Acclimatization of micropropagated mature avocado

J.C.A. Hiti Bandaralagea, A. Hayward, C. O’Brien, C. Beveridge and N. Mitter

The University of Queensland, Brisbane, Queensland, Australia.

Abstract
The morphology, anatomy and physiology of in vitro cultured plants do not
facilitate survival when transferred to dynamic ex vitro environments. The benefits of
a micropropagation system completely depend on successful transfer of in vitro
plants to septic ex vitro conditions. Avocado (Persea americana Mill.) is a luxurious
tropical fruit becoming very popular with increasing consumption around the globe
due to its high nutritional value. It is a “micropropagation worthy” plant species, to
meet the high industry demand for clonal propagules. Being a woody perennial, in the
category of recalcitrant species for micropropagation, avocado has consistently
proven to be very difficult to induce roots in vitro. Even if rooted, plant survival during
acclimatization is very poor due to few, mostly unbranched roots produced at the
rooting stage. The current study focused on optimising the last stage of the
micropropagation process; acclimatization of in vitro regenerated avocado plantlets
to achieve higher survival rates. The methods tested included mist spray application,
root soaking treatments and a new method where plants were acclimatized in the
tissue culture jar itself. The mist spray durations tested (1, 2 and 3 days) produced
similar results. Highest survival rate was 74% with 3 days mist spray. Root soaking
treatments and the duration of humidity dome-cover positively influenced plant
survival. Root soaking for 2-3 days followed by a 4-week dome-cover period gave very
high survival of 97%. The method of acclimatizing plantlets in the tissue culture jar
with lids open (under a dome cover for 2 weeks followed by no dome cover for 2
weeks) also resulted in 96% survival of plantlets even after 24 weeks under
glasshouse conditions. The optimised acclimatization methods are convenient and
expected to be effective on large scale. The methods will likely be transferrable to
other woody species encountering similar problems as avocado during
acclimatization.

Keywords: avocado, acclimatization, micropropagation

INTRODUCTION
Acclimatization, or hardening, is the final phase of micropropagation where careful
optimisation is essential for survival and successful establishment of in vitro cultured plants.
Transition from controlled in vitro conditions to a dynamic ex vitro environment puts
immense pressure on micropropagated plants and it is a bottlenecking step in many
micropropagation processes (Soukup et al., 2004). Culture of plants in a nurturing, stress-
free and rich artificial environment induces physiological and anatomical characteristics that
necessitate the gradual training of plants to ex vitro environments. Plants under in vitro
conditions have less available CO2 due to enclosed sterile containment and are supplied with
a saccharide based carbon source forcing them to be semi autotrophic (Lesar et al., 2012).
Many anatomical features required to survive in the harsh natural environment, such as a
fully developed cuticle and adequate amount of stomata, as well as basic plant functional
units for photosynthesis such as a well-defined palisade and amount of spongy parenchyma,
are altered under in vitro conditions (Fabbri et al., 1986; Wetzstein and Sommer, 1982).
Avocado (Persea americana Mill.) is an important horticultural crop of high economic
value. It is grown as a grafted plant, combining important agronomical traits of a selected
rootstock with a scion selected for postharvest and fruit quality characteristics (Silva and

a
E-mail: jayeni.hitibandaralage@uq.net.au

Acta Hortic. 1224. ISHS 2018. DOI 10.17660/ActaHortic.2018.1224.3 13

Proc. VII Int. Symp. on Production and Establishment of Micropropagated Plants
Eds.: R. Paiva et al.
Ledesma, 2014; Castro et al., 1995). Rootstocks may be produced from seed of a pollinated
tree or through clonal propagation, which unlike seed conserves the genetic quality of the
source tree. To date clonal plants are produced from rooted cuttings of a mature tree
through a lengthy and arduous ex vitro process, causing limited availability and high prices
of clonal avocado plants (Ernst, 1999; Platt, 1976). In vitro propagation, or
micropropagation, is a desirable avenue for clonal avocado rootstock production to produce
large numbers of true-to-type plantlets from fewer explants in a space-efficient, sterile, soil-
free and stress-free environment (Mohamed-Yasseen, 1993). However, similar to many
woody plant species, mature material of avocado is highly recalcitrant to tissue culture
conditions and suffers from poor and inconsistent rooting (Bandaralage et al., 2015; Salih
Duhoky et al., 2012; Zirari and Lionakis, 1994; Kadman and Ben-Ya’acov, 1965). If rooted,
additional losses during the acclimatization process amplify inefficiency of the system.
Therefore, a successful hardening practice with minimum mortality is vital for avocado
micropropagation system. To date, the necessity of an effective acclimatization practice has
been left unaddressed, possibly due to the difficulty of sourcing large number of
micropropagated avocado plantlets.
The main objective of this study was to test different acclimatization methods to
improve survival rate of avocado plantlets (‘Reed’ rootstock). The growth rate of
acclimatised plants was also evaluated under glasshouse conditions. The findings of this
study will also benefit other tissue culture systems experiencing similar problems during
acclimatization phase.

MATERIAL AND METHODS

‘Reed’ avocado plantlets, micropropagated, were subjected to different treatments in
independent experiments for acclimatization. All plantlets had at least one root with a
minimum root length of 1 cm. Unless specified, plantlets were submerged in a 1 g L-1
solution of Fongarid (Systemic fungicide) for 10 min before planting in soil.

Mist-spray application treatments

Plantlets were placed between two layers of two moistened tissues in a tray at 25°C,
16 h light in the laboratory (Figure 1). Mist spray was applied every 3 h from 8.00 am to 4.00
pm for a duration of 1, 2 or 3 days using a hand spray bottle (n=35). After this, plants were
planted in 50-mL pots using pasteurised soil as substrate media (1:1 mixture of UQ-23
glasshouse potting mix and perlite) and placed in trays covered with a sealed plastic
humidity dome for two weeks in a glasshouse mist chamber (temperature in the mist
chamber was 28-32°C and under natural day length during summer). Dome covers were
then removed and plants left for another 2 weeks in the mist chamber. Plantlet survival was
recorded at 2 and 4 weeks from the start of acclimatization. Plant height and number of
leaves produced were also recorded at 4 weeks.

Figure 1. De-flasked rooted ‘Reed’ plantlets placed on a moistened tissue during mist spray
treatment.

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Root soaking and dome cover treatments
Plantlets were subjected to different durations of root soaking (0, 1, 2, and 3 days;
n=25) in sterile distilled water in a closed container prior planting in soil. At the time of
planting root number and presence of lateral roots was recorded for each plant. Plantlets
were potted in an equal mixture of peat:perlite:sand:Osmocote (Scotts) seed raising mix in
50-mL pots, covered with a plastic dome for 1 week and incubated in a growth cabinet at
25°C, 16 h light under 78% relative humidity (RH). Dome covers were removed after 1 week
while maintaining the plants in the growth cabinet. Plantlet survival data were recorded 4
weeks from de-flasking. The treatment that resulted in highest percentage of plant survival
was repeated using 52 plantlets.
A similar experiment was done with plantlets subjected to root soaking for 3 days
(n=35) followed by different dome cover durations (1, 2, 3, 4, 5 and 6 weeks). Plants were
incubated for another 2 weeks after removing the dome and survival rate recorded.

In-jar hardening
Individual culture jars with rooted plants were selected and lids were removed.
Without de-flasking plantlets, 5 mL of 1 g L-1 solution of Fongarid (Systemic fungicide) was
added to each jar and jars were placed under a plastic dome and incubated in the growth
cabinet at 25°C, 16 h light under 78% RH. Every 3 days plants were sprayed with the same
fungicide solution. Three treatments (T) were carried out (n=80); T1 – 3 weeks in-jar (under
closed dome for 1 week, open dome for 2 weeks); T2 – 4 weeks in-jar (under closed dome
for 2 weeks, open dome for 2 weeks); and T3 – 5 weeks in-jar (under closed dome for 3
weeks, open dome for 2 weeks). Domes were kept open for 1, 2, 3 h, respectively, every day
for up to two weeks after the end of the continuous cover before being left completely open.
Plantlets were then potted in 100-mL pots and incubated for another 4 weeks in the growth
cabinet until transfer to the glasshouse mist chamber. Plants were retained in the mist
chamber for 4 weeks before transferring to open bench within the glasshouse. Data recorded
included number of surviving plants, plant height and number of leaves at every 4 weeks
from the start of the acclimatization process.

Data analysis
Data analysis was carried out using statistical software IBM SPSS 23. Pearson Chi-
Square test was performed to analyse the significance of survival percentages of different
treatments in independent treatments. ANOVA was performed at confidence level 0.05. Least
significant difference and Mann-Whitney U tests were also carried out during data analysis.

RESULTS AND DISCUSSION

In avocado plantlet acclimatization, a gradual increase in relative humidity starting
from 100% under greenhouse conditions is recommended (Barceló -Muñ oz and Pliego-
Alfaro, 2003). Once de-flasked both shoots and roots experience stress under reduced
relative humidity and absence of proper contact with culture. Different strategies such as,
use of different potting media, application of anti-transpirants, gradual reduction of relative
humidity, gradual increase of light conditions, use of mist chambers have been tested
including pre-conditioning strategies such as supplementing medium with high sucrose
doses, paclobutrazol and desiccants during the rooting stage (Hazarika, 2003, 2006). The
current study tested different methods; 1) mist spray, 2) root soaking, and 3) in-jar
hardening, with the aim to increase plant survival. The different methods tested showed
variable results with respect to plant survival and growth rate but all increased the plant
survival percentages with the use of root soaking treatment, dome cover and in-jar
acclimatization.

Mist-spray application
The duration of mist spray did not significantly influence the survival percentage of
plantlets either at 2 weeks or at 4 weeks (p=0.05). The survival percentages recorded at 2
weeks for 1, 2 and 3 days mist spray were 62.86, 77.14 and 82.86%, respectively. However,

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after 4 weeks incubation in the mist chamber survival rates reduced to 51.4, 68.57 and,
74.28%, respectively. Further, plants acclimatized using different mist spray durations
showed no significant difference (data not shown) in their mean height and mean number of
leaves produced at 1 month after transplanting in soil.

Root soaking
Under root soaking conditions less water stress and humidity stress are imposed on
plants. Roots are trained to undergo active absorption while shoots are under de-flasking
stress due to low atmospheric RH.
Plant survival percentages recorded for 0, 1, 2 and 3 days, root soaking, followed by 1
week dome-cover, were 32, 28, 52 and 60%, respectively. Soaking roots in water for 3 days
resulted in significantly higher survival percentage compared to no soaking (p=0.045) and 1
day soaking (p=0.022). However, both 2 and 3 days of root soaking resulted in significantly
similar percentages with regards to plant survival.
Taking the best soaking treatment of 3 days and varying the duration of dome cover
revealed 4 weeks to be significantly the best among treatments, recording 97% survival (1
week – 74%, 2 weeks – 71%, 3 weeks – 74%, 5 weeks – 71%, 6 weeks – 74%) (Figure 2).
Though we expected prolonged dome cover to increase the plant survival, going beyond 4
weeks was not optimum for avocado. This could be due to the anoxic conditions created by
prolonged dome cover and regular watering practices. In an avocado acclimatisation study
using rooted plants with juvenile origin by Azcó n-Aguilar et al. (1992) plant survival was
also improved when incubated in completely closed jars for 4 weeks. However, they
recorded less survival (75%) compared with the method used in this study.

Figure 2. Survival percentage at 8 weeks form the start of acclimatization using 3 days of
root soaking treatment followed by different dome cover durations; a and b are
significantly different values, n=35, p<0.05).

In-jar hardening
In this method, shoots become hardened with gradual exposure to open environment
before the root system is trained to establish in soil.
Survival percentages were significantly different for different in-jar durations
(p=0.05). An in-jar incubation period of 3 weeks (86.25%) resulted in significantly less
survival after 1 month compared to both 4 (96.25%) and 5 (97.5%) weeks (Figure 3). After 2
months however, survival was not enhanced by 5-week in-jar treatment.

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Figure 3. Survival percentage at 4 and 8 weeks form the start of acclimatisation with in-jar
acclimatization periods of 3, 4 and 5 weeks, a and b are significantly different
values, n=80, p<0.05).

The mean height of plantlets subjected to 5 weeks in-jar incubation was significantly
greater than other treatments (Figure 4), while the mean number of leaves produced was
significantly greater in the 3 weeks in-jar treatment (Figure 5). The height increase at 5
weeks may result from the longer dome cover period, which maintains favourable
conditions to grow with least amount of stress. The higher number of leaves in the 3 weeks
in-jar treatment could be due to the early exposure to the low RH conditions, which require
the plantlets to become fully autotrophic. Hence this requires new leaves with fully
functional stomata and completely developed palisade and spongy parenchyma layers.

Figure 4. Mean height (cm) of plantlets at 4 weeks under different in-jar treatments. Data
are shown as the mean ± SE for n=80 per treatment. Letters a, b and c indicate
significantly different means at p=0.05.

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Figure 5. Mean number of leaves produced at 4 weeks under different in-jar treatments.
Data are shown as the mean ± SE for n=80 per treatment. Letters a, b and c
indicate significantly different means at p=0.05.

At 8 weeks, survival percentages remained unchanged for the 3 and 4 week in-jar
treated plants while plants treated for 5 weeks showed reduced survival of 83.75% (Figure
3). Therefore, the 4 week in-jar treatment was significantly better with respect to plant
survival at 8 weeks (96.25%) (Figure 6). Increased mortality of 5 weeks treated plants could
be due to failure in withstanding transpiration loss or poor root establishment. Prolonged
dome cover period may have produced fragile leaves. On the other hand, prolonged growth
in the tissue culture jar may lead to accumulation of water in the bottom, creating anexic
conditions to roots which cause root damage and poor root establishment before
transplanting to soil.

Figure 6. ‘Reed’ plantlets in the mist chamber after 8 weeks during in-jar hardening process.

Previous studies have shown low survival percentages and poor growth of tissue-
cultured avocado plants of juvenile origin (Azcó n-Aguilar et al., 1992; Vidal et al., 1992). This
has been attributed to the absence of mycorrhizal fungus association with micropropagated
avocado plants (Vidal et al., 1992). Therefore, exogenous inoculation of mycorrhizal fungus
has been tested as a strategy for successful acclimatization. However, Azcó n-Aguilar et al.

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(1992) reported only 85% plant survival (juvenile plants) with inoculation of vesicular-
arbuscular mycorrhizae Glomus fasciculatum. Further, plant height and number of leaves
produced at 12 weeks in that study were comparatively less than the results here following
the 4 week in-jar hardening method (Figure 7). A similar experiment inoculating Glomus
fasciculatum by Vidal et al. (1992) also showed only 72% avocado plantlet survival. Our
results show that there is no absolute requirement of mycorrhizal fungal inoculation for
survival and healthy growth of in vitro propagated avocado plantlets.

Figure 7. Mean height (cm) and mean number of leaves produced (n=80) over 24 weeks
from start of acclimatization in the best in-jar hardening treatment (4 weeks in-
jar).

In this study, we identified two successful methods to achieve more than 96% plantlet
survival after acclimatization. A 2- to 3-day root soaking treatment followed by a 4-week
dome cover period for plantlets transplanted in soil was the fastest successful method of
acclimatization. Meanwhile, a 4-week in-jar hardening (including 2 weeks under dome and 2
weeks open dome) followed by transplant to soil showed equal success for avocado plant
acclimatization. These simple procedures will enable practicality at a large scale operational
level.

CONCLUSIONS
Root soaking treatment and duration of dome cover period influenced the survival
rate of micropropagated avocado plants. A 2- to 3-day root soaking and 4-week dome cover
incubation was successful to reduce mortality to 3%. A new method of acclimatization, ‘in-
jar hardening’ was also developed which resulted in 96% survival and higher plant growth
rate than previously reported acclimatizing practices for avocado, even after 24 weeks under
glasshouse conditions.

ACKNOWLEDGEMENTS
This project is jointly funded by the Australian Research Council Linkage Programme,
grant no. LP130100870 (2013), the Department of Agriculture and Fisheries, Australia and
the University of Queensland, Australia collaborating with industry partners; Primary
Growth Pty Ltd., Jasper Farms Holdings Pty Ltd., Millwood Holdings Pty Ltd., T/A Delroy
Orchards and Anderson Horticulture Pty Ltd., J.C.A. Hiti Bandaralage is supported by an
Australian Postgraduate Award.

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