Revista Mexicana Ciencias Agrícolas volume 10 number 7 September 28 - November 11, 2019
DOI: https://doi.org/10.29312/remexca.v10i7.1627
Article
Salicylic acid induces tolerance to cryogenic stress in Solanum tuberosum
Diana Daniela Ayala-Hernández1
Diana Rocío Ruiz-Saénz1
Esmeralda Judith Cruz-Gutiérrez2
Humberto Antonio López-Delgado1§
1National Institute of Agricultural and Livestock Forestry Research-SEDAGRO Joint. Metepec, State of Mexico. CP. 52140. Tel. 722 2329833. (ayaladany84@hotmail.com; druizsaenz@hotmail.com; lopez.humberto@inifap.gob.mx). 2National Center for Genetic Resources-INIFAP. Tepatitlán de Morelos, Jalisco, Mexico. CP. 47600. Tel. 378 1065020, ext. 5102. (cruz.esmeralda@inifap.gob.mx).
§Corresponding author: lopez.humberto@inifap.gob.mx.
Abstract
A common problem in cryogenic processes is survival at ultra low temperature (-196 °C). Based on the physiological effects of salicylic acid (AS) on the signaling of stress tolerance responses, the objective was to evaluate the effect of AS on increasing survival and on the growth of potato buds undergoing cryogenics. Microplants of the advanced clone 06-27 of the Germplasm Bank in vitro of the Potato Program in Metepec State of Mexico, Mexico, were incubated for 28 days in AS (0, 10-6 and 10-5 M) and subsequently subjected to cryogenic process. Effect of AS in plant regeneration: a significant increase in survival was observed in plants pretreated with AS 10-5 and 10-6 M with respect to the control (1.66-2.04 times respectively). Fresh weight, stem length and root significantly increased in microplants pretreated with AS with respect to the control. Cryogenesis: both concentrations of 10-5 and 10-6 M AS induced significantly greater survival (2.17-3.21 times respectively) of explants to cryogenics. Plants preincubated in AS and regenerated after being subjected to cryogenics for 1 h significantly increased root length. The control showed callus formation, absent in the plants treated with AS. The AS-cryogenic combination favored the development of plants subjected to methods that employ ultra-low temperatures in potatoes for the purpose of germplasm cryopreservation or cryotherapy to obtain virus-free materials.
Keywords: in vitro regeneration, potato, ultra low temperature.
Reception date: June 2019
Acceptance date: October 2019
Introduction
An important factor for food security is the adequate conservation of available genetic resources and the preservation of plant diversity. The conservation of genetic resources can be done by short or long term strategies. One of these long-term strategies is the use of cryogenic procedures, which involve the storage of plant materials such as corn (Zea mays) beans, yam (Sphenostylis stenocarpa), cowpea (Vigna unguiculata), peanut bambara (Vigna subterranea), soybean (Glicine max), oil palm (Elaeis guineensis), cocoa (Theobroma cacao), coconut (Cocos nucifera), avocado (Persea americana), mango (Mangifera indica) and coffee (Coffea spp.) among other species (Sánchez-Chiang and Jiménez, 2010).
This storage is done at ultra low temperatures (-196 °C) using liquid nitrogen (NL) (Day et al., 2008; Hamilton et al., 2009). At this temperature all cellular functions are suspended and therefore the tissue can be stored without changes or deterioration for long periods (Kaczmarczyk et al., 2012). In Solanum tuberosum, various cryogenic methods have been successfully tested, such as encapsulation-dehydration, vitrification and D-cryoplaque (Hao et al., 2002; Halmagyi et al., 2005; Hirai, 2011; Yamamoto et al., 2015). Wang et al. (2006), proposed the use of cryogenic methods for the long-term conservation of potato accessions and for the elimination of viruses.
However, the physiological response to cryogenics will depend on the genotype, with the optimization of methods to increase survival (Rivera et al., 2008) being necessary. During cryogenics cell tissues are susceptible to various types of stress such as mechanical, osmotic and temperature, which lead to oxidative stress. (Baek and Skinner, 2012). Exposures to extreme temperatures such as cold (0-20 °C) or freezing (<0 °C) result in decreased survival (Chinnusamy et al., 2007), producing oxidative damage (Horvath et al., 2007).
The formation of reactive oxygen species (EROS) in cryogenics can occur during several of the steps involved, (Kaczmarczyk et al., 2012). It has been shown that the addition of exogenous antioxidants such as ascorbic acid, vitamin E and glutathione in blackberry and citrus fruits during cryopreservation, resulted in greater survival (Wang and Deng, 2004; Uchendu et al., 2010). Plants have enzymatic and non-enzymatic antioxidant systems to resist stress, enzymatic such as catalase (CAT), peroxidase (POX), super oxide dismutase (SOD) and non-enzymatic such as ascorbic acid, glutathione and α tocopherol (Gill and Tuteja, 2010).
It has been reported that salicylic acid participates in signaling antioxidant activity in potatoes under extreme temperatures (Mora-Herrera et al., 2005; Aguilar-Camacho et al., 2016). Salicylic acid (AS) induces an increase in antioxidant enzymes during periods of pre-acclimatization at low temperatures in treated plants (Sasheva et al., 2010; López-Delgado et al., 2018). Mora-Herrera and López-Delgado (2006), found that AS induces freezing temperature tolerance (-6 °C) in potatoes. AS has also been tested in cryogenics in various species, inducing tolerance to freeze stress (Wang and Valkonen, 2009b; Li et al., 2011; Pathirana et al., 2016).
In embryonic axes of Persian lilac (Melia azedarach L.) they reported that the AS significantly increased the viability percentages after being subjected to cryopreservation (Bernard et al., 2002). The objective of this work was to test the potential effect of AS in increasing survival and growth responses in potato buds undergoing cryogenic process.
Materials and methods
Solanum tuberosum L. plants of advanced clone 06-27 with late blight tolerance of the in vitro Germplasm Bank of the INIFAP Potato Physiology and Biotechnology Laboratory in Metepec State of Mexico, Mexico, were selected based on their low tolerance to cryogenesis observed in preliminary laboratory work. The plants were incubated for 28 days in MS propagation medium (Murashige and Skoog, 1962) under treatment of AS, 0, 10-6 and 10-5 M (Mora-Herrera et al., 2005; Aguilar-Camacho et al., 2016). Nodal buds (1-2.5 mm) of these microplants were evaluated under the following conditions:
a) Effect of AS on plant regeneration after treatment: twenty buds were subcultured to MS medium without AS and incubated for 15 days, to assess survival, fresh weight, plant height and root length. Survival was assessed considering oxidation and turgidity.
b) Cryogenics: twenty buds were subcultured to MS medium without AS and incubated for 3 days, to be subjected to cryogenics. The cryogenic method used was Dehydration-cryoplate (D-cryoplate) (Yamamoto et al., 2015, modified by Arizaga et al., 2017) which consists of the following steps.
1) a volume of 2µL of sodium alginate (2 % w/v sodium alginate/0.4 M sucrose in a basal solution of MS medium), was placed in each of the 10 wells of a cryoplate (7 x 37 x 5 mm).
2) each of the buds were transferred individually to each of the wells of the cryoplate, to later cover them with the sodium alginate solution.
3) the cryoplate with the buds was covered with a sterile sheet of BEMCOT paper (7X30 mm).
4) 1 mL of calcium chloride solution (0.1 M calcium chloride/0.4 M sucrose in basal MS solution) was added to cover the cryoplate. Polymerization of sodium alginate/calcium chloride was completed after 15 min at room temperature, removing excess calcium chloride solution.
5) the cryoplate with the buds and the adhered paper were transferred to a loading solution (SC) (2 M glycerol/1 M sucrose in basal MS solution), for 45 min, the excess SC was removed.
6) the cryoplate with the buds and the attached paper were transferred to a petri dish with 35 g of silica gel for the dehydration process, for 90 min at 24 °C.
7) after dehydration, the cryoplates were transferred to cryotubes and immersed directly in liquid nitrogen (NL) for 60 min.
8) the cryoplates were removed from the NL and transferred to cryotubes with 2 mL of sucrose solution (1 M in basal MS medium) for 15 min at room temperature.
9) The buds attached to the Bemcot paper were removed from the cryoplate and placed in petri dishes with MS medium for 24 h.
10) the sodium alginate capsule was subsequently removed leaving the bud exposed, which was subcultured to fresh MS medium.
11) 15 days after the subculture, the survival of the regenerated plants was evaluated, considering those that resumed their growth alive; as well as callus formation and root length (n= 8-20).
Statistical analysis was performed through analysis of variance (Anova) and Duncan test (Duncan, 1955) in all experiments, using the Statgraphics Centurion XVI program. The established level of reliability was p< 0.05. The experiments were performed in triplicate.
Results and discussion
Effects of AS on plant regeneration after treatment.
After the treatment of AS in the microplants treated with AS 10-6 and 10-5 M, physiological responses were observed such as greater survival, root length and plant height. A significant increase in survival of 2.04 and 1.66 times respectively compared to the control (Figure 1).
Figure 1. Effect of AS on the survival of potato clone 06-27 after treatment. Variables evaluated 15 days after subculture. Data obtained from 3 experiments (n= 20). Data analyzed with Anova and Duncan test (p< 0.05). Different letters indicate significant differences from the control.
Similar effects on increased survival by AS have been found in various species when used in different concentrations, such as in Astragalus adsurgens (1.64 times) (Luo et al., 2001) and Coffea arabica (1.57 times) (Quiroz-Figueroa et al., 2001).
Microplants pretreated with AS 10-6 and 10-5 M showed a significant increase in root length of 3.28 and 2.87 times respectively, as well as in plant height of 2.60 and 1.87 times compared to the control (Figures 2a and 2b).
Figure. 2 Effect of AS on the growth of potato clone 06-27 after treatment. a) root length; b) plant height; and c) fresh weight. Variables evaluated 15 days after subculture. Data obtained from 3 experiments (n= 20). Data analyzed with Anova and Duncan test (p< 0.05). Different letters indicate significant differences from the control.
The results obtained in this research are of high interest, because greater root development is important for the establishment in both in vitro and ex vitro conditions of any species, because it is directly related to the absorption of nutrients and their It also induces an increase in growth (Ruiz, 2000; Selles et al., 2003; Callejas-Rodríguez et al., 2012).
This coincides with that reported by Larqué-Saavedra and Martin-Mex (2007), since they found an increase in density and root length of plants treated with AS. According to Sakhabutdinova et al. (2003), AS treatments can increase cell division in seedling roots by increasing their overall growth, the above may be related to the effect of AS on the increase in root length observed in this work in potato microplants (Figure 2a).
Other works that agree with the results obtained in this investigation corresponding to the effects of AS in root length and plant height (Figure 2a, 2b), have been reported in different crops such as Hibiscus (varieties acetocela and moscheutos) (Sakhanokho and Kelley, 2009), corn (Gunes et al., 2007), soy (Gutiérrez-Coronado et al., 1998) and wheat (Shakirova et al., 2003). Pre-treated microplants with AS 10-6 and 10-5 M showed a significant increase in fresh weight of 2.58 and 1.96 times respectively, compared to the control (Figure 2c). Paez-García et al. (2015), mention that the application of AS induces an increase in biomass due to a greater development in the radical system, which leads to a better absorption of water and nutrients.
Cryogenesis
The microplants that resumed their growth from the treatments of AS 10-6 and 10-5 M showed an increase in cryogenic survival of 3.21 and 2.17 times respectively, compared to the control (Figure 3).
Figure 3. Effect of AS on the survival of potato clone 06-27 after cryogenesis. Variables evaluated 15 days after subculture. Data obtained from 3 experiments (n= 8-20). Data analyzed with Anova and Duncan test (p< 0.05). Different letters indicate significant differences from the control.
These results contrast with the values obtained by Rivera et al. (2008), who obtained up to 52% survival in the absence of pretreatment of AS. The observed results may be related to a reduction in oxidative stress due to AS, as has been demonstrated in potatoes under different types of stress, such as greater survival under thermotherapy (up to 2 times) (Aguilar-Camacho et al., 2016).
Higher tuber weight in plants infected with phytoplasma (1.88 times) (Sánchez-Rojo et al., 2011), especially greater survival at low temperatures (1.77-2.35 times) (Mora-Herrera et al., 2005; López-Delgado et al., 2018). After the cryogenic process, the absence of callus was observed in the microplants from both AS treatments while the control showed 68% callus formation without stem bud formation (Figure 4).
Figure 4. Effect of AS on the development of potato clone 06-27 after cryogenesis. a) Callus formation without stem bud development; and b) Root formation. Evaluations made 15 days after the cryogenic process. Data obtained from 3 experiments (n= 8-20). Data analyzed with Anova and Duncan test (p< 0.05). Different letters indicate significant differences from the control.
During plant regeneration after cryogenic techniques, direct regeneration without callus formation is very desirable, because it ensures the genetic stability of the plants obtained (Reed, 2008). The results in this investigation demonstrate the potential of AS to increase survival in cryogenesis in potato genotypes that have low survival in these processes, in addition to preventing callus formation in regeneration, since the risk of somaclonal variation is reduced, favoring conservation of phenotypic and genotypic characteristics of plant material.
In addition to the absence of callus in the regenerated explants, a significant increase in root length was obtained in plants pretreated with AS 10-6 and 10-5 M of 3.35 and 2.36 times respectively after cryogenesis (Figure 5).
Figure 5. Effect of AS on the development of potato clone 06-27 after cryogenesis. a) callus formation without stem bud development; and b) root formation. Evaluations made 15 days after the cryogenic process. Data obtained from 3 experiments (n= 8-20). Data analyzed with Anova and Duncan test (p< 0.05). Different letters indicate significant differences from the control.
Compared to the control, the success of optimal regeneration after exposure to liquid nitrogen is based on an adequate physiological and morphological state of the explant that was subjected to the cryogenic method (Engelmann et al., 2008).
Pretreatments with AS can be an important tool for a better survival to cryopreservation or cryotherapy techniques in potatoes, in genotypes with low survival at ultra low temperatures since it is a signaling molecule of stress tolerance induction and plant growth regulator (Horvath, 2007), also involved in the development of roots in conditions of stress due to low temperatures (Huang and Villanueva, 1993; Melkonian et al., 2004).
Conclusions
In this investigation, the effect of AS was observed as an inducer of tolerance to ultra-low temperatures used in cryogenic processes in potato plants that have low survival to cryogenics, increasing survival rates.
In addition to this there is also a promoter effect of AS in the development of explants subjected to the cryogenic process since a significant increase in the development of the roots of these explants after their regeneration was obtained. AS avoided callus formation, which is an important factor in maintaining genetic stability in crops such as Solanum tuberosum.
Cited literature
Aguilar-Camacho, M.; Mora-Herrera, M. E. and López-Delgado, H. A. 2016. Potato virus X (PVX) elimination as short and long term effects of hydrogen peroxide and salicylic acid is differentially mediated by oxidative stress in synergism with thermotherapy. Am. J. Potato Res. 93(4):360-367.
Arizaga, M. V.; Navarro, O. F. V.; Martínez, C. R. C.; Gutiérrez, E. J. C.; Delgado, H. A. L.; Yamamoto, S. I.; Watanabe, K. and Niino, T. 2017. Improvement to the D cryo-plate protocol applied to practical cryopreservation of in vitro grown potato shoot tips. The Hortic. J. 86(2):222-228.
Baek, K. H. and Skinner, D. Z. 2012. Production of reactive oxygen species by freezing stress and the protective roles of antioxidant enzymes in plants. Journal of Agricultural Chemistry and Environment. 1(01):34-40.
Bernard, F.; Shaker-Bazarnov, H. and Kaviani, B. 2002. Effects of salicylic acid on cold preservation and cryopreservation of encapsulated embryonic axes of Persian lilac (Melia azedarach L.). Euphytica. 123(1):85-88.
Callejas-Rodríguez, R.; Rojo-Torres, E.; Benavidez-Zabala, C. y Kania-Kuhl, E. 2012. Crecimiento y distribución de raíces y su relación con el potencial productivo de parrales de vides de mesa. Agrociencia. 46(1):23-35.
Chinnusamy, V.; Zhu, J. and Zhu, J. K. 2007. Cold stress regulation of gene expression in plants. Trends in Plant Sci. 12(10):444-451.
Day, J. G.; Harding, K. C.; Nadarajan, J. and Benson, E. E. 2008. Cryopreservation: conservation of bioresources at ultra low temperatures. In: molecular biomethods handbook. Humana Press. 52(2):917-947.
Duncan, D. B. 1955. Multiple range and multiple F tests. Biometrics. 11(1):1-42.
Engelmann, F.; Arnao, M. T. G.; Wu, Y. and Escobar, R. 2008. The development of encapsulation dehydration. In: plant cryopreservation: A practical guide. Springer. 4(1):59-75.
Gill, S. S. and Tuteja, N. 2010. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol. Biochem. 48(12):909-930.
Gunes, A.; Inal, A.; Alpaslan, M.; Eraslan, F.; Bagci, E. G. and Cicek, N. 2007. Salicylic acid induced changes on some physiological parameters symptomatic for oxidative stress and mineral nutrition in maize (Zea mays L.) grown under salinity. J. Plant Physiol. 164(6):728-736.
Gutiérrez-Coronado, M. A.; Trejo-López, C. and Larqué-Saavedra, A. 1998. Effects of salicylic acid on the growth of roots and shoots in soybean. Plant Physiol. Biochem. 36(8):563-565.
Halmagyi, A.; Deliu, C. and Coste, A. 2005. Plant regrowth from potato shoot tips cryopreserved by a combined vitrification-droplet method. CryoLetters. 26(5):313-322.
Hamilton, K. N.; Ashmore, S. E. and Pritchard, H. W. 2009. Thermal analysis and cryopreservation of seeds of Australian wild Citrus species (Rutaceae): Citrus australasica, C. inodora and C. garrawayi. CryoLetters. 30(4):268-279.
Hao, Y. J.; You, C. X. and Deng, X. X. 2002. Analysis of ploidy and the patterns of amplified fragment length polymorphism and methylation sensitive amplified polymorphism in strawberry plants recovered from cryopreservation. CryoLetters. 23(1):37-46.
Hirai, D. 2011. Gelled droplet vitrification improves recovery of cryopreserved potato germplasm. CryoLetters. 32(4): 287-296.
Horváth, E.; Szalai, G. and Janda, T. 2007. Induction of abiotic stress tolerance by salicylic acid signaling. J. Plant Growth Reg. 26(3):290-300.
Huang, H. and Villanueva, V. R. 1993. Amino acids, polyamines and proteins during seed germination of two species of Dipterocarpaceae. Springer. Trees Structure and Function. 7(3):189-193.
Kaczmarczyk, A.; Funnekotter, B.; Menon, A.; Phang, P. Y.; Al-Hanbali, A.; Bunn, E. and Mancera, R. L. 2012. Current issues in plant cryopreservation. In: current frontiers in cryobiology. InTech. 14(1):418-438.
Larqué-Saavedra, A. and Martin-Mex, R. 2007. Effects of salicylic acid on the bioproductivity of plants. In Salicylic acid: A Plant Hormone. Springer. 2(1):15-23.
Li, Y.; Liu, C.; Li, T.; Wang, C.; Xiao, Y.; Zhang, L.; Jin, D.; Zhao, Y.; Wang. Z.; Cao, J. and Hao, L. 2011. Regulatory role of exogenous salicylic acid in the response of Zoysia japonica plants to freezing temperatures: a comparison with cold-acclimatisation. The J. Hortic. Sci. Biotechnol. 86(3):277-283.
López-Delgado, H. A.; Martínez-Gutiérrez, R.; Mora-Herrera, M. E. and Torres-Valdés, Y. 2018. Induction of freezing tolerance by the application of hydrogen peroxide and salicylic acid as tuber-dip or canopy spraying in Solanum tuberosum L. Plants. Potato Res. 61(3):195-206.
Luo, J. P.; Jiang, S. T. and Pan, L. J. 2001. Enhanced somatic embryogenesis by salicylic acid of Astragalus adsurgens Pall: relationship with H2O2 production and H2O2-metabolizing enzyme activities. Plant Science. 161(1):125-132.
Melkonian, J.; Yu, L. X. and Setter, T. L. 2004. Chilling responses of maize (Zea mays L.) seedlings: root hydraulic conductance, abscisic acid, and stomatal conductance. J. Exp. Bot. 55(403):1751-1760.
Mora Herrera, M. E. y López Delgado, H. A. 2006. Tolerancia a baja temperatura inducida por ácido salicílico y peróxido de hidrógeno en microplantas de papa. Rev. Fitotec. Mex. 29(2):81-85.
Mora‐Herrera, M. E.; López‐Delgado, H.; Castillo‐Morales, A. and Foyer, C. H. 2005. Salicylic acid and H2O2 function by independent pathways in the induction of freezing tolerance in potato. Physiol. Plantarum. 125(4):430-440.
Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plantarum. 15(3):473-497.
Paez-García, A.; Motes, C.; Scheible, W. R.; Chen, R.; Blancaflor, E. and Monteros, M. 2015. Root traits and phenotyping strategies for plant improvement. J. Plants. 4(2):334-355.
Pathirana, R.; McLachlan, A.; Hedderley, D.; Panis, B. and Carimi, F. 2016. Pre-treatment with salicylic acid improves plant regeneration after cryopreservation of grapevine (Vitis spp.) by droplet vitrification. Acta Physiologiae Plantarum. 38(12):1-11.
Quiroz-Figueroa, F.; Méndez-Zeel, M.; Larqué-Saavedra, A. and Loyola-Vargas, V. 2001. Picomolar concentrations of salicylates induce cellular growth and enhance somatic embryogenesis in Coffea arabica tissue culture. Plant Cell Reports. 20(8):679-684.
Reed, B. M. 2008. Cryopreservation: Practical Considerations. In: plant cryopreservation: a practical guide. Springer. 1(1):3-13.
Rivera, Á.; Valbuena, R.; Hidalgo, R. y Moreno, J. 2008. Crioconservación de yemas de microtubérculos de papa Solanum tuberosum ssp. andigena mediante desecado de tejidos. Corpoica. Ciencia y Tecnología Agropecuaria. 9(2):37-44.
Ruiz, S. 2000. Dinámica nutricional en cinco parrones de diferente productividad del valle central regado de Chile. Agric. Téc. 60(4):379-398.
Sakhabutdinova, A. R.; Fatkhutdinova, D. R.; Bezrukova, M. V. and Shakirova, F. M. 2003. Salicylic acid prevents the damaging action of stress factors on wheat plants. Bulgarian J. Plant Physiol. 21(1):314-319.
Sakhanokho, H. F. and Kelley, R. Y. 2009. Influence of salicylic acid on in vitro propagation and salt tolerance in Hibiscus acetosella and Hibiscus moscheutos (cv ‘Luna Red’). Afr. J. Biotechnol. 8(8):1474-1481.
Sánchez-Chiang, N. y Jiménez, V. M. 2010. Técnicas de conservación in vitro para el establecimiento de bancos de germoplasma en cultivos tropicales. Agron. Mesoam. 21(1):193-205.
Sánchez-Rojo, S.; López-Delgado, H. A.; Mora-Herrera, M. E.; Almeyda-León, H. I.; Zavaleta-Mancera, H. A. and Espinosa-Victoria, D. 2011. Salicylic acid protects potato plants-from phytoplasma-associated stress and improves tuber photosynthate assimilation. Am. J. Potato Res. 88(2):175-183.
Sasheva, P.; Szalai, G.; Janda, T. and Popova, L. 2010. Study of the behaviour of antioxidant enzymes in the response to hardening and freezing stress in two wheats (Triticum aestivum L.) varieties. Acta Agron. Hungarica. 61(2):161-172
Sellés, G. V. S.; Ferreyra, R. E.; Contreras, G. W.; Ahumada, R. B.; Valenzuela, J. B. and Bravo, R. V. 2003. Drip irrigation management in table grapes cv. Thompson seedless grown on fine textured soils. Agric. Téc. 63(2):180-192.
Shakirova, F. M.; Sakhabutdinova, A. R.; Bezrukova, M. V.; Fatkhutdinova, R. A. and Fatkhutdinova, D. R. 2003. Changes in the hormonal status of wheat seedlings induced by salicylic acid and salinity. Plant Sci. 164(3):317-322.
Uchendu, E. E.; Muminova, M.; Gupta, S. and Reed, B. M. 2010. Antioxidant and anti-stress compounds improve regrowth of cryopreserved Rubus shoot tips. In Vitro Cellular Developm. Biol. Plant. 46(4):386-393.
Wang, Q. and Valkonen, J. P. 2009b. Cryotherapy of shoot tips: novel pathogen eradication method. Trends Plant Sci. 14(3):119-122.
Wang, Q.; Liu, Y.; Xie, Y. and You, M. 2006. Cryotherapy of potato shoot tips for efficient elimination of potato leafroll virus (PLRV) and potato virus Y (PVY). Potato Res. 49(2):119-129.
Wang, Z. C. and Deng, X. X. 2004. Cryopreservation of shoot-tips of citrus using vitrification: effect of reduced form of glutathione. CryoLetters. 25(1):43-50.
Yamamoto, S. I.; Rafique, T.; Arizaga, M. V.; Fukui, K.; Gutiérrez, E. J. C.; Martínez, C. R. C. and Niino, T. 2015. The aluminum cryo-plate increases efficiency of cryopreservation protocols for potato shoot tips. Am. J. Potato Res. 92(2):250-257.