First report on cryopreservation of mature shoot tips of two avocado (Persea americana Mill.) rootstocks

Abstract

Cryopreservation combined with in vitro culture offers a safe and cost-effective method to conserve germplasm. Conservation of Persea spp. has been limited to heterozygous somatic embryos that are not true-to-type. A method for shoot-tip cryopreservation is vital to preserve the exact gene pool of interest. For the first time cryopreservation protocols for mature shoot tips of two avocado cultivars (cvs) ‘Velvick’ and ‘Reed’, were established. In vitro shoots were subjected to two different optimised pre-treatments; (1) cv ‘Velvick’—high sucrose (0.3 M) or (2) cv ‘Reed’—low temperature (10 °C) incubation, over a 2-week period prior shoot tip dissection. Two different plant vitrification solutions, plant vitrification solution 2 (PVS2) and vitrification solution L (VSL) were tested at 0 °C for 0, 10, 20, 30 and 40 min. Vitrified shoots were evaluated for survival and regrowth at 2 and 8 weeks after vitrification treatment and either with or without liquid nitrogen exposure. The study revealed that the optimal exposure time for each cultivar varied with the cryoprotectant used. After liquid nitrogen cv ‘Velvick’ highest regrowth levels were observed with 20 min exposure to either PVS2 or VSL, however, vigorous plants were produced only from VSL treated shoots. In the case of cv ‘Reed’ highest regrowth levels were observed with 10 min exposure to PVS2 however only morphologically normal plants were recovered from VSL treated shoots.

Key message

Cryopreservation of avocado shoot tips was successful using PVS2 and VSL with both recording similar recovery rates for ‘Velvick’ and ‘Reed’; although only vigorous and morphologically normal plants were developed from VSL treatments.

Introduction

Avocado (Persea americana Mill.), the most economically important plant in the Lauraceae family, is a dense tropical evergreen tree, reaching a height of ~ 20 m (Bergh and Ellstrand 1986). Three recognised ecological races of P. americana (Furnier et al. 1990) are Mexican (var. drymifolia), Guatemalan (var. guatamalensis) and West Indian (var. americana); although at least three additional races are suggested to exist (Ben-Ya'acov and Michelson 1995; Furnier et al. 1990; Silva and Ledesma 2014). The centres of origin for these three races are distinct, making their own characteristics and hence making them distinct from one another (Chen et al. 2008). A prime example of this, is cold tolerance. Avocado displays a wide variation in cold tolerance amongst the three ecological races with the West Indian race growing best in temperatures from 15 to 29 °C, while the Guatemalan race can tolerate cooler temperatures of − 3 to − 1 °C. Yet, the best of all, the Mexican race can withstand temperatures as low as − 7 °C (Crane et al. 2007; Krezdorn 1973; Mickelbart and Arpaia 2002).

Consumer demand for avocado has exploded in recent years due to its popularity as a healthy eating option. It has been reported to reduce inflammation, risk of heart disease, diabetes, metabolic syndrome and certain types of cancer (Ding et al. 2007; Fulgoni et al. 2013; Sales-Campos et al. 2013; Wang et al. 2015). The estimated world avocado production for 2017 was 4.1 million tons with Mexico, Peru, and Indonesia being the main producers (Fresh Plaza 2020). In Australia, avocados are produced almost all year round due to rotation of favourable climatic conditions within the eight major avocado growing regions accounting for ~ $543 m in farm gate value (Avocados Australia 2018). As climate change and threats of new diseases face agriculture, an important focus within the industry should be breeding new varieties. Conservation of diversity in a plant germplasm collection is necessary to maintain a broad gene base for crop improvement, with Persea spp. remaining untapped in terms of breeding and having a large amount of diversity within the genus (Ben-Ya'acov et al. 1992; O’Brien et al. 2018b). However, at present, this diversity is maintained only in field collections, which are threatened by climate changes and the spread of disease, both with the potential to reduce diversity.

With the development of tissue culture technology, a new era of plant conservation through ‘cryopreservation’ allows preservation of living tissues at ultra-low temperatures generally around − 196 °C in liquid nitrogen (LN), offering an attractive method for in vitro storage of avocado germplasm (Engelmann 2011). At this temperature all chemical and enzymatic activities are arrested, and samples can be stored safely with negligible risk of genetic variation (Engelmann 2011) requiring minimal space and maintenance (Benson et al. 2007; Engelmann 2011).

For plant tissues to undergo ultra-cooling, it is critical that most or all intracellular freezable water is removed, thus reducing or avoiding detrimental intracellular ice formation (Elliott 2013; Engelmann 2004). Intracellular ice formation at ultra-low temperatures causes alterations to cell structure and its colligative integrity (Kaczmarczyk et al. 2012). These alterations to the cell can cause damage to cellular components leading to cell death (Kaczmarczyk et al. 2012). Vitrification-based protocols are particularly effective for complex organs such as embryos and shoot tips as these types of tissue usually contain a variety of cell types (Engelmann 2011). Differences in cell types require specific conditions to be optimized before freeze induced dehydration can be acquired (Engelmann 2011). Tolerance is achieved by exposure to concentrated cryoprotective media and/or air desiccation and is performed before rapid freezing in LN (Engelmann 2004).

Droplet vitrification is a cryogenic process and involves adequate dehydration of tissue in cryoprotectant solution and placing in a droplet of cryoprotectant on an alfoil strip before immersion in LN (Panis et al. 2005). Dehydration time is a critical factor for tissue survival (Benson et al. 2007; Mathew et al. 2018). This technique has been successful with shoot tip cryopreservation of several plant species displaying varying degrees of tolerance to abiotic stresses; Eucalyptus spp. with regrowth rates of 38–85% (Kaya et al. 2013); Malus spp. with regrowth of 70% (Condello et al. 2011; Halmagyi et al. 2010); Prunus spp. with regrowth of 30% (Vujović et al. 2011); Rubus spp. with regrowth of 18% (Condello et al. 2011) and regrowth of 30% (Vujović et al. 2011) and Vitis spp. recording regrowth rates of 50% (Marković et al. 2013; Pathirana et al. 2016) and regrowth at least 43% for 12 Vitis species (Bettoni et al. 2019).

To date, cryopreservation of avocado has been limited to somatic embryos, which are genetically diverse to mother plant, thus not appropriate for preserving true genotypes (O’Brien et al. 2018b). Shoot tips from mature avocado plants preserve the exact genotype of the mother plant but have never been successfully cryopreserved (Vargas 2008; Vidales-Fernandez et al. 2011). Avocado displays high susceptibility to osmotic stresses caused by cryopreservation solutions, leading to browning and eventual death of tissue (O’Brien et al. 2018a). In our previous work (O’Brien et al. 2018a) we successfully eliminated tissue browning with the use of ascorbic acid (ASA) at 100 mg L−1 in regrowth media, as a preparatory step for cryopreservation. In another study by O’Brien et al. (2020) the best pre-treatment conditions; high sucrose or cold pre-treatments were identified, which are effective to withstand the cryoprotectant treatments and increased survivability of shoot tips.

The current study is focused on selecting the best cryoprotectant and exposure time durations for shoot tip cryopreservation of two avocado cultivars, ‘Velvick’ and ‘Reed’ to optimise a full cryopreservation protocol able to successfully maintain them in LN and regrow from cryopreserved material to obtain intact plants.

Materials and methods

Material

In vitro mature shoot cultures of avocado (Persea americana Mill.), cvs ‘Velvick’ and ‘Reed’, were grown in a culture room at 26 ± 1 °C with a photoperiod of 16 h (Hiti-Bandaralage 2019; Hiti-Bandaralage et al. 2019). Plantlets were sub-cultured once every 4 weeks. All liquid media used [liquid incubation, loading, plant vitrification solution 2 (PVS2), vitrification solution L (VSL) and unloading] were filter sterilised, solid media were autoclaved at 121 °C for 15 min. Heat sensitive chemicals such as ASA were filter sterilised and added to media after autoclaving (O’Brien et al. 2020).

Pre-treatment of avocado donor plants

Stock cultures of cvs ‘Velvick’ and ‘Reed’ were submitted to two separate pre-treatments for a period of 2 weeks; (1) high sucrose 0.3 M for cv ‘Velvick’ and (2) cold incubation at 10 °C for cv ‘Reed’ (O’Brien et al. 2020). Except during cold pre-treatment, cultures were incubated at 26 ± 1 °C at all times. The photoperiod used for incubation was 16 h photoperiod under LED lights (Valoya, L-series model L18).

Excision of shoot tips for cryopreservation experiments

Apical shoot tips of 1.5 × 1.5 mm in size (leaving the last 2 leaf primordia intact) were excised from 2-week-old pre-treated donor plants under a dissection microscope in an aseptic laminar flow cabinet according to the method described by O’Brien et al. (2020). The excised shoot tips were immediately placed into a liquid incubation medium containing a high concentration of sucrose (0.3 M) supplemented with 100 mg L−1 ASA, (O’Brien et al. 2020) in a 50 mL falcon tube. Tubes were kept wrapped in alfoil to impose dark conditions until the completion of shoot tip excisions.

Optimization of cryoprotectant type and exposure time

After excision, the liquid incubation medium was replaced with loading solution [2 M glycerol + 0.4 M sucrose, 100 mg L−1 ASA, pH 5.65], (O’Brien et al. 2020) for 20 min at room temperature. After loading time had expired, solution was replaced with ice cold (0 °C) cryoprotectants; either PVS2 [30% (w/v) glycerol, 15% (w/v) dimethyl sulfoxide (DMSO), 15% (w/v) ethylene glycol (EG), and 0.4 M sucrose] (Sakai et al. 1990) or VSL [20% (w/v) glycerol, 10% (w/v) DMSO, 30% (w/v) EG, 5% sucrose and 10 mM CaCl2] (Suzuki et al. 2008) supplemented with 100 mg L−1 ASA, and subjected to different incubation times 0, 10, 20, 30 and 40 min on ice (n = 20).

After cryoprotectant treatment one set of shoot tips from each treatment was washed three times with sterilised unloading solution for 20 min [1.2 M sucrose, pH 5.65], (O’Brien et al. 2020) at room temperature, and plated on sterile filter paper then placed on regrowth medium to study the effect of cryoprotectant exposure minus liquid nitrogen treatment.

The other set of shoot tips was prepared to go through cryopreservation. Five minutes prior to expiration of cryoprotectant treatment time, shoot tips were placed on alfoil strips (autoclaved and chilled, 0.8 cm × 3 cm) and placed in a droplet of respective cryoprotectant solution, PVS2 or VSL (Panis et al. 2005). Alfoil strips were placed in a sterile petri dish kept on an ice block to maintain cold conditions. Prior to freezing in LN, cryo tubes (Nunc®) of 1.8 mL in size were precooled in LN. After cryoprotectant exposure time had expired alfoil strips containing the shoot tips were placed inside the precooled cryo tubes, which were then immersed into LN. After 30 min in LN, samples were rewarmed into unloading solution for 20 min at room temperature and shoot tips placed on recovery media as described above for − LN treatment.

Shoot tips on recovery media were maintained for one day in dark at 26 ± 1 °C followed by transferring to fresh regrowth medium (no filter paper) on plates wrapped in alfoil to maintain dark conditions for 2 weeks before placing in a growth room at 26 ± 1 °C. Sub-culture onto fresh regrowth media was performed at 28 days. Regrowth data was collected and shoot tips of each cultivar were sub-cultured for a further 16 weeks on the same regrowth medium with fresh sub-culturing taking place at every 28 days. Shoots regenerated from individual shoots tips were maintained and individual rooted plants were generated as per Hiti Bandaralage and Jayeni Chathurika Amarathunga (2019).

Assessment of survival and regrowth, experiment design and statistical analyses

The effect of the two different cryoprotectants, PVS2 and VSL; and their optimum exposure times were evaluated ± LN by assessing the percentage of surviving and regrowing meristem shoots. Shoot tips that appeared to be white/greenish after 2 weeks dark incubation were recorded as surviving shoots tips and shoot tips grown with 3 fully developed expanded leaves after 8 weeks were considered as regrowing shoots (O’Brien et al. 2020).

Individual experiments were carried out for the two different avocado cvs ‘Velvick’ and ‘Reed’. For each cultivar, cryoprotectant type (PVS2 or VSL) was used in an independent experiment to test the exposure time and effect on survival and regrowth ± LN treatments. Each treatment was composed of 2 petri dishes with 10 shoot tips per treatment and duplicated in independent experiments to calculate the percentage survival and regrowth. ANOVA was performed using SPSS23 statistical software and multiple comparisons were through Tukey test.

Results

Cryoprotectant exposure time was critical for survival and regrowth of shoot tips. Shoot tips treated with PVS2 for 10 and 20 min and without LN exposure (− LN) recorded best survival of 85% and regrowth of 75% with cv ‘Velvick’ (Fig. 1a and Supplementary data Fig. 1a). There was no statistical difference between 10 and 20 min exposure of PVS2. Longer exposure times of either 30 or 40 min reduced the survival to 55 and 35% respectively, and regrowth to 35 and 15% respectively (Fig. 1a). PVS2 treatment duration was linked with the morphological and vigour differences of shoots when regenerated after 24 weeks regrowth (Fig. 2a–e). Shoot tips that had been exposed to PVS2 for 30 and 40 min had large callus at basal ends. Even though some shoots were produced from these treatments they lacked the vigour of the 0–20 min treatments. Cultivar ‘Velvick’ shoot tips exposed to PVS2 for 10 and 20 min had a normal growth habit similar to shoots with no PVS2 treated shoots (0 min exposure time). Shoots exposed to 0–20 min PVS2 treatment showed vigorous growth possessing dark green leaves and continuous reddish new flush.

Fig. 1
figure1

Regrowth of shoot tips excised from donor plants of cv ‘Velvick’, 8 weeks after inoculation on recovery medium a cv ‘Velvick’ pre-treated (0.3 M sucrose for 2 weeks), loading 20 min and PVS2 at 0 °C for 0–40 min with (+ LN) or without (− LN) LN treatment, b cv ‘Velvick’ (0.3 M sucrose for 2 weeks), loading 20 min and VSL at 0 °C for 0–40 min with (+ LN) or without (− LN) LN treatment. Means are presented, different letters indicate significantly different values for survival or regrowth for ± LN

Fig. 2
figure2

Regrowth of shoot tips excised from donor plants of cv ‘Velvick’, 24 weeks after inoculation on recovery medium a-e cv ‘Velvick’ pre-treated (0.3 M sucrose for 2 weeks), loading 20 min and PVS2 at 0 °C for 0–40 min − LN

Shoot tips subjected to PVS2 and were exposed to LN (+ LN) showed variation in survival and regrowth percentages after different durations of PVS2 treatment. For cv ‘Velvick’, the highest values were recorded with 20 min PVS2 treatment; 65% survival and 35% regrowth (Fig. 1a and Supplementary data Fig. 1a). There was no statistical difference between 10 and 20 min exposure of PVS2. Longer exposure times of 30 and 40 min of PVS2 did not reduce mortality, and recorded only 45% and 25% survival and 20% and 15% regrowth respectively. Even though a high survival and moderate regrowth was achieved when using PVS2 for 20 min, the + LN treated shoot tips displayed less vigour and abnormal leaf growth as compared to shoots that did not go through the LN treatment (Fig. 3a). Further sub-culturing of these shoots did not improve the quality of the plants and a lot of callus overgrowth of the shoots was observed (Fig. 3b).

Fig. 3
figure3

Regrowth of shoot tips excised from donor plants of cvs ‘Velvick’ and ‘Reed’, 8 weeks a and d which show basal callus growth and stunted shoots and 24 weeks after inoculation b and e which show callus overgrowth and stunted growth and c and f which show morphological normal plants on recovery medium a cv ‘Velvick’ pre-treated (0.3 M sucrose for 2 weeks), loading 20 min and PVS2 at 0 °C for 20 min with LN treatment (+ LN), b cv ‘Velvick’ pre-treated (0.3 M sucrose for 2 weeks), loading 20 min and PVS2 at 0 °C for 20 min with LN treatment (+ LN), c cv ‘Velvick’ (0.3 M sucrose for 2 weeks), loading 20 min and VSL at 0 °C for 20 min with LN treatment (+ LN), d) cv ‘Reed’ pre-treated (10 °C for 2 weeks), loading 20 min and PVS2 at 0 °C for 10 min with LN treatment (+ LN), e cv ‘Reed’ pre-treated (10 °C for 2 weeks), loading 20 min and PVS2 at 0 °C for 10 min with LN treatment (+ LN) and f cv ‘Reed’ pre-treated (10 °C for 2 weeks), loading 20 min and VSL at 0 °C for 10 min with LN treatment (+ LN)

VSL as the cryoprotectant for cv ‘Velvick’ was promising as PVS2 and the exposure time was again shown to be critical (Fig. 1b and Supplementary data Fig. 1b). Shoot tips survival levels were between 100 and 90% across the five VSL exposure times for non-LN treated shoot tips. Shoot tip regrowth levels without LN were 0%, 25%, 35%, 15% and 0% for 0, 10, 20, 30, and 40 min VSL (Fig. 1b). With LN exposure, ‘Velvick’ shoot tip had higher levels of survival and regrowth with 20 min of VSL exposure (70 and 35%, respectively) (Fig. 1b). Any exposure times beyond 20 min reduced regrowth percentages (Fig. 1b). Both 10 and 20 min exposure of VSL had similar shoot regeneration vigour and appearance of dark green colour and new leaf growth with reddish flush, as with 0 min exposure time. There was no statistical difference between 10 and 20 min exposure of VSL. Contrastingly, 30 and 40 min treatments with VSL reduced the growth, produced large callus at basal ends and shoots were not as vigorous as seen with shoots treated for 0, 10 and 20 min exposure times. Shoot tips on regrowth media were more vigorous, greener and appeared morphologically normal after 8 weeks compared to that of PVS2 treated shoots. Further sub-culturing of these shoots for another 16 weeks resulted in normal shoot development, producing elongated shoots (Fig. 3c). Longer exposure times of 30 and 40 min of VSL did not improve survival and regrowth of shoot tips during LN treatment (Fig. 1b).

Similar to cv ‘Velvick’, when PVS2 was used as a cryoprotectant for cv ‘Reed’, exposure time was critical for the survival and regrowth of shoot tips. PVS2 treatment for 10 or 20 min without LN exposure (− LN) recorded the best survival, at 100%, and regrowth of 80% with cv ‘Reed’ (Fig. 4a). When shoot tips were subjected to longer exposure times of 30 and 40 min, survival and regrowth were reduced, survival recorded as 75 and 45% and regrowth recorded as 45 and 35% respectively (Fig. 4a). Cultivar ‘Reed’ shoot tips that had been exposed to PVS2 for 10 and 20 min had a similar growth to shoots with 0 min exposure time. These showed vigorous growth possessing dark green leaves and continuous reddish new flush.

Fig. 4
figure4

Regrowth of shoot tips excised from donor plants of cv ‘Reed’, 8 weeks after inoculation on recovery medium a cv ‘Reed’ pre-treated (10 °C for 2 weeks), loading 20 min and PVS2 at 0 °C for 0–40 min with (+ LN) or without (− LN) LN treatment, b cv ‘Reed’ pre-treated (10 °C for 2 weeks), loading 20 min and VSL at 0 °C for 0–40 min with (+ LN) or without (− LN) LN treatment. n = 20 for each treatment. Means are presented, different letters indicate significantly different values for survival or regrowth for ± LN

The ‘Reed’ shoot tips that were PVS2 exposed and LN treated had high survival (85%) and regrowth (55%) after 10 min cryoprotectant treatment; for 20 min exposure (80%) and regrowth (40%) after LN were achieved (Fig. 4a). Longer exposure times of 30 and 40 min of PVS2 did not reduce the damage caused by LN, which resulted in only 45% and 15% survival and 25% and 0% regrowth, respectively. Even though a high survival and moderate regrowth was achieved when using PVS2 for 10 and 20 min, the + LN treated shoot tips displayed less vigour and abnormal leaf growth, large hard callus at basal ends and callus overgrowth. Further sub-culturing of these shoots only slightly improved the quality of the plants and growth was slow with low multiplication (Fig. 3e).

Both PVS2 and VSL showed good regrowth percentages for both cultivars after exposure to cryoprotectant and being treated with LN. However, for both cultivars the regrowth after PVS2 exposure and treated with LN lacked vigour as compared to shoots treated with VSL. When VSL was used as a cryoprotectant exposure time was again shown to be critical. The ‘Reed’ shoot tips that were VSL exposed and non-LN (− LN) treated had 100% survival and 85% regrowth after VSL exposure for 10 and 20 min (Fig. 4b). Any exposure times beyond 20 min reduced survival and regrowth percentages (Fig. 4b). Exposing cv ‘Reed’ shoot tips to VSL for 10 and 20 min, as with 0 min exposure time, produced similarly good shoot vigour and appearance, dark green colour, new leaf growth with reddish flush. Contrastingly, 30 and 40 min treatments with VSL reduced the growth, produced large callus at basal ends and shoots were not as vigorous as shoots from 0, 10 and 20 min exposure times. The ‘Reed’ shoot tips that were not exposed to LN (− LN) had higher average survival and regrowth levels with VSL exposure times compared with the corresponding LN treatment (+ LN). Shoot tips that were exposed to LN (+ LN) had the highest values for survival (65%) and regrowth (45%) after VSL exposure for 10 min (Fig. 4b and Supplementary data Fig. 2b). Shoot tips on regrowth media after VSL treatment were more vigorous, greener and appeared morphologically normal after 8 weeks compared to PVS2 treated shoots. Further sub-culturing of these shoots for another 16 weeks resulted in normal shoot development, producing elongated shoots (Fig. 3f). Longer exposure times of 30 and 40 min of VSL did not improve survival and regrowth of shoot tips + LN.

Discussion

The droplet-vitrification technique for cryopreservation is currently used in many plant species and more recently woody crops e.g. Malus domestica, Borkh (Condello et al. 2011), Vitis spp. (Pathirana et al. 2016), Magnolia spp. (Folgado and Panis 2019) and Castanea dentata (Liu 2019). However, it has not been reported as being successful for avocado shoot tip cryopreservation. Two previous attempts by Vargas (2008) and Vidales-Fernandez et al. (2011) on avocado shoot tips adopted other techniques i.e. dehydration-vitrification where shoot tips were dehydrated with sterile air before being treated with cryoprotectants, and resulted in no survival after LN treatment. For the first-time using the droplet-vitrification technique, this study has produced positive results with two avocado cultivars belonging to different ecological races; (1) cv ‘Velvick’ from the West Indian race (no cold tolerance) and (2) cv ‘Reed’ from the Guatemalan race (moderate cold tolerance).

Cryopreservation protocols can cause several stresses both osmotic and temperature related, i.e. exposure to low temperature, exposure to highly concentrated cryoprotectants and freezing injury (Reed 2012). This can lead to an increase in reactive oxygen species (ROS) within the plant, affecting viability. As reported by O’Brien et al. (2018a), the addition of ASA to each stage of the avocado cryopreservation protocol is necessary to avoid browning of shoot tips. The major site of cryo-injury is the cell membrane (Streczynski et al. 2019) and the maintenance of the integrity and stability of the cell membrane under water stress is paramount in cryopreservation and drought tolerance respectively (Bajji et al. 2002). Water stress is caused by desiccation or freezing during the cryopreservation process (Streczynski et al. 2019). Desiccation of plant material on a high sucrose media has been used to overcome toxicity of cryoprotectants. Pre-culture of shoot tips in sucrose can increase their viability during LN treatment, by better pre-conditioning the meristem to cryoprotectants (Feng et al. 2013; Kaczmarczyk et al. 2008; Park and Kim 2015). Pre-conditioning of shoot tips causes changes in lipid composition in cell membranes, accumulation of sugars, and production of membrane protecting polypeptides (Thomashow 1999). High sucrose levels in culture media of apple and blackberry plants has been reported to help, significantly increasing cold tolerance (Caswell et al. 1986; Palonen and Junttila 1999).

In another study (O’Brien et al. 2020), pre-treatment of avocado shoot tips was necessary to withstand the stress caused by cryoprotectants. Tolerance to cryoprotectants is an essential step of the cryopreservation process especially for tropical species (Souza et al. 2016). Sucrose stimulates the production of other elements such as proline, glycine betaine, glycerol and polyamines, which have colligative as well as non-colligative effects (Antony et al. 2013; Hirsh 1987). Folgado et al. (2015) reported that accumulation of sugar especially sucrose and the raffinose family oligosaccharides (RFOs) can be linked to increased cold tolerance in potato shoots; meaning a higher cryopreservation success level. Increasing the endogenous sugar of a plant provides stability of the membranes and promotes intracellular dehydration by increasing the osmotic pressure and the tolerance to PVS2 vitrification, thus reducing injuries that may cause cell death after freezing (Grapin et al. 2007; Sakai et al. 2008).

This study revealed that the type of cryoprotectant and the duration of cryoprotectant treatment had a great influence on shoot tip survival and regrowth of avocado shoot tips. When using PVS2 as a cryoprotectant, high survival and regrowth percentages were achieved with both cultivars − LN. The effect of PVS2 exposure time was more evident after liquid nitrogen treatment, with survival and regrowth levels reduced as compared to the − LN. After LN treatment the maximum regrowth of 35% and 55% were recorded for cvs ‘Velvick’ and ‘Reed’, respectively (Figs. 1a, 4a and Supplementary data). Shoots that survived in LN after PVS2 treatment showed much slower growth compared to controls (− LN) for both cultivars. The shoot tips that were LN treated (+ LN) also contained large callus overgrowth at basal ends and some leaf abnormalities, which could suggest some damage to shoot tips after LN treatment when using PVS2 due to incomplete cryoprotection. Shoot tips that were − LN treated appeared morphologically normal with exposure times of 0, 10 and 20 min. However, shoots exposed to PVS2 for 30 and 40 min without LN treatment (− LN) showed abnormalities in growth i.e. stunted, callus overgrowth at basal ends, leaf shape and colour.

Cryoprotectant toxicity remains the greatest obstacle in developing cryopreservation protocols while exposure time and conditions (temperature) (Volk et al. 2014) also determines good post-cryopreservation survival and recovery of shoot tips (Azimi et al. 2005; Sakai et al. 2008). We showed that long exposure times of 30 and 40 min had detrimental effects on survival and regrowth of avocado shoot tips (Figs. 1, 4). In such cases, it may be that meristems are damaged during the longer exposure times and cannot regenerate into differentiated shoots. This could lead to the production of callus at basal ends and eventually a callus overgrowth, suggesting the cells in these tips have lost their regenerative capacity to develop into normal shoots. This hypothesis should be further explored by the use of histology of avocado shoot tips ± LN. It is, therefore, important to establish minimum exposure time to cryoprotectant solutions in order to dehydrate tissue sufficiently yet avoid toxic effects (Azimi et al. 2005). Chemicals such as DMSO, a frequent component in cryoprotectants, has been reported to be toxic to cells (Aronen et al. 1999). The same is suggested for another component, glycerol (Wang et al. 2014). Some authors have reported that toxicity of PVS2 is the major barrier limiting vitrification protocols (Best 2015; Kim et al. 2009) especially when it is used at room temperature (Bettoni et al. 2019). Several studies have aimed at reducing cryoprotectant toxicity including sequential ‘loading’ and ‘unloading’ to prevent osmotic shock (Day et al. 2008), optimising temperature and timing (Kaczmarczyk et al. 2012; Pence 2014) and using mixtures of cryoprotectants (Hughes and Mancera 2014).

VSL has been used successfully in previous studies as a cryoprotectant to cryopreserve tips of Pinus kesiya Royle ex. Gord (73% regrowth) (Kalita et al. 2012), Gentiana scabra Bunge var. buergeri Maxim (73–78% regrowth) (Suzuki et al. 2008) and Hladnikia pastinacifolia Rchb (60% regrowth) (Ciringer et al. 2018). In our study, VSL as a cryoprotectant was very effective for both cultivars, conferring high survival and regrowth levels, especially − LN (Fig. 1b, 4b and Supplementary data). The effect of VSL exposure time was only evident after treatment + LN, where survival and regrowth levels were reduced as compared to − LN. The maximum regrowth of 35% was recorded for cv ‘Velvick’ and 45% for cv ‘Reed’ + LN when combined with VSL. Interestingly, morphological differences were evident between the VSL and PVS2 shoot tips at the regrowth stage when treated + LN. VSL + LN plants were morphologically normal when recovered after LN (Fig. 3c, f). Thus, VSL was the best for both avocado cultivars tested after 24 weeks regrowth.

Positive results of vigorous clumps after LN treatment obtained when using VSL may be attributed to the faster penetration level into the cells (Suzuki et al. 2008). This is because VSL is not as viscous as PVS2, which may lead to faster dehydration levels of tissue. On the other hand, VSL is rich in ethylene glycol (30% w/v) and contains less DMSO, glycerol and sucrose as compared to PVS2 (Suzuki et al. 2008). Suzuki et al. (2008) reported some toxic effects of VSL solution on plant tissues and states that the effect of toxicity depends on the type of material being preserved. Survival of material may be due to VSL containing less DMSO as compared to PVS2, however the higher content of ethylene glycol may offset the reduced effects of DMSO (Suzuki et al. 2008). In our case avocado shoots treated with both cryoprotectants at 10 and 20 min without LN treatment (− LN) showed no toxic effects of cryoprotectant. However, after the LN exposure, shoot tips treated with PVS2 showed poor survival and regrowth levels. Thus, the meristems that did not survive LN treatment may be attributed to tissue being damaged by the LN itself. As previously reported, this indicates there may be either insufficient penetration of cryoprotectant into the shoot tips or inadequate dehydration for PVS2 (Kalita et al. 2012). A low amount of cryoprotectant penetrating the shoot tip could theoretically reduce the cryo tolerance of avocado shoots after LN treatment if the dividing stem cells deep within the meristem are not exposed and protected. Previous studies by Engelmann (2011) have shown that cryopreservation procedures can lead to destruction of large zones of the meristems after LN treatment, and callusing or transitory callusing is often observed before organised regrowth starts (Engelmann 2011). The overgrowth of callus may be a result of only partial survival of meristematic areas. Consistent with this we saw higher callus regrowth in avocado shoot tips after LN treatment when using PSV2 (Engelmann 2011). For the shoot tips that survived LN we observed comparable regrowth percentages for both cultivars when PVS2 and VSL was used as a cryoprotectant. The major difference between the two cryoprotectants used was the superior ability of VSL treated shoots exposed to LN to regrow into full vigorous plants 24 weeks after treatment. In the case of PVS2 treated shoots lacked vigour. Further optimizations of the regrowth step may improve the quality of these shoots.

Conclusion

This manuscript for the first time reports the successful cryopreservation protocol for mature material of two avocado cultivars using the droplet-vitrification technique (Fig. 5). This will provide a useful tool for further optimizations of the species and other woody plant species facing similar challenges in conservation. It was evident that the two cultivars perform differently and require that cryoprotectants are optimized for each cultivar. Not only the type of cryoprotectant but the exposure time to the cryoprotectant was critical in obtaining morphologically normal and vigorous plants. This reinforces the outcomes from other studies that each cultivar and plant species are dependent on protocol readjustments in order to achieve high survival and regrowth levels after LN treatment (Souza et al. 2016). These findings, in combination with previous studies (O’Brien et al. 2018a, 2020) are a critical breakthrough to develop protocols for other avocado cultivars. It is envisioned that the work will pave way for setting up a world’s first germplasm repository to store a core collection of Persea spp. for true-to-type avocado shoot tip preservation.

Fig. 5
figure5

Flow diagram of the avocado droplet-vitrification procedure

Abbreviations

ASA:

Ascorbic acid

cv/s:

Cultivar/s

DMSO:

Dimethyl sulfoxide

EG:

Ethylene glycol

LN:

Liquid nitrogen

min:

Minutes

PVS2:

Plant vitrification solution 2

VSL:

Vitrification solution L

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Acknowledgements

The Queensland Alliance for Agriculture and Food Innovation (QAAFI) is a research institute of The University of Queensland (UQ), supported by the Queensland Government 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).

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CO: Conceptualization, Methodology, Visualization, Investigation, Writing—Original Draft. JHB: Writing—Review & Editing, Visualization, Formal Analysis. RF: Conceptualization, Supervision, Writing—Review & Editing. SL: Project Administration, Resources. AH: Supervision, Writing—Review & Editing. JF: Resources, Funding Acquisition. NM: Supervision, Resources, Writing—Review & Editing, Validation.

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O’Brien, C., Hiti-Bandaralage, J.C.A., Folgado, R. et al. First report on cryopreservation of mature shoot tips of two avocado (Persea americana Mill.) rootstocks. Plant Cell Tiss Organ Cult 144, 103–113 (2021). https://doi.org/10.1007/s11240-020-01861-y

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Keywords

  • Avocado
  • Cryopreservation
  • PVS2
  • Shoot tips
  • Vitrification
  • VSL