A cryopreservation protocol for embryos of the endangered species Zizania texana

Abstract

Seeds of the endangered species Zizania texana are recalcitrant, making it difficult to preserve the remaining genetic diversity of this species in genebanks. Excised embryos can be cryopreserved using solution-based cryoprotection protocols. Survival following cryoexposure increased from less than 5% to about 75% by preculturing embryos in high concentrations of sugars, bathing them in cryoprotectant solutions, and partially drying them to water contents of about 0.6 g H2O/g dry mass.

Full text

A CRYOPRESERVATION PROTOCOL FOR EMBRYOS OF
THE ENDANGERED SPECIES ZIZANIA TEXANA
Christina Walters1*, Darren H. Touchell1, Paula Power2,
James Wesley-Smith3 and Michael F. Antolin4
1USDA-ARS National Center for Genetic Resources, 1111 S. Mason Street, Fort Collins,
Colorado, USA
2US Fish and Wildlife Service, San Marcos National Fish Hatchery and Technology Center,
San Marcos, Texas, USA
3 Electron Microscope Unit, Univ of Natal, Durban, South Africa
4Department of Biology, Colorado State University, Fort Collins, Colorado, USA
* for correspondence (email: chrisv@lamar.colostate.edu)
Summary
Seeds of the endangered species Zizania texana are recalcitrant, making it difficult to preserve
the remaining genetic diversity of this species in genebanks. Excised embryos can be
cryopreserved using solution-based cryoprotection protocols. Survival following cryoexposure
increased from less than 5% to about 75% by preculturing embryos in high concentrations of
sugars, bathing them in cryoprotectant solutions, and partially drying them to water contents of
about 0.6 g H2O/g dry mass.
Keywords: cryopreservation, desiccation tolerance, embryo, recalcitrant, vitrification, wildrice,
Zizania
INTRODUCTION
Zizania species are aquatic grasses that produce recalcitrant seeds (13,16,20). Z. texana
grows in a 4 mile stretch of the San Marcos River in Texas and is a Federally-listed endangered
species (2). Preserving Z. texana seeds in ex situ genebanks will forestall the continued erosion
of genetic diversity of this species and provide a wider genetic base for improving its close
relative, Z. palustris, which produces gourmet wildrice.
As with all recalcitrant species, Z. texana cannot be stored in seed genebanks using
conventional protocols, which require drying to about 5% water content and storing at !18°C (5).
Recalcitrant seeds of many species have been successfully cryopreserved by optimizing water
contents (reviewed by 3, also 10,18,19,20) and cooling rates (23) to cryogenic temperatures so
that both freezing and desiccation damage are limited. For unknown reasons, this approach has
not been effective with Z. texana, giving less than 20% survival for seeds dried to 0.2 to 0.4 g/g
and cooled at about 500C/sec (Wesley-Smith et al., unpublished).
In this paper, we report successful cryopreservation of embryos of Z. texana using
solution-based cryoprotection protocols (frequently called “vitrification” because of the glass-
forming properties of the cryoprotectants glycerol and DMSO) more typically used for shoot
apices (e.g., 1,3). These protocols involve incubating tissues in a protective sugar solution,
exposing them to cryoprotectants, and then drying them to appropriate water contents before
CRYOLETTERS 23 (5): 291-298 SEP-OCT 2002

rapidly plunging them into LN (1,3). The optimum combination of sugar, incubation time,
cryoprotectant concentration, and final water content vary among species, cultivars, cell types
and provenance. Solution-based cryoprotection is sometimes used with systems that are
relatively tolerant of desiccation (e.g. 6,8,17), although this type of application is usually
unnecessary since the cells survive drying to water contents where water freezing is not observed
or is not damaging (11,19,20,23).
MATERIALS AND METHODS
Plant material and viability assay
Zizania texana seeds rarely mature in the wild (12). Plants were grown in containers
offsite at Southwest State University, San Marcos, Texas. Seeds were harvested weekly or
biweekly from April through July, packaged in moist paper towels and sent immediately by
courier mail to Fort Collins, Colorado, where they were stored in moist paper at 5°C until used
(within 8-9 months). Seeds from all harvests were pooled before experiments were begun in
September to ensure adequate supply of similar plant material for a complete set of experiments.
Embryos used for cryopreservation studies were excised from fully matured grains, which
were usually green with a hard endosperm, according to maturity classifications developed
previously for Zizania (13,20). Embryos germinated about 16 h after excision. To preclude the
confounding effects of germination, experiments were started within 3 h of excising embryos.
To prevent imbibitional damage in dried embryos, embryos were slowly rehydrated on damp
filter paper before viability assessments. Viability assays consisted of surface-sterilizing embryos
with 1.0% commercial sodium hypochlorite solution for 5 min, washing 3 times in sterile distilled
water, and placing embryos in culture on Linsmaier and Skoog (LS) basal salts, supplemented
with 60 mM sucrose and solidified with 0.8% agar (7). Cultures were incubated at 25 °C in the
dark for 4 d before moving them under cool white fluorescent lights (100 µ Einsteins m-2s-1)
according to Touchell and Walters (18). Embryo survival was scored when the coleoptile
elongated to half the length of the embryo (approx. 4 d). Experiments were conducted within a
4 month period, with assessments of sugars and sugar alcohols conducted simultaneously within
the first 2 months and preculture, PVS2 exposure time and dry time manipulated in the third
month. Water relations studies and survival following the established procedures was verified in
the forth month, and these studies exhausted the seed supply. Five replicates of 10 embryos were
used for each trial.
Drying and exposure to liquid nitrogen
Following application of cryoprotective solutions, embryos were dried to different
moisture contents using “flash drying “ procedures developed previously (23,19,20,21). Excised
embryos were held in a stream of nitrogen gas flowing at approximately 10 liters/min, and
moisture content was manipulated by drying time (0 to 300 min).
Embryos, dried to different water contents, were mounted on ultramicrotomy specimen
holders (Leica, Austria) using a thin film of glycerol as an adherent. Specimen holders with up
to 5 embryos attached were mounted in a compressed-air device, and individually injected into
LN subcooled to a slush (c. !210°C) within 10 s of axes contacting the glycerol solution. Cooling
rates achieved using this procedure were about 250 °C/s as measured by the data recorder
described by Wesley-Smith et al. (23). Embryos remained in LN for 30 min, after which they
were warmed by submerging naked axes into water at 40 °C with vigourous mixing to enhance
warming.
Cryoprotective solutions
Developing the optimum cryoprotective treatment required determining the most effective
sugar or sugar alcohol, and its optimum concentration (0 to 1.2 M). Optimum times of
incubation (0 to 4 d), exposure to a modified plant vitrification solution, PVS2 (14) (0 to 60 min),

0 50 100 150 200 250 300
DRYING TIME (min)
0
0.5
1
1.5
2
WATER CONTENT (g HOH/g dry wt)
after cryoprotection
before cryoprotection
and drying (0 to 60 min) were also assessed. Optimization experiments were done sequentially,
with a 48 hr preculture on a concentrated sugar medium, a 30 min exposure to PVS2 at 0°C, and
a 30 minute drying time used as the default treatment unless that variable was specifically
studied.
The efficacy of various sugars and sugar alcohols in protecting Z. texana embryos during
LN exposure was measured by incubating freshly excised embryos for 48 h on standard culturing
medium (LS salts solidified with 0.8% agar and containing 60 mM sucrose) further supplemented
with 0 to 1.2 M solutions of sorbitol, mannitol, xylitol, ribitol, sucrose, glucose or raffinose. The
water potential of supplemented medium was measured using a thermocouple psychrometer and
varied from –0.45 to –3.85 MPa, depending on the sugar concentration. Embryos were then
exposed to PVS2 (30 min) and partially dried (30 min) (as described in the previous paragraph)
before LN exposure.
Embryos used in experiments to determine the optimum time of preculture, PVS2
exposure and drying were precultured on the standard LS medium further supplemented with 0.8
M sorbitol.
RESULTS
Freshly excised (e.g. no exogenous cryoprotectants) embryos of Z. texana contained about
2.0 ± 0.1 g H2O/g dry mass (= 0.66 g H2O/ g fresh mass). Embryos were dried to water contents
less than 0.3 g/g dm within 150 min of flash drying (Fig 1). Survival decreased abruptly for
embryos dried to water contents less than 0.3 g/g (Fig 2). Embryos showed # 5% survival after
cryogenic exposure, irrespective of partial drying treatments (Fig 2), necessitating the use of
cryoprotective solutions
.
Figure 1. Drying time course
for Z. texana embryos before
and after a cryoprotective
treatment of 48 h preculture in
0.8 M sorbitol followed by 30
min exposure to modified PVS2
solution. Lines are exponential
decay curves fit to time course
data and are used to aid the
eye.
Generalized procedures for solution-based cryoprotection (e.g., 1,3) were applied to
excised Z. texana embryos. All of the steps (preculture with sugars, exposure to a vitrifying
solution, partial drying) were necessary for survival. Preculture for 48 hours on LS medium
supplemented with additional sugar alcohols (Fig 3) or sugars (Fig 4) gave increased survival,
though the concentration giving maximum benefit varied among the different sugars and sugar
alcohols. Raffinose gave the best protection at the lowest concentration (46% survival at 0.2 M),
but was relatively ineffective at higher concentrations. Maximum survival (> 70%) was achieved
using 0.8 and 1.0 M sorbitol. Most sugars and sugar alcohols gave optimum concentrations, with
survival decreasing if concentration exceeded 0.4 M (raffinose), 0.6 M ( mannitol, xylitol,
glucose) or 1.0 M (sucrose, sorbitol).

0 0.5 1 1.5 2
WATER CONTENT (g water/gdw)
0
25
50
75
100
SURVIVAL (%)
LN exposed, +
cryoprotection
no cooling
LN exposed, -
cryoprotection
SUGAR ALCOHOL CONCENTRATION (M)
SURVIVAL (%)
A - sorbitol B - ribitol
D - xylitol
C- mannitol
10.5010.50
100
0
50
0
100
0
50
0
SUGAR ALCOHOL CONCENTRATION (M)
SURVIVAL (%)
A - sorbitol B - ribitol
D - xylitol
C- mannitol
10.5010.50
100
0
50
0
100
0
50
0
Figure 2. Survival of Z. texana
embryos dried to different water
contents (circles) and then
exposed to LN with (open
squares) and without (closed
squares) cryoprotection. Data
for cryoprotected embryos are
replotted from data in Fig 5C
using the time course from Fig
1.
Exposure times for preculturing, bathing in PVS2, and flash-drying were optimized using
embryos precultured on LS medium additionally supplemented with 0.8 M sorbitol (Fig 5).
Survival was highest if embryos were precultured for 1-2 d, with longer times giving poor
survival (Fig 5A). Embryos precultured for 2 d in 0.8 M sorbitol gave highest survival when
bathed in PVS2 for 30 min, with toxic effects becoming evident after longer exposure times (Fig
5B). Partial drying was required for embryo survival following LN exposure, with 30 min drying
giving highest levels (Fig 5C). The cryoprotection procedure lowered the initial water content
of embryos from about 2 to about 1.6 g/g and increased drying rates (Fig 1). Highest survival
was achieved when embryo water contents were reduced to about 0.6 g/g, with further reductions
in water content giving reduced survival (Fig 2).
Figure 3. Survival of Z.
texana embryos following
48 hours preculture in LS
medium further
supplemented with varying
concentrations of sorbitol
(A), ribitol (B), mannitol (C)
and xylitol (D), followed by
30 min exposure to PVS2,
30 min flash drying
(circles), and
cryo-exposure (squares).
The bars represent the
standard error of 5
replicate treatments, each
replicate consisting of 10
embryos.

0
25
50
75
100
0
25
50
75
100
0 0.25 0.5 0.75 1
0
25
50
75
100
SURVIVAL (%)
SUGAR CONCENTRATION (M)
C - glucose
B - raffinose
A - sucrose
Figure 4. Survival of Z. texana embryos
following 48 hours preculture in LS medium
further supplemented with varying
concentrations of sucrose (A), raffinose (B)
and glucose (C), followed by 30 min
exposure to PVS2, 30 min flash drying
(circles), and cryo-exposure (squares). The
bars represent the standard error of 5
replicate treatments, each replicate
consisting of 10 embryos.
DISCUSSION
This paper describes a protocol that can be used to cryopreserve excised embryos of the
endangered species Z. texana. Embryos of Z. texana do not survive drying to water contents less
than 0.3 g/g (Fig 2) and are therefore termed “recalcitrant” because they do not survive standard
storage protocols used in genebanks for orthodox seeds (5). Unlike its congener Z. palustris,
fully mature Z. texana embryos do not survive typical cryopreservation in the absence of
cryoprotectants. Protectant solutions can usually be avoided if cellular water is made unavailable
for freezing by either sufficient drying (reviewed by 3, also 5,18,19,20) or by restricting the time
available for ice crystal growth at higher water contents (23). Exogenous application of
cryoprotectants to excised embryos of Z. texana increased survival following LN exposure from
< 5% to about 70% (Fig 2).
The methodology to cryoprotect Z. texana embryos using exogenous solutions may be
improved further through a better understanding of how the variables of the treatment interact
[i.e. sugar, concentration, preculture time, PVS2 exposure, and drying time] as well as how the
condition and provenance of embryos affect the response to cryoprotecting treatments. For
example, 0.8 M sorbitol was deemed optimum for a 2 day preculture treatment, though it is
possible that a shorter exposure time would reveal that sugars, rather than sugar alcohols, gave
superior performance. Additionally, survival may be further enhanced if embryos were treated
immediately after harvest rather than after a few months storage. Our experiments did not
exclude the possibility that variables interact, but we had a limited number of seeds and so were
unable to carry out all combinations of treatments.

SURVIVAL (%)
0
50
100
TIME (d)
TIME (min)
015 30 7545
0
50
100
C- flash- drying
A- preculture
012 4
3
60
0
50
100 B- PVS2 exposure
SURVIVAL (%)
0
50
100
TIME (d)
TIME (min)
015 30 7545
0
50
100
C- flash- drying
A- preculture
012 4
3
60
0
50
100 B- PVS2 exposure
Figure 5. Effects of various exposure times during
cryoprotective treatments on survival of Z. texana
embryos cooled to liquid nitrogen. In A, embryos are
precultured for 0-4 d on LS medium further
supplemented with 0.8 M sorbitol and then bathed in
PVS2 for 30 min followed by 30 min flash drying. In
B, embryos previously precultured for 2 d in 0.8 M
sorbitol are exposed to a modified PVS2 soution for
0-60 min. In C, embryos, precultured for 2 d in 0.8 M
sorbitol and bathed for 30 min in PVS2, are flashed
dried for 0-60 min. The bars represent the standard
error of 5 replicate treatments, each replicate
consisting of 10 embryos.
Preculturing embryos in nutrient media additionally supplemented with sugars or sugar
alcohols was required for survival of Z. texana embryos exposed to LN (Figs 3-4). Different
sugars gave variable effects on the overall survival achieved, the concentration that gave
maximum survival, and possibly (though not tested) the exposure time that gave maximum
benefit. The mechanism by which sugars protect embryos may provide insights into the
specificity of different sugars, concentrations and kinetics. Sugars and sugar alcohols may play
a direct role in protecting cells from damage (e.g. 15), and so preculture in high concentrations
may be regarded as a period when protectants are loaded into cells. Alternatively, the high
concentrations of sugars and sugar alcohols during the preculture period may affect the metabolic
status of embryos with sugars serving either as substrates or osmotica. In this case, the
concentration and kinetic effects are critical since embryos begin to germinate at higher water
potentials (> !0.8 MPa), suffer from “pathological” metabolism at lower water potentials (< -3
MPa), and produce protective proteins and carbohydrates at intermediate water potentials
(9,21,22). The bimodal effect of water potential on embryo metabolism is consistent with the low
survival of embryos precultured at both low and high sugar concentrations (Rw = !0.45 or !3.9
MPa in media with 0 or 1.2 M sugar added, respectively) (Figs 3-4) or for longer incubation
times (Fig 5A). Endogenous production of protectants may account for the higher survival of
embryos precultured in intermediate sugar concentrations [0.4 to 0.8 M sugar solutions gave !1.4
to !2.5 MPa, with only slight differences observed among sugars (data not shown)]. Whether
sugars are directly protective or induce the production of protectants through an osmotic effect,
the specificity among different sugars and concentrations is probably related to the rate at which
they penetrate into the cytoplasm and are metabolized within cells.
Exposure to PVS2 and partial drying were also necessary for survival of embryos exposed
to LN. Glycerol and DMSO are potent inhibitors of ice formation, as is desiccation which
increases intracellular viscosity (22,23). However, PVS2 can be toxic (4) and prolonged

exposure results in diminished survival (Fig 5B). Also, a comparison of water content versus
survival of embryos before cryoprotecting procedures and following protection and cryoexposure
(Fig 2) shows lower survival at water contents < 0.5 g/g in the treated samples, suggesting that
desiccation damage is exacerbated by exposure to low temperatures (Fig 2). Interestingly,
cryoprotection did not appear to increase the desiccation tolerance of Z. texana embryos though
it clearly increased the rate at which embryos dried (Fig 1).
It is unknown why embryos of Z. texana required exogenous application of
cryoprotectants to survive cryoexposure. The water-content-limits for drying without damage
and for cooling without freezing transitions were coincident at about 0.30 g/g (Fig 2 and 20,
respectively), giving the false impression that water content and cooling rates could be balanced
to minimize both freezing and desiccation damage, as has been achieved in many other species
(reviewed by 3, also 5,18,19,20,23). The experiments presented here provide no insights into
whether unprotected Z. texana embryos were damaged by freezing or desiccation. If damage
resulted from freezing injury, then we hypothesize that the relationship between water content
and intracellular viscosity [which dictates required cooling rates for successful cryoexposure
(23)] differs in Z. texana compared to other recalcitrant embryos that are more amenable to
cryoexposure. Differences in intracellular viscosity among tissues have been reported previously
(11) and were predicted for Z. palustris embryos excised from brown and green seeds based on
calorimetric measurements of water transitions (20). In this case, exogenous cryoprotection
presumably increased intracellular viscosity to levels which prevented freezing damage when
embryos were cooled at 250°C/sec. Alternatively, if damage to cryoexposed Z. texana embryos
resulted from desiccation damage, then we hypothesize that low temperature exposures
exacerbate desiccating stresses in unprotected Z. texana embryos as was demonstrated for
immature Z. palustris embryos (20). In this case, exogenous cryoprotectants either directly
stabilized cell structures or induced metabolism which gave Z. texana embryos a similar cryo-
physiology as mature brown Z. palustris embryos.
CONCLUSION
Embryos of Z. texana survive exposure to liquid nitrogen following cryoprotective
treatments which include 1-2 d preculture in high concentrations of sugar or sugar alcohols, brief
exposure to a cryoprotectant solution and partial drying. Each step in the methodology must be
optimized as over-exposure has detrimental effects. The mechanisms of protection during
cryogenic exposure are unknown, but may be elucidated with additional experiments that explore
the interaction among cryoprotective steps.
Acknowledgments
The authors acknowledge Dr. Kathyrn Kennedy (formerly of US Fish and Wildlife
Service and now with Center for Plant Conservation) for inspiring research on this species.
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