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Cryopreservation of Woody Crops: The Avocado Case

Centre for Horticultural Science, Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St Lucia, QLD 4072, Australia
The Huntington Library, Art Museum, and Botanical Gardens, 1151 Oxford Road, San Marino, CA 91108, USA
Author to whom correspondence should be addressed.
Plants 2021, 10(5), 934;
Submission received: 14 April 2021 / Revised: 29 April 2021 / Accepted: 29 April 2021 / Published: 7 May 2021
(This article belongs to the Special Issue Plant Cryopreservation)


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.

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 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 [17,18,19]. 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.

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 [35,36,37,38] 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).

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 [50,51,52,53].
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 [54,58,59,60,61]. The most commonly used cryoprotectants for plant cells are PVS2 [59] and PVS3 [58].

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].

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 [96,97,98] 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 [101,102,103]. 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 [122,123,124]. 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.

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.

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 [83,165,166,167,168,169]. 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 [173,174,175] 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 [177,178,179,180], 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 pre-culture 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 sub-tropical 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 [202,203,204]. 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 [202,203,204] have shown that in vitro cryopreservation using droplet-vitrification 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−1 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 pre-treatment 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.

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.


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.


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.


  1. Wilson, E. The Diversity of Life; Penguin: London, UK, 1992. [Google Scholar]
  2. Harlan, J.R.; de Wet, J.M. Toward a rational classification of cultivated plants. Taxon 1971, 20, 509–517. [Google Scholar] [CrossRef]
  3. Engels, J.; Ebert, A.; Thormann, I.; De Vicente, M. Centres of crop diversity and/or origin, genetically modified crops and implications for plant genetic resources conservation. Genet. Resour. Crop Evol. 2006, 53, 1675–1688. [Google Scholar] [CrossRef]
  4. Maxted, N.; Guarino, L.; Myer, L.; Chiwona, E. Towards a methodology for on-farm conservation of plant genetic resources. Genet. Resour. Crop Evol. 2002, 49, 31–46. [Google Scholar] [CrossRef]
  5. Haidet, M.; Olwell, P. Seeds of success: A national seed banking program working to achieve long-term conservation goals. Nat. Areas J. 2015, 35, 165–173. [Google Scholar] [CrossRef]
  6. Engelmann, F.; Engels, J. Technologies and strategies for ex situ conservation. In Managing Plant Genetic Diversity; Engels, J.M., Ramanatha, R.U., Brown, A.H.D., Jackson, M.T., Eds.; CAB International: Wallingford, UK, 2002; pp. 89–103. ISBN 9780851995229. [Google Scholar] [CrossRef]
  7. Panis, B.; Nagel, M. Challenges and Prospects for the Conservation of Crop Genetic Resources in Field Genebanks, in In Vitro Collections and/or in Liquid Nitrogen. Plants 2020, 9, 1634. [Google Scholar] [CrossRef]
  8. Tsioumani, E. The State of the World’s Biodiversity for Food and Agriculture: A Call to Action? Environ. Policy Law 2019, 49, 110–112. [Google Scholar] [CrossRef]
  9. Migicovsky, Z.; Warschefsky, E.; Klein, L.L.; Miller, A.J. Using living germplasm collections to characterize, improve, and conserve woody perennials. Crop Sci. 2019, 59, 2365–2380. [Google Scholar] [CrossRef] [Green Version]
  10. Normah, M.N.; Sulong, N.; Reed, B.M. Cryopreservation of shoot tips of recalcitrant and tropical species: Advances and Strategies. Cryobiology 2019, 87, 1–14. [Google Scholar] [CrossRef]
  11. Berjak, P.; Pammenter, N. Seed recalcitrance-current perspectives. S. Afr. J. Bot. 2001, 67, 79–89. [Google Scholar] [CrossRef] [Green Version]
  12. Niino, T.; Arizaga, M.V. Cryopreservation for preservation of potato genetic resources. Breed. Sci. 2015, 65, 41–52. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  13. Engelmann, F. Use of biotechnologies for the conservation of plant biodiversity. Vitr. Cell. Dev. Biol. Plant 2011, 47, 5–16. [Google Scholar] [CrossRef]
  14. Acedo, V.; Arradoza, C. In vitro conservation of yam germplasm. Philipp. J. Crop Sci. 2005. [Google Scholar] [CrossRef] [Green Version]
  15. Reed, B.; Bell, R. In Vitro Tissue Culture of Pear: Advances in Techniques for Micropropagation and Germplasm Preservation. In Proceedings of the VIII International Symposium on Pear 596, Ferrara—Bologna, Italy, 31 December 2002; pp. 412–418, ISBN 9066058668. [Google Scholar] [CrossRef]
  16. Bertrand-Desbrunais, A.; Noirot, M.; Charrier, A. Slow growth in vitro conservation of coffee (Coffea spp.). Plant Celltissue Organ Cult. 1992, 31, 105–110. [Google Scholar] [CrossRef]
  17. Engelmann, F. In vitro conservation methods. In Biotechnology and Plant Genetic Resources: Conservation and Use; Callow, J.A., Ford-Lloyd, B.V., Newburry, J.H., Eds.; CABI: Wallingford, UK, 1997; pp. 119–162. ISBN 0851991424. [Google Scholar]
  18. George, E.F.; Sherrington, P.D. Plant propagation by tissue culture: Handbook and directory of commercial laboratories. In Journal of Basic Microbiology; Schmauder, H.P., Ed.; Exegetics Ltd.: Eversley, UK, 1984; ISBN 0950932507. [Google Scholar] [CrossRef]
  19. Reed, B.M.; Gupta, S.; Uchendu, E.E. In vitro genebanks for preserving tropical biodiversity. In Conservation of Tropical Plant Species, 1st ed.; Normah, M.N., Chin, H.F., Reed, B.M., Eds.; Springer New York: New York, NY, USA, 2013; pp. 77–106. ISBN 978-1-4614-3776-5. [Google Scholar] [CrossRef]
  20. Bhojwani, S.S.; Razdan, M.K. Plant Tissue Culture: Theory and Practice; Elsevier: Amsterdam, The Netherlands, 1986; p. 0444596151. [Google Scholar]
  21. Benson, E.E. Cryopreservation Theory. In Plant Cryopreservation: A Practical Guide; Reed, B.M., Ed.; Springer: New York, NY, USA, 2008; pp. 15–32. ISBN 978-0-387-72275-7. [Google Scholar] [CrossRef]
  22. Harding, K. Genetic integrity of cryopreserved plant cells: A review. CryoLetters 2004, 25, 3–22. [Google Scholar]
  23. Engelmann, F. Cryopreservation of embryos: An overview. In Plant Embryo Culture, Methods in Molecular Biology (Methods and Protocols.); Thorpe, T.A., Yeung, E.C., Eds.; Humana Press: Totowa, NJ, USA, 2011; Volume 710, pp. 155–184. ISBN 978-1-61737-987-1. [Google Scholar] [CrossRef]
  24. Bi, W.L.; Pan, C.; Hao, X.Y.; Cui, Z.H.; Kher, M.; Marković, Z.; Wang, Q.C.; Teixeira da Silva, J. Cryopreservation of grapevine (Vitis spp.)—A review. Vitr. Cell. Dev. Biol. Plant 2017, 53, 449–460. [Google Scholar] [CrossRef]
  25. Benelli, C.; de Carlo, A.; Engelmann, F. Recent Advances in the cryopreservation of shoot-derived germplasm of economically important fruit trees of Actinidia, Diospyros, Malus, Olea, Prunus, Pyrus and Vitis. Biotechnol. Adv. 2013, 31, 175–185. [Google Scholar] [CrossRef]
  26. Engelmann, F. Germplasm collection, storage, and conservation. In Plant Biotechnology and Agriculture. Prospects for 21st Century; Altman, A., Hagegawa, A., Eds.; Academic Press: San Diego, CA, USA, 2012; pp. 255–267. [Google Scholar] [CrossRef]
  27. Bettoni, J.C.; Costa, M.D.; Gardin, J.P.P.; Kretzschmar, A.A.; Pathirana, R. Cryotherapy: A new technique to obtain grapevine plants free of viruses. Rev. Bras. De Frutic. 2016, 38. [Google Scholar] [CrossRef] [Green Version]
  28. Wang, Q.; Valkonen, J.P. Cryotherapy of shoot tips: Novel pathogen eradication method. Trends Plant Sci. 2009, 14, 119–122. [Google Scholar] [CrossRef]
  29. Barrientos-Priego, A.F.; Borys, M.W.; Escamilla-Prado, E.; Ben-Ya’acov, A.; De La Cruz-Torres, E.; Lopez-Lopez, L. A study of the avocado germplasm resources, 1988–1990. IV. Findings in the Mexican Gulf Region. In Proceedings of the Second World Avocado Congress, Orange, CA, USA, 21–26 April 1992; pp. 551–558. [Google Scholar]
  30. Benz, B. The conservation of cultivated plants. Nat. Educ. Knowl. 2012, 3, 4. Available online: (accessed on 18 January 2021).
  31. GRIN-Global, U.S. National Plant Germplasm System. Available online: (accessed on 4 January 2020).
  32. NTBG Breadfruit Institute. To Promote the Conservation, Study, and Use of Breadfruit for Food and Reforestation. Available online: (accessed on 5 January 2021).
  33. Hanke, M.V.; Höfer, M.; Flachowsky, H.; Peil, A. Fruit Genetic Resources Management: Collection, Conservation, Evaluation and Utilization in Germany; International Society for Horticultural Science (ISHS): Leuven, Belgium, 2014; pp. 231–234, 2406–6168. [Google Scholar] [CrossRef]
  34. National-Fruit-Collection National Fruit Collection held at Brogdale Farm. Available online: (accessed on 4 January 2020).
  35. Withers, L.A.; Engelmann, F. In vitro conservation of plant genetic resources. In Agricultural Biotechnology; Altman, A., Ed.; Marcel Dekker: New York, NY, USA, 1998; pp. 57–88. ISBN 0-8247-9439-7. [Google Scholar]
  36. Ashmore, S.E. Status Report on the Development and Application of In Vitro Techniques for the Conservation and Use of Plant Genetic Resources; International Plant Genetic Resources Institute: Rome, Italy, 1997; ISBN 9290433396. [Google Scholar]
  37. Mandal, B.; Tyagi, R.; Pandey, R.; Sharma, N.; Agrawal, A. In Vitro Conservation Of Germplasm of Agri-Horticultural Crops at NBPGR: An Overview. In Conservation of Plant Genetic Resources in Vitro; Razdan, M.K., Cocking, E.C., Eds.; Science Publishers Inc.: Hauppauge, NY, USA, 2000; Volume 2, pp. 297–307. [Google Scholar]
  38. Reed, B.M.; Denoma, J.; Wada, S.; Postman, J. Micropropagation of pear (Pyrus spp.). Methods Mol. Biol. 2013, 11013, 3–18. [Google Scholar] [PubMed]
  39. FAO. Genebank Standards: For Plant Genetic Resources for Food and Agriculture; Food and Agriculture Organization of the United Nations: Rome, Italy, 2014; ISBN 978-92-5-108262-1. [Google Scholar]
  40. Reed, B.M.; Engelmann, F.; Dulloo, M.E.; Engels, J.M.M. Technical Guidelines for the Management of Field and In vitro Germplasm Collections. In IPGRI Handbooks for Genebanks No. 7; International Plant Genetic Resources Institute: Rome, Italy, 2004; Volume 472, ISBN 9290436409. [Google Scholar]
  41. Benson, E.; Harding, K.; Debouck, D.G.; Dumet, D.; Escobar, R.; Mafla, G.; Panis, B.; Panta, A.; Tay, D.; Houwe, I. Refinement and Standardization of Storage Procedures for Clonal Crops. Global Public Goods Phase 2: Part 1. Project Landscape and General Status of Clonal Crop in Vitro Conservation Technologies; System-Wide Genetic Resources Programme (SGRP): Rome, Italy, 2011; ISBN 929043905X. [Google Scholar]
  42. Benson, E.; Harding, K.; Debouck, D.G.; Dumet, D.; Escobar, R.; Mafla, G.; Panis, B.; Panta, A.; Tay, D.; Houwe, I. Refinement and Standardization of Storage Procedures for Clonal Crops. Global Public Goods Phase 2. Part 2: Status of in Vitro Conservation Technologies for: Andean Root and Tuber Crops, Cassava, Musa, Potato, Sweetpotato and Yam; System-Wide Genetic Resources Programme (SGRP): Rome, Italy, 2011; ISBN 9290439068. [Google Scholar]
  43. CGAIR Cassava Diversity. Available online: (accessed on 5 January 2021).
  44. Gutierrez, B.; (The Agricultural Research Service United States Department of Agriculture, Geneva, NY, USA). Personal Communication, 2020.
  45. Walters, C.; (The United States Department of Agriculture, Fort Collins, CO, USA). Personal Communication, 2020.
  46. Reed, B. Choosing and applying cryopreservation protocols to new plant species or tissues. In Proceedings of the I International Symposium on Cryopreservation in Horticultural Species 908, Leuven, Belgium, 5–8 April 2009; pp. 363–372. [Google Scholar]
  47. Reed, B.M. Plant Cryopreservation: A Practical Guide. Reed, B.M., Ed.; Springer New York: New York City, NY, USA, 2008; ISBN 978-0-387-72275-7. [Google Scholar]
  48. Kaczmarczyk, A.; Funnekotter, B.; Menon, A.; Phang, P.Y.; Al-Hanbali, A.; Bunn, E.; Mancera, R. Current issues in plant cryopreservation. In Current Frontiers in Cryobiology; Katkov, I.I., Ed.; In Tech: Rijeka, Croatia, 2012; pp. 417–438. ISBN 978-9535101918. [Google Scholar]
  49. Streczynski, R.; Clark, H.; Whelehan, L.M.; Ang, S.-T.; Hardstaff, L.K.; Funnekotter, B.; Bunn, E.; Offord, C.A.; Sommerville, K.D.; Mancera, R.L. Current issues in plant cryopreservation and importance for ex situ conservation of threatened Australian native species. Aust. J. Bot. 2019, 67, 1–15. [Google Scholar] [CrossRef] [Green Version]
  50. Panis, B.; Lambardi, M. Status of cryopreservation technologies in plants (crops and forest trees). In The Role of Biotechnology in Exploring and Protecting Agricultural Genetic Resources; Ruane, J., Sonnino, A., Eds.; Food & Agriculture Organization: Rome, Italy, 2006; pp. 61–78. ISBN 92-5-105480-0. [Google Scholar]
  51. Pérez, R.M. Cryostorage of Citrus embryogenic cultures. In Somatic Embryogenesis in Woody Plants; Jain, S.M., Gupta, P.K., Newton, R.J., Eds.; Springer: Dordrecht, The Netherlands, 2000; Volume 67, pp. 687–705. ISBN 978-90-481-5508-8. [Google Scholar] [CrossRef]
  52. Sakai, A.; Engelmann, F. Vitrification, encapsulation-vitrification and droplet-vitrification: A review. CryoLetters 2007, 28, 151–172. [Google Scholar] [PubMed]
  53. Tanino, K.K.; Chen, T.H.H.; Fughigami, L.H.; Weiser, C.J. Metabolic alterations associated with abscisic acid-induced frost hardiness in bromegrass suspension culture cells. Plant Cell Physiol. 1990, 31, 505–511. [Google Scholar] [CrossRef]
  54. Kim, H.-H.; No, N.-Y.; Shin, D.-J.; Ko, H.-C.; Kang, J.-H.; Cho, E.-G.; Engelmann, F. Development of alternative plant vitrification solutions to be used in droplet-vitrification procedures. Acta Hortic. 2011, 908, 181–186. [Google Scholar] [CrossRef]
  55. Azimi, M.; O’Brien, C.; Ashmore, S.; Drew, R. Cryopreservation of papaya germplasm. Acta Hortic. 2005, 692, 43–50. [Google Scholar] [CrossRef]
  56. Yamada, T.; Sakai, A.; Matsumura, T.; Higuchi, S. Cryopreservation of apical meristems of white clover (Trifolium repens L.) by vitrification. Plant Sci. 1991, 78, 81–87. [Google Scholar] [CrossRef]
  57. McGann, L.E. Differing actions of penetrating and nonpenetrating cryoprotective agents. Cryobiology 1978, 15, 382–390. [Google Scholar] [CrossRef]
  58. Nishizawa, S.; Sakai, A.; Amano, Y.; Matsuzawa, T. Cryopreservation of asparagus (Asparagus officinalis L.) embryogenic suspension cells and subsequent plant regeneration by vitrification. Plant Sci. 1993, 91, 67–73. [Google Scholar] [CrossRef]
  59. Sakai, A.; Kobayashi, S.; Oiyama, I. Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification. Plant Cell Rep. 1990, 9, 30–33. [Google Scholar] [CrossRef]
  60. Uragami, A.; Sakai, A.; Nagai, M. Cryopreservation of asparagus (Asparagus-officinalis L.) cultured in vitro. Jpn. Agric. Res. Q. 1993, 27, 112–115. [Google Scholar]
  61. Uragami, A.; Sakai, A.; Nagai, M.; Takahashi, T. Survival of cultured cells and somatic embryos of Asparagus officinalis cryopreserved by vitrification. Plant Cell Rep. 1989, 8, 418–421. [Google Scholar] [CrossRef] [PubMed]
  62. Matsumoto, T. Studies on Cryopreservation of In Vitro Grown Apical Meristems of Wasabi (Wasabia japonica M.). Doctorial Thesis, Bulletin of Shimane Agricultural Experiment Station, Shimane, Japan, 1999. [Google Scholar]
  63. Suzuki, M.; Tandon, P.; Ishikawa, M.; Toyomasu, T. Development of a new vitrification solution, VSL, and its application to the cryopreservation of gentian axillary buds. Plant Biotechnol. Rep. 2008, 2, 123. [Google Scholar] [CrossRef] [Green Version]
  64. Langis, R.; Schnabel, B.; Earle, E.; Steponkus, P. Cryopreservation of Brassica campestris L. cell suspensions by vitrification. Cryoletters 1989. Available online: (accessed on 21 February 2021).
  65. Towill, L.E. Cryopreservation of isolated mint shoot tips by vitrification. Plant Cell Rep. 1990, 9, 178–180. [Google Scholar] [CrossRef]
  66. Fahy, G.M.; MacFarlane, D.; Angell, C.; Meryman, H. Vitrification as an approach to cryopreservation. Cryobiology 1984, 21, 407–426. [Google Scholar] [CrossRef]
  67. Engelmann, F. Importance of cryopreservation for the conservation of plant genetic resources. Cryopreservation of tropical plant germplasm: Current research progress and application. In Proceedings of the an International Workshop, Tsukuba, Japan, 31 October – 1 November 1998; International Plant Genetic Resources Institute (IPGRI): Maccarese, Italy, 2000; pp. 8–20. ISBN 9290434-287. [Google Scholar]
  68. Yamamoto, S.-i.; Rafique, T.; Priyantha, W.S.; Fukui, K.; Matsumoto, T.; Niino, T. Development of a Cryopreservation Procedure Using Aluminium Cryo-plates. Cryoletters 2011, 32, 256–265. [Google Scholar] [PubMed]
  69. Adu-Gyamfi, R.; Wetten, A. Cryopreservation of cocoa (Theobroma cacao L.) somatic embryos by vitrification. CryoLetters 2012, 33, 494–505. [Google Scholar]
  70. de Oliveira Prudente, D.; Paiva, R.; Nery, F.C.; de Oliveira Paiva, P.D.; Alves, J.D.; Máximo, W.P.F.; Silva, L.C. Compatible solutes improve regrowth, ameliorate enzymatic antioxidant systems, and reduce lipid peroxidation of cryopreserved Hancornia speciosa Gomes lateral buds. Vitr. Cell. Dev. Biol. Plant 2017, 53, 352–362. [Google Scholar] [CrossRef]
  71. Shibli, R.; Al-Juboory, K. Cryopreservation of ’Nabali’ olive (Olea europea l.) somatic embryos by encapsulation-dehydration and encapsulation-vitrification. CryoLetters 2000, 21, 357–366. [Google Scholar] [PubMed]
  72. Nadarajan, J.; Mansor, M.; Krishnapillay, B.; Staines, H.J.; Benson, E.E.; Harding, K. Applications of differential scanning calorimetry in developing cryopreservation strategies for Parkia speciosa, a tropical tree producing recalcitrant seeds. CryoLetters 2008, 29, 95–110. [Google Scholar]
  73. Soliman, H.I. Cryopreservation of in vitro-grown shoot tips of apricot (Prunus armeniaca L.) using encapsulation-dehydration. Afr. J. Biotechnol. 2013, 12, 1419–1430. [Google Scholar]
  74. Ballesteros, D.; Pence, V.C. Survival and growth of embryo axes of temperate trees after two decades of cryo-storage. Cryobiology 2019, 88, 110–113. [Google Scholar] [CrossRef] [PubMed]
  75. Ibrahim, S.; Normah, M. The survival of in vitro shoot tips of Garcinia mangostana L. after cryopreservation by vitrification. Plant Growth Regul. 2013, 70, 237–246. [Google Scholar] [CrossRef]
  76. Yamamoto, S.; Rafique, T.; Sekizawa, K.; Koyama, A.; Ichihashi, T.; Niino, T. Development of an effective cryopreservation protocol using aluminum cryo-plates for in vitro-grown shoot tips of mulberries (Morus spp.) originated from the tropics and subtropics. Sanshi Konchu Biotec (J. Insect Biotechnol. Sericology) 2012, 81, 57–62. [Google Scholar]
  77. Matsumoto, T.; Yamamoto, S.-i.; Fukui, K.; Rafique, T.; Engelmann, F.; Niino, T. Cryopreservation of persimmon shoot tips from dormant buds using the D cryo-plate technique. Hortic. J. 2015. [Google Scholar] [CrossRef] [Green Version]
  78. Kim, H.H.; Kim, J.B.; Baek, H.J.; Cho, E.G.; Chae, Y.A.; Engelmann, F. Evolution of DMSO concentration in garlic shoot tips during a vitrification procedure. CryoLetters 2004, 25, 90–100. [Google Scholar]
  79. Kartha, K.; Leung, N.; Mroginski, L. In vitro growth responses and plant regeneration from cryopreserved meristems of cassava (Manihot esculenta Crantz). Z. Für Pflanzenphysiol. 1982, 107, 133–140. [Google Scholar] [CrossRef]
  80. Bettoni, J.C.; Bonnart, R.; Volk, G.M. Challenges in implementing plant shoot tip cryopreservation technologies. Plant Cell Tissue Organ Cult. 2020. [Google Scholar] [CrossRef]
  81. Panis, B.; Piette, B.; Swennen, R. Droplet vitrification of apical meristems: A cryopreservation protocol applicable to all Musaceae. Plant Sci. 2005, 168, 45–55. [Google Scholar] [CrossRef]
  82. Benson, E.; Harding, K.; Debouck, D.G.; Dumet, D.; Escobar, R.; Mafla, G.; Panis, B.; Panta, A.; Tay, D.; Houwe, I. Refinement and Standardization of Storage Procedures for Clonal Crops. Global Public Goods Phase 2. Part 3: Multi-Crop Guidelines for Developing in Vitro Conservation Best Practices for Clonal Crops; System-Wide Genetic Resources Programme (SGRP): Rome, Italy, 2011; ISBN 9290438339. [Google Scholar]
  83. Mathew, L.; McLachlan, A.; Jibran, R.; Burritt, D.J.; Pathirana, R. Cold, antioxidant and osmotic pre-treatments maintain the structural integrity of meristematic cells and improve plant regeneration in cryopreserved kiwifruit shoot tips. Protoplasma 2018, 255, 1065–1077. [Google Scholar] [CrossRef]
  84. Niino, T.; Sakai, A. Cryopreservation of alginate-coated in vitro-grown shoot tips of apple, pear and mulberry. Plant Sci. 1992, 87, 199–206. [Google Scholar] [CrossRef]
  85. Paul, H.; Daigny, G.; Sangwan-Norreel, B. Cryopreservation of apple (Malus× domestica Borkh.) shoot tips following encapsulation-dehydration or encapsulation-vitrification. Plant Cell Rep. 2000, 19, 768–774. [Google Scholar] [CrossRef]
  86. Wang, Q.; Tanne, E.; Arav, A.; Gafny, R. Cryopreservation of in vitro-grown shoot tips of grapevine by encapsulation-dehydration. Plant Celltissue Organ Cult. 2000, 63, 41–46. [Google Scholar] [CrossRef]
  87. Wang, Q.; Batuman, Ö.; Li, P.; Bar-Joseph, M.; Gafny, R. Cryopreservation of in vitro-grown shoot tips of’Troyer’citrange [Poncirus trifoliata (L.) Raf.× Citrus sinensis (L.) Osbeck.] by encapsulation-dehydration. Plant Cell Rep. 2002, 20, 901–906. [Google Scholar] [CrossRef]
  88. Wang, Q.; Batuman, Ö.; Li, P.; Bar-Joseph, M.; Gafny, R. A simple and efficient cryopreservation of in vitro-grown shoot tips ofTroyer’citrange [Poncirus trifoliata (L.) Raf.× Citrus sinensis (L.) Osbeck.] by encapsulation-vitrification. Euphytica 2002, 128, 135–142. [Google Scholar] [CrossRef]
  89. Niino, T.; Yamamoto, S.; Matsumoto, T.; Engelmann, F.; Valle Arizaga, M.; Tanaka, D. Development of V and D Cryo-Plate Methods as Effective Protocols for Cryobanking; International Society for Horticultural Science (ISHS): Leuven, Belgium, 2019; pp. 249–262, 2406–6168. [Google Scholar] [CrossRef]
  90. Kopp, L. A taxonomic revision of the genus Persea in the Western Hemisphere (Persea: Lauraceae). Revisión taxonómica del género Persea en el hemisferio occidental (Persea: Lauraceae). Garden 1966, 14, 1–120. [Google Scholar]
  91. Scora, R.W.; Bergh, B.O. Origin of and Taxonomic Relationships within the Genus Persea. In Proceedings of the Second World Avocado Congress, Orange, CA, USA, 21–26 April 1992; Volume 2, pp. 505–574. [Google Scholar]
  92. Bergh, B.; Ellstrand, N. Taxonomy of the avocado. Calif. Avocado Soc. Yearb. 1986, 70, 135–145. [Google Scholar]
  93. Rohwer, J.G.; Li, J.; Rudolph, B.; Schmidt, S.A.; van der Werff, H.; Li, H.-w. Is Persea (Lauraceae) monophyletic? Evidence from nuclear ribosomal ITS sequences. Taxon 2009, 58, 1153–1167. [Google Scholar] [CrossRef]
  94. Storey, W.; Bergh, B.; Zentmyer, G. The origin, indigenous range and dissemination of the avocado. Calif. Avocado Soc. Yearb. 1986, 70, 127–133. [Google Scholar]
  95. Pliego-Alfaro, F.; Palomo-Ríos, E.; Mercado, J.; Pliego, C.; Barceló-Muñoz, A.; López-Gómez, R.; Hormaza, J.; Litz, R. Persea americana avocado. Biotechnol. Fruit Nut Crop. 2020, 258–281. [Google Scholar] [CrossRef]
  96. Ben-Ya’acov, A.; Bufler, G.; Barrientos-Priego, A.; De La Cruz-Torres, E.; López-López, L. A Study of Avocado Germplasm Resources, 1988–1990. I. General Description of the International Project and its Findings. In Proceedings of the Second World Avocado Congress, Orange, CA, USA, 21–26 April 1992; Volume 2, pp. 535–541. [Google Scholar]
  97. Ge, Y.; Zhang, T.; Wu, B.; Tan, L.; Ma, F.; Zou, M.; Chen, H.; Pei, J.; Liu, Y.; Chen, Z.; et al. Genome-wide assessment of avocado germplasm determined from specific length amplified fragment sequencing and transcriptomes: Population structure, genetic diversity, identification, and application of race-specific markers. Genes 2019, 10, 215. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  98. O’Brien, C.; Hiti-Bandaralage, J.C.H.; Hayward, A.; Mitter, N. Avocado (Perse, americana Mill.). In Step Wise Protocols for Somatic Embryogenesis of Important Woody Plants; Jain, S.M., Gupta, P.K., Eds.; Springer Cham: cham, Switzerland, 2018; Volume 85, pp. 305–328. ISBN 978-3-319-79086-2. [Google Scholar] [CrossRef]
  99. Furnier, G.; Cummings, M.; Clegg, M. Evolution of the avocados as revealed by DNA restriction fragment variation. J. Hered. 1990, 81, 183–188. [Google Scholar] [CrossRef]
  100. Popenoe, W. The avocado—a horticultural problem. Trop Agric. 1941, 18, 3–7. [Google Scholar]
  101. Crane, J.H.; Balerdi, C.F.; Maguire, I. Avocado growing in the Florida home landscape. Hort. Sci. Dept. Fla. Coop. Ext. Serv. Inst. Food Agric. Sci. Univ. Florida. Circ. 2007, 1034, 1–12. [Google Scholar]
  102. Krezdorn, A. Influence of rootstock on cold hardiness of avocados. Proc. Fla. State Hort. Soc. 1973, 86, 346–348. [Google Scholar]
  103. Mickelbart, M.V.; Arpaia, M.L. Rootstock influences changes in ion concentrations, growth, and photosynthesis of ’Hass’ avocado trees in response to salinity. J. Am. Soc. Hortic. Sci. 2002, 127, 649–655. [Google Scholar] [CrossRef] [Green Version]
  104. Chen, H.; Morrell, P.L.; Ashworth, V.E.; de La Cruz, M.; Clegg, M.T. Tracing the geographic origins of major avocado cultivars. J. Hered. 2009, 100, 56–65. [Google Scholar] [CrossRef] [Green Version]
  105. Barrientos-Priego, A.F.; López-López, L. Historia y genética del aguacate. Télizd. Y Moraa.(Comps.). El Aguacate Y Su Manejo Integrado. 2ª (Ed.) Ed. Mundi-Prensa. Df México 2000, 19–31. Available online: (accessed on 12 February 2021).
  106. Ayala-Silva, T.; Ledesma, N. Avocado history, biodiversity and production. In Sustainable Horticultural Systems; Nandwani, D., Ed.; Springer: Cham, Switzerland, 2014; pp. 157–205. ISBN 978-3-319-06903-6. [Google Scholar] [CrossRef]
  107. Köhne, S. Selection of Avocado Scions and breeding of rootstocks in South Africa. In Proceedings of the New Zealand and Australia Avocado Grower’s Conference, Tauranga, New Zealand, 20–22 September 2005. [Google Scholar]
  108. Rendón-Anaya, M.; Ibarra-Laclette, E.; Bravo, A.M.; Lan, T.; Zheng, C.; Carretero-Paulet, L.; Perez-Torres, C.A.; Chacón-López, A.; Hernandez-Guzmán, G.; Chang, T.-H. The avocado genome informs deep angiosperm phylogeny, highlights introgressive hybridization, and reveals pathogen influenced gene space adaptation. Proc. Natl. Acad. Sci. USA 2019, 116, 17081–17089. [Google Scholar] [CrossRef] [Green Version]
  109. Avocados Australia. Facts at a Glance 2019/20 for the Australian Avocado Industry. Available online: (accessed on 11 February 2021).
  110. Dreher, M.L.; Davenport, A.J. Hass avocado composition and potential health effects. Crit. Rev. Food Sci. Nutr. 2013, 53, 738–750. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  111. Hiti-Bandaralage, J.C.; Hayward, A.; Mitter, N. Micropropagation of avocado (Persea americana Mill.). Am. J. Plant Sci. 2017, 8, 2898–2921. [Google Scholar] [CrossRef] [Green Version]
  112. Alcaraz, M.; Hormaza, J. Selection of potential pollinizers for ‘Hass’ avocado based on flowering time and male–female overlapping. Sci. Hortic. 2009, 121, 267–271. [Google Scholar] [CrossRef]
  113. Davenport, T. Avocado flowering. Hortic. Rev. 1986, 8, 89. [Google Scholar]
  114. Lavi, U.; Lahav, E.; Degani, C.; Gazit, S.; Hillel, J. Genetic variance components and heritabilities of several avocado traits. J. Am. Soc. Hortic. Sci. 1993, 118, 400–404. [Google Scholar] [CrossRef] [Green Version]
  115. Arpaia, M.L. Avocado Brainstorming 2018, Towards a Sustainable Future; Fairview Hotel: Tzaneen, South Africa, 2018. [Google Scholar]
  116. Bergh, B. Avocado research in Israel. Calif. Avocado Soc. Yearb. 1975, 58, 103–126. [Google Scholar]
  117. Parkinson, L.; Geering, A. Biosecurity capacity building for the Australian avocado industry. Available online: (accessed on 11 February 2021).
  118. Darvas, J.; Kotze, J. Avocado fruit diseases and their control in South Africa. S. Afr. Avocado Grow. Assoc. Yearb. 1987, 10, 117–119. [Google Scholar]
  119. Vega, D.E.S. Propagation in vitro of Rootstocks of Avocado. Calif. Avocado Soc. Yearb. 1989, 73, 196–199. [Google Scholar]
  120. Pliego-Alfaro, F.; Murashige, T. Possible rejuvenation of adult avocado by graftage onto juvenile rootstocks in vitro. HortScience 1987, 22, 1321–1324. [Google Scholar]
  121. Lorea Hernández, F.G. La familia Lauraceae en el sur de México: Diversidad, distribución y estado de conservación. Boletín De La Soc. Botánica De México 2002, 71. [Google Scholar] [CrossRef] [Green Version]
  122. Ben-Ya’acov, A. Avocado rootstock-scion relationships. S. Afr. Avocado Grow. Assoc. Yearb. 1987, 10, 30–32. [Google Scholar]
  123. Wolstenholme, B. Theoretical and applied aspects of avocado yield as affected by energy budgets and carbon partitioning. S. Afr. Avocado Grow. Assoc. Yearb. 1987, 10, 58–61. [Google Scholar]
  124. Wolstenholme, B. Avocado rootstocks: What do we know; are we doing enough research. S. Afr. Avocado Grow. Assoc. Yearb. 2003, 2003, 106–112. [Google Scholar]
  125. Smith, L.; Dann, E.; Pegg, K.; Whiley, A.; Giblin, F.; Doogan, V.; Kopittke, R. Field assessment of avocado rootstock selections for resistance to Phytophthora root rot. Australas. Plant Pathol. 2011, 40, 39–47. [Google Scholar] [CrossRef]
  126. Mickelbart, M.V.; Bender, G.S.; Witney, G.W.; Adams, C.; Arpaia, M.L. Effects of clonal rootstocks on ‘Hass’ avocado yield components, alternate bearing, and nutrition. J. Hortic. Sci. Biotechnol. 2007, 82, 460–466. [Google Scholar] [CrossRef]
  127. New Zealand avocado Varieties and rootstocks. Available online: (accessed on 6 February 2021).
  128. Folgado, R.; (The Huntington Library, Art Museum, and Botanical Gardens, San Marino CA, USA). Personal Communication, 2020.
  129. Arpaia, M.L.; Focht, E.; (Department of Botany and Plant Sciences, University of California, Riverside, CA, USA). Personal Communication, 2020.
  130. Manosalva, P.; (Department of Microbiology and Plant Pathology, University of California, Riverside, CA, USA). Personal Communication, 2020.
  131. Goenaga, R.; (Agricultural Research Service, United States Department of Agriculture, Miami, FL, USA). Personal Communication, 2020.
  132. Álvarez, S.P.; Quezada, G.Á.; Arbelo, O.C. Avocado (Persea americana Mill). Cultiv. Trop. 2015, 36, 111–123. [Google Scholar] [CrossRef]
  133. Barrientos-Priego, A.; (Departamento de Fitotecnia, Texcoco de Mora, Mexico). Personal Communication, 2020.
  134. Nkansah, G.; Ofosu-Budu, K.; Ayarna, A. Avocado germplasm conservation and improvement in Ghana. In Proceedings of the VII World Avocado Congress 2011, Cairns, Australia, 5–9 September 2011. [Google Scholar]
  135. Alcaraz, M.; Hormaza, J. Molecular characterization and genetic diversity in an avocado collection of cultivars and local Spanish genotypes using SSRs. Hereditas 2007, 144, 244–253. [Google Scholar] [CrossRef]
  136. Dann, E.; (Centre for Horticultural Science Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St Lucia, Queensland, Australia). Personal Communication, 2020.
  137. Borokini, T.I. Conservation Science. Int. J. Conserv. Sci. 2013, 4, 197–212. [Google Scholar]
  138. Cantuarias-Avilés, T.; (Department of Plant Production, University of Sao Paulo, Piracicaba SP, Brazil). Personal Communication, 2020.
  139. Encina, C.L.; Parisi, A.; O’Brien, C.; Mitter, N. Enhancing somatic embryogenesis in avocado (Persea americana Mill.) using a two-step culture system and including glutamine in the culture medium. Sci. Hortic. 2014, 165, 44–50. [Google Scholar] [CrossRef]
  140. Guan, Y.; Li, S.-G.; Fan, X.-F.; Su, Z.-H. Application of somatic embryogenesis in woody plants. Front. Plant Sci. 2016, 7, 938. [Google Scholar] [CrossRef] [Green Version]
  141. Kulkarni, V.; Suprasanna, P.; Bapat, V. Plant regeneration through multiple shoot formation and somatic embryogenesis in a commercially important and endangered Indian banana cv. Rajeli. Curr. Sci. 2006, 842–846. Available online: (accessed on 4 April 2021).
  142. Márquez-Martín, B.; Barceló-Muñoz, A.; Pliego-Alfaro, F.; Sánchez-Romero, C. Somatic embryogenesis and plant regeneration in avocado (Persea americana Mill.): Influence of embryogenic culture type. J. Plant Biochem. Biotechnol. 2012, 21, 180–188. [Google Scholar] [CrossRef]
  143. Jain, S.M.; Ishii, K. Micropropagation of Woody Plants and Fruits; Kluwer Academic Publishers: Amsterdam, The Netherlands, 2003; Volume 75, ISBN 1-4020-1135-0. [Google Scholar]
  144. O’Brien, C.; Constantin, M.; Walia, A.; Yiing, J.L.Y.; Mitter, N. Cryopreservation of somatic embryos for avocado germplasm conservation. Sci. Hortic. 2016, 211, 328–335. [Google Scholar] [CrossRef]
  145. Pliego-Alfaro, F.; Murashige, T. Somatic embryogenesis in avocado (Persea americana Mill.) in vitro. Plant Celltissue Organ Cult. 1988, 12, 61–66. [Google Scholar] [CrossRef]
  146. Litz, R.; Litz, W. Somatic embryogenesis of avocado (Persea americana) and its application for plant improvement. Int. Symp. Trop. Subtrop. Fruits 2000, 575. [Google Scholar] [CrossRef]
  147. Mujib, A.; Šamaj, J. Somatic Embryogenesis; Springer Science & Business Media: Berlin/Heidelberg, Germany, 2006; Volume 2, p. 3540287175. [Google Scholar]
  148. Mooney, P.; Staden, J.V. Induction of embryogenesis in callus from immature embryos of Persea americana. Can. J. Bot. 1987, 65, 622–626. [Google Scholar] [CrossRef]
  149. Ammirato, P.V. Organizational events during somatic embryogenesis. Plant Biol. 1986. Available online: (accessed on 7 January 2021).
  150. Raharjo, S.; Litz, R.E. Rescue of Genetically Transformed Avocado by Micrografting. In Proceedings of the V World Avocado Congress (Actas V Congreso Mundial del Aguacate), Granada-Málaga, Spain, 19–24 October 2003; pp. 119–122. [Google Scholar]
  151. Witjaksono, Y.; Litz, R. Maturation and germination of avocado (Persea americana Mill.) somatic embryos. Plant Cell Tissue Organ Cult 1999, 58, 141–148. [Google Scholar] [CrossRef]
  152. Efendi, D.; Litz, R.E. Cryopreservation of Avocado. In Proceedings of the V Congreso Mundial del Aguacate, Actas; Consejería de Agricultura y Pesca, Junta de Andalucía, Sevilla, Spain; 2003; Volume 1, pp. 111–114. [Google Scholar]
  153. Guzmán-García, E.; Bradaï, F.; Sánchez-Romero, C. Cryopreservation of avocado embryogenic cultures using the droplet-vitrification method. Acta Physiol. Plant. 2013, 1–11. [Google Scholar] [CrossRef]
  154. Vargas, V.M. Efecto Fisiológico de Brasinoesteroides y Crioprotectores Sobre Yemas Axilares de Aguacate Criollo Producidas in Vitro. 2008. Available online: (accessed on 24 February 2021).
  155. Vidales-Fernandez, I.; Larios-Guzman, A.; Tapia-Vargas, L.M.; Guillen-Andrade, H.; Villasenor-Ramirez, F. Criopreservación de germoplasma de aguacate. In Proceedings of the VII World Avocado Congress, Cairns, Australia, 5–9 September 2011. [Google Scholar]
  156. Panis, B.; Piette, B.; André, E.; Van den houwe, I.; Swennen, R. Droplet Vitrification: The First Generic Cryopreservation Protocol for Organized Plant Tissues? International Society for Horticultural Science (ISHS): Leuven, Belgium, 2011; pp. 157–162, 2406–6168. [Google Scholar] [CrossRef]
  157. Engelmann, F. Plant cryopreservation: Progress and prospects. Vitr. Cell. Dev. Biol. Plant 2004, 40, 427–433. [Google Scholar] [CrossRef]
  158. Burke, M.J. The glassy state and survival of anhydrous biological systems. In Membranes, Metabolism and Dry Organisms; Cornell University Press: Ithaca, NY, USA, 1986; pp. 358–363. ISBN 978-0801419799. [Google Scholar]
  159. Krishna, H.; Sairam, R.; Singh, S.; Patel, V.; Sharma, R.; Grover, M.; Nain, L.; Sachdev, A. Mango explant browning: Effect of ontogenic age, mycorrhization and pre-treatments. Sci. Hortic. 2008, 118, 132–138. [Google Scholar] [CrossRef]
  160. Uchendu, E.E.; Paliyath, G.; Brown, D.C.; Saxena, P.K. In vitro propagation of North American ginseng (Panax quinquefolius L.). Vitr. Cell. Dev. Biol. Plant 2011, 47, 710–718. [Google Scholar] [CrossRef]
  161. Preece, J.; Compton, M. Problems with Explant Exudation in Micropropagation. In High-Tech and Micropropagation I. Biotechnology in Agriculture and Forestry; Bajaj, Y.P.S., Ed.; Springer: Berlin, Germany, 1991; Volume 17, pp. 168–189. ISBN 978-3-642-76417-2. [Google Scholar] [CrossRef]
  162. Akram, N.A.; Shafiq, F.; Ashraf, M. Ascorbic Acid-A Potential Oxidant Scavenger and Its Role in Plant Development and Abiotic Stress Tolerance. Front. Plant Sci. 2017, 8, 613. [Google Scholar] [CrossRef]
  163. Shao, H.-B.; Chu, L.-Y.; Lu, Z.-H.; Kang, C.-M. Primary antioxidant free radical scavenging and redox signaling pathways in higher plant cells. Int. J. Biol. Sci. 2007, 4, 8. [Google Scholar] [CrossRef] [PubMed]
  164. Smirnoff, N.; Wheeler, G.L. Ascorbic acid in plants: Biosynthesis and function. Crit. Rev. Plant Sci. 2000, 19, 267–290. [Google Scholar] [CrossRef]
  165. González-Benito, M.E.; Kremer, C.; Ibáñez, M.A.; Martín, C. Effect of antioxidants on the genetic stability of cryopreserved mint shoot tips by encapsulation–dehydration. Plant Celltissue Organ Cult. 2016, 127, 359–368. [Google Scholar] [CrossRef] [Green Version]
  166. Johnston, J.W.; Harding, K.; Benson, E.E. Antioxidant status and genotypic tolerance of Ribes in vitro cultures to cryopreservation. Plant Sci. 2007, 172, 524–534. [Google Scholar] [CrossRef]
  167. Reed, B.M. 4. Are antioxidants a magic bullet for reducing oxidative stress during cryopreservation? Cryobiology 2012, 65, 340. [Google Scholar] [CrossRef]
  168. Uchendu, E.E.; Leonard, S.W.; Traber, M.G.; Reed, B.M. Vitamins C and E improve regrowth and reduce lipid peroxidation of blackberry shoot tips following cryopreservation. Plant Cell Rep. 2010, 29, 25. [Google Scholar] [CrossRef] [PubMed]
  169. Wang, Q.; Laamanen, J.; Uosukainen, M.; Valkonen, J. Cryopreservation of in vitro-grown shoot tips of raspberry (Rubus idaeus L.) by encapsulation–vitrification and encapsulation–dehydration. Plant Cell Rep. 2005, 24, 280–288. [Google Scholar] [CrossRef]
  170. O’Brien, C.; Hiti-Bandaralage, J.C.A.; Folgado, R.; Lahmeyer, S.; Hayward, A.; Mitter, N. Developing a Cryopreservation Protocol for Avocado (Persea americana Mill.) Apical Shoot Tips Using Different Antioxidants; International Society for Horticultural Science (ISHS): Leuven, Belgium, 2020; pp. 15–22, 2406–6168. [Google Scholar] [CrossRef]
  171. Sharma, S.D. Cryopreservation of Somatic Embryos—An Overview. 2005. Available online: (accessed on 12 January 2021).
  172. Chang, Y.; Reed, B.M. Pre-culture conditions influence cold hardiness and regrowth of Pyrus cordata shoot tips after cryopreservation. HortScience 2001, 36, 1329–1333. [Google Scholar] [CrossRef]
  173. Feng, C.-H.; Cui, Z.-H.; Li, B.-Q.; Chen, L.; Ma, Y.-L.; Zhao, Y.-H.; Wang, Q.-C. Duration of sucrose pre-culture is critical for shoot regrowth of in vitro-grown apple shoot-tips cryopreserved by encapsulation-dehydration. Plant Celltissue Organ Cult. 2013, 112, 369–378. [Google Scholar] [CrossRef]
  174. Kaczmarczyk, A.; Shvachko, N.; Lupysheva, Y.; Hajirezaei, M.-R.; Keller, E.R.J. Influence of alternating temperature pre-culture on cryopreservation results for potato shoot tips. Plant Cell Rep. 2008, 27, 1551–1558. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  175. Park, S.U.; Kim, H.H. Cryopreservation of sweet potato shoot tips using a droplet-vitrification procedure. CryoLetters 2015, 36, 344–352. [Google Scholar] [PubMed]
  176. Ashmore, S.E.; Azimi, M.; Drew, R.A. Cryopreservation trials in Carica papaya. Acta Hortic. 2001, 560, 117–120. [Google Scholar] [CrossRef]
  177. López-López, L.; Barrientos-Priego, A.; Ben-Ya’acov, A. Variabilidad genética de los bancos de germoplasma de aguacate preservados en el Estado de México. Rev. Chapingo Ser. Hortic. 1999, 5, 19–23. [Google Scholar]
  178. Mikuła, A. Comparison of three techniques for cryopreservation and reestablishment of long-term Gentiana tibetica suspension culture. CryoLetters 2006, 27, 269–282. [Google Scholar]
  179. Mikuła, A.; Tykarska, T.; Kuraś, M. Ultrastructure of Gentiana tibetica proembryogenic cells before and after cooling treatments. CryoLetters 2005, 26, 367–378. [Google Scholar]
  180. Pritchard, H.; Grout, B.; Short, K. Osmotic stress as a pregrowth procedure for cryopreservation: 1. Growth and ultrastructure of sycamore and soybean cell suspensions. Ann. Bot. 1986, 57, 41–48. [Google Scholar] [CrossRef]
  181. Panis, B.; Strosse, H.; van den Hende, S.; Swennen, R. Sucrose pre-culture to simplify cryopreservation of banana meristem cultures. CryoLetters 2002, 23, 375–384. [Google Scholar]
  182. Lynch, P.T.; Siddika, A.; Johnston, J.W.; Trigwell, S.M.; Mehra, A.; Benelli, C.; Lambardi, M.; Benson, E.E. Effects of osmotic pre-treatments on oxidative stress, antioxidant profiles and cryopreservation of olive somatic embryos. Plant Sci. 2011, 181, 47–56. [Google Scholar] [CrossRef] [PubMed]
  183. Malabadi, R.B.; Nataraja, K. Cryopreservation and plant regeneration via somatic embryogenesis using shoot apical domes of mature Pinus roxburghii sarg, trees. Vitr. Cell. Dev. Biol. Plant 2006, 42, 152. [Google Scholar] [CrossRef]
  184. Crowe, L.M. Lessons from nature: The role of sugars in anhydrobiosis. Comp. Biochem. Physiol. Part A Mol. Integr. Physiol. 2002, 131, 505–513. [Google Scholar] [CrossRef]
  185. Antony, J.J.J.; Keng, C.L.; Mahmood, M.; Subramaniam, S. Effects of ascorbic acid on PVS2 cryopreservation of Dendrobium Bobby Messina’s PLBs supported with SEM analysis. Appl. Biochem. Biotechnol. 2013, 171, 315–329. [Google Scholar] [CrossRef]
  186. Hirsh, A.G. Vitrification in plants as a natural form of cryoprotection. Cryobiology 1987, 24, 214–228. [Google Scholar] [CrossRef]
  187. Herbert, R.; Vilhar, B.; Evett, C.; Orchard, C.; Rogers, H.; Davies, M.; Francis, D. Ethylene induces cell death at particular phases of the cell cycle in the tobacco TBY-2 cell line. J. Exp. Bot. 2001, 52, 1615–1623. [Google Scholar] [CrossRef]
  188. Williams, W.P.; Quinn, P.J.; Tsonev, L.I.; Koynova, R.D. The effects of glycerol on the phase behaviour of hydrated distearoylphosphatidylethanolamine and its possible relation to the mode of action of cryoprotectants. Biochim. Biophys. Acta Bba Biomembr. 1991, 1062, 123–132. [Google Scholar] [CrossRef]
  189. Burritt, D.J. Proline and the cryopreservation of plant tissues: Functions and practical applications. In Current Frontiers in Cryopreservation; Katkov, I.I., Ed.; InTech: Rijeka, Croatia, 2012. [Google Scholar]
  190. Cleland, D.; Krader, P.; McCree, C.; Tang, J.; Emerson, D. Glycine betaine as a cryoprotectant for prokaryotes. J. Microbiol. Methods 2004, 58, 31–38. [Google Scholar] [CrossRef]
  191. Janská, A.; Maršík, P.; Zelenková, S.; Ovesná, J. Cold stress and acclimation—What is important for metabolic adjustment? Plant Biol. 2010, 12, 395–405. [Google Scholar] [CrossRef] [PubMed]
  192. Arora, R. Freezing Tolerance and Cold Acclimation in Plants; Deparment of Horticulture, Iowa State University: Ames, IA, USA, 2010. [Google Scholar] [CrossRef]
  193. Gusta, L.; Trischuk, R.; Weiser, C.J. Plant cold acclimation: The role of abscisic acid. J. Plant Growth Regul. 2005, 24, 308–318. [Google Scholar] [CrossRef]
  194. Reed, B. Pre-treatment strategies for cryopreservation of plant tissues. In In Vitro Conservation of Plant Genetic Resources; Normah, M.N., Narimah, M.K., Clyde, M.M., Eds.; Universiti Kebangsaan: Bangi Selangor, Malaysia, 1996; pp. 73–87. [Google Scholar]
  195. Janmohammadi, M.; Zolla, L.; Rinalducci, S. Low temperature tolerance in plants: Changes at the protein level. Phytochemistry 2015, 117, 76–89. [Google Scholar] [CrossRef] [PubMed]
  196. Thomashow, M.F. Plant cold acclimation: Freezing tolerance genes and regulatory mechanisms. Annu. Rev. Plant Biol. 1999, 50, 571–599. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  197. Dumet, D.; Chang, Y.; Reed, B.M.; Benson¹, E.E. Replacement of cold acclimatization with high sucrose pretreatment in black currant cryopreservation. Satoshi Katomasaya Ishikawamiwako Ito Tatsuo Matsumoto 338 2000, 17, 393. [Google Scholar]
  198. Coelho, N.; González-Benito, M.E.; Martín, C.; Romano, A. Cryopreservation of Thymus lotocephalus shoot tips and assessment of genetic stability. CryoLetters 2014, 35, 119–128. [Google Scholar]
  199. Fki, L.; Bouaziz, N.; Chkir, O.; Benjemaa-Masmoudi, R.; Rival, A.; Swennen, R.; Drira, N.; Panis, B. Cold hardening and sucrose treatment improve cryopreservation of date palm meristems. Biol. Plant. 2013, 57, 375–379. [Google Scholar] [CrossRef]
  200. Kushnarenko, S.V.; Romadanova, N.V.; Reed, B.M. Cold acclimation improves regrowth of cryopreserved apple shoot tips. CryoLetters 2009, 30, 47–54. [Google Scholar]
  201. Reed, B.M. Responses to ABA and cold acclimation are genotype dependent for cryopreserved blackberry and raspberry meristems. Cryobiology 1993, 30, 179–184. [Google Scholar] [CrossRef]
  202. O’Brien, C.; Hiti-Bandaralage, J.; Folgado, R.; Lahmeyer, S.; Hayward, A.; Folsom, J.; Mitter, N. A method to increase regrowth of vitrified shoot tips of avocado (Persea americana Mill.): First critical step in developing a cryopreservation protocol. Sci. Hortic. 2020, 266, 109305. [Google Scholar] [CrossRef]
  203. O’Brien, C.; Hiti-Bandaralage, J.C.; Folgado, R.; Lahmeyer, S.; Hayward, A.; Mitter, N. Developing a cryopreservation protocol for avocado (Persea americana Mill.) shoot tips. Cryobiology 2018, 85, 171. [Google Scholar] [CrossRef]
  204. O’Brien, C.; Hiti-Bandaralage, J.C.A.; Folgado, R.; Lahmeyer, S.; Hayward, A.; Folsom, J.; Mitter, N. First report on cryopreservation of mature shoot tips of two avocado (Persea americana Mill.) rootstocks. Plant Celltissue Organ Cult. 2020. [Google Scholar] [CrossRef]
  205. Sánchez-Romero, C.; Márquez-Martín, B.; Pliego-Alfaro, F. Somatic and zygotic embryogenesis in avocado. In Somatic Embryogenesis; Springer: Berlin/Heidelberg, Germany, 2006; pp. 271–284. [Google Scholar]
  206. Hiti-Bandaralage, J.; Hayward, A.; O’Brien, C.; Gleeson, M.; Nak, W.; Mitter, N. Advances in Avocado Propagation for the Sustainable Supply of Planting Materials. In Achieving Sustainable Cultivation of Tropical Fruits; Burleigh dodds Science Publishing: Cambridge, UK, 2019; pp. 215–238. ISBN 9781786762849. [Google Scholar]
Figure 1. One of the 56 avocado accessions being maintained in The Huntington Botanical Gardens [in San Marino, California USA] living germplasm collection.
Figure 1. One of the 56 avocado accessions being maintained in The Huntington Botanical Gardens [in San Marino, California USA] living germplasm collection.
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Figure 2. Shoot tips of cv ‘Reed’ treated with VSL and revived from LN growing in a glasshouse.
Figure 2. Shoot tips of cv ‘Reed’ treated with VSL and revived from LN growing in a glasshouse.
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Table 1. Some examples of field repositories maintaining living collections of economically important crops.
Table 1. Some examples of field repositories maintaining living collections of economically important crops.
CountryField RepositoriesGenus/SpeciesReference
USAUSDA—Geneva NY, Davis CA, Riverside CAMalus domestia Borkh. (apple)
Vitis vinifera L. (grape)
Actinidia deliciosa (kiwifruit)
Diospyros spp. (persimmon)
Ficus carica L. (fig)
Juglans spp. (walnut)
Olea europaea L. (olive)
Pistacia vera L. (pistachio)
Punica granatum L. (pomegranate)
Citrus spp. (citrus)
Prunus spp. (plum)
USATropical Botanical GardenArtocarpus altilis (breadfruit)[32]
GermanyGerman Fruit Gene bankMalus spp. (apple)
Prunus avium (cherry)
Prunus domestica (plum)
Rubus spp. (raspberry)
United KingdomNational Fruit CollectionMalus domestica Borkh. (apple)
Prunus domestica (plum)
Pyrus communis L. (pear)
Prunus avium (cherry)
Table 2. Some examples of cryo-storage gene banks maintaining collections of economically important crops.
Table 2. Some examples of cryo-storage gene banks maintaining collections of economically important crops.
CountryGene BankGenus/SpeciesAccessions HeldReference
FranceInstitute of Research DevelopmentCoffea spp. (coffee)~500[12]
ColumbiaInternational Centre for Tropical AgricultureManihot esculenta (cassava)5690[43]
JapanNational Institute of Agrobiological SciencesMorus spp. (mulberry)
Juncus effusus (rush)
JapanShimane Agriculture Research CentreWasabi japonica M. (Japanese horseradish)40[12]
USANational Clonal Germplasm RepositoryMalus spp. (apple)
Pyrus spp. (pear)
Rubus spp. (raspberry)
Vitis spp. (grape)
BelgiumBioversity International Transit Centre Musa spp. (banana)1600[7]
Table 3. Methods to reduce water content.
Table 3. Methods to reduce water content.
Dehydration MethodUses
Desiccation(1) Air drying of explants in laminar flow hood or using flow of compressed air.
(2) Dehydration of explants in a desiccator with silica gel.
Cryoprotectants(1) Penetrating cryoprotectants, e.g., dimethyl sulfoxide (DMSO) and glycerol act by replacing intracellular water.
(2) Non-penetrating cryoprotectants, e.g., sucrose, polyvinylpyrrolidone (PVP) and polyethylene glycol (PEG), display different osmotic potential inside and outside the cells.
Freeze-induced dehydrationPreferential freezing of extracellular water by slow cooling at a rate of 0.5–2 °C per min creates a hypotonic surrounding for the cell, resulting in outflow of cellular water.
Pre-conditioning of donor plant or explantIncluding DMSO abscisic acid, sucrose, polyols or proline in the pre-culture medium or low temperature treatment to induce tolerance to dehydration and freezing.
Table 4. Some examples of cryoprotectants used for plant tissue.
Table 4. Some examples of cryoprotectants used for plant tissue.
PVS130% w/v glycerol, 15% w/v EG, 5% w/v sucrose, 15% w/v DMSO [61]
PVS230% w/v glycerol, 15% w/v DMSO, 15% w/v EG and 15% sucrose [59]
PVS350% w/v glycerol and 50% w/v sucrose [58]
PVS435% w/v glycerol, 20% w/v EG and 20.5% M sucrose [62]
VSL+20% w/v glycerol, 10% w/v DMSO, 30% w/v EG, 15% sucrose and 10 mM CaCl2 [63]
VSL20% w/v glycerol, 10% w/v DMSO, 30% w/v EG, 5% sucrose and 10 mM CaCl2 [63]
Steponkus50% w/v EG, 15% sorbitol, 6.0% bovine serum albumin, 13.7% sucrose [64]
Towill35% EG, 6.8% w/v DMSO, 10% PEG 8000 and 13.7% sucrose [65]
Fahy20% DMSO, 20% formamide, 15% propylene glycol [66]
Table 5. Some examples of cryopreservation methods, techniques and applications used.
Table 5. Some examples of cryopreservation methods, techniques and applications used.
VitrificationPre-culture of cultures on basal medium supplemented with cryoprotectants, pre-treatment with loading solution, dehydration with PVS, rapid freezing rewarming.Cocoa secondary somatic embryos74.5% survival with 5- day pre-culture on 0.5 M sucrose followed by 60 min dehydration in PVS2 treatment for 1 h at 0 °C.[69]
Droplet-vitrificationResembles vitrification in all steps with only difference that materials are cryopreserved on foil strips in drops of vitirification solution.Hancornia speciosa Gomes (rubber tree) shoot tips43% regrowth with pre-culture on basal + proline (0.193 M) for 24 h in the dark at 25 °C and PVS2 15 min at 0 °C.[70]
Encapsulation-vitrificationSodium alginate beads are formed and explants are encapsulated in them and dehydrated in PVS before freezing.Olea europaea (olive)
somatic embryos

Parkia speciosa Hassk. (stink bean) shoot tips
64% regrowth after 4 day pre-culture in sucrose; PVS2 treatment for 3 h treatment and rapid freezing.
Pre-culture on MS + trehalose (5% w/v) for 3 days; PVS2 for 1 h at 0 °C.

Encapsulation-dehydrationSodium alginate-encapsulated cultures are dehydrated osmotically with high concentrations of sucrose for 1–7 days and/or desiccated in an air current before slow cooling to –80 °C and then immersed in LN.Olea europaea (olive) somatic embryos

Prunus armeniaca (apricot) shoots
40% regrowth following 4 days of sucrose pre-growth, desiccation and freezing.
Recovered after treated with 0.5 M sucrose for 2 days followed by air dehydration for 2 h and frozen in LN.

DehydrationSamples are dehydrated by either air current, silica gels, or incubation with cryoprotectant, followed by rapid freezing or two-step freezing.Juglans nigra (walnut) embryo axesDried in a laminar flow hood until 5–15% moisture content and 100% recovery after LN. [74]
Pre-growth and pre-growth-dehydrationSamples are cultured on media containing cryoprotectants such as DMSO, dehydrated and then frozen slowly or rapidly.Garcinia mangostana L. (mangosteen) shoot tips50% MS + sucrose (0.6 M) + 5% DMSO for 2 days[75]
V-cryoplateModification of encapsulation-vitrification and droplet-vitrification. Dehydration is performed using vitrification solution PVS2.Morus alba (mulberry) shoot tips87% regrowth, 13 lines pre-cultured at 25 °C for 1 day on MS medium containing 0.3 M sucrose. PVS2 solution for 30 min at 25 °C.[76]
D-cryoplateModification of encapsulation-vitrification and droplet-vitrification. Dehydration is achieved using the air current of the laminar flow cabinet or silica gel.Diospyros kaki (persimmon) shoot tipsAverage 87% regrowth, 10 lines 1–3 months cold acclimatization, 3 °C pre-cultured on 0.3 M sucrose, 2 days at 25 °C, laminar flow 30 min at 25 °C.[77]
Table 6. Avocado germplasm maintained as field repositories throughout the world.
Table 6. Avocado germplasm maintained as field repositories throughout the world.
CountryGermplasm RepositoriesNo. of AccessionsReferences
USAThe Huntington San Marino CA56 Persea americana accessions
4 wild Persea spp (6 accessions)
USARiverside University CA~230 avocado scion accessions[129]
~15 wild Persea spp.
~246 avocado rootstock accessions[129,130]
USANational Genetic Resources Program, Miami, FloridaP. americana (167 accessions) and P. schiedeana (1 accession)[44,131]
USAThe Sub-Tropical Horticulture Research Station, Miami, Florida~400 avocado accessions[132]
MexicoNational Research Institute of Forestry and Livestock in Guanajuato500 accessions belonging to P. americana: Mexican and Guatemalan races. Related species: P. schiedeana, P. cinerascens, P. floccosa, P nubigena[133]
MexicoState of Mexico of the Fundación Salvador Sanchez Colin-CICTAMEX, S.C.800 accessions of avocado and related species. Mexican, Guatemalan, West Indian races, P. americana var. costaricensis race materials.[133]
MexicoCoatepec Harinas and Temascaltepec; State of MexicoWild relatives: Beilschmiedia anay, B. miersii, P. schiedeana, P. longipes, P. cinerascens, P. hintonni, P. floccosa, P. tolimanensis, P. steyermarkii, P. nubigena, P. lingue, P. donnell-smithii, P. parvifolia, P. chamissonis, Persea spp.[133]
GhanaUniversity of Ghana Forest and Horticultural Crops Research Centre110 local land races and 5 varieties from South Africa (‘Hass’, ‘Fuerte’, ‘Ryan’, ‘Ettinger’ and ‘Nabal’[134]
IsraelVolcanic Centre in Bet Dagan194 trees, propagated from 148 accessions[96]
SpainThe Experimental Station ‘La Mayora’ in Malaga75 avocado accessions[132,135]
CubaN/A210 genotypes[132]
ChileN/A4 botanical breeds of P. americana: var. drymifolia, var. guatemalensis, var. jacket and var. costaricencis[132]
AustraliaMaroochydore Research Station46 avocado accessions[136]
Nigeria 8 avocado accessions[137]
BrazilBrasilia, in the Federal District, depending on the Embrapa Research Institute30 avocado accessions[138]
BrazilConceicao do Almeida and Juazeiro collections, both in the Bahia State22 avocado accessions[138]
BrazilPiracicaba, in the Sao Paulo State33 avocado accessions[138]
BrazilJaboticabal, in the Sao Paulo State7 avocado accessions[138]
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.
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.
Cryopreservation TechniqueCultivars* Recovery Percentages
‘Fuerte’5% [152]
‘Duke 7’80% [144]
Slow freezing‘Suardia’60–80%
‘Fuerte’73–75% [152]
Droplet vitrification‘A10’100%
‘Velvick’93% [144]
Two lines of ‘Duke 7’78–100% [153]
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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.

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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(5):934.

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O’Brien, Chris, Jayeni Hiti-Bandaralage, Raquel Folgado, Alice Hayward, Sean Lahmeyer, Jim Folsom, and Neena Mitter. 2021. "Cryopreservation of Woody Crops: The Avocado Case" Plants 10, no. 5: 934.

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