Pretreatment with abscisic acid accompanied by sucrose improves callus survival after cryopreservation of hazel (Corylus avellana L.) by desiccation

In the current study, a simple cryopreservation method (desiccation) was applied to Corylus avellana L. callus. Accordingly, the effects of abscisic acid (ABA) concentration, pretreatment duration on MS medium containing ABA + 10% sucrose, and storage length in liquid nitrogen (LN) on the callus survival rate were investigated. Calli’s survival was assessed 8 weeks after exiting from LN. Callus survival after cryopreservation was significantly affected by the concentration and duration of the treatment with ABA-supplemented media. However, storage duration in LN did not have an impact on the callus survival rate. In the present study, the highest survival rate (45.79%) was obtained in both treatments of 20 days preculture on medium containing 2 mg l− 1 ABA following 2 h desiccation-one day storage in LN and 2.5 h desiccation-thirty days storage in LN. The desiccation method with the help of ABA and sucrose was an effective method in the successful cryopreservation of Corylus avellana L. callus.


Introduction
Paclitaxel, a powerful drug to treat cancer, was derived in small amounts from the bark of the yew tree (Taxus baccata) (Hoffman and Shahidi 2009). Finding a new source of taxol can be, therefore, one of the best solutions to preserve this limited population (Gibson et al. 1993). The common hazel, Corylus avellana L., is an economically valuable nut tree native to Europe and Asia, and commonly grows in moderate climates such as Turkey, Spain, and Italy, in which paclitaxel is found (Torello Marinoni et al. 2018;Bestoso et al. 2006). Plant cell culture is a promising, eco-friendly technology for producing paclitaxel in massive amounts (Espinosa-Leal et al. 2018). In comparison with taxus, hazel cell cultures are a promising alternative to taxol-producing supply because they are readily available, rapid-growing, and easier to in vitro culture (Bestoso et al. 2006).
As plant genetic resources are lost and genetically eroded due to biotic and abiotic stresses or human errors, the conservation of these resources has turned into a serious challenge (Ochatt et al. 2021). Undifferentiated tissues such as callus are genetically unstable and may be susceptible to epigenetic changes that result in secondary metabolite synthesis (Meijer et al. 1991). Fortunately, cryopreservation is regarded as a reliable method of conserving plant resources by which samples are stored safely for a long time in LN (− 196 °C) (Engelmann 2004). The most destructive event during cryo-storage is the formation of ice crystals in plant cells. Desiccation is the easiest way to make cells contain less water (Dumet and Benson 2000;Panis et al. 2001).
Most cells are susceptible to physical and chemical damage during the desiccation process (Takagi 2000). The success of cryopreservation protocols depends on the tolerance 1 3 of the plant's germplasm to the stresses resulting from the protocols used for dehydration (Reed et al. 2005). This tolerance can be achieved by preculturing plant materials on media including high concentrations of sucrose (Engelmann et al. 2003;Suzuki et al. 2006), or ABA that contributes to protein synthesis and compatible solutes, which are significant in developing drought and freezing tolerance, thus leading to the increased survival rate of specimens (Stewart and Voetberg 1985;Edesi et al. 2020). An increase in proline content can be induced by exogenous ABA. A higher accumulation of proline under ABA treatment might also consider for an increased freezing tolerance .
Cryopreservation has been done successfully on hazel's embryogenic axes, seeds, and pollens using desiccation, shoot tips by vitrification and axillary buds by droplet vitrification (Gonzalez-Benito and Perez 1994;Michalak et al. 2013;Shukla et al. 2016;Nebot et al. 2018;Sgueglia et al. 2021). However, to date no study has focused on the cryopreservation of C. avellana callus. Therefore, this study was designed to investigate the possibility of the successful cryopreservation of C. avellana callus as a precious secondary source of taxol.

Induction and maintenance of callus culture
A stable 6-year-old diploid callus was used to establish new cultures of C. avellana callus. Principally, seed cotyledons were cultured on MS medium (Murashige and Skoog 1962) enriched with 0.2 mg l − 1 6-benzylaminopurine (BAP), and 2 mg l − 1 2,4-dichlorophenoxyacetic acid (2,4-D) and solidified with 8 g l − 1 agar agar (pH 5.8). All cultures were placed in the dark at 25 ± 2 °C until the calli appeared. The calli were then subcultured every 25 days on the same medium (Salehi et al. 2017).

Pretreatment and desiccation
Corylus avellana calli were cryopreserved using a modified version of the desiccation procedure published by Popova et al. (2009). Two supplementary experiments were conducted. In the first experiment, callus fragments with 50 ± 5 mg of fresh weight were separated on the day 24 of callus culture and incubated for 7 days on MS solidified medium without plant growth regulators. The 7-day-old calli were moved on MS solidified medium supplemented with 1, 2 and 3 mg l − 1 ABA, 100 g l − 1 sucrose and 8 g l − 1 agar agar for 5, 10 and 15 days (pretreatment) to induce desiccation endurance. Desiccation was then performed at a relatively constant temperature (27 ± 1 °C) by placing the calli in 100 mm open Petri dishes inside the laminar airflow cabinet and exposing them to sterile airflow for 0-3 h with 30 min intervals.
In the second experiment, after 7 days of culture on hormone-free MS medium, the calli were transferred to the MS medium supplemented with 2 mg l − 1 ABA and 100 g l − 1 sucrose solidified with 8 g l − 1 agar agar for 10, 15, 20, and 25 days. After pretreatment, the calli were subjected to sterile airflow for 0-5 h with 30 min intervals at 27 ± 1 °C.

Cryopreservation
After desiccation, as the control, half of the calli were transferred to the MS medium including 0.2 mg l − 1 BAP, 2 mg l − 1 2, 4-D, and 8 g l − 1 agar agar; they were sealed and kept at 25 ± 1 °C. The remaining half of the calli were placed in cryovials (4 ml, Simport Company), sealed and then plunged immediately in LN for one day in the first experiment and for one day and 30 days in the second experiment.

Rewarming and recovery
For rewarming, the samples were instantly plunged into a warm water bath (40 °C) for 1 min; this was then followed by 1 min rewarming in cold tap water. Cryovials were surfacesterilized with 70% ethanol. To evaluate the survival rate, the calli were transferred to the MS medium containing 0.2 mg l − 1 BAP and 2 mg l − 1 2, 4-D and 8 g l − 1 agar agar in order to evaluate survival rate. Cryostoraged calli were kept 8 weeks at 25 ± 1 °C in the dark. Subcultures were performed every 2 weeks during the recovery period.

DATA analysis
Data were analyzed using a factorial experiment in a completely randomized design with three replications (10-12 callus clumps in each replication). The survival rate was assessed by the number of fresh callus clumps emerging from cryo-storaged calli after 8 weeks of culturing on the recovery medium and presented as a percentage. A normality test was performed on the residuals before analyzing the variance of the data. As the residuals did not have a normal distribution, the data were transformed using the formula (y = ARSIN (SQRT (y/100)) before being analyzed in SAS software (version 9.4) using the general linear model (GLM) procedure. The anti-transformed means (± standard errors) were input into an Excel spreadsheet, and the charts were created with this software. All treatments with a 0% survival rate have been removed from the charts.

Results
All the calli turned brown during the desiccation and freezing process. Eight weeks after cryopreservation, yellowish calli emerged from survived calli (Fig. 1); however, the dead calli still remained brown (Fig. 2). The survival rate of the desiccated calli was influenced by the ABA concentration and preculture duration on medium containing this hormone.
Based on the results obtained from the first experiment, the calli cultured on the media containing 2 and 3 mg l − 1 ABA had more desiccation tolerance, as compared to those cultured on the medium with 1 mg l − 1 ABA. However, without performing desiccation, extending the pretreatment duration of calli on MS medium containing 3 mg l − 1 ABA to 15 days suppressed their growth from 100 to 75%. The calli that were grown in the medium with 1 mg l − 1 ABA could not survive being dessicated for more than 2 h. After cryopreservation, the survival rate of these calli was decreased dramatically. In contrast, the survival rate was gradually increased by culturing the calli on the media containing 2 and 3 mg l − 1 ABA. Meanwhile, increased preculture duration to 15 days for 2 mg l − 1 ABA, and 10 days for 3 mg l − 1 ABA showed a positive impact on the callus survival after both desiccation and cryopreservation.
Although the calli cultured on the medium with 2 mg l − 1 ABA for 15 days could survive desiccation up to 3 h before and after storing in LN, the highest survival rate after cryopreservation belonged to the calli which were exposed to 2 h of desiccation (37.99%). As a result, extending the preculture period on the medium containing 2 mg l − 1 ABA appeared to be more effective in term of calli survival after cryopreservation (Fig. 3).
According to the results of the second experiment, the 20 days culture of the calli on the medium with 2 mg l − 1 ABA enhanced the callus survival rate to 45.79% with 2 h desiccation and 1 day of LN storage, and 43.30% and 45.79% with 2.5 h desiccation and 1 and 30 days of LN storage, respectively.
The preculture of calli for 25 days significantly reduced the growth of calli from 100 to 33%. In addition, no survival was observed after cryopreservation of these calli. Extension of the desiccation duration more than 2.5 h did not enhance the survival rate after LN storage. No significant difference in the callus survival rate was observed between 1 day and 30 days of storage in LN. However, some exceptions were observed in the treatments with the 20 days of preculture followed by 1 and 2 h desiccation (Fig. 4).

Discussion
In this study, we evaluated the effect of the desiccation procedure on the C. avellana callus survival rate after cryopreservation by examining the effects of ABA concentration, duration of preculture on ABA containing medium, desiccation periods and LN storage length. In both experiments, every 2 week subculture was crucial to obtain the optimal growth of calli after cryopreservation. Fig. 1 a, Cryopreserved callus at 100 × magnification, 6 weeks after removal from LN (2 mg l − 1 ABA, 15 days preculture, 2 h desiccation); b, cryopreserved callus at 100 × magnification, 6 weeks after removal from LN (2 mg l − 1 ABA, 20 days preculture, 2.5 h desiccation, 1 day storage in LN)

Fig. 2
Calli survived after 1 day of storage in LN, followed by 20 days of preculture on the medium containing 2 mg l − 1 ABA, and 2.5 h of desiccation. a. callus growth, 6 weeks after removal from LN; b. callus growth, 10 weeks after removal from LN Basically, the amount of water in cells must be declined to an adequate level to enable cells to tolerate the very cold temperature of LN (Bian et al. 2002). However, Popova et al. (2010) stated that the appropriate water content for cryopreservation depends on the species and type of plant specimen and should be found through different experiments.
The ability to elongate the desiccation period without affecting the survival of samples is particularly advantageous for cryopreservation since the cells will lose water gradually until they reach the desired water content ). Desiccation-based cryopreservation has been found to be successful on the calli of different species, such as the embryogenic calli of cassava (Danso and Ford-Lloyd 2004), Ginkgo biloba ), and Schisandra chinensis (Turcz.) Baill (Sun et al. 2016). In our experiments, we were also able to effectively use this approach for C. avellana calli.
ABA has been used as a pretreatment to increase sucrose absorption and accumulation from the medium, causing the formation of a glassy state in cells (Giladi et al. 1977;Wolkers and Hoekstra 2003). Besides, this hormone is involved in the expression of specific genes which produce specific proteins and antioxidant enzymes that are responsible for cell protection and oxidative stress tolerance, respectively (Cutler et al. 2010). Burch and Wilkinson (2002) reported high recovery compared to control on cryopreservation of Ditrichum cornubicum (Paton) by using both ABA and sucrose; also, a high recovery was recorded on the cryopreservation of Brassavola nodosa (L) Lind. (Orchidaceae), by applying ABA alone as the pretreatment (Mata-Rosas and Lastre-Puertos 2015).
Based on the results of our first experiment, the callus growth was reduced by increasing the pretreatment period on medium containing 3 mg l − 1 ABA to15 days (Fig. 3). These findings are in line with the study of Burritt (2008) who reported a 15-23% decrease in the growth of adventitious shoots of Begonia x erythrophylla by enhancing the ABA concentration from 0.5-1 mg l − 1 up to 1.76 mg l − 1 . We also observed that prolonging pretreatment duration up to 20 days on medium with 2 mg l − 1 ABA had a positive effect on calli survival after either desiccation or cryopreservation. In other words, increasing the pretreating time caused the calli to tolerate longer desiccation times; however, the highest growth rate was observed within 2, 2.5, and 3 h of desiccation. Even in the control conditions, a 25-day preculture inhibited calli development (Fig. 4). ABA can cause the accumulation of sucrose and glucose in the cells (Lu et al. 2009); as a consequence, pretreating samples with high concentrations of ABA, as well as extending the preculture period on ABAcontaining media, may result in a decrease in the growth rate due to excessive sugar accumulation.
Cryopreservation is an acceptable method for the longterm storage of genetic resources because all biological activity of cells is stopped at this temperature, and materials can be preserved without alteration or modification for an extended period of time (Gonzalez-Arnao et al. 2008;Engelman 2011). Our findings proved that the length of the LN-storage did not affect the calli's survival as no significant difference was observed in regard to the survival of calli between one and 30 days of LN-storage. This result is in agreement with some previous studies on the cryopreservation of other plant species such as Dioscorea deltoidei, sugarcane (Saccharum sp.), and Pinus radiata (Martinez-Montero et al. 1998;Hargreaves et al. 2002;Mandal and Dixit-Sharma 2007;Turner et al. 2001). However, in regard to the effect of the LN-storage period on the survival rate, there are some exceptions. Germination of Hevea pollens has been decreased from 20% with one month of storage to 2% after 5 months (Hamzah and Leen 1986). Conversely, in some Prunus mume cultivars, the germination of pollens kept in LN for one to four years was significantly increased, as compared to unfrozen pollens (Withers and Engels 1990).

Conclusion
The present work demonstrated the successful cryopreservation of C. avellana callus for the first time. Our study showed that desiccation-based cryopreservation was easy to apply. We also examined the effects of ABA concentration, preculture duration on the medium containing ABA, and desiccation length to achieve the highest survival rate, which was obtained to be 45.79% in our study. However, further studies into many parameters and even other protocols are required to improve higher survival rates; also, as the sample was callus, genetic stability testing is essential.