Storage of Pineapple Shoot Tips in Liquid Nitrogen for Three Years Does Not Modify Field Performance of Recovered Plants


 Pineapple is the third most important tropical fruit traded after banana and mango. In recent years, pineapple producers have faced production challenges due to unpredictable weather patterns as a consequence of climate change. In order to develop new genotypes with improved tolerance to these biotic and abiotic stresses, it is necessary to have access to a diverse gene pool, however, the germplasm currently stored in field gene banks are at risk from prevailing environmental conditions. Therefore, there is a need to develop and implement supplementary strategies to conserve plant germplasm under controlled conditions in the laboratory and cryopreservation represents the most viable option for this. To date, methods have been developed for cryopreservation of pineapple shoot tips but there is a lack of information on whether extended periods of storage in liquid nitrogen (LN) has any effect on the regeneration capacity and field performance of cryostored germplasm. Hence, the current study investigated the field performance of pineapple shoot tips after storage for 24 h, one, two and three years. Results were collected following nine months for plant morphological characteristics and 14 months for evaluation of fruit and nutritional characteristics. The results indicated that storage of pineapple shoot tips for up to three years did not have any adverse effects on field performance of plants or on fruit characteristics. This provides evidence that cryopreservation offers a suitable tool for the long term storage of germplasm.


Introduction
The cultivation of tropical and subtropical fruit makes a valuable contribution to the Gross Domestic Product in many developed and developing countries. In terms of volume, pineapple (Ananas comosus L. Merr.) represents third most traded tropical fruit after banana and mango (Leal and d'Eckenbrugge 2018).
In 2019, global production of pineapples was approximately 28.18 metric tons with Costa Rica, the Philippines and Brazil being the leading producers (Shahbandeh 2020). Pineapples are consumed primarily as fruit and juice (fresh and canned). The esh is rich in vitamins, antioxidants and phenolic compounds with notable anti-in ammatory bene ts (Gil et al. 2006 climate change has emerged as a global environmental concern with impacts predicted in many sectors including agriculture. Unpredictable weather patterns mean that annual rainfall events are no longer occurring as normal and water scarcity is becoming a major problem. In addition, global warming leading to higher temperatures mean that fruit are expose to higher temperatures for extended periods of time leading to deterioration in fruit quality parameters, for example sunburn, changes in texture, nutritional components, etc. (Bindi et al. 2001). Secondary effects include changes in the migration patterns of pests, increased incidence of both pests and diseases, changes in weed pressure, etc. (Prasad and Chakravorty 2015). Hence, climate change is threatening the sustainability of production systems and efforts need to be focused towards the development of mitigation strategies to improve the resilience of agricultural systems (reviewed by Parajuli et al. (2019)). This includes the adoption of a holistic approach encompassing the development of improved management practices coupled with the production of superior genotypes capable of withstanding the imposed stresses.
In order to generate improved genotypes, it is necessary to have access to a diverse gene pool. With regard to pineapple, germplasm is stored in eld gene banks, greenhouses or as in vitro cultures (Souza et al. 2007). Brazil is considered one of the centers of origin for pineapple and houses the Pineapple Active

Materials And Methods
Pineapple buds (cultivar MD-2) from eld-grown plants were initiated in vitro as per Daquinta and Benegas (1997). For cryopreservation, the droplet vitri cation technique was performed as outlined by For exposure to the vitri cation solution, shoot tips were transferred to Petri dishes with lter paper moistened with PVS3 solution [50% glycerol (m:v) and 50% sucrose (m:v)] at 0°C. There was 5 mL of PVS3 supplied to each Petri dish. The Petri dishes were placed on ice for 60 min. Following this, shoot tips were transferred to strips of aluminum foil (4 x 0.5 x 0.005 cm with 5 shoot tips per foil strip) containing micro-drops (0.1 mL) of PVS3 solution. The aluminum foil strips were maintained on ice until transfer to cryovials which were subsequently immersed in LN for 24 h, one, two or three years.
Shoot tips were recovered at room temperature as follows: PVS3 solution was discarded and replaced with 1 mL of MS medium supplemented with 1 M sucrose for 20 min. For re-growth after cryostorage, shoot tips were cultured on MS salts and vitamins, 100 mg l − 1 myo-inositol, 0.1 mg l − 1 thiamine-HCl, 30 g l − 1 sucrose, 4.4 µM 6-benzyladenine (BA) and 5.3 µM naphthaleneacetic acid (NAA) (Daquinta and Benegas 1997). Plants from shoot tips that were not immersed in LN but were exposed to the cryopreparative stages were used as a control. To conduct simultaneously the acclimatization and eld growth of the ve groups of plants, in vitro procedures were not performed at the same time.

Results And Discussion
The current study reports on the eld performance of shoot tips of pineapple stored in LN for up to three years. The results showed that more than 90% of shoot tips retrieved from LN regenerated shoots following 45 d of re-growth. This regeneration rate was similar to the results obtained from the control (non-cryopreserved) shoot tips. Souza et al. 2016 reported 44-86% survival in cultivated and wild pineapple following cryopreservation using droplet vitri cation with PVS2. Following re-growth in vitro, plants were successfully hardened-off with 97% survival across all treatments (i.e. shoot tips stored for 0, 24 h, one, two or three years in LN). Similarly, Souza et al. (2016) also reported high survival (100%) following hardening-off of cultivated and wild pineapple after droplet vitri cation. Furthermore, all plants survived transplanting into the eld environment (data not shown). Table 1 summarizes the results obtained for the eld performance of shoot tips that were cryostored and for control plants. The phenotypic characteristics evaluated included plant traits after nine months of growth in the eld and fruit characteristics at harvest (14 months). Overall, the results indicated no signi cant differences in the eld performance of plants regenerated from cryopreserved shoot tips, irrespective of the storage duration in the cryogen. Furthermore, the characteristics of plants regenerated after cryostorage were similar to those that had not been cryostored. The abovementioned observations were recorded for the plant morphological indicators assessed after nine months of eld growth (i.e. plant height, characteristics of the D leaf, stem base diameter and plant fresh weight). This rst level of analysis indicated that cryopreservation did not alter plant physical characteristics. Following an additional ve months, it was con rmed that the fruit characteristics (size and mass) and quality parameters (Brix, ascorbic acid, total titratable acids, overall acidity and pH) were also similar in plants stored in LN for different durations (and in the control).   2019) noted that cryopreservation of wild rose shoot tips did not affect biochemical attributes or pollen characteristics following plant growth in the eld. Furthermore, in the present study, the results from eld trials showed that not only were the fruit characteristics of pineapples regenerated from cryostored and non-stored shoot tips similar, but also the nutritional components of fruit were similar (Table 1). In this regard, the ascorbic acid content was similar in fruit stored for 0, 24 h, one, two and three years, i.e. 76.44 ± 0.12 to 76.53 ± 0.05 mg/100 ml juice (Table 1). This provides evidence that cryopreservation did not alter the nutritional characteristics of stored germplasm.
In theory, explants stored cryogenically can be maintained for an inde nite period of time (Engelmann 1997 To the best of our knowledge, this is the rst publication evaluating pineapple agricultural traits after cryopreservation of shoot tips for up to three years. Previously, our group has studied the effects of LN on the subsequent germination and growth of common bean, tomato, maize, tobacco, sorghum, chickpea, neonotonia and teramnus seeds. These studies showed that cryostorage induced transient morphological, physiological and biochemical changes that gradually disappeared as plants developed (Cejas et