Efficacy of Passion Fruit Cryopreservation Using Cryopotectant Agents

ABSTRACT

This study aimed to evaluate the effects of cryoprotectant agents on Passiflora eichleriana, Passiflora crystallina and Passiflora nitida seeds undergoing cryopreservation. The experiment was conducted with non-cryoprotected or non-cryopreserved seeds (control), cryopreserved seeds without cryoprotectant and cryopreserved seeds treated with the following cryoprotectants: 1.73, 2.28, 2.60 or 2.71 mol glycerol; 0.37, 0.46, 0.54 or 0.61 mol sucrose; or 0.37, 0.72, 1.04 or 1.35 mol dimethylsulfoxide (DMSO). After treatment, the seeds were cryopreserved in liquid nitrogen (LN₂) for 7 days and then defrosted in 37°C water for 5 min. The seeds were submitted to germination and vigor tests consisting of four replicates of 50 seeds. Percentage of germination (PE) and germination velocity index, percentage of emergence velocity index, shoot and root length, and shoot and root dry mass were evaluated. After cryopreservation, all of germination and vigor values were higher without the use of cryoprotectants. The cryoprotectant glycerol had a deleterious effect on seed viability and vigor for all Passiflora species. Glycerol inhibit seedling emergence and concentrations higher than 2.60 mol seed germination on P. cristalina. Higher concentrations of DMSO decrease seed viability and vigor in P. crystalina and P. nitida.

KEYWORDS:

• Liquid nitrogen
• glycerol
• sucrose
• dimethylsulfoxide

Introduction

Passiflora is the largest genus within the Passifloraceae family comprised of 450 to 537 species. Most species are native to the tropical Americas, and Brazil is the center of origin for at least one-third of them (Vanderplank, 2007). Brazil is the world’s largest producer and consumer of yellow and sour passion fruit (Passiflora edulis Sims), the species with highest commercial value (Meletti, 2011). Passiflora can be used as a food, ornamental plant or herbal medicine, and it is economically important for the juice industry (Paiva et al., 2014). Some passion fruit species from north central Brazil are resilient to disease and pests and have higher longevity and adaptability, as well as other properties and characteristics important for the improvement of P. edulis (Meletti et al., 2007). The use of new areas for agriculture in north central Brazil have, however, resulted in the eradication of passion fruit native species and loss of many genotypes with potential for genetic improvement. Consequently, such initiatives as creation, expansion, and maintenance of germplasm banks are an essential part of species conservation (Faleiro et al., 2011).

Seeds from different species of Passiflora have low and irregular germination; thus, there is a long period between the start and the end of seed germination with consequential irregular seedling production (Souza and Meletti, 1997). Seeds from Passiflora sp. can also exhibit dormancy, which is an obstacle for large-scale seedling production (Grzybowski et al., 2019). Seeds from many passion fruits are recalcitrant and rapidly lose their viability; consequently, more wide-ranging seed germination and storage studies considering the genetic variability of passion fruit accessions and cultivars within each species are necessary (Faleiro et al., 2019).

Seed cryopreservation is a germplasm conservation technique considered ideal to provide almost unlimited seed preservation by reducing seed metabolism and, consequently, its deterioration (Tarré et al., 2007). However, to be successful as a means of storing genetic resources, cryopreservation technology must be based on protocols that will prevent the formation of intra- and extracellular ice crystals through cell vitrification and immersion of explants in liquid nitrogen (Panis and Lambardi, 2006). To reduce cell damage, chemical substances, termed cryoprotectants, can protect tissues from the harmful effects caused by ice crystals that are formed inside and outside cells during the freezing and defrosting processes (Carvalho and Otoni, 2010).

Therefore, the present paper aimed to examine the effects of different cryoprotectant agents and concentrations on Passiflora eichleriana, Passiflora cristalina and Passiflora nitida cryopreserved seeds.

Material and Methods

Passiflora eichleriana, Passiflora cristalina and Passiflora nitida seeds were obtained from the Active Germplasm Bank of the experimental field at Mato Grosso State University, Cáceres, Mato Grosso State, Brazil. The seeds were obtained from mature fruits in good phytosanitary and morphological condition. The fruits were cut to remove the pulp and seeds, which were then cleaned on a sieve with hydrated lime, washed in tap water to remove all seed mucilage, and dried for 24 h at room temperature in the laboratory (27.7°C ± 1.9) as described by Araújo et al. (2016). The seed water content was determined in an oven, using the forced air circulation method at 105 ± 3°C for 24 h, according to the Guidelines for Seed Analysis (Brasil, 2009). P. eichleriana seeds were soaked in KNO₃ solution (1%) for 24 h at 30°C, and P. nitida seeds were soaked in 1,000 mg L⁻¹ GA₃ (gibberellic acid, 99.0%, VETEC©, Brazil) solution for 6 h at 25°C in the absence of light (Marostega et al., 2017) to overcome dormancy. P. cristalina was not submitted to any process to overcome dormancy.

The experiment was conducted with non-cryoprotected or non-cryopreserved seeds (control), cryopreserved seeds without cryoprotectant and cryopreserved seeds treated with the cryoprotectants, as follows: 1.73, 2.28, 2.60 or 2.71 mol glycerol (99.0%, Kasvi©, Brazil); 0.37, 0.46, 0.54, or 0.61 mol sucrose (99.0%, Synth©, Brazil); or 0.37, 0.72, 1.04, or 1.35 mol dimethylsulfoxide (DMSO, 99.9%, Synth©, Brazil), totaling 14 treatments. The seeds were immersed for 3 h in cryoprotectant solutions. Chemicals and reagents used were of analytical grade.

The seeds were packaged in aluminum “pets”, set in canisters and immersed in liquid nitrogen (LN₂). Cryopreservation was performed in liquid nitrogen (−196°C) for 168 h (7 days), and defrosting was carried out in a water bath at 37°C for 5 min (Araújo et al., 2016). Control seeds were kept in cold rooms at 10ºC and 65% humidity.

The seeds from all treatments were sown in “Gerbox” transparent acrylic boxes with paper blotted with distilled water (two and a half times the mass of dry paper) as a substrate and placed in a germination chamber (B.O.D.) for the germination test. Experimental temperatures alternated between 20°C and 30°C with a photoperiod of 12 h. Evaluations were performed daily over a 30-day period. Seeds with tegument rupture and primary root 2 mm in length were evaluated, and the percentage of germination and germination speed index were calculated according to Maguire (1962). The emergence test was carried out at room temperature (19.2°C to 31.6°C), using plastic pots containing Vivatto Plus® as a substrate. Substrate moisture was maintained with irrigation (4 to 5 times a day) with suspended sprinklers, and evaluation was carried out 30 days after sowing. Only plantlets with fully expanded cotyledonary leaves were considered as seedlings. The emergence speed index was carried out together with the percentage of emergence using Maguire’s formula (1962). After 30 days, the length of shoots and roots was measured. The seedlings were cut into roots and shoots, weighed for fresh mass, and put into a forced-air oven at 70°C for 72 h for dry mass.

The experimental design was completely randomized with 4 replicates of 50 seeds per treatment at all stages of the study. Data in percentage were transformed with arc sine √ ((X)%/100). The germination and emergence test data were submitted to analysis of variance, and the averages were compared by Dunnett’s test (p ≤ 0.05) using the statistical program ASSISTAT, version 7.7 beta.

Results and Discussion

Initial seed moisture of P. eichleriana, P. crystallina, and P. nitida was 7.1%, 6.0%, and 7.4%, respectively. P. eichleriana seeds showed no difference in variable percentage of germination. The germination speed index of treatments with sucrose and 0.37, 0.72 and 1.35 mol DMSO showed no statistical difference (p ≤ 0.05) when compared to seeds cryopreserved without cryoprotectant and control. P. eichleriana seeds presented slower germination when treated with glycerol and 1.04 mol DMSO (Table 1). P. eichleriana seeds presented low percentage of emergence and loss of vigor with glycerol, showing, in addition, possible phytotoxicity. The variables emergence speed index, shoot and root length, and root dry mass showed the same results; hence, these variables are associated with the percentage of emergence. Glycerol might increase the water content in seeds, contributing to the decrease of seed physiological quality, as seen in cryopreserved onion seeds that had a lower germination rate with glycerol at 50% (5.42 mol) (Molina et al., 2006). Low concentrations of 1.73 to 2.71 mol resulted in toxicity that can compromise the germination and vigor of Passiflora seeds. P. eichleriana seeds without cryoprotectant maintained the same germination, emergence, and vigor as cryoprotected seeds and seeds not cryopreserved or cryopreserved (control). Passiflora cryopreservation without the use of cryoprotectants could be used for the long-term preservation of P. mucronate, P. suberosa, and P. edulis seeds (Araújo et al., 2016). DMSO is indicated for larger explants or more complex structures because it presents superior penetration capacity (Jaganathan and Liu, 2014); however, it was not efficient for the cryopreservation of P. eichleriana seeds. Plants can respond differently to cryopreservation as Chrysanthemum apices are less sensitive to osmotic stress produced by glycerol and sucrose and very susceptible to toxic action of permeating cryoprotectants DMSO and EG (Popova et al., 2015).

Table 1. Percentage of germination (PG), germination speed index (GSI), percentage of emergency (PE), emergency speed index (ESI) of Passiflora eichleriana seeds and shoot length (SL), root length (RL), shoot (SDM) and root (RDM) dry mass of P. eichleriana seedlings submitted to different cryoprotectants

Treatments	PG (%)	GSI	PE (%)	ESI	SL (cm)	RL (cm)	SDM (g)	RDM (g)
Without Cryoprotection	56^ns	4.04 a*	91 a*	4.76 a*	6.9 a*	12.3 a*	0.22 a*	0.06 a*
Control	51	3.26 a	83 a	4.34 a	6.1 a	11.1 a	0.22 a	0.08 a
Gly 1.73	54	1.97 b	2 b	0.07 b	0.6 b	2.0 b	0.02 b	0.01 b
Gly 2.28	56	2.40 b	1 b	0.04 b	0.2 b	0.5 b	0.01 b	0.01 b
Gly 2.60	47	1.71 b	4 b	0.17 b	1.0 b	2.9 b	0.04 b	0.02 b
Gly 2.71	51	1.76 b	2 b	0.07 b	0.4 b	1.2 b	0.01 b	0.01 b
Suc 0.37	67	5.29 a	85 a	4.35 a	6.2 a	12.6 a	0.20 a	0.09 a
Suc 0.46	64	4.70 a	90 a	4.91 a	6.4 a	12.4 a	0.22 a	0.08 a
Suc 0.54	64	4.45 a	86 a	4.60 a	6.9 a	12.8 a	0.24 a	0.10 a
Suc 0.61	72	5.14 a	76 a	3.64 a	5.9 a	12.6 a	0.21 a	0.09 a
DMSO 0.37	66.5	3.93 a	82 a	4.25 a	6.7 a	12.1 a	0.24 a	0.10 a
DMSO 0.72	56.0	3.93 a	84 a	4.24 a	6.5 a	11.3 a	0.19 a	0.09 a
DMSO 1.04	37.0	2.28 b	85 a	4.32 a	6.12 a	11.3 a	0.17 a	0.08 a
DMSO 1.35	52.0	2.86 a	82 a	3.96 a	6.0 a	11.2 a	0.18 a	0.09 a
CV %	11.72	10.95	7.82	6.83	6.3	8.3	2.74	1.11

*Means within same column followed by same letter in the column do not differ by Dunnett’s test at the 5% probability level; ^ns not significant. Gly – Glycerol; Suc – Sucrose. Concentrations in mol. Transformed data into arc sin $(X\mathrm{\%}/100{)}^{1/2}$ for percentage data.

Treatments with sucrose and cryopreserved seeds without cryoprotection did not statistically differ from control for percentage of germination and germination speed index of P. crystalline seeds. Sugars, such as sucrose, trehalose and glucose, have been used as cryoprotective substances because they are not cytotoxic, even when accumulated in large quantities in the cytoplasm (Vendrame et al., 2014). Sucrose can protect seeds by stabilizing cell membranes during freezing. This can be seen during cold acclimatization of temperate climate plants when plants are more tolerant to freezing owing to the maximum sugar accumulation of soluble carbohydrates (Imanishi et al., 1998). DMSO (1.35 mol) and glycerol decreased the percentage of germination and germination velocity index of P. cristalina (Table 2). Glycerol was ineffective for cryoprotection and/or toxic to P. cristalina seeds, as also observed in the cryopreservation of mature rice calli (Miranda et al., 2009) evidencing its toxicity. P. cristalina was more sensible to DMSO during the germination process (low percentage of germination and germination speed index) and with higher DSMO concentration (1.35) during the emergence process. Even in very low concentrations, DMSO can be toxic to some biological systems, owing to its high solubility in water, damaging the cell membrane and consequently compromising its function (Hubálek, 2003). The treatment of cryopreserved seeds without any cryoprotection agent had a higher percentage of germination (66%); however, it was not statistically different from that of control (60%), indicating the viability of cryopreservation without the application of any cryoprotection agents for these species. The viability of cryopreservation without cryoprotectant was also verified for seeds of Amaryllidaceae species. In this study, the cryoprotective agents sodium alginate and PVS₂ did not improve cryopreservation (Tombolato et al., 2009). Treatments with glycerol showed the lowest values for the variables percentage of emergence, emergence speed index, shoot, and root length, shoot, and root dry mass, reinforcing the toxic nature of glycerol for seeds of these species. The other treatments showed no statistical difference compared to treatments with cryopreserved seeds without cryoprotection, except the highest dosage of DMSO (1.35 mol), which showed results analogous to those of glycerol (Table 2). Sucrose treatments did not statistically differ from cryopreserved seeds without cryoprotection and control. How sugar acts in tissues to acquire tolerance to dehydration and freezing remains elusive, but sugars may act as external osmotic agents, removing the excess of intracellular water through an osmotic gradient (Barbas and Mascarenhas, 2009). Sugar can also replace the water removed from biomolecules, maintaining hydrophilic structures, even after the water is removed (Stegani et al., 2017).

Table 2. Percentage of germination (PG), germination speed index (GSI), percentage of emergency (PE), emergency speed index (ESI) of Passiflora cristalina seeds and shoot length (SL), root length (RL), shoot (SDM) and root (RDM) dry mass of P. cristalina seedlings submitted to different cryoprotectants

Treatments	PG (%)	GSI	PE (%)	ESI	SL (cm)	RL (cm)	SDM (g)	RDM (g)
Without Cryoprotection	66 a*	1.73 a*	44 a*	1.07 a*	1.8 a*	5.7 a*	0.07^ns	0.03 a*
Control	60 a	1.49 a	41a	1.01 a	1.7 a	4.5 a	0.07	0.03 a
Gly 1.73	0 b	0.00 b	0 b	0.00 b	0.0 b	0.0 b	0.00	0.00 b
Gly 2.28	8 b	0.21 b	0 b	0.00 b	0.0 b	0.0 b	0.00	0.00 b
Gly 2.60	0 b	0.00 b	0 b	0.00 b	0.0 b	0.0 b	0.00	0.00 b
Gly 2.71	0 b	0.00 b	0 b	0.00 b	0.0 b	0.0 b	0.00	0.00 b
Suc 0.37	57 a	1.41 a	32a	0.81 a	1.6 a	4.8 a	0.08	0.03 a
Suc 0.46	58 a	1.34 a	15 a	0.33 a	0.7 a	2.0 a	0.03	0.01 a
Suc 0.54	57 a	1.46 a	41 a	1.01 a	1.8 a	5.6 a	0.07	0.03 a
Suc 0.61	58 a	1.43 a	25 a	0.62 a	1.4 a	3.9 a	0.21	0.02 a
DMSO 0.37	45 b	0.92 b	38 a	0.94 a	1.9 a	5.2 a	0.08	0.03 a
DMSO 0.72	25 b	0.47 b	44 a	1.11 a	2.0 a	6.2 a	0.087	0.03 a
DMSO 1.04	10 b	0.20 b	46 a	1.12 a	2.0 a	6.9 a	0.09	0.04 a
DMSO 1.35	7 b	0.14 b	2 b	0.05 b	0.6 b	0.8 b	0.05	0.01 b
CV %	13.14	5.89	30.40	14.64	15.8	21.6	6.67	0.96

*Means within same column followed by same letter in the column do not differ by Dunnett’s test at the 5% probability level; ^ns not significant. Gly – Glycerol; Suc – Sucrose. Concentrations in mol. Transformed data into arc sin $(X\mathrm{\%}/100{)}^{1/2}$ for percentage data.

Percentage of germination and germination speed index of Passiflora nitida seeds (Table 3) showed that treatments with sucrose and cryopreserved seeds without cryoprotection and control had higher values than other treatments (p ≤ 0.05). Treatments with glycerol and DMSO negatively affected germination and vigor of seeds, showing the lowest averages for germination percentage and germination speed index. The lowest concentration of DMSO (0.37 mol) was not statistically different from all sucrose treatments, cryopreserved seeds without cryoprotection and control for the variables emergence percentage, emergence speed index, shoot length, root length, shoot and root dry mass. Passiflora micropetala and P. edulis seeds treated with 7% DMSO showed a lower percentage of germination and germination speed index (Araújo et al., 2016). The achievement of sucrose treatments can be explained by their ability to maintain the crystalline liquid state of membrane bilayers and stabilize proteins under freezing conditions with no toxicity to plant cells, even when accumulated in large amounts in the cytoplasm (Carvalho and Otoni, 2010). The lower percentages of germination and emergence of P. nitida seeds may be related to seed dormancy (Marostega et al., 2017). Passiflora spp. might exhibit dormancy, and the main mechanisms through which this phenomenon occurs are embryo dormancy (endogenous) and dormancy imposed by the outer layers of the seed (exogenous) (Grzybowski et al., 2019). Passiflora spp. can be further divided into two major groups according to dormancy type: chemical dormancy (responsive to chemical compounds, such as GA₃ and/or KNO₃, and mechanical dormancy combined/or not with chemical dormancy (Marostega et al., 2017). However, no studies have reported on physical treatments of seeds such as light (Asghar et al., 2016), laser (Shafique et al., 2017) and magnetism (Kehinde et al., 2017) in relation to seed pretreatment to enhance seed germination and seedling growth. Future studies should be focused on overcoming seed dormancy and increasing the emergence and development of seedlings with plant growth regulators, chemical and mechanical scarification (Faleiro et al., 2019) and techniques (Abbas et al., 2017; Faleiro et al., 2019), as well as post-germination events, such as nutrient content, enzymatic activity, and chlorophyll content (Asghar et al., 2016).

Table 3. Percentage of germination (PG), germination speed index (GSI), percentage of emergency (PE), emergency speed index (ESI) of Passiflora nitida seeds and shoot length (SL), root length (RL), shoot (SDM) and root (RDM) dry mass of P. nitida seedlings submitted to different cryoprotectants

Treatments	PG (%)	GSI	PE (%)	ESI	SL (cm)	RL (cm)	SDM (g)	RDM (g)
Without Cryoprotection	22 a*	0.76 a*	23 a*	0.54 a*	2.6 a*	7.7 a*	0.30 a*	0.10 a*
Controle	29 a	0.93 a	21 a	0.51 a	2.8 a	7.0 a	0.31 a	0.09 a
Gly 1.73	0.5 b	0.01 b	0 b	0.00 b	0.0 b	0.0 b	0.00 b	0.00 b
Gly 2.28	0.5 b	0.01 b	0 b	0.00 b	0.0 b	0.0 b	0.00 b	0.00 b
Gly 2.60	1 b	0.02 b	0 b	0.00 b	0.0 b	0.0 b	0.00 b	0.00 b
Gly 2.71	0 b	0.00 b	0 b	0.00 b	0.0 b	0.0 b	0.00 b	0.00 b
Suc 0.37	23 a	0.77 a	16 a	0.38 a	1.9 a	5.5 a	0.25 a	0.07 a
Suc 0.46	25 a	0.69 a	13 a	0.32 a	1.9 a	4.8 a	0.25 a	0.07 a
Suc 0.54	22 a	0.68 a	11 a	0.26 a	1.6 b	4.4 a	0.21 a	0.06 a
Suc 0.61	26 a	0.73 a	14 a	0.25 a	2.3 a	6.7 a	0.28 a	0.08 a
DMSO 0.37	17.5 b	0.51 b	13 a	0.30 a	1.9 a	4.7 a	0.23 a	0.07 a
DMSO 0.72	9.0 b	0.20 b	1 b	0.03 b	0.3 b	0.4 b	0.02 b	0.01 b
DMSO 1.04	1.0 b	0.02 b	0 b	0.00 b	0.0 b	0.0 b	0.00 b	0.00 b
DMSO 1.35	1.0 b	0.23b	0 b	0.00 b	0.0 b	0.0 b	0.00 b	0.00 b
CV %	19.74	7.72	36.51	8.78	16.77	25.62	5.97	2.30

*Means within same column followed by same letter in the column do not differ by Dunnett’s test at the 5% probability level; ^ns not significant. Gly – Glycerol; Suc – Sucrose. Concentrations in mol. Transformed data into arc sin $(X\mathrm{\%}/100{)}^{1/2}$ for percentage data.

Conclusion

In conclusion, we have demonstrated that seeds of P. eichleriana, P. cristalina, and P. nitida can be deep-frozen at −196°C. Our results also showed those seeds can be cryopreserved without the use of cryoprotectants. The cryoprotectant glycerol had a deleterious effect on seed viability and vigor for all Passiflora species. Glycerol inhibit seedling emergence and concentrations higher than 2.60 mol seed germination on P. cristalina. Higher concentrations of DMSO decrease seed viability and vigor in P. crystalina and P. nitida.

Acknowledgments

The authors would like to thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for financial support (Processo: 471856/2013-4).

Additional information

Funding

This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico [471856/2013-4].