Plant material and in vitro culture

Sugarcane cultivars N12, N19, N58, and NCo376 infected with SCYLV and NCo376 co-infected with SCYLV and SCMV were visually identified in-field at the South African Sugarcane Research Institute (SASRI), Mount Edgecombe, KwaZulu-Natal, Republic of South Africa (RSA) (29°42′11′′S, 31°02′34′′E). Leaf samples were collected and processed for RNA extractions to confirm the presence of virus by RT-PCR. Apical shoot tips (5 mm) were excised and used to establish in vitro shoot cultures for cryo-therapy experiments (as per Banasiak and Snyman 2017, medium composition given below).

For cryo- and osmo-therapy experiments, in vitro shoot tips (3 mm) were excised and cultured on initiation medium (IM comprising Murashige and Skoog (1962) basal salts and vitamins (Duchefa Biochemie, Haarlem, The Netherlands), 1.0 mg L−1 methylene blue (antimicrobial activity; Merck KGaA, Darmstadt, Germany), 0.015 mg L−1 kinetin (Duchefa), 0.1 mg L−1 benzyl aminopurine (Duchefa), 20.0 g L−1 sucrose (table sugar; Tongaat Huletts Sugar, Durban, RSA), 8.0 g L−1 plant agar (Duchefa), pH 4.5) in the dark for 1 d, followed by preconditioning on Murashige and Skoog (MS; Murashige and Skoog 1962) medium (MS basal salts and vitamins, 171.0 g L−1 (0.5 M) sucrose (Tongaat Huletts Sugar), and 8.0 g L−1 agar (Duchefa)) in the dark for 1 d. Subsequent to cryo- and osmo-therapy treatments, all shoots above 4 cm in height were transferred to rooting medium (RM) (MS salts and vitamins (Duchefa), 20.0 g L−1 sucrose (Tongaat Huletts Sugar), 1.0 mg L−1 indole-3-butyric acid (IBA; Duchefa)) all added pre-sterilization (autoclaved at 121 °C for 20 min), at a pH 5.3 (adjusted with 1 M KOH (Merck) or 1.0 M HCl (Merck) in Magenta vessels (Merck)) and placed in the photoperiod growth room with sub-culturing every 2 wk.

Hardening of plantlets was achieved by planting single rooted shoots into a 105-well (40 mm × 120 mm) polystyrene seedling tray (HygroTech (Pty) Ltd., Pretoria, RSA) containing an autoclaved potting mixture of vermiculite and sphagnum peat moss (1:1; Coastal Farmers Co-op Ltd, Umhlali, RSA) and placed in the SASRI insect-free quarantine glasshouse for 4 mo. The planting layout was 28 plants per treatment, replicated three times. The plants were watered manually twice per week and fertilized with Nutrifeed (1.0 g L−1, Starke Ayres, Pietermaritzburg, RSA) every 2 wk for 4 mo. Shoot and root dry mass (g) were recorded after 4 mo. Dry mass was obtained after drying fresh material in a drying oven (IncoTherm Incubator, Labotec, Durban, RSA) for 48 h at 70 °C.

Virus detection via RT-PCR

Plant genomic RNA was extracted using the GeneJET plant RNA purification mini kit (Thermo Fisher Scientific, Waltham, MA). The presence of SCYLV and SCMV in samples was confirmed using the Titan One-Step RT-PCR kit (Merck). For SCMV detection, the forward primer SCMV-F4: 5′-GTTTTYCACCAAGCTGGAACAGTC-3′ and reverse primer SCMV-R3: 5′-AGCTGTGTGTCTCTCTGTATTCTC-3′ (Alegria et al. 2003) were used. The RT-PCR reaction mix (25.0 µL) consisted of the following: 0.5 to 1.0 µg µL−1 RNA, 5 × RT-PCR buffer, 0.2 mM dNTP, 5.0 mM DTT solution, 1 U Titan enzyme, 1.5 mM MgCl2, 0.4 µM of each primer, and DEPC-treated water. The RT-PCR cycling program comprised of 50 °C for 30 min, 95 °C for 15 min (1 cycle); 94 °C for 1 min, 55 °C for 1 min, 72 °C for 30 s (35 cycles); and 72 °C extension for 10 min. The amplified product (900 bp) was detected by gel electrophoresis in Tris–acetate (TAE) buffer (40 mM Tris–acetate (Merck), 1.0 mM EDTA (Univar, Modderfontein, RSA), and pH 8), using a 1.2% (w/v) SeaKem® LE agarose (Lonza Biosciences, Basel, Switzerland) gel stained with SYBR™ safe (Thermo Fisher Scientific).

The forward primers SCYLV-F1: 5′-GACAGACTCGGCCAGTGGTCGTG-3′ and reverse primer SCYLV-R1: 5′-GTAAGCCATTGTTGAACGCTGCG-3′ (Girard et al. 2010) were used for the detection of SCYLV. The Titan One-Step RT-PCR kit (Merck) was used with the reaction mix as described above except for the exclusion of MgCl2, and the program was as follows: 45 °C for 45 min, 94 °C for 2 min (1 cycle); 94 °C for 1 min, 71 °C for 1 min, 72 °C for 30 s (35 cycles); and 72 °C extension for 10 min. The amplified product (219 bp) was detected by gel electrophoresis, as above.

Virus indexing using RT-PCR was carried out at four stages: (a) material from the field prior to shoot tip in vitro culture; (b) multiplied in vitro plantlets prior to therapy treatments; (c) plants recovered in vitro after the therapy treatments; and (d) plants hardened for 4 mo. Each therapy experiment was replicated three times, each comprising of 12 (for the control), 24 (for the osmo-therapy), and 36 (for the cryo-therapy) shoot tips. The virus eradication (ER) value observed for each treatment was calculated as follows:

$$\mathrm=\left(\frac\;in\mathit\;vitro\;\mathrm}\;in\mathit\;vitro\;\mathrm}\right)\times100$$

Osmo-therapy and cryo-therapy

The D-V protocol (described by González-Arnao et al. (2020a) and modified from Panis et al. (2005)) was applied to in vitro shoot tips as follows: preconditioned shoot tips were placed in loading solution (LS; MS salts and vitamins (Duchefa), 1.6 M sucrose (Merck), and 2.0 M glycerol (Merck)) for 20 min, followed by dehydration with plant vitrification solution 2 (PVS2; 30.0% (w/v) glycerol, 15.0% (w/v) dimethyl sulfoxide (Merck), 15.0% (w/v) ethylene glycol (Merck), 0.4 M sucrose (Merck)) at room temperature for 30 min before being placed in a drop of PVS2 within the wells of an aluminium V-cryoplate (7 mm × 37 mm × 0.5 mm with 12 wells; Taiyo Nippon Sanso Corp., Tokyo, Japan (Yamamoto et al. 2011)). Cryoplates were directly immersed in liquid nitrogen (LN) for 30 min. For warming, the cryoplates were removed from LN and plunged into liquid MS medium supplemented with 1.0 M sucrose for 30 min at room temperature. Re-culture involved placing the shoot tips upside-down with the base above the surface of the semi-solid IM (with 4.0 g L−1 activated charcoal) in the dark for 7 d. The osmo-therapy protocol was as above without the LN steps.

Following both osmo- and cryo-therapy procedures and after the first 7 d of re-culture in the dark, shoot tips were transferred to semi-solid IM and placed under low light conditions for 7 d, followed by transfer to standard photoperiod conditions (16 h day/8 hr night). Once recovered and when the shoots were approximately 3 cm in height, they were transferred to liquid IM (without agar) and sub-cultured every 2 wk. After 16 wk, the following was determined for both osmo-therapy (preconditioned shoot tips subjected to treatments with loading and PVS2 solutions without the LN steps) and cryo-therapy (pre-treated shoot tips followed by their immersion in LN) procedures: (a) the percentage (%) of shoots that recovered and multiplied (for example, regeneration) and (b) the presence of a virus. Shoot tips not exposed to cryoprotection or cooling served as the control. The experimental approach is summarized in Fig. 1.

Figure 1.

Summary of the experimental approach for the cryo- and osmo-therapy of sugarcane (Saccharum spp. hybrids) shoot tips infected with SCMV and SCYLV. LS, loading solution; PVS2, plant vitrification solution 2; LN, liquid nitrogen; SCMV, sugarcane mosaic virus; SCYLV, sugarcane yellow leaf virus.

Data analysis

Data analysis was done using Genstat software version 19 (VSN International, Hemel Hempstead, UK). All the data were examined for normality using the Shapiro–Wilk test. A one-way analysis of variance (ANOVA) and a Sidak test were performed to compare the percentage of shoot tip recovery and virus elimination. To determine whether there was a significant difference in the mean values at p < 0.05, the least significant difference (LSD) test was employed.