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Article

Cryopreservation and Cryotolerance Mechanism in Zygotic Embryo and Embryogenic Callus of Oil Palm

by
Qiuen Wei
1,2,†,
Peng Shi
2,3,†,
Faiza Shafique Khan
3,4,†,
Yin Min Htwe
3,4,
Dapeng Zhang
2,3,
Zhiying Li
2,3,
Xueke Wei
2,3,
Qun Yu
3,4,
Kaibing Zhou
1 and
Yong Wang
2,3,*
1
School of Horticulture, Hainan University, Haikou 570228, China
2
Hainan Key Laboratory of Tropical Oil Crops Biology/Coconut Research Institute of Chinese Academy of Tropical Agricultural Sciences, Wenchang 571339, China
3
Hainan Key Laboratory for Biosafety Monitoring and Molecular Breeding in Off-Season Reproduction Regions/Sanya Research Institute of Chinese Academy of Tropical Agricultural Sciences, Sanya 572000, China
4
Hainan Yazhou Bay Seed Laboratory, Sanya 572025, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Forests 2023, 14(5), 966; https://doi.org/10.3390/f14050966
Submission received: 1 April 2023 / Revised: 30 April 2023 / Accepted: 4 May 2023 / Published: 7 May 2023
(This article belongs to the Special Issue Biotechnology in the Propagation of Forest Trees)
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Abstract

:
Oil palm (Elaeis guineensis) is the highest oil-yielding commercially grown perennial tree. Oil palm germplasm conservation and in vitro clonal propagation strengthened the world’s efforts to ensure future food security. Cryopreservation provides long-term storage for germplasm. The storage of plant material at cryogenic temperatures (−196 °C) following dehydration causes cryoinjury. The cryotolerance mechanism has rarely been studied in oil palm zygotic embryos (ZE) and embryogenic calli (EC). A simple and effective cryopreservation method was established for ZE. ZE surrounded by endosperm was air-dried for 3 days without any complicated chemical pre-treatments before cryopreservation, while the viability rate and following germination rate could reach up to 96.67% and 90.88%, respectively. As for EC, the preferred method could be pre-culture in liquid MS medium with 0.3 M sucrose for 12 h and PVS2 treatment for 5 min prior to cryopreservation, and the viability rate reached 68.33%. SSR markers were used to verify the genetic stability after cryopreservation. In addition, changes in enzyme activities (CAT, POD, and SOD) showed a consistent trend with H2O2 production among ZE samples, indicating that these antioxidants were involved in ROS scavenging. Furthermore, differently expressed genes (DEGs) related to ROS, osmotic, and cold stress responses were selected for correlation network analysis. Most genes involved in ROS production (RBOH, PAO, and PRX) and ROS scavenging (APX, PER, SOD, CAT, GPX, and AOX) showed higher expression levels in EC, suggesting that EC was more sensitive to oxidative stress than ZE. The cryotolerance mechanism was further summarized accordingly. These results contributed to cryopreservation methods and provided a better understanding of cryotolerance in oil palm.

1. Introduction

Oil palm (Elaeis guineensis Jacq.) is the highest oil-yielding crop and palm oil is the world’s most consumed and traded vegetable oil [1]. Oil palm is a perennial tree with a long breeding cycle and a single growing apex [2]. Generally, oil palm propagates through seeds, whereas seed propagation is hampered by a long germination period and low germination rates. Oil palm does not produce any offshoots, so in vitro tissue culture is the only means of vegetative propagation [3,4,5]. In recent years, micropropagation via somatic embryogenesis has become an efficient way for the in vitro propagation of commercial tree crops, such as palms [6,7]. Oil palm is potentially propagated by somatic embryogenesis; 10 million in vitro cultured oil palm plantlets were produced worldwide [8]. The most common cryopreservation material in oil palm is somatic embryos (SE). The survival rate of cryopreserved SE displayed an average value of 19.1% out of 29 clones, whereas the survival rate of 20 years was 33.2% out of 28 clones. Unfortunately, the in vitro clonal propagation of SE has limitations; 28 survived clones were lost due to re-growth decline [9], low in vitro regeneration, and long-term subcultures that can produce flower abnormalities and somaclonal variations [5,10,11]. In contrast, zygotic embryos (ZE) are ideally suitable for germplasm conservation [12,13,14].
The early attempts at the in vitro conservation of oil palm germplasm focused on ZE, polyembryoids, seeds, kernels, embryogenic cell suspensions, and pollens [4,9,15,16]. Due to their relatively small size compared to seeds and kernels, ZE is preferred for oil palm cryopreservation. Larger tissues are more confined by freezing and desiccation sensitivity [17]. The in vitro regeneration of oil palm polyembryoids has been successfully established via a vitrification-based cryopreservation method with a 45% survival rate [3]. Cryopreservation techniques are successfully used for the germplasm storage of commercial palms, such as oil palm, date palm, and coconut [18,19,20]. Until now, no further reports have been published on the improved cryopreservation protocols with a high survival or viability rate.
Cryopreservation methods are different and include the older classic methods based on the freeze-induced dehydration of cells. However, classical techniques have been applied to callus and undifferentiated cells [21]. Cryopreservation involves storage at ultra-low temperatures, such as LN (liquid nitrogen) at −196 °C. All metabolic changes are stopped at such a temperature and no genetic changes occur during storage. As a result, further research can be conducted with plant tissues [16]. The plant cryopreservation method requires several steps, such as osmotic protection, rapid cooling, and recovery [22]. The critical tool for successful cryopreservation by vitrification is to induce tissue tolerance to dehydration with a highly concentrated vitrification solution [23]. Vitrification has been successfully used in the Coffea arabica zygotic embryo, and increased the germination rate (90%) after dehydration with plant vitrification solution 2 (PVS2) for 10 min at 0 °C before LN treatment [24]. LN treatment is linked with cryoinjury associated with stress-associated factors, such as the failure of tissue to tolerate dehydration and temperature. ROS (reactive oxygen species) induced oxidative stress can significantly impact the cryopreserved material’s success or total failure. The acute effects of oxidation and dehydration may be reduced by combining antioxidants in cryopreservation methods. Antioxidants and antioxidant enzymes, such as catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), and ascorbic peroxidase (APX), play an essential role in ROS-scavenging [25].
RNA sequencing is used to analyse the molecular changes of plant material subjected to cryopreservation. In grapes, RNA sequencing was performed to evaluate the gene expression pattern during zygotic embryo cryopreservation. The expression profiles showed the up- and down-regulations of genes related to osmotic and freezing injury and tolerance [26]. Stress-responsive transcription factors (HSFs, WRKY, NAC, bZIP, and AP2/ERF) are highly expressed during sequential stages of cryopreservation in the banana shoot apical meristem [27]. The antioxidant enzyme genes SOD, POD, CAT, and APX served as key indicators of oxidative stress-induced genes in cryopreservation [28]. Further, the success of the cryopreservation method could be obtained only after evaluating the cryopreserved material’s genetic stability. The maintenance of genetic stability is essential for the long-term storage of plant material. It requires genetic tools for evaluating genetic stability. Various studies have reported the genetic stability assessment via DNA marker technology, such as simple sequence repeat (SSR), random amplified polymorphism DNA (RADP), and methylation-sensitive amplified polymorphism (MSAP) [1,29,30]. The Inter Simple Sequence Repeat (ISSR) markers are used to study genetic stability. The ISSR-PCR method is easy and rapid and contains most other markers’ (SSR, RADP, and MSAP) benefits. RADP and ISSR detected no variability in in vitro clonal multiplied plantlets of Alpinia calcarata Rosc. [31]. The ISSR marker technique was used for the genetic stability of cryopreserved mulberry (Morus species). ISSR markers detected no genetic difference between regenerated plantlets [32]. ISSR markers have been used to detect the genetic stability of citrus [33], apple [34], pineapple [35], and lotus tenuis [36]. There is a need to develop an improved cryopreservation method for oil palm conservation. In this study, cryopreservation methods were developed for the ZE and embryogenic calli (EC) of oil palm, respectively; the genetic stabilities of both rooted seedlings from ZE and regenerated plantlets from EC were evaluated with simple sequence repeats markers. We used transcriptome analysis to investigate the gene expression pattern on sequential stages of cryopreservation. Meanwhile, during cryopreservation and recovery, changes in the ROS content (H2O2) and enzyme activity of several antioxidants and genes involved in ROS, in osmotic and cold stress, were investigated, and the mechanisms for cryopreservation and cryotolerance were predicted.

2. Materials and Methods

2.1. Plant Material

ZE and EC of oil palm were selected for cryopreservation. Mature fruits were collected from a 15-year-old Tenera oil palm tree to prepare ZE and EC samples. The tree was located in the National Germplasm Nursery for Tropical Palms (19° 33′ N, 110° 47′ E; Wenchang, China).

2.2. Cryopreservation Methods

ZE was submitted to the cryopreservation procedure as follows (Figure 1a): ZE surrounded by part of endosperm (about 4 mm × 4 mm) was cut from mature fruits and then treated with 4% sodium hypochlorite (v/v) for 20 min to obtain sterile materials [37]. Fresh ZE (E1) with endosperm was air-dried on filter paper at room temperature for 3 days to dehydrate ZE (E2) with endosperm for cryopreservation. E2 samples were put into 2 mL cryotubes and then immersed in liquid nitrogen for cryopreservation for 1 d, 3 d, 7 d, 20 d, 40 d, and 60 d, respectively. After thawing in a 40 °C water bath for 8 min, ZE (E3) was extracted from the endosperm with a tweezer and scalpel. Inoculate ZE (E3) in solid Y3 medium was supplemented with 4 g/L of phytagel (P8170, Solarbio Co., Ltd. Beijing, China), 30 g/L of sucrose (S8270, Solarbio, China), and 2.5 g/L of activated charcoal (C7261, Solarbio, China) [38]. Germinated ZE (E4) was obtained 7 days later and rooted seedling (E5) was obtained 80 days later. The culture condition was dark and the temperature was 27 °C.
EC was induced from the stripped ZE in the Y3 medium supplemented with 4 g/L of phytagel, 30 g/L of sucrose, 2.5 g/L of activated charcoal, and 120 mg/L of 2,4-dichlorophenoxyaceticacid (2,4-D) (D8100, Solarbio Co., Ltd. Beijing, China) for 3–6 months [39]. Embryogenic callus was submitted to the cryopreservation procedure as follows (Figure 1b): Fresh EC (C1) was pre-cultured in liquid MS medium containing different concentrations of sucrose (0.3 M, 0.5 M, 0.7 M, and 0.9 M) for 12 h, treated with MS liquid medium containing 10% DMSO and 0.7 M sucrose for 30 min, and then treated with PVS2 (MS + 0.4 mol/L Sucrose + 30% (w/v) glycerol + 15% (w/v) ethylene glycol + 15% (w/v) DMSO) for different times (5 min, 10 min, 30 min, 60 min) [30]. Dehydrated EC (C2) were put into 2 mL cryotubes, immersed in LN for more than 12 h, and then rapidly thawed in the 40 °C water bath for 3 min and washed in an unloading solution containing 1.2 M sucrose for 20 min to get thawed EC (C3) [1]. Part of the C3 samples was submitted to a triphenyl tetrazolium chloride (TZ) test according to Copeland and McDonald [40] as an early viability test, and the rest were transferred onto the same medium used in EC induction for recovery. Recovered EC (C4) with a new fresh callus was obtained around 30 days later, and then submitted to tissue culture procedures according to previous methods [39]. Regenerated plantlets (C5) were obtained 180 days later via somatic embryo (SE) and regenerated shoot. All the ZE and EC samples obtained from the above-mentioned cryopreservation procedures were shown together (Figure 2). Samples were plunged immediately in liquid nitrogen and stored at −80 °C for subsequent experiments.

2.3. Tissue Sectioning and Microscopy

Alive and dead samples from both thawed ZE (E3) and thawed EC (C3) were collected for tissue sectioning and microscopy according to our previous work [39]. Samples were fixed overnight in (Coolaber SL1620-500ML) formalin-aceto-alcohol (FAA) fixation fluid (45% ethanol, 6% acetic acid, 5% formaldehyde) [41]. Tissue blocks (<5 mm3) were placed onto a pre-labeled tissue base mold and were then embedded into OCT (Optimal Cutting Temperature) compound cryo-embedding media (BL557A, Biosharp, China) and frozen at −20 °C in the cryostat of a cryotome (CryoStar NX50 OPD, Thermo Fisher, Waltham, MA, USA). The frozen tissues were sectioned into 20 μm and placed onto a standard glass slide. Samples were stained with Saframin O/fast green kit (G1375, Solarbio Co., Ltd. Beijing, China) and observed using an epifluorescence microscope (Olympus BX53, Olympus, Tokyo, Japan).

2.4. Detection of H2O2 Content and Antioxidant Enzyme Activities

In order to investigate the changes in ROS and antioxidant enzyme activities during cryopreservation and recovery, the content of hydrogen peroxide (H2O2) and the enzyme activities of CAT, POD, SOD, GPX, and APX among these samples (E1, E2, E3, E4 and C1, C2, C3, C4) were determined using kits (BC3590, BC0175, BC4785, BC0090, BC0220, and BC1195, respectively, Solarbio Co., Ltd. Beijing, China) according to the manufacturer’s instructions.

2.5. Genetic Stability Detection

In order to investigate possible genetic variation before and after cryopreservation, gene polymorphism was detected in two groups: fresh ZE (E1) and rooted seedling (E5) from zygotic embryos, as well as fresh EC (C1) and regenerated plantlet (C5) from embryogenic callus, using SSR markers and PAGE analysis. SSR primers (Table S1) were selected according to a previous publication [42] and were synthesized by BGI, China. According to the manufacturer’s protocol, the total genomic DNA of these samples was extracted using a rapid plant genomic DNA isolation kit (Sangon Biotech, Shanghai, China) and then diluted to a working concentration of 20 ng/μL with Tris-EDTA (TE) buffer for SSR analysis.

2.6. Transcriptome Analysis

Samples of ZE (E1, E2, E3, E4) and EC (C1, C2, C3) were selected for RNA-seq. However, recovered EC (C4) was not included because we had mainly focused on changes just before and after cryopreservation, while C4 was about 30 days later than the previous sample, C3, and the changes could be too complicated to investigate. Total RNA extraction was performed using a Trizol reagent kit (Invitrogen, USA) according to the manufacturer’s instructions. The extracted total RNA was treated with DNase I (TaKaRa, Japan) to remove contaminated genomic DNA. RNA sequencing and standard transcriptome analysis were performed by Gene Denovo Biotechnology Company (Guangzhou, China).

2.7. qPCR Validation

Samples (E1, E2, E3, E4 and C1, C2, C3, C4) were used for qPCR analysis. Total RNAs were extracted using a Trizol reagent kit (Invitrogen, USA) and cDNA were prepared using a RevertAidTM first-strand cDNA synthesis kit (Fermentas, Lithuania) according to the manufacturer’s instructions. Primers (Table S2) for qPCR were designed by NCBI Primer-BLAST and were synthesized by BGI Tech (Shenzhen, China). The qPCR experiments were performed on ABI QuantStudio™ 3 (Applied Biosystems, Foster City, CA, USA) using 2× PowerUp SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA) according to our previously published procedure [43].

2.8. Statistical Analysis

The multiple comparisons were performed by ANOVA analysis. Heatmaps were generated using R 4.0.3 [44] and Rstudio 1.4.1103 [45]. A correlation network was generated using Cytoscape 3.3.0 [46].

3. Results

3.1. Effect of Dehydration and Cryopreservation Time on Viability and Germination of ZE

Dehydration time is important for the ZE to reach suitable water content (around 30%) for the subsequent cryopreservation procedure (Figure 1a). Different treatments on dehydration time were compared and the results showed that the viability rate and following germination rate reached 96.67% and 90.88%, respectively, at a dehydrating time of 3 d (Table 1). None of the samples survived under a too short (0 d) or too long (10 d) dehydration time. Furthermore, more samples were tested under the same conditions to verify the feasibility of this cryopreservation procedure. Statistical analysis of 8 repeated experiments with up to 498 samples showed that the viability rate was 97.39%, and the following germination rate reached 93.98% (Table S3).
Moreover, the viability and germination rates of the samples stored in liquid nitrogen for 1 d, 3 d, 7 d, 20 d, 40 d, and 60 d were investigated. The results showed no significant difference among different cryopreservation times (Table 2), indicating that it is a reliable procedure for the long-term preservation of the zygotic embryo.

3.2. Effect of Pre-Culture and PVS2 Treatments on the Viability Rate of EC

Treatment with the protective agent PVS2 is a critical step in the cryopreservation of embryogenic callus. EC samples were treated with vitrification solutions containing various sugar as osmoprotectants, and the viability rate was investigated by the presence of recovered EC (C4). The results showed that with the prolongation of PVS2 treatment time, the viability rate of EC decreased gradually from 68.33% to 25.00% at a lower concentration of sucrose (0.3 M), while it increased gradually from 38.33% to 76.67% during pre-culture at 0.5 M sucrose. On the other hand, the viability rate declined rapidly to less than 25.00% at a higher concentration of sucrose (0.9 M), indicating that over-dehydration may cause damage. Among all these treatments, three of them with viability rates over 60% (in bold), were selected for multiple comparisons, and there was no significant difference (Table 3), suggesting that the preferred method could be pre-culture in liquid MS medium with 0.3 M sucrose for 12 h and PVS2 treatment for 5 min prior to cryopreservation. Furthermore, the statistics of six repeated experiments for this optimized treatment on embryogenic callus showed an average viability rate of 65.20 ± 4.44% (Table S4).

3.3. Analysis of Cell Viability, H2O2 Content, and Antioxidants Activity

Cryopreservation in low temperatures may have some deleterious effects. To investigate possible damages, alive and dead samples from both thawed ZE (E3) and thawed EC (C3) were collected for tissue sectioning and microscopy. The staining results showed the presence of both cell nuclei and cytoplasm (Figure 3a) and the plumule with intact structure in alive E3 (Figure 3b). On the contrary, few cell nuclei were observed (Figure 3c) and the plumule structure was damaged in dead E3 (Figure 3d), indicating that the protection of the plumule against previous dehydration and cryopreservation procedures could be essential for ZE. On the other hand, although there was no clear key structure such as plumule in EC, both cell nuclei and cytoplasm were also visible in alive C3 (Figure 3e,f), while empty cells without nuclei were observed in dead C3 (Figure 3g,h), suggesting that severe damage was caused by PVS2 treatment on EC.
Cryopreservation procedures (dehydrating, freezing, and thawing) may result in physiological and biochemical changes, which can lead to increased ROS production and oxidative damage. The content of H2O2 and the enzyme activities of five antioxidants (CAT, POD, SOD, GPX, and APX) at different cryopreservation stages were also detected accordingly. As expected, significant increases in H2O2 content against E1 or C1 were observed in both ZE and EC dehydrated samples. The H2O2 production at the dehydration stage of E2 was much higher than at the C2 stage (Figure 3i), indicating that ZE experienced intense dehydration by air drying and EC suffered relatively gentle osmotic stress from dehydration by sucrose. As for corresponding ROS scavenging by antioxidants, changes in the enzyme activities of CAT, POD, and SOD showed a consistent trend with H2O2 production among ZE samples, indicating that these antioxidant enzymes may play more important roles than GPX and APX. However, no obvious rule was found among EC samples.

3.4. Genetic Stability of Cryopreserved Material

Both rooted seedling (E5) from the zygotic embryo (ZE) and regenerated plantlet (C5) from embryogenic callus (EC) after cryopreservation, together with their corresponding non-cryopreserved fresh ZE (E1) and EC (C1), were used to assess the genetic stability by comparing their PCR products from eight SSR markers. Almost all the PAGE results of eight SSR markers showed identical bands between the regenerated plantlet (C5) and its original callus (C1) (Figure 4), indicating no variation resulting from EC cryopreservation. As for ZE cryopreservation, most PCR products showed polymorphism to a certain extent, as expected, between rooted seedlings (E5) and fresh ZE (E1) before cryopreservation. Unlike how C5 was regenerated from C1 by tissue culture (asexual propagation) with the same genotype, E5 was not originated from the same zygotic embryo of E1, which was completely destroyed by DNA extraction. E5 and E1 were from the same oil palm bunch, but could be different genotypes due to sexual hybridization. These results suggested that cryopreservation may not affect genetic stability and could be used for germplasm preservation.

3.5. Transcriptome Changes

Transcriptional changes in samples of ZE (E1, E2, E3, E4) and EC (C1, C2, C3) were analyzed by RNA-seq and a total of 160.08 Gb clean data were obtained. Principal Component Analysis (PCA) based on gene expression levels showed good reproducibility among replicates for all samples (Figure 5a). Differentially expressed genes (DEGs) were obtained from pairwise comparisons and showed little change between E2 and E3 (50 up and 63 down), possibly due to the heavy protection of the surrounding endosperm. In contrast, dramatic changes were observed (Figure 5b) between E3 and E4 (9790 up and 4333 down), probably resulting from the obvious morphological changes from embryo to seedling. To confirm the reliability of RNA-seq data, nine DEGs were randomly selected for qPCR validation (Figure S1).
Correlation analysis between RNA-Seq and qPCR validation (Figure 5c), according to their Fragments per Kilobase of transcript Per Million mapped reads (FPKM) values and relative expression levels, showed high consistency (R2 = 0.7145), indicating that the transcriptome data obtained by RNA-Seq were reliable for further analysis. DEGs were submitted to trend analysis, and 20 profiles among ZE samples (Figure S2) and 8 profiles among ZE samples (Figure S3) were observed. KEGG enrichment and expression heatmap of the abiotic stress-response genes involved in expected profiles (3, 6, 16) among ZE samples and in profiles (1, 5, 6) among EC samples were shown in Figure S4 and Figure S5, respectively.

3.6. Response of DEGs in Cryotolerance

Differentially expressed genes during cryopreservation can help to explain the reason for the increased cryotolerance. In order to investigate the relationship of the genes involved in cryotolerance of both ZE and EC samples, a total of 37 genes related to ROS, osmotic, and cold stress response were selected (FPKM >10) for correlation network construction. The pairs that significantly correlated (p < 0.05) were included in the network, in which 16, 15, and 6 genes were involved in ROS, osmotic, and cold stress pathways (Figure 6a), respectively. The expression patterns of these 16 genes involved in ROS production (RBOH, PAO, PRX), ROS scavenging (APX, PER, SOD, CAT, GPX, and AOX), and related transcription factors (HSF, WRKY, MYB, etc.) in the ROS pathway were shown in detail by heatmaps accordingly (Figure 6b). Most genes involved in ROS production and ROS scavenging had high expression levels from C1-C3, while low expression levels from E1-E3 indicated that EC was more sensitive to oxidative stress than ZE.
The expression pattern of the genes involved in the pathway of osmotic stress (15 genes) and cold stress (7 genes) was also investigated, respectively. Most of the genes’ responses to osmotic stress were up-regulated in both ZE and EC (Figure S6). Some osmo-sensors (BAK and CNGC) were significantly expressed in E2 compared to E1 (Figure S6). In contrast, different osmo-sensors (RALF, LRX, FER) were significantly expressed in C2 compared to C1 (Figure S6), indicating that a different sensor mechanism occurred between the zygote embryo and embryogenic callus. Cryopreservation needs to undergo an extremely low temperature of −196 °C; the cold tolerance of plant tissues depends on cellular signal transduction pathways. The expression of DEGs in the pathway of the cold stress response (Figure S7) showed that cold sensor ANN was highly expressed during liquid nitrogen preservation in EC compared to ZE, while CNGC barely responded to liquid nitrogen treatment. Downstream core response genes, such as DREB/CBF and COR, barely responded after cryopreservation, which could be due to extremely rapid freezing in liquid nitrogen.

4. Discussion

ZE and EC are effective materials for the cryopreservation of plant germplasm conservation [16]. A successful cryopreservation method involved the sequential management of ROS-induced oxidative stress. Numerous cryopreservation-induced stresses (cold and osmotic stress) can damage the organ and cell, resulting in a decreased survival rate or viability. However, explant dehydration is the most critical step in cryopreservation [47,48,49]. Plants are susceptible to dehydration and low temperature and require cryogenic protective treatments to ensure their survival and cell structural integrity [15]. Cryoprotectants are commonly used to increase the survival rate of cryopreserved material [33].
In the present study, a high EC survival rate of 76.67% was observed during pre-culture at 0.5 M sucrose along with PVS2 treatment for 60 min, an improvement over the earlier report [3]. According to previously reported results, EC was successfully cryopreserved by pre-culture and PVS2 treatment. The sucrose concentration varies depending on how these species acclimate to dehydration, cold, and in vitro propagation conditions. PVS2 treatment at a suitable time could reduce the freezing point of plant cells and avoid ice crystal formation in the LN environment [50]. It has been demonstrated that EC could survive after being subjected to cryoprotectant treatments [1,51]. However, a high survival rate has rarely been observed due to EC’s fragile nature and increased susceptibility to dehydration and freezing. A survival rate of 63% was observed in cacao (Theobroma cacao L.) SE after pre-culture for 7 days with 1 M sucrose [52]. Pre-culture with 70 g/dm3 sucrose and PVS2 pre-treatment significantly affected the date palm regeneration rate [53]. In a past study, olive (Olea europaea L.) somatic embryos pre-treatment with 0.75 M sucrose and cryoprotectant for 3 d enhanced the survival rate [54]. Pre-culture with 0.7 M sucrose for 1 day at 5 °C following PVS2 treatment at 25 °C increased 80% shoot formation in apples (Malus domestica borkh. cv. Fuji) [55]. Similarly, stepwise droplet vitrification (12 h in 0.5 M sucrose and 10 min in PVS2) before direct immersion in LN for the cryopreservation of oil palm polyembryoids reached a 68% survival rate [30]. A high survival rate of oil palm somatic embryos clumps was reported with 16–18 h dehydration (30% water content) [56]. Chinese fan palm (Livistona chinensis) post-thaw embryos show an increase in the viability rate with 30%–18% (water content), whereas more extended dehydration with 13% (water content) causes severe damage to cells [57]. In contrast, the high survival and germination rate showed that ZE underwent protection by the endosperm and survived cryopreservation. Generally, endosperms play an important role in nurturing and protecting the ZE. Moreover, we optimized and improved the dehydration step (30% water content) with a high survival rate of 96.67% and a germination rate of 90% in comparison with the previously reported survival rate of 63% with 0.3 g H2O/g DW (dry weight) [15].
The positive accumulation of antioxidants in the improved dehydration procedure may increase cryotolerance to obtain a high survival rate. Increased antioxidant enzyme activities may serve as a signaling mechanism responsible for activating cryotolerance and ROS scavenging [25,58]. ROS-induced oxidative stress reduced the ability of the plant to survive following cryopreservation, eventually resulting in cryopreservation failure [59]. Higher tolerance in Agapanthus praecox cryopreservation was associated with high activities of CAT, SOD, and POD [60]. Moreover, H2O2 is a vital ROS that ameliorates oxidative stress damage in EC and affects cell viability during cryopreservation [60]. H2O2 induced oxidative stress at the step of dehydration and rapid warming is the major cause of cryo-injury during the cryopreservation of Arabidopsis seedlings [25]. H2O2 was found to be one of the major causes of oxidative damage in Paeonia suffruticosa pollen during cryopreservation [61]. This may be the result of osmotic stress and increasing ROS activity, which have also been described in vitrification-based cryopreservation [25].
Various studies reported no instability in the molecular analysis of cryopreserved plants [62,63,64,65]. SSR markers are used to determine genetic changes within one species and to identify the somaclonal variability. SSR makers have been used to analyze oil palm genetic diversity because of their high rate of polymorphism, multi-allelic nature, and locus-specific nature [66]. Similar results were reported in the embryogenic callus of oil palm [51]. In the present work, SSR marker band patterns revealed similar results. The results showed that there was no alteration in the genotype and determined the genetic stability of cryopreserved plant material [51].
Except for changes in physiology and cytology, transcriptomic changes also occurred during stress responses [67]. The cryopreservation protocol employed in this study primarily exhibits the up- and down-regulation of genes related to oxidative stress and tolerance mechanisms. ROS signaling was delivered to downstream TFs by Ca2+ and MAPK pathways, such as HSF, NPR, RAV, WRKY, and MYB. The expression level of these TFs altered sharply and then responded to osmotic and cold stress by activating target genes (Figure 6). During cryopreservation, MYB, WRKY, and HSF were mainly up-regulated in Arabidopsis seedlings. H2O2 commonly up-regulates these transcription factors, while MYB is specifically regulated by 1O2 (singlet oxygen) [68]. Mitogen-activated protein kinases (MAPKs) are involved in oxidative stress signal transduction during cryopreservation [69]. MAPK was mainly up-regulated in the EC and ZE dehydration and rapid freezing. The CBF/DREB genes are involved in a regulatory network that responds to cold stress [70], which includes various cold sensors, calcium signals, calcium-binding proteins, and the C-repeat binding factor/dehydration-responsive element binding pathways [71]. Similarly, cold sensors also showed different expression profiles. ANN up-regulated after LN preservation in the EC, while CNGC up-regulated after LN preservation in the ZE. The genes involved in ROS production are expressed differentially in different stages of cryopreservation. Polyamine oxidase is highly expressed in E2-E4, and RBOH in E4. On the other hand, studies have shown that PAO produces H2O2, regulates the enzyme activities of RBOH, CAT, and SOD, and plays an essential role in ROS homeostasis [72]. Most ROS scavenging genes were up-regulated in E4, such as APX, SOD, CAT, POD, and AOX (Figure 6). Inducing cryotolerance by reducing oxidative damage or ROS inhibition is an efficient way to enhance cell survival during and after cryopreservation.

5. Conclusions

This study explores cryopreservation methods and cryotolerance mechanisms for ZE and EC in oil palm (Figure 7). We selected plant material while considering essential parameters, such as the stage of explant (ZE, EC), tolerance, and viability. ROS-induced oxidative stress is a primary factor in viability loss. EC showed a high expression of ROS, osmotic, and cold-responsive genes. The low viability rate may be caused by EC fragility and sensitivity. Conversely, higher levels of antioxidant enzyme accumulation and endosperm protection are demonstrated to be responsible for the cryotolerance and high viability rate of ZE. Therefore, the cryopreservation methods optimized in this study are practical and efficient. The results achieved, especially with ZE, regarding the viability rate, genetic stability, and cryotolerance, reveal that the optimized cryopreservation protocol can be easily used for germplasm conservation and elite clonal propagation.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/f14050966/s1, Figure S1: qPCR verification of gene expression. Figure S2: Profiles ordered by the p-value significance among ZE samples. Figure S3: Profiles ordered by the p-value significance among EC samples. Figure S4: KEGG enrichment and expression heat map of genes involved in expected profiles among ZE samples. Figure S5: KEGG enrichment and expression heatmap of genes involved in expected profiles among EC samples. Figure S6: Pathway of osmotic stress response. Figure S7: Pathway of cold stress response. Table S1: Primers for SSR. Table S2: Primers of qPCR. Table S3: Effect of repeated experiments on cryotolerance of zygotic embryo. Table S4: Statistics of repeated experiments for the optimized treatment on embryogenic callus.

Author Contributions

Performed the experiment, Q.W. and P.S.; data curation, Y.M.H., D.Z., Z.L., X.W. and Q.Y.; writing—original draft preparation, Y.W., F.S.K. and Q.W.; writing—review and editing, F.S.K. and Y.W.; supervision, Y.W. and K.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

The RNA-seq data for this study can be found in the National Genomics Data Center-Genome Sequence Archive (GSA accession: CRA009437). Please access it from the following link: https://bigd.big.ac.cn/gsa/browse/CRA009437 (accessed on 5 January 2023).

Acknowledgments

This work was supported by the Hainan Provincial Natural Science Foundation (320RC739), the National Natural Science Foundation of China (No. 32071740), the earmarked fund for CARS-14 (China Agriculture Research System-Specific Oilseed Crops), the Foreign Youth Talent Program (QN2021034002L), and the post-doc project of Hainan Yazhou Bay Seed Laboratory (No. B21Y10301). We are very grateful to the National Germplasm Nursery for Tropical Palms for providing oil palm materials.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Cryopreservation procedures for ZE (a) and EC (b).
Figure 1. Cryopreservation procedures for ZE (a) and EC (b).
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Figure 2. Materials used for cryopreservation and genetic stability analysis. Fresh ZE (E1), dehydrated ZE (E2), thawed ZE (E3), germinated ZE (E4), and rooted seedling (E5) from zygotic embryos; Fresh EC (C1), dehydrated EC (C2), thawed EC (C3), recovered EC (C4), and regenerated plantlet (C5) from embryogenic callus.
Figure 2. Materials used for cryopreservation and genetic stability analysis. Fresh ZE (E1), dehydrated ZE (E2), thawed ZE (E3), germinated ZE (E4), and rooted seedling (E5) from zygotic embryos; Fresh EC (C1), dehydrated EC (C2), thawed EC (C3), recovered EC (C4), and regenerated plantlet (C5) from embryogenic callus.
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Figure 3. Analysis of cell viability, H2O2 content, and antioxidant enzyme activity. (a) Longitudinal section of alive thawed ZE (E3) stained with safranin O/fast green. The red color showed the presence of cell nuclei while the green color showed the presence of cytoplasm; (b) plumule with intact structure in alive thawed ZE (E3); (c) longitudinal section of dead thawed ZE (E3) stained with safranin O/fast green. Few cell nuclei were observed in red color while the majority of cytoplasm was shown in green color; (d) plumule with damaged structure in dead thawed ZE (E3); (e) section of alive thawed EC (C3) stained with safranin O/fast green; (f) visible cell nuclei in red color in alive thawed EC (C3); (g) section of dead thawed EC (C3) stained with safranin O/fast green; (h) empty cells in dead thawed EC (C3); (i) H2O2 and activities of enzymes: CAT, POD, SOD, GPX, and APX. The values with the same letters are not significantly different (p < 0.05).
Figure 3. Analysis of cell viability, H2O2 content, and antioxidant enzyme activity. (a) Longitudinal section of alive thawed ZE (E3) stained with safranin O/fast green. The red color showed the presence of cell nuclei while the green color showed the presence of cytoplasm; (b) plumule with intact structure in alive thawed ZE (E3); (c) longitudinal section of dead thawed ZE (E3) stained with safranin O/fast green. Few cell nuclei were observed in red color while the majority of cytoplasm was shown in green color; (d) plumule with damaged structure in dead thawed ZE (E3); (e) section of alive thawed EC (C3) stained with safranin O/fast green; (f) visible cell nuclei in red color in alive thawed EC (C3); (g) section of dead thawed EC (C3) stained with safranin O/fast green; (h) empty cells in dead thawed EC (C3); (i) H2O2 and activities of enzymes: CAT, POD, SOD, GPX, and APX. The values with the same letters are not significantly different (p < 0.05).
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Figure 4. SSR analysis of plantlets obtained from cryopreserved ZE and EC. M: DNA 2kb marker, E1 (fresh ZE) and E5 (rooted seedling) from zygotic embryos; C1 (fresh EC) and C5 (regenerated plantlet) from embryogenic callus.
Figure 4. SSR analysis of plantlets obtained from cryopreserved ZE and EC. M: DNA 2kb marker, E1 (fresh ZE) and E5 (rooted seedling) from zygotic embryos; C1 (fresh EC) and C5 (regenerated plantlet) from embryogenic callus.
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Figure 5. General information of transcriptome among biological replicates and qPCR verification of gene expression. (a) Principal Component Analysis (PCA) of seven samples. (b) DEGs in pairwise comparisons among different samples. (c) Correlation analysis between RNA-Seq and qPCR validation.
Figure 5. General information of transcriptome among biological replicates and qPCR verification of gene expression. (a) Principal Component Analysis (PCA) of seven samples. (b) DEGs in pairwise comparisons among different samples. (c) Correlation analysis between RNA-Seq and qPCR validation.
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Figure 6. Correlation network of genes involved in stress response during cryopreservation (a) and genes involved in ROS pathway (b).
Figure 6. Correlation network of genes involved in stress response during cryopreservation (a) and genes involved in ROS pathway (b).
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Figure 7. Graphical representation of cryopreservation and cryotolerance mechanism.
Figure 7. Graphical representation of cryopreservation and cryotolerance mechanism.
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Table
Table 1. Effect of dehydrating time on viability and germination rate of zygotic embryo.
Table 1. Effect of dehydrating time on viability and germination rate of zygotic embryo.
Dehydrating Time (d)Viability (%)Germination (%)
000
396.67 ± 0.03 a90.88 ± 0.03 a
480.00 ± 0.03 b69.58 ± 0.04 b
1000
Note: The values with the same letters are not significantly different (p < 0.05).
Table
Table 2. Effect of cryopreservation time on viability rate of zygotic embryo.
Table 2. Effect of cryopreservation time on viability rate of zygotic embryo.
Cryopreservation Time (d)Viability (%)Germination (%)
191.11 ± 0.02 a85.56 ± 0.03 a
390.00 ± 0.03 a83.33 ± 0.05 a
791.11 ± 0.04 a83.33 ± 0.03 a
2090.00 ± 0.03 a81.11 ± 0.04 a
4090.00 ± 0.03 a82.22 ± 0.06 a
6092.22 ± 0.03 a84.44 ± 0.02 a
Note: The values with the same letters are not significantly different (p < 0.05).
Table
Table 3. Effect of pre-culture in liquid MS medium with different sucrose concentrations and PVS2 treatment time on viability rate (%) of embryogenic callus.
Table 3. Effect of pre-culture in liquid MS medium with different sucrose concentrations and PVS2 treatment time on viability rate (%) of embryogenic callus.
+LN/
−LN		PVS2 Treatment
Time (min)		Sucrose Concentration (M)
0.30.50.70.9
+LN568.33 ± 0.02 a38.33 ± 0.1255.00 ± 0.1225.00 ± 0.04
−LN80.53 ± 0.1154.57 ± 0.2173.57 ± 0.0550.54 ± 0.10
+LN1063.33 ± 0.10 a43.33 ± 0.0846.67 ± 0.0623.33 ± 0.06
−LN77.54 ± 0.1458.57 ± 0.0861.39 ± 0.0850.61 ± 0.03
+LN3046.67 ± 0.0548.33 ± 0.0658.33 ± 0.1621.67 ± 0.06
−LN64.48 ± 0.0966.40 ± 0.0468.51 ± 0.1246.38 ± 0.14
+LN6025.00 ± 0.0476.67 ± 0.05 a55.00 ± 0.166.67 ± 0.02
−LN53.48 ± 0.1285.56 ± 0.1474.49 ± 0.120.52 ± 0.12
Note: Only viability rates over 60% with LN treatment (in bold) were selected for multiple comparisons. The values with the same letters are not significantly different (p < 0.05). +LN or −LN means with or without LN treatment after pre-culture and PVS2 treatment.
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MDPI and ACS Style

Wei, Q.; Shi, P.; Khan, F.S.; Htwe, Y.M.; Zhang, D.; Li, Z.; Wei, X.; Yu, Q.; Zhou, K.; Wang, Y. Cryopreservation and Cryotolerance Mechanism in Zygotic Embryo and Embryogenic Callus of Oil Palm. Forests 2023, 14, 966. https://doi.org/10.3390/f14050966

AMA Style

Wei Q, Shi P, Khan FS, Htwe YM, Zhang D, Li Z, Wei X, Yu Q, Zhou K, Wang Y. Cryopreservation and Cryotolerance Mechanism in Zygotic Embryo and Embryogenic Callus of Oil Palm. Forests. 2023; 14(5):966. https://doi.org/10.3390/f14050966

Chicago/Turabian Style

Wei, Qiuen, Peng Shi, Faiza Shafique Khan, Yin Min Htwe, Dapeng Zhang, Zhiying Li, Xueke Wei, Qun Yu, Kaibing Zhou, and Yong Wang. 2023. "Cryopreservation and Cryotolerance Mechanism in Zygotic Embryo and Embryogenic Callus of Oil Palm" Forests 14, no. 5: 966. https://doi.org/10.3390/f14050966

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