*Review* **Vitrification Solutions for Plant Cryopreservation: Modification and Properties**

**Jiri Zamecnik \*, Milos Faltus and Alois Bilavcik**

Crop Research Institute, Drnovska 507, 16106 Prague, Czech Republic; faltus@vurv.cz (M.F.); bilavcik@vurv.cz (A.B.)

**\*** Correspondence: zamecnik@vurv.cz

**Abstract:** Many plants cannot vitrify themselves because they lack glassy state-inducing substances and/or have high water content. Therefore, cryoprotectants are used to induce vitrification. A cryoprotectant must have at least the following primary abilities: high glass-forming property, dehydration strength on a colligative basis to dehydrate plant cells to induce the vitrification state, and must not be toxic for plants. This review introduces the compounds used for vitrification solutions (VSs), their properties indicating a modification of different plant vitrification solutions, their modifications in the compounds, and/or their concentration. An experimental comparison is listed based on the survival or regeneration rate of one particular species after using more than three different VSs or their modifications. A brief overview of various cryopreservation methods using the Plant Vitrification Solution (PVS) is also included. This review can help in alert researchers to newly introduced PVSs for plant vitrification cryoprotocols, their properties, and the choice of their modifications in the compounds and/or their concentration.

**Keywords:** cryoprotectant; ultra-low temperature; glassy state; toxicity

#### **1. Introduction**

The cryopreservation of plant genetic resources aims to ensure the long-term storage of viable and genetically stable plant material at an ultra-low temperature using liquid nitrogen (LN, −196 ◦C) or liquid nitrogen vapour (LNV, −165 to −190 ◦C). At these temperatures, plant tissues are preserved in a state where cellular divisions and metabolic activities are minimized [1,2], thus preserving the genetic integrity for a longer duration [3,4]. The process of cryopreservation ensures the viability of plant tissues for a theoretically unlimited period [5].

Vitrification—glass formation without crystallization [6,7]—is one of the basic principles used in plant cryopreservation methods. Only a few plants can form a vitreous state naturally [8]. Most plants cannot vitrify themselves because they lack glassy state inducing substances and/or have high water content. The cryoprotectant used for vitrification should have at least three primary abilities: a high glass-forming ability, dehydration strength on a colligative basis to dehydrate plant cells to induce the vitrification state, and the cryoprotectant concentration used must not result in excessive toxicity to the plants. Despite the toxicity of Plant Vitrification Solution 2 (PVS2), it remains a highly effective vitrification solution for plant shoot tip systems [9].

Vitrification cryoprotective solutions reduce the risk of damage of the organelle structures by avoid forming ice crystals [10]; this is achieved by increasing the cell viscosity to the point at which ice formation is inhibited both inside and outside the cell [11]. Thus, cryoprotective solutions protect cell membranes with a gelatinous fluid and can form a glassy state in the cells, which helps plants survive at ultra-low temperatures. In addition, they can prevent further lethal water loss and maintain the percentage of regeneration after cryopreservation [12].

**Citation:** Zamecnik, J.; Faltus, M.; Bilavcik, A. Vitrification Solutions for Plant Cryopreservation: Modification and Properties. *Plants* **2021**, *10*, 2623. https://doi.org/10.3390/ plants10122623

Academic Editors: Carla Benelli, Petronia Carillo and Stephen O. Amoo

Received: 16 September 2021 Accepted: 24 November 2021 Published: 29 November 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Cryopreservation through the application of vitrification solutions was first reported in plant cells [13–15]. Currently, the vitrification-based methods that use vitrification solutions are considered the most widely applied for plant cryo-biologists [16,17]. There are more than 800 papers on shoot tip cryopreservation using vitrification solutions in the literature [18,19]. In addition, several vitrification solutions with different compositions have been reported, but most of the vitrification-based protocols use only a few key solutions [19–22].

Cryopreservation procedures are currently available for many essential plant species [23–25]. For successful vitrification protocols using Cryo Protective Agent (CPA), all the freezable water must be removed from the cells through the use of Plant Vitrification Solution (PVS) before LN exposure. Furthermore, other steps are essential to the success of cryopreservation protocols such as preconditioning, hardening, pre-loading, loading, osmoprotection with various substances before cryopreservation [26,27] and unloading after cryopreservation [28,29]. PVS treatment time, concentration, temperature, as well as shoot tip size, age, its physiology are also very important [30]. All these points are beyond the scope of this review.

The issues addressed by this review are the comparison of different PVSs, their differences in the concentration of the substances used, or the modification of their composition graphically in tables for a quick orientation. Modification is a way to improve previously used PVSs for new genotypes or genotypes with low regeneration rates. A case study on 13 different plant species using at minimum four different PVSs and their modifications is listed. In addition, a brief overview of cryopreservation methods using the PVSs is also included. We assume that this review will also help better select PVSs and their modifications for plant cryopreservation progress.

#### **2. Cryoprotective Substances**

The successful cryoprotection by vitrification is based on eliminating the formation of ice crystals and on reducing the toxicity of cryoprotective substances [31]. Cryopreservation is a reversible process, as long as the optimal combination and concentration of cryoprotective solution effective enough to form vitrified plant tissues are used. Vitrification refers to the physical process of supercooling a liquid to low temperatures and finally solidifying into a metastable glass without undergoing crystallization at a applied cooling rate [32]. The basic characteristics of substances used in cryopreservation are summarized in Table 1. The glass transition temperature tends to increase with increasing the relative molecular mass as opposed to the melting point [33]. In this order, the most commonly used cryoprotective substances are glycerol (Gly), dimethyl sulfoxide (DMSO), ethylene glycol (EG), propylene glycol (PG), sucrose (Suc), and sorbitol (Sor), at different concentrations in combination with MS medium [34] to allow sufficient dehydration of plant material and also induce cryoprotective processes to the cells [35]. DMSO was later discovered to be an active cryoprotective substance, the alkaline in the main, a good ligand, easily alkalized by a strong base, and subject to deprotonation. Some key features of cryoprotective substances are mentioned below and in the Table 1.

The cryoprotective substances are classified into several groups according to the way of penetrating the cells: (a) small substances penetrating the cells through the cell wall and plasma membrane, such as EG, PG, Gly and DMSO; (b) substances penetrating only through the cell wall, e.g., oligosaccharides such as sucrose (Suc), sorbitol (Sor), mannitol (Man), or amino acids such as proline (Pro), and relatively low molecular mass polymers such as polyethylene glycol (PEG1000); (c) substances that do not penetrate through the cell walls or the plasma membrane, such as relatively high molecular mass polymers (soluble proteins, polysaccharides and, polyethylene glycol PEG 6000) [36–38].

#### *2.1. Substances That Can Penetrate through the Cell Wall and into the Protoplast* 2.1.1. Glycerol

Gly was used as the first for mouse embryo cryopreservation [39]. Gly was also the first cryoprotectant to see widespread use in human cryobiology [40]. Due to its higher relative molecular mass and viscosity, it penetrates membranes [35,41,42], but slower than DMSO and EG [42]. Gly occurs in plants in relevant quantities. Fifteen crop plants grown under field conditions had leaf concentrations of Gly between 10 and 39 μg g−<sup>1</sup> wet weight of tissue [43]. Gly is a polar molecule, freely miscible with water and simple alcohols [44]. Gly shows lower toxicity because it has a low ability to penetrate the membrane, which at high concentrations can lead to osmotic shock [45]. Gly concentration used for cryopreservation is dependent on the specific vitrification solution and ranges from 20% (weight in volume, *w*/*v*) [46] to 50% (*w*/*v*) [47].

**Table 1.** Characteristics of commonly used vitrification solutions and their chemical and physical properties in PVS.


§—the glass transition is depending on the rate of cooling [60];. Abr.—abbreviation; Mr—relative molecular mass; Tg—glass transition temperature; Tm—temperature of melting point equilibrium; LD50—median dose (*dosis letalis* media) \* <5000, \*\* 5001–20,000, \*\*\* >20,001 mg kg<sup>−</sup>1, mL kg−<sup>1</sup> (mouse, rat, rabbit). The data in columns 3–5 are from the citations mentioned in column No. 1 as upper index; data in columns 6–7 are from the safety sheets.

#### 2.1.2. Dimethyl Sulfoxide

Another common cryoprotectant is DMSO [61], which is used, like Gly, not only for freezing both animal and plant tissues but also in the cryopreservation of microorganisms [41]. DMSO enhances the passage of water molecules across the cell wall and cytoplasmic membrane [62]. The uptake dynamics of DMSO, Gly, and sucrose during dehydration of garlic shoot tips displayed a biphasic nature, with an initial rapid influx followed by a slower, gradual increase in DMSO [63], Gly, and sucrose (Suc) [64]. Room temperature increased the membrane permeability in contrast to a temperature close to zero. The reverse efflux pattern during unloading was similarly temperature dependent. DMSO is commonly used in combination with glycol-type compounds as this combination interacts with water and biological materials slightly different way than these components

alone. DMSO is hydrophobic at higher temperatures, meaning it is less toxic at lower temperatures, which leads to lower, slower, and easily controllable tissue dehydration and oxidation of sulphide groups [65]. In addition, DMSO is an effective solvent and has a high osmolality. In contrast, it is harmful and may cause somaclonal variability or mutagenesis. Due to the persisting uncertainties regarding mutagenesis [66,67] or high acute and chronic toxicity, as shown in the case of rhesus monkeys [68] and low order phytotoxicity, some current cryobanks do not use vitrification solutions containing DMSO, as a precaution, not denying the fact that DMSO can be an excellent cryoprotective substance.

#### 2.1.3. Ethylene Glycol

EG acts as a dehydration substance before cryopreservation. Due to its low freezing point, EG is used for its rapid penetration into the cells and its ability to block the ice crystal formation. EG in cryoprotective solutions is usually used at a half concentration in comparison with Gly [69,70]. EG is metabolized to oxalic acid, which increases the acidity of the organism and it is harmful. The oxalic acid reacts with the calcium contained mainly in the cell wall and forms insoluble calcium oxalate crystals stored in vacuoles [71]. High concentrations of the calcium oxalate, crystallized in various crystals, form raphides, druses, or others [72]. EG is commonly used in combination with glycol-type compounds as its combination interacts with water and biological materials [73].

#### 2.1.4. Propylene Glycol

PG is also a commonly used liquid in the cryopreservation process. PG is metabolized to oxalic acid in the metabolic process and acts like EG. However, as a cryoprotectant in warm conditions, PG is non-toxic while EG is metabolized to toxic elements [74].

#### 2.1.5. Polyethylene Glycol

PEG is a liquid or solid, depending on the molecular mass (which typically ranges from 300 g mol−<sup>1</sup> to 10,000,000 g mol<sup>−</sup>1). The chemical properties are almost the same, but forms with different molecular mass (from 600 to 8000 g mol−1), and different physical properties are usually used for cryopreservation [75]. PEG used for plant cryopreservation, e.g., PEG 6000 normally has a concentration of 15% (*w*/*v*) [76].

#### *2.2. Substances That Can Penetrate through the Cell Wall*

High levels of carbohydrates and sugar alcohols occur in plants as natural, non-toxic cryoprotective substances [77]. Monosaccharides are readily dissolved in cryoprotective solutions and can vitrify plant tissues at a lower concentration level than disaccharides. Therefore, the disaccharide sucrose is often used as an antifreeze agent. Compared to cryoprotection using monosaccharide glucose, sucrose has a higher efficiency [78]. In addition, carbohydrates contribute to the dehydration of samples and are added to the cryoprotective mixture to increase the protection of the membrane integrity in a dehydrated state [70,79,80].

#### 2.2.1. Glucose

Glucose belongs to a group of monosaccharides that reducing sugar due to the presence of an aldehyde group, which is oxidized to a carboxylic acid group to form D-gluconic acid [81]. On the contrary, reducing the aldehyde group of glucose to the primary alcoholic group forms D-Glucitol, called sorbitol. D-Glucose monohydrate is produced in green plants during the photosynthesis process, which is a fast and basic energy supply. Due to its molecular size, it penetrates the cell membrane faster than sucrose, but in an experimental comparison of plant regeneration after cryopreservation, sucrose, as well as the sugar alcohol mannitol, proved to be more useful [69].

#### 2.2.2. Sorbitol

Sorbitol is used in cryopreservation because of its lower melting point [82]. It provides less energy than sucrose (1 g of sorbitol gives up to 10,886 kJ of energy). Göldner et al. [83] introduced a range of carbohydrates to increase the frost resistance of plants (*Digitalis lanata*) used for plant pre-cultivation. They showed that the most damaged plant cells were cultured on sorbitol and proline medium. In contrast, the smallest cell damage occurred when sucrose was used. The concentration of 0.4 M and 0.8 M sorbitol in the pre-cultivation embryogenic tissues of hybrid firs (*Abies alba* × *A. cephalonica*, *Abies alba* × *A. numidica*) has been found acceptable for subsequent survival and regeneration of the plants [84]. High levels of sugars and sugar alcohols are found in many polar plants, insects, fungi, etc., as non-toxic cryoprotectants [85].

#### 2.2.3. Sucrose

Sucrose, composed of fructose and glucose molecules, is the most widespread disaccharide. It is easily hydrolyzable, dissociated by glycosidase invertase to the laevorotatory glucose and dextrorotatory fructose. These translocated sugars are photosynthetically metabolized in the Calvin cycle. Due to their molecular size, these carbohydrates are preferable for the transport assimilated over long distances. Sucrose is energetically abundant (1 g of sucrose provides 16,747 kJ of energy), and it acts as an energy source in heterotrophic nutrition after plant rewarming during its regeneration. The disaccharide sucrose is more effective than the monosaccharide glucose for vitrification [47,86–88]. Sucrose is used to promote dehydration before and/or during cryopreservation. Sucrose is normally membrane-impermeable and has low toxicity. The concentration of sucrose used in cryopreservation processes varies from 5% (*w*/*v*) [46] to 50% (*w*/*v*) [47], but most often 40% sucrose (*w*/*v*) [89,90] is used. For mint shoot tips, sucrose reduces the toxicity of ethylene glycol and DMSO at 22 ◦C and Gly at 0 ◦C [65].

#### 2.2.4. Amides

Amides are weak cryoprotectants compared to polyols (formamide is too weak to vitrify itself, but can assist vitrification by other cryoprotectants). Adding methyl groups increases the effectiveness of cryoprotectants [91]. Amides, compared to polyols, generally have weak cryoprotective effects [45].

#### 2.2.5. Bovine Serum Albumin (BSA)

BSA decreases the kinetic constant value determined for concentrated EG solutions. However, BSA's effect was small compared to that which could be produced by a slight increase in EG concentration [92]. On the contrary, Rall [93] suggest that the inclusion of BSA in vitrification solutions may be an effective means of increasing the stability of the amorphous state of vitrification solutions.

#### **3. Substances That Do Not Penetrate through the Cell Wall**

Substances that do not penetrate through the cell wall are polymers with high molecular weight such as soluble proteins, polysaccharides, mucilage, PEG1000.

Turner et al. [94] proposed that the mode of action of polyalcohols (in our enumeration Gly, mannitol, sorbitol) is not based on molarity, but rather on the total number of hydroxyl (OH) groups present in the medium. Furthermore, based on their results, they propose that the orientation of OH groups is a determining factor in effective cryopreservation [94].

The development of cryogenic technologies is facilitated by biophysical studies capable of monitoring glass stability during cryopreservation [33]. The glass transition temperature of substances depends on the concentration of an aqueous solution, cooling/warming rates, annealing temperature, and type of mixture. For example, three different glass transitions were found in the subzero temperature range of −163, −138, and −93 ◦C at 20% BSA (*w*/*w*) [60]. Sucrose has also glass transitions at the three different temperatures ranges; Tg1 (−50 to −45 ◦C), Tg (−36 ◦C), and Tg (−83 to −57 ◦C); all

sucrose glass transitions are concentration-dependent and the first two are cooling rateindependent [88]. The thermal analysis of plant vitrification solution: PVS1, PVS2, Towill's, Fahy's, or Steponkus' vitrification solutions reveals only a small water peak detected in shoot tips after 120 minutes dehydration duration. Still, recovery of cryopreserved garlic shoot tips exposure to these vitrification solutions was low (from 0 to 25%), in comparison to 80% regeneration after PVS3 [95].

#### **4. Vitrification Solutions and Modifications**

Many PVS solution variations have been reported to be suitable for cryopreservation of several plants. During their testing, a number of their successful modifications were published. In this section, an attempt is made to give an overview of the most important of them. The original Plant Vitrification Solution 1 (PVS1) was firstly used by Uragami [14] for cultured cells and somatic embryos derived from the mesophyll tissue of asparagus (*Asparagus officinalis* L.). The original composition of PVS1 is in Table 2.

**Table 2.** The concentration of substances of the original Plant Vitrification Solution numbered one (PVS1) uses Uragami [14].


DMSO—dimethyl sulfoxide, Suc—sucrose, Gly—glycerol, EG—ethylene glycol, PG—propylene glycol, PEG—polyethylene glycol 8000 m.w., Sor—sorbitol, Total—total concentration of all substances. Significant composition changes added or omitted substances and/or modification in the concentrations of original PVS1 in % (*w*/*v*) used in plant cryopreservation. The shaded area expresses no changes concerning the original PVS1.

> The PVS1 was used in modifications PVS1-M1 to PVS1-M3 and PVS1-M8 with a lower concentration of DMSO at a concentration up to 6% and in modifications PVS1-M4 with a higher concentration of DMSO (10%). Sucrose was not used in the original PVS1, but it was used at a concentration of 13.7% (*w*/*v*) in the modifications (PVS1-M3 and PVS1-M8). The Gly was in the modification PVS1-M2, PVS-M6, and PVS1-M8 in lower concentrations and higher concentrations in the PVS1-M6 than in the original PVS1. EG was used less concentrated (13% *w*/*v*) in (PVS1-M1 to PVS1-M4) and more concentrated (PVS1-M7) in comparison with the original. PG was used less concentrated in PVS1-M1 to PVS1-M3 and without change in modifications PVS1-M4 to PVS1-M8. Sorbitol was omitted in PVS1-M1 and PVS1-M3 to PVS1-M8. These modifications were also used for shoot tip cryopreservation e.g., *Rauvolfia serpentine* [102] and *Cocos nucifera* L. [103].

> Among several PVSs, PVS2 (Table 3) and PVS3 (Table 4) are the most frequently used vitrification solutions. The PVS2 was firstly used at a concentration of 60% [22]. The PVS2 in full-strength [13] (first row in Table 3) is also widely used. Several modifications of PVS2 with different substances and their concentration used have been published: DMSO was used in a lower concentration, from 7.5 to 13% (*w*/*v*), in modifications PVS2-M2 to PVS2-M6 (Table 3). The exact concentration of sucrose (0.4 M) used in the original PVS2 was also in modifications from PVS2-M1 to PVS2-M3 and in PVS2-M10. Sucrose was used in a higher concentration from 15 to 34.2% (*w*/*v*) in the modifications from PVS2-M6 to PVS2-M9 and no sugar was used in PVS2-M4 and PVS2-M5. Gly was used in the modification PVS2-M3, PVS2-M6 in a lower concentration, and PVS2-M8 higher than in the original PVS2. The concentration of EG was unchanged. PG was added in PVS2-M1 and PVS2-M2 at 15 and 7.5% (*w*/*v*), respectively. PEG 8000 was added in PVS2-M3 and PVS2-M10 as 3% (*w*/*v*) solution and PVS2-M6 as 2% (*w*/*v*) solution. Instead of sucrose, sorbitol in PVS2-M5 was added at 15% (*w*/*v*) (Table 3).

PVS2-M1 is a widely used plant vitrification solution [104] in several cryoprotocols for various plant species e.g., *Photinia* × *fraseri* Dress. [105], *Allium sativum* L. [95,106], *Rauvolfia serpentine* [102], *Cocos nucifera* L. [103], *Porphyra yezoensis* [94], *Mentha piperita* L. [65], *Dioscorea* spp. [15]. PVS2-M3 was used for cryopreservation of e.g. *Prunus avium* L. [107], and PVS2-M6 was used for cryopreservation of *Bromus inermis* Leyss [46]. PVS2-M8 was used for cryopreservation of *Clinopodium odorum* [108].

Incubation time in PVS2 varies according to species, temperature conditions, shoot tip size, pretreatment, preculture and, cryopreservation protocol. Therefore, there is no generic time for PVS2. For example, in apple the droplet-vitrification method had the highest regrowth percentage after 30–50 min PVS2 exposure at room temperature [109]; in potato droplet-vitrification had the highest regrowth percentage after 50 min PVS2 exposure at 0 ◦C [110]; in shallot droplet-vitrification had the highest regrowth percentage after 40–60 min PVS2 exposure at 0 ◦C [111]; in grapevine droplet-vitrification had the highest regrowth percentage after 90 min PVS2 exposure at 0 ◦C [112], and in yacon droplet-vitrification had the highest regrowth percentage after 60 min PVS2 exposure at 0 ◦C [22,113]. DMSO and Gly penetrate the cell wall membrane and increase cellular osmolality avoiding ice formation [7,38,114].

Volk and Walters [9] proposed according to their differential scanning calorimeter measure that the PVS2 operates through two cryoprotective mechanisms: (a) it replaces cellular water, and (b) it changes the freezing behaviour of any water remaining in the cells. They expressed the theory that the penetration of some of the components (e.g., DMSO) of PVS2 into the cell is essential to its cryoprotective efficacy. Significantly, the assumption that the mode of action of PVS2 is primarily caused by osmotic dehydration cannot explain its high effectiveness. Cell-penetrating constituents of PVS2 replace water as the cells become dehydrated and prevent injurious cell shrinkage caused by dehydration [9].

**Table 3.** Composition and modification in concentration of substances of Plant Vitrification Solution 2 (PVS2) Sakai [13].


Important composition changes, added or omitted substances, and/or modification in the concentrations of original PVS2 in % (*w*/*v*) used in the plant cryopreservation. All substances were dissolved in MS medium with 0.4 M of sucrose. The sucrose concentration in PVS2 was approximately 0.15 M. The shaded area expresses no changes concerning the original PVS2. DMSO—dimethyl sulfoxide, Suc—sucrose, Gly—glycerol, EG—ethylene glycol, PG—propylene glycol, PEG—polyethylene glycol, Sor—sorbitol, Total—total concentration of all substances. § termed '100%' of PVS2; §§ termed PVS2-A3 [61]; §§§—'60%' of PVS2; \*—PEG 8000 m.w., \*\*—PEG 4000 m.w.

> The first use of PVS3 was reported on *Asparagus officinalis* L. by Nishizawa et al. [47]. The original PVS3 plant vitrification solution contained 50% to 50% (*w*/*v*) sucrose and Gly (Table 4).

**Table 4.** The concentration of substances of the original Plant Vitrification Solution 3 (PVS3) [47]. Important composition changes added or omitted substances, and/or modification in the concentrations of original PVS3 in % (*w*/*v*) used in plant cryopreservation.



**Table 4.** *Cont*.

DMSO—dimethyl sulfoxide, Suc—sucrose, Gly—glycerol, EG—ethylene glycol, Total—total concentration of all substances. The shaded area expresses no changes concerning the original PVS3.

> Sakai [119] and Benson [11,22] indicated 40% to 40% ratio like an original PVS3. We label this ratio as modification 4 to the original PVS3; this modification is listed as PVS3-M4 in Table 4. Since the first description of PVS3, there have been many reports of vitrification methods using PVS3. PVS3 was used for 136 different plant species till 2007 [22]. The published modifications concern is lowering the concentration of sucrose, mostly in the same ratio, e.g., [22,69,121], to Gly, e.g., PVS3-M3 [121] and PVS3-M4 [22]. PVS3 is the plant vitrification solution without DMSO and EG in the original composition compared to PVS2; curiously, the first modification, PVS3-M1, contains 5% (*w*/*v*) of DMSO in addition to the basic substances. In PVS3-M2, there is only a change in Gly as a 30% (*w*/*v*) concentrated solution [69]. PVS3-M5 contains 20% (*w*/*v*) of ethylene glycol in addition together with an increase of the content of sucrose to 60% (*w*/*v*) and a decrease of glycerol to 35% (*w*/*v*).

> The original PVS3 is also widely used in the cryopreservation of *Lithodora rosmarinifolia* (Ten.) [122], *Photinia* x *fraseri* Dress. [105], *Allium sativum* L. [95], *Rauvolfia serpentine* [102], *Cocos nucifera* L. [103], *Bromus inermis* Leyss [103], *Porphyra yezoensis* [94], *Mentha piperita* L. [65], *Dioscorea* spp. [15], *Malus* [96] and other species, but somewhat less than PVS2, the most frequently used vitrification solution [22].

> The original PVS4 is without DMSO (Table 5). The two following modifications (PVS4-M1) and PVS4-M2) are with the addition of DMSO. In PVS4-M1 there are only two compounds, DMSO and Gly, at 5% (*w*/*v*); this low concentration of cryoprotectants combined with a slow cooling rate (0.1–0.2 ◦C min−1) act rather as dehydration solution than vitrification solution.

**Table 5.** Plant Vitrification Solution 4 (PVS4), Steponkus', Towill's and their modifications in concentration, composition, and some omitted and added substances in % (*w*/*v*).


DMSO—dimethyl sulfoxide, Suc—sucrose, Gly—glycerol, EG—ethylene glycol, PG—propylene glycol, PEG—polyethyleneglycol, Sor sorbitol, BSA—bovine serum albumin, Total—total concentration of all substances. *\** PEG 8000 m.w., *\*\** known also as VSL, *\*\*\** known also as VSL+ [46]. The shaded area expresses no changes concerning the original PVS.

> The PVS proposed by Steponkus' vitrification solution is without sucrose. Sucrose is used in the first modification of Steponkus' vitrification solution (Steponkus-M1) in the concentration of 13.7% (*w*/*v*). The PVS proposed by Towill's is slightly modified by increasing the DMSO to 10% and decreasing PEG 8000 to a half. Sucrose is used in the second modification of Towill's vitrification solution (Towill-M2) in the concentration of 13.7% (*w*/*v*) together with 6.8 % (*w*/*v*) of DMSO influencing the regeneration up to 39% of *Allium sativum* shoot tips [126].

> Thermal analysis of PVSs revealed that increasing Gly concentration reduced endothermic peaks, indicating the ice-blocking property of Gly [61]. Increasing sucrose

concentration in PVSs also decreased endothermic enthalpies by decreasing explant moisture content and increasing the influx of cryoprotectants [64]. Therefore, balancing the Gly and sucrose concentration in the design of PVSs is also crucial to increase recovery. A limitation of the use of PVS3 is the high osmotic stress increasing during the action; therefore, induction of desiccation tolerance during preconditioning of samples is essential if they are not inherently tolerant [61,128].

#### **5. Comparison of Vitrification Solutions on Regeneration**

Evaluation of different cryoprotectant solutions and their modification are ordered in Table 6. There is a comparison of three or more different cryoprotective vitrification mixtures.

**Table 6.** Regeneration rate (%) after application of Plant Vitrification Solutions and their modifications (M1\_M4 for details see Tables 2–5). Three or more Plant Vitrification Solutions or their modifications at one particular species.


§ according to Watanabe and Steponkus [134]; §§§§ 88% of PVS3; \* PVS N (1 M sucrose + 15% glycerol + 14% ethylene glycol [133]; \*\* R—stands for regeneration, regrowth, S—stands for survival after cryopreservation. The PVS3, PVS4, PVS5 [96], VSL [46], and Fahy's vitrification solution [31] are unmodified compared to other PVSs.

> PVS3, PVS4, PVS5, and Fahy are without modification in Table 6, even though they have several modifications (see Tables 3 and 4). Among the PVSs, PVS2 and PVS3 are the most frequently used [104]. In some cases, PVS3 may be less toxic to plant species sensitive to PVS2, such as *Allium* sp. [111]. Based on the results presented over the years and also from Tables 2–6, it is evident that the optimum cryoprotectant solution treatment is species or cultivar-specific. Furthermore, the PVSs exposure duration and temperature conditions during incubation are related to the size of the shoot tip, as well as to the preculture and pretreatment conditions [19,135].

> The original composition is listed in the first row in each table (Tables 2–5) and its modifications in concentration and/or in compounds used are followed. Sakai et al. [82] were the first to report a PVS2-vitrification cryopreservation protocol for nucellar cells of *Citrus sinensis*. The modifications of PVS2 were applied on other plants as presented in the paper by Uragami et al. [14] and Maruyama et al. [117].

> Modifications of PVS and its influence on the viability of explants have been reported. Suzuki [46] in addition to the three original vitrification solutions (PVS1, PVS2, VSL) (Table 6) presented the effect of 12 other combinations of cryoprotective substances on gentian axillary buds. The best regeneration of 79.7% after liquid nitrogen treatment was achieved with the original VSL. Cho et al. [99] used four original PVSs (PVS1, PVS2, VSL, and VSL+) (Table 6). The best one for *Citrus madurensis* embryonic axes survival after liquid nitrogen treatment was PVS2. Kim [61] modified the PVS2 in nine modifications and PVS3 in four modifications in concentrations of substances in the droplet-vitrification procedure. The best one was the PVS3 without any modifications for shoot tips harvested from in vitro conditions of *Dendranthema grandiflora* T. and garlic clove shoot apices of *Allium sativum* L.

#### **6. Vitrification Solution and Cryopreservation Methods**

Increased vitrification method efficiency was achieved by treating plants in a pretreatment and preculture steps before cryopreservation of plant shoot tips [22,89,136–139]. Pretreatment/preculture increases tolerance to PVSs during the dehydration process. Pretreatment conditioning differs by species, and then preculture for some species is crucial [140]. During pretreatments the sucrose intake mostly takes place, and increased content of proline and other protective substances accumulates in the plant shoot tips while growing in the carbohydrate enriched culture medium. The temperature during the incubation of plants in PVSs is important for both toxicity and dehydration. The temperature close to 0 ◦C for plants treated in PVS2 is crucial and had significantly lower lethality than at 22 ◦C [65,141–143]. When the temperature is subsequently lowered, the penetrating components of PVS2 cryoprotect the cells by restricting the molecular mobility of water molecules and preventing them from nucleating ice crystals [11].

In PVSs vitrification-based methods, most or all of the freezable water is removed by using highly concentrated and viscous cryoprotectant mixtures which, after rapid cooling in LN, form a glass [11,144]. The amount of water in the cells is decreasing due to an accumulation of these substances, and the central vacuole is divided into several smaller ones.

Cryoprotective substances help ensure the stability of membranes and enzymes in subsequent dehydration by vitrification solutions and avoid the formation of ice crystals [145,146]. In this case, the samples are exposed to minutes–a few hours long treatment by several cryoprotective substances before LN exposure. The effect of cryoprotective solution composition for plant regeneration was studied in different plant species [11,61,126,139,147,148]. The published results indicate the importance of PVS compositions, the vitrification protocol, pre-culture, regrowth media, and the application of an appropriate vitrification technique to achieve optimum post-cryopreservation recovery [105,131].

With the combination of the composition of cryopreservation solution (15% DMSO + 3% sucrose) and subsequent slow cooling, a droplet-freezing method was developed for cassava shoot tips [146].

The droplet-vitrification method is derived from the DMSO droplet methods proposed by Kartha et al. [146], and Kaczmarczyk et al. [149] and Schaefer-Menhur et al. [150]. The procedure is similar to the droplet method but with highly concentrated cryoprotective solution PVS2 [104,147,150–152] or PVS3 either in original or in its modification PVS3- M4 [122] or both PVS2 and PVS3 [97,113,126] before ultra-fast cooling. Rewarming of the samples is usually done in unloading solution temperated in a 40 ◦C sterile water bath for 1–2 min. When using potentially phytotoxic DMSO, the cryoprotective mixture is washed out in unloading solution temperated in a water bath with solutions of decreasing concentrations of sucrose or sorbitol as unloading solutions. This method was successfully applied many of plant species and is widely used in genebanks for cryopreserving vegetatively propagated crop collections [153,154].

Other cryopreservation methods use the PVS for inducing vitrification, such as encapsulation-dehydration and encapsulation-vitrification method with PVS2, PVS3 [139,155] foil-vitrification, droplet-vitrification, and droplet-freezing methods with PVS1, PVS2, PVS3, and VSL [105]. In addition other methods use the vitrification solutions PVS2 and PVS3 in V cryo-plate and D cryo-plate methods [27], PVS2 in cryo-mesh method [156,157], and PVS2 in vacuum infiltration vitrification method (VIV) [158].

The determination of plant survival and regeneration level is done by a visual evaluation of growing the plants in vitro conditions. The ratio of regenerated to the total number of cryopreserved plants is expressed as a success of the cryoprotocol (see Table 6).

Cryoprotectants can change the biophysical properties of plant parts. Cryoprotectants are selected based on their potential non-toxicity, high osmolality, and ability to penetrate as a particular component of vitrification solution into the cell. A low survival and regeneration of plants can also be caused by insufficient osmotic adjustment of plant material, excessive shrinkage of cells in hypertonic conditions, the toxicity of the vitrification solution, low penetration ability of the cryoprotective solution into the plant tissue, low dehydration of the plant tissue and subsequent formation of intracellular ice crystals during freezing [6]. During slow cooling, the sample may reduce its cell surface due to

the loss of cytoplasmic membrane, and the cell lysis can occur upon returning to a normal state [64,79]. The toxicity of cryoprotective substances can be associated with the denaturation of proteins, which are damaged either by low temperature or high cryoprotectant concentration necessary for plant tissue vitrification. The strongest vitrification is achieved with cryoprotective substances, which can bind hydrogen bonds to water molecules. They make the interaction of hydrogen bonds water-water, which is the basis for forming ice crystal structure. These substances can be bound by hydrogen bonds in proteins, causing their denaturation [35]. Reduction of water bound to proteins can damage the cells. Dehydration to the level of bound water is essential for successful cryopreservation without the use of cryoprotective substances.

The following steps are recommended for cryopreservation of a new plant species. First, the new PVS should be chosen from cryoprotective solutions close to the species family with the highest regeneration ability. The second possibility is to use the PVS widely used for most plants, e.g., PVS2, PVS3, etc. After choosing the PVS, it is necessary to test the toxicity level following the growing test and level of dehydration [159] according to their regeneration. If the thermal analysis is available, it will help a lot at this step [160]. The difference between the regeneration rate of control and ultra-low temperature treated plants is the potential to improve regeneration by improved vitrification solution.

#### **7. Conclusions**

Cryopreservation methods allow long-term storage of genetically unique plant material in the vitreous state at ultra-low temperatures of LN, which leads to the suppression of all biochemical reactions. Vitrification solutions as a mixture of two to seven substances induce a glassy state in plant tissues and prevent the ice crystal formation during the cooling and warming process. The cryoprotective mixture toxicity can be reduced by an appropriate combination or decrease in the concentration of cryoprotective substances and/or physical condition, mainly low temperature at which those are applied. The best cryoprotective solutions can reduce the toxicity of the vitrification mixture. Easier and faster cryoprotectant penetration into the cells and tissue dehydration to the optimal level for cryopreservation will increase the survival and regeneration of plants and extend cryopreservation methods for other plant species and genotypes. The widely used vitrification solutions meet these demands for high regeneration (over the minimum standard of cryobank) after cryopreservation.

**Author Contributions:** Writing and review of literature, J.Z.; writing—review and editing, A.B. and M.F.; project and funding acquisition, J.Z. and M.F. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Ministry of Agriculture of the Czech Republic, projects number MZERO0418, QK1910476, and QK1910277.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** The authors would like to acknowledge Renata Kotkova for first establishing the requirement of this topic during her doctoral studies and to Stacy Hammond Hammond for the English corrections.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Review* **Cryopreservation of Woody Crops: The Avocado Case**

**Chris O'Brien 1,\*, Jayeni Hiti-Bandaralage 1, Raquel Folgado 2, Alice Hayward 1, Sean Lahmeyer 2, Jim Folsom <sup>2</sup> and Neena Mitter <sup>1</sup>**


**Abstract:** Recent development and implementation of crop cryopreservation protocols has increased the capacity to maintain recalcitrant seeded germplasm collections via cryopreserved in vitro material. To preserve the greatest possible plant genetic resources globally for future food security and breeding programs, it is essential to integrate in situ and ex situ conservation methods into a cohesive conservation plan. In vitro storage using tissue culture and cryopreservation techniques offers promising complementary tools that can be used to promote this approach. These techniques can be employed for crops difficult or impossible to maintain in seed banks for long-term conservation. This includes woody perennial plants, recalcitrant seed crops or crops with no seeds at all and vegetatively or clonally propagated crops where seeds are not true-to-type. Many of the world's most important crops for food, nutrition and livelihoods, are vegetatively propagated or have recalcitrant seeds. This review will look at ex situ conservation, namely field repositories and in vitro storage for some of these economically important crops, focusing on conservation strategies for avocado. To date, cultivar-specific multiplication protocols have been established for maintaining multiple avocado cultivars in tissue culture. Cryopreservation of avocado somatic embryos and somatic embryogenesis have been successful. In addition, a shoot-tip cryopreservation protocol has been developed for cryo-storage and regeneration of true-to-type clonal avocado plants.

**Keywords:** vitrification; ex situ conservation; long-term conservation; embryogenic; shoot tips; plant biodiversity

#### **1. Introduction**

Globally plants are recognized as a vital component of biodiverse ecosystems, the carbon cycle, food production and the bioeconomy. An estimated 7000 species of plants provide food, fiber, fuel, shelter and medicine [1]. Plant genetic diversity is the foundation of crop improvement [2] and a primary target of conservation efforts. The two major approaches to conserve plant genetic resources are ex situ and in situ conservation [3]. In situ conservation involves the designation, management and monitoring of target taxa where they are encountered [4]. It protects an endangered plant species in its natural habitat. In situ techniques are described as protected areas, e.g., genetic reserve, on-farm and home garden conservation. Ex situ conservation involves the sampling, transfer and storage of target taxa from the collecting area [4]. Ex situ techniques include seed, in vitro (tissue culture and cryopreservation), DNA and pollen storage; field gene banks and botanic garden conservation. In vitro storage using tissue culture and cryopreservation techniques can deliver valuable tools to achieve a positive conservation outcome for genetic resources.

The majority of conservation programs focus on seed storage [5]. Many of the world's major food plants produce orthodox seeds which undergo maturation drying and are

**Citation:** O'Brien, C.; Hiti-Bandaralage, J.; Folgado, R.; Hayward, A.; Lahmeyer, S.; Folsom, J.; Mitter, N. Cryopreservation of Woody Crops: The Avocado Case. *Plants* **2021**, *10*, 934. https://doi.org/ 10.3390/plants10050934

Academic Editor: Carla Benelli

Received: 14 April 2021 Accepted: 29 April 2021 Published: 7 May 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

tolerant to extensive desiccation and can be stored dry at low temperature [6]. Seed storage under dry and cool conditions is the most widely adopted method for long-term ex situ conservation at relatively low costs [7]. About 45% of the accessions stored as seeds are cereals, followed by food legumes [15%], forages [9%] and vegetables [7%] [8]. However, seeds of many woody perennial plants are recalcitrant, e.g., *Juglans* spp. (walnut) [9], *Hevea brasiliensis* (rubber tree) and *Artocarpus heterophyllus* (jackfruit) [10]. Thus, they are difficult to maintain in seed banks. Additionally, seed-based conservation efforts miss clonal lineages that form the foundation of woody perennial agriculture [9]. Crops such as *Persea americana* Mill. (avocado), have recalcitrant seeds that are shed at relatively high moisture content, thus cannot undergo drying to facilitate long-term storage [6,11]. In addition, species that are seedless, e.g., *Musa* spp. (edible banana); or crops vegetatively propagated as their seeds are not true-to-type, e.g., *Manihot esculenta* (cassava), *Malus domestica* Borkh (Apple). and *Citrus* spp. (citrus)*;* are not storable through seeds. Field, in vitro and cryopreserved collections provide an alternative [7].

Field gene banks maintain living collections [12]. They are advantageous as physiological attributes and characteristics of the accessions such as plant habit, yield, tree height and disease resistance can be evaluated periodically [13]; however, there are several limitations posed; high maintenance cost, intensive labor and land requirements, pressure of natural calamities, risk of biotic and abiotic stresses as well as funding sources and economic decisions limiting the level of accession replication to maintain genetic diversity.

Tissue culture maintains plant material collections employing growth retardants [14], reduced light [15] or reduced temperature [16] to achieve slow growth, normally in sterile conditions. Plant germplasm storage via these methods has been increased with more in vitro protocols being developed for a vast number of plant species [17–19]. These approaches are used for large-scale micropropagation, reproduction purposes including embryo rescue, ploidy manipulations, protoplast fusions and somatic embryogenesis and are appropriate tools for short- and mid-term storage of plant genetic resources [7]. These methods allow for physical evaluation of material, rapid multiplication and plant establishment when needed, still, very costly to maintain due to space, consumables and labor inputs [20].

Plant cryopreservation (storage at −196 ± 1 ◦C) is a technique whereby plant tissues are preserved at ultra-low temperatures without losing viability [21]. It is the most relevant technology that provides safe long-term conservation of biological material as it maintains ex vivo biological function, does not induce genetic alterations [22] and provides long-term stable storage. Thus, it serves as an ultimate back-up of plant accessions for long-term storage, and material is generally not withdrawn from cryotanks unless it is necessary to use for research such as genetic manipulations [23] or in vitro culture [24]. A wide range of plant tissue can be cryopreserved, e.g., pollen, seeds, shoot tips, dormant buds, cell suspensions, embryonic cultures, somatic and zygotic embryos and callus tissue [25,26]. Recent uses of cryopreservation including cryotherapy to eradicate pathogens, such as phytoplasmas, viruses and bacteria in plants [27,28] is gaining a lot of attention [23]. Samples are normally given a short exposure to LN and surviving cells are regenerated from meristematic tissue which is pathogen free [28]. Cryotherapy has been used successfully in eradicating virus infections in several species with economic importance, such as *Prunus* spp. (plum), *Musa* spp. (banana), *Vitis vinifera* (grape), *Fragaria ananassa* (strawberry), *Solanum tuberosum* (potato), *Rubus idaeus* (raspberry) and *Allium sativum* (garlic) [28]. This review will look at conservation approaches for woody plants, focusing on avocado as a case study.

#### **2. Field Repositories of Woody Crops**

Field based germplasm conservation maintains living plants and serves as a source of plant genetic variation. Plants represented in these collections are current and historic cultivars, breeding material, landraces and sometimes wild relatives [9]. All of these are important to maintain for future development of new cultivars with superior growth

characteristics or resistance to pest and diseases. Field repositories have the advantage that researchers can physically evaluate and characterize the accessions for parameters such as yield, tree height and disease resistance [29,30]. Table 1 summarizes some examples of woody crops that are held as field repositories. However, the field repositories require an adequate area of land and continuous maintenance as well as on-going funding. They are also vulnerable to loss from natural disasters and damage caused by pests and diseases. This makes it important to potentiate field germplasm conservation with other methods which address some of these concerns.


**Table 1.** Some examples of field repositories maintaining living collections of economically important crops.

#### **3. In Vitro Conservation**

Different in vitro storage methods are employed depending on the storage duration required [17,35], i.e., in vitro culture for short- and medium-term storage and cryopreservation for long-term storage. Many reviews have been carried out to determine success [35–38] and standards established for managing field and in vitro germplasm gene banks [39,40]. These standards ensure effective, safe and efficient conservation of genetic resources. Due to the success of in vitro conservation techniques, many in vitro gene banks have been established nationally and internationally [41,42] (Table 2).

**Table 2.** Some examples of cryo-storage gene banks maintaining collections of economically important crops.


#### **4. Plant Cryopreservation of Somatic Embryos and Shoot Tips**

Cryopreservation of plants covers the entire plant kingdom from herbs and vines to shrubs and trees. The growth may be annual, biennial or perennial and the climate arctic; temperate, sub-tropical or tropical. A range of responses can occur within these groups and they are not always useful groupings for evaluating cryopreservation strategies [46]. The choice of material used, depends on the conservation goal, e.g., seeds and embryos capture species diversity; whereas shoot tips and dormant buds capture specific genotypes [47]. The most commonly used material to cryopreserve is apical meristems. They are at less risk of genetic variations due to their organized structure and are made up of small unvacuolated cells generally having a small vascular system [48]. In species that are recalcitrant and maintained in living field repositories, long-term cryopreservation storage of shoot tips can offer an alternative back-up as compared to seed storage which is only short-term [24].

Cryopreservation has several steps: (1) initial excision of the germplasm; (2) desiccation or pre-culture on osmotic media to reduce water content; (3) cryoprotection through exposure to cryoprotective agents; (4) cryopreservation in LN; (5) re-warming; and (6) unloading of cryoprotective agents and recovery of germplasm after cryopreservation [49]. The most critical step of cryopreservation is avoiding the intracellular and extracellular water that can lead to damage of cells during freezing [21]. Crystal formation, without extreme reduction of cellular water, can only be prevented though 'vitrification' i.e., the physical process of transition of an aqueous solution into an amorphous and glassy state (non-crystalline state) [50].

#### *4.1. Methods to Reduce Water Content*

Concentrated intracellular solute is a pre-requisite for successful cryopreservation and can be achieved with the following methods (Table 3) either individually or in combination [50–53].


**Table 3.** Methods to reduce water content.

Cryopreservation protocols using vitrification solutions typically involve a two-step cryoprotection process: (1) loading sometimes called osmoprotection is achieved by incubation in loading solution; and (2) dehydration using vitrification solution [52]. Loading solutions are commonly used to improve permeation of the cryoprotectant through cell membrane, it also induces tolerance to dehydration, which will be imposed by vitrification solutions. A common loading solution used is 2 M glycerol + 0.4 M sucrose [52]. Vitrification solutions contain chemicals that are high in concentration, e.g., ethylene glycol, glycerol and DMSO which have been reported as toxic to plant tissue [54]. It is therefore important to establish minimum exposure time to vitrification solutions in order to dehydrate tissue sufficiently to undergo cryopreservation and avoid damage effects to plant tissue [55,56].

Application of cryoprotectants is the most widely used method in cryopreservation protocols. Cryoprotectants that are penetrating in nature are able to reduce cell water at temperatures sufficiently to minimize the damaging effect of the concentrated solutes on the cells [57]. Whereas non-penetrating cryoprotectants osmotically "squeeze" water from the cells during the initial phases of freezing at temperatures between −10 and −20 ◦C [57]. Many authors have developed mixtures of cryoprotectants (Table 4) since the discovery of their benefits in protecting cells during the cryogenic process [54,58–61]. The most commonly used cryoprotectants for plant cells are PVS2 [59] and PVS3 [58].


**Table 4.** Some examples of cryoprotectants used for plant tissue.

#### *4.2. Cryopreservation Methods*

Presently there is no one method of cryopreservation that can be applied to a diverse range of plant species. Many cryopreservation methods (Table 5) have been developed for shoot tips and somatic embryos depending on the plant species used [17]; namely, vitrification, droplet-vitrification, encapsulation-vitrification, encapsulation-dehydration, dehydration, pre-growth, pre-growth-dehydration and D-cryoplate and V-cryoplate, a modification of the encapsulation-vitrification and droplet-vitrification [52,67,68].

**Table 5.** Some examples of cryopreservation methods, techniques and applications used.



**Table 5.** *Cont.*

#### 4.2.1. Vitrification

Vitrification can include the pre-culture of samples on medium supplemented with sucrose, then treated with a loading solution normally high in sucrose molarity [52] (e.g., a mixture of sucrose and glycerol), dehydration with a vitrification solution such as PVS2 or PVS3, rapid cooling, rewarming, and plant recovery by removing cryoprotectants [78].

#### 4.2.2. Droplet-Vitrification

Droplet-vitrification is a modification of vitrification [79]; treating explants with loading (usually 2 M glycerol and 0.4 M sucrose) and vitrification solutions; cooling them ultra-rapidly in a droplet of vitrification solution either PVS2 or PVS3 placed on an alfoil strip [49] with a droplet of cryoprotectant added before immersion in LN. The alfoil strip helps with the ultra-rapid cooling (about 4000–5000 ◦C min−1) and re-warming (3000–4500 ◦C min−1) of samples due to the good conductivity of thermal current of aluminum [80]. The removal of the cryoprotectant is achieved during re-warming stage by using an unloading solution usually with high level of sucrose 1.2 M, then transferred to recovery and regeneration media [25,55]. Droplet vitrification combines the use of highly concentrated vitrification solutions with ultra-fast cooling and re-warming rates [81] shown to be critical for survival [82]. For high success in survival and recovery of shoot tips after LN it is vital that samples are sufficiently dehydrated by the vitrification solution in order to vitrify while rapidly cooling in LN [83].

#### 4.2.3. Encapsulation-Vitrification and Encapsulation-Dehydration

Encapsulation-vitrification and encapsulation-dehydration have been successfully applied to cryopreserve shoot tips of woody species of crops, such as, *Malus* (apple) [84,85], *Pyrus* (pear), *Morus* (mulberry) [84], *Vitis* (grape) [86] and *Poncirus trifoliata* × *Citrus sinensis* (Chinese bitter orange) [87,88]. Dissected shoot tips or somatic embryos are suspended in a solution of sodium alginate. Beads (4–5 mm in size) are then formed using a truncated pipette tip and pipetted into a solution of CaCl2 where they are allowed to set for 30 min [52]. For encapsulation-vitrification, once beads are formed with explant inside,

they are then dehydrated in PVS solutions such as PVS2 or PVS3 prior to immersion in LN. Although encapsulation is time-consuming, it eases manipulation due to alginate beads being relatively large in size [52]. For the encapsulation-dehydration technique instead of dehydration with PVS solutions beads are dehydrated in a laminar flow hood or under silica gel before immersion in LN [52].

#### 4.2.4. Dehydration

Of all the methods explained, dehydration is the simplest, as it involves just the dehydration of explants followed by direct immersion in LN. Embryonic axes or zygotic embryos extracted from seeds are mainly used. Desiccation is usually achieved by the air current of a laminar airflow cabinet or over silica gel. Dehydration using a vitrification solution removes intracellular water from cells and permits intracellular solution to undergo phase transition from liquid phase into an amorphous phase upon rapid cooling [52]. Cryoprotectant mixtures are commonly used as vitrification solution, such as PVS2 and PVS3.

#### 4.2.5. Pre-Growth and Pre-Growth-Dehydration

In pre-growth and pre-growth-dehydration, explants are first exposed and grown on media containing cryoprotectants, dehydrated by air under a laminar flow cabinet or with silica gel, and then frozen rapidly. Depending on the plant species optimal conditions can vary greatly.

#### 4.2.6. D-cryoplate and V-cryoplate

D-cryoplate and V-cryoplate use special aluminium cryoplates which have been developed (length 37 mm, width 7 mm and a thickness of 0.5 mm with 10 wells). An alginate solution containing 2% (*w*/*v*) sodium alginate in calcium-free MS basal medium with 0.4 M sucrose is poured over the cryo -plate. Samples are placed in wells and more sodium alginate solution is poured over the top to cover them. In V-cryoplate, dehydration is performed using the vitrification solution PVS2, while in D cryo-plate, dehydration is achieved using the air current of the laminar flow cabinet or silica gel [89]. After dehydration cryo-plates are immersed in LN. The main advantages of V-cryoplate and D-cryoplate is that handling of specimens is easy and quick because only the cryo-plates are manipulated [89].

#### **5. The Avocado Case**

#### *5.1. Background*

Avocado (*Persea americana* Mill.), a high-value fruit found in almost all tropical and sub-tropical regions of the world [90,91] belongs to the plant family Lauraceae [92], genus *Persea* [93]. Mexico is thought to be the center of origin of the species [94]. The genus *Persea* has about 400 to 450 species consisting of the currently often recognized genera *Alseodaphne* Nees, *Apollonias* Nees, *Dehaasia* Blume, *Machilus* Nees, *Nothaphoebe* Blume, *Persea* Mill. and *Phoebe* Nees. There are eight sub-species of *P. americana* including *P. americana* var. nubigena (Williams) Kopp, *P. americana* var. steyermarkii Allen, *P. americana* var. zenymyerii Schieber and Bergh, *P. americana* var. floccosa Mez, *P. americana* var. tolimanensis Zentmyer and Schieber, *P. americana* var. drymifolia Blake, *P. americana* var. guatemalensis Williams, *P. americana* var. americana Mill. [91,95]. Genetic diversity within the genus *Persea*, the sub-genera *Persea* and *Eriodaphne* and the species *P. americana* is large and is threatened by the progressive loss of tropical and sub-tropical forests [95]. This genetic diversity can serve as a resource in crop improvement [96–98] and plays an important role both ecologically and culturally.

The three recognized ecological races of *P. americana* [99]; are the Mexican race, *P. americana* var. drymifolia, adapted to the tropical highlands; the Guatemalan race, *P. americana* var. guatemalensis, adapted to medium elevations in the tropics; and the West Indian race, *P. americana* var. americana, adapted to the lowland humid tropics [100]. The ability

of the three main races to withstand cold conditions varies; the West Indian race cannot tolerate temperatures below 15 ◦C, the Guatemalan race can tolerate cooler temperatures of −3 to −1 ◦C, and the Mexican race withstands temperatures as low as −7 ◦C exhibiting the highest cold tolerance [101–103]. They have distinctive characteristics; e.g., plant habit, leaf chemistry, peel texture, fruit color, disease and salinity tolerance [104]. The Guatemalan and Mexican races and their hybrids are very important for conservation and future breeding programs [97]. Cultivars classified as pure Guatemalan and Mexican races and Mexican × Guatemalan hybrids have been shown to have more diversity than those of pure West Indian race and Guatemalan × West Indian hybrid cultivars [97]. In Mexico and Central America, avocado trees grow under highly varied ecological conditions and natural selection over thousands of years has produced vast populations [97]. This serves as an essential source of varied attributes that are not among horticulturally available items [105].

The main avocado sold throughout the world, 'Hass', is a medium sized pear-shaped fruit with dark purplish black leathery skin [106]. Its commercial value is due to its superior taste, size, shelf-life, high growing yield, and in some areas, year-round harvesting [107]. The precise breeding history of 'Hass ' is unknown however, it is reported to be 61% Mexican and 39% Guatemalan [108]. This finding is supported by a study that analyzed the complete genome sequences of a 'Hass' individual and a representative of the highland Mexican landrace, *Persea americana* var. drymifolia; as well as genome sequencing data for other Mexican individuals, Guatemalan and West Indian accessions [108]. Analyses of admixture and introgression highlighted the hybrid origin of 'Hass', pointed to its Mexican and Guatemalan progenitor races and showed 'Hass' contained Guatemalan introgression in approximately one-third of its genome [108]. In Australia, 'Hass', represents 80% of total production [109] with 2019/20 producing 87,546 tonnes of avocados, an increase of 2% more than the previous season's 85,546 tonnes [109]. This increased consumer demand is due to its popularity as a healthy food; often referred to as a superfood due to its beneficial nutrients, vitamins, minerals, fiber and healthy fats [110,111]. Consumer market value of Australian fruit sold domestically was worth ~\$845 m in 2019/20 [109].

Due to the vast range of climates and conditions in our eight major avocado growing regions, avocados are produced all year round [109]. Avocado trees propagated by seed, take approximately 4–6 years to bear fruit, in some cases they can take 10 years to come into bearing [111]. Avocado trees are partially able to self-pollinate. Their flowers behave in synchronous dichogamy, flowers are perfect, bearing both male and female parts, however the periods of maleness and femaleness are temporarily distinct to enhance the likelihood of outcrossing [112,113]. The resultant progeny is highly heterozygous in the desirable parent tree characteristics [114]. New cultivars are normally derived from chance seedlings or mutations due to the difficult nature of breeding programs, which are costly, time-consuming and under threat of abiotic and biotic stresses. Nevertheless, the avocado industry's goal is to preserve superior cultivars for commercial production. Thus, to meet this goal, avocado is propagated clonally through grafting with breeding programs based on both scion and rootstock cultivars. The threat of Ambrosia beetle species and its symbiont fungus Laurel Wilt disease to the avocado field gene banks and commercial industry in Florida, California, and Israel is a glaring example of a biotic stress that could destroy the industry [115]. For scion cultivars the focus is on high yield [116], extending harvest season, regular bearing tendencies and disease resistance e.g., Anthracnose [117], Cercospora spot [118] and Verticillium wilt [117]. Rootstocks are often selected for dwarf size [119], salinity tolerance, adaptation to alkaline soil [119,120] and pest and disease resistance [120] such as *Phytophthora cinnamomi* Rands and *Rosellina necatrix* [121]. Clonal rootstocks are thought to be the only rootstocks for the future for achieving sustainable productivity gains [122–124]. These influence the total productivity of the plant in terms of yield and health. Rootstocks from Mexico, 'Orizaba 3', 'Antigua' and 'Galvan', show a universal adaptation to multiple soil stress problems. The last two, also, have tolerance to *P. cinnamomi* [96]. Many breeding programs have concentrated on the development of

new rootstocks such as 'Dusa', 'Bounty' and 'Velvick' [125] to help the industry overcome these threats [126]. 'Dusa's popularity has increased significantly since the mid-2000s. It is a common standard against which other *P. cinnamomi* tolerant rootstocks are compared in international breeding programs. It has been reported to bear fruit even under heavy *P. cinnamomi* disease pressure and has higher yields than many other rootstocks [127]. 'Bounty' is often selected for its *P. cinnamomi* tolerance and ability to survive in wet soils [127].

#### *5.2. Avocado Conservation*

#### 5.2.1. Global Germplasm Repositories

Field living germplasm collections (Table 6) and (Figure 1), are currently the most used conservation method, but funding and threats from natural calamities; pest and diseases are a problem.



**Figure 1.** One of the 56 avocado accessions being maintained in The Huntington Botanical Gardens [in San Marino, California USA] living germplasm collection.

#### 5.2.2. Cryopreservation of Avocado Somatic Embryos

To preserve global avocado diversity; development of improved technologies for avocado conservation, breeding/improvement and propagation is essential. In vitro somatic embryogenesis has direct importance to these objectives [139,140]. Somatic embryogenesis is the process by which somatic cells give rise to totipotent embryogenic cells capable of becoming complete plants [141]. Somatic embryogenesis can be a robust tool to regenerate genetically clonal plants from single cells chosen from selected plant material, or genetically engineered cells [142]. Somatic embryogenic cultures are generally highly heterogeneous since they consist of embryos at different developmental stages [143]. Though heterozygous in nature when regenerated using zygotic embryos as explants, cryopreservation of avocado somatic embryos offers an attractive pathway to conserve avocado germplasm. Recovery of plantlets from somatic embryos and clonal multiplication in vitro is an essential step for commercial application of this technology to crop improvement [144].

Somatic embryogenesis in avocado was first achieved using immature zygotic embryos of cv 'Hass' [145]. Studies have reported that the embryogenic capacity of avocado was highly genotype dependent [146]. To improve somatic embryogenesis previous studies have shown that several factors are vital for success, (1) composition of media, (2) hormone type and concentration, (3) type and concentration of gelling agent and (4) light intensity [147]. Morphogenic competence of somatic embryos has been reported to be lost 3–4 months after induction depending on the genotype [145,148]. In addition, the main factor limiting conversion of somatic embryos into plantlets is incomplete maturation [149]. Studies have found that there are two types of regeneration that occur after maturation; unipolar (only shoot apex or root) and bipolar (both shoot apex and root).

Shoots regenerated from unipolar embryos can either be rooted or rescued using in vitro micrografting [150]. Studies have shown that the percentage of high-quality bipolar embryos from avocado somatic embryos was extremely low at 2–3% and was genotype dependent [145,150,151]. This low rate of somatic embryo conversion is currently the main bottleneck in avocado regeneration via somatic embryogenesis [144]. A study described an in vitro induction and multiplication system for somatic embryos of avocado, across four cultivars, which remained healthy and viable for 11 months, on a medium used for mango somatic embryogenesis [139]. Furthermore for one of the cultivars, cultivar 'Reed', a two-step regeneration system was developed that resulted in 43.3% bipolar regeneration [139].

Cryopreservation of avocado somatic embryos has been successful for various cultivars (Table 7). The effect of cryogenic storage on five avocado cultivars ('Booth 7', 'Hass', 'Suardia', 'Fuerte' and 'T362') using two cryopreservation protocols (controlled-rate freezing and vitrification) was investigated [152]. In terms of controlled-rate freezing, three out of five embryogenic cultivars were successfully cryopreserved with a recovery of 53 to 80%. Using vitrification, cultivar 'Suardia' showed 62% recovery whereas 'Fuerte' had only a 5% recovery. When the droplet-vitrification technique was used, two 'Duke-7' embryogenic cell lines showed viability ranging from 78 to 100% [153]. Protocols employed in both studies cannot be applied in general to multiple cultivars and optimization of loading sucrose concentrations and plant vitrification solution 2 (PVS2), temperature and times need more intensive research.


**Table 7.** Summary of successfully applied cryopreservation techniques to avocado somatic embryos. \* Recovery is defined as any somatic embryo clump which was proliferating into new callus clumps.

#### 5.2.3. Shoot-Tip Cryopreservation of Avocado

Cryopreservation is a secure and cost-effective method for long-term storage of avocado. It provides a high degree of genetic stability in maintaining avocado collections for the long-term compared to other conservation methods. Shoot-tip cryopreservation conserves 'true-to-type' avocado plant tissue. It is ideal for preserving a core selection of avocado genotypes, for example, with superior characteristics, disease and pest resistance, rarity, drought and salinity tolerance. In one study, it was shown that axillary buds of Mexican and Guatemalan races were viable through fluorescein diacetate staining after dehydration with sterile air and being treated with cryopreservation solutions; however, shoot regeneration was not achieved with the cryopreserved material [154]. Another study, showed that dehydration at 60 min with sterile air and 30 min in PVS4 at 0 ◦C produced normal plant development and 100% survival was obtained after 30, 45 and 60 days [155].

#### 5.2.4. Critical Factors Identified for Successful Cryopreservation of Avocado Shoot-Tips

Although still cultivar-dependent, in vitro protocols have been established for multiple cultivars of avocado [111] advancing cryopreservation of avocado. Droplet vitrification can be considered as a "generic" cryopreservation protocol for hydrated tissues, such as in vitro cultures [49,156]. Vitrification-based procedures offer practical advantages in comparison to classical freezing techniques and are more appropriate for complex organs e.g., avocado shoot tips, which contain a variety of cell types, each with unique requirements under conditions of freeze-induced dehydration [157]. A problem associated with cryopreservation is formation of lethal ice crystals. To overcome this vitrification makes use of the physical phase called 'vitrification', i.e., solidification of a liquid forming an amorphous 'or glassy' structure [7] to avoid ice crystal formation of a watery solution. Glass is viscous and stops all chemical reactions that require molecular diffusion, which leads to dormancy and stability over time [158]. Samples can be vitrified and rapidly supercooled at low temperatures and form in a solid metastable glass with crystallization [66]. For procedures that involve vitrification, cell dehydration occurs using a concentrated cryoprotective media and/or air desiccation and is performed first before rapid freezing in LN [157]. It is important that cells are not damaged or injured during the vitrification process and are vitrified enough to sustain immersion in LN [24]. As a result, all factors that affect intracellular ice formation are avoided [157].

Oxidative stress is a common and often severe problem in plant tissue [159,160] of most woody plant species, such as avocado. Therefore, it is important to optimize regrowth conditions of extracted avocado shoot tips to prevent browning when developing an in vitro cryopreservation protocol. Browning of cell tissue takes place as the cytoplasm and vacuoles are mixed and phenolic compounds readily become oxidized by air, peroxidase or polyphenol oxidase. Oxidization of phenolic compounds inhibit enzyme activity and result in darkening of the culture medium and subsequent lethal browning of explants [161]. The antioxidant ascorbic acid (ASA) or vitamin C (ASA) occurs naturally in plants, in plant tissue and meristems [162]. It has many roles in a plant's physiological processes but mainly in its defense against oxidative damage resulting from aerobic metabolism, photosynthesis, pollutants and other stresses caused by the environment [163]. Wounding of avocado tissue can lead to an increase in reactive oxygen species (ROS) within the shoot therefore affecting the viability. ROS are highly reactive molecules and have been shown to cause damage in cells. Many molecules are considered as ROS, some of which include oxygen-free radical species and reactive oxygen non-radical derivatives [48]. The most common ROS species found in plants are superoxide (O2 −), hydroperoxyl (OOH), hydroxyl radical (OH) and singlet oxygen (O2) [48]. ASA has an important role in the detoxification of ROS species both enzymatically or non-enzymatically [164]. It can do this by scavenging a singlet oxygen, hydrogen peroxide, superoxide and hydroxyl radical [163].

It has been reported by several authors that the addition of antioxidants can help increase the viability of plants by suppressing browning which leads to shoot tip death [83,165–169]. By maintaining a higher antioxidant level protection improved post cryopreservation [166]. It has been reported that in *Actinidia* spp. (kiwifruit) the addition of ASA in regrowth media improved the survival after cryopreservation by reducing lipid peroxidation [83]. The addition of ASA to pre-culture media, loading solution, unloading solution and regrowth media significantly increased regrowth of shoot tips of *Rubus* spp. (raspberry) [168]. A recent study found treating *Persea americana* cv 'Reed' (avocado), with varying concentrations of different antioxidants (ASA, polyvinylpyrrolidone [PVP], citric acid and melatonin) reduced browning caused when extracting shoot tips. The type of antioxidant and concentration had an effect on viability, vigor and health of the shoots [170].

Avocado is highly susceptible to osmotic stresses imposed by cryoprotectants which are high in osmolarity. Cold sensitive species such as avocado are likely to be positively responsive to vitrification treatments during cryopreservation if optimizations are done carefully [171]. In order to improve on tolerance to cryoprotectants and increase permeation of the cryoprotectant through the cell membrane and induce tolerance to dehydration

caused by vitrification solutions, a pre-step called 'loading' is used [52]. Loading is achieved by incubating tissues for 10−20 min in solutions composed of glycerol and sucrose [48]. This loading step is particularly useful for plant species, that are sensitive to direct exposure to cryoprotectants due to dehydration intolerance and osmotic stresses [48]. However, use of loading solution alone for avocado shoot tips is not adequate to induce tolerance to cryoprotectants, and other pre-treatments/pre-culture such as osmotic conditioning with sugars and cold acclimatization are necessary [172].

Pre-culturing shoot tips with a high sugar enriched media has been reported previously by several authors [173–175] to increase the viability post-cryopreservation by better pre-conditioning the shoot. Also, time of incubation in pre-culture solutions was critical to ensuring survival and high regrowth rates [55,176]. There have been attempts to use alternative sources of sugar in pre-culture media, such as, sorbitol or mannitol [177–180], glucose and fructose; all have shown no negative effects on post-cryopreservation survival [181]. However, most researchers prefer to use sucrose as the sugar source when adding to pre-culture media [181]. Sucrose has been found to be more beneficial in preculture as compared to sorbitol and mannitol as these two sugars were unable to support regrowth of olive somatic embryos [182]. However, when 0.2 M sorbitol was combined with 5% DMSO it was an effective cryoprotectant for embryogenic tissue of *Pinus roxburghii* Sarg. (chir pine) [183]. Sucrose is an excellent glass former and is able to stabilize membranes and proteins [184]. Sucrose stimulates the production of other elements such as proline, glycine betaine, glycerol and polyamines, which have colligative as well as non-colligative effects [185,186]. Of the above-mentioned sugars [187], glycerol [188], proline [189] and glycine betaine [190] have proved their cryoprotectant ability, whereas polyamines are known for their antioxidant properties. Therefore, these compounds play a vital role in protecting the cells during cryopreservation. It has also been shown that pre-culturing in high sucrose media enhances the acclimatization process to low temperature and stimulates osmotic dehydration [47].

Water availability and temperature are influenced by environmental variables and are major determinants of plant growth and development [191]. Most tropical and subtropical species have little to no freezing tolerance, however, temperate plant species have evolved some form of cold tolerance [191,192]. It has been shown in temperate plants that they have the genetic ability to increase cold tolerance significantly when exposed to environmental cues that signal the arrival of winter [193]. Many plants can increase their tolerance to the cold by exposure to lower temperatures, generally with temperatures below 10 ◦C [193]. This process is referred to as cold hardening or cold acclimatization (CA) and requires days to weeks for full development [50,193,194]. Several biochemical, physiological and metabolic functions are altered in plants by low temperature as well as gene expression [195]. Expression of cold induced genes include those that control the function of cell membranes to stabilize and protect themselves against freezing injury [196]. Freezing tolerance can be increased by 2–8 ◦C in spring annuals, 10–30 ◦C in winter annuals and 20–200 ◦C in tree species [193]. Cold acclimatization can help improve the regrowth rates of in vitro plants, improve regeneration rates [197]. Cold acclimatization has been used as an in vitro pre-treatment on donor plants before shoot tip extraction [198] in developing cryopreservation protocols in plants such as *Malus domestica* Borkh (apple), *Malus sieversii* (Ledeb.) (wild apple) and *Phoenix dactylifera* (date palm) [199,200]. Cold acclimatization with or without ABA significantly improved the survival of *Rubus* spp. [201]. Abscisic acid (ABA) pre-treatment alone could not increase the survival of plants grown under warm conditions after cryopreservation, but the survival tripled when cold acclimatization was combined with ABA pre-treatment [201]. High sucrose (0.3 M) or low temperature (10 ◦C) incubation treatments primed in vitro plants of cvs 'Reed' and 'Velvick' shoot tips to tolerate cryoprotectant (PVS2) treatments but was cultivar-specific [202].

#### **6. Conclusions**

Field living germplasm collections are currently the only conservation method for avocado, but funding and threats from natural calamities; pest and diseases are a problem. Cryopreservation is an invaluable tool that could be utilized in conjunction with field repositories to securely preserve this important horticultural crop. There have been significant improvements within the cryopreservation platform to preserve *Persea* spp. germplasm [202–204]. Studies have shown that cryopreservation of somatic embryos offers usefulness in conserving *Persea* germplasm biodiversity [144,152,153]. An important factor for somatic embryos is that regeneration can be achieved after exposure to LN to ensure that protocols can be effectively applied for conservation programs [176]. Cryopreservation of somatic embryos is valuable as it is readily retrievable for further biotechnology manipulations as well as storage of biotechnology products such as genetically transformed lines [23,205].

To date, although cultivar-dependent, in vitro multiplication protocols have been established for maintaining multiple avocado cultivars in tissue culture from mature glasshouse cuttings [111]. This can be used to supply new plants to avocado farmers, meeting a critical issue that is preventing the expansion of industry, the shortage of available avocado trees. Twenty thousand in vitro plants can be maintained in a 10 sqm tissue culture room saving on land, fertilizer, pesticides promoting an environmentally sustainable and efficient method of multiplication of avocado plants.

Development of the in vitro shoot-tip cryopreservation protocol was highly dependent on the availability of this reliable in vitro multiplication and regeneration protocol. For the first time studies [202–204] have shown that in vitro cryopreservation using dropletvitrification for mature material of two avocado cultivars have been successful. Correctly treating avocado shoot tips with the ideal pre-treatment before LN is vital for a successful outcome [202]. It was identified that the use of 100 and 250 mg L−<sup>1</sup> of ASA can effectively reduce browning of freshly extracted avocado shoot tips [170,202]. High sucrose and cold pre-treatments are effective in increasing survivability following cryoprotectant incubation of avocado shoot tips. While pre-treatments are effective for avocado, the type of pretreatment needed and the degree of effectiveness was cultivar-specific [202]. This can be directly linked to the genetics of the two cultivars which display varying tolerance to cold and salinity in their natural growing environments; namely, cv 'Velvick' from West Indian race (no cold tolerance) and cv 'Reed' from Guatemalan race (moderate cold tolerance) [204]. The type of cryoprotectant and exposure time to the cryoprotectant was also essential in obtaining morphologically normal and vigorous plants [204]. Avocado shoots that survived LN grew into full plants ready for rooting after 24 weeks [204]. Cultivar 'Reed' shoots were successfully rooted [206] and after 8 weeks, plantlets were ready to be acclimatized in a University of Queensland glasshouse (Figure 2). These plants will be screened for growth parameters and yield in a field trial at Duranbah, Queensland. Shoot tips from cv 'Velvick' are currently in the rooting stage.

In vitro multiplication and in vitro cryopreservation protocols provide another set of tools that can be used to preserve global avocado diversity to improve conservation germplasm collections, breeding and propagation. Somatic embryogenesis, cryopreservation of somatic embryos and shoot tips, have the ability to be adapted to lead to the establishment of a global Cryo-Bank conserving avocado biodiversity and offering a source of disease-free genetic material. They provide useful tools for further optimization of the species and other woody plant species facing similar challenges in conservation. Shoot tip cryopreservation is ideal for preserving a core selection of avocado genotypes, for example, with superior characteristics, disease and pest resistance, rarity, drought and salinity tolerance. Shoot tip cryopreservation of avocado is a major breakthrough and this work can pave the way for storing a core collection of *Persea* spp. for true-to-type avocado shoot tip preservation.

**Figure 2.** Shoot tips of cv 'Reed' treated with VSL and revived from LN growing in a glasshouse.

**Author Contributions:** C.O.: Writing and review of literature — original draft, Drafting and production. J.H.-B.: Drafting and production. R.F.: Drafting and production. S.L.: Drafting and production. A.H.: Drafting and production. J.F.: Drafting and production. N.M.: Drafting and production. All authors have read and agreed to the published version of the manuscript.

**Funding:** Chris O'Brien is supported by an Australian Commonwealth Government Research Training Program (RTP) Scholarship and funding from The Huntington Library, Art Museum, and Botanical Gardens as well as funding from Advance Queensland Innovation Partnerships Project Avocado Tissue-Culture: From Lab-to-Orchard (AQIP06316-17RD2).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The Queensland Alliance for Agriculture and Food Innovation (QAAFI) is a research institute of The University of Queensland (UQ), supported by the Queensland Government Department of Agriculture and Fisheries. Chris O'Brien is supported by an Australian Commonwealth Government Research Training Program (RTP) Scholarship and funding from The Huntington Library, Art Museum, and Botanical Gardens as well as funding from Advance Queensland Innovation Partnerships Project Avocado Tissue-Culture: From Lab-to-Orchard (AQIP06316-17RD2). The authors would also like to acknowledge the following people for providing information on avocado germplasm collections Mary Lu Arpaia, Eric Focht, Patricia Manosalva, Alejandro Barrientos-Priego, Tatiana Cantuarias, Elizabeth Dann, Ricardo Goenaga. We also acknowledge Christina Walters, Kim E. Hummer and Gan-Yuan Zhong for information on clonal germplasm repositories.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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