Note: Descriptions are shown in the official language in which they were submitted.
CA 02451941 2003-11-28
DESICCATION-TOLERANT GYMNOSPERM EMBRYOS
RELATED APPLICATIONS
This application is a division of Canadian Patent Application
Serial No. 2, 238, 373, which is itself a division of Canadian Patent
Application Serial No. 2,125,410 filed December 18, 1992. For
convenience, the inventions of both parent applications and of the
present divisional application are described herein; the term
"invention" used in this specification may as the context requires
refer to the invention of the parent or of the earlier division
or of this division, or both.
FIELD OF THE INVENTION
The invention relates to mature viable desiccation-tolerant
gymnosperm embryos, optionally encapsulated.
BACKGROUND OF THE INVENTION
Somatic embryogenesis offers the potential to produce
clonally large numbers of low cost plants of many species . Somatic
embryos develop without the surrounding nutritive tissues and
protective seed coat, so considerable research has been devoted
to causing somatic embryos to functionally mimic seeds with regard
to efficient storage and handling qualities. The development of
techniques for somatic embryogenesis in conifers has greatly
improved the ability to culture conifer tissues in vitro and now
offers the means to propagate clonally commercially valuable
conifers of a number of species. However, all conifer species
suffer from poor plantlet vigor.
It has been suggested to use abscisic acid (ABA) or osmoticum
for enhancing storage levels in plant cells. For
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CA 02451941 2003-11-28
example-, it Was shown that somatic embryos of Theobroma cacao
could be induced to accumulate fatty acids approaching the
composition of commercial cocoa butter by increasing the sucrose
concentration of the culture medium. Modifying the culture
conditions by varying osmotic concentration and/or ABA content
similarly improved lipid accumulation in Brassica _napus L.
somatic and microspore derived embryos as well as somatic embryos
of carrot and celery. Also, the level of storage lipids in P.
abies somatic embryos was improved by optimizing the ABA level
to between 10-20 ~Cm, but the somatic embryos contained about 4%
of the lipid level obtained by zygotic embryos.
Also, Japanese laid-open patent publication No. 1-218520
describes a process for producing plant body regenerative tissue.
The process. includes a step of cultivating a plant body
regenerative tissue in a medium containing ABA and having an
osmotic pressure of 180 to 2500. In order to control the osmotic
pressure within the specific range, a non-toxic substance such
as sugar, alcohol, an amino acid or glycol is added.
Water stress plays an important role in maintaining embryos
in a maturation state (Kermode 1990, Crit. Res. Plant Sci. 9,
155-194). Low water content rather than ABA prevents precocious
germination during later stages of development. This is
important because precocious germination of embryos during
development in seeds would be lethal durirag desiccation.
A conventional way to water stress plant cells grown in
vi tro is to increase the osmotic concentration of the culture
medium through the use of plasmolysing osmotica. For example,
increased concentrations of plasmolysing osmotica such as sucrose
have been used to promote somatic rmbryo maturation of many plant
species. Sucrose at levels up to 6s was found to improve somatic
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CA 02451941 2003-11-28
3.
t. ,
embryo development of some conifers but a smaller increase in
sucrose from 1 to 3% was previously considered optimal for the
maturation of white and Norway spruce somatic embryos. It seems
that a higher concentration generally led to repressed embryo
S development. 3% sucrose is the concentration most commonly used
for conifer somatic embryo maturation. Mannitol had a similar
effect on maturation of conifer somatic embryos (Roberts 1991).
Low levels of mannitol (2-60) led to a doubling of the number of
mature embryos recovered at the end of the maturation period;
l0 however, the treatment could only be applied as a short pulse
(1 week) as prolonged maturation treatment with mannitol became
detrimental to further embryo maturation.
Poor response using sucrose and mannitol or other simple
15 sugars and salts may be because such plasmolysing osmotica are
absorbed by the symplast of plant cells. Such absorption
facilitates adjustment of tissue osmotic potential (osmotic
recovery) without lowering the tissue water content.
Additionally, direct or indirect metabolic effects on specific
20 plant metabolites can occur, due to utilization of the solute or
its toxic effects.
Alternatives to plasmolysing osmotica are non-permeating
high molecular weight compounds such as polyethylene glycol (PEG)
25 or dextran. These compounds are usually available in a wide
range of molecular weights. For example, PEG is available in
molecular weights ranging from 200 to 35,000. However, only
those with a molecular weight above 1000 to 3000 would be non-
permeating (Carpita et al, 1979). This is because the large
30 molecular size of these solutes excludes their passage through
plant cell walls, so preventing entry into cells and plasmolysis,
while still removing water. Consequently, their non-plasmolysing
effect reduces tissue water content in a manner similar to the
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,a
y
effects of water stress observed in cells of plants subjected to
drought conditions. The effect is constant and cell turgor can
only be restored by cells actively increasing their cellular
solute concentrations. PEG has been most commonly used to apply
water stress to whole plants, to osmoti.cally prime whole seeds
to synchronize germination and improve seedling vigor.
Embryo drying occurs naturally in most seeds, and has a role
to play in the developmental transition between maturation and
germination. Thus, desiccation led to enhanced germination of
both zygotic and somatic embryos. Desiccation of whole somatic
embryos is also an alternative method of germplasm storage.
Somatic embryos produced continuously year-round could therefore
be dried and stored until the appropriate planting season, or
shipped .to new locations.
A number of prior publications describe methods for the
desiccation of angiosperm somatic embryos. Senaratna et al., in
EP application 0300730, describe a method through which in vitro
formed plant embryos are desiccated fol~.owing the application of
ABA or other types of environmental stress inducing desiccation
tolerance. The angiosperm embryos are induced at the torpedo
shaped stage with the source of ABA for a sufficient period of
time to cause expression of desiccation tolerance. The induced
embryos are then dried to provide stable, viable artificial
seeds. in EP 0300730, Senaratna et al. emphasize on the
importance of stimulating the embryo at the appropriate stage by
the use of signals to develop tolerance to desiccation. It is
stressed that if the signals are applied at the incorrect stage
of development, the tissue will not respond properly. Angiosperm
embryos can undergo maturation ir~ the absence of ABA and it is
suggested that ABA be supplied as late as possible during the
maturation protocol and applied for a relatively short period of
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CA 02451941 2003-11-28
time. Hence, the timing and duration of ABA application seem to
be critical.
Japanese laid-open patent publication No. 2-31624 discloses
the use of ABA in plant cultures. ABA is used as part of a
process for drying embryos prior to storage.
In published PCT specification No. WO 89/05575, a method for
the production o~ synthetic seeds comprising dehydrated somatic
embryos is described. The method, which is applicable to
monocotyledonous and dicotyledonous embryos, comprises
maintaining the somatic embryos in an atmosphere having a
relative humidity (r.h.) of from about 30 to about 85's for a
period of time sufficient to reduce the moisture content of the
embryos from about 85 to 65o to about 4 to 15a. The use of
osmotically active materials, once the embryos are matured, is
suggested.
Senaratna et al., in 1989, Plant Science, 65; pp. 253-259,
describe the induction of desiccation tolerance in alfalfa
somatic embryos by exogenous application of ABA in the form of
a short pulse. Embryos are then dried to 10 to 15% of their
moisture content and stored for at least 3 weeks in the dry
state. Senaratna et al. also describe a method by which
tolerance to desiccation is induced by exposing the somatic
embryos to sub-lethal levels of low temperature, water, nutrient
or heat stress prior to desiccation. However, it was
demonstrated that some of these stress pre-treatments had
deleterious effects on embryo maturation and seedling vigor.
Hence, the prior literature on somatic embryos and
artificial seeds shows that desiccation tolerance has been
achieved in some angiosperm plant species such as alfalfa,
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CA 02451941 2003-11-28
,s
. 3
geraniums, celery, brassica, carrots, corn, lettuce; orchardgrass
and soybeans. Various methods have been suggested, which all
appear to evolve around promoting desiccation tolerance by
applying ABA and other stresses late in maturation and
subsequently reducing the water content of the embryos. However,
survival following desiccation of conifer somatic embryos has,
at present, not been reported, as these methods are not
applicable to conifers.
The creation of artificial seeds in which somatic embryos
are encapsulated in.a hydrated gel has also been described. The
encapsulated embryos may then be planted using traditional seed
planters. The major drawback of encapsulation in a hydrated gel
is the fact that it allows only limited storage duration. The
following are examples of hydrated gels for encapsulation.
Japanese laid-open patent publication No. 2-46240 discloses
a method by which an oxygen supplying substance is used to coat
a plant embryo. The document also refers to the possible use of
a water-soluble polymeric. substance together with the oxygen
supplying compound. Preferred oxygen supplying compounds a.re
calcium perchlorate or barium perchlorate. The water soluble
polymeric substances referred to are hydrated gels of sodium
alginate, gelatin, mannan, polyacrylamide ,and carboxymethyl
cellulose.
In Japanese laid-open publication No. 63-133904, a method
is described to coat plant embryos and nutrients with a water-
soluble polymeric substance such as alginic acid and
polyacrylamide. Polyethylene glycol is mentioned as an example
of polymeric substance that can be used together with the water-
soluble polymeric substances.
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CA 02451941 2003-11-28
Japanese laid-open patent publication No. 61-40708 describes
a technique through which an embryo is encapsulated with
nutrients, an anti-bacterial agent and a water-soluble polymeric
substance which may include cross-linked polyethylene glycol.
The role of the water-soluble polymer appears to be to keep
moisture during storage of the encapsulated embryo.
In U.S. Patent No. 4,615,141, Janick and Kitto describe a
method for encapsulating asexual plant embryos. Tn this method,
the embryos are pre-treated by increasing the sucrose
concentration of the maintenance medium from normal levels to
high levels, or by applying ABA. The hydrated embryos are then
encapsulated in a hydrated coating material. The coating material
dries to form a thin,. non-toxic film enclosing one or more
embryos, protecting the embryos during storage but readily
redissolving in an aqueous solution. The use of ABA and
increased sucrose is suggested to improve survival of ,the
encapsulated embryos. Once the embryos have been encapsulated,
they are dried at a temperature ranging from 20 to 30°C for a
period of at least 5 hours.
In U.S. Patent No. 4,777,762, Redenbaugh et al. describe a
method for producing desiccated analogs of botanic seeds which
are created by removing a portion of the water by;slow or fast
drying so that the plant tissue is no longer saturated with
water. The method also involves encapsulating meristematic
tissue in a hydrated gel or polymer and removing water by slow
or fast drying. The formation of somatic embryos is induced and
the embryos are then encapsulated in the gel or polymer followed
by drying. Alternatively, the somatic embryos are desiccated to
less than complete tissue saturation during, or at the end of,
embryo development then encapsulated.
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CA 02451941 2003-11-28
' 4'v
a
When the gels described above are used to encapsulate the
somatic embryos either before or after dehydration, preferred
gels are selected from hydrogels or polymers which contain water
within the confines of the gel matrix but which can be dried as
the plant tissue is being desiccated. One of the disadvantages
of such a method is that controlled drying of the encapsulated
embryos is difficult to achieve. In most instances double drying
of embryos is necessary. Thus, desiccated embryos are
encapsulated in the hydrogel, which leads to rehydration, then
the embryos are redesiccated. Recently published data shows that
somatic embryos encapsulated in hydrated gel without desiccation
have a storage life restricted to a few months, even when
refrigerated at above freezing temperatures.
In a 1991 review article concerning somatic embryogenesis
and development of synthetic seed technology (Critical Reviews
in Plant Sciences 10:33-61, 1991), Gray et al. mention that
synthetic seed technology for the forest products industry would
be extremely beneficial. This is because forest conifers can be
propagated economically only from natural seed and since
improvement via conventional breeding is extremely time consuming
due to the long conifer life cycle.
There has been a trend for using increasingly higher
concentrations of ABA to promote the maturation of conifer
somatic embryos. This trend probably results from a need to
inhibit precocious germination which has become more apparent
following the increasingly longer maturation times being used.
Thus ABA was first successfully used by Hakman and von Arnold
1988 (Physiol. Plant. 72:579-587) and von Arnold and Hakman 1988
(J. Plant Physiol. 132:164-169), at 7.6 ~.cM. Dunstan et al. 1988
(Plant Sci. 58:77-84) subsequently found 12 ~M ABA to be better.
Shortly after, Attree et al. 1990 (Can. J. Bot. 68:2583-2589)
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CA 02451941 2003-11-28
,a
reported that 16 ,uM was optimal. Roberts et al. 1990
(Physiologia Plantarum 78; 355-360) have shown that for some
species of spruce, ABA at 30-40 ,uM could be used to promote
maturation and yield mature embryos with storage protein
polypeptides comparable to zygotic embryos. Such high levels
were. necessary to prevent precocious germination and allow
maturation to proceed. Dunstan et a1. 1991 (Plant Sci. 76:219-
228) similarly found that high levels could permit embryo
maturation. Unfortunately, high ABA levels also increased the
frequency of developmentally abnormal embryos. In the above
reports concerning conifers, increased osmoticum was not included
with the ABA.
Conifer somatic embryos appear different to somatic embryos
of monocotyledonous and dicotyledonous angiosperm species in that
ABA should be supplied as early as possible in maturation
protocols in order to promote embryo maturation. Merely reducing
or eliminating auxin and cytokinin levels, as has been successful
for maturation of somatic embryos of many angiosperm species
(Amnirato 1983, Handbook of Plant Cell Culture; Vol. 1, pp. 82
123) led to infrequent or poor maturation in conifer embryos and
mare often resulted in browning and death of the immature somatic
embryos. Furthermore,~it appears that ABA should be applied for
longer periods and at higher levels than generally applied to
angiosperm somatic embryos.
In U.S. Patent No. 5,036,382, Gupta et al. describe a method
for developing tissue culture induced coniferous somatic embryos
into well-developed cotyledonary embryos. The method comprises
a multi-stage culturing process in which early stage embryos are
cultured on a late stage medium comprising a significantly higher
osmotic potential along with ABA and an absorbent material to
gradually reduce the level of available ABA over time. A
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CA 02451941 2003-11-28
critical aspect of this method lies in the inclusion of the
absorbent material in the embryo development medium. Absorbent
materials suggested include activated charcoal and silicates.
The absorbent is used to slowly reduce the ABA and remove
metabolic waste products.
Gupta also suggests the use of osmoticants to control
osmotic potential: A preferred osmoticant suggested is sucrose
in amounts in the range of 2 to 3°s. Another osmoticant that is
suggested by Gupta et al. is PEG. Gupta et al. mention that PEG
8000 was evaluated and found to be a superior osmoticant, stating
that the reasons for its superior performance compared with other
materials is not entirely clear. Gupta et al. also suggest that
polyethylene or polypropylene glycols of other molecular weights
are believed to be equally useful. According to U.S. Patent No.
5,036,007,, the combination of osmoticants is to be modified at
some point during the developmezit stage. In fact; the osmotic
concentration is gradually increased during development.
In U.S. Patents Nos. 4,957,866 and 5,041,832; Gupta et al.
describe a method for reproducing coniferous trees by somatic
embryogenesis using plant tissue culture techniques. The method
consists of placing coniferous somatic embryos in a maturation
medium initially comprising no ABA and a low osmoticant
concentration. ABA is then added and the levels of osmoticant
are raised for the final stage of development. The osmoticants
suggested by Gupta et al. are sugars such as sucrose, myo-
inositol, sorbitol and mannitol.
Tn U.S. Patent No. 5,034,326, Pulman et al. describe a
method for reproducing coniferous plants by somatic embryogenesis
using adsorbent materials in the development stage media. The
adsorbent material (activated charcoal being a preferred
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CA 02451941 2003-11-28
t
embodiment) is used to gradually reduce the concentration of ABA
present in the medium used in the development stage. The purpose
of this reduction in ABA is to follow the natural tendency in
embryo development. As development approaches completion, the
presence of lesser amounts of ABA is required.
In PCT published specification WO 91/01629, Roberts
describes a process for assisting germination of spruce somatic
embryos that comprises partially drying the embryo at humidifies
of less than about 99.9%. Also described is a process to
differentiate somatic embryos of conifers that comprises
contacting embryogenic calli with a medium containing ABA.
Roberts also suggests that a medium having a sucrose
concentration of 2 or 3.4%t., which is used between the maturation
treatments and the germination media, promotes root and shoot
elongation. Roberts mentions that the humidity range that can
be used for partial drying of somatic embryos without lethal
effect is about 85 to 99.9% which results in only a very small
(5-10%) moisture loss.
In a °study published in Can. J. Bot., Vol. 68, 1990,
pp. 1086-1090, Roberts et al. mention that conifer somatic
embryos (interior spruce) do not survive desiccation at room
humidity, but that partial drying at very high humidity promoted
germination up to 90%. Roberts et al. also refer to the fact
that drying embryos over a range of r.h. indicated that r.h. of
810 or lower was lethal to.conifer embryos. This can be further
visualized at Table 3 of the report where the effects of partial
drying at different r.h. on germination are shown. It can be
seen that very small levels of germination are obtained following
drying at a r.h. of 90% and that no germination is observed when
r.h. of 81% and 75% are used. Based on those results, Roberts
concluded that only a mild drying of the somatic embryos was
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CA 02451941 2003-11-28
possible to permit normal germination and that the spruce somatic
embryos did not tolerate desiccation to zygotic levels. Spruce
somatic embryos did survive and undergo improved vigor following
a partial drying treatment in an environment of very high
humidity (over 95% humidity) during which time .only 5% of
moisture was removed.
Later, Roberts et al. {J. Plant Physiol., 138, pp. 1-6,
1991) emphasize that somatic embryos from a number of species,
l0 including spruce, are sensitive to severe water loss and show
decreased survival following desiccation. In this paper, Roberts
shows that Sitka spruce somatic embryos do not survive
desiccation, even though high frequency and synchronized
germination could be obtained following partial drying of the
embryos.
Hence, despite attempts to desiccate conifer somatic embryos
following ABA maturation, survival has not been described.
Desiccation of conifer somatic embryos would be desirable
to enable somatic embryos to be stored for very long periods.
Storage times of desiccated embryos may be further extended by
storing frozen embryos. The ability to suxvive prolonged storage
is important considering the long life cycles of conifers and the
length of time required to evaluate in vitro produced trees.
This would then be an alternative method of germplasm storage,
from which somatic embryos could later be re-induced. Tissues
able to survive freezing in liquid nitrogen are considered to be
capable of survival following storage for indefinite periods.
For nearly all plant species, in vitro techniques are more
costly in comparison to traditional methods of seeding. Somatic
embryos also usually require pre-germination and greenhouse
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,o
4 ,iy
acclimatization prior to planting in the field. To overcome
these problems, several methods have been suggested. Fluid
drilling has been used for pre-germinated seeds. However,
fluid drilling requires new planting techniques, specialized
machinery and does not allow for precision at planting of
embryos or plants.
In conclusion, the prior art would appear to suggest that
currently available techniques have failed in providing strong
conifer somatic embryos and desiccated conifer somatic embryos
suitable for encapsulation. Conifer somatic embryos require
particular plant growth regulator conditions in order to
develop, and do not follow the developmental pattern of the
more advanced angiosperms. Furthermore,. permeating osmotica
have been shown to be detrimental to late embryo stages.
Therefore, applying short-term ABA and osmotic treatments late
in embryo development to .achieve desiccation tolerance is not
feasible for conifers, and other methods are required.
SUNa~IARY OF THE INVENTION
In accordance with the present invention, there is
provided a plant embryo encapsulated in a non=hydrated water
soluble compound having a melting point ranging between 20°C
and 70°C, said coating having been applied to the embryo in
molten form.
The invention may employ a desiccation-tolerant
mature viable gymnosperm somatic embryo characterized in
that it has a moisture content of less than about 55~/wt.
The embryo may have a dry weight and per embryo lipid
content higher than the per embryo- lipid content and
dry weight of its corresponding gymnosperm zygotic embryo.
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CA 02451941 2003-11-28
Preferred desiccated gymnosperm somatic embryos that fall
within the scope of the present invention are characterized by
a moisture content ranging between 10 and. 36o/wt. They may
have a dry weight between 30 and 6000 higher than the
corresponding desiccated zygotic embryo and an amount of
storage lipid between
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CA 02451941 2003-11-28
~r
50 and 700% higher than.this corresponding zygotic embryo. The
desiccated embryos of the present invention may be stored at room
temperature or frozen in the freezer or 'in liquid nitrogen either
before or after encapsulation, encapsulation being optional.
The term "desiccated" is used in different senses in the
somatic embryogenesis technology. In some contexts it can, when
applied in a very general way to gymnosperm somatic embryos,
designate mature gymnosperm somatic embryos that are sufficiently
dried and mature to be viable, i.e., that have good to excellent
chances of survival. In most cases, such embryos may be expected
to have a moisture content that is sir~nificantly lower (at least
about 5% lower) than the moisture content of corresponding mature
gymnosperm zygotic embryos from imbibed seed, the latter being
usually greater than 60%. More specifically, the viable mature
somatic embryos obtained pursuant to the present invention can
be either "mildly" or "severely" desiccated. Mildly desiccated
embryos are characterized-by having a moisture content equal to
or less than about 55%, and by being tolerant of further
desiccation to moisture content levels.belaw 55%, preferably to
at least about the 45% range, and more preferably to at least
about the 36s level. Such embryos that have a moisture content
of about 55% or below and that withstand further desiccation
without loss of viablity are referred to in the appended claims,
and from time, to time in this specification, as "desiccation-
tolerant°'. By contrast, "severely desiccated" embryos prepared
according to the invention are characterized by having either a
moisture content equal to or less than the moisture content of
corresponding zygotic embryos grown from mature seeds, or by
being sufficiently devoid of free unbound water to permit the
embryos to survive freezing. Usual water content levels for
severely desiccated embryos range from about 10% to about 36%.
Desiccated embryos prepared according to the invention having
CA 02451941 2003-11-28
0.r
moisture content values less than about 36% may be classified as
severely desiccated embryos.
Typically in the preparation of viable severely desiccated
embryos according to the invention, one begins with immature
embryos having a relatively high moisture content that are water
stressed under controlled conditions as described in this
specification .to achieve desiccation tolerance. When their
moisture content has been reduced to about the 55% level, such
embryos axe viable and are tolerant to further desiccation to
moisture content levels well below 550. Upon further desiccation
to the severely desiccated stage of about 360 or less moisture
content, such embryos can be.stored for protracted periods, and
will typically germinate successfully and grow normally when
subjected to normal germination conditions.
Also within the scope of the present invention are
encapsulated mature gymnosperm somatic embryos . The embryos are
coated with a non-hydrated water soluble compound having a
melting point ranging between 20 and 70°C. The compound is then
solidified to yield hardened capsules containing the embryo.
This yields coated 'embryos having an enhanced resistance to
attacks from organisms such as fungi and bacteria and animal
pests.
The fact that the water can~ent of the severely desiccated
embryos is reduced to a lower level than that of mature dry seeds
improves embryo quality and long-term starage. In fact, the
water content is sufficiently reduced that the embryos can be
3o stored for extended periods of time in the frozen state without
damage due to ice formation.
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CA 02451941 2003-11-28
sa
Furthermore, reductions in water content allow long-term
storage of germlines without need for complex cryopreservation
equipment, whereby somatic embryogenesis may be recaptured from
stored mature somatic embryos. Also, encapsulation of the
desiccated embryos of the present invention in a non-hydrated
polymer allows for machine handling of the coated embryos as the
polymer coating enhances resistance to shock.
Embryos of the present invention are preferably prepared by
the combined use of a non permeating water stress,agent such as
polyethylene glycol of molecular weight at least about 1,000 and
a plant growth regulator having an influence on embryo
development such as abscisic acid (ABA) and/or analogs,
precursors or derivatives thereof.
This summary is necessarily abbreviated; the full scope of
the invention is as presented in the claims. The present
invention will be more completely understood by referring to the
following description. For convenience, the present product
invention and a companion method invention are described
together, and aspects of both or either may be referred to herein
as "the invention".
IN TFiE DRAWINGS
Figure 1 represents the influence of PEG concentration and
osmotic potential on the number of mature white spruce somatic
embryos recavered per replicate and on the percentage of
maturation frequency.
Figure 2A represents the dry weight of white spruce somatic
embryos directly following maturation in the presence of
different PEG concentrations (all with 16 E,cM ABA).
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CA 02451941 2003-11-28
l ~ ' ~8
Figure 2B represents the moisture content of white spruce
somatic embryos directly following maturation in the presence of
different PEG concentrations (all with 16 uM ABA).
Figure 3A shows shrunken dry somatic embryos immediately
following desiccation for 14 d in an environment of 81% relative
humidity.
Figure 3B shows the somatic embryo photographed after.
2 hours of imbibitian.
Figure 3C shows regenerating plantlets 7 d after rehydration
following desiccation in an environment of 810 relative humidity.
Figure 3D shows an aberrant typical plantlet matured in the
presence of polyethylene glycol (PEG), then germinated for 28
days without a prior desiccation treatment.
Figure 4 shows the high frequency survival of cotyledonary
stage white spruce somatic embryos following 8 weeks on
maturation medium containing 16 ~M ABA and 7.5 PEG, then
desiccated by treatment in low relative humidity (r.h.).
Figure 5 shows three week old white spruce somatic plantlets
regenerated from somatic embryos matured for 8 weeks on
maturation medium containing 16 ~.cM ABA and 7.5°s PEG, then
desiccated by treatment in low r.h.
Figure 6 shows three-week-old white spruce zygotic
seedlings.
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CA 02451941 2003-11-28
Figures 7-14 show sectioned material of white spruce. All
electron micrographs are of cells adjacent to'the shoot apical
meristem.
Figure 7A is a light micrograph showing the shoot apical
meristem (black arrow) and procambial cells (white arrows) of a
mature zygotic embryo dissected from a seed imbibed for 16 hours.
Figure 7B is an electron micrograph of cells in the zygotic
embryo shown in Figure 7A.
Figure 8 is an electron micrograph of cells in a zygotic
embryo dissected from a seed imbibed for 65 hours.
Figure 9 is an electron micrograph of cells in a non
desiccated somatic embryo immediately following maturation for
8 weeks with 1.6 uM ABA and 7.5% PEG.
Figure 10A is a light micrograph showing a median section
through the shoot apical meristem of a 2 h imbibed somatic embryo
following maturation for 8 weeks with 16 ~M ABA and 7.5% PEG,
then desiccation by treatment in low r.h.
Figure 10B is an electron micrograph of cells in the somatic
embryo of Figure 10A.
Figure 11A is a light micrograph showing a median section
through a shoot apical meristem of a somatic embryo matured for
4 weeks with l6 ~M ABA but without PEG.
Figure 11B is an electron micrograph of cells in the somatic
embryo of Figure 11A.
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CA 02451941 2003-11-28
a
9
Figure 12A is a light micrograph showing a median section
through a shoot apical meristem of a somatic embryo matured for
4 weeks with 16 ~M ABA and 7.5°s PEG.
Figure 12B is an electron micrograph of cells in the somatic
embryo of Figure 12A.
Figure 13A is a light micrograph'showing a median section
through the shoot apical meristem of a 4 week old zygotic
seedling grown from an isolated embryo.
Figure 13B is an electron micrograph of cells in the zygotic
seedling of Figure 13A.
Figure 14A is' a light micrograph showing a median section
through the shoot apical meristem (large arrow) of a 4 week old
somatic plantlet regenerated from a somatic embryo matured for
8 weeks.with 16 ~,cM ABA and 7.5s PEG, then desiccated.
Figure 14B is an electron micrograph of cells in the somatic
plantlet of Figure 14A.
Figure 15 represents the desiccation tolerance of white
spruce somatic embryos as a function of maturation time.
DETAILED DESCRIPTION OF THE INVENTION
The inventions of this application and a companion
application relate to desiccated mature gymnosperm somatic
embryos and to a method for producing desiccated mature
gymnosperm somatic embryos. The preferred method generally
comprises developing immature somatic embryos in a medium
comprising at least one non-permeating water stress agent, a
_ 1g _
CA 02451941 2003-11-28
,a
metabolizable carbon source and a plant growth regulator such as
ABA and/or analogs, precursors or derivatives thereof for a
period of time sufficient to yield mildly desiccated mature
viable somatic embryos tolerant to further desiccation and having
a moisture content ranging between 32 and 55o/wt, preferably
between 35 and 45%/wt. '
If it is desired to obtain further desiccation of the
somatic embryos, as for storage, the desiccated mature somatic
embryos obtained previously are submitted to continuing
desiccation treatment involving further water stress, which can
be, for example, either osmotic stress or a controlled
environment such as one having a low r.h. for a period of time
sufficient to yield severely desiccated somatic embryos having
a moisture content ranging between about 10 and about 36o/wt.
The resulting desiccated somatic embryos can then optionally be
coated in a non-hydrated water-soluble compound and stored either
frozen or at room temperature.
. There is a striking.similarity in design of embryos of all
gymnosperm species. Indeed, there is a basal plan of embryo
development which is more or less common to all gymnosperms. At
first; there is a free nuclear phase of ~crarying derivation. Then
there is a wall formation followed by the organization of two
tiers of which the upper remains open toward the archegonium.
After this usually the cells of the latter divide once more
resulting in the upper tier which again remains open and the
middle tier which functions as the suspensor. Somatic
embryogenesis involves a reactivation of much of the development
program of normal embryogeny, and to date, the same range of
conditions found to promote induction, proliferation and
maturation of white spruce are the same as all other conifers and
are distinct from the methods developed for angiosperms. Zygotic
20 -
CA 02451941 2003-11-28
embryos of all gymnosperms, the group to which conifers belong;
display similarity in their mode of development, which is unique
from all other plant groups, particularly the angiosperms.
Hence, although the following description refers specifically to
methods used to produce desiccated conifer somatic embryos, it
is to be understood that these methods have a broader field of
application which includes all gymnosperms.
The present invention requires the understanding and control
of certain critical factors such as the concentration of ABA and
the nature and concentration of the non-permeating water stress
agent used in the development of the mildly desiccated mature
embryo, the environment and method by which the mildly desiccated
mature somatic embryos can be further desiccated and the method
by which the desiccated somatic embryos are subsequently
encapsulated. Each of these aspects will be discussed separately
along with more detailed considerations on the maturation and
desiccation methods.
.Abscisic acid
The period during which abscisic acid is supplied in the
development stage varies according to plant species. For
example, as immature conifer somatic embryos do not develop into
functional mature embryos on hormone-free medium, ABA must be
supplied at least at the beginning of the maturation period even
if the application of ABA can be interrupted for a portion of the
development. Preferably, ABA should be initially present in the
medium in sufficient concentration so as to have a ffinal
concentration of ABA of at least O.1 ,uM at the end of the period
during which the embryos are developed. The presence of high
levels of ABA throughout most of the maturation period maximizes
development whsle limiting precocious germination. It is
- 2T
CA 02451941 2003-11-28
generally preferred that a substantially constant ABA
concentration be maintained during the majority of the maturation
period, but levels may be gradually raised and lowered at the
start or end of maturation, particularly if a bioreactor is used
as a culture vessel.
Maturation of the somatic embryos can be initiated
immediately following transfer from induction or multiplication
medium to maturation medium but maturation may be improved if
immature embryos are precultured on reduced growth regulator, or
growth regulator free multiplication medium, or multiplication
medium containing reduced auxin or cytokinin alone: Additional
details on preculturing of the embryos is provided further below.
Development of the embryos takes place for a period of time
usually ranging from 1 to 15 weeks. Commercial ABA consists of
racemic forms but only the (+) form is effective in promoting
maturation. The concentrations of (+) ABA that can be used
during development, whether for maturation purposes or ultimately
for desiccation purposes, range from 0.1 to 100 ~.iM.
Concentrations of (~) ABA between 12 ~cM to 60 ACM are preferred.
Optimal results are obtained when maturing conifer somatic
embryos for 6 to 8 weeks on a medium containing 16-24 ,uM (~) ABA.
Non-permeating water stress agent
As mentioned previously, two types of osmotic stress can be
applied to plant cells. The first type is a permeating osmotic
stress usually induced by low molecular weight compounds such as
sucrose or mannitol. In this instance the permeating osmotic
agent crosses the cell wall and causes water to exit from the
symplast (cell cytoplasm) by osmosis. However, the permeating
agent is free to enter the symplast of the cell. Over time,
- 22 -
CA 02451941 2003-11-28
sufficient permeating agent may enter to alter the cells osmotic
potential which leads to water then reentering the cell by
osmosis. Thus, tissue water contents are not necessarily lowered
during prolonged incubation with permeating osmotica.
Furthermore, the internalized osmotica may directly or indirectly
affect cellular metabolism. For example, simple sugars and salts
may be absorbed and utilized by the plants cells, resulting in
nutritional or osmotic adjustment. Toxic effects on metabolism
may also result.
In the case of a non-permeating osmotic stress, compounds
such as PEGs or dextrans should have a sufficiently. high
molecular weight to avoid penetration of the agent through the
matrix of the cell wall. Non-permeating osmotica similarly
remove water from the cell by osmosis, however, the osmotic agent
is not free to enter the cytoplasm. The effects are therefore
long lasting and simulates a non-osmotic (e. g., drought) stress
at the cellular level. When non-penetrating or less readily
penetrating solutes are used, the more negative osmotic potential
of the external medium due to_these..solutes can only be counter-
balanced by tissue dehydration, or active uptake of other
external solutes and the biosynthesis of organic osmotica. The
latter may then be converted to stored product.
As the diameter of pores in the walls of living plant cells
through which molecules can freely pass has been determined by
a solute exclusion technique to be between 30 and 45 angstroms,
depending on plant species and type of tissue (e. g. root or leaf,
etc.), it seems that molecules with diameters larger than these
pores would be restricted in their ability to penetrate such a
cell wall. It would therefore~appear that molecules having a
diameter above 30 angstroms could be used either alone or in
combination with other types of osmotica to induce a non-
- 23 -
CA 02451941 2003-11-28
permeating water stress in conifer somatic embryos. Polyethylene
glycols having a molecular weight above 1000 and dextrans having
a molecular weight above 4000 are preferred non-permeating water
stress agents, although it is to be understood that the present
invention is not to be restricted to the use of these products.
In fact, the class of solutes or compounds that could be used to
induce a non-permeating water stress could include any water
soluble high molecular weight compound having a molecular size
above 30 angstroms. Suitable alternatives include but are not
restricted to: complex carbohydrates such as celluloses, pec.tins,
galactans, polysucrose such as that sold under the trademark
Ficoll, polypropylene glycols, agars, gums and oligosaccharides
as well as proteins, amino acids (especially polyamino acids),
lipoproteins, nucleotides, oligonucleotides and
lipopolysaccharides.
The use of non-permeating solutes to cause non-osmotic
moisture stress in whole soil grown plants to compensate salt
effects or to effect osmotic priming of seeds has been widely
documented. High molecular.. weight compounds have also been
suggested as components in hydrated gels to encapsulate
previously desiccated meristematic tissue, somatic embryos or
tissue cultured plants. However; the specific use of non-
permeating solutes such as PEGS or dextrans in combination with
ABA- for the purpose of reducing moisture contents, enhancing
maturation and ultimately permitting severe desiccation of
somatic embryos is described for the first time in the context
of the present invention.
In the context of the present invention, concentrations of
non-permeating compounds ranging between 1 and of 30o/wt have
been found useful to promote embryo maturation. The use of
polyethylene glycol (PEG) having a molecular weight ranging
- 24 -
CA 02451941 2003-11-28
~ °
between 1000 and 35,000, preferably PEG having a molecular weight
ranging between 3500 and 10,000 and most preferably PEG 4000 to
8000 in concentrations of 1 to 30o/wt is preferred. Most
preferred PEG 4000 and PEG 8000 concentrations are in the range
of 2 to 15.0~/wt. A 7.5%/wt concentration of PEG 4000 led to a
threefold increase in maturation frequency when compared to
controls and was optimal for storage reserve accumulation.
It was observed with PEG that the higher molecular weight
varieties needed to be applied in greater amounts to achieve
comparable 'osmotic potentials than lower molecular weight
varieties. Thua, very high molecular weight PEGS can occupy a
greater proportion of the medium which prevents gelling of the
medium. When culturing embryos on agar gelled media, PEG 4000
is preferred for applying a non-permeating water stress while
enabling gelling of the medium at appropriate concentrations.
A continuous flow bioreactor enables a greater range of high
molecular weight compounds to be used.
20~ With regard to dextran, dextran with a molecular weight up
to 80,000 has been found suitable with a molecular weight above
6000 being preferred. Dextrans should generally be present in
the medium in amounts ranging between 1 and 30%/wt, with 5 to
20%/wt being preferred and l0o/wt being most preferred.
It is required to use a non-permeating compound in such a
concentration as to reach the desired osmotic potential in the
medium. Generally, the osmotic potential of the medium can vary
between -0.3 and -2.0 MPa, with -0.6 to -1.0 MPa being preferred.
- 25 -
CA 02451941 2003-11-28
~a
Processfor the Production of desiccated somatic embryos
Preculturinq of somatic embr5ros
Pre-culture with multiplication medium containing cytokinin
as the sole growth regulator, then transfer to ABA containing
maturation medium, promoted maturation of somatic embryos from
lines previously found recalcitrant to standard ABA maturation
treatments. Similarly, the inclusion of cytokinin with ABA
during the first few weeks of maturation, prior to transfer to
maturation medium contain ABA alone, prevented precocious
germination during the maturation phase. This resulted in
improved maturation frequencies, and also led to the recovery of
mature embryos from cell lines previously found to be
recalcitrant to maturation treatments.
Investigations of white spruce indicate that preculture for
one week in plant growth regulator free liquid multiplication
medium promoted subsequent maturation frequencies, frequently
doubling the recovery' of mature embryos. Subsequent embryo
development on ABA containing maturation medium was also faster,
as early cotyledonary stage embryos were evident up toga week
sooner than ABA cultures given no pretreatment. This optimum
duration of the pre-culture period seemed to vary with age of the
suspension culture. Thus, a newly established suspension culture
( < 1-3 months) following cryostorage, benefitted from a plant
growth regulator free culture of 1-3 days while a one-week pre-
culture led to reduced recovery of mature embryos. However, the
same cell line recovered frorr~ cryostorage 18 months earlier,
required a pre-culture of at least 1 week for optimal maturation.
The best and most consistent method of pre-culturing was to
reduce by at least 1/10 the auxin from the multiplication medium
for a 1 week period, prior to plating on medium containing ABA
and non permeating osmoticum. An alternative method is to reduce
- 26 -
CA 02451941 2003-11-28
,a
all growth regulator levels without eliminating them entirely.
These pre-treatments, plus subsequent ABA/moisture stress had a
synergistic effect on somatic embryo maturation frequencies,
which were at least doubled compared to no pretreatment.
Thus, even though the pre-culturing of the.somatic embryos
prior to desiccation remains optional in the process of the
present invention, it may, in some 'instances, be useful to
enhance maturation of selected cell lines which are not as
to responsive as others to direct ABA maturation.
Maturation and mild desiccation of somatic embr~ros
Desiccated mature somatic embryos are obtained by developing
immature somatic embryos in a medium comprising at least one non-
permeating water stress agent, a metabolizable carbon source such
as sucrose and ABA and/or analogs, -precursors~or derivatives
thereof for a period ranging from 1 to Z5 weeks, with 4 to 10
weeks being preferred and 6 to 8 weeks being most preferred. As
mentioned previously, the concentration of (+) or (-) ABA used
during the maturation process may range from 0.1 to 100 ~.cM but
(~) ABA should preferably range from 12 to 60 E.cM. With regard
to the non-permeating water stress agent, PEG 4000 to 8000 is
preferred in concentrations of l to 30o/wt, with a 1-15S/wt
concentration being preferred. It is important to mention that
the temperature at which maturation is effected can influence the
time required to complete maturation. The process is especially
suitable for maturing conifer somatic embryos.
Characteristics of mildly desiccated mature somatic embryos
The somatic embryos obtained by the process described above
are characterized by having a moisture. content ranging between
- 27 -
CA 02451941 2003-11-28
a
32 and 55%/wt, preferably between 35 and 45%/wt and a total per
embryo lipid content and dry weight which are higher than the per
embryo lipid content and dry weight of corresponding zygotic
embryos. In fact, the weight of total lipid and triacylglycerols
(TAG) per embryo can be up to 5 times higher than in
corresponding zygotic embryos.
In the case of mildly desiccated mature conifer somatic
embryos, the moisture content usually ranges between 32 and
55o/wt, with a moisture content between 35 and 45o/wt being
preferred. With regard to TAG, they can be present in amounts
ranging between 70 and 350 ~,cM, with 70 to 150 ~.cM being usually
obtained.
Dry weights of conifer somatic embryos usually varies
between 0.2 and 1.5 mg, with 0.2 to 0.8 mg being usually
obtained. Preferred conifer somatic embryos are those from the
family Pinaceae.
For example,. when comparing white spruce somatic embryos
matured with 7.5% PEG and 16 ~M AHA for 4-8 weeks to
corresponding zygotic embryos, both have similar TAG fatty acid,
and storage polypeptide profiles, similar structure and similar
desiccated and imbibed moisture contents. However, somatic
embryos after just 4 weeks maturation are larger as demonstrated
by their greater dry weights, and by the 8th week of maturation
contain considerably more storage reserves such as lipids. Thus,
by 6 weeks levels of TAG per embryo have almost doubled compared
to zygotic embryos, and by 8 weeks levels have at least tripled,
as shown in Table 1 below. Secondary desiccation treatments to
achieve lower moisture contents may increase values further.
TABLE 1
- 28 -
CA 02451941 2003-11-28
v
Embryo type Dry wt TAG
mg/embryo ~g/embryo
Somatic
maturation time
(weeks )
4 0.27 36
6 0.4 72
8 0.7 143
Zvgotic 0.15 44
Severe desiccation of mature somatic embryos to low moisture
contents
The method for desiccating somatic embryos provided by the
present invention is carried out in two major steps. The first
step consists in reducing the water content of immature somatic
embryos during their development by maturing these embryos in a
medium comprising at least one non-permeating water stress agent,
a metabolizable carbon source and ABA and/or analogs, precursors
or derivatives thereof for a period of time sufficient to yield
mildly desiccated mature somatic embryos having a moisture
content ranging between 32 and 55%/wt, preferably between 35 and
45o/wt. The second step consists of a late stage desiccation
process. The mildly desiccated mature somatic embryos are then
submitted to a secondary desiccation treatment which involves
either submitting the embryos to further osmotic stress or to ut
least one environment having a low r.h. to yield severely
desiccated somatic embryos having a final moisture content
ranging between l0 and 36%/wt, with a 20 to 30o/wt moisture
content being the most preferred range.
- 29 -
CA 02451941 2003-11-28
t
1° Reduction of the water content and increase of storage
reserves of immature somatic embryos.
In orderto successfully achieve severe desiccation to low
moisture contents of mature somatic embryos and particularly
conifer somatic embryos, it is necessary to first reduce the
moisture content of the embryos during maturation to a percentage
between 32 and 55%/wt, ideally between 35 and 45%/wt. Reducing
the water content of the embryos during maturation leads to
enhanced tolerance to further severe desiccation for the
following reasons. Tolerance to desiccation to low moisture
contents appears to be closely related to the level of storage
reserves. Treatments that promote storage reserve accumulation,
such as ABA, non-plasmolysing moisture stress, and increased
maturation time, also promote desiccation tolerance. This is
because vacuolate cells containing little reserve material~may
undergo mechanical disruption and tearing of membranes during
severe water loss, while the presence of sufficient reserves
limits such changes. Additionally precocious germination is
inhibited which further enhances severe desiccation tolerance.
Treatment of the embryos with a non-permeating water stress
agent improves the maturation frequencies of the embryos. The
promotive effect is considered to be a consequence of the induced
non-plasmolysing water stress. As will be demonstrated later,
non-permeating water stress agents such as high molecular weight
PEGs and dextrans, when used in appropriate concentrations, that
is generally in concentrations of 1 to 30%/wt, stimulate
substantial increases in maturation frequency when compared to
controls. In fact , in one of the preferred features of the
present invention, 5 to 7.5% PEG 4000 or 10% dextran 80,000
stimulated a threefold increase in maturation frequency of
conifer somatic embryos compared with controls.
- 30 -
CA 02451941 2003-11-28
The moisture content of mature severely desiccated somatic
embryos treated by the process of the present invention is
similar to that of mature zygotic embryos. However, regenerated
plantlets from non-permeating water stress treated then severely
desiccated somatic embryos are of better quality than the non-
osmotically treated controls. A possible reason for this is that
somatic embryos, matured in the absence of water stress agents,
germinate precociously in the first few days of secondary stage
desiccation, while moisture contents are still high. It is
probable that in these instances subsequent survival of tissues
such as root and/or shoot meristems, hypocotyl and cotyledons in
somatic embryos was non-uniform, leading to irregular plantlets.
By comparison, somatic embryos matured in the presence of
non-permeating water stress agents had a lower moisture content
and were therefore already considerably ~drier~ prior to further
severe desiccation. These embryos remain quiescent following
transfer from the ABA medium, and desiccation of each embryo is
more uniform, thereby improving plantlet quality. However,
somatic embryos remain quiescent. under low osmotic conditions
only when ABA is present. Thus, for conifer somatic embryos; a
combination of both ABA and non-permeating water stress agent is
more effective in promoting maturation and preventing precocious
germination than when ABA and, the non-permeating water stress
agent are taken individually. There seems to be a synergistic
effect occurring when ABA, and PEG are used concurrently.
During prolonged maturation (e.g. 8 weeks maturation) the
non-permeating water stress becomes increasingly important in
preventing precocious germination, which improves survival
following further desiccation. Precocious germination is
especially evident for treatments with low ABA concentration; and
low water stress, and during secondary desiccation treatments,
- 31 -
CA 02451941 2003-11-28
' .9.
leading to limited or no survival for all of these treatments.
After 8 weeks maturation, the increased tendency for precocious
germination during prolonged maturation treatments may be because
somatic embryos undergo a reduction in ABA sensitivity during
S maturation. It was also observed that a reduction in tolerance
to rapid severe desiccation (e. g. on the lab bench) occurs late
in maturation (i.e. 8 weeks maturation) which corresponds with
the time at which somatic embryos display a greater tendency for
precocious germination.
Both ABA and asmoticum promote the accumulation of storage
reserves in embi~yos. The trend of increasing dry weight and
decreasing moisture content of osmotically treated white spruce
somatic embryos indicates that storage reserves are deposited
within cells while water is displaced. As mentioned previously,
the osmotically treated somatic embryos accumulate more storage
reserves (e.g. proteins and lipids) when compared to the
untreated controls.
Embryos of many plant species germinate normally only if
desiccated first, suggesting activation of new genes. In the
case of conifer somatic embryos, it has been shown that further
severe desiccation of somatic embryos is necessary to promote
subsequent plaritlet development only when the somatic embryos are
matured using elevated osmotic concentrations. Embryos matured
under low osmotic conditions subsequently develop without the
need for further desiccation, but show a tendency towards
precocious germination.
The effect of PEG concentration on osmotic potential, is
different from that of solutions of permeating water stress
agents such as salts and sugars . For instance, a negatively
curvilinear decrease in osmotic potential, occurs with increasing
- 32 -
CA 02451941 2003-11-28
PEG concentration and is apparently related to structural changes
in the PEG polymer. The application of sucrose at similar
osmotic potentials to 5.0-7.5% PEG 4000 does not readily promote
the maturation of conifer somatic embryos, possibly because'
absorption of this solute by the tissues leads to an altered
metabolism.
Thus, the application of a non-permeating water stress agent
to the maturation medium leads to somatic embryos that resemble
zygotic counterparts, in terms of low moisture content, and high
degree of quiescence. In addition, the non-permeating water
stress agent stimulates maturation frequencies, and improves
storage product. accumulation.
In order to maximize water loss (mild desiccation) and
maturation during the development stage. of the somatic'embryos,
various experiments have been set up to observe the effects of
different culture conditions on maturation and water loss. It
seems that the embryo should be maintained for a minimal period
2o of 1 week and.a maximal period of 15 weeks, preferably for 4 to
8 weeks and most preferably for 6 to 8 weeks on a medium
containing preferably between 12 and 60 ~.cM ABA and preferably
between 1 and 30°s/wt of non-permeating water stress agent. The
concentration of the non-permeating water stress agent may vary
depending upon its nature.
For example, in the case of PEG, having an average molecular
weight of 4000, concentrations of 7.5~ with an osmotic potential
of -0.7 MPa were determined to be optimal for maturation on agar
gelled medium. Once the desired water content has been achieved
through maturation of the somatic embryos, severe desiccation is
effected to further reduce moisture levels, thereby enhancing
- 3f -
CA 02451941 2003-11-28
long term storage, enhancing resistance of the embryos to frost
damages and improving subsequent plantlet vigor.
Maturation of somatic embryos using a continuous-flow ' solid-
s support bioreactor
Experiments to date usually involve maturation of somatic
embryos on semi-solidified medium, or on supports over liquid
media, within.petri dishes. This is labor-intensive for large
l0 scale propagation, particularly when frequent media changes are
required. Recovery of mature embryos directly from submerged
liquid suspensions permits easier handling of large quantities
of material; however, to date, there have been no reports of
successful maturation in submerged culture. It seems that in all
15 reports where embryos are submerged in liquid or agar, or merely
enclosed within surrounding embryogenic tissues, maturation is
inhibited. Conifer somatic embryos have been cultured in liquid
suspensions during initial maturation stages, but they then
required transfer onto solid supports over media to complete the
20 maturation process. Thus, it is likely that good gas transfer
to and from the developing embryos, in addition to a moisture
stressing environment, is important. These parameters are,
however, difficult to achieve in a liquid environment. An
alterna-tive method for yielding large numbers of mature conifer
25 embryos requiring minimal handling, is the use of continuous-flow
solid-support bioreactors. Such a system was described in
JP 87123756, hereby incorporated by reference. The bioreactor
comprises a culture chamber having medium inlet and outlet means
for continuous supply of fresh medium in. the culture chamber.
30 It also comprises a support for maintaining the immature embryos
above the surface of the medium and means for providing and
controlling air flow in the chamber. Thus, conifer somatic
embryos may be matured within a large chamber supported above
- 34 -
CA 02451941 2003-11-28
. >
liquid medium. Fresh liquid culture medium is pumped into one
end of the vessel, while spent medium exits from the opposite
end. Also, growth regulator and osmotic changes can be applied
gradually as the liquid medium is added, and the air space in the
chamber may also be controlled to provide the optimal gaseous
environment. The large culture chamber enables large numbers of
somatic embryos to be cultured per run, which reduces the costs
of using petri dishes.
2° Secondary desiccation treatment of mature somatic embryos
to low moisture contents.
It was initially believed that the desiccation tolerance of
somatic embryos to severe desiccation to low moisture content,
particularly conifer somatic embryos, was influenced by the rate
of desiccation. Hence, it was thought that slow desiccation
rates increased survival under all osmotic treatments, especially
for incompletely matured embryos. I~owever, it has been
demonstrated that optimally matured conifer embryos obtained
2C according to the method of the present invention can be
desiccated further either using rapid or slow drying., Other
desiccation methods using controlled humidity cabinets providing
air circulation can also be employed and when doing so, the
treatment times outlined below may vary. Severe desiccation can' w
also be achieved by prolonging exposure of .the embryos to high
concentrations of osmoticum. When used herein, the term ~~low
moisture content~~,is intended to designate somatic embryos having
a moisture content ranging- between 10 and 36% following
maturation and subsequent desiccation.
a) Gradual secondary desiccation treatment
- 35 -
CA 02451941 2003-11-28
z
Embryos are matured on filter paper supports. Gradual
desiccation of the embryos to low moisture contents may be
accomplished by transferring mature somatic embryos on their
filter paper supports through a series of environments of
progressively lower r.h. This technique is described by
Senaratna et al. in 1989, Plant Science 65, 253-259 which is
hereby incorporated by reference. It was initially believed that
a gradual water loss allowed sufficient time for the protective
changes to occur in cells and hence increased the embryos'
resistance to severe dehydration. Further investigations have
shown that gradual desiccation is not an absolute requirement
even though the technique can be successfully used.
The rate at which gradual desiccation is to be conducted may
vary substantially. For example, embryos on moist filter paper
supports placed in 81s r. h. chambers usually cause an initial
increase in r.h. The r.h. then declines over the next few days
to the desired value, thereby producing a very gradual
desiccation. If desiccation at a lower r.h. is desired, the rate
at which cultures should be transferred to successively lower
relative humidity environments may vary substantially, but
generally speaking, the matured embryos should be transferred
successively to lower r.h. desiccators at 1 to 7 day intervals.
The time left at the final required humidity depends on. the rate
at which the embryos were previously transferred to the lower
r.h. Hence, the cultures can be maintained for a minimum of 1
to 7 days at the final required r.h. It is to be noted that r.h.
can range between 30 and 95o at a temperature ranging from 20 to
30°C. Total secondary desiccation treatment times can range
between 7 and 21 days.
The r.h. can be visually checked within the desiccation
chambers by meter. Following stabilization of the meter, a
- 36 -
CA 02451941 2003-11-28
. .
period of I to 7 days is allowed at the desired r.h. A visual
inspection of the embryos can readily confirm that they are
desiccated to low moisture contents as they change from swollen
embryos of a pale cream colour, to a shrunken and distorted
outline and a yellowish, waxy translucent appearance.
b~ Rapid secondary desiccation
Experiments have demonstrated that conifer somatic embryos
survive slow secondary desiccation at high frequency, preferably
when retained with the callus upon the filter paper support.
Those removed from the callus and placed horizontally upon the
support led to recovered plantlets that were abnormal (the
embryos did not elongate normally, but remained stunted). It
seems that during slow secondary desiccation treatment, conifer
somatic embryos need to be retained within the whole callus in
order to subsequently develop normally. Somatic embryos can also
survive rapid secondary desiccation which may, in some instances,
be more practical than gradual secondary desiccation:
, In the case of rapid secondary drying, the technique
involves an ambient r.h. ranging between 5 and 95%. An ambient
r.h. ranging between 20 and 63% at an ambient temperature ranging
between 20 and 25~C is preferred. An ambient r:h. ranging
between 30 and 40%, at a temperature of 25°C has been found to
be suitable. Matured somatic embryos from mild desiccation
treatments retained upon filter paper supports and submitted to
rapid secondary drying usually desiccate to low water contents
within a period of time ranging from 24 to 48 hours but should
be maintained at ambient r.h. for a period of at least 3 days,
which can extend to 1 week or more if .prolonged storage is
desired. The tendency seems to be that somatic embryos must be
matured in mild desiccation treatment for at least 6 to 8 weeks
- 37 -
CA 02451941 2003-11-28
in order to survive rapid secondary drying. This will be
demonstrated in further detail later on.
characteristics of low moisture content severely desiccated
somatic embryos
First, severely desiccated somatic embryos exhibit a
moisture content that is lower than the moisture content of
corresponding zygotic embryos from dried seed. Hence, the
moisture content of desiccated somatic embryos obtained according
to the present invention usually ranges between 10 and 36%/wt%.
In fact, the important moisture content that removes all free
water is that which permits freezing without injury, that is
preferably below about 36%. The level of desiccation achieved
depends on the method of secondary desiccation used. Bench dried
embryos may have much lower moisture, preferably between 10 and
30/wt%, depending on ambient r.h. and temperature. Furthermore,
the dry weight of conifer somatic embryos following secondary
desiccationvis usually 30 to 600% higher than the weight of
corresponding zygotic embryos. Also, the amount of storage
lipids found in severely desiccated somatic embryos is 50 to 700%
higher than that of corresponding zygotic embryos while
demonstrating fatty acids and polypeptide storage reserves which
are similar to~those of corresponding zygotic embryos. Also, the
25, secondary desiccated somatic embryos have large protein bodies
as well as abundant lipid bodies.
Freezina tolerance of severel~r desicr~ted embryos
An analogy exists between tolerance to desiccation and
tolerance to freezing. Tissues able to survive freezing in
liquid nitrogen are considered to be capable of survival
following storage for indefinite periods. For example, somatic
- 38 -
CA 02451941 2003-11-28
embryos matured for 8 weeks with 7.5% PEG and 16 ~,cM ABA-were
placed in 81% or 63% r.h. environments to achieve severe
desiccation. Somatic embryos from the 63% environment were first
given 1 week at 81%. Total secondary desiccation treatment times
were 2-3 weeks. Following these treatments, somatic embryos on
their filter paper supports were imbibed with 1/2 strength
culture medium, stored overnight in a -20°C freezer, or plunged
into liquid nitrogen then removed and immediately transferred to
the freezer overnight. Frozen embryos were imbibed the next day.
Survival frequencies have been used to determine the
effectiveness of some of the treatments referred to in the
present application. Hence, the term survival, when used herein,
is defined as: embryos that became green or commenced elongation
within the first week of culture. Embryos matured for 8 weeks
survived severe desiccation in the 81% and 63% environments at
similar high frequencies (e. g. 70-100%). Embryos also survived
freezing at -20°C, but frequencies were better for embryos
desiccated in the 63% r.h. environment (96%) compared to 44% for
embryos desiccated only in the 81% r.h. environment. Embryos
frozen in liquid nitrogen survived at slightly lower frequencies,
as about 1-4% of the embryos split or shattered during the rapid
freezing process. This problem may be overcome by transferring
embryos to liquid nitrogen after initially freezing them to -
20°C. Normal plantlets were recovered following all freezing
methods.
Characteristics of imbibed somatic embryos.
Imbibed somatic embryos have a moisture content usually
ranging between 59 and 65%.
- 39 -
CA 02451941 2003-11-28
r .
'Enca lat' n desi sate om a r o ~n a on- dr
colder
One of the novel elements of the present invention resides
in the fact that PEG is not to be used as a hydrated gel for
encapsulation, but is to be molten and used to encapsulate mature
somatic embryos, zygotic embryos or desiccated somatic or zygotic
embryos without causing re-hydration. Mature conifer somatic
embryos, conifer zygotic embryos as well as desiccated somatic
or zygotic embryos, preferably from the family Pinaceae and most
preferably from the genus Picea can be encapsulated using the
method of the present invention. Other compounds having
properties similar to PEG can be used. Tt is required that the
compound used for encapsulation be a non-hydrated water soluble
compound having a melting point ranging between 20 and 70
although polymers such as PEG are preferred.
PEG is a water-soluble wax-like polymer which is non- toxic,
poorly metabolized and highly resistant to attack by organisms
(e. g. fungi, bacteria, animal pests, etc.). It is currently used
to promote seedling vigor by osmotic priming of seeds, so should
be ideal as an encapsulation agent.
Before testing PEG as a suitable agent for encapsulation,
the effect of high concentrations of PEG on embryo germination
was first tested. This was done using the technique of osmotic
priming. Osmotic priming is a method of controlled hydration in
which the physiological process-of germination is initiated but
stopped before radicle emergence.. Natural seeds lose vigor
during storage, and cell deaths may occur as a result of rapid
water uptake during the first minutes of imbibition. PEG and
other osmotica have been used to osmotically prime whole seeds
to synchronize germination and improve seedling vigor. The
- 40 -
CA 02451941 2003-11-28
method involves soaking seeds in osmotic solutions of sufficient
osmotic strength to allow seeds to take up water and metabolism
to be restored, but germination is prevented. Imbibition injury
may be reduced or prevented, and any cellular damage repaired.
Thus, full vigor is restored upon removal of osmoticum (Powell
and Mathews 1978; Bodsworth and Bewley 1979; Woodstock and Tao
1981) .
A study of sectioned white spruce~~somatic and zygotic
embryos using transmission electron microscopy showed that
desiccated zygotic embryos take up to several days to fully
imbibe, as they are enclosed within seed coats and other
structures. .Imbibition of somatic embryos desiccated to low
moisture contents, by contrast, occurs within 1-2 h. Such rapid
imbibition may lead to injury. Severely desiccated somatic
embryos were osmotically primed by imbibing them in liquid medium
containing 30% PEG for 3 days prior to transfer to PEG-free
medium. Survival frequencies were similar to non-primed
treatments, showing the absence of toxicity of high PEG levels
on germination; furthermore, root elongation of the PEG treated
embryos appeared to be improved.
PEG of different molecular ranges vary in melting point.
Highest is only about 66°C. PEG was considered suitable as an
encapsulation agent as it could be molten and applied to
desiccated embryos without causing embryo re-hydration. PEG of
different molecular weights were used singly or mixed to achieve
desired properties, or different types applied in different
layers (e. g. an embryo coated in a soft wax surrounded by a hard
wax layer).
TABLE 2
- 41 -
CA 02451941 2003-11-28
PEG mol wt melt pt wa:x type viscosity
< 1000 < 23°C soft or liquid very low
- 1000 - 37°C soft low
- 4000 - 59°C hard medium
> 6000 60-66°C very hard high
Therefore, PEG with molecular weights over 1000 and
preferably between 1000 and 3000 may be used. Embryos desiccated
to low moisture contents are considered to be tolerant to
temperature extremes so should not be harmed by brief exposure
to molten PEG; however, the PEG types with lower temperature
melting points may be preferable, and are less viscous so flow
and coat embryos more readily. PEG 1000-400.0 and mixtures
thereof are ideal. PEG 1000 is soft and pliable. PEG 4000 is
harder and more brittle. Embryos encapsulated in PEG should also
be protected from imbibition injury similar to osmotic priming.
Thus, as the PEG capsule dissolves into solution around the
embryo it creates an osmotic pressure. This pressure would
approach zero as the PEG becomes fu~.ly dispersed, and would have
the effect of preventing the more rapid imbibition, that would
otherwise occur in the absence of PEG encapsulation. Capsules
take up to 8 hours to dissolve when placed on agar gelled medium.
Various adjuvants may be added to the PEG to assist in
seedling establishment. Such adjuvants may include a carbon
source such as sucrose or glucose, etc., (myo-inositol was found
to be least resistant to caramelizing during prolonged heating
~ with higher melting point PEG), activated charcoal, Psilium seed
powder to bind water after re-hydration, fungicides and
insecticides added in powder form, microorganisms, organic energy
reserves and enzymes such as starch and a-amylase (capsules may
- 42 -
CA 02451941 2003-11-28
be surrounded by other polymers, such as gelatin, or mixed with
insoluble waxes, both of which would perhaps give a slow timed
release of embryos from the capsules), amino acids (e. g.,
glycine), plant growth regulators. An example of the
encapsulation method of the present invention is outlined below.
Moulds were prepared by drilling shallow wells into a 4 mm
thick sheet of silicone rubber (other types of mould may be
suitable). Prior to use for encapsulation, the rubber mould was
sterilized with alcohol, then evaporated dry.
PEG 6000, 4000, and 1000 and an equal mixture of 4000:1000
have been tested, PEG 1000 being preferred. PEG was heated to
above melting point. The molten PEG was heat sterilized by
maintaining it at its boiling port, or just below, for at least
1/2 h. PEG should be cooled to just above its melting point prior
to embryo encapsulation. One of the key elements of the
encapsulation method is to assure that the embryos are
encapsulated with the non-hydrated water soluble compound at a
temperature slightly above the melting point of the non-hydrated
water soluble compound so as to provide rapid solidifying of the
coating to yield hardened capsules containing the embryos.
A small drop of PEG was first added to the wells of the
mould. Somatic embryos, desiccated slowly to low moisture
content at a r.h. of &3%, were removed from the filter paper
supports and placed singly in~the wellsr and a drop of nnolten PEG
added to enclose the embryo. The volume of the synthetic seeds
was approximately 30 ,u1. After the PEG had solidified the
encapsulated desiccated embryos were removed from the mould and
stored at room temperature, or in the freezer. For germination,
the synthetic seeds were placed on filter papers placed on
solidified plantlet regeneration medium. Survival following
- 43 -
CA 02451941 2003-11-28
encapsulat ton is 93%. After one week in the freezer, the same
batch had survived at frequency 83%. Normal plantlets.have been
recovered. Recovery of plantlets after planting the capsules
directly in soil has not yet been tested.
DESCRIPTION OF PREFERRED EMBODIMENTS
Study of the effects of a non-plasmolysing induced moisture
stress on the maturation and desiccation survival of white spruce
somatic embryos and determination of lipid composition of matured
and desiccated embryos
A. MATURATION AND DESICCATION.
Source of material andculxure media
The white spruce (line WSI) liquid culture was initiated and
maintained on basal medium (BM) as reported previously (Attree,
Dunstan and Fowke, 1989; Attree et al., 1990).
The BM used for maintenance was that of von Arnold and
Eriksson (1981), and also contained 1% sucrose, 9 ~M 2,4-
dichlorophenoxyacetic acid (2,4-D) and 4.5 ,uM benzyladenine (BA).
The maturation medium consisted of half-strength BM containing
90 mM sucrose and 16 uM (f) ABA (Sigma, product number A 2784)
solidified with 0.8% agar (Difco Bitek). A stock solution of ABA
was filter sterilized and added to cooled medium after
autoclaving. The plantlet regeneration medium consisted of half-
strength BM with 60 mM sucrose 0.6% agar and lacked plant growth
regulators (PGRs). All of the above media were adjusted to pH
5.7. Plastic petri dishes (10 cm) containing 15-20 ml medium
were used. Dishes were sealed with Parafilm (American Can Co.)
and cultures were incubated at 25°C. Osmotic potentials of media
- 44 -
CA 02451941 2003-11-28
were determined using a vapour pressure psychrometer (model no.
5130, Wescor Inc., Logan, Utah) due to its greater accuracy with
PEG solutions (Michel and Kaufmann, 1973).
Somatic embryo maturation and mild desiccation
Maturation of somatic embryos was carried out using methods
modified from Attree et al. (1990). Suspension cultured somatic
embryos were washed and resuspended (20a w/v) in half-strength
PGR-free BM cozztaining 3% sucrose, to remove the previous PGRs,
then 0.75 ml aliquots were pipetted onto filter paper supports
(Whatman no: 2) on the surface of maturation medium. The
supports facilitated subsequent~transfe:rs to fresh media. To
determine the mean number of somatic embryos plated, 10 ,u1
samples of the 20o suspension were stained with acetocarmine
(B.D.H.) and counted (repeated _15 times). The plated somatic
embryos were maintained on the maturation medium for 4 weeks in
the dark. To test the effect of ir.~creased osmoticum, the
following concentrations of PEG-4000 (Fluka AG) were included in
the maturation medium; 0, 2.5, 5.0, 7.5 and 10% (w/v) (25
replicates per treatment). Maturation frequencies per treatment
were calculated as the percent mean number of somatic embryos
that matured to normal-looking cotyledonary embryos. Maturation
frequency results are shown in Figure 1.(~ confidence limits, P
- 0.05) .
The application of PEG-4000 in the presence of 16 ~.cM ABA for
28 d promoted the maturation of white spruce somatic embryos
(Fig. 1). The mean number of immature somatic embryos plated per
replicate was 560, and maturation results are based upon a total
of 4087 mature embryos recovered. The o~>timal PEG concentration
was within the range of 5.0 - 7.51. PEG at 7.5°s led to a
threefold increase in the maturation frequency, compared to the
- 45 -
CA 02451941 2003-11-28
s: '
control, giving an overall mean maturation frequency of 9%. In
addition, maturation in the presence of 5% PEG or greater ied to
the absence of sustained embryogenic callus proliferation, which
occurred in the 0 and 2.5% PEG treatments despite the presence
of ABA.
In preliminary experiments sucrose was tested at 6 and 9%.
Visual comparisons showed that 6% sucrose yielded lower
maturation frequencies than at sucrose alone, while 9% sucrose
led to no growth; therefore, elevated sucrose was not tested
further. The osmotic potential of the PEG media decreased non-
linearly, falling less sharply at the higher concentrations
tested (Fig. 1). The osmotic potential of the 7.5% PEG
maturation medium (which also contained media salts and 3%
sucrose) was -0.7 MPa; equivalent to the osmotic potential of
maturation medium containing 9% sucrose. The osmotic potential
of -0.61 MPa for maturation medium containing 5% PEG was
approximately equivalent to that containing 7% sucrose.
Determination of moisture content and dry weight of mildly
desiccated mature somatic embryos
Somatic embryos from various PEG treatments were weighed
(hydrated weight), dried in an oven at 60°C for 3-4 d then their
dry weights recorded. The dry weights were used to determine the
moisture contents of the mildly desiccated somatic embryos.
Measurements were repeated 6 to 12 times depending on the
availability of somatic embryos, and 20 somatic embryos were used
per replicate. Zygotic embryos were also dissected from
unimbibed mature seed, weighed, imbibed in distilled water, then
weighed again (repeated three times with 40 embryos per
replicate). The hydrated (somatic) unimbibed (zygotic) and dry
weights were used to determine the moisture contents of the
zygotic and somatic embryos.
- 46 -
CA 02451941 2003-11-28
As it can be seen in Figure 2, the dry weights of mature,
mildly desiccated somatic embryos increased with increasing PEG
concentration, while moisture contents decreased (~ confidence
limits, P - 0.05). Dry weights of PEG treated somatic embryos
increased from 0.17 to 0.27 mg per embryo. Zygotic embryos
possessed dry weights of 0.15 ~ 0.01 m~3 per embryo. Hydrated
somatic embryos matured with PEG had mean, moisture contents prior
to further desiccation of 41-45%. The controls by comparison,
possessed mean moisture contents of 57%.
Post-maturation. secondary desiccation to low moisture content,
and plantlet regeneration
To determine the effects of PEG, ABA, and secondary
desiccation t.o low moisture content on somatic embryo survival
and plantlet regeneration, i.e. desiccation tolerance, somatic
embryos matured with or without 7.5o PEG were treated as follows
(repeated five times per treatment)e directly germinated; given
a post-maturation treatment (no ABA, see below) and then
germinated; given a post-maturation treatment, secondary
desiccated at 81% r.h. (see below), then germinated; or secondary
desiccated directly (i.e. no post-maturation) then germinated
(see Table 3).
Post-maturation
Post-maturation was achieved by transferring whole somatic
embryo cultures, by their filter-paper ;supports, onto plantlet
regeneration medium (which contains no ABA) for 14 d in the dark;
as described previously (Attree et al., 1990). Post-maturation
of cultures matured with PEG was carried out using plantlet
regeneration medium containing the same PEG concentration as that
for maturation.
- 47 -
CA 02451941 2003-11-28
Slow secondary desiccation
The effect of PEG on desiccation tolerance was tested by
subjecting 4-week matured somatic embryos from all PEG
concentrations to the 8l, 63; and 43% r.h. environments (repeated
7 to 15 times per treatment).
Secondary desiccation to different degrees of moisture
content was accomplished by transferring matured somatic embryos
through a series of environments of progressively lower r.h. as
described by Senaratna et al. (1989). The following saturated
salt solutions contained in desiccators were used to generate the
respective r.h. (NH4)ZS04, r.h. 81%; N'H4NO3, r.h. 63%; KZC03,
r.h. 43%. Matured mildly desiccated somatic embryos were
transferred on their filter paper supports to unsealed petri
dishes which were then placed within the 81% r.h. desiccator.
For r.h. below 81%, petri dishes containing the cultures were
transferred successively to the lower r.h. desiccators at 3-4 d
intervals to reduce the desiccation rate. All cultures were
maintained for a minimum of 7-10 d at the final required r.h.
Total secondary desiccation treatment times were 14 d.
Following secondary desiccation, unimbibed and imbibed
somatic embryos had moisture contents in the range 20-31% and 56-
65%, respectively (Table 3). Mean moisture contents directly
following the 81% r.h. treatment were marginally higher than
those following the 43% r.h. treatment, and closely approximated
those for unimbibed zygotic embryos. Somatic embryos from the
different osmotic treatments had similar moisture contents after
secondary desiccation. The controls matured without PEG
underwent the greatest moisture loss during secondary
desiccation. Imbibed zygotic embryos ha.d moisture contents of
62% and imbibed somatic embryos had moisture contents of 56-65%.
- 48 -
CA 02451941 2003-11-28
TABLE 3
Moisture content (% ~ confidence limits, P = 0.05)
of desiccated unimbibed; and desiccated imbibed
white spruce somatic and zygotic embryos.
The somatic embryos were matured in different PEG
concentrations (%) then given a 43 or 81%
relative humidity desiccation treatment.
Somatic
PEG concentration (o
Zygotic r.h. , CI 5 7.5
Unimbibed 32.5~3.0 81 25.7~9.9 29.4~3.0 31.2~6.9
43 21.9~7_0.5 20.1~3.7 27.8~8.7
Imbibed 61.9~3.1 81 58.0~10.8 64.7~4.1 61.5~4.9
43 59.1~9.1 56.6~5.0 59.1~2.4
Plantlet regeneration following slow secondary desiccation
Plantlet generation was studied from white spruce somatic
embryos matured for 28 days with 16 ACM and 5.0% PEG. Somatic
embryos desiccated to low moisture contents were imbibed in the
Petri dishes by flooding the filter paper supports with liquid
plantlet regeneration medium. The dishea were then sealed with
Parafilm and placed under low light [2 'W m-2, 12 h photoperiod,
20 W cool-white fluorescent lamps (Westi.nghouse)]. Those that
survived and commenced development to plantlets were scored and
transferred to fresh solidified plantlet regeneration medium 7-
14 d after re-hydration.
Somatic embryos that were not given a secondary desiccation
treatment were separated individually from the cultures following
the maturation/postmaturation treatments. They were placed
horizontally on fresh plantlet regeneration medium, and
maintained at low light intensity (as above).
- 49 -
CA 02451941 2003-11-28
v : g
0, 5.O and 7.5% PEG matured somatic embryos from the 81 and
43% r.h. treatments were weighed (unimbibed weight), imbibed for
h with germination medium, then gently blotted and weighed
again ( imbibed weight) , prior to detei~mining the dry weights .
5 These measurements were repeated three to six times per treatment
with 2o somatic embryos per replicate.
Post-maturation and slow secondar3r desiccation
The appearance of mature somatic embryos and regenerated
~plantlets is shown in Fig. 3. All secondary desiccation
treatments led to somatic embryos of a dry and shrunken
appearance as shown in Figure 3A (bar: 2mm). After the
application of liquid medium, somatic embryos imbibed water; and
within 2 h had regained a swollen appearance (Fig. 3B).
Survivors placed under Iow light developed into plantlets within
d as seen in Figure 3C (bar: 5 mm) . The timing of the slow
secondary desiccation treatment was critical (Table 4).
TABLE 4
Overall effects of maturation treatment
(16 ~M ABA ~7.5°s PEG for 28 d), ABA-free
post-maturation (14 d), and 81% relative humidity
secondary desiccation treatment (14 d) on white
spruce somatic embryo survival and plantlet regeneration.
ABA 1°mild Fresh (F) or
desiccation ABA-free severely Plantlet
maturation post-maturation desiccated (D) regeneration
treatment
PEG absent No D Poor
No F +
Yes . D -
Yes F +
- 50 -
CA 02451941 2003-11-28
7.50 PEG No D +
No F -
Yes D -
Yes F Poor
(+) Plantlets regenerated;.(-) no embryo survival.
Somatic embryos initially survived the 81o r.h. treatmen.t
only if the treatment was applied directly following transfer of
the somatic embryos from the ABA maturation media (with or
without PEG). Hence, when somatic embryos matured with 7.5% PEG
were further desiccated directly, somatic embryos developed to
plantlets. PEG-matured somatic embryos did not develop further
in the absence of secondary desiccation treatment but swelled and
became vitrified. As it can be seen in Figure 3D, the axes of
the somatic embryos have failed to elongate normally, the
plantlet is vitrified; and the root is necrotic (bar: 3 mm).
Those PEG matured embryos given post-maturation instead of
secondary desiccation developed to plantlets with swollen bases
and no roots. Thus, normal regeneration of PEG matured embryos
occurred only if the embryos were subsequently desiccated to low
moisture contents. Furthermore,_secondary desiccation following
post-maturation in the absence of ABA (with or without PEG) was
lethal to all embryos.
Effect of slow secondary desiccation upon filter~aper supports
Somatic embryos survived secondary desiccation at high
frequency when the embryos were desiccated while retained as
whole callus on the filter paper supports and placed in an
unsealed petri dish in an 8l~ r.h. environment. Also, it was
noted that somatic embryos did not survive secondary desiccation
in an unsealed petri dish placed in an 81o r.h. environment, if
- 51 -
CA 02451941 2003-11-28
. .
they were removed from the main callus and filter paper supports.
As slower desiccation occurred in the former, then these
experiments suggested that somatic embryos were intolerant to
rapid desiccation to low moisture contents. However, it was,
subsequently found that somatic embryos can survive rapid
secondary desiccation to low moisture contents; furthermore; when
somatic embryos matured for 8 weeks with 16 ACM ABA and 7.5% PEG
were separated from the main callus but placed on the same filter
paper beside the whole callus, somatic embryos survived slow
secondary desiccation (81% r.h.) at high frequency, but recovered
plantlets were abnormal - the embryos did not elongate normally,
but remained stunted. This was overcome if the mature somatic
embryos were removed from the callus and washed in culture medium
containing ABA and PEG, then placed on filter paper moistened
with the same medium, prior to further desiccation.
Survival and plantlet regeneration following secondary slow
desiccation to low moisture contents
Table 5 shows that somatic embryo survival and plantlet
regeneration generally diminished with increasing severity of the
secondary desiccation treatments, for - somatic embryos matured
for only 4 weeks.
TABLE 5
Regeneration {% ~ s.e.) Of plantlets from white spruce
somatic embryos that were matured in the presence of
different % concentrations of PEG {all with 16 ~M ABA),
then further desiccated in climates of different % relative
humidity (r. h.)
PEG concentration (%)
r.h.
(%) f 0 I 2.5 ~ 5 ~ 7.5 ~ 10
- 52 -
CA 02451941 2003-11-28
81 44.3~7.8 61.9~10.2 35.2~7.2 33.9~6.6 37.6~10.1
63 34.5~11.6 21.9~7.4 28.3~8.1 17.34.1 12.6~4.8
43 8.3~8.2 9.9~8.9 7.9~4.5 8.6~4.6 0.2~0.2
Controls (no PEG treatment or secondary desiccation)
developed to plantlets at a frequency of 43% (s.e. ~ 120). The
inclusion of PEG during the maturation phase did not greatly
influence the desiccation survival of the somatic embryos;
however, results within treatments-were very variable. Highest
mean survival and plantlet regeneration at 81% r.h. occurred with
the 2.5% PEG matured somatic embryos (62%). Survival of the
other osmotic treatments was within the range 34-44%. Following
the 43% r.h. treatment, survival was less than 10% for all
osmotic treatments, and less than 1% for the 10%-PEG matured
embryos. Although somatic embryos matured without PEG
regenerated to plantlets, often these were aberrant, especially
following the more severe secondary desiccation treatments of low
r.h. For example, rooting was retarded, not all cotyledons
elongated, and hypocotyls were often curled following elongation.
It was also observed that the somatic embryos matured without PEG
did not remain quiescent after transfer from the ABA media, but
greened and underwent precocious germination during the first few
days of the secondary desiccation treatment. Somatic embryos
from the other osmotic treatments remained quiescent throughout
secondary desiccation.
Effect of culture time on tolerance to slow and rapid secondary
desiccation
It was of interest to examine whether these somatic embryos
were tolerant to rapid secondary desiccation, such as drying on
the lab bench at ambient r.h. Bench drying is simpler to perform
- 53
CA 02451941 2003-11-28
:m
than drying in controlled environments. It was also of interest
to determine if there was a particular maturation period that was
optimal for survival. Therefore, somatic embryos matured for 4,
5, 6, 7 and 8 weeks on medium containing 16 uM ABA and 7.5% PEG,
were each transferred on their filter paper supports either to
sterile petri dishes, which were left unsealed on the lab bench
for three days or 50 to 81% r.h. desiccator for slow drying for
2 weeks. The ambient r.h, was about 35°s during the time of the
air drying experiments, and the laboratory temperature was 20-
25°C. 10-17 replicates were prepared per treatment. After
secondary desiccation, the somatic embryos were hard and dry.
Somatic embryos were then imbibed as before with half-strength
PGR-free medium and scored for plantlet: regeneration after 2-3
weeks. Results (~ standard errors) are shown in Figure 15.
It can be seen from Figure 15 that somatic embryos survived
slow secondary drying treatments after 4 weeks of maturation, and
regenerated to plantlets at a frequency of about 60%. This
frequency improved slightly further following 6, then 8 weeks
maturation, achieving in the order of 80'~ plantlet regeneration:
Somatic embryos matured for just 4 weeks then further dried more
rapidly at ambient r.h., did not regenerate to plantlets. Often
with these embryos, the root meristem survived and developed a
root, but the shoot apex had died, so remained white and did not
elongate. Rapidly air dried somatic embryos were capable of
regenerating to plantlets after 5 and 6 weeks maturation, but at
low frequency (less than 10 and 25% respectively): By the 7th
week of maturation these somatic embryos survived and regenerated
to plantlets at a frequency of just oven: 60%. Thus, from this
graph it is clear that optimal desiccation tolerance to drying
to low moisture levels is achieved after a minimum of 7 weeks of
maturation in the presence of ABA and non-permeating osmoticum.
The quality of the regenerated~,plantlets following the rapid
- S4 -
CA 02451941 2003-11-28
0
secondary drying treatment was-good, and it appeared that root
elongation often appeared earlier and was more vigorous following
rapid drying.
Evaluation of the molecular size threshold of solutes for
promoting maturation of white spruce somatic embryos during
primary treatment of mild desiccatior~ and maturation
1.0 In order to determine the effective molecular weight and
size range of solutes that promote the maturation of immature
white spruce somatic embryos, they were matured in half-strength
LP maturation medium containing,a range of different solutes of
differing molecular weight. Five replicates were prepared per
treatment, and treatments were repeated twice. All media
contained 20 ~cM ABA and a base level of 3% sucrose. The
supplemented solutes were included at 7.5~ (w/v), which
supplemented solutes constituted an addit3_on to the 3% base level
of sucrose. The treatment time was 9 weeks. The sucrose,
mannitol, and PEGS 200, and 400 treatments were also tested at
2.5 and 5% (w/v), but as these cancentrations did not result in
recovery of any mature embryos, results for these concentrations
are not presented. Similarly, PEG 8000 led to poor maturation
as this solute prevented effective gelling of the medium. The
somatic embryos remained wet throughout the maturation treatment
which led to poorly formed embryos at low frequency. Therefore,
results for PEG 8000 are excluded here; however, PEG 8000 was
tested in a liquid flow bioreactor where ~_t effectively promoted
maturation (see later). For all treatments, only normal looking
cotyledonary stage opaque white embryos were scored and used for
lipid analyses . Levels of lipid triacyl_glycerols (TAGS) were
analyzed as described later and results provided are means of two
replicates.
- 55 -
g
2~, '
CA 02451941 2003-11-28
A low recovery of mature control (no PEG) somatic embryos
was recorded. Many more mature embryos were initiated in this
treatment but the majority underwent precocious germination
during later phases of the treatment, so were not scored or used
for lipid determinations. It can be seen from Table 6 that the
molecular weight of PEGS capable of pramoting maturation is
initiated at the 600-1000 molecular weight range. PEGs of 200
and 400 were toxic and no callus growth was observed throughout
the maturation treatments, similar to the mannitol and sucrose
treatments. PEG 600 was somewhat toxic; the mean number of
mature embryos recovered was similar to the control in the
absence of any precocious germination, and lipid. levels were
lower than control levels. PEGS of greater than 1000 were the
more effective at promoting maturation. PEG 1000 led to recovery
of a large number of mature somatic embryos. Visual comparison
showed these embryos were smaller than other effective
treatments, giving .a high o DW of TAG, but lower total lipid
content. Visual comparisons of their tolerance to further
desiccation to lower moisture levels (at 81o r.h.) and
development to plantlets, showed a variable response among
replicates; furthermore the quality of regenerated plantlets was
more variable than with the higher molecular weight osmotica.
Thus, it is likely that PEG 600 and to a lesser extent PEG 1000,
do slowly penetrate the cell walls of the somatic embryos. It
therefore appears that the threshold molecular size of osmotica
that effectively promotes maturation is in the region of 30-35
A, and it is considered that this is achieved due to their
exclusion from entering the cell through pores in the cell walls,
so exerting a non-toxic moisture stress.
- 56 -
CA 02451941 2003-11-28
P d
~Q
TABLE 6
Comparison of the effect of molecular weights and
molecular sizes of solutes; at 7.5°s concentration
with 20 ~cM ABA, on maturation response (mature embryos
per replicate ~ SE, and lipid contents) of white
spruce somatic embryos.
Solute Molecular Molecular Mature TAG (% TAG (mg)
weight size (A) embryos embryo per
per DW ~ , embryo
replicate
Control 342 10 6f1.0 24.4 2 O1
(3s
sucrose)
Sucrose 342 10 0 - --- ---
Mannitol 182 8 0 --- ---
PEG 2000 -200 < 30 0 --- ---
PEG 400 -400 < 30 0 --- ---
PEG 600 -600 < 30 8.22.6 22.3 123.8
PEG 1000 > 950 > 30 44.65.5 34.8 201
PEG 4000 > 3000 > 30 31.52.6 31.1 250.5
Dextran -6000 > 30 17.02.6 28.8 2 60
6000
Dextran -80000 > 30 16.21.9 25.9 213.2
80,000
Maturation of cultured white spruce somatic embryos
A one-week-old white spruce suspension culture, previously
grown in liquid medium containing 10 ACM 2, 4-D and 5 ~,cM BA, was
collected by filtration. The somatic embryos were rinsed in
growth regulator free liquid medium, 3-6 g o~ somatic embryos
were transferred to a fresh 250 ml flask containing 50 ml of
half-strength liquid medium (1% sucrose) containing 5 ,uM BA with
or without auxin reduced by 1/10. The somatic embryos were then
_ 5~7 _
CA 02451941 2003-11-28
cultured for l week. After this time they were again collected
by filtration, and a 20% suspension (w/v) of somatic embryos was
prepared in fresh BA containing medium. This was inoculated
(0.75 ml aliquots) onto filter paper supports overlying
maturation medium. The embryos were then cultured following the
description above. Both the pre-treatments enhanced maturation
substantially and can be said to have a synergistic effect with
PEG/ABA treatments on maturation frequencies. Inclusion of low
auxin was beneficial.
Maturation of white sbruce somatic embryos using a continuous
flow, solid-support bioreactor
A bioreactor was fabricated out of a high density plastic
container 15 x 21.x 6 cm, with air tight lid. One entrance and
one exit port were situated at opposite corners of the chamber
base. The inside base on the chamber was overlaid with a
cotton-wool pad, on which way placed a filter paper support
(Whatman no 1; cut to 15 x 21 cm) . Liquid maturation medium
(half strength LP medium with 7.5% PEG 8000, 3% sucrose and 20
,uM ABA) was supplied from an 8 L vessel containing 4 L of culture
medium. The whole apparatus was autoclaved. The bioreactor
retained approximately 450 ml of liquid medium within the cotton
pad. Culture medium was pumped through the bioreactor chamber
at a flow rate of 20 ml per h., for 3 h per day. This provided
the equivalent to approximately one full medium change per week.
Suspension-cultured immature somatic embryos were inoculated onto
the filter paper support as a 20% suspension (w/v) in growth-
regulator-free medium. Approximately 10-l5 ml of suspension was
distributed over the filter-support surface. The system was run
for 7 weeks. The filter paper supporting the mature mildly
desiccated somatic embryos was then removed, and cut into smaller
pieces for easier handling. The mature embxyos were then further
- 58 -
CA 02451941 2003-11-28
' s
a
desiccated to low moisture contents either on the supports in an
environment of 63% r.h. for two weeks then imbibed in half
strength hormone free medium, or air dried and analyzed for
storage lipid (TAG). Lipid analyses were conducted as described
in section B of preferred embodiments, using two replicates of
100 embryos each.
Somatic embryos underwent maturation within the bioreactor
chamber yielded high quality mature embryos. The embryos had
well developed cotyledons. The bioreactor yielded approximately
500 mature embryos of this quality. It is expected that this
number can. be improved with optimizing of the conditions within
the bioreactor, and the size of the b:ioreactor itself can be
increased if desired. The lipid levels .are shown in Table 7 and
these levels compare favourably to the levels observed in 6-8
week somatic embryos matured on agar medium. The somatic embryos
survived further desiccation at high frequency and germinated
vigorously into normal looking plantlets. Thus, using this
method, large numbers of embryos of excellent quality have been
produced with minimal cost and manipulation. Using this method
it is possible to slowly increase ABA and/or osmotic
concentrations over the first few days of maturation, and
similarly to modify their concentrations prior to conclusion of
the production run. However, it is envisaged that levels of ABA
will be maintained at a substantially constant level throughout
the majority of the maturation period. As the medium is supplied
regularly while spent medium is removed, it may also be possible
to provide a modified culture medium with more suitable levels
of nutrients than are presently provided by agar cultures:
Additionally osmotic and ABA concentrations may need to be re-
optimized. It is to be noted that other combinations of flow
ratio and flow times of the medium can be used efficiently.
- 59 -
CA 02451941 2003-11-28
TABLE 7
Characteristics of white spruce somatic embryos matured
for 7 weeks in a continuous flow,
solid support bioreaictor.
DW(mg) s DW of TAG TAG/TL o Plantlet
TAG (FAMES) regeneration
( FAMES ) (fig per frequency
embryo) (%)
0.56 25.2 140 75.95 84.4
Desiccation of black and Norwav spruce somatic embryos
Somatic embryos of white spruce, Norway spruce and black
spruce have been matured and desiccated to a low moisture
contents and regenerated to plantlets using these methods. The
conditions found suitable for white spruce were tested on Norway
and black spruce. Thus, suspension cultured black and Norway
spruce somatic embryos were washed in growth-regulator-free
medium, and a 20% suspension (w/v) was prepared in fresh growth-
regulator-free medium. Aliquots (0.75 ml) were.pipetted onto
half strength culture medium containing 7.5o PEG 4000 and 16 (~)
,uM ABA. The somatic embryos were matured for 4 weeks then
further desiccated on their filter paper supports in an
environment of 81% r.h. for 2 weeks. The Norway and black spruce
somatic embryos survived and regenerated to plantlets at
frequencies of about 35 and 65%, respectively. It is likely that
these values could be improved further following optimization of
the ABA and osmotic concentrations, and increasing the length of
the maturation period to at least 7 weeks. Norway spruce somatic
embryos survived at lower frequency, however, the~culture was not
well established in suspension culture which was only 3 weeks
old, so overall maturation was poor. Suspension cultures usually
- 60 -
CA 02451941 2003-11-28
, y ' ~'
require up to 3 months establishment before embryos undergo
effective maturation.
B. DETERMINATION OF LIPID COMPOSTTIONS OF MATURED AND DESICCATED
WHITE SPRUCE SOMATIC EMBRYOS.
Somatic embryo maturation
Maturation of the immature suspension cultured white spruce
somatic embryos (line WS1) was carried out using the methods
described previously in Part A (Maturation and Dessication) of
this Description of Preferred Embodiments.
Experiments were set up to observe the effects of different
culture conditions on somatic embryo lipid biosynthesis. Control
somatic embryos were matured for 4 weelts on maturation medium
containing 16 ~cM ABA (~ racemic, product number A 2784; Sigma,
St Louis, USA). To observe the effects of osmoticum, PEG-4000
(Fluka AG) was included in the maturation medium at
concentrations of 2.5, 5.0, 7.5, and lOn (w/v), all with 16 ~.cM
ABA. Somatic embryos were maintained on these media for 4 weeks
in the dark prior to lipid analysis.
To test the effect of culture time on lipid biosynthesis
somatic embryos were maintained on maturation medium which
contained 16.,uM ABA and 7.5% PEG, for 2, 4, 6, or 8 weeks prior
to lipid analysis. Cultures that were matured for longer than
4 weeks were transferred to fresh medium after this time. The
lipid contents of immature somatic embryos from suspension
culture were also determined.
Investigations were conducted to observe the effects of
different ABA concentrations, in the presence of PEG; on lipid
- 61~ -
CA 02451941 2003-11-28
. ;~ ' ~ b
biosynthesis. Somatic embryos were, therefore, maintained on
maturation medium containing 7.5~ PEG and 12, 16, 24, or 32 fcM
ABA for 8 weeks. To observe the effects of slow secondary
desiccation on lipid biosynthesis, somatic embryos matured in
these treatments were also transferred into an environment of 81%
r.h., as described previously to achieve further desiccation to
low moisture levels. Matured somatic embryos were transferred
on their filter-paper supports to unsealed petri dishes which
were then placed within the 81% r.h. desiccator. Total secondary
desiccation treatment time was 2 weeks. Somatic embryos were
analyzed for lipid after imbibing in liquid medium for 1-2 h, in
order to free~them from the filter-paper supports. Results were
compared to those of somatic embryos matured under the same
conditions but not further desiccated.
For comparisons with somatic embryos, zygotic embryos were
dissected from the megagametophytes of mature seeds after they
had been imbibed for 16 h in distilled water. In addition, whole
seed was analyzed for lipid.
Plantlet regeneration
Following maturation and further desiccation white spruce
somatic embryos intended for further culture were imbibed in
liquid plantlet regeneration medium, using a method modified
slightly from the method described previously in A., in order to
reduce the rate of water uptake. Thus, instead of flooding the
liquid medium directly onto the somatic embryos, 1-2 ml was added
to the petri dishes which were then maintained on a slope with
the filter-paper carriers dipped into the liquid. The medium was
first absorbed by the filter paper and conveyed to the somatic
embryos. After imbibition, somatic embryos were maintained under
low light intensity as described before. One week later,
- 62 -
CA 02451941 2003-11-28
,r ' '
regenerating plantlets were separated and cultured individually.
They were placed horizontally on fresh plantlet regeneration
medium, and maintained at the same low light intensity. Four
weeks after imbibing, they were analyzed for storage lipid and
the results compared to those of fully expanded zygotic seedlings
derived from embryos dissected from the megagametophytes of
mature seed and grown in vitro for 4 weeks.
Lipid_analxsis
IO
The whole white spruce seeds, isolated zygotic embryos,
somatic embryos from the various treatments, and regenerated
plantlets and zygotic seedlings were counted, blotted dry, and
fresh weights determined. The samples were then placed in an
oven at 80°C for 24 h and dry weights recorded. Lipids were
extracted from fresh tissues by the hexsme/isopropane method of
Hara and Radin (1978), after first placing the samples in boiling
isopropane for 10 min. TAGS were separated from total lipid
extracts by thin layer chromatography using HPTLC-Fertigplatten
Kieselgel 60 plates (Mandel Scientific Co., Toronto, Canada).
Plates were developed in a solvent system containing petroleum
ether: diethyl ether: acetic acid (82:18:1). TAGS were
identified using authentic standards and scraped from the plates
using a razor blade. Fatty acid methyl esters (fames) were
prepared from TAGS and total lipid extracts, as previously
described (Pomeroy et al. 1991). Sample sizes consisted of 50-
180 somatic or zygotic embryos, 25 whole seeds, and 5 zygotic
seedlings or regenerated somatic plantleta. Each lipid extract
sample was divided into 2-3 replicates for analysis, and
experiments were repeated three~times. Results shown are means
of one experiment.
Microsconv
- 63 -
CA 02451941 2003-11-28
Somatic and zygotic embryos of white spruce were prepared
for transmission electron microscopy (TEM) according to
previously published methods (Fowke 1984). Mature dry seeds were
imbibed in tap water for 16, or 65 h prior to zygotic embryo
S removal and fixation. Somatic embryos desiccated to low moisture
contents were rapidly imbibed by complete immersion in liquid
plantlet regeneration medium for 2 h prior to fixation. Somatic
embryos were first cut longitudinally to ensure subsequent
penetration of fixatives and resin. Thick sections (i.e., l ;um)
were cut from the same plastic embedded material and stained. with
toluidine blue (1% w/v in 1~ borax solution) for observations by
light microscopy.
RESULTS
A. Lipid composition
Fatty acid compositions were determined for both TL and TAG,
but since values were similar throughout, only TAG fatty acid
compositions are provided with the exception of the data for
zygotic embryos and seeds (Table 8.).
- 64 -
CA 02451941 2003-11-28
. ,
TABLE 8
A, TL (fatty acid methyl esters (fames)) and
TAG (fames) contents, and B, fatty acid
compositions, of white spruce mature whole seed and
isolated zygotic embryos.
A
TL TAG TAG/TL
0
~g ( i.ndivi a d wt E.cg ( indivi % d wt
- -
dual ) -1 dual ) '1
Seed 688 29 372 16 54
embryo 62 51 44 36 71
B
Fatty acid comt~osition of TL anal TACK (%)
0.67 0.67 0.75 0.75 0.251 0.75 18:2b 0.75 EC-20,22
TL 2:4 0.2 1.3 15.8 4.5 45.0 29.6 0.2 1.1
seed .
TAG 2.7 0.2 1.3 16.9 4.7 42.8 28.6 0.2 2.4
embryo TL 5.1 1.0 1.6 18.4 3.3 48.9 20.7 0.3 0.8
TAG 4.9 0.9 1.6 19.5 3.0 49.1 19.4 0.60 0.9
Double bond in the C-9.
C-7
position
instead
of
the
Double bond at the and C-9 positions.insteadof the C-9 and C-
C-5
12 positions.
Represents the sum all identified C-20 C-22 fatty acids.
of and
- 65 -
CA 02451941 2003-11-28
Zvctotic embxyos and seeds
A large proportion of the dry weight of zygotic embryos was
due to lipid (Table 8A). They consisted of 51% TL by dry weight,
36% of the dry weight (16% imbibed fresh weight; not shown) was
attributed to TAG; therefore, the ratio of TAG to TL was 71% .
Isolated zygotic embryos contained only about 12% of the TAG
present in whole seed. .Thus, TAG was distributed between the
megagametophyte and the zygotic embryo at a ratio of 7.5 . 1,
respectively. The low % dry weight value of lipid from whole seed
compared to isolated zygotic embryos was due in part to the
inclusion of the seed coats during analysis. The fatty acid
analysis of TL and TAG for isolated zygotic embryos and whole
seeds showed that the compositions were ,similar (Table 8B?. The
predominant fatty. acids in both 'zygotic embryos and whole seeds
of white spruce were two separate molecular species of 18:2,
comprising around 70% of total fatty acids. The most abundant
species of 18:2 in both embryos and seeds has double bonds at the
usual C-9 and C-12 positions 09.12). However, an unusual 18:2
fatty acid with double bonds at the.C-5 and C-9 positions (~5,9)
comprised 20-30% of total fatty acids. The total content of 18:1
was about 20% of total fatty acids, with around 80% of the 18:1
with the double bond at the C-9 position. The 16:2, 18:0 , 18:3,
and longer chain fatty acids were each present at less than 2%.
Somatic embryos
The effect of PEG concentration on lipid biosynthesis
and fatty acid composition after 4 weeks culture with 16 uM ABA
are shown in Table 9.
- 66 -
CA 02451941 2003-11-28
TABLE 9
Influence of PEG concentrations on A, TL (fames) and TAG
(fames)
accumulation and B, fatty composition of white spruce
somatic embryos. These were matured for 4 weeks with 16 ~.cM
ABA.
A
TL TAG TAG/TL
PEG
0 0
I~g (embryo) % d wt ~.cg~ (embryo) % d wt
-1 -1
0 control 32 21 23 15 72
2.5 ~ 33.2 30 20.8 19 63
44 31 29 21 66
7.5 40.6 30 28 21 69
37 27 26 20 70
B
Fatty acid composition of TAG (%)
PEG % 0.667 0.668 0.75 0.75 0.251 0.75 18:2b 0.75 EC-20,22
0 1.9 2.4 23.3 2.2 45.6 11.31.1 3.2
control
9
2.5 9.1 1.9 2.4 23.4 2.3 45.2 11.41.1 3.3
5 8.5 1.6 2.6 24 2.1 46.4 11 1.1 2.7
7.5 8.5 1.6 2.7 24.3 2.4 45 11.11.2 3.2
10 7 1.2 2.5 23.3 3.4 41 16.60.9 4.3
.
Double bond the C-7 insteadof the
in position C-9.
Double bond the C-5 C-9 insteadof and C-
at and positions the
C-9
l2 positions.
Represents sum of all identified 20 and C-22fatty acids.
the C-
PEG increased the quantity of SAG in somatic embryos (Table
9A), but they did not achieve levels as high as those recorded
for zygotic embryos (c.f., Table 8A), either on a per embryo, or
- 67 -
CA 02451941 2003-11-28
% dry weight basis. In the absence of PEG somatic embryos
contained about 50% of the amount of TL and TAG present in the
zygotic embryos. TL and TAG per somatic embryo increased with
5.0 and 7.5% PEG compared to the control, and reached close to
70% of the amount of TAG observed in zygotic embryos. The % dry
weight TAG increased by 40% with 5 and 7.5% PEG, achieving 58%
of the dry weight value observed in zygotic embryos. TAG fatty
acid composition was not influenced to any great extent by
different concentrations of osmoticum after 4 weeks of culture
(Table 9B). Furthermore, at all PEG concentrations, the somatic
embryos contained the same predominant fatty acids, as zygotic
embryos (Table 8B), although the proportion of 18:1 was higher
and that of 18:2 (A5,9) was lower in the somatic embryos.
The effect of culture time and 7.50. PEG on lipid
biosynthesis and fatty acid composition is shown in Table 10.
68 _
CA 02451941 2003-11-28
TABLE 10
Influence of culture time on A, TL (fames) and TAG (fames)
accumulation and B, fatty acid composition of white spruce
somatic embryos. These were matured with 16 ~.M ABA, and
0% or 7.5% PEG.
A
Time TL TAG TAG/TL °s
( Weeks ) I~g ( embryo ) ' 1 ~ d wt ~,cg ( embryo ) -1 °s d wt
0 ND 6 LsfD 2 3 8
2 +PEG ND 8 ND 3 ~ 30
4 +PEG 57.2 28 36.1 18 63
6 +PEG 128.7 30 72.7 17 57
8 +PEG 238.6 36 172.7 26 72
8 -PEG 173.3 21 113.3 14 65
ND, not determined.
- 69
CA 02451941 2003-11-28
n in a
TABLE 10 (cont' d)
Influence of culture time on A, TL (fames) and TAG (fames)
accumulation and B, fatty acid composition of white spruce
somatic embryos. These were matured with 16 E.cM ABA, and
0% or 7.5% PEG.
B
Fatty amid composition of TAG (%)
Time 0.667 0.668 0.75 0.75 0.251 0.75 18:2° 0_75 EC-20,22
(Weeks)
0 8.8 2.4 2.8 29.1 6.4 31.5 7.8 5.1 6.1
2 +PEG 9 2.4 2.9 29.4 6.2 31.5 7.8 4.5 6.3
4 +PEG 7.9 1.5 3.2 25.2 2.3 45 11.3 1.2 2.4
6 +PEG 6.2 1.2 2.2 23.3 3.1 46 13.8 0.9 3:2
8 +PEG 4.3 0.7 1.3 24.6 3.9~ 47.2 15:8 0.4 1.7
8 -PEG 6.3 0.1 1.8 23.9 2.7 48.9 14.1 0.9 1.4
Double bond in the C-7 position instead of the C-9.
Double bond at the C-5 and C-9 positions instead of the C-9 and C-
12 positions.
Represents the sum of all identified C-20 and C-22 fatty acids.
- 70 -
a.
CA 02451941 2003-11-28
Somatic embryos continued to accumulate TL and TAGS
throughout the 8-week culture period (Table 10A). For example,
during 4-6 weeks with PEG the. weight of TL and TAG per embryo
increased to levels greater than those recorded for zygotic
embryos and by 8 weeks the somatic embryos had four times more
TAG compared to zygotic embryos. The increase was more modest
when expressed as % dry weight, achieving 720 of the level
recorded for zygotic embryos; even so, somatic embryos contained
45o more TAG at 8 weeks compared to those at 4 weeks. The TAG
component of the somatic embryos was 26% dry weight (11% fresh
weight; not shown) by the 8th week of culture. The effect of PEG
on TAG accumulation was clearly evident after 8 weeks° culture.
At this time somatic embryos matured with 7.5% PEG had
accumulated 50o more TAG per embryo compared to non-PEG-treated
somatic embryos, and contained almost twice as much TAG on a
dry weight basis. The o of TAG to TL increased during maturation
with PEG, and resulted in a higher rati~a of TAG to TL compared
to somatic embryos matured without PEG. The TAG fatty acid
composition of somatic embryos changed with culture time (Table
9B) and by 8 weeks had reached ratios that closely approXimated
zygotic levels (c. f., Table 8B). The most abundant fatty acids
present in immature suspension cultured somatic embryos were 18:1
(n9) and 18:2 (09,12). The 7.5o and ~Oo PEG treated somatic
embryos had similar fatty acid composition values, which again
showed that the PEG osmoticum had little effect on fatty acid
composition even after 8 weeks culture. During the 8-week study
period, the trend was for the 18:2 (09,12 and o5,9) fatty acids
to increase while the other. fatty acids decreased
proportionately, resulting in fatty acid compositions similar to
mature zygotic embryos (Table 7B).
- 71 -
CA 02451941 2003-11-28
The effects of ABA concentration arid secondary desiccation
treatments on lipid biosynthesis and fatty acid composition after
8 weeks with 7.5a PEG are shown in Table 11.
CA 02451941 2003-11-28
c
TABLE 10
Influence of culture time on A; TL (farces) and TAG (farces)
accumulation and B, fatty acid composition of white spruce
somatic embryos. These were matured with 16 ~cM ABA, and
0% or 7.5% PEG.
A
Time TL TAG TAG/TL
%
( Weeks ~g ( embryo % d wt ~.cg ( embryos d wt
) ) ' 1 ) -1
0 ND 6 . ND 2 38
2 +PEG ND 8 ND 3 30
4 +PEG 57.2 28 36.1 18 63
6 +PEG 128.7 30 72.7 17 57
8 +PEG 238.6 . 36 172.7 26 72
8 -PEG 173.3 21 113.3 14 65
ND, not determined.
- 73 -
CA 02451941 2003-11-28
TABLE 10 s:o~r,~~ d)
Influence of culture time on A, TL (farces) and TAG (farces)
accumulation and B, fatty acid composition of white spruce
somatic embryos. These were matured with~l6 ~M ABA, and
0% or 7.5% PEG.
B
Fatty acid composition f TAG %l
o f
Time 0.667 0.668 0.75 0.75 0.251 0.?5 18:26 0.75 EC-20,22
(weeks)
0 8.8 2.4 2.8 29.1 6.4 31.5 7.8 5.1 6.1
2 +PEG 9 2.4 2.9 29.4 6.2 . 31.57.8 4.5 6.3
4 +PEG 7.9 1.5 3.2 25.2 2.3 45 11.3 1.2 2.4
6 +PEG 6.2 I.2 2.2 23.3 3.1 46 13.8 0.9 3.2
8 +PEG 4.3 0.7 1.3 24.6 3.9 47.2 15.8 0.4 1.7.
8 -PEG 6.3 0.1 1.8 23.9 2.7 48.9 14.1 0.9 1.4
Double bond in the C-7 position instead of the C-9.
Double bond at the C-5 and C-9 positions instead of the C-9 and
C-12 positions.
Represents the sum of all identified C-20 and C-22 fatty acids.
- 74
CA 02451941 2003-11-28
Somatic embryos continued to accumulate TL and TAGS
throughout the 8-week culture period (Table 10A). For example,
during 4-6 weeks with PEG the weight of TL and TAG per embryo
increased to levels greater than those recorded for zygotic
embryos and by 8 weeks the somatic embryos had four times more
TAG compared to zygotic embryos. The increase was more modest
when expressed as % dry weight, achieving 72% of the level
recorded for zygotic embryos; even so, somatic embryos contained
45% more TAG at 8 weeks compared to those at 4 weeks. The TAG
component of the somatic embryos was,26% dry weight (11% fresh
weight; not shown) by the 8th week of culture. The effect of PEG
on TAG accumulation was clearly evident after 8 weeks' culture.
At this time somatic embryos matured with 7.5% PEG had '
accumulated 50% more TAG per embryo compared to non-PEG-treated
somatic embryos, and contained almost twice as much TAG on a
dry weight basis. The % of TAG to TL increased during maturation
with PEG, and resulted in a higher ratio of TAG to TL compared
to somatic embryos matured without PEG. The TAG fatty acid
composition of somatic embryos changed with culture time (Table ,
9B) and by 8 weeks had reached ratios that closely approximated
zygotic levels (c.f., Table 8B). The most abundant fatty acids
present in immature suspension cultured somatic embryos were 18:1
(09) and 18:2 (n9,12). The 7.5% and 0% PEG treated somatic
embryos had similar fatty acid composition values, which again
showed that the PEG osmoticum had little effect on fatty acid
composition even after 8 weeks culture. During the 8-week study
period, the trend was for the 18:2 (09,12 and n5,9) fatty acids
to increase while the other fatty acids . decreased
proportionately, resulting in fatty acid compositions similar to
mature zygotic embryos (Table 7B).
- 75.
CA 02451941 2003-11-28
The effects of ABA concentration and secondary desiccation
treatments on lipid biosynthesis and fatty acid composition after
8 weeks with 7.5% PEG are shown in Table 11.
'75 _
CA 02451941 2003-11-28
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CA 02451941 2003-11-28
In somatic embryos not subjected to secondary,desiccation,
TL increased with increasing ABA concentration (Table 11A). ABA
at 16 ~,cM, however, yielded the highest accumulation of TAG per'
embryo (143 fig), but on a % dry weight basis 24 ,uM ABA was higher
(20%). Following secondary desiccation somatic embryos from all
ABA concentrations displayed higher TL and TAG - those from the
16 and 24 ~c.M ABA treatments~contained over 30% more - showing
that lipid accumulation continued during the secondary
desiccation treatment. On a'per embryo basis; 16 ~M ABA yielded
the most TAG per embryo (214 ,ug). This is five times the zygotic
value (Table 7A), and about a nine fold increase over the
original controls (Table 9A). On a % dry weight basis 24 uM ABA
was optimal producing somatic embryos containing 30% TAG. This
is 83% of the zygotic value, and twice the value of the initial
control somatic embryos. On a % ,fresh weight basis all ABA
treatments led to somatic embryos containing approximately 11%
TAG (not shown), which is about 70% of the zygotic level. The
TAG fatty acid composition was not modified appreciably by ABA
at the concentrations tested (Table 48), however, following
desiccation the proportion of 18:1 (n9) consistently decreased
slightly, while the proportion of 18:2 (n9,12) underwent a slight
increase, resulting in values that more closely approximated
zygotic values compared to somatic embryos not desiccated to low
moisture contents.
Somatic ~lantlets and n,~rcotic seedl,ing~
The TL and TAG content for regenerated somatic plantlets
matured for 6 weeks with 16 ~rM ABA, 7.5% PEG, then further
desiccated, and expanded zygotic seedlings grown from isolated
zygotic embryos are compared in Table 12.
_ 79 _
CA 02451941 2003-11-28
TABLE 12
A, TL (fames) and TAG (fames) contents, and
B, fatty acid compositions, of white spruce
expanded seedling and somatic plantlet following maturation
for 6 weeks on medium containing 16 ~cl~I ABA and 7.5% PEG
then further desiccated. The somatic plantlet and
zygotic seedling were both 4 weeks old.
Time TL ' TAG TAG/TL %
~g ( embryo ) -1 % d wt ,ug ( embryo ) '' d % wt
somatic
plantlet 26 2.3 8 0.7 ~31
zygotic
seedling 20 2.1 6 0.63 30
ND, not determined
Fatty acid composition of TAG f%)
0.667 0.668 0.75 0.75 0.251 0.75 18:2° 0.75 EC-20,22°
somatic
plantlet 9.3 . 1 4.1 23.I 4.9 34.9 12.9 3.2 6.8
zygotic
seedling 14.8 0.6 6 37.9 2.9 23 7.8 3.2 3.8
Double bond in the C-7 position instead of the C-9
Double bonds at the C-5 and C-9 positions instead of the C-9 and C-12
positions
Represents the sum of all identified C-20 and C-22 fatty acids
_ 8p _
CA 02451941 2003-11-28
After 4 weeks' growth, the TL and TAG contents were similar
(Table 12A). Low levels of lipid were present in both plant
types, confirming the storage function of the TAGs, and their
utilization for post-gezminative growth. The data for the TAG
fatty acid compositions showed similar trends (Table 12B). Thus,
with both plant types the 18:2 (e9,12 and ~5,9) decreased, while
the proportions of the other fatty acids increased, in comparison
to mature zygotic embryo levels (c.f., Table 8B). The somatic
plantlets which were matured with 16 ~cM ABA had not achieved the
degree of change observed for the zygotic seedlings. However,
these results were inconsistent, and, furthermore, the level to
which these changes occurred for zygotic seedlings varied greatly
among experiments. Thus, it appears that the synthesis of 16:0,
18:0, and longer chain fatty acids in the. seedlings and plantlets
occurs at the expense of 18:2 (n9,12 and n5,9), which is the
reverse of events observed during maturation (c.f., Table 10B).
B. Plantlet conversion
During culture for 4-8 weeks with 7.5°s PEG and 12-32 ~cM ABA,
white spruce somatic embryos matured without germinating
precociously. Somatic embryos desiccated to low moisture
contents were dry and shzvnken and had a translucent appearance.
During secondary desiccation, however, many somatic embryos
matured for 8 weeks with 12 pH ABA had undergone slight greening
prior to drying. Precocious germination during the secondary
desiccation treatment was more pronounced with somatic embryos
matured for 8 weeks with 0 and 2.5' PEG, especially the former
where considerable greening and hypocotyl elongation approached
loot and survival did not occur. Thus, following prolonged
maturation treatments, the higher ABA and PEG concentrations
prevented the onset of precocious gezmi.nation that otherwise
occurred once ABA was removed for secondary desiccation. As
- 81 -
CA 02451941 2003-11-28
shown in Figure 4, fully imbibed, norn~al somatic embryos regained
their predesiccated swollen opaque-white appearance, and
converted to plantlets at high frequency. Embryos at this stage
are light green and have commenced elongation (X 3.0 bar: 0.5
cm) . For example, after 4, 6 or 8 week: treatment .with 16 ~cM
ABA, a total of 700-800 normal-looking cotyledonary somatic
embryos matured per treatment. ~ As seen in Figure 5, somatic
plantlets regenerated from the 16-24 ~.cM ABA treatments underwent
root and hypocotyl elongation (X 2.7; bar 0.5 cm): Elongation
is comparable in extent to zygotic seedlings grown in-vitro from
isolated embryos (see Figure 6). The zygotic seedlings shown in
Figure 6 were obtained from mature embryos separated from the
megagametophyte of mature seed and grown in vitro for 3 weeks
under the same conditions as the somatic embryos of'Figure 5 (X
2.7, bar: 0~.5 cm) .
C. Micrpscopv
Mature white spruce zygotic embryos had distinct cotyledon
and apical meristem regions, and procambium was evident as shown
in Figure 7A (X 76, bar: 0.2 mm). Lipid bodies (L) were abundant
within the cells of the root, hypocotyl and areas adjacent to the
shoot apical meristem, some apparently fusing together (arrow)
as seen in Figure ?B. Zygotic embryos dissected from mature dry
seeds imbibed for 16 h also had numerous mature-protein bodies
(Figure 78 (X 6500, bar: 3 ,um) ) . However, the protein bodies
within cells of zygotic embryos dissected from seeds imbibed for
65 h had enlarged, and the protein deposits had dispersed as seen
in Figure 8. The cells shown in Figure 8 also contain numerous
tightly packed lipid bodies (L) some apparently fusing together
(arrow) (N: nucleus, X 6500, bar: 3 E.cm) .
' - 82 ._
CA 02451941 2003-11-28
Somatic embryos matured for 8 weeks with 16 ACM AHA and 7.5%
PEG as seen in Figure 9 contained large amounts of lipid (L) and
compact protein bodies (P) similar to zygotic embryos from 16 h
imbibed seed (X 6500, bar: 3 um). After secondary desiccation
and rapid imbibition for 2 h, the somatic embryos shown in
Figure 9 contained abundant lipid bodies comparable in
distribution and frequency to the mature zygotic embryos from 65
h_ imbibed seed as seen in Figure 10A. The cells are densely
cytoplasmic and storage reserves are evident (small arrows).
Note the rather flat meristem (large arrow) and procambial cells
(white arrow) (X 80, bar: 2 mm). Figure 10B shows that the cells
are packed with lipid bodies (L). Also, the severely desiccated
and imbibed somatic embryos exhibited enlarged protein bodies (P)
containing dispersed protein deposits after just 2 h imbibation,
similar to the zygotic embryos from 6S h imbibed seed (N:
nucleus, X 6000, bar: 3 ,um) : Somatic embryos had a distinct
apical meristem, procambium and well developed cotyledons, and
were generally larger than zygotic embryos.
In contrast, somatic embryos matured for only 4 weeks
without PEG (Fig. 11) or with 7.5% PEG (Fig. 12), contained
considerably fewer lipid bodies than observed in~8-week treated
somatic embryos (Fig. 9). The level of 20 lipid accumulation was
also distinctly lower than in zygotic embryos (c.f. Fig. 7b).
Cells of somatic embryos matured for 4 weeks with PEG were more
densely cytoplasmic when compared to somatic embryos matured
without PBG (for which most cells of the'hypocotyl and cotyledons
are vacuolate so are not mildly desiccated) which are shown in
Figure 11A (X 135, bar: 0.1 mm).
As shown in Figure 11B (N: nucleus; X 7500, bar 3 E.cm), the
cytoplasm of cells from somatic embryos matured for 4 weeks with
16 uM but without PEG contain fewer and smaller lipid bodies (L)
_ g3 ._
CA 02451941 2003-11-28
than in cells from somatic embryos matured for 8 weeks with both
ABA and PEG (c.f. Figure 10B). The cells shown in Figure 12A (X
130, bar: 0.1 mm) are not vacuolate, but are more densely
cytoplasmic and contain more storage reserves (arrows) than cells
in embryos matured for the same time in the absence of PEG (c.f.
Figure IlA). The inclusion of PEG during maturation has
increased the size and number of lipid bodies (L) , starch (S)
deposits and mature protein bodies (P) as shown in Figure 12B
(X 6500, bar: 3 ~cm). However, lipids are not as abundant as in
somatic matured for 8 weeks with ABA and PEG as seen in Figure 9.
Following germination and 4 weeks' growth of zygotic
seedlings, most cells had enlarged and ,undergone vacuolation.
As seen in Figure 13A, vascular traces (large arrow), apical
meristems (small arrow) and vacuolate cells were well defined (X
72, bar: 0.2 mm). The electron macrograph shown in Figure l3B
illustrates that lipid bodies were infrequent throughout the
seedling and appeared almost empty (arrows) due to utilization
of the contents. Protein bodies are absent (N: nucleus, X 6000,
bar: 3 um). This pattern of development also occurred in
similarly aged somatic plantlets, regenerated from somatic
embryos matured for 8 weeks with 7.5% PEG then further
desiccated. However, in some instances plantlets regenerated
from the latter treatment had r.uzdergone epicotyl (E) elongation
and needle development around the apical-meristem by 4 weeks as
seen in Figure 14A (X 54, bar: 0:2 mm), The small arrow
indicates the original cotyledon. This degree of development was
not observed in the zygotic seedlings of equivalent age. In
Figure 14B, the lipid bodies and protein bodies were not
3 0 observed. The cells are characterized by many small vacuoles and
differentiated chloroplasts (arrows) (N: mucleus, V ~ vacuole,
X 60.00, bar: 3 ,um) .
- 84 -
CA 02451941 2003-11-28
s
a
Discussion
By manipulation of the culture conditions for white spruce
somatic embryos it was possible to attain storage lipid levels
S and fatty acid compositions higher than those observed in zygotic
embryos. Such manipulations produced somatic embryos that
survived desiccation to low moisture contents then regenerated
to plantlets at high frequency_ The maturation conditions that
resulted in somatic embryos with a fatty acid composition which
most closely approximated the mature zygotic embryos were 6-8
weeks with 16-24 ~,~M ABA and 7.5% PEG, followed by further
desiccation. These concentrations also led to optimal storage
protein deposition in white spruce somatic embryos. The latter
5 study also showed that 5.0-7.5% PEG afforded protection to
storage proteins which were otherwise degraded during further
desiccation. In addition, this PEG concentration stimulated a
doubling of lipid levels and a threefold increase in the
maturation frequency of white spruce somatic embryos, and the
somatic embryos also possessed lower moisture levels than zygotic
embryos from mature dry seed.
Synchronous maturation of the immature white spruce somatic
embryos occurred following their transfer from proliferation
medium containing 2,4-D acid and BA, to the moisture stressing
medium containing PEG-and ABA. No maturation occurred in the
absence of PEG' and ABA. The concentration of ABA and PEG, and
maturation period, had an effect on TAG accumulation; whilst
fatty acid composition was mostly modified by the latter. More
minor modifications to fatty acid composition occurred fol lowing
3 0 further desiccation. TAG levels - as % dry weight - increased
from 42% of zygotic levels in the original controls (4 weeks with
0% PEG) to 83% after 8 weeks maturation with 7.5% PEG and 16-24
~M ABA followed by further desiccation, while TAG levels per
- 85 -
CA 02451941 2003-11-28
somatic embryo increased from half that observed in zygotic
embryos to almost five times the zygotic levels. This led to
somatic embryos with roughly 9 times the level of TAG observed
in the controls, and 6 times the fresh weight level recorded by
~Feirer et al. (1989? for Norway spruce somatic embryos: Vigorous
root and shoot elongation was evident in the regenerated somatic
plantlets.~ These results show that althbugh the total amount of
TAG for somatic embryos was greater than f-_or zygotic embryos, a
lower lipid density resulted from the larger size of the somatic
embryos. The increase in dry weight and decrease in moisture
content in the presence of PEG as observed in A was, therefore,
indicative of increased storage reserves.
The results for lipid accumulation, fatty acid composition,
and the TEM and Regeneration studies, together indicate that a
4 week treatment with ABA - as is often used for maturation. of
conifer somatic embryos, did not allow sufficient time for
optimal accumulation of TAG by white spx:~uce somatic embryos,
resulting in somatic embryos that were not of comparable maturity
to zygotic embryos. A large amount of TAG was synthesized during
the 4~ to 8th week of culture . The TFsM study provided further
evidence for stimulated lipid biosynthesis with 7.5% PEG and
extended maturation time, illustrating the well developed
structure of the somatic embryos. Storage reserves were
previously shown to accumulate initially in the root regions of
white spruce somatic embryos, and then subsequently in the later
developing shoot meristem and cotyledon regions. The
cotyledonary and shoot meristem regions of the somatic embryos
appeared after the third -week of culture, so additional
development of these regions would be necessary before lipid
could be deposited.
- 86 -
CA 02451941 2003-11-28
In order to achieve slow secondary desiccation to low
moisture contents somatic embryos were transferred to the 81%
r.h. desiccators. The filter-paper supports on which they were
transferred were saturated with culture medium, therefore, the
moisture stressing environment and initial availability of
nutrients appears to have enabled further lipid accumulation,
prior to the supply of nutrients drying and the moisture contents
of the somatic embryos becoming too low t:o support metabolism.
A non-phasmolysing moisture stress was influential in
preventing precocious germination of white spruce somatic embryos
during prolonged maturation and desiccation treatments thereby
promoting survival following further desiccation. Optimal TAG
accumulated using 7.5% PEG and 16-24 ~cM ABA. Maturing embryos
underwent an increased tendency for precocious germination with
increased maturation time leading to poor survival following
further desiccation to low moisture content. The increasing
tendency for precocious germination suggests a decreased
sensitivity to ABA with increased maturation time. Precocious
germination was prevented.by PEG treatments. In the absence of
high moisture stressing treatments, concentrations of up to 60
~eM applied throughout the maturation period have been used to
inhibit precocious germination during maturation of conifer
somatic embryos. However, such concentrations increased the
incidence of abnormal somatic embryos.
The plantlet conversion frequencies of 72-81% reported here
for somatic embryos matured for 6-8 weeks, may be because they
have entered a more desiccation tolerant phase. Desiccation
tolerance appears closely related to levels of storage reserves.
Thus, treatments that promoted storage reserve accumulation, such
as PEG, ABA and increased maturation time, also promoted
desiccation tolerance. This is because vacuolate cells
- g7 ..
CA 02451941 2003-11-28
s
s ,
containing little reserve material may undergo mechanical
disruption and tearing of membranes during severe water loss,
while the presence of sufficient reserves limits such changes.
Severely desiccated somatic embryos appear to undergo very
rapid imbibition and hence sustain injury, unlike zygotic embryos
which are protected within seeds. Protein bodies within the
cells of dry seeds swell and take up water during imbibition;
thus, as evidenced ~by protein body ultrastructure, rapidly
imbibing somatic embryos by immersing them in liquid medium for
just 2 h, was comparable to 65 h of seed imbibition. Therefore,
the alternative slower imbibition method used probably reduced
injury, so promoted plantlet conversion.
High osmoticum stimulates TAG biosynthesis and influences
the quantity and/or composition of the fatty acids; sucrose being
the customary osmoticum of choice (e. g. Pence et al. 1981; Janick
et al. 1982; Avjioglu and Rnox 1989; Dutta and Appelqvist 1989).
Fatty acids are fo~ned by converting sucrose into acetyl
Coenzyme A, from which palmitic (16:0) and oleic (18:0) acids are
formed and used in the synthesis of unsaturated and longer chain
fatty acids (Stymne and Stobart 1987). It has been suggested
that sucrose stimulates lipid biosynthesis either by influencing
the chemical intermediates of the tricarboxylic acid cycle, or
by eliciting osmotic alterations in the cell in response to the
low water potential of the culture medium (Pence et al. 1981).
The stimulation of lipid biosynthesis in the white spruce somatic
embryos using PEG shows that the effect wa:~ due to the induced
moisture stress and not to a limiting sucrose substrate.
Consequently, for maturation of white spruce somatic embryos the
optimal osmoticum concentration was higher for PEG than for
sucrose. For maturation of white spruce somatic embryos the
_ 88 _
CA 02451941 2003-11-28
m.
optimal osmotic potential of the culture medium, which contained
7.5% PEG and 3% sucrose, was -0.7 Mpa.
The oil reserves of seeds are rapidly mobilized back to
sucrose following germination to provide energy and carbon
skeletons for the post-germinative embryo growth. Lipid reserves
are depleted during growth of the white spruce somatic embryos
to plantlets in a manner similar to in vitro cultured zygotic
seedlings.
_ 8g _
~ 02451941 2003-11-28
~ , °..~ s,.. .. , _ .. . _;:: - .-.. .__ . ..._ , :. , .. ... _ . . .
,. ", . _ ,.,
References
Ammirato, P.V., 1983. Embryogenesis, eds. D.A. Evans, W.R.
Sharp, P.V. Ammirato and Y. Yamada, In Handbook of Plant Cell
Culture, Vol. 1, pp. 82-123, Macmillan, New York.
Anandarajah; K. and McKersie, B.D. , 1990. Enhanced vigor of dry
somatic embryos of Medicago Sativa L. with increased sucrose.
Plant Science 71, 261-266.
15
Anandarajah, K. and McKersie, B.D., 1990. Manipulating the
desiccation tolerance and vigor of dry somatic embryos of
lKedicago Sati va L. with sucrose, heat shock and abscisic acid.
Plant Cell Reports 9, 451-455.
Arnoid, R.L.B., Fenner, M., Edwards, P.J. (1991) Changes in
Germinability, ABA content and ABA embryonic sensitivity in
developing seeds of Sorghum bicolor (L.) Moench. induced by water
stress during grain filling. New Phytol. 118, 339-347.
Attr2e, S.M., Dunstan, D.I., and Fowke, L.C., 1989. Initiation
of embryogenic callus and suspension cultures, and improved
embryo regeneration from protoplasts of white spruce (Picea
glauca). Canadian Journal of Botany 67, 1790-1795.
Attree, S.N., Tautorus, T.$., Dunstan, D.I., Fowke, L:C. (1990)
Somatic embryo maturation, germination, and soil establishment
of plants of black and white spruce (P, icea mariana and Pic a
g~iauca). Can J. Bot. 68, 2583-2589.
Attree, S.N., Fowke, L.C. (1991) Micropropagation through somatic
embryogenesis in conifers. In. Biotechnoloav in ag ricul~ure and
_ 90 _
CA 02451941 2003-11-28
-< a
forestry, "High-tech ~n Micropropgaation", vol 17; pp. 53-70,
Bajaj Y.P.S. ed. Springer-Verlag, Berlin.
Attree, S.M., Dunstan, D.I., Fowke, L.C. (1991 a) White spruce
[Picea glauca (Moench) Voss~ and black spruce [P_ icea mariana
(Mill) B.S.P.]. In: Trees III. Biotechno7.omr in agriculture and
forestr~r, vol 16, pp. 423-445, Bajaj Y.~P.S. ed. Springer-Verlag,
Berlin.
Avjioglu, A., Knox, R.B. (1989) Storage lipid accumulation by
zygotic and somatic embryos in culture. Ann. Bot. 63, 409-420.
Barratt, D.H.P., Whitford, P.N., Cook, S.K., Butcher, G. and
Wang, T.L., 1989, Analysis of seed developments in Pisum sati yam
L. VIII. Does abscisic acid prevent precocious germination and
control storage protein synthesis? Journal of Experimental
Botany 40, 1990-1014.
Becwar, M.R., Noland, T.L.,~ Wyckoff, J.L. (1989) Maturation
germination, and conversion of Norway spruce (Picea allies L.)
somatic embryos to plants. In Vitro Cell. Devel. Biol. 25, 575-
580.
Becwar, H.R., Nagmani, R., Wann, S.R. (1990) Initiation of
embryogenic cultures and somatic embryo development in loblolly
pine (P_inus taeda). Can. J. For. Res. 20, 810-817.
Bewley, J.D., Black, M. (1984) Seeds Physiol~ Qf develo meat
and germination 367 pp. Plenum press, New York.
Bodsworth, S. and Bewley, J.D., 1981. Osmotic priming of seeds
of crop species with polyethylene glycol as a means of enhancing
- 91 -
CA 02451941 2003-11-28
a
c 7 ,
early and synchronous germination at cool temperatures. Can. J.
Bot. 59, 672-676_
Brown, C., Brooks, F.J., Pearson, D. and Mathias R.J., 1989.
Control of embryogenesis.and organogenesis in immature wheat
embryo callus using increased medium osmolarity and abscisic
acid. J. Plant. Physiol., Vol. 133, pp. 727-733.
Boulay, M.P., Gupta, P.K., Krogstrup, P. and Durzan, D.J., X988.
Development of somatic embryos from cell suspension cultures of
Norway spruce (Picea abies Karst.). Plant Cell Reports 7, 134-
13 7'.
Carpita, N., Sabularse, D., Montezinos, D. and Delmer, D., 1979.
Determination of the pore size of cell walls of living plant
cells. Science 205, 1144-1147.
Ching, T.M. (1963) Fat utilization in germinating Douglas fir
seed. Plant Physiol 38, 722-728.
Ching, T.M. (1966) Compositional changes of Douglas fir seed
during germination. Plant Physiol. 41, 1313-1319.
Cress, W.A. and Johnson, G.V., 1987. The effect of three osmotic
agents on free proline and amino acid pools in.Atriplex canescens
and Hilaria James II~. Canadian Journal of Botany 65, 799-801.
Dunstan, D.I., Bethune, T.D., Abrams, S.R. (1991) Racemic
abscisic acid and abscisyl alcohol promote maturation of white
spruce (Pi'cea giauca) somatic embryos. Plant Science 76, 219-
228.
- 92 -
CA 02451941 2003-11-28
Dunstan, D.I., Bekkaoui, F., Pilon, M., Fowke, L.C. and Abrams,
S.R., 1988_ Effects of abscisic acid and analogues on the
maturation of white spruce (Picea glauca) somatic embryos. Plant
Science 58, 77-84.
Dutta, P.C., Appelqvist, L.A. (1989) The effects of different
cultural conditions on the accumulation of depot lipids notably
petroselinic acid during somatic embryogenesis in Daucus scrota
L. Plant Science 64, 167-177.
Feirer, R.P., Conkey, J.H., S.A. (1989) Triglycerides in
embryogenic conifer calli: a comparison with zygotic embryos.
Plant Cell Rep. 8, 207-209.
Finkelstein, R.R., Crouch, M.L. (1986) Rapeseed embryo
development in culture an high osmoticum is similar to that in
seeds. Plant Physiol. 81, 907-912.
Florin, B. and Petiard, v., Canadian Patent Application
2,020,572.
Florin, B., Lecouteux, C. and Petiard, V., Canadian Patent
Application 2, 013, 821.
Fowke, L.C. (1984) Preparation of cultured cells for transmission
electron microscopy. In: Cell culture and somatic cell genetics
of plants. vol. 1, Laboratory Procedures ~n their agplicatioxis,
pp. 728-737, Vasil, I.K. ed. Academic Press, Inc. Orlando.
Gates, J.C., Greenwood, M.S. (1993, The physical and chemical
environment of the developing embryo of P~'.n~ red' n~ osa . Am . J .
Bot. 78, 1002-1009.
- 93 -
CA 02451941 2003-11-28
' t
W
5
Gomez, J., Sanchez-Martinet, D., Stiefel, V., Rigau, J.,
Puigdomenech, P. and Pages, M., 1988. A gene induced by the
plant hormone abscisic acid in response to water stress encodes
a glycine-rich protein. Nature 334, 262-264.
Gray, D.J., Conger, B.V. and Songstad, D.D., 1987. Desiccated
quiescent somatic embryos of orchardgrass for use as synthetic
seeds. In Vitro Cellular and Developmental Biology 23, 29-33.
Gray, D.J. and Conger, B.V., PCT Application W088/03934.
Gray, D.J. and Purohit, A., 1991. Somatic embryogenesis and
development of synthetic seed technology. Critical Review in
Plant sciences 10(1), 33-61.
Gupta, P.K. and Pullman, G., U.S. Patent 4,957,866.
Gupta, P.K. and Pullman, G., U.S. Patent 5,036;007.
Gupta, P.K. and Pullman, G., U.S. Patent 5,041,382.
Hakman, I., and Fowke, L.C., 1987. Somatic embryogenesis in
Picea glauca (white spruce) and Picea mariana (black spruce).
Canadian Journal of Botany 65, 656-659.
Halanan, I., von Arnold, S. (1988) Somatic embryogenesis and plant
regeneration from suspension cultures of Pi a auc (white
spruce). Physiol. Plant. 72 , 579-587:
Hakman, I., von Arnold, S. and Eriksson, T., 1985. The
development of somatic embryos in tissue cultures initiated from
immature embryos of Picea abies (Norway spruce). Plant science
38, 53-59.
- 94 -
CA 02451941 2003-11-28
a ~ .. s
10
Hakrnan, I., Stabel, P., Engstrom, P., Eriksson, T. (1990) Storage
protein accumulation during zygotic and somatic embryo
development in Picea , ies (Norway spruce). Physiol. Plant. 80,
441-445.
Hammatt, N. and Davey, M.R., 1987. Somatic embryogenesis and
plant regeneration from cultured zygotic embryos of soybean
(Glycine max L. Merr.). Journal of Plawt Physiology 128, 219-
22.6.
Hara, A., Radin, N.S. (1978) Lipid extraction of tissues with a
low toxicity solvent. Anal. Biochem. 90, 420-426.
Heyser, J.W. and Nabors, M.W., 1981. Growth, water content, and
solute accumulation of two tobacco cell lines cultured on sodium
chloride, dextran, and polyethylene glycol. Plant Physiolagy 68,
1454-1459.
Hohl, M. and Schopfer, P., 1991. Water relations of growing
maize coleoptiles. Plant Physiology 95, 716-722.
Janick, J., Wright, D.C., Hasegawa, P:M. (1982) in vitro
production of cacao seed lipids. J. Amen. Soc. Hort. Sci. 107,
919-922. '
Janick,_J. and Kitto, S.L., U.S. Patent 4,615,141.
Joy, R.W., Yeung, E.C., Kong, L., Thorpe, T. (1991) Development
of white spruce somatic embryos: 1. Storage product deposition.
In vitro Cell_ Devel. Biol. 27P, 32-41.
Kartha, K.K., Fowke, L.C., Leung, N.L., Caswell, K.L. and
Hakman, I., 1988. Induction of somatic embryos and plantlets
- 95
CA 02451941 2003-11-28
c
from cryopreserved cell cultures of white spruce (Picea glauca)
. J. Plant Physiol. 132, 529-539.
Kermode, A.R. (1990) Regulatory mechanisms involved in the
transition from seed development to germination. CRC Crit. Rev.
Plant Sci. 9, 155-195.
Kermode, A.R. and Bewley, D.J., 1985. T'he role of maturation
drying in the transition from seed development to germination.
Journal of Experimental Botany 36, 1916-1927.
Kermode, A.R. and Bewley, 1989. Developing seeds of Riccinus
communis L., when detached and maintained in an atmosphere of
high relative humidity, switch to a germinative mode without the
requirement for complete desiccation. Plant Physiology 90, 702-
707. .
Kim, Y-H , Janick, J. (1991) Abscisic acid and praline improve
desiccation tolerance and increase fatty acid content of celery
somatic embryos. Plant Cell Tissue Organ Culture. 24, 83-89.
Kim, Y-H. and Janick, J., 1989 . ABA and polyox-encapsulation or
high humidity increases survival of desiccated somatic embryos
of celery. HortScience 24 , 574-675.
Kishor, P.B.K., 1987. Energy and osmotic requirement for high
frequency regeneration of rice plants from long-term cultures.
Plant Science 48, 189-194.
Kitto, S.L., Pill; W.G. and Molloy, D.M., 1991. Fluid drilling
as a delivery system for.somatic embryo-derived plantlets of
carrot (Daucus carota L. ). Scientia Horticulturae 47, 209-220_
- 96 -
CA 02451941 2003-11-28
v 9
Konar, R.N. (1958) A quantitative survey of some nitrogenous
substances and fats in the developing embryos and-gametophytes
of Pinus roxburShii Sar. Phytomorphology 8, 174-176.
Krizec, D.T. , 1985. Methods of inducing 'water stress in plants.
HortScience 20, 1028-1038.
Krogstrup, P. (1990) Effect of culture densities on cell
proliferation and regeneration from embryogenic cell suspensions
of Pic~~ sitchensi~. Plant Science 72, 115-123.
Laine, E., David, A. (1990) Somatic embryogenesis in immature
embryos and protoplasts of Pinus caribaea. Plant Science 69,
215-224.
Lawlor, D.W., 1979. Absorption of polyethylene glycols in plants
and their effects on plant growth. New Phytologist 69, 914-916.
Lawlor, D.W., 1970. Absorption of. polyethylene glycols by plants
and their effects on plant. growth. New Priytol. 69, 501-513.
Leopold, A.C., 1991. Stress responses in Plants: Adaptation and
acclimation mechanisms_ Pages 37-56, Wiley-Liss, Tnc.
Lott. N.A. (1980) Protein Bodies. In: The biochemistrv Qf,
plants, _a comprehensive treatise, vol. 1, pp. 589-623, Tolbert
N.E. ed. Academic Press, New York.
Mexal, J., Fisher, J.T., Osteryoung, J. and Reid, C.P.P., 1975.
Oxygen availability in polyethylene glycol solutions and its
implications in plant-water relations. Plant Physiol. 55, 20-24.
- 9~ -
CA 02451941 2003-11-28
d
Marsolais, A.A., Wilson, D.P.M., Tsujita, M.J. and Senaratna, T.,
1991: Somatic embryogenesis and artificial seed production in
Zonal (Pelargonium x hortorum) and Regal (Pelargonium X
domesticum) geranium. Can. J. Bot. 69, 1188-1193.
Misra, S., Green, M.J. (1990) Developmental gene expression in
conifer embryogenesis and germination. 1. Seed proteins and
protein composition of mature embryo and the megagametophyte of
white spruce (Picea glauca [Moench] Voss.). Plant Science 68;
163-173.
Misra, S., Kexmode, A. and Bewley, D.J., 1985. Maturation drying
as the 'switch' that terminates seed development and promotes
germination. eds. L. van Vloten-Doting, G.S.P. Groot and T:C.
Hall, In Molecular form and Function of the Plant Genome, pp.
113-128.
Nato ASI series, Plenum Press, New York, London.
Oertli, J.J. , 1985. The response of plant cells to different
fortes of moisture stress, Journal of Plant Physiology 121, 295-
300.
Parrott, W.A., Dryden G., Wogt, 5.; Hilderbrand, D.F.,
Collies, G.B. and Wiliiams, E.G., 1988. Optimization of somatic
embryogenesis and embryo germination in soybean. In Vltro
Cellular and Development Biology 24, 817-820. .
Pence, V.C., Hasegawa, P.M., Janick, J. (1981) Sucrose mediated
regulation of fatty acid composition in asexual embryos of
Theabroma cacao. Physiol. Plant. 53, 378-384.
- 98
CA 02451941 2003-11-28
s
s ~ f
Pomeroy, M.K., Kramer, J.K.D., Hunt, D:J., Keller, W.A. (1991)
Fatty acid changes during development of zygotic and microspore
derived embryos of Brassica nanr~. Physiol. Plant. 81, 447-454.
Pullman, G.S. and Gupta, P.K., U.S. Patent 5,034,326.
Redenbaugh, K, Viss, P., Slade, D. and Fujii, J.A., 1987. Scale-
up: artificial seeds. Plant Tissue and Cell Culture. 473-493.
Redenbaugh, K., Slade, D. and Fujii, J.A., U.S. Patent 4,777,762.
Roberts, D.R., 1991. Abscisic acid and mannitol promote early
development maturation and storage protein accumulation in
somatic embryos of interior spruce. Physiologia plantarum 83,
247-254.
Roberts, D.R., Lazaroff, N.R. and Webster, F.B., 1991.
Interaction between maturation and high relative humidity
treatments and their effects on germination of sitka spruce
somatic embryos. J. Plant Physiol. 138, 7..-6.
Roberts., D R., Flinn, B.S., Webb, D.T., Webster, F.B., Sutton,
B.C.S. (1990) Abscisic acid and indole-3-butyric acid regulation
of maturation and accumulation of storage proteins in somatic
embryos of interior spruce. Physiol. Plant. 78, 355-360.
Roberts, D.R., Sutton, B.C.S. and Flinn, B.S., 1990b.
Synchronous and high-frequency germination of interior spruce
somatic embryos following partial drying at high relative
3 0 humidity. Canadian Journal of Botany 68, 1086-1090.
Roberts, D.R., PCT Application CA90/00241.
_ gg ._
CA 02451941 2003-11-28
s
r
Saranga, Y. and Janick, J., 1991. Celery somatic embryo
production and regeneration: improved protocols. Hortscience
26(10) , 1335.
Senaratna, T., McKersie, B.D., Bowley, S., Bewley, J.D. and
Brown, D., European Patent Application 0 300 730.
Senaratna, T., McKersie, B.D. and Bowley, S.R., 1989.
Desiccation tolerance of alfalfa (Medicago sativa L:) somatic
embryos. Influence of Abscisic acid, stress pretreatments and
drying rates. Plant Science 65, 253-259.
Senaratna, T., McKersie B.D. and Bowley, S.R., 1989. Desiccation
tolerance of alfalfa (Medicago sativa L.) somatic embryos.
Influence ~of abscisic acid, stress pret,reatments and drying
rates. Plant Science 65, 253 259.
Senaratna, T., Kott, L., Beversdorf, W.D., McKersie, B.D., 1-991.
Desiccation of microspore derived embryos of oilseed rape
(Brassica napus L.). Plant Cell Reports 10, 342-344.
Shimonishi, K., Ishikawa, M., Suzuki, S. and 0osawa, K., 1991.
Cryopreservation of melon somatic embzyos by desiccation method.
Japan. J. Breed. 41, 347-351.
Stymne, S., Stobart, A.K. (1987) Triacylglycerol biosynthesis.
In: _T_he biochemistry of Plants, ~ r,~omorehen iv treatise, vol.
9, pp. 175-214, Stumpf P.K_ ed. Academic Press, New York.
Taylor, D.C., Weber, N., Underhill, E.W., Po~meroy, M.K., Keller,
W.A., Scowcroft, W.R., Wilen, R.W., Maloney, M.M., Holbrook, L.A.
(1990) Storage protein regulation and lipid accumulation in
microspore embryos of Brassica napus L. Pl.anta 183, 18-26.
- 100 -
CA 02451941 2003-11-28
Von Arnold, S., Eriksson, T. (1981) in vi r studies of
adventitious shoot forn~ation in Pinus ,~ontorta. Can. J. Bat. 59,
870-874.
Von Arnold, S. and Hakman, I., 1988. Regulation of somatic
embryo development in Picea ba yes by abscisic acid (ABA).
Journal of Plant Physiology 132, 164-169.
Webster, F.B., Roberts, D.R., McInnis, S.N., Sutton, B.C.S.
(1990) Propagation of interior spruce by somatic embryogenesis.
Can. J. Res. 20, 1759-1765.
Woodstock, L.W. and Tao, K.-L. J., 1981. Prevention of
imliibitional injury in low vigor soybean embryonic axes by
osmotic control of water uptake. Physiol. Plant 51, 133-139.
Xu, N., Bewley, D.J. (1991) Sensitivity to abscisic acid and
osmoticum changes during embryogenesis in alfalfa (M~~.is~go
sativa) J. Exp. Bot. 42 ,821-826
Xu, N., Coulter, K.M. and Bewley, D.J., 1990. Abscisic acid and
osmoticum prevent germination of developing alfalfa embryos, but
only osmoticum maintains the synthesis of developmental proteins.
Planta 182, 382-390.
Zeevaart, J.A.D. and Creelman, R.A., 1988. Metabolism and
physiology of abscisic acid. Annual Review of Plant Physiology
and Plant Molecular Biology 39, 439-473.
- 101 -
