Cryopreservation of Arachis hypogaea L. varieties, from the IN-IAP-Ecuador Germplasm Bank.

: The peanut ( Arachis hypogaea L.) is recognized as one of the most important legume crops globally for its use in human food; it is widely distributed and cultivated in tropical and subtropical regions. The purpose of this study was to evaluate the cryopreservation of five peanut varieties conserved in the INIAP Germplasm Bank, testing cryopreservation methods, evaluating the germination percentage of whole seeds and embryonic shoots. Subsequently, two quantitative variables, shoot length and root, were evaluated. The average germination percentage of varieties and treatments was higher when embryonic axes were isolated with 99.31% than 86.06% seeds. The best germination percentage of the five varieties for seeds and embryonic shoots was obtained by the Peruvian variety with 88.13% and 92.50%. The best treatments by variety for the germination of whole seeds and embryonic axes were obtained by the treatment (desiccation and NL) for whole seeds (GS2) with 95.42% and embryonic axes with 92.83%. Ageing and cryopreservation treatments positively affected germination and seedling vigor in whole seeds and embryonic axes. The two quantitative variables, shoot and root length showed variability between the five varieties; significant differences were observed between the four treatments evaluated for whole seeds and embryonic axes. The three treatments for whole seeds (GS1, GS2 GS3) and the non-cryopreserved control treatment (GSC), as well as the treatments for embryonic axes (GEA1, GEA2 GEA3) and the non-cryo-preserved control treatment (GEAC), obtained good survival. They germinate whole seeds and embryonic axes with sprout development (aerial part) and root formation. With the most effective treatments for whole seeds (GS2) and embryonic axes (GEA2), the cryopreservation of the national peanut collection of the INIAP Germplasm Bank could be started.


Introduction
The peanut (Arachis hypogaea L.) is native to South America, where the Arachis genus is widely distributed [1] and is currently cultivated in tropical and subtropical regions [2]. This herbaceous species with erect, decumbent, or creeping vegetative growth presents two branching patterns: sequential with bushy and compact growth or alternate with prostrate or decumbent vegetative growth [3].
Although A. hypogaea seeds have been classified as orthodox by Hanson et al. [6], viability losses frequently occur even under optimal storage conditions [7]. This behavior may result from the high-fat content of their storage tissues [8,9] that can undergo autooxidation and generate free radicals, which damage proteins and nucleic acids [10]. Therefore, Arachis seeds can be more appropriately classified as sub-Orthodox, a subdivision of seeds, including species whose seeds can be stored under the same conditions as Orthodox, but for shorter periods [8,9,11].
Many crop varieties are in danger of extinction due to habitat loss and global changes. For example, climate change will be responsible for the 50% loss in the range of distribution of the wild populations of groundnut (Arachis sp.), potato (Solanum sp.) and cowpea (Vigna sp.) Moreover, due to its effects, 16-22% of these species will be extinct by 2055 [12]. Therefore the conservation of some species in germplasm banks such as seeds, field collections or in vitro is promoted [13]. However, in vitro storage can lead to somaclonal variation, which is a drawback in conservation programs. For this reason, cryopreservation at low temperatures for germplasm storage is considered the best method for conservation for indefinite periods, with lower risks of inducing genetic alterations [14,15] and fewer periodic tests of viability, reducing conservation costs [8].
There are several cryopreservation techniques; one of them is the desiccation technique that is based on exposing the tissues to a stream of air, compressed air stream or silica gel before rapid freezing by direct immersion in liquid nitrogen [16][17][18]. This approach has been used for orthodox seed embryonic axes [19][20][21][22][23][24] and recalcitrant seeds [23,[25][26][27][28]. On the other hand, the vitrification technique, in which the explants are pretreated with high concentrations of cryoprotectants, has been successfully applied to embryonic axes [29][30][31] of genera like orchids, peas and jack fruit. Furthermore, Runthala et al. [32] and Abdulmalik et al. [18] published protocols to preserve the embryonic axes of peanuts using cryoprotectants, with which they obtained variable levels of survival (40-90%) according to the genotype. Several methods have been developed for the cryopreservation of peanuts, such as desiccation in a laminar flow chamber [18] or the vitrification technique in embryonic axes [33], or both techniques in cultivated and wild species [34][35][36].
In Tacán et al. [37], the effects of accelerated ageing and cryopreservation were measured in seeds and embryonic axes of Phaseolus vulgaris L and Arachis hypogaea L. On the germination and vigor of the seedlings, they were using whole seeds and embryonic axes of a commercial variety of peanuts. It was observed that the ageing and cryopreservation treatments positively affected the germination and vigor of the peanut seedlings. In another study [38], our results show the important differences, attributable to the genotype, concerning the content of proline and the rest of amino acids, about the stress conditions related to desiccation tolerance and cryopreservation. The present research aimed to determine the effect of cryopreservation with three treatments and control for five varieties existing in the peanut collection conserved at the BG-INIAP to evaluate the method's usefulness for the conservation of the peanut Arachis hypogaea collection.

Plant Material
The INIAP germplasm bank provided the seeds of A. hypogaea. The selection of eight morphotypes that are detailed in Table 1 was made based on the taxonomic and morphological characterization carried out in the National Department of Plant Genetic Resources (DENAREF) of the INIAP, considering the following parameters: groups, species, subspecies, province (geographic distribution) and altitude. (Table 1). The previous process for this research was refreshing and multiplication to have an adequate number of seeds per treatment and repetition. Then, conditioning was carried out, considering the protocols of Monteros-Altamirano et al. [39]. The already conditioned materials remained in seed lots stored at 5 °C until use. In a laminar flow chamber, the embryonic axes of the previously disinfected seeds were extracted. For the disinfection process, the seeds were placed 5 min in 70% alcohol, 20 min in 10% NaOCl (commercial bleach) plus 2 to 3 drops of tween 20, rinsed three times with sterile distilled water and immersed in 5 NaOCl % plus 2 to 3 drops of tween 20 for 10 min. With occasional shaking and rinsing twice with sterile distilled water. Subsequently, the seeds were soaked in sterile distilled water for 3 h.
2.2 Seedling germination and growth tests The whole seed germination tests were carried out by placing four replicates of 10 seeds in 9 cm Petri dishes on two sheets of filter paper (previously moistened with 3.5 ml of distilled water, which was added periodically) in the dark 25 0C. The criterion for germination was the protuberance of the radicle of 1 mm, and it was quantified on day 10. The results are expressed as the germination percentages (%) and the number of days necessary to reach 50% of germination final (T50). In addition, the length of the radicle (mm) and the length of the shoots (mm) were also determined. The embryonic axes were cultured in sterile glass containers with MS medium [40] supplemented with 0.3 M sucrose. The germination temperature was 25 °C with illumination and a light / dark photoperiod of 16: 8 h. cold white fluorescent light (40 µmol m -2 s -1 ). Four 10-axis replicas per bottle were studied, quantified at ten days, and the percentage of germinated embryos, the T50, the length of the radicles (mm) and the length of (mm) were measured. For whole seeds, it was carried out in Treatment 2 (direct immersion in NL) and Treatment 4 (desiccation and immersion in NL); the seeds were introduced into cryovials and were introduced directly into liquid nitrogen. NL was removed from the cryovials after 1 h at room temperature (20 °C), and they were immediately placed in Petri dishes under the germination conditions indicated above. For the embryonic axes, the whole peanut seeds were immersed in liquid nitrogen for 5 minutes, then they were placed in a Petri dish at room temperature 180 °C, for one hour. After dissecting the embryonic axes under sterile conditions in a laminar flow chamber, ten embryonic axes were placed in each container; in total, there were four containers and 40 embryonic axes; each container contains a solid MS base culture medium supplemented with 20 g l -1 of sucrose.

Experimental Unit
The experimental unit for whole seeds (GS) consisted of a 10 cm × 6 cm petri dish containing ten whole seeds. For the embryonic axes (GEA), the experimental unit consisted of a 10 cm × 2 cm flask containing 30 mL of culture medium with ten embryonic axes.

Treatments
The GS and GEA were subjected to the following treatments: Control (C): untreated, Treatment 1: direct immersion in liquid Nitrogen (NL), Treatment 2: desiccation, Treatment 3: desiccation and immersion in NL.

Experimental Design
A completely randomized experimental design (DCA) with ten observations was used. For each treatment, four replicas of 10 whole seeds (GS) and embryonic axes (GEA) were made.

Data Analysis
The statistical program InfoStat version 2008 was used [41]. An analysis of variance and the Tukey test was performed to compare means (α = 0.05).

Handling the experiment 2.7.1 Desiccation: silica gel
For the drying of the seeds, fifty seeds were placed in Petri dishes (9 cm in diameter) containing a layer of dehydrated silica gel of approximately 5 g and covered by a filter paper disk in which the seeds were placed, which, once sealed, were kept for 3 hours at 20 °C.

Cryoconservasion
The embryonic axes were cultured in sterile glass containers with MS medium supplemented with 0.3 M sucrose. Germination temperature was 25 °C with a light / dark photoperiod of 16: 8 h with cold white fluorescent light illumination (40 µmol m -2 s -1 ). Four 10-axis replicas per bottle were studied, quantified at ten days, and the percentage of germinated embryos, the T50, the length of the radicles (mm) and the length of (mm) were measured. For whole seeds, it was carried out in Treatment 2 (direct immersion in NL) and Treatment The seeds of all the studied peanut varieties tolerated desiccation and cryopreservation treatments, both for whole seeds and for embryonic axes, since, in all cases, the treated seeds germinated. The four treatments (GSC, GS1, GS2 and GS3,) on the germination of whole seeds (at ten days) for the five varieties were evaluated. For the hypogaea variety ( Figure 1-black bar, Appendix 1), it was observed that the GSC germination percentages were higher than 87.50% with the Ecuadorian variety, and differences were detected with the other varieties. In GS1, the germination was higher than 62.50% of the Peruvian variety, and differences with the other varieties were also detected. The GS2 presented a germination superior to 88.75% with the hypogaea variety and presented differences between varieties. For GS3, the germination percentages were higher than 70.00% with the fastigiata variety. The GS1 and GS3 treatments delay germination start, although T50 values do not differ significantly from the control and each of the varieties.
In the same way, the effect of the four treatments on the germination of embryonic axes of the five varieties of A. hypogaea was analyzed ( Figure 1-gray bar, Appendix 2). In the four treatments (GEAC, GEA1, GEA2 and GEA3), it was observed that, in all treatments, the germination percentages of the embryonic axes at ten days. GEAC were higher than 95.00% with the fastigiata variety, and differences were detected with the other varieties. In GEA1, germination was higher than 84.17% of the Ecuadorian variety and differences were detected with the other varieties. The GEA2 presented a germination superior to 87.50% with the hirsute variety and presented differences between varieties. For GEA3, the germination percentages were higher than 77.50% with the fastigiata variety. The T50 values do not differ significantly from the treatments and each of the varieties. The black bar is data from evaluating whole peanut seeds, and the grey bar is data from embryonic axes.  The black bar is data from evaluating whole peanut seeds, and the grey bar is data from embryonic axes.
A comparison was made between whole seeds and embryonic axes for root length. For whole seeds and embryonic axes, a significant effect was observed in the treatments used (p ≤ 0.05), whereas for treatment 1 of whole seeds (GSC), there was a greater root length of var. 3 with 39.   (Fig. 2, Appendix 3). Also, a comparison was made between whole seeds and embryonic axes for aerial length. For whole seeds and embryonic axes, a significant effect was observed in the treatments used (p ≤ 0.05), where for treatment 1 of whole seeds (GSC), there was a greater aerial length in var. 4 with 30.82 mm. In embryonic axes (GEAC), var. 2 with 14.57 mm, followed by var. 5 with 14.27 mm. For aerial length in whole seeds treatment 2 (GS1), var.

Discussion
Authors should discuss the results and how they can be interpreted from the perspective of previous studies and of the working hypotheses. The findings and their implications should be discussed in the broadest context possible. Future research directions may also be highlighted.
The seeds of the five varieties of A. hypogaea provided by the INIAP-DENAREF seed bank were refreshed and multiplied to avoid problems due to storage times for the recovery of whole seeds and embryonic axes. As a result, all the shoots developed roots and aerial parts, confirming the results obtained by Tacán et al. [37]. Gagliardi et al. [13] mention that the age of the seed does not influence the recovery of the plant from its embryonic axes.
The whole seeds and embryonic axes of the Arachis varieties showed high survival (germination) after ten days after sowing. The explant was not interfered with by the growth or presence of callus. This is confirmed by Ishikawa et al. [29] and Kuranuki et al. [42] in that the vitrification procedure allows the direct development of meristem shoots without callus formation).
The whole seeds of the peanut varieties showed average germination between treatments of 85.6% and an average moisture content of the seed of 6.4%. The embryonic axes showed average germination of 90.3% with a moisture content of the seeds at the beginning of each of the treatments of 6.4%, which confirms the results of previous work about the germination percentages and moisture content of whole seeds and embryonic axes [38]. In the case of embryonic axes, it agrees with the results obtained by Gagliardi et al. [35], where it indicates that embryonic axes dried for 1 hour (up to a moisture content of 18%) and submerged in NL for 24 h produced outbreaks in 80%. In wild Arachis species in species of the Arachis, Triseminatae and Erectoid Sections, the regenerative response of the embryonic axes was similar to our study and ranged between 78 and 100% [43].
The minimum differences of the peanut varieties depend on the morphological characteristics, such as the size of the leaflets, plant height, and periods of emergence and ripening of the fruits [44]. However, the differences found between the varieties in this study revealed a similarity between the data obtained between whole seeds and embryonic axes for aerial and root length. Therefore, it can be indicated that these differences found between the varieties could have been due to the characteristics of the seeds concerning color and size, which are characteristics of the domestication of A. hypogaea [45].
Recovery after freezing was influenced by the treatments for whole seeds and embryonic axes, not the culture medium. This confirms the results of other authors about the culture medium [38,[46][47][48][49]. All BV, with whole seeds and embryonic axes, developed aerial parts and roots. The results confirm that the cryopreservation of whole seeds and embryonic axes of the A. hypogaea varieties have the tolerance to the osmotic stress that is required for successful cryopreservation, which is related to the amino acid composition of peanut embryonic axes [23,37]. Cryopreservation of organized tissues such as shoot apices and embryonic axes is often appropriate for conserving genetic resources. In vitro plants derived from these tissues can ensure a reduced genetic change, although these structures could be recovered after cryopreservation [46].
In summary, we demonstrate and confirm the possibility of cryopreserving whole seeds and embryonic axes of the five peanut varieties studied using desiccation and rapid cooling with NL. This methodology is advantageous compared to the previously described methods for the cryopreservation of A. hypogaea, providing recovery rates significantly like those obtained by Tacán et al. [38] and Bajaj et al. [50]. Furthermore, unlike the method reported by Runthala et al. [32], this protocol does not require cryoprotectants or programmable freezers.

Conclusions
This section is not mandatory but can be added to the manuscript if the discussion is unusually long or complex.
The variables aerial and root length showed significant variations between treatments and varieties. These differences indicate that the five varieties conserved within the peanut collection in the INIAP Germplasm Bank can be cryopreserved. Furthermore, these results would indicate that cryopreservation could be successfully used in other intermediate (semi-orthodox) species.
This study used two cryopreservation strategies according to the type of explant (whole seed and embryonic axes) for each of the five genotypes. The behavior of the five varieties of Arachis suggests that characteristics related to each have been physiologically conserved.
All varieties showed excellent results with GS2 treatments for whole seeds and GEA2 in embryonic axes. In addition, an important point to consider is that none of the treatments existed in the presence of the callus phase, and therefore, the risk of somaclonal variation is reduced.
In this study, a cryopreservation and regeneration protocol for explants has been generated and developed for five varieties of Arachis, using whole seeds and embryonic axes, which can be replicated in other germplasm banks desiccation and rapid cooling with NL. The five varieties of peanuts present in the INIAP Germplasm Bank showed excellent results with the GS2 treatments for whole seeds and GEA2 in embryonic axes. In addition, an important point to keep in mind is that none of the treatments had the presence of the callus phase, and therefore, the risk of somaclonal variation is reduced.