1. Cleo Henderson
10344240
Survival and recovery of Zea mays var. everta seeds following
cryopreservation treatments with direct vitrification
A protocol for cryopreservation with the use of direct vitrification or preloading of
PVS2 was established and tested on Zea mays var. everta seeds with direct
immersion in liquid nitrogen (-196°C). The aim of the investigation was to test
viability of explants 7 days post 4 different treatments (6 including controls). This
was deduced by survival and recovery. Seeds were pre-cultured on M&S solid media
beforehand. Treatments using PVS2 were also tested without immersion in liquid
nitrogen. Survival 7 days post was ≤10% in explants that were immersed in liquid
nitrogen and ≥90% in explants that were not. Mean recoveryfor PVS2 treatments at
room temperature was in the range of 20-21mm for roots and 21-29mm for shoots.
INTRODUCTION
The causes of prehistoric extinctions are relatively less significant in comparison to the
modern anthropogenic causes of extinction, which include, but are not limited to, habitat
destruction, over-exploitation, pollution and invasive species introduction (Gurevitch &
Padilla, 2004; Hogan, 2014). Plants provide a multiplicity of resources and many are also
paramount to maintaining biodiversity, essential for plant breeding (Panis & Lambardi,
2005). Additionally, there is a demand for high quality, high quantity crops (Wang et al.
2009). Species depletion can be observed all aroun. As an example, the species
Hygrophila albobracteata, of the Acanthaceae family, has suffered due to agricultural
pressure and mining activity and is now listed as endangered (Luke et al. 2015). Plant
conservation aims to prevent extinction of endangered plants (Botanic Gardens
Conservation International (BGCI), 2007). H. albobracteata is currently being protected in
the Katavi National Park and the Ruaha National Park (Luke et al. 2015). Establishing seed
banks is an ex situ means of conservation, which can be more advantageous as it’s
economically efficient (BGCI, 2005). It also allows the storage of genetically diverse
samples that remain viable for a long duration (BGCI, 2005). In light of a potential food
sustainability crisis, landraces and wild type cultivated crops have been stored in gene
banks (Panis & Lambardi, 2005). The genetic make up of valuable crop species are
reservoirs for high yield potential, which must be protected (Wang et al. 2009). At ultra low
temperatures, seeds can be desiccated and freeze preserved, usually using liquid nitrogen
(-196°C), a practice called ‘cryopreservation’ (Panis & Lambardi, 2005). However, some
species of seed are sensitive to desiccation, termed ‘recalcitrant’, which incidentally also
describes seeds that may have difficulty germinating due to dormancy and possess release
mechanisms that are not fully understood (Walters et al. 2008). Plants that tolerate
desiccation effectively, termed ‘orthodox’, avoid ice crystal formation through synthesizing
substances, such as sugars and proteins, which maintain integrity and allow ‘super-cooling’
to occur. (Panis & Lambardi, 2005). Consequently, for recalcitrant seeds and many others,
a transition of aqueous solution to an amorphous state is required, which can be achieved
using vitrification, a method that relies on a concentrated cellular solution and rapid freezing
rates (Panis & Lambardi, 2005; Walters et al. 2008).
Aims and objectives
The aim of this practice is to deduce the effectiveness of vitrification methods in terms of
their ability to produce explants that are still viable following treatment and freezing. It is of
relevant importance due to current issues regarding food scarcity and sustainability (Wang
et al. 2009). The study will also serve to emphasize the potential beneficial input of plant
biotechnology in valuable sepcies.
Abbreviations
2. Cleo Henderson
10344240
Control at room temperature (CRT), control with liquid nitrogen (CLN), preloaded then liquid
nitrogen (PLLN), direct vitrification then liquid nitrogen (DVLN), preloaded at room
temperature (PLRT), direct vitrification at room temperature (DVRT), Plant vitrification
solution (PVS2).
METHOD
Zea mays var. everta seeds were precultured and grown on solid Murashige and Skoog
(M&S) media, as utilised by Usman & Abdulmalik (2010). Conditions were maintained at
20°C for a 16 hour photoperiod. Six Z. mays seeds were exposed to different treatments,
including controls, some of which using the plant vitrification solution, PVS2. This chemical
was also used by Usman & Abdulmalik (2010) in their methods of cryopreservation with Z.
mays L.. The treatments are displayed in Table 1.
Table 1:
The controls and treatments of individual Z. mays seeds
Seed ID Preloaded with
PVS2
Direct vitrification
with PVS2
Liquid nitrogen
CRT
CLN
PLLN
DVLN
PLRT
DVRT
Preloading
The 2 seeds (PLLN & PLRT) were individually immersed in 20% PSV2 for 30 minutes at
24°C. The 20% PSV2 was then removed and the seeds were immersed in 60% PSV2 and
incubated for 15 minutes on ice. The 60% PSV2 was then removed and the seeds were
immersed in ice cold 100% PSV2. PLLN was kept on ice before being plunged into liquid
nitrogen for 30 minutes.
Direct Vitrification
The 2 seeds (DVLN & DVRT) were individually immersed in ice cold 100% PSV2 for 5
minutes. DVLN was kept on ice before being plunged into liquid nitrogen for 30 minutes.
Thawing
Following 30 minutes of liquid nitrogen immersion, cryo-tubes containing the individual
explants were transferred into a 35°C water bath for 1 minute. The 100% PSV2 was
removed and replaced with 60% PSV2 and explants were immersed for 5 minutes. 60%
PSV2 was replaced with 20% PSV2 and seeds were immersed for 30 minutes at room
temperature.
Surface sterilization
After treatments of PLLN, DVLN, PLRT & DVRT, the explants were surface sterilized in a
70% alcohol wash, then immersed in 10% bleach for 5 minutes, followed by a rinse in
autoclaved distilled water.
Replicates
This method was repeated 10 times. This resulted in 60 explants all together.
Viability testing
After treatments and post treatment procedures were carried out, (excluding CRT) all
explants were plated onto solid M&S media using an aseptic technique. This was to
stimulate germination and assess which seeds were still viable after 7 days following the
3. Cleo Henderson
10344240
treatments. Survival, root growth and shoot growth were all measured when applicable after
7 days.
Data analysis
Analysis and manipulation of the data was conducted using Minitab®
Express (2014) and
Microsoft Excel®
(2011). 2-sample T tests were carried out to indicate statistical significance
in the data. The number of seeds to survive out of the 10 replicates was converted into
percentages before being reproduced in figures. Means were calculated for the root growth
and shoot growth data before being presented graphically.
RESULTS
The results obtained from this study indicate that the treatments involving immersion in
liquid nitrogen were ineffective at reproducing viable explants after 7 days. Recovery was
indicated by the extent of root and shoot growth when applicable (Fig. 2). CLN, PLLN and
DVLN exhibited no recovery and little to no survival 7 days post treatment (Fig. 1 & Fig. 2).
It comes as little surprise therefore that when datasets for treatments with the inclusion of
liquid nitrogen immersion (PLLN & DVLN) were compared to those without (PLRT &
DVRT), there was significance (P =<0.001 for both).
Explants that had direct vitrification and preloading carried out on them without being
immersed in liquid nitrogen survived, with many indicating recovery (Fig 1 & Fig. 2).
However, greater recovery (in terms of both root and shoot growth) was exhibited in the
controls maintained at room temperature than those following treatments with PVS2. In
particular, explants that were preloaded with PVS2 but not subjected to liquid nitrogen
exhibited an average root growth far less than that of the room temperature control (Fig. 2),
a noticeable trend with which further analysis agreed (P= <0.05). Average root growth was
also far less in DVRT than CRT, although this showed no significance (P= >0.05).
0
20
40
60
80
100
120
CLN PLLN DVLN CRT PLRT DVRT
Precentagesurvival(%)
Treatment
Percentage of survival of explants 7 days
post treatments
Figure 1: The
percentages of
survived
explants 7
days post all
treatments
4. Cleo Henderson
10344240
Comparatively, average shoot growth was less impacted by the treatments of direct
vitrification (P=>0.5) and preloading (P=>0.1), exhibiting no statistical significance when
comparing each room temperature treatment with CRT. This was supported by
observations in Fig. 2. There was no significant difference between the two room
temperature treatments (PLRT & DVRT) for both root growth (P=>0.5) and shoot growth
(P=>0.1).
DISCUSSION & CONCLUSIONS
The lack of recovery exhibited in those explants exposed to the liquid nitrogen indicates that
the treatments of direct vitrification with PVS2 treatment and preloading with PVS2 were
ineffective at preserving viability in Z. mays after freezing and thawing. Although it is
already established that there are difficulties in the preservation of recalcitrant species, it
was also suggested that cryogenic applications have been successful with the treatment of
direct vitrification (Panis & Lambardi, 2005). This method prevents crystal formation whilst
avoiding the extreme loss of cellular water. This is because the PVS2 allows the transition
of the cellular solution state to an amorphous one (Panis & Lambardi, 2005). Additionally,
Matsumoto (et al. 1994, 2002 in: Usman & Abdulmalik, 2010) praised its advantages in
terms of the greater recovery of explants. Despite this, the outcomes of this investigation
disagree. It is also believed that immersion in the PVS2 incurred some form of a negative
impact on the recovery of explants, as results indicated that the overall control had the
greatest recovery of all explants. This suggests that if even when direct vitrification with
cryopreservation is successful and produces viable explants post treatment, these explants
may not demonstrate the full growth potential of those undergoing no treatment at all. This
agreed with results produced by Nogueira’s study (et al. 2013), which reported less shoot
growth in sugar cane treated with PVS2, but not immersed in liquid nitrogen, than the
control.
In this investigation, cryopreservation did not produce viable explants, with or without direct
vitrification or preloading. This could be due to the extent to which the species variant was
recalcitrant or orthodox, as it was known to be “semi-recalcitrant”. Further investigation into
characterising the variant used could shed more light on its state of recalcitrant
identification. Following this, the method used could be manipulated. Panis emphasized this
Figure 2: The
average root and
shoot growth of
explants 7 days
post all
treatments
0
5
10
15
20
25
30
35
40
45
50
CLN PLLN DVLN CRT PLRT DVRT
Averagegrowth(mm)
Treatment
Average growth of roots and shoots 7 days post
treatment
Roots
Shoots
5. Cleo Henderson
10344240
(et al. 2005 in: Nogueira et al. 2013), by claiming that depending on the species, the
development of the appropriate technique and protocol could take years to perfect.
In regards to Panis and Lambardi’s needs for vitrification (2005), it was also recommended
that higher cooling rates could be achieved (60°C/sec) by enclosing meristems in semen
straws or using a droplet freezing method, with the use of aluminium foil (130°C/sec).
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(2011) (Version 14.4.1) Microsoft®
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