study.txt

ABSTRACT
The current threat of myrtle rust (Austropuccinia psidii) to New Zealand
Myrtaceae, including a number of indigenous and socio-economically
important species, requires that ex situ conservation is used to
complement in situ populations. New Zealand’s Myrtaceae have
received little attention in terms of ex situ conservation. In this study,
we assessed the integrated ex situ conservation strategies for
selected New Zealand Myrtaceae. We particularly investigated seed
banking options by assessing seed desiccation tolerance, in vitro
culture, pollen cryopreservation and zygotic embryo
cryopreservation of the recalcitrant Syzygium maire. A desiccation
trial was conducted on six Myrtaceae species: Lophomyrtus bullata,
L. obcordata, Metrosideros diffusa, M. umbellata, M. bartlettii and
Syzygium maire. S. maire seeds and embryos showed extreme
sensitivity to desiccation confirming its recalcitrant behaviour. The
seeds of the other species were desiccation tolerant i.e. orthodox.
Zygotic embryos of S. maire were successfully cryopreserved using
an encapsulation-dehydration technique. Pollen cryopreservation
was successful for M. excelsa following desiccation to about 5%
moisture content, rapid freezing and rapid thawing. For M. bartlettii,
one of the most endangered and a nationally critical Myrtaceae
species in New Zealand, we tested the efficacy of hand pollination in
producing viable seeds. Our assessment confirmed that one of the
M. bartlettii trees at Otari is self-incompatible, and successful hand
pollination using pollen obtained from different genotypes growing
in the gardens at the University of Auckland resulted in seed
production with c. 20% germination. Tissue culture protocols were
successfully developed for selected Myrtaceae. In addition,
photoautotrophic micropropagation techniques were developed for
the first time for L. scoparium. This paper highlights the importance
of holistic conservation strategies to ensure future access to New
Zealand’s unique Myrtaceae germplasm as a key component of
long-term management response to the threat posed by A. psidii.

Introduction
To mitigate the effects of biotic and abiotic threats on wild populations, ex situ germplasm
conservation has been widely applied in many species using protocols for conservation of
pollen, seed and clonal germplasm, complemented by in vitro propagation and cryopre-
servation (Sakai 2004; Pence 2014). As the myrtle rust threat is advancing at an alarming
rate in New Zealand (Biosecurity New Zealand: MPI Myrtle Rust Update March 2019;
Toome-Heller et al. 2020), there is an urgent need to investigate ex situ conservation strat-
egies for New Zealand’s Myrtaceae species, as little information is available on long-term
ex situ conservation. A combination of both in situ and ex situ conservation has been
identified as a key to conserving threatened and critically endangered species where estab-
lishment of tissue culture protocols in addition to seed and pollen conservation and cryo-
preservation were identified as critical components of ex situ conservation (Sarasan et al.
2006; Engelmann 2011; Nadarajan et al. 2018a). The aim of this work was to develop a
research-led conservation strategy to efficiently and cost-effectively conserve New
Zealand Myrtaceae species with the ultimate future goal of conserving maximum
genetic diversity for repatriation to the environment.
Seeds are the most preferred plant propagule for ex situ germplasm conservation
because they are easy to handle, relatively inexpensive to store and enable the regeneration
of whole plants from genetically diverse materials, provided seed is collected from geneti-
cally diverse parents. Seed banking is aimed at preservation of genetic variation within and
among populations for future use in breeding programmes and for germplasm conserva-
tion purposes. The internationally recommended standard for seed banking is −18°C and
15% relative humidity (FAO 2014). However, not all seeds can be dried and stored at low
temperatures. Seeds are divided into three main categories (orthodox, intermediate and
recalcitrant) based on their storage behaviour and their sensitivity to desiccation and
temperature (Roberts 1973; Ellis et al. 1993). Orthodox seeds are those that can tolerate
drying to very low moisture contents (≤3%–7% fresh weight), and whose longevity
increases as moisture content and temperature are reduced (Roberts 1973). Intermediate
seeds tolerate partial desiccation (∼10% moisture content) but longevity is reduced at low
moisture content in low temperature storage. Lastly, recalcitrant seeds are very sensitive to
desiccation and will lose viability after only the slightest amount of drying. Therefore, con-
ventional seed banking can be used to store orthodox and intermediate (to some extent)
seeds but not recalcitrant seeds. For long-term storage of recalcitrant seeds, cryopreserva-
tion, a process by which living tissues are conserved in liquid nitrogen at −196°C, is rec-
ommended. Seed storage behaviour for most of New Zealand Myrtaceae is still unknown,
with some Myrtaceae species are considered orthodox (Royal Botanic Gardens Kew 2020)
and some members of this family are expected to exhibit recalcitrant seed storage behav-
iour e.g. Syzygium maire as indicated in our pilot study (Nadarajan et al. 2018b).
In vitro techniques have been increasingly used in the conservation of threatened plants
in the recent years (Benson 1999; Sarasan et al. 2006). Several critical steps are involved in
establishing plants in vitro including initiation, multiplication, rooting, weaning and storage.
Genebanking through in vitro culture is another conservation option we tested in this
research. The application of photoautotrophic micropropagation technique which was
reported to be superior to conventional tissue culture technique in producing healthy
plants was also investigated in this study (Aitken-Christie et al. 1995; Zobayed et al.
2004). Once, optimised, tissue culture methods can be used to establish in vitro repositories
for conservation and also can be used to source explants for cryopreservation.
Metrosideros bartlettii is one of the most endangered Myrtaceae species in New
Zealand. Its current conservation status is ‘Threatened – Nationally Critical’ (de Lange
et al. 2018), down from 31 in 2000 (Drummond et al. 2000) to only 13 adult trees in
the wild as of 2018 (Lehnebach and van der Walt 2018). Unlike many species of Metrosi-
deros, flowering of M. bartlettii is erratic, lessening opportunities for natural regeneration
from seed. Despite its rarity in the wild, M. bartlettii is present in cultivation in both
private and public gardens. For instance, there are three trees at Otari Native Botanic
Garden and Wilton’s Bush Reserve (Otari) (Wellington). These trees flowered for the
first time in 2017, almost 25 years after they were planted (Lehnebach and van der
Walt 2018), which gave us the opportunity to evaluate the efficacy of controlled pollination
in producing viable seeds for this species.
Given the paucity of information on Myrtaceae conservation strategies and techniques,
germplasm conservation technologies developed in other families either as in vitro cul-
tures or as cryo-preserved shoot tips, seeds, embryos and pollen have been tested,
modified and adopted in this study for use on various New Zealand threatened Myrtaceae
species. This article presents an overview of our current work in development of integrated
ex situ germplasm conservation strategies for selected New Zealand Myrtaceae species.
Materials and methods

Seed, pollen and plant material
Seeds of Lophomyrtus bullata, L. obcordata, M. diffusa, M. umbellata, M. bartlettii and S. maire
were collected and supplied by New Zealand’s Department of Conservation (DOC) and Otari
Native Botanic Garden, Wellington (Otari). S. maire seeds collected from Taranaki District
were used in cryopreservation and desiccation studies. L. bullata, M. diffusa and
L. obcordata produce around 6–7 mm long berries in bright to dark red colour with numerous
pale brown testa, glossy, smooth and very hard seeds, M. bartletii seeds are formed in capsules
∼1.5–2.5 mm in diameter, M. umbellata fruits have woody capsule ∼ 6 mm in diameter which
releases very fine hair like exalbuminous seeds and S. maire has fleshy fruits ∼10–15 mm in
diameter with a single embryo, although polyembryony is recorded sporadically (Sanewski
2010; New Zealand Plant Conservation Network 2020).
For the tissue culture experiment, seedlings of M. bartlettii and L. obcordata growing in
water agar plates were received from Otari. Lophomyrtus ‘Red Dragon’ – a hybrid of
L. bullata × L. obcordata, M. excelsa and M. perforata seedlings were received from
Ardmore Nurseries Ltd., Papakura, Auckland and maintained in the field at the New
Zealand Institute for Plant and Food Research Limited (PFR), Palmerston North. Leptos-
permum scoparium seedlings were sourced from the PFR collection at Palmerston North.
Pollen of M. excelsa was collected from Victoria Esplanade, Palmerston North.

Seed germination
Seed germination was carried out on fresh and following seed desiccation sensitivity
assessment for L. bullata, L. obcordata, M. diffusa, M. umbellata, M. bartlettii and
S. maire seeds. For germination, the seeds were first extracted from their capsules or
fruits. Seeds were placed on water agar (7% w/v) and incubated at alternating temperature
(13°C/25°C) with a 12-h photoperiod. Fifty seeds in four replicates each were used for this
test. A seed was considered germinated when radicle and plumule growth were observed
(>2 mm). Germination tests were carried out for 25 days and at the end of the germination
trial, a cut test was conducted to confirm whether non-germinated seeds were dead (ident-
ified by their soft and off-white colour); if so, these were excluded from the experiment.
Seed desiccation sensitivity assessment
This assessment was conducted on L. bullata, L. obcordata, M. diffusa, M. umbellata,
M. bartlettii and S. maire seeds. Initial seed moisture content (MC) was determined grav-
imetrically by drying four replicates of 25 seeds using the ISTA (2018) method of drying
the seeds at 103°C (±2°C) for 17 h and calculated on a fresh weight basis. The seeds were
then equilibrated to six relative humidity (RH) environments (5%, 15%, 30%, 55%, 75%
and 100%). These RH environments were created using lithium chloride (Sigma-
Aldrich, NZ) salt solutions with different concentrations (Kate and Fiona 2014).
Around 100–200 seeds were used for each treatment. Seeds were placed in an airtight con-
tainer, and initial seed weight recorded. Seed weight loss or increase was monitored at
regular intervals until there were no changes recorded in the weight indicating the
seeds had equilibrated with their environment. Upon reaching equilibrium, seed MC
and germination was assessed as previously described.
Cryopreservation of Syzygium maire zygotic embryos
Desiccation sensitivity assessment of the excised embryos
Seeds of S. maire were surface washed with 20% Janola® (commercial bleach with 5%
sodium hypochlorite) for 2 min and the embryonic axes (referred to as embryos from
here onwards) were excised aseptically in a laminar flow hood. The embryos were
surface sterilised with 20% Janola® and 50% (v/v) ethanol for 1 min, respectively followed
by three rinses in sterile water. To assess the sensitivity of the excised embryos to desicca-
tion, the embryos were desiccated for various durations (0, 1, 2, 3, 4, 5 and 6 h) in the air
flow of a laminar flow bench. MCs of the embryos were determined as previously
described for seed at the end of each desiccation period. For each treatment, 10
embryos in four replicates were germinated on solid Murashige and Skoog (MS) (1962)
medium supplemented with 3% (w/v) sucrose and incubated in a growth room set at
25°C with 16/8 h photoperiod provided by cool white fluorescent tubes supplying a
PFD of 30–50 μmol/m 2
/s. Survival was recorded as percentage of radicle emergence (>2
mm) and complete germination i.e. when both radicle and plumule growth were observed.
Cryopreservation of excised non-encapsulated embryos
Protocols as described above were followed for seed surface washing, embryo excision and
sterilisation. The excised embryos were desiccated for 2, 3, 4 and 5 h in the air flow of a
laminar flow bench. After each desiccation period, MCs of the embryos were determined
as previously described and 10 embryos in four replicates were packed into cryovials and cryo-
preserved by rapid freezing in liquid nitrogen. The embryos were stored in liquid nitrogen for
1 h and then thawed in a water bath at 40 ± 2°C for 2 min. Following thawing, the embryos
were plated on solid MS medium supplemented with 3% (w/v) sucrose and incubated in the
growth environment specified above to assess their germination as described before. Germi-
nation of desiccated and non-cryopreserved embryos was recorded as control.
Cryopreservation of encapsulated embryos
The sterilised embryos were placed in 3% (w/v) sodium-alginate in half-strength MS medium
without calcium at pH 5.7. Beads were formed by suspending embryos in sodium-alginate and
dripping them into a calcium chloride solution (half-strength MS medium with 1% (w/v)
CaCl2). Beads were allowed to polymerise for 20 min. Beads were then blotted dry on
sterile filter paper, transferred to an open Petri dish and dehydrated for between 0 and 6 h
in a laminar air flow cabinet (Figure 1). After each dehydration period, MC and germination
before and after cryo-storage for 1 h were determined as previously described.
Cryopreservation of Metrosideros excelsa pollen
Open flowers with indehiscent anthers with powdery pollen were collected from an adult
M. excelsa tree. The flowers were bagged and pollen was collected by brushing the
anthers. Initial pollen germination was tested on BK medium (Brewbaker and Kwack
1963) consisting of 300 mg/L Ca(NO3)2.4H2O (hydrated calcium nitrate), 200 ppm MgSO4.-
7H2O (hydrated magnesium sulphate) and 100 ppm KNO3 (potassium nitrate) added with
10% sucrose and 150 ppm boric acid. Pollen was considered germinated when the length of
pollen tube was at least equal to or greater than the grain diameter (Weinbaum et al. 1984).
Fresh pollen germination was compared with non-dried pollen stored at room temperature
for 2 days, pollen dried to c. 5% MC (achieved in equilibrium with 15% RH), dried pollen
stored at room temperature for 2 days and dried-cryopreserved pollen. For cryopreservation,
dried pollen was packed in filter paper sachets, placed in cryovials and then rapidly cooled in
liquid nitrogen. The pollen was stored in liquid nitrogen for 1 h and rapidly thawed in a
water bath at 40 ± 2°C for 2 min before assessing their germination.
Metrosideros bartlettii hand pollination and assessment of viable seed
production
To ensure production of genetically diverse seeds, hand pollination was performed on one of
the M. bartlettii trees at Otari. The three trees at Otari are clones and therefore genetically
identical; we chose the healthiest tree with the most flowers. Before anthesis, 200 flowers
were covered with a fine mesh bag and checked weekly for further development. When
the style started protruding among the petals, both stamens and petals were removed follow-
ing a methodology described by Seguel et al. (1999). After emasculation, flowers were bagged
again until the style was fully expanded. Flowers from an adult M. bartlettii tree growing in
the gardens of The University of Auckland, and originally from a distinct provenance (Peter
de Lange, per. comm. 3 November 2017) to the tree at Otari, were collected and their pollen
was used to cross-pollinate emasculated flowers using a fine brush. To ensure the flowers
were successfully pollinated, pollen was applied on the stigmas again 4 days later. After
both cross-pollination events, the flowers were bagged and kept isolated until the style
started to wither and the ovary to swell. To control for agamospermy some of the emascu-
lated flowers were left bagged and unpollinated. Bags were only removed after the style had
withered. Capsules were collected before dehiscence and stored in paper bags at 20°C for a
maximum of 2 weeks. Fresh seed germination (n = 1250) was determined after surface ster-
ilising the seed for 20 min in 5 g/L sodium dichloroisocyanurate (NaDCC), followed by three
rinses in distilled sterile RO water. Since capsules contained a mixture of filled and empty
Figure 1. Sodium alginate (Na-alginate) encapsulation cryopreservation procedure. A, Excised embryos
were treated with 3% Na-alginate followed by 1% calcium chloride (CaCl2) solution for polymerisation
of the beads. B, Encapsulated embryos. C, Embryos encapsulated in the beads were desiccated to
various moisture contents. D, Following desiccation, the embryos were packed in cryovials. E, Cryovials
were attached to the cryo-cane. F, The cryo-canes were rapidly cooled by submerging in liquid nitrogen
(LN) and stored in LN for 1 h. G, following LN storage, embryos were rapidly rewarmed and plated on
Murashige and Skoog medium.
seeds, 100 filled seeds were selected under a stereo microscope and germination was deter-
mined using the methodology described above.
In vitro culture methods
Newly formed shoots (3–5 cm) were excised from field-grown plants, leaves were removed
and the shoot surfaces sterilised by immersion in 75% ethanol (45 s) followed by shaking
(70 rpm) in a solution of NaDCC, (5 g/L containing 0.1% (v/v) Tween 20®) for 30 min on
an orbital shaker and then washed in sterile RO water (3×). Shoot tips and nodal segments
were dissected aseptically and cultured on medium consisting of half-strength MS macro
elements, full-strength MS microelements, and B5 vitamins (Gamborg et al. 1968), 3% (w/
v) sucrose solidified with agar (7.5% w/v) (basal medium – BM). The culture medium pH
was adjusted to pH 6 prior to autoclaving at 121°C for 15 min. If contamination was
observed during the first week of culture, the explants were rinsed in 75% ethanol for
40 s and cultured in the same medium as above, supplemented with NaDCC 70–
100 mg/L for 2–4 days after which the plantlets were transferred to NaDCC-free
medium. To induce axillary shoot formation for micropropagation, two-nodal shoot
pieces were transferred to BM supplemented with 4.44 μM 6-benzylamino purine
(BAP) and 0.5 μM indole-3-butyric acid (IBA). The shoots arising were separated and
transferred back to BM for maintenance. Rooting of shoots was tested in two ways: (a)
by dipping cut ends of shoots in IBA (0.74–1.5 mM) for 30 s and growing in BM, (b)
by supplementing the solid BM with IBA (2.5–5.0 μM). The cultures were maintained
at 24 ± 1°C with a 16/8 h photoperiod and a photosynthetic photon flux density of 30–
50 μmol/m2
/s. For photoautotrophic micropropagation, shoot tips (2–5 cm) sterilised as
described above were embedded in sterile rock wool cubes aseptically, before placing in
sterile 250 mL plastic tissue culture vessels. After adding 10 mL of sterile liquid BM
without sucrose, the tubs were placed on a tilting device that enabled liquid feeding for
10 min followed by 20 min of draining. The cultures received 70–80 μmol/m2
/s light
(16 h per day) supplied by Sylvania Grow-Lux 58w/GRO-T8 (Germany) lights. Rooting
of shoots was tested using 7.4 μM IBA in the same growth solution. Regenerated plants
with roots were acclimatised in the laboratory and greenhouse conditions. Healthy
growing M. bartlettii seedlings were then transferred to Otari nursery for further growing.
Data analysis
The statistical software GenStat 17th edition (VSN International) was used to perform an
analysis of variance (ANOVA) on the germination data. Prior to analysis, data were trans-
formed and checked for normality. Where significant effects were detected in the ANOVA
(P = 0.05), means were compared using Fisher’s protected least significant difference test.
Results
Initial seed germination
The initial germination (average percentage ± standard deviation) for the studied species
was as follow; L. bullata (91 ± 2), L. obcordata (89 ± 3.9), M. diffusa (88 ± 3.2), M. bartlettii
(91 ± 1.2), M. umbellata (92 ± 3.3), and S. maire (93 ± 6).
Seed desiccation sensitivity assessment
Figure 2 summarises the seed MC and germination following equilibration at the selected
RH. The seed MC ranged from 2% to 46% following equilibration at 5% and 100% RH,
respectively. However, the germination was still high (>70%) even for the seeds dried to
around 2% MC for L. bullata, L. obcordata, M. diffusa, M. umbellata and M. bartlettii,
indicating that all these species seeds are orthodox. Syzygium maire seeds showed clear
evidence of desiccation sensitivity as the germination declined rapidly with reduced MC
and no germination was recorded for MC below 20% (Figure 2).
Cryopreservation of Syzygium maire zygotic embryos
Desiccation sensitivity assessment of the excised embryos
The embryo MC following drying and their corresponding radicle emergence and germina-
tion are summarised in Table 1. The results reconfirmed that S. maire is a recalcitrant species
as embryos show sensitivity to desiccation with viability critically reduced when the MC falls
below 20% and no germination at MC c. 12% (Table 1). An interesting observation was
noted where not all embryos that showed radicle emergence completed full germination.
Excised non-encapsulated embryo cryopreservation
Only four desiccation treatments (2, 3, 4 and 5 h) were used for this experiment based on
the results of the previous experiment. Embryos desiccated to 36% and 24% (following
drying for 2 and 3 h) showed germination of 55% and 40%, respectively (Table 2).
Further drying caused significant decline in radicle emergence and embryo germination
confirming the sensitivity of S. maire embryos to desiccation. Similar observation as
before was noted where not all embryos showed complete germination despite showing
radicle emergence. There was no radicle emergence or germination noted for the desic-
cated and cryopreserved embryos (Table 2).
Encapsulated embryo cryopreservation
The initial moisture content of the embryos encapsulated in the beads was c. 68% (fresh
weight basis), it rapidly decreased to c. 37% within the first 3 h of air drying and then
gradually reduced to 26% after 6 h (Figure 3). The MC of the encapsulated embryos
was higher than that of non-encapsulated embryos for the same duration of desiccation
(Table 2 and Figure 3). Radicle emergence and germination of the dehydrated but not
cryopreserved (–LN) embryos decreased from 96% and 90% to c. 30%, respectively,
after 4 h dehydration (embryo MC c. 30%) and then dropped to c. 20% when the
embryos reached 26% MC (Figure 4). The desiccated and cryopreserved (+LN)
embryos showed radicle emergence following drying to 37% and 31% MC. However,
these cryo-storage surviving embryos, despite showing radicle elongation, did not form
complete plantlets (Figure 4).
Cryopreservation of Metrosideros excelsa pollen
M. excelsa fresh pollen had high germination c. 80% at collection. However, germination
declined very rapidly, i.e. within 48 h at room temperature (Table 3). Desiccation to c. 5%
MC reduced the pollen germination slightly to 77%. This desiccated pollen also lost via-
bility within 48 h at room temperature. Desiccated and cryopreserved pollen retained a
similar level of germination as freshly collected pollen (Table 3).
Figure 2. Desiccation sensitivity profiles for A, Lophomyrtus bullata. B, Lophomyrtus obcordata.
C, Metrosideros diffusa. D, Metrosideros umbellata. E, Metrosideros bartlettii. F, Syzygium maire seeds.
80 J. NADARAJAN ET AL.
Metrosideros bartlettii hand pollination and assessment of viable seed
production
More than 600 flowers were used in this pollination study. From these, only the hand
cross-pollinated flowers set fruit (Table 4). Flowers that underwent other pollination treat-
ments wilted within weeks and ovaries detached within a month. The ovaries of hand
cross-pollinated flowers quickly swelled up but the fruits took almost 5 months to
mature. A change in colour, from light green to dull brown, indicated the capsules were
mature. The harvested capsules contained a mixture of filled and empty seeds, a charac-
teristic common of many Myrtaceae species. Mean seed germination was c. 21% with all of
the filled seeds germinated. The first germination was observed after 3 days, with germi-
nation completed within 7 days.
Establishment of in vitro cultures
Surface sterilisation using NaDCC as the sterilant and the subsequent culture in media
supplemented with NaDCC gave 20% clean explants of Lophomyrtus ‘Red Dragon’ and
M. perforata, and 42% L. scoparium. Shoot tip culture of M. bartlettii produced almost
100% sterile plants with low rates of contamination following initiation. Inclusion of
BAP and IBA at 10:1 ratio enabled proliferation of axillary shoots and IBA alone was
effective in producing roots (Figure 5). These plantlets with roots were easily acclimated
to the green house by first holding them in non-soil media for 2 weeks in a bottom
heated (27°C) fog tent, followed by misting in a mist bed. These plants were then
potted and transferred to Otari Gardens for planting (Figure 5).
Table 1. Syzygium maire embryo moisture content and corresponding germination rate following
desiccation (n = 40).
Desiccation duration (h)
Embryo moisture content (%)
(average ± SD)
% Radicle emergence
(average ± SD)
% Germination
(average ± SD)
0 75.3 ± 2.5 a 100.0 ± 0.0 a 100.0 ± 0.0a
1 54.7 ± 1.5 b 80.0 ± 2.0 a.b 72.0 ± 2.0a.b
2 38.6 ± 1.1 b,c 56.0 ± 2.0 c 52.0 ± 2.0c
3 24.4 ± 1.0 c 44.0 ± 2.0 c.d 38.0 ± 2.0c,d
4 20.0 ± 1.0 c 30.0 ± 1.2 d 20.3 ± 1.2d
5 15.3 ± 0.6 c,d 10.0 ± 2.0 e 8.0 ± 2.0e
6 12.6 ± 1.0 cd 0 ± 0 e 0 ± 0 e
Notes: The different letter on each column indicates the statistical significance at P < 0.05 based on Fisher’s protected LSD
test.
Table 2. Radicle emergence and germination before (–LN) and after cryopreservation (+LN) following
desiccation for excised embryos of Syzygium maire (n = 40).
Desiccation
duration (h)
Embryo moisture
content (%)
(average ± SD)
% Radicle emergence (–LN)
(average ± SD)
% Germination
(-LN)
(average ± SD)
% Radicle emergence/
germination (+LN)
2 36.40 ± 1.05a 60.0 ± 0.2a 55.0 ± 0.6 a 0
3 24.63 ± 1.0 b 40.0 ± 2.0c 40.0 ± 2.0 b,c 0
4 20.18 ± 0.39b,c 24.00 ± 1.5d,e 18.0 ± 1.5 d,e 0
5 15.16 ± 0.63c 10.00 ± 1.5e,f 6.0 ± 1.5 f 0
Notes: –LN (non-cryopreserved); +LN (cryopreserved). The different letter on each column indicates the statistical signifi-
cance at P < 0.05 based on Fisher’s protected LSD test.

Discussion
The desiccation sensitivity assessment conducted on six New Zealand Myrtaceae species
revealed that apart from S. maire, the seeds of the remaining five species are desiccation
tolerant. Though seed banking can be recommended as the rule of thumb for storing
these seeds for long-term, further investigation is needed to confirm the seeds sensitivity
to low temperature and their ageing kinetics i.e. if they are short lived and therefore would
benefit from cryostorage for long-term storage (Ballesteros and Pence 2014). Seeds of S.
maire are desiccation sensitive, as none of the seeds dried to moisture contents below
c. 20% remained viable. The level of desiccation sensitivity displayed by S. maire seeds
is typical of many recalcitrant seeds i.e. viability is lost at 20%–30% moisture content
(Pritchard 2004). Hong and Ellis (1997) investigated patterns of response to seed
Figure 4. A,B, Sodium alginate encapsulated embryos of Syzygium maire regenerating on Murashige
and Skoog medium 4 weeks after cryopreservation.
Figure 3. Radicle emergence and germination of sodium alginate encapsulated embryos of Syzygium
maire following desiccation ( –LN) and following desiccation and cryopreservation (+LN).
desiccation in various species and found that species with desiccation-sensitive seeds typi-
cally occur in moist areas, particularly rainforest, and produce large (>1 g), round fleshy
seeds, which are shed at high moisture contents. These observations are aligned with the
ecology of S. maire, which is also known as swamp maire due to its natural habitat, i.e.
swamp or waterlogged areas.
We explored the potential application of cryopreservation to the recalcitrant New
Zealand Myrtaceae species, S. maire, using its embryos with or without sodium-alginate
encapsulation. Plant cryopreservation has advanced rapidly in the last 25 years thanks
to increased understanding of the low temperature biology. Innovations in cryopreserva-
tion method development and methodological improvement covering encapsulation-
dehydration, vitrification, encapsulation-vitrification, droplet vitrification and the inno-
vation of V- and D-cryoplates (Engelmann 2011; Benelli et al. 2013) have resulted in suc-
cessful cryopreservation of various crops and wild germplasm. In our study, encapsulated
embryos outperformed the non-encapsulated embryos in post-cryopreservation survival,
with no survival recorded for the non-encapsulated embryos. Peran et al. (2006) specu-
lated that the poor performance of non-encapsulated embryos could be due to one or
both of the following two reasons; (1) the apical meristem of the embryonic axes of
many species lacks any protective covering layer and, during the partial drying treatment,
may dry to a lower water concentration than other parts of the axis, consequently suffering
more desiccation damage; and (2) the process of excision could inflict substantial mech-
anical damage on the meristem. Embryo survival (radicle emergence) between 20% and
30% following cryopreservation was recorded when the encapsulated embryos were
dried to moisture contents of 37% and 31% respectively. Moisture content above 37%
was lethal as no embryos survived cryopreservation, possibly due to ice crystallisation.
It is a well-known fact that alginate protects tissues from injury during dehydration and
freeze–thaw treatments, and alleviates excessively rapid dehydration (Fabre and
Dereuddre 1990; Engelmann 1997; Benson 1999; Grospietsch et al. 1999). The success
of the method is largely dependent upon the embryo’s desiccation tolerance and the
ability to circumvent ice nucleation during cooling and warming.
Table 3. Germination of Metrosideros excelsa pollen following desiccation and cryopreservation.
Treatment
Germination (%)
(mean ± SD)
Fresh pollen 80.0 ± 1.8 a
Fresh pollen stored at room temperature for 2 days 0 ± 0b
Desiccated pollen (not stored) 77.5 ± 1.9 a
Desiccated pollen stored at room temperature for 2 days 0 ± 0b
Desiccated and cryopreserved pollen 76.0 ± 0.8 a
Notes: The different letter indicates the statistical significance at P < 0.05 based on Fisher’s protected LSD test.
Table 4. Pollination treatments performed on Metrosideros bartlettii, number of flowers used in each
treatment and number of fruits developed.
Pollination treatment No. of flowers No. of fruits (%)
Agamospermy 20 0
Autonomous self-pollination 183 0
Hand cross-pollination 107 73 (68.2)
Hand self-pollination 69 0
Natural pollination 244 0
Although encapsulated S. maire embryos showed post-cryopreservation survival
(radicle emergence and elongation), the embryos did not form complete plantlets follow-
ing radicle elongation. This has been reported previously: Farrant et al. (1986), Goveia
et al. (2004), Kioko et al. (1998) and Pence (1992) showed the lack of or poor capacity
for shoot formation from desiccated axes before and after cryopreservation. In many
cases, axes surviving after cryo-storage may have produced roots or callus, but often did
not form plantlets (Gonzalez-Benito et al. 2002). Cryopreservation of large (>5 mm)
and heterogeneous recalcitrant embryonic tissues has always been problematic (Nadarajan
and Pritchard 2014). Uniform dehydration of the tissue is often difficult to achieve with
desiccation tolerance varying between different tissues. Non-uniform dehydration could
lead to insufficient drying of the embryo tissues, increasing the risk of intracellular ice for-
mation and thus tissue death (Wang et al. 2015; Wesley-Smith et al. 2015).
Information on pollen viability and longevity is crucial in maximising the possibilities
of using viable pollen during artificial or controlled pollination. This is especially true for
Myrtaceae since seed production through natural pollination is typically low in this family
(Schmidt-Adam et al. 1999). Our examination of M. excelsa pollen storability established
that pollen at room temperature lost viability within two days. The combination of high
temperature and humidity at room temperature could have resulted in high respiration
and metabolic activities that in combination with rapid moisture loss could have led to
Figure 5. In vitro culture, micropropagation and greenhouse acclimation of propagated Metrosideros
bartlettii material. A, Seedlings of M. bartlettii in agar/water plates as received. B, Multiple shoots
induced. C, Rooted plantlet ready for the green house. D, Healthy plants after acclimation in the
green house ready for planting.
rapid decline of pollen viability. The pollen however retained high germination following
cryopreservation. The dehydration associated with cryo-storage protocols may have
delayed ageing of the pollen. Thus, for long-term preservation, low temperature storage
including cryogenic temperatures are recommended (Hanna and Towill 1995). Page
et al. (2006) reported that pollen of Kunzea pomifera (Myrtaceae) can be stored for up
to 370 days at 4°C and 10% relative humidity without any significant loss of viability.
Similar to our study, Page et al. (2006) conducted the study on only one accession and
hence recommended assessment of possible genotype variation in pollen longevity.
Our pollination experiments confirmed that one of the M. bartlettii trees at Otari is a
self-incompatible individual that depends on pollen from unrelated individuals to set
fruit. Self-incompatibility has been reported in other species of Metrosideros in New
Zealand and overseas (Carpenter 1976; Schmidt-Adam et al. 1999) and was suspected
to occur in M. bartlettii, but never experimentally confirmed. These findings contradict
reports of abundant fruit-set and the production of viable seeds observed in single
M. bartlettii trees growing in other botanic gardens or private gardens. It is possible
that, similar to M. excelsa (Schmidt-Adam et al. 1999), self-incompatibility in
M. bartlettii is incomplete and self-compatible individuals may also exist. Alternatively,
M. bartlettii trees may be capable of hybridising with other Metrosideros species
growing nearby. Past and recent genetic studies have confirmed hybridisation, and intro-
gression, are possible between closely related species of Metrosideros (e.g. Gardner et al.
2004; Melesse 2019). Unfortunately, none of the trees flowered the season following our
study and we have been unable to further investigate these hypotheses. Although the
average germination of seed obtained from the controlled pollination of M. bartlettii
was generally low which is consistent with findings in other Myrtaceae species such as
M. excelsa (Schmidt-Adam et al. 1999) and Leptospermum scoparium (Herron et al.
2000), hand pollination is beneficial in in situ conservation where trees are too far apart
for pollination to occur and when no pollinators are present on site.
Vegetative propagation using cuttings of several Myrtaceae species has been demon-
strated but is slow, often difficult, and season and genotype dependent. Therefore micro-
propagation is the preferred method of propagation. Once optimised, tissue culture
methods can be used to establish in vitro repositories for conservation and also can be
used to source explants for cryopreservation for long-term conservation. Establishment
of axenic cultures is often challenging when explants are sourced from field-grown
plants, such as when rescuing endangered Myrtaceae from the wild. We introduced
NaDCC into our Myrtaceae tissue culture establishment protocols and optimised it for
surface sterilisation of plant material from the field. Parkinson et al. (1996) showed that
NaDCC is more effective than commercial bleach (sodium hypochlorite) for disinfestation
of shoots in a range of species contaminated predominantly with Pseudomonas, Xantho-
monas and Actinomycetes. Indeed, we were able to show for the first time the possibility of
disinfesting contaminated field tissue using NaDCC in culture medium. Combination of
two steps (surface sterilisation and culture in NaDCC-supplemented media) enabled us to
obtain 20%–42% clean cultures in the three Myrtaceae species studied.
A first attempt at photoautotrophic micropropagation for selected Myrtaceae species
using a tilting device under high light intensity without sucrose in the culture medium
seemed promising. Studies by Zobayed et al. (2004) and Aitken-Christie et al. (1995) high-
lighted the advantages of photoautotrophic micropropagation in a sugar-free medium
where better growth, higher quality, lower contamination rate and higher percentage sur-
vival ex vitro was achieved compared to the conventional tissue culture system. Appli-
cation of forced ventilation coupled with carbon dioxide enrichment and increased light
intensity was shown to be effective in achieving high efficiency in autotrophic system.
We did not measure the growth of plants or increase CO2 concentration as required for
efficient photosynthesis under photoautotrophic conditions (Xiao et al. 2011), however,
culture vessels appeared to have sufficient air exchange due to the corrugated edge of
the lid, another requirement for such a system (Xiao et al. 2011). Rooting was initiated
without difficulty in our photoautotrophic micropropagation condition by incorporating
IBA in the culture medium. Hence, this relatively simple method has a huge potential in
contributing to growing and maintaining healthy plants in tissue culture.
Conclusions
This study identified or verified various ex situ conservation strategies including seed
storage, in vitro propagation systems, and cryopreservation (embryonic axes and
pollen) for selected Myrtaceae species. This will contribute to the development of inte-
grated in situ and ex situ conservation strategies for conservation of threatened New
Zealand Myrtaceae species.
Acknowledgements
We gratefully acknowledge Otari Native Botanic Garden, Wellington City Council (WCC) for con-
tributions to this project; Department of Conservation (DOC), New Zealand for supplying
seeds and plant materials. Funding for this project by the Ministry of Primary Industries (MPI),
New Zealand (Project 18608) is greatly appreciated.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Funding
This work was supported by the Ministry of Primary Industries (MPI), New Zealand [Project
18608].
ORCID
Jayanthi Nadarajan http://orcid.org/0000-0002-2132-5395
Carlos A. Lehnebach http://orcid.org/0000-0001-7368-013X
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