Journal of Applied Botany and Food Quality 92, 94 - 99 (2019), DOI:10.5073/JABFQ.2019.092.013 1Laboratory for Plant Breeding and Conservation of Genetic Resources, Centro de Bioplantas, Universidad de Ciego de Ávila, Ciego de Ávila, Cuba 2Escuela Superior Politécnica Agropecuaria de Manabí Manuel Félix López (ESPAMMFL), Campus Politécnico El Limón, Carrera de Ingeniería Agrícola, Calceta, Manabí, Ecuador 3School of Life Sciences, University of Kwazulu-Natal, Durban, South Africa Cryopreservation of sorghum seeds modifies germination and seedling growth but not field performance of adult plants Ariel Villalobos1, Melissa Arguedas1, Doris Escalante1, Julia Martínez1, Byron E. Zevallos2, Inaudis Cejas1, Lourdes Yabor1, Marcos Edel Martínez-Montero1, Sershen3, José Carlos Lorenzo1 (Submitted: November 22, 2018; Accepted: February 7, 2019) Summary Climate change poses risks to both wild and crop plant biodiversity, which can be mitigated by cryopreservation (usually at -196 °C in liquid nitrogen [LN]) of crop germplasm. Cryopreservation is widely regarded as a reliable method for the ex situ conservation of plant genetic resources but its effects on subsequent field performance of popular crop species such as sorghum are largely unknown. This hampers the large-scale implementation (i.e. germplasm banks) of cryostorage for such species. This short communication describes the early stages of germination and field performance of plants derived from cryopreserved sorghum seed. Compared with the control, cryopreservation significantly increased seed electrolyte leakage and from 24 to 120 hours, percentage of germination of the control was ~2.6 folds higher than cryopreserved seeds. At 0 days, chlorophyll a/b rate was ~1.7 folds higher in the control and at 7 and 14 days, chlorophyll a level (~1.5 folds) and chlorophyll a/b rate (~1.8-1.9 folds) were higher in the control. Contrastingly, at 7 days, seedlings derived from cryopreserved seeds (treatment seed- lings) showed ~1.5 folds more superoxide dismutase activity and ~1.9 folds more peroxidase activity. In contrast, treatment and con- trol adult plants were statistically comparable in terms of chloro- phylls, proteins, superoxide and peroxidase activities, plant architec- ture, and yield components. The fact that differences in biochemical indicators observed between control and treatment seedlings did not persist in adult plants validates the use of seed cryopreservation for the conservation of sorghum genetic resources. Keywords: field performance; germplasm preservation; liquid nitrogen; Sorghum bicolor (L.) Moench. Introduction Sorghum, an African grass related to sugarcane and maize, is the fifth most important cereal crop globally and is known to tolerate low- nutrient soils and drought. Cultivated varieties are phenotypically diverse (Kayodé et al., 2006; Labeyrie et al., 2016) and are grown for food, feed, fiber and fuel due to the high soluble sugar content of their stems (breeze, 2018; ManzeLLi et al., 2006; Mathur et al., 2017; Paterson et al., 2009). In 2016, the world area harvested for sorghum reached 44 771 056 ha and the world production, 63 930 558 t (Food and Agriculture Organization of the United Nations Statistics (Faostat, 2017)). Access to sorghum seed is crucial for farming and shortfalls are common in many countries. Farmer-farmer exchange is important for providing locally-adapted seed to fill this gap but access varies considerably among households, affecting quantities supplied and terms of exchange (McGuire, 2008; otieno et al., 2018; sMaLe et al., 2018). In the context of climate change with risks to lose diversity, cryopreservation of plant materials in liquid nitrogen (LN) has been described as a suitable technology to conserve genetic re- sources of several species (Panis, 2018; berjaK et al., 2010), such as Actinidia deliciosa (Mathew et al., 2018), Solanum betaceum Cav. (Graça et al., 2018), Elaeis guineensis Jacq. (beuLé et al., 2018) and Lilium (Pan et al., 2018). However, the potential effects of cryostsorage of explants and seeds on subsequent plant growth in the field must be established before large-scale implementation in cryobanks (enGeLMann and raManatha, 2012). In this regard, studies have shown that cryostorage of seed-derived germplasm can compromise the vigor of recovered plants (berjaK et al., 2010). Studies have also shown that recovery after cryopreservation of excised embryonic axes also seldom results in the production of true- to-type plants (hardinG, 2004; MycocK, 1999; steinMacher et al., 2007; wesLey-sMith et al., 2001). It has been known for some time now that exposure to different types of stress can alter subsequent plant responses (bruce et al., 2007), but there is little understanding of how stresses imposed at the embryonic stage for example, are translated or manifested during subsequent plant growth. A few reports suggest that there exists with- in cryopreserved plant materials some ‘memory’-based mechanism that senses environmental signals (Forsyth and staden, 1983; KvaaLen and johnsen, 2008), which in turn influence adaptive traits in the seedlings they give rise to. With this background, this short communication describes the early stages of germination, seedling growth and field performance of plants produced from cryopreserved sorghum seed. Materials and methods Plant material Harvested sorghum seeds (cv. CIAP2) were air dried at room temperature from 15% to 6% moisture content and then stored for 4 months at 4 °C in the dark in hermetically sealed containers. Seeds with 6% moisture content (fresh weight basis) (ista, 2005) were used in subsequent experiments. Seed cryopreservation and recovery One batch of the seeds was placed in cryo-vials (volume: 2 ml; 5 seeds per cryo-vial) and directly plunged in LN for 24 h (referred to hereafter as treated/cryopreserved seeds). Recovery of seeds from LN was performed according to Stanwood and Bass (1981). Another batch of seeds was stored at 4 °C until further use (referred to hereafter as control seeds). Cryopreservation of sorghum seeds 95 Seed electrolyte leakage, germination and seedling growth from 0 to 144 hours Electrolyte leakage from seeds was measured as recommended by Martínez-Montero (2002) immediately after seed recovery from LN. Percentage of seed germination (0-144 hours) and seedling weight (0-72 hours) were also evaluated by incubating seeds on filter papers in Petri dishes (Ø: 10 cm; 15 mL distilled water; five replicates of 10 seeds/dish). These parameters were also measured for control seeds. Studies of seed and seedlings from 0 to 14 days A second group of control and treated seeds was set out to germinate as described above (five replicates of 10 seeds/dish) and evaluated at 0, 7 and 14 days for chlorophyll pigments (Porra, 2002), total proteins (bradFord, 1976), and activities of superoxide dismutase (KuMutha et al., 2009) and peroxide oxidase (saGiv and bar- aKiva, 1972) were evaluated in the seeds (0 day) or the primary leaves (7 and 14 days). Hypocotyl length, primary leaf length, radicle length, total plantlet fresh weight, and total plantlet dry weight were also recorded. Growth of adult plants in a plant bed until harvest at 110 days Ninety treated and control seeds were randomly selected and sown in a plant bed as described in Fig. 1. Technical instructions pro- vided by the Cuban Ministry for Agriculture to cultivate sorghum were applied. Border plants, which had more space to grow, were not considered. Levels of chlorophyll pigments (Porra, 2002), total proteins (bradFord, 1976), and activities of superoxide dismutase (KuMutha et al., 2009) and peroxide oxidase (saGiv and bar- aKiva, 1972) were evaluated in middle-aged leaves at 62 days after planting on soil (anthesis). Additionally, the following agricultural traits were evaluated according to Peacock (1990) at 62 days of growth: plant height, number of leaves per plant, middle-aged leaf length and width, number of stems per plant, and fresh and dry weight of plants. All plants were harvested after 110 days of growth and the following traits were recorded: panicula length and width, number of branches per panicula, number of grain per panicula branch, number of grains per panicula, fresh weight of 1000 grains and dry weight of 1000 grains. Statistical analysis SPSS (Version 17.0 for Windows, SPSS Inc.) was used to compare growth and physiological parameters between control and treated plants/seeds using a Students t-test (p≤0.05). Results Seed electrolyte leakage, germination and seedling growth from 0 to 144 hours Compared with the control, cryopreservation significantly increased electrolyte leakage from seeds (~2 folds: 19.1% / 9.4%, Fig. 2A) im- mediately after recovery from LN. Although percentage of germina- tion was similar at 144 hours, from 24 to 120 hours, percentage of germination of the control was ~2.6 folds higher (average, Fig. 2B). Seedling weight of control group was also higher (average ~1.2 folds, Fig. 2C). Studies of seed and seedlings from 0 to 14 days Tab. 1 shows the effects of cryopreservation on early stages of germination (0, 7, 14 days). Except for chlorophyll a + b on 0 day, statistically significant differences were observed on all sampling Fig. 1: Superior view of the useful area of the plant bed, made of Ferralytic- red soil and filter-cake-sugarcane ashes (1:1, v:v). Dots symbolize seeds (70 cm × 25 cm apart). The broken arrow in the middle of the plant bed represents the microject irrigation system, which watered the plants for 5 min every 8 h. The plant bed was 50 cm high and its bottom contained a 10 cm-high stones layer to facilitate drainage. days (Tab. 1): At 0 day, chlorophyll a/b rate was ~1.7 folds higher in the control treatment (1.13 / 0.65); at 7 days the level of chlorophyll a was ~1.5 folds higher in the control (21.37 μg g-1 fresh weight / 14.02 μg g-1 fresh weight); and chlorophyll a/b rate was ~1.9 folds higher in the control (1.31 / 0.71). Contrastingly, at 7 days, plantlets derived from cryopreserved seeds showed ~1.5 folds (0.42 U mg-1 proteins / 0.27 U mg-1 proteins) more specific superoxide dismutase activity and ~1.9 folds (0.71 U mg-1 proteins / 0.37 U mg-1 proteins) more specific peroxidase activity. At 14 days, plantlet chlorophyll a levels was ~1.5 folds higher in the control (25.31 μg g-1 fresh weight / 17.13 μg g-1 fresh weight) and chlorophyll a/b rate was ~1.8 folds (1.38 / 0.75) higher in this group (Tab. 1). Contrastingly, the specific peroxidase activity was ~1.7 folds higher in cryopreserved seed-derived seedlings (0.60 U mg-1 proteins / 0.36 U mg-1 proteins). 96 A. Villalobos, M. Arguedas, D. Escalante, J. Martínez, B.E. Zevallos, I. Cejas, L. Yabor, M.E. Martínez-Montero, Sershen, J.C. Lorenzo Growth of adult plants in a plant bed until harvest at 110 days The data shown in Tab. 2 indicates that seed cryopreservation has no significant effects on adult plants relative to the control. Interes- tingly, ex vitro, 100% of seed germination was recorded in both treat- ments. Differences in biochemical indicators between treatment and control seedlings reported above (Tab. 1) did not persist in adult plants (Tab. 2), e.g. levels of chlorophylls and proteins, and superoxide dis- mutase and peroxide oxidase activities. Discussion Cryopreservation imposes a series of stresses on plant material both during storage and upon recovery, and this can induce modifications in plants produced from cryopreserved explants/seeds (benson, 2008a; benson, 2008b). For example, partial dehydration and cryo- preservation reduced the number of Amaryllis belladonna excised zygotic embryos that produced seedlings, as well as the subsequent biomass of these seedlings relative to non-cryopreserved embryos (berjaK et al., 2010). Those authors showed that A. belladonna seedling produced from cryopreserved explants also exhibited lower CO2-assimilation rates and stomatal density, abnormal root growth, damage to the photosynthetic apparatus and less efficient adjustment of leaf water potential relative to control seedlings. Other studies have also reported phenotypic variation in in vitro recovery times, plant heights and modes of development (steinMacher et al., 2007) and regeneration in plants recovered from cryopreserved germplasm (hardinG, 2010). Similarly, our results for sorghum suggest that cryopreservation increased electrolyte leakage from seeds (Fig. 1A), and delayed germination (Fig. 1B) and seedling growth (Fig. 1C). Cell membranes are one of the main targets of numerous stress events, including cryopreservation (berjaK et al., 2010; cLose, 1996, 1997; hardinG, 2010; sanGwan et al., 2002; ueMura and stePonKus, 1994). Previously, our group has studied the effects of LN on the subsequent germination and growth of common bean, tomato, tobacco, maize and Teramnus labialis (L.F.) Spreng seeds. In brief, these studies showed that cryopreservation induced some morphological, physio- logical and biochemical (e.g. chlorophyll, carotenoids, proteins, malondialdehyde, other aldehydes, soluble and cell wall-linked phe- nolics, and peroxidase and superoxide dismutase activity) changes B C A Fig. 2: Effect of seed cryopreservation on seed electrolyte leakage (A), germination (B) and seedling growth (C) from 0 to 144 hours. In each moment of eva- luation, results with the same letters are not statistically different (t-test, p>0,05). Vertical bars represent SE. Cryopreservation of sorghum seeds 97 that gradually disappeared as the plants grew (acosta et al., 2018; arGuedas et al., 2018a; arGuedas et al., 2018b; cejas et al., 2012; Pérez-rodríGuez et al., 2017; zevaLLos et al., 2014). This supports our present findings for sorghum where cryopreservation-induced declines in seedling chlorophyll a levels and consequently chloro- phyll a/b rate, and enhanced superoxide dismutase and peroxidase activities did not persist in adult plants. Various markers, including electrolyte efflux, peroxide and superoxide oxidations, reflect the structural and functional integrity status of cell membranes after ex- posure to such stressful events (duMet and benson, 2000). Various abiotic stresses decrease the chlorophyll content in plants (ahMad et al., 2012) and this decline is believed to be due to inhibition of important enzymes, such as δ-aminolevulinic acid dehydratase and protochlorophyllide reductase associated with chlorophyll biosyn- thesis (van assche and cLijsters, 1990). However, a major finding of the present study is that cryopreservation did not appear to affect the phenotype of adult sorghum plants in terms of growth and per- formance significantly. This report therefore demonstrates the value of seed cryopreservation to conserve sorghum genetic resources, although in large-scale field experiments, the reduced germination speed, seedling weight and the increase of enzyme activities may result in inhomogeneous field establishment and lower yield. Author contribution: AV, MA, DE, JM, BEZ, IC, LY, MEMM, S and JCL designed the research; AV, MA, DE and JM conducted the experiment; AV, BEZ, IC, LY, MEMM, S and JCL analyzed data; S and JCL wrote the paper; JCL had primary responsibility for the final content. All authors have read and approved the final manuscript. Acknowledgements This research was supported by the University of Ciego de Avila (Cuba), the Escuela Superior Politécnica Agropecuaria de Manabí Manuel Félix López (Ecuador), and the University of KwaZulu-Natal (South Africa). 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In each moment of evaluation (0, 7, 14 days after exposure to LN), results with the same letter are not statistically different (t-test, p>0.05). Intervals represent average ± SE. 0 days 7 days 14 days (seeds were evaluated) (primary leaves were evaluated) (primary leaves were evaluated) Non- Cryopreserved Non- Cryopreserved Non- Cryopreserved cryopreserved seeds cryopreserved seeds cryopreserved seeds seeds seeds seeds Chlorophyll a (μg · g-1 fresh weight) 16.85 ± 0.39 a 12.37 ± 0.27 b 21.37 ± 0.28 a 14.02 ± 0.18 b 25.31 ± 0.25 a 17.13 ± 0.30 b Chlorophyll b (μg · g-1 fresh weight) 14.97 ± 0.30 b 19.17 ± 0.40 a 16.39 ± 0.31 b 19.95 ± 0.47 a 18.36 ± 0.36 b 22.93 ± 0.57 a Chlorophyll a + b (μg · g-1 fresh weight) 31.82 ± 0.49 a 31.53 ± 0.43 a 37.76 ± 0.34 a 33.97 ± 0.58 b 43.66 ± 0.40 a 40.06 ± 0.56 b Chlorophyll a/b 1.13 ± 0.03 a 0.65 ± 0.02 b 1.31 ± 0.04 a 0.71 ± 0.02 b 1.38 ± 0.03 a 0.75 ± 0.03 b Protein content 5.06 ± 0.16 a 4.13 ± 0.17 b 6.76 ± 0.22 a 5.12 ± 0.15 b 7.51 ± 0.17 a 6.23 ± 0.16 b (mg proteins · g-1 fresh weight) Superoxide dismutase activity 1.70 ± 0.04 b 1.88 ± 0.02 a 1.83 ± 0.02 b 2.12 ± 0.02 a 1.88 ± 0.00 b 2.15 ± 0.01 a (mg · g-1 fresh weight) Specific superoxide dismutase activity 0.34 ± 0.02 b 0.46 ± 0.02 a 0.27 ± 0.01 b 0.42 ± 0.01 a 0.25 ± 0.01 b 0.35 ± 0.01 a (U mg-1 proteins) Peroxidase activity 2.43 ± 0.08 b 2.79 ± 0.07 a 2.49 ± 0.07 b 3.60 ± 0.05 a 2.71 ± 0.06 b 3.71 ± 0.03 a (mg · g-1 fresh weight) Specific peroxidase activity 0.48 ± 0.01 b 0.68 ± 0.02 a 0.37 ± 0.01 b 0.71 ± 0.02 a 0.36 ± 0.01 b 0.60 ± 0.01 a (U mg-1 proteins) Hypocotyl length (cm) --- --- 2.79 ± 0.01 a 2.46 ± 0.04 b 6.93 ± 0.04 a 6.44 ± 0.04 b Primary leaf length (cm) --- --- 2.91 ± 0.02 a 2.71 ± 0.03 b 7.26 ± 0.06 a 6.69 ± 0.03 b Radicle length (cm) --- --- 4.98 ± 0.06 a 4.61 ± 0.12 b 8.00 ± 0.10 b 8.19 ± 0.13 a Total plantlet fresh weight (mg) --- --- 0.24 ± 0.00 a 0.20 ± 0.00 b 0.51 ± 0.00 a 0.41 ± 0.00 b Total plantlet dry weight (mg) --- --- 0.08 ± 0.00 a 0.07 ± 0.00 b 0.17 ± 0.00 a 0.13 ± 0.00 b 98 A. 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Chlorophyll a (μg · g-1 fresh weight) 85.17 ± 0.37 85.63 ± 0.44 Chlorophyll b (μg · g-1 fresh weight) 56.10 ± 0.31 55.63 ± 0.44 Chlorophyll a + b (μg · g-1 fresh weight) 141.27 ± 0.41 141.26 ± 0.88 Chlorophyll a/b 1.52 ± 0.01 1.54 ± 0.00 Protein content (mg proteins · g-1 fresh weight) 8.97 ± 0.03 9.05 ± 0.03 Superoxide dismutase activity (mg · g-1 fresh weight) 3.36 ± 0.07 3.39 ± 0.07 Specific superoxide dismutase activity (U mg-1 proteins) 0.38 ± 0.01 0.37 ± 0.01 Peroxidase activity (mg · g-1 fresh weight) 15.03 ± 0.19 15.11 ± 0.18 Specific peroxidase activity (U mg-1 proteins) 1.68 ± 0.02 1.67 ± 0.02 Agricultural traits evaluated at 62 days after planting on soil (anthesis). Plant height (cm) 168.75 ± 0.53 169.15 ± 0.49 Number of leaves per plant 10.38 ± 0.15 10.25 ± 0.15 Middle-aged leaf length (cm) 97.99 ± 0.24 97.79 ± 0.18 Middle-aged leaf width (cm) 7.72 ± 0.03 7.70 ± 0.01 Number of stems per plant 1.00 ± 0.00 1.00 ± 0.00 Stem diameter (cm) 5.89 ± 0.02 5.88 ± 0.02 Fresh weight of plants (g) 1.10 ± 0.02 1.09 ± 0.03 Dry weight of plants (g) 0.27 ± 0.01 0.28 ± 0.01 Agricultural traits evaluated at at 110 days after planting on soil (harvest). Panicula length (cm) 17.91 ± 0.03 17.88 ± 0.02 Panicula width (cm) 6.07 ± 0.02 6.06 ± 0.01 Number of branches per panicula 29.80 ± 0.15 29.83 ± 0.09 Number of grains per panicula branch 37.43 ± 0.77 37.93 ± 0.67 Number of grains per panicula 1119.15 ± 27.93 1131.58 ± 20.96 Fresh weight of 1000 grains (g) 27.98 ± 0.70 28.29 ± 0.52 Dry weight of 1000 grains (g) 5.13 ± 0.07 5.15 ± 0.07 Cryopreservation of sorghum seeds 99 Meena, r., 2009: Waterlogging induced oxidative stress and antioxidant activity in pigeonpea genotypes. Biologia Plantarum 53, 75-84. DOI: 10.1007/s10535-009-0011-5 KvaaLen, h., johnsen, Ø., 2008: Timing of bud set in Picea abies is regu- lated by a memory of temperature during zygotic and somatic em- bryogenesis. New Phytol. 177, 49-59. 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Zevallos, Escuela Superior Politécnica Agropecuaria de Manabí Manuel Félix López (ESPAMMFL), Campus Politécnico El Limón, Carrera de Ingeniería Agrícola, Calceta, Manabí, Ecuador Sershen, School of Life Sciences, University of Kwazulu-Natal, Durban, 4001, South Africa © The Author(s) 2019. This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creative- commons.org/licenses/by/4.0/deed.en).