In vitro establishment and propagation
In vitro establishment and propagation of A. peacockii were successfully achieved using rhizomatous shoots as starting explant. The significantly (p≥0.05) higher number (87.00±17.18) of shoot generation was obtained by the interaction of the highest concentrations of both hormones BAP (26.6 μM) and KIN (27.84 μM) added to MS basal culture medium. On the other hand, root formation in the new proliferated shoots was only detected when the media without growth regulator or supplemented with KIN were used (Table 1).
The effect of cytokinins alone or in combination with auxins has been previously tested for micropropagation of different Agave species as A. cantala, A. fourcroydes and A. sisalana (Binh et al. 1990); A. parrasana (Santacruz-Ruvalcaba et al. 1999); A. inaequidens (Aureoles-Rodríguez et al. 2008); A. cupreata, A. difformis, A. karwinskii, A. obscura and A. potatorum (Dominguez et al. 2008); A. marmorata (Aguilar and Rodríguez, 2018). However, until now, the use of only two cytokinins has never been reported for the same purpose in Agave. It is well known that each species responds differently to growth regulators used as supplements (Domínguez et al. 2008). Based on the auxin-cytokinin interactions, Garriga et al. (2010) found that combining Thidiazuron with BAP and IBA in the MS basal medium allowed improving shoot multiplication percentage in A. fourcroydes. Another efficient micropropagation protocol was achieved in A. americana via indirect organogenesis using 2,4-D and BAP (Lara-Hidalgo et al. 2017). According to Pérez-Molphe-Balch et al. (2012), A. peacockii has shown a higher shoot production rate (up to 17.1 shoots per explant) than other species in the medium containing 0.5 µM BAP. However, in the present study, we have remarkably increased the results by combining BAP and KIN to generate multiple shoots proliferation (87.00±12.93). During our experiments, the combined effect of these two cytokinins significantly improved the morphogenetic response compared to those obtained by only using BAP or KIN in the culture medium. Figure 1 (a to d) illustrates different stages of the micropropagation process of A. peacockii shoots.
Rooting and acclimatization
The rooting medium with 3% sucrose and supplemented with the highest concentration (29.25 µM) of IBA, allowed obtaining the significantly (p≥0.05) higher (8.6±1.01) number of formed roots (Table 2). In vitro developed roots were classified as thick, a common characteristic detected in the roots formed on any IBA-supplemented media. By contrast, longer (up to 20 cm in length) and thinner roots were obtained only using sucrose as an additive (Figure 2).
The observed differences in thickness during the radicular development led to further investigations to determine roots' anatomy and cellular structure. The histological analysis showed that thin roots did not present a well-organization regarding epidermis, cortex, endodermis, pericycle and secondary root formation, unlike what was observed in the thick roots' structural organization after the same culture time.
Nevertheless, regardless of the anatomical characteristics of roots, all rooted plantlets were successfully acclimated with 100% survival and normal morphological development. After 120 d of acclimatization, a more remarkable evolution of both the aerial part and root system was observed in those plants previously rooted in culture media with IBA and sucrose than in plants rooted in sucrose-free media (Table 3). It is well known that sugars usually serve as carbon source and energy during in vitro culture, but they can also change the water potential of medium (Lipavská and Vreugdenhil,1996). This might explain the experimented stimulus in root elongation by the effect of this component, which, combined with IBA, accelerated the early vegetative growth during the greenhouse performance of plants.
Cryopreservation of apical meristems by Droplet-Vitrification
The preliminary assessment of the critical osmoprotective step associated to dehydration with the vitrification solution PVS2, demonstrated a decrease in regrowth of agave shoot-tips from 100 % at 15 min down to 60% after 90 min of exposure at 25 °C (Figure 3). The detrimental effect was progressively detected by the increase of the exposure time longer than 30 min. Therefore, treatment for 15 min was selected as the best condition to dehydrate A. peacockii shoot-tips prior to cryopreservation.
Once the best dehydration time was determined with PVS2, the following cryoprotective experiments focused on optimizing the preculture duration of shoot-tips on a semisolid medium with 0.3 M sucrose. Before liquid nitrogen immersion, results revealed no significant differences in regrowth whatever the preculture time (3 h or 1 d) used or whether the mother plantlets from which shoot-tips were isolated had been pretreated with sucrose for 15 d before dissection. However, 1d of preculture resulted in the most effective treatment in both stages, before (-LN) and after (+LN) cryopreservation, because it allowed obtaining significantly higher regrowth (98% and 96%, respectively) without the 15 d-preconditioning of the donor-plantlets (Figure 4). Therefore, following the droplet-vitrification procedure, the best protocol for cryopreservation of A. peacockii shoot tips involved: 1d-preculture on semisolid medium with 0.3 M sucrose, treatment for 20 min in loading solution containing MS medium with 0.4 M sucrose and 1.6 M glycerol, exposure to cold vitrification solution PVS2 for 15 min and then, ultra-rapid cooling and warming. Recovery and regrowth of shoot-tips after cryopreservation using the optimized protocol is presented in Figure 5.
The cryogenic protocol defined for A. peacockii shoot tips included modifications that simplified and shortened the process's duration compared to the first report on cryopreservation of A. sobria shoot-tips (Tin and Folgado, 2019). The highest (87%) post-cryopreservation regrowth of A. sobria shoot-tips was achieved by pretreating donor-plantlets for 15 d in medium enriched with 0.3 M sucrose. By contrast, the highest (96%) post-cryopreservation regrowth of A. peacockii shoot tips was achieved using 1d-preculture of shoot-tips on semisolid medium with 0.3 M sucrose and without requiring the pretreatment of donor plants. Therefore, 1d-preculture of shoot-tips on semisolid medium resulted more effective than extended pretreatment of donor plants at the same concentration of sucrose and replaced the use of liquid medium with 20 mg L-1 of ascorbic acid during dissection as Tin and Folgado (2019) previously reported.
Preculture for 1d on semisolid medium supplemented with 0.3M sucrose is usually the most applied pretreatment of any cryopreservation procedure for organized tissues like shoot-tips (Gonzalez-Arnao et al., 2014). This conditioning step also allows explants to recover from the dissection stress and, followed by a loading treatment in a sucrose-glycerol solution, has proved to enhance the acquisition of tolerance to dehydration with PVS2 (Sakai and Engelmann, 2007). Therefore, this combination's beneficial effect has led to increased post-cryopreservation survival of shoot-tips subjected to different vitrification-based procedures (Takagi et al. 1997; Matsumoto et al. 2014; Valle Arizaga et al. 2017). Another modification of the first reported protocol (Tin and Folgado 2019) was performing dehydration at room temperature using a pre-chilled solution of PVS2, instead of dehydration at 0°C placing the samples on ice.
We can assume that Agave spp., have a high tolerance to cryopreservation, since with the two species (A. sobria and A. peacockii) tested until now, post-cryopreservation results have exceeded 80% in the first case (Tin and Folgado, 2019), and 90% in our studies. Different protocols following droplet-vitrification approach have been successfully applied to cryopreserve organized structures of several endemic and endangered plant species such as adventitious shoot-tips of Paraisometrum mileense (Lin et al. 2014); shoot-tips of Castilleja levisecta Greenm (Salama et al. 2018); shoot apices and axillary buds of Dianthus taxa (Halmagyi et al. 2020); shoot-tips of Pogostemon yatabeanus (Lee at al. 2021).
A process of cryopreservation imposes several stressful conditions which can affect survival and potentially alter genetic stability (Harding, 2004), therefore, optimizing the composition of the post-cryopreservation reculture medium is also essential to prevent induction of somaclonal variation. In this sense, cryopreserved shoot tips were firstly recovered on medium with low (0.44 μM) concentration of BAP for 30d, and then, transferred and maintained in culture using a modified (NH4NO3 reduced to 5 mM) MS semisolid medium with 3% sucrose and devoid of growth regulators. After 60 d of culture, the new plantlets spontaneously developed roots and were transferred to greenhouse culture conditions. After 120 d of acclimatization, the features (number, length, dry weight of roots, leaves number, and total leaf area) compared between plants derived from cryopreserved shoot-tips and propagated non-cryopreserved plants are summarized in Table 4.
Evaluation of vegetative growth of greenhouse-grown plants showed no significant differences in four out of five compared characteristics between plants regenerated from micropropagated shoots and the plants obtained after cryopreservation of shoot-tips. Therefore, the general analysis indicates no significant effect of cryopreservation altering the vegetative growth of agave plants. Our results agree with other authors' reports using droplet-vitrification with PVS2 to cryopreserve shoot tips of different plant species. Zhang et al. (2015) found that root formation of Argyranthemum during an early stage of greenhouse-performance was less in cryo-derived plants than in plants regenerated after micropropagation; however, this initial difference in rooting performance did not influence the genetic stability and other morphological characteristics during the further development of plants. On the other hand, no significant differences were detected in the vegetative growth of in vitro and cryo-derived plants of Actinidia spp. under greenhouse culture conditions (Zhang et al. 2020). In this regard, Pawłowska et al. (2019) found that cryopreservation of wild rose shoot-tips did not have any adverse effect on biochemical attributes or pollen characteristics in field-grown plants.
Field or greenhouse performances are critical to validate the success of a micropropagation process and the effectiveness of a cryopreservation protocol. The results presented here provide new verified biotechnological approaches up to greenhouse culture conditions. Their implementation will allow further signs of progress addressed to the propagation and safe conservation of plant germplasm, besides supporting new research on Agave spp.