalog numbers indicated. For propagation, microplant cuttings were placed on agar-solidified MS (Murashige and Skoog 1962) nutrient medium (#M5519) with 2.2 μM 6-Benzylaminopurine (BA, #B3408), 0.2 μM Indole-3-butyric acid (IBA, #I5386), and 3% (w/v) sucrose (#S7903) (Wang et al. 2005). The initial medium included 7 g L−1 agar (#A1296) with the pH adjusted to 5.8 using 0.1 N KOH before autoclaving at 121 °C for 20 min. Growth room conditions were 16-h photoperiod (25 μM m−2 s−1) with cool-white tubular fluorescent lamps (OSRAM T8L36W/640G13 1200 mm, OSRAM, Munich, Germany) at 23 °C (standard growth room conditions). Cultures were subcultured at 4-wk. intervals.

Cryopreservation and regrowth

All reagents for cryopreservation and regrowth were obtained from Sigma-Aldrich®, St. Louis, MO, with catalog numbers indicated. The droplet vitrification method described by Panis et al. (2005) with modifications (Table 3) was used for cryopreservation of 12 red raspberry cultivars. Isolated shoot tips 1.1–1.8-mm long were put into liquid MS medium without growth regulators during isolation and were transferred into a filter sterilized loading solutions (LS) containing MS basal liquid medium with 2 M glycerol and 0.4 M sucrose (Matsumoto et al. 1994), for 20 min at 23–25 °C. The shoot tips were further incubated in filter sterilized Plant Vitrification Solution 2 (PVS2), consisting of MS basal liquid medium with 3.26 M glycerol, 2.42 M ethylene glycol (EG), 1.9 M dimethyl sulfoxide (DMSO), and 0.4 M sucrose (Sakai et al. 1990), for 30 min on ice. Five minutes before the end of the PVS2 treatment, the explants were put into individual droplets (3 μL) of PVS2 on sterile aluminum foil strips, and quickly plunged into cryovials filled with liquid nitrogen (LN). The cryovials were then plunged into LN. After 1 h in LN on aluminum foil, the explants were rewarmed in filter sterilized rewarming solution (RS), pH 5.8, consisting of MS basal liquid medium with 1.2 M sucrose, (Sakai 1997) for 15 min at 23–25 °C. The rewarmed explants were put on the solidified basic MS recovering medium, pH 5.8, containing 1.4 μM trans-zeatin-riboside (Sigma-Aldrich®, #Z0375), 2.9 μM 3-indoleacetic acid (IAA, #I2886), and 0.6 μM gibberellic acid (GA, #G7645) which were dissolved in distilled water and sterilized through a 0.22-μm Millipore filter (Guangzhou Jet Bio-filtration Co, Guangzhou, China) and added to autoclaved MS medium and 30 g L−1 sucrose (Towill 1983). The recovering medium included 7 g L−1 agar (#A1296) with the pH adjusted to 5.8 using 0.1 N KOH before autoclaving. The recovering medium was dispensed into sterile plastic Petri dishes (60 mm × 10 mm) after autoclaving at 121 °C, 152 kPa for 20 min. Ten explants were cultured per Petri dish and cultivated for regeneration in standard growth room conditions.

Table 3. Comparison of droplet-vitrification (DV) procedure applied for red raspberry cryopreservation compared to original DV method of Panis et al. (2005) developed for banana meristem
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Each step of cryopreservation was performed with three replications containing 20 experimental explants per step. In total, three replications of 60 explants per accession were cryopreserved to assess the regeneration rate. Simultaneously, 10 control explants per replication were treated with all the solutions (LS, PVS2, and RS), but not immersed in liquid nitrogen. In total, there were three replications of 30 control explants per accession.

Regeneration (percent of explants which restarted growth and formed shoots) was determined on the 3rd and the 6th wk after rewarming. Data are presented as a percentage of the total number of cryopreserved explants.

Each accession for long-term storage in a cryobank (in liquid nitrogen) was processed with 90 explants, and distributed in 9 cryogenic vials, with 10 explants in each vial. This made it possible to periodically remove individual cryogenic vials to monitor the ability of the cryopreserved shoot tips to recover.

DNA extraction

All chemicals for DNA extraction and separation of amplified DNA fragments were obtained from Carl Roth® (Karsruhe, Germany, https://www.carlroth.com). Assessment of trueness to type of post-cryopreservation recovered plants to the initial in vitro plants was performed for two cultivars: Barnaulskaya and Samarskaya Plotnaya. All microplants of each cultivar were represented by the same clone. DNA was extracted from the donor in vitro plants, and from ten recovered cryo-regenerants of each cultivar. DNA was extracted by a modified cetyl trimethylammonium bromide (CTAB) extraction method as described by Gavrilenko et al. (2013).

PCR analysis

All chemicals for PCR analysis were obtained from Dialat (Moscow, Russia http://dialat.ru/). Two ISSR and two SCoT primers were selected from Graham et al. (1994), Collard and Mackill (2009), respectively (Table 4). Primers were synthesized by Evrogen, Moscow (http://evrogen.ru/). PCR was performed in 20-μL reaction mixtures containing 40 ng of raspberry DNA, × 1 reaction buffer (Dialat, Moscow, Russia http://dialat.ru), 2.5 mM MgCl2, 300 μM of each of deoxynucleotides, 0.8 μm of primer and, 1 unit of Taq-polymerase (Dialat). The PCR procedure included the following steps: 94°С for 4 min, followed by 37 cycles [94°С - 45 s, for 1.5 min., 72°С for 2 min] and finally 72°С for 5 min.

Table 4. Primers used in genetic stability assessment of red raspberries plants regrown after cryopreservation
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Separation of amplified DNA fragments

The products from the polymerase chain reaction were separated on horizontal 2.5% (w/v) agarose gels in ТВЕ buffer (Tris 0.089 M; H3BO3 0.089 M; EDTA 0.002 M; pH 8.2) at about 5 V cm−1 of gel length. After electrophoresis, the gels were stained with ethidium bromide. The DNA fragments were visualized by transmitted UV light.

Statistical analysis

The statistical analysis of the results was conducted using conventional statistical methods (Student’s t test). Statistical dispersion analysis was also carried out using the Program StatSoft Statistica™ 6.0 (Palo Alto, CA). Analysis of variance (ANOVA) was performed by Tukey’s test. Significance was determined at p ≤ 0.05.

Results and discussion

Cryopreservation of red raspberry cultivars

Control explants of all 12 cultivars had better regrowth results than post-cryogenic explants (Table 5).

Table 5. Shoot recovery after cryopreservation of 12 red raspberry cultivars (apical shoot tips of in vitro plants) on 6th wk after rewarming
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Out of the 12 cryopreserved red raspberry cultivars, the maximum shoot recovery after rewarming was recorded for explants of cv. Meteor (89.3%), and the minimum shoot recovery was for those of cv. Skromnitsa (24.2%). The average recovery rate for all subsets was 58.8% (Table 5; Fig. 1). According to the dispersion analysis, the influence of the genotype on the regeneration of shoot tips after thawing was statistically significant (p = 0.0003).

Figure 1.
figure 1

Shoot recovery of apical buds of red raspberry ‘Barnaulskaya’—6 wk after rewarming

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The literature reports a significant influence of genotype on shoot regeneration after cryopreservation for almost all studies of representatives of different taxa, including Rubus accessions with 50–85%, (average 68%) (Wang et al. 2005), and 60–100% (average 85%) (Gupta and Reed 2006; Reed 2008). Therefore, the current results of post-cryogenic recovery of red raspberry cultivars were comparable with those obtained by other laboratories and genebanks (Table 1).

Several modifications of the droplet vitrification protocol were applied for the cryopreservation of red raspberry (Rubus idaeus L.) accessions (Nukari et al. 2011; Condello et al. 2011) (Tables 1and 3). It should be noted that the current modified protocol makes it possible to considerably shorten the cryopreservation procedure compared with the original protocol by Panis et al. (2005) from 14 to 11 wk.

The other differences between the original droplet vitrification method (Panis et al. 2005) and proposed protocol are described in Table 3 in detail along with other droplet vitrification protocols developed previously for cryopreservation of raspberry accessions (Nukari et al. 2011; Condello et al. 2011).

The current droplet vitrification procedure contained the following modifications:

  • First, the changed plant growth regulator composition of the micropropagation medium was different from that suggested in the above-cited publications (Table 3).

  • Second, a lengthy (4-wk-long) pretreatment of the initial microplants on the sucrose-rich (60 g L−1) medium was omitted in contrast to the protocol by Panis et al. (2005) (Table 3). Other Rubus protocols use different pretreatments. It was shown that 1 wk of cold acclimation (22 °C 8-h day and 1 °C 16-h night) of the initial microplants, and a combination of the cold acclimation with 50 μM ABA, significantly improved regrowth for several Rubus genotypes from slow freezing (Reed 2008). Nukari et al. (2011) made the pretreatment of initial Rubus microplants by cold acclimation and sucrose-rich medium (0.25–0.75 M, 86–256 g L−1) for droplet vitrification. Condello et al. (2011) did not use any pretreatment and shortened the total duration of the droplet vitrification protocol (Table 3). However, only one raspberry accession was used in the study. The droplet vitrification modified protocol of Condello et al. (2011) was noted when developing the modifications in the present study.

  • Third, the LS medium used from 5–7 h during pretreatment and the osmoprotection stage in the Panis et al. (2005) protocol was replaced by incubating the isolated apical buds in the liquid MS hormone-free medium with 30 g L−1 of sucrose for 1 h, followed by incubating the explants in the LS medium for 20 min. In contrast to the protocol by Condello et al. (2011), incubation was completed in the light instead of darkness (Table 3). As was shown by other authors, the treatment timing in LS and PVS2 vary for different Rubus cryopreservation methods. For example, in the encapsulation and vitrification (EV) protocol, the treatment timings are 90 min (LS) and 180 min (PVS2) (Wang et al. 2005), whereas in the PVS2-vitrification protocol by Gupta and Reed (2006), the treatment timings are 20 min (LS) and 20 min (PVS2).

  • Fourth, the droplet vitrification protocol developed by Panis et al. (2005) for banana meristems was used with some modifications for cryopreservation of many plant species. These modified droplet vitrification protocols include various recovery media with different plant growth regulators (BA, kinetin, and zeatin) (Reed 2008; Wang et al. 2014; Panis et al. 2016). In the present study, MS medium with 1.4 μM of zeatin-riboside, 2.9 μM of IAA, and 0.6 μM of GA (Towill, 1983) was used at the final stage of post-cryogenic recovery (Table 3) because zeatin was superior for in vitro propagation of Rubus accessions compared to other cytokinins (Debnath 2004; Nicuţă et al. 2014; Zayova et al. 2016).

Assessment of the trueness to type in recovered plants

Both ISSR primers [(АG)8С, (GT)8С] generated 11 fragments in cultivar Barnaulskaya and 14 fragments in cv. Samarskaya Plotnaya (Fig. 2, Table 6). In total, both SCoT primers [SCoT4, SCoT12] generated 10 fragments in cv. Barnaulskaya. Of the two SCoT primers used, PCR products were obtained only with SCoT4 in the case of cv. Samarskaya Plotnaya (Table 6). The analysis of electropherograms of all fragments did not detect any differences between the DNA bands in the donor microplants and in the 10 post-cryopreservation regenerants of each cultivar (Fig. 2, Table 6). These results corresponded to published data. The majority of research on cryopreservation of apical shoot tips or dormant buds demonstrates stability of the analyzed DNA loci in the post-cryopreservation regenerants (Schäfer-Menuhr et al. 1997; Mix-Wagner et al. 2002; Keller et al. 2006; Kaczmarczyk et al. 2010; Castillo et al. 2010; Zhang et al. 2015; Solov’eva et al. 2016; Matsumoto 2017). Polymorphism detected in a few cases (Castillo et al. 2010) was due to the initial heterogeneity of the donor microplants, which served as the source of explants for cryopreservation.

Figure 2.
figure 2

Comparison of electrophoregrams of PCR-products generated with ISSR primer (GT)8С in initial in vitro plant and in 10 microplants recovered after rewarming in red raspberry ‘Samarskaya Plotnaya’. M molecular weight marker (100 bp + 1500, http://russia.sibenzyme.com), 1 initial in vitro plant used as a source of explants of ‘Samarskaya Plotnaya’ for cryopreservation, 2–7 and 9–12 plants regenerated after rewarming, 8 H2O

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Table 6. Number of amplified bands with ISSR and SCoT primers in initial donor plants and in 10 plantlets regenerated from shoot tips of each red raspberry cultivar following droplet vitrification cryo-procedure
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Conclusions

The modifications to the droplet vitrification protocol included removal of the initial microplant pretreatment stage. Modified media were used at the stages of initial micropropagation, explant isolation, and post-cryogenic regeneration. The modifications resulted in a reduction in the duration of some cryo-conservation stages compared to the initial protocol by Panis et al. (2005), and reduced the total duration from 14 to 11 wk. The modified cryopreservation method developed demonstrated a level of post-cryogenic regeneration for 12 raspberry cultivars that was comparable with the literature. The results of ISSR and SCoT marker analysis confirmed the genetic stability of the analyzed loci of post-cryopreservation regenerants compared to donor in vitro plants of two analyzed raspberry cultivars.

VIR will further expand cryopreservation of the raspberry and blackberry collections using the modified droplet vitrification method.