Exploring in vitro germplasm conservation options for sugarcane (Saccharum spp. hybrids) in South Africa M. Banasiak & S. J. Snyman
In Vitro Cellular & Developmental Biology - Plant ISSN 1054-5476 Volume 53 Number 4 In Vitro Cell.Dev.Biol.-Plant (2017) 53:402-409 DOI 10.1007/s11627-017-9853-2
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Author's personal copy In Vitro Cell.Dev.Biol.—Plant (2017) 53:402–409 DOI 10.1007/s11627-017-9853-2
CRYOPRESERVATION
Exploring in vitro germplasm conservation options for sugarcane (Saccharum spp. hybrids) in South Africa M. Banasiak 1 & S. J. Snyman 1,2
Received: 23 March 2017 / Accepted: 13 August 2017 / Published online: 12 September 2017 / Editor: Barbara Reed # The Society for In Vitro Biology 2017
Abstract In vitro plantlets of sugarcane cultivar NCo310 were maintained in slow growth conditions at both 18 and 24°C and on four semi-solid media: SG1—Murashige and Skoog (MS) salts and vitamins with 20 g L−1 sucrose, SG2—½ MS with 10 g L −1 sucrose, SG3—MS with 20 g L−1 sucrose and 1 mg L−1 abscisic acid (ABA), and SG4—½ MS with 10 g L−1 sucrose and 1 mg L−1 ABA. After 8, 12, 24, 36, and 48 mo shoot multiplication rates were recorded, shoots were removed from storage and subcultured every 2 wk on SG1 with 0.015 mg L−1 kinetin and 0.1 mg L−1 benzyl aminopurine for 2 mo. At 18°C, all media supported storage for 48 mo with subculturing every 12 mo. Shoot multiplication post-retrieval was significantly higher on the SG2 medium compared with the non-stored control (362 ± 84 and 126 ± 26 shoots per recovered shoot after 2 mo, respectively). In addition, shoots could be maintained for 48 mo on SG2 medium with one subculture without compromising post-storage multiplication ability. At 24°C, storage on all four media supported recovery and multiplication of shoots for 8 mo and only SG2 medium facilitated survival for 12 mo. There was no advantage to incorporating ABA into the storage media, regardless of the temperature and storage time. Cryopreservation of cultivar NCo376 in vitro-derived shoot meristems using the V-cryo-plate method demonstrated that the sucrose concentration in the loading solution (0.8–1.8 M) had no
* S. J. Snyman
[email protected] 1
South African Sugarcane Research Institute, Private Bag X02, Mount Edgecombe, KwaZulu-Natal 4300, South Africa
2
School of Life Sciences, University of Kwa-Zulu Natal, Westville Campus, Private Bag X54001, Durban 4000, South Africa
significant effect on survival of the meristems, which ranged from 41.7 ± 4.8 to 69.4 ± 10%. Keywords Slow growth . Cryopreservation . V-cryo-plate
Introduction Traditionally, sugarcane germplasm collections of commercial lines (Saccharum spp. hybrids) and ancestral breeding material are kept in the field. Sugarcane is vegetatively propagated, so these assemblages are costly to maintain and vulnerable to environmental hazards. Consequently, ex situ conservation options have been developed for this crop using in vitro storage methodologies through slow growth and cryopreservation (Engelmann and Engels 1996; Snyman et al. 2011). These are viewed as complementary to field collections, as they are aseptic and are stored under controlled conditions with minimal space and manpower requirements. Although one of the main concerns for storage of in vitro material was the propensity for somaclonal variants, the agronomic and genetic fidelity of sugarcane plants has been repeatedly assessed during field transfer and found to be indistinguishable from the parent material (Taylor and Dukic 1993; Watt et al. 2009). There may be an epigenetic effect on stalk appearance, with an increased tiller number and decreased stalk diameter compared with non-cultured plants. This was first mentioned in Taylor and Dukic (1993) when they discussed phenotypic characteristics in field-evaluated plants stored in vitro, with annual subculturing, for several years in CIRAD, France. However, these features normalized after the first vegetative planting. These findings were also reported when characteristics of in vitro micropropagated sugarcane germplasm were assessed (Lourens and Martin 1987; Lorenzo et al. 2001).
Author's personal copy SUGARCANE IN VITRO GERMPLASM CONSERVATION
Medium-term storage is one option for sugarcane plantlets through slow growth using low temperatures, minimal media, and short photoperiods than used for regular multiplication. Plantlets derived from apical meristem tissue or axillary buds can be stored for periods of 2 mo (Lemos et al. 2002), 6 mo (Sreenivasan and Sreenivasan 1985), and 12 mo (Taylor and Dukic 1993), at a range of temperatures between 15 and 18°C without the need to subculture onto fresh medium. To prolong the period between subcultures, various growth retardants have been used including mannitol (Sawar and Siddiqui 2004; Chandran 2010), abscisic acid (Lemos et al. 2002), or by reducing the Murashige and Skoog (MS) salts and vitamins by one half and reducing the carbohydrate source (Lersrutaiyotin et al. 1993; Taylor and Dukic 1993; Watt et al. 2009). Long-term storage of sugarcane germplasm using cryopreservation of shoot apices derived from in vitro plantlets was first reported by Paulet et al. (1993). Some researchers describe the cryostorage of somatic embryos (Eksomtramage et al. 1992; Martínez-Montero et al. 2008; Distabanjong et al. 2015), but apices are viewed as genetically stable and are generally the preferred explant for cryostorage. The latest sugarcane research focuses on using aluminum cryo-plates, as described by Yamamoto et al. (2011), on which the apical meristem is embedded in a sodium alginate-filled well using either the V (vitrification) method (Rafique et al. 2015) or the D (dehydration) method (Rafique et al. 2016), with an average regrowth of 70.3 and 52.1% of explants 7 d after retrieval, respectively. The approach for both of these methods involves the following steps: (a) excision of the meristems, (b) 24 h recovery, (c) 24 h pre-conditioning with sucrose, (d) adhesion of meristems to an aluminum cryo-plate with sodium alginate, (e) osmoprotection with loading solution, (f) dehydration using either a vitrification solution or air drying in a laminar flow hood, (g) cryopreservation in liquid nitrogen (LN), (h) rewarming, and (j) recovery. The goals of this study were to present options for slow growth of sugarcane plantlets at either 18 or 24°C, with deployment dependent on the facilities available, and the cryostorage of in vitro-derived apical meristems, the latter using the recently published V-cryo-plate method of Rafique et al. (2015). This is the first published report of maintaining sugarcane plantlets in slow growth conditions at 18°C for 4 yr with subculturing, either annually or every 2 yr.
Materials and Methods Production of in vitro plants In vitro plants of cultivars NCo310, NCo376, and N41 were produced from apical meristems isolated from field-grown plants at the South African Sugarcane Research Institute (SASRI), Mount Edgecombe, KwaZulu Natal, South Africa, following the methods of
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Ramgareeb et al. (2010). Excised meristems were cultured on an initiation medium for 1 wk [MS salts and vitamins (Murashige and Skoog 1962), 0.1 mg L−1 benzyl aminopurine (BAP), 0.015 mg L−1 kinetin (KIN), 4 g L−1 activated charcoal (all from Sigma-Aldrich®, St Louis, MO), 20 g L−1 sucrose (table sugar), and 8 g L−1 agar (Plant Tissue Culture Agar, Lab M Ltd., Lancashire, UK), all added presterilization (autoclaved at 121°C for 20 min), at a pH 4.5 (adjusted with 1 M KOH or 1 M HCl)] in the dark at 28°C. This was followed by culturing on semi-solid shoot multiplication medium (same as initiation medium but with 1 mg L−1 methylene blue and without activated charcoal) in a growth room at 28°C with a 16 h photoperiod. After 4–6 wk, once shoot clusters had been established, these were subcultured every 2 wk in Magenta™ vessels (GA-7, Sigma-Aldrich®) containing 60–100 mL of liquid shoot multiplication medium (as above but pH 5.3). All cultures were maintained in a growth room at 28°C and 16 h photoperiod at a photon flux density of 160 μM m−2 s−1 (Biolux®, Osram Licht AG, Munich, Germany). Slow growth conditions For slow growth conditions, in vitro plants of cultivar NCo310 were maintained in Magenta™ vessels (five plants per vessel with 100 mL medium) at 18 and 24°C, the former in a Fitotron® growth chamber (Weiss Technik, Königswinter, Germany) and the latter in a separate growth room. In both cases, the photoperiod was 12 h and the light source and intensity was as described in the section above with subculturing every 12 mo. Semi-solid slow growth (SG) media 1–4 were comprised of MS salts and vitamins (Sigma-Aldrich®), 8 mg L−1 agar (Plant Tissue Culture Agar, Lab M Ltd.), and sucrose (table sugar) as follows: SG1, full-strength MS with 20 g L−1 (0.06 M) sucrose; SG2, ½ strength MS with 10 g L−1 (0.03 M) sucrose; SG3, fullstrength MS with 1 mg L−1 abscisic acid [(ABA) (SigmaAldrich®)] and 20 g L−1 sucrose; and SG4, ½ strength MS with 1 mg L−1 ABA and 10 g L−1 sucrose. Media were chosen on the basis of previous studies (Watt et al. 2009) and the requirement to be simple and cost-effective. The control was not stored and was cultured on shoot multiplication medium as described above. After 8, 12, 24, 36, and 48 mo, the number of green tillers per original shoot were counted directly after retrieval, and single shoots were removed and subcultured every 2 wk on shoot multiplication medium for 2 mo. Thereafter, leaf chlorophyll content [measured using a SoilPlant Analyses Development (SPAD) device (SPAD-502 Plus, Konica Minolta Inc., Sakai, Osaka, Japan)], shoot multiplication (number shoots produced per stored shoot), and shoot dry mass (post multiplication) were determined. Material was dried in a drying oven (IncoTherm Incubator, Labotec, South Africa) for 48 h at 70°C. Samples were cooled down in a desiccator for 15 min prior to weighing (balance PS750.R2, Radwag, Radom, Poland). The SPAD readings
Author's personal copy 404
BANASIAK AND SNYMAN
were taken from the fourth leaf of each plant with 10 readings per leaf (averaged) and on 5–10 randomly chosen plants per original multiplied shoot. Cryopreservation The V-cryo-plate procedure for cryopreservation of shoot meristems followed the method of Rafique et al. (2015) with some modifications. Apical meristems from ex vitro shoots (from germinated setts—a section of stalk with a bud and root primordia) of sugarcane cultivar NCo376 were excised and cultured for 1 wk on meristem initiation medium before trimming to 1.5–2 mm. Apical meristems of the same size were also taken from in vitro shoots, and both were placed on meristem initiation medium for 24 h followed by 24 h preconditioning on an osmoprotection medium [MS salts and vitamins (Sigma-Aldrich®), 0.5 M sucrose (table sugar), 8 g L−1 agar (Plant Tissue Culture Agar, Lab M Ltd.), pH 5.4 prior to autoclaving]. Meristems were then placed in the wells of an aluminum cryo-plate [made in-house at SASRI using the design number 2 of Yamamoto et al. (2011)], and encapsulated for 30 min in sodium alginate [2.2 μL of a 2% (w/v) solution (Sigma-Aldrich®)] polymerized with CaCl2 [100 mM (Sigma-Aldrich®)] in MS medium. The cryo-plate containing the meristems was placed in an Eppendorf microfuge tube (Merck, Darmstadt, Germany) with loading solution [2 mL; MS salts and vitamins (Sigma-Aldrich®), 2 M glycerol (Merck), and 0.8, 1.2, 1.6, and 1.8 M sucrose (table sugar) for 10, 20, 30, and 40 min at 24°C, then transferred to the plant vitrification solution number 2 (PVS2) [2 mL; 30% (v/v) glycerol (Merck), 15% (w/v) dimethyl sulfoxide (Sigma-Aldrich®), 15% (v/v) ethylene glycol (ACE Chemicals, Johannesburg, South Africa), and 0.4 M sucrose (table sugar)] for 30 min at 24°C. After this osmotic dehydration, the plate was placed in an uncapped cryogenic vial (Corning® Inc., Corning, NY) held on a cryo-cane and directly immersed in LN. Vials were capped and stored in a Dewar (Biocane 34, Thermo-Fisher Scientific®, Waltham, MA) for 24 h. After removal of the cryogenic vials from LN, the aluminum plate was quickly transferred to a tube with thawing solution [2 mL; MS salts and vitamins (Sigma-Aldrich®) and 1 M sucrose (table sugar)] for 30 min at 24°C. Meristems were removed from the alginate beads under aseptic conditions and placed on meristem initiation medium for 1 wk in the dark followed by subculture onto meristem initiation medium without activated charcoal and cultured under photoperiod conditions as described for the production of in vitro plants. Meristems were subcultured every 2 wk and their survival rates were determined after 7 wk. Cultivar N41 meristems were cryopreserved using the above protocol with 10-min exposure to two loading solutions containing 1.6 and 1.8 M sucrose. Statistical analyses For the slow growth studies, a randomized complete block design was used, with 4 ‘time blocks’
(12, 24, 36, and 48 mo) consisting of 24 Magenta™ vessels (6 replicates for each medium within each ‘time block’), from which 5 were removed for shoot recovery (and subsequent multiplication for 2 mo) every 12 mo for a period of 48 mo. There was one spare Magenta™ vessel per medium per time interval in case of microbial contamination. Statistical analysis was completed using (a) t test when comparing each treatment with a non-stored control and (b) analysis of variance (ANOVA) for balanced data and restricted maximum likelihood (REML) for unbalanced data to evaluate the effects of storage time and media, and the interaction between the medium and time. Some of the data were log10 transformed for analyses, but untransformed data are presented in the tables for ease of presentation. GenStat® version 18 (VSN International Ltd., Hemel Hempstead, UK) software was used for performing the analyses. Results were presented as mean ± standard error (SE) and significant differences were compared using a post hoc Fisher’s protected least significance difference (LSD) test at a 95% confidence level. For cryopreservation studies, each experiment was replicated three to four times and the number of meristems used was 7–32, with the final number depending on the level of microbial contamination. The following statistical analyses were used depending on the data (balanced or unbalanced) and type of comparison required: t test, ANOVA, and REML. Results were presented as mean ± SE and significant differences were compared using LSD at a 95% confidence level.
Results Storage at 18°C A number of green tillers were counted when the plants were removed from storage at 18°C at annual intervals over a 4-yr period. Plantlets stored for 36 and 48 mo tillered less profusely than those stored for 12 and 24 mo with the latter time interval resulting in significantly more tillers, regardless of the medium (Table 1). The highest number of tillers per original stored shoot was observed in the SG2 medium at 24, 36, and 48 mo. Plants stored on the SG3 medium had the least number of tillers, except at 24 mo (Table 1). On SG3 and SG4 media, some plants had curled leaves and shoots appeared vitrified, suggesting that ABA was not an ideal component for slow growth. Medium SG2 supported the highest tiller production, which although not favorable for slow growth purposes is advantageous when shoot multiplication is required post-retrieval for multiplication purposes. The chlorophyll content (SPAD units) of plants measured 2 mo after storage as an indication of general plant health showed that there were some significant differences between the control plants (non-stored) and those retrieved from
Author's personal copy SUGARCANE IN VITRO GERMPLASM CONSERVATION Table 1.
A comparison of NCo310 plantlets retrieved after 12, 24, 36, and 48 mo of medium-term storage at 18°C on four media
Storage time (mo)
Storage medium
0 non-stored control
–
12
SG1 SG2 SG3
24
36
48
405
Number of green tillers per stored shoot
Leaf chlorophyll content (SPAD units)
Number of shoots per stored shoot after 2 mo
Shoot dry mass (mg)
–
26.0 ± 0.5
126.0 ± 26.0
2.1 ± 0.2 fgh
24.1 ± 0.4*, defghi
89.0 ± 12.0
20.9 ± 1.7 fgh
2.2 ± 0.3 fgh 1.3 ± 0.2 cde
25.7 ± 0.7 defghi 24.5 ± 0.8*, cdefgh
120.0 ± 18.0 90.0 ± 8.0
19.6 ± 1.2 bfgh 21.8 ± 2.8 bfgh
SG4
2.8 ± 0.4 fgh
26.6 ± 0.6 ei
88.0 ± 15.0
SG1 SG2
2.3 ± 0.5 efgh 4.2 ± 0.6 i
27.1 ± 0.5*, i 26.2 ± 0.5 ehi
70.0 ± 13.0 183.0 ± 24.0
33.0 ± 2.7 bdefgh 6.0 ± 1.3*, bc
SG3 SG4
3.0 ± 0.5 fhi 2.9 ± 0.5 fgh
25.8 ± 0.7 deghi 22.6 ± 1.0*, abc
185.0 ± 25.0 147.0 ± 28.0
8.0 ± 1.0*, a 11.3 ± 3.3 bcdef
SG1
1.5 ± 0.3 cde
25.2 ± 0.6 defghi
87.0 ± 15.0
SG2 SG3
2.4 ± 0.5 defg 0.4 ± 0.1 a
23.6 ± 0.6*, bcdeg 24.3 ± 0.8*, cdefg
146.0 ± 16.0 124.0 ± 18.0
15.5 ± 1.1*, bcde 11.7 ± 1.4*, bc
SG4 SG1
0.9 ± 0.5 ab 0.7 ± 0.3 a
21.2 ± 0.7*, a 25.0 ± 0.6 cdefghi
122.0 ± 20.0 144.0 ± 37.0
14.1 ± 1.2 bcd 18.8 ± 1.0 befgh
SG2 SG3 SG4
2.6 ± 0.7 def 0.3 ± 0.1 a 1.1 ± 0.5 abc
21.8 ± 0.7*, ab 24.1 ± 3.1*, abcde 22.6 ± 1.6*, abcd
394.0 ± 84.0* 412.0 ± 186.0 361.0 ± 135.0
11.5 ± 2.5*, bc 10.5 ± 2.1*, ab 11.8 ± 2*, abc
22.7 ± 2
30.6 ± 3.6 i
25.2 ± 2.0 hi
The number of green tillers per original stored shoot was counted directly after removal from storage, while other measurements were taken on postretrieval shoots cultured on multiplication medium for 2 mo. Means ± SE (n = 3–32) followed by asterisk are significantly different from the non-stored control (t test; p < 0.05). Values followed by the same or no letters in each column are not significantly different (LSD; p < 0.05)
several of the treatments with lower SPAD readings from plants stored on a medium containing ABA (SG3 and SG4) at 36 and 48 mo (Table 1). When comparing plants derived from the experimental treatments, analyses showed significant effects of storage time, media, and time plus media interaction. There were no differences in chlorophyll content between plants stored for 12 mo regardless of the medium (Table 1). A comparison of shoot multiplication 2 mo post-retrieval showed no significant interaction between media and the length of time in storage (Table 1). The highest number of shoots was observed after retrieval and multiplication of plants from 48-mo storage regardless of the medium (Table 1). At that time point, a significant difference was only detected between the control (non-stored shoots) and medium SG2, although the mean number of shoots from that medium and SG3 and SG4 media were similar (394 ± 84, 412 ± 186, and 361 ± 138 shoots per retrieved shoot, respectively). There were no significant differences between control and SG3 and SG4 (Table 1; t test, p = 0.261, p = 0.150, respectively). This is likely due to the low number of replicates in SG3 and SG4 due to losses from microbial contamination, which caused high variability in the number of shoots from these two media, and consequently no significant differences from the control (Table 1). The dry mass of recovered shoots, measured as an indication of the ‘thickness’ of the multiplied shoots after 2 mo, was significantly lower in media SG2, SG3, and SG4 for storage
time intervals of 36 and 48 mo compared with the control nonstored shoots (Table 1). This result coincides with the multiplication rates of which significant differences were observed for media and time intervals when multiplication was higher than in the control (average shoot was thinner than the control) (Table 1). When comparing treatments, there was a significant effect of time and treatment on the dry mass of multiplied plants, but it was difficult to identify an optimum time and medium for further use. Plantlets stored for 12 mo regardless of the medium and on SG1 medium regardless of time had the greatest dry mass, but this was presumably because these were the plants that had a low multiplication rate, as seen in Table 1. Storage for a longer period than 24 mo without subculturing was possible. Due to space constraints in the growth chamber, the only medium evaluated in these experiments was SG2. From previous experiments, it was concluded that it was preferable not to use ABA-containing media due to abnormalities associated with leaves, and the SG2 medium supported significantly higher numbers of green tillers and shoots after storage for 48 mo compared with SG1 and the non-stored control (Table 1). When plantlets were subcultured every 24 mo, there was no difference in tillering whether stored for 24 or 48 mo (Table 2). However, at 24 mo, there was a significant difference in the green tiller number under the different subculturing intervals (2 × 12 versus 1 × 24 mo), with a significantly lower number at the longer time interval.
Author's personal copy 406 Table 2. A comparison of NCo310 plantlets retrieved after 24 and 48 mo of medium-term storage at 18°C on medium SG2 with subculture intervals of either 12 or 24 mo
BANASIAK AND SNYMAN
Storage time (mo)
Subculturing interval
Number of green tillers per stored shoot
Number of shoots per stored shoot after 2 mo
Shoot dry mass (mg)
0 non-stored control 24
–
–
126.0 ± 26.0 a
22.7 ± 2.0 a
48
12 mo
3.7 ± 0.3 a
183.0 ± 18.0 a
13.5 ± 1.3 b
24 mo
1.6 ± 0.3 b
54.0 ± 24.0 b
31.2 ± 18.1 c
12 mo 24 mo
2.1 ± 0.3 b 2.1 ± 0.6 b
394.0 ± 52.0 c 362.0 ± 84.0 c
11.5 ± 2.5 b 12.7 ± 1.0 b
The number of green tillers per original stored shoot was counted directly after removal from storage, while other measurements were taken on post-retrieval shoots cultured on multiplication medium for 2 mo. Means ± SE (n = 17–32) followed by the same letter are not significantly different (LSD; p < 0.05). For analyses, data were square root transformed for dry mass and log10 transformed for shoot number, but untransformed data are presented
At 48 mo, there was no difference between the subculturing regimes (4 × 12 versus 2 × 24 mo) on the green tiller number from retrieved shoots (Table 2). For those plants stored for a total of 48 mo with a single subculture after 24 mo, there were significant differences between the measured parameters (shoot number and dry mass after 2 mo of multiplication) when compared with the nonstored control, with a higher shoot multiplication and a concomitant lower dry mass per shoot at 48 mo (Table 2). At 24 mo, this trend was reversed (lower shoot number and higher associated shoot mass). The subculturing regime of every 12 or 24 mo did not have any effect on shoot multiplication and dry mass after 48 mo of storage. These results support the practice of maintaining cultures in slow growth conditions at 18°C for 48 mo with subculturing only every 24 mo. Slow growth at 24°C After 8 mo of storage at 24°C, visible leaf senescence was observed on all storage media (results not shown), and at 12 mo, the only medium that supported viable material was SG2. There were no significant differences in the number of green tillers on the 4 media at 6 and 8 mo, but all of these values were significantly higher than at 12 mo on SG2 medium (Table 3). When compared with the control, shoot numbers after 2 mo multiplication post-retrieval were significantly higher on SG2 medium after 8 and 12 mo, and significantly lower on SG3 medium stored for 6 and 8 mo (Table 3). The dry mass of shoots, except on SG3 for 8 mo, was significantly lower than the non-stored control (Table 3). The only medium that supported survival of plants for 12 mo at 24°C was SG2, on which the highest number of shoots was produced, although the shoots on SG2 were thinner than the control shoots (Table 3). Cryopreservation using the V-cryo-plate method The cryopreservation protocol followed was the V-cryo-plate procedure of Rafique et al. (2015). Although each step of the procedure was optimized, the following pre-treatments had no effect on the survival of meristems: osmoticum pre-treatment
(MS, 0.5 M sucrose, 8 g L−1 agar for 24 h), or encapsulation on aluminum-cryo-plates [2% (w/v) alginate in MS, 0.6 M sucrose, polymerization with 100 mM CaCl2 in MS] (results not shown). There was no significant effect of the sucrose concentration in the loading solution on survival of cryopreserved meristems from either in vitro- or ex vitro-derived explants (Table 4). For statistical evaluation of the in vitro-derived meristems, REML analysis was used because the data was unbalanced, and for the rest of the variables, ANOVA was used. The high variability between replicates per treatment likely influenced the statistical analysis. For example, the percentage survival of cryopreserved in vitro-derived meristems pre-treated with 1.6 M sucrose in loading solution was 65.6%, the mean of four values—83.3, 70.8, 79.2, and 29.2%. The survival of non-cryopreserved (control) and cryopreserved meristems initiated from germinated sett shoots was significantly lower than for those from in vitro shoots (for each sucrose concentration a t test was performed; Table 4). After treatment with loading solution containing 0.8 M sucrose, survival was 51.3% (control) and 13.9% (cryopreserved) of ex vitro explants versus 85.8 and 68.9% from in vitro-derived meristems, respectively. High and variable levels of microbial contamination (0–100%, with an average of 54.1 ± 11.7%; results not shown) hampered the optimization of the protocol for meristems excised from ex vitro-derived explants. Exposure to LN did not compromise the survival of meristems from either explant source at any of the sucrose concentrations with two exceptions at 1.2 M for ex vitro- and 1.8 M for in vitro-derived meristems (t test). Neither the exposure time (10–40 min) nor the sucrose concentration of the loading solution (1.2–1.8 M) had an effect on the survival of cryopreserved meristems derived from in vitro shoots (Table 5). The protocol was tested on a commercial cultivar N41, and the survival was not significantly different from NCo376 (Table 5; t test). For future work, it is likely that loading solution containing 1.8 M sucrose and 10min exposure time will be used.
Author's personal copy SUGARCANE IN VITRO GERMPLASM CONSERVATION Table 3. A comparison of NCo310 plantlets retrieved after medium-term storage at 24°C on media SG1–4 with subculture intervals of 6, 8, and 12 mo
407
Storage time (mo)
Storage medium
Number of green tillers per stored shoot
Number of shoots per stored shoot after 2 mo
Shoot dry mass (mg)
0 non-stored control 6
–
–
126.0 ± 26.0
22.7 ± 2.0
SG1
2.8 ± 0.4#
SG2
#
1.4 ± 0.2
SG3 SG4 SG1 SG2 SG3 SG4 SG1 SG2
2.2 ± 0.4# 2.7 ± 0.4# 2.6 ± 0.4# 2.4 ± 0.3 1.5 ± 0.3# 2.0 ± 0.4# Senesced 0.8 ± 0.1
SG3 SG4
Senesced Senesced
8
12
90.0 ± 16.0#, ab
14.0 ± 1.5*, a
85.0 ± 130#, ab 59.0 ± 10.0#, *, ab 113.0 ± 21.0#, ab 155.0 ± 22.0#, abc
10.6 ± 0.7#, *, a 12.7 ± 1.1*, a 12.9 ± 2.2*, a 16.1 ± 1.1*, a
239.0 ± 41.0*, c 48.0 ± 8.0#, *, a 176.0 ± 29.0#, bc
12.7 ± 1.6*, a 21.2 ± 1.9#, b 11.4 ± 1.0*, a
Senesced 314.0 ± 40.0*
Senesced 13.2 ± 0.8*
Senesced Senesced
Senesced Senesced
The number of green tillers per original stored shoot was counted directly after removal from storage, while other measurements were taken on post-retrieval shoots cultured on multiplication medium for 2 mo. Means ± SE (n = 17–32) followed by the same letter or none are not significantly different (LSD; p < 0.05). Means in a column followed by asterisk are significantly different from the control (not stored) (t test; p < 0.05). Means in a column followed by pound sign are significantly different from SG2 medium at 12 mo (t test; p < 0.05)
Discussion Slow growth options This report of low-temperature in vitro storage of sugarcane plantlets at 18°C with subculturing every 12 mo on a minimal medium, SG2, comprising ½ MS with 10 g L−1 sucrose without the addition of plant growth regulators (Tables 1 and 2), is by no means the first. Taylor and Dukic (1993) described storage of multiple cultivars for 12 mo. This study included novel aspects such as storage for 48 mo with subculturing either every 12 or 24 mo, suggesting that improved efficiencies with respect to resources and time can be made to storage protocols. Table 4. The effect of sucrose concentration in the loading solution on survival of cryopreserved NCo376 meristems that were excised from either ex vitro germinated setts or in vitro plantlets
Sucrose concentration in loading solution (M)
The addition of ABA to media SG3 and SG4 did not provide any advantage impacting tiller number, number of shoots multiplied, or size (dry mass) (Tables 1 and 2). Similar observations were made by Bello-Bello et al. (2014) and Nogueira et al. (2015), in which ABA incorporation in the medium resulted in a decrease in the percentage survival of stored sugarcane plantlets in slow growth conditions compared with other media modifications such as the addition of sorbitol. Reports on photosynthetic characterization of stored plants are limited, and although SPAD readings provide a rapid measurement of chlorophyll content and offer a potential method to assess plant health after storage (Capuana and Di Lonardo
Survival (%) of meristems excised from two different explants Ex vitro setts
0.8 1.2 1.6 1.8
In vitro plantlets
−LN
+LN
−LN
+LN
51.3 ± 15.6 54.8 ± 14.2 31.4 ± 16.5 39.2 ± 8.1
13.9 ± 8.3 16.4 ± 5.9# 11.1 ± 5.5 17.1 ± 8.6
85.8 ± 4.7 66.3 ± 7.0 67.8 ± 19.0 88.5 ± 3.9*
68.9 ± 7.2* 77.5 ± 5.6* 65.6 ± 12.4* 73.5 ± 4.6*#
Control non-cryopreserved meristems were treated in the same way without exposure to LN. Results were recorded after 7 wk of culturing on multiplication medium. Means ± standard error (SE) of 3–4 replicated experiments with 7–12 meristems per replicate (REML). Values followed by asterisk are significantly different between meristems obtained from ex vitro setts and in vitro plants at each sucrose treatment (t test; p < 0.05). Values followed by pound sign are significantly different between meristems exposed to LN and those not exposed to LN (t test; p < 0.05) LN liquid nitrogen
Author's personal copy 408 Table 5. The effect of exposure time to loading solution with 1.2– 1.8 M sucrose concentration on survival of cryopreserved NCo376 in vitro-derived meristems
BANASIAK AND SNYMAN
Exposure time (min)
Sucrose concentration in loading solution (M) 1.2
1.6
1.8
−LN
+LN
−LN
+LN
−LN
+LN
10 20
39.4 ± 19.0 63.3 ± 13.3
51.3 ± 2.8 41.7 ± 4.8
88.1 ± 8.1 91.7 ± 4.2
69.4 ± 10.0 68.1 ± 13.2
87.8 ± 5.1 93.5 ± 3.0
68.0 ± 11.0 65.5 ± 11.2
30 40
56.1 ± 8.4 34.3 ± 4.9
41.7 ± 8.3 53.8 ± 14.6
80.0 ± 8.1 83.7 ± 2.7
51.7 ± 14.4 61.4 ± 7.3
90.6 ± 2.6 81.1 ± 6.9
62.5 ± 7.1 59.9 ± 3.5
Cultivar N41 (10 min)
NT
NT
76.1 ± 3.9
46.4 ± 1.9
61.6 ± 15.5
67.3 ± 8.6
Means ± SE of three replicates (n = 8–32 meristems) showed no significant differences between treatments (ANOVA). Results were recorded after 7 wk of culturing NT not tested, LN liquid nitrogen
2013), the present results did not identify a medium or storage time that was any better than the rest (Table 1). The quality of the plants as assessed by the number green tillers counted directly after retrieval from the storage medium and the multiplication of retrieved shoots post-storage (Tables 1 and 2) suggested that these more standard measurements are sufficient to determine the success of a protocol. Room temperature storage at 24°C is also an option for germplasm conservation for those subtropical plants where cold storage is unsuccessful, or if low-temperature facilities are not available. The only medium that supported the survival of stored sugarcane shoots for 12 mo at 24°C was SG2, and although these shoots were significantly smaller than the control, they multiplied well poststorage (Table 3). The current study confirmed 8 mo as the optimal time point for sugarcane storage at 24°C (Watt et al. 2009), but this could be extended to 12 mo if SG2 medium is used. Other subtropical crops such as banana and cocoa can be stored in vitro at this temperature for 1 and 2 yr, respectively (Cha-um and Kirdmanee 2007). For medium-term storage of sugarcane, both storage temperatures of 18 and 24°C are suitable. The choice would be dependent on storage facility availability. The SG2 medium resulted in high multiplication post-storage and this was associated with thinner shoots (Tables 1 and 3). However, this result could be overcome by prolonging the post-retrieval culturing on medium for an additional 2 wk without the addition of plant growth regulators (Jalaja et al. 2008). These observations suggest that this step may be necessary to halt multiplication and allow shoots to ‘thicken up’ prior to rooting. Testing a cryostorage protocol Longer-term storage options using published cryopreservation protocols were tested in the present study to complete the assessment of available methods. In vitro-derived meristems were a preferable source of material compared with those from an ex vitro source due to the low survival rates of the latter (Table 4). This result could
be attributed to in vitro material responding better to penetration of pre-cryostorage solutions to cells due to both less waxy and under-developed cuticles (George et al. 2008). This, in combination with lower microbial contamination associated with in vitro-maintained material, makes these explants preferable for a routinely applied laboratory protocol. Survival of meristems after cryostorage was 60–77%, and although only two cultivars were tested, there was no apparent genotype effect (Tables 4 and 5). This result was similar to the study of Rafique et al. (2015), who recorded survival of 57–80% in 9 of the 11 cultivars tested using the V-cryo-plate. An important aspect of field evaluation with respect to agronomic traits from plants derived from storage has not been reported in this manuscript, although those studies are underway. In a study in which stalk characteristics and yield were assessed in plants derived from cryopreserved apices versus those not stored, no differences were recorded (González-Arnao et al. 1999), reinforcing the findings of others in which similar studies with tissue cultured plants were compared with conventional vegetative material (Lourens and Martin 1987; Taylor and Dukic 1993; Lorenzo et al. 2001; Watt et al. 2009).
Conclusions It is advantageous to have several options for germplasm storage as the need to access material varies. Therefore, the multiple storage options identified in this study allow for flexible options including (a) whole plantlet medium-term storage for 12 mo on SG2 medium at 24°C that does not require any specialized facilities; (b) whole plantlet medium-term storage at 18°C for 48 mo with a single subculture at 24 mo on the same medium, SG2; and (c) cryostorage of in vitro-derived shoot apices with survival and recovery rates of 60–68% using the V-cryo-plate method.
Author's personal copy SUGARCANE IN VITRO GERMPLASM CONSERVATION Acknowledgements Funding from SASRI and the National Research Foundation of South Africa (grant 85414) is gratefully acknowledged. Thanks to Ms. C Sewpersad (Biometrician, SASRI) for the assistance with statistical analyses.
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