Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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USE OF POROUS MEMBRANE TO SUPPORT
DEVELOPING CONIFER SOMATIC EMBRYOS
FIELD OF THE INVENTION
The present invention relates to methods for producing plant embryos in vitro,
and
optionally producing plants from the plant embryos.
BACKGROUND OF THE INVENTION
The demand for coniferous trees, such as pines and firs, to make wood products
continues to increase. One proposed solution to the problem of providing an
adequate
supply of coniferous trees is to identify individual coniferous trees that
possess desirable
characteristics, such as a rapid rate of growth, and to produce numerous,
genetically
identical, clones of the superior trees by somatic cloning.
Somatic cloning is the process of creating genetically identical trees from
tree
somatic tissue. Tree somatic tissue is tree tissue other than the male and
female gametes.
In one approach to somatic cloning, tree somatic tissue is cultured in an
initiation medium
which includes hormones, such as auxins and/or cytokinins, that initiate
formation of
embryogenic cells that are capable of developing into somatic embryos. The
embryogenic cells are then further cultured in a maintenance medium that
promotes
multiplication of the embryogenic cells to form pre-cotyledonary embryos
(i.e., embryos
that do not possess cotyledons). The multiplied embryogenic cells are then
cultured in a
development medium that promotes development of cotyledonary somatic embryos
which can, for example, be placed within artificial seeds and sown in the soil
where they
germinate to yield conifer seedlings. The seedlings can be transplanted to a
growth site
for subsequent growth and eventual harvesting to yield lumber, or wood-derived
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products. The cotyledonary somatic embryos can also be germinated in a
germination
medium, and thereafter transferred to soil for further growth.
A continuing problem with somatic cloning of conifer embryos is stimulating
efficient formation of somatic embryos that are capable of germinating to
yield plants.
Preferably conifer somatic embryos, formed in vitro, are physically and
physiologically
similar, or identical, to conifer zygotic embryos formed in vivo in conifer
seeds. There is,
therefore, a continuing need for methods for producing viable conifer somatic
embryos from
conifer embryogenic cells.
SUMMARY OF THE INVENTION
The present inventors have discovered that a porous membrane, such as a nylon
membrane, can be used to support plant tissue during the development phase of
plant
somatic embryo production. The developing somatic embryos are disposed on a
porous
membrane which is either continuously or intermittently contacted with liquid
development
medium. For example, the porous membrane may be placed on an absorbent pad
which is
soaked in development medium so that the development medium passes through the
nylon
membrane and contacts the embryos. The nylon membrane bearing embryos is
typically
enclosed within a sealed space which contains a humid atmosphere that ensures
that the
embryos remain moist. The embryos should not be completely submerged in
development
medium. The present application describes a representative system for
intermittently
wetting the lower surface of a membrane that bears developing somatic embryos
on its
upper surface. Preferred porous membranes are sufficiently strong to resist
tearing when the
membranes are lifted in order to transfer somatic embryos from the development
stage to
subsequent stages of the somatic embryo production process.
Accordingly, in one aspect, the present invention provides a method for
developing
conifer, cotyledonary, somatic embryos, the method comprising the steps of (a)
disposing
conifer pre-cotyledonary somatic embryos on an upper surface of a porous
membrane
wherein the porous membrane does not absorb liquid development medium; (b)
intermittently contacting the lower surface of the porous membrane with liquid
development
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medium wherein the liquid development medium wets a portion of each somatic
embryo
disposed on the porous membrane but does not completely immerse the somatic
embryos in
liquid development medium; and (c) culturing the conifer pre-cotyledonary
somatic embryos
on the porous membrane for a period of time sufficient to develop conifer,
cotyledonary,
somatic embryos from the pre-cotyledonary somatic embryos.
In another aspect, the present invention provides a method for developing
conifer,
cotyledonary, somatic embryos, the method comprising the steps of: (a)
culturing conifer
somatic cells in, or on, an induction medium to yield embryogenic cells; (b)
culturing the
embryogenic cells prepared in step (a) in, or on, a maintenance medium to form
pre-
cotyledonary conifer somatic embryos; (c) disposing pre-cotyledonary conifer
somatic
embryos formed in step (b) on an upper surface of a porous membrane that does
not absorb
liquid development medium; (d) intermittently contacting the lower surface of
the porous
membrance with liquid development medium wherein the liquid development medium
wets
a portion of each somatic embryo disposed on the porous membrane but does not
completely
immerse the somatic embryos in liquid development medium; and (e) culturing
the conifer
pre-cotyledonay somatic embryos on the propous membrane for a period of time
sufficient
to develop conifer, cotyledonary, somatic embryos from the pre-cotyledonary
somatic
embryos.
BRIEF DESCRIPTION OF THE DRAWINGS
The foregoing aspects and many of the attendant advantages of this invention
will
become more readily appreciated as the same become better understood by
reference to the
following detailed description, when taken in conjunction with the
accompanying drawings,
wherein:
FIGURE 1 shows a representative system for intermittently or continuously
wetting
a porous membrane with liquid development medium, wherein the membrane
supports
developing plant somatic embryos.
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DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
Unless specifically defined herein, all terms used herein have the same
meaning as
they would to one skilled in the art of the present invention.
Unless stated otherwise, all concentration values that are expressed as
percentages
are weight per volume percentages.
In one aspect the present invention provides methods for developing conifer
cotyledonary somatic embryos. The methods of this aspect of the invention each
include the
step of culturing conifer pre-cotyledonary somatic embryos on a porous
membrane, that is at
least intermittently contacted with liquid development medium, for a period of
time
sufficient to produce conifer, cotyledonary, somatic embryos from the pre-
cotyledonary
somatic embryos.
The methods of the invention can be used to produce cotyledonary somatic
embryos
from any conifer, such as members of the genus Pinus, such as Loblolly pine
(Pinus taeda)
and Radiata pine. Again, by way of example, Douglas-fir cotyledonary somatic
embryos
can be produced by the methods of the invention.
Membranes that are useful in the practice of the present invention are porous,
have
no matrix potential (or substantially no matrix potential), and are
sterilizable.
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Examples of useful membranes include nylon membranes, nylon fiber, wire mesh,
plastic
mesh and polymeric fibers that do not absorb development medium. Useful pore
diameters in the porous membranes are in the range of from about 5 microns to
about 1200 microns, such as from about 50 microns to about 500 microns.
The development medium is a liquid medium. The development medium contains
nutrients that sustain the somatic embryos. Maltose may be included in the
development
medium as the principal or sole source of sugar for the somatic embryos.
Useful maltose
concentrations are within the range of from about 1 % to about 2.5 %. Suitable
development media typically do not include multiplication hormones, such as
auxins and
cytokinins.
The osmolality of the development medium can be adjusted to a value that falls
within a desired range, such as from about 250 mM/Kg to about 450 mM/Kg.
Typically,
an osmolality of 350 mM or higher is advantageous. An example of a suitable
development medium is described in Example 1 herein.
By way of example, pre-cotyledonary conifer somatic embryos may be cultured
on a nylon membrane, that is at least intermittently contacted with
development medium,
for a period of from 5 weeks to 12 weeks, such as from 8 weeks to 10 weeks, at
a
temperature of from 10 C to 30 C, such as from 15 C to 25 C, or such as from
20 C
to 23 C.
Liquid development media can, for example, be applied to an absorbent
substrate,
such as a substrate made from cellulose (e.g., cellulose fibers), such as one
or more filter
papers, or some other absorbent paper material. The substrate absorbs the
liquid
development medium which passes through a porous membrane disposed on the
substrate
and contacts conifer precotyledonary somatic embryos disposed on the nylon
membrane.
The development medium promotes the development of the conifer precotyledonary
somatic embryos to form cotyledonary somatic embryos.
Again, by way of example, the porous membrane can be contacted with liquid
development medium using an atomiser which sprays the porous membrane with
development medium. Typically, the somatic embryos are disposed on an upper
surface
of the membrane and the opposite, lower, surface of the membrane is sprayed
with liquid
development medium. By way of further example, the porous membrane bearing
somatic
embryos can be disposed over liquid development medium that includes a
rotating stir bar
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which rotates sufficiently fast to spray liquid development medium up onto the
lower
surface of the porous membrane.
FIGURE 1 shows a representative system 10 for continuously or intermittently
wetting a porous membrane with development medium, wherein the membrane
supports
plant somatic embryos. System 10 includes a first chamber 12 including a lower
surface 14, an upper surface 16, a front end 18, a rear end 20, a first side
22, and a second
side 24. Rear end 20 defines three air vents 26. First chamber 12 is supported
by four leg
members 28 that each extend vertically from lower surface 14 of first chamber
12 to a
base plate 30. Each leg member 28 includes a proximal end 32, attached to
lower surface
14 of first chamber 12, and a distal end 34 that rests upon base plate 30.
Distal end 34 of
each leg member 28 can be rotatably adjusted about the longitudinal axis of
leg member
28 to adjust the height of first chamber 12 relative to base plate 30.
Upper surface 16, lower surface 14, front end 18, rear end 20, first side 22,
and
second side 24 together define a first chamber cavity 36. Disposed within
first chamber
cavity 36 are two frames 38 that each include a frame body 40 that is
supported by four
vertically oriented frame legs 42. Stretched across each frame 38 is a
horizontally
oriented, porous, nylon membrane 44.
System 10 includes a second chamber 12' that includes the same components as
first chamber 12. Components of second chamber 12' have the same number as the
corresponding component in first chamber 12, except that the component number
used in
connection with second chamber 12' includes a prime (').
Thus, second chamber 12' includes a lower surface 14', an upper surface 16', a
front end 18', a rear end 20', a first side 22', and a second side 24'. Rear
end 20' defines
three air vents 26'. Second chamber 12' is supported by four leg members 28'
that each
extend vertically from lower surface 14' of first chamber 12' to a base plate
30'. Each leg
member 28' includes a proximal end 32', attached to lower surface 14' of
second chamber
12', and a distal end 34' that rests upon base plate 30'. Distal end 34' of
each leg member
28' can be rotatably adjusted about the longitudinal axis of leg member 28' to
adjust the
height of second chamber 12' relative to base plate 30'.
Upper surface 16', lower surface 14', front end 18', rear end 20', first side
22', and
second side 24' together define a second chamber cavity 36'. Disposed within
second
chamber cavity 36' are two frames 38' that each include a frame body 40' that
is supported
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by four vertically oriented frame legs 42'. Stretched across each frame 38' is
a
horizontally oriented, porous, nylon membrane 44'.
System 10 also includes a development medium reservoir 46 that includes a
reservoir body 48 that defines a cavity 50. An amount of liquid development
medium 52
is disposed within cavity 50. An air vent 54 penetrates medium reservoir body
48.
System 10 also includes a medium outlet tube 56 that includes a proximal end
58 that is
submerged within development medium 52 within reservoir cavity 50. Outlet tube
56 is
connected to a pump 60 that is controlled by a timer 61. Timer 61 is
optionally
programmable.
Outlet tube 56 bifurcates to form an outlet tube first portion 62 and an
outlet tube
second portion 64. Outlet tube first portion 62 is connected to a first
development
medium outlet 66 that penetrates lower surface 14 of first chamber 12 and
extends into
first chamber cavity 36. Outlet tube second portion 64 connects to a second
development
medium outlet 66' that penetrates lower surface 14' of second chamber 12' and
extends
into second chamber cavity 36'.
System 10 also includes a first drainage tube 68 having a proximal end 70
located
within medium reservoir cavity 50. First drainage tube 68 bifurcates to form a
first
drainage tube first portion 72 and a first drainage tube second portion 74.
First drainage
tube first portion 72 connects to a first medium outlet 76 that penetrates
lower surface 14
of first chamber 12. First drainage tube second portion 74 connects to a first
medium
outlet 76' that penetrates lower surface 14' of second chamber 12'.
System 10 also includes a second drainage tube 78 having a proximal end 80
located within medium reservoir cavity 50. Second drainage tube 78 bifurcates
to form a
second drainage tube first portion 82 and a second drainage tube second
portion 84.
Second drainage tube first portion 82 connects to a second medium outlet 86
that
penetrates (and is flush with) lower surface 14 of first chamber 12. Second
drainage tube
second portion 84 connects to a second medium outlet 86' that penetrates (and
is flush
with) lower surface 14' of second chamber 12'.
Developing pine somatic embryos 88 are shown disposed on nylon membrane 44.
In operation, pump 60 moves development medium 52 from reservoir 46 through
medium outlet tube 56 into first chamber 12 and second chamber 12' via outlet
tube first
portion 62 and outlet tube second portion 64, respectively. Pumped development
medium
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52 rises to the level of first medium outlet 76 and 76', and drains
therethrough into first
drainage tube first portion 72 and first drainage tube second portion 74,
respectively,
which direct the development medium back into medium reservoir 46. Pump 60 may
operate continuously or intermittently. The level of development medium 52
within first
chamber 12 and second chamber 12' is typically sufficiently high so that
development
medium 52 contacts nylon membrane 44, and thereby wets a portion of each
somatic
embryo 88 disposed on nylon membrane 44. Somatic embryos 88 should not be
completely immersed in development medium 52. A humid atmosphere within
reservoir
cavities 50 and 50' continuously moistens somatic embryos 88.
When pump 60 is inactivated, development medium 52 drains from first
chamber 12 and second chamber 12' through second medium outlets 86 and 86' and
is
directed back into reservoir 46 via second drainage tube 78.
First medium outlets 76 and 76' are each is set at a height within first
chamber 12
and second chamber 12', respectively, that corresponds to the desired level of
development medium 52 within first chamber 12 and second chamber 12'.
Typically the
height of first medium outlets 76 and 76' is the same as, or slightly greater
than, the
distance of nylon membranes 44 and 44' from first chamber lower surface 14 and
second
chamber lower surface 14', respectively. Consequently, while pump 60 is
activated, at
least a portion of each developing somatic embryo 88 is contacted with
development
medium 52. Pump 60 includes a programmable timer that activates and
deactivates
pump 60.
Second medium outlets 86 and 86' are flush with first chamber lower surface 14
and second chamber lower surface 14', respectively, so that when pump 60 is
inoperative
development medium 52 completely, or almost completely, drains from first
chamber 12
and second chamber 12'.
In some embodiments, the present invention provides methods for producing
conifer cotyledonary somatic embryos, wherein the methods each include the
steps of:
(a) culturing conifer somatic cells in, or on, an induction medium to yield
embryogenic
cells; (b) culturing the embryogenic cells prepared in step (a) in, or on, a
maintenance
medium to form pre-cotyledonary conifer somatic embryos; and (c) culturing pre-
cotyledonary conifer somatic embryos formed in step (b) on a porous membrane,
that is
at least intermittently contacted with liquid development medium, for a period
of time
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sufficient to produce conifer, cotyledonary, somatic embryos from the pre-
cotyledonary
somatic embryos. The conifer somatic cells, and resulting cotyledonary somatic
embryos, can be genetically-identical.
Thus, in some embodiments, conifer somatic cells are cultured in, or on, an
induction medium to yield embryogenic cells. Embryogenic cells are capable of
producing one or more cotyledonary conifer somatic embryos. Examples of
embryogenic
cells are embryonal suspensor masses (ESMs). The induction medium typically
includes
inorganic salts and organic nutrient materials. The osmolality of the
induction medium is
typically about 160 mM/kg or even lower, but it may be as high as 170 mM/kg.
The
induction medium typically includes growth hormones. Examples of hormones that
can
be included in the induction medium are auxins (e.g., 2,4-
dichlorophenoxyacetic acid
(2,4-D)) and cytokinins (e.g., 6-benzylaminopurine (BAP)). Auxins can be
utilized, for
example, at a concentration of from 1 mg/L to 200 mg/L. Cytokinins can be
utilized, for
example, at a concentration of from 0.1 mg/L to 10 mg/L.
The induction medium may contain an adsorbent composition, especially when
very high levels of growth hormones are used. The adsorbent composition can be
any
composition that is not toxic to the embryogenic cells at the concentrations
utilized in the
practice of the present methods, and that is capable of adsorbing growth-
promoting
hormones, and toxic compounds produced by the plant cells during embryo
development,
that are present in the medium. Non-limiting examples of useful adsorbent
compositions
include activated charcoal, soluble poly(vinyl pyrrolidone), insoluble
poly(vinyl
pyrrolidone), activated alumina, and silica gel. The adsorbent composition may
be
present in an amount, for example, of from about 0.1 g/L to about 5 g/L. The
induction
medium is typically solid, and may be solidified by inclusion of a gelling
agent.
Conifer somatic cells are typically cultured in, or on, an induction medium
for a
period of from 3 weeks to 10 weeks, such as from 6 weeks to 8 weeks, at a
temperature of
from 10 C to 30 C, such as from 15 C to 25 C, or such as from 20 C to 23 C.
The maintenance medium may be a solid medium, or it may be a liquid medium
which can be agitated to promote growth and multiplication of the embryogenic
tissue.
The osmolality of the maintenance medium is typically higher than the
osmolality of the
induction medium, typically in the range of 120-180 mM/kg. The maintenance
medium
may contain nutrients that sustain the embryogenic tissue, and may include
hormones,
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such as one or more auxins and/or cytokinins, that promote cell division and
growth of
the embryogenic tissue. Typically, the concentrations of hormones in the
maintenance
medium is lower than their concentration in the induction medium.
It is generally desirable, though not essential, to include maltose as the
sole, or
principal, metabolizable sugar source in the maintenance medium. Examples of
useful
maltose concentrations are within the range of from about 1 % to about 3.0 %.
Conifer
embryogenic cells are typically transferred to fresh maintenance medium once
per week.
Useful development media are described supra. After being cultured in
continuous, or periodic, contact with a development medium, the cotyledonary
somatic
embryos can optionally be transferred to a maturation medium, and then to a
stratification
medium, for a further period of culture.
The methods of the invention can be used, for example, to produce clones of
individual conifer trees that possess one or more desirable characteristics,
such as a rapid
growth rate. Thus, in one aspect, the present invention provides methods for
producing a
population of genetically-identical, conifer, cotyledonary, somatic embryos.
The
methods of this aspect of the invention each include the step of culturing
genetically-
identical, conifer, precotyledonary somatic embryos on a porous membrane
(e.g., porous
nylon membrane) that is in continuous, or periodic, contact with a development
medium,
for a period of time sufficient to produce genetically-identical, conifer,
cotyledonary,
somatic embryos from the precotyledonary somatic embryos, wherein the
development
medium passes through the porous membrane and contacts the somatic embryos.
The conifer cotyledonary somatic embryos produced using the methods of the
invention can optionally be germinated to form conifer plants which can be
grown into
coniferous trees, if desired. The cotyledonary embryos may also be disposed
within
artificial seeds for subsequent germination. The conifer cotyledonary somatic
embryos
can be germinated, for example, on a solid germination medium, such as the
germination
medium described in Example 1 herein. The germinated plants can be transferred
to soil
for further growth. For example, the germinated plants can be planted in soil
in a
greenhouse and allowed to grow before being transplanted to an outdoor site.
Typically,
the conifer cotyledonary somatic embryos are illuminated to stimulate
germination.
Typically, all the steps of the methods of the invention, except germination,
are
conducted in the dark.
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In another aspect, the present invention provides systems for developing plant
somatic embryos, wherein each system includes: (a) a medium reservoir
containing
liquid development medium; (b) a culture chamber comprising a body defining a
development medium inlet and a development medium outlet, wherein the
development
medium outlet is connected to the medium reservoir; (c) a porous membrane
disposed on
a membrane support within the culture chamber; and (d) a pump that is
connected to the
medium reservoir and to the development medium inlet of the culture chamber,
wherein,
in operation, the pump moves development medium from the reservoir to the
culture
chamber through the development medium inlet, and the development medium
drains
from the culture chamber through the development medium outlet and returns to
the
development medium reservoir. An example of a system of the present invention
is
shown in FIGURE 1. The system may optionally include a timer (e.g., a
programmable
timer) that is connected (e.g., electrically connected) to the pump, and that
activates and
deactivates the pump.
In the systems of the present invention, the development medium outlet is
spaced
relative to the membrane support so that the development medium does not
completely
cover developing plant somatic embryos disposed on the porous membrane (i.e.,
the
development medium outlet permits the development medium to drain out of the
culture
chamber before the medium completely submerges the embryos disposed on the
porous
membrane).
The following examples are provided for the purpose of illustrating, not
limiting,
the invention.
EXAMPLE 1
This Example shows a representative method of the invention for producing
somatic pine embryos from loblolly pine.
Female gametophytes containing zygotic embryos are removed from seeds four to
five weeks after fertilization. The seed coats are removed but the embryos are
not further
dissected out of the surrounding gametophyte other than to excise the nucellar
end. The
cones were stored at 4 C until used. Immediately before removal of the
immature
embryos the seeds are sterilized utilizing an initial washing and detergent
treatment
followed by a ten minute sterilization in 15% H202. The explants were
thoroughly
washed with sterile distilled water after each treatment.
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Tables 1 and 2 set forth the compositions of media useful for producing pine
somatic embryos.
TABLE 1
Pinus Taeda Basal Medium (BM)
Constituent Concentration (mg/L)
NH4NO3 150.0
KNO3 909.9
1042104 136.1
Ca(NO3)2.4H20 236.2
CaC12.4H20 50.0
MgSO4.7H20 246.5
Mg(NO3)2.61-120 256.5
MgC12.6H20 50.0
KI 4.15
H3B03 15.5
MnSO4.H20 10.5
ZnSO4.7H20 _________________________ 14.4
NaMo04.2H20 0.125
CuSO4.5H20 0.125
CoC12.6H20 0.125
FeSO4.7H20 27.86
Na2EDTA 37.36
Maltose 30,000.
myo-Inositol 200
Casamino acids 500
L-Glutamine 1000
Thiamine.HC1 1.00
Pyridoxine.HC1 0.50
Nicotinic acid 0.50
Glycine 2.00
GelriteTM 1600
pH adjusted to 5.7
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+Used if a solid medium is desired.
TABLE 2
Composition of Media for Different Stage Treatments
BMi¨Induction Medium BM+2,4-D (15 M)+Kinetin (2 M)+BAP (2 M).
BM2¨Maintenance Medium BM+2,4-D (5 M)+Kinetin (0.5 M)+BAP (0.5 M).
GELRITE (1600 mg/L) is added when a solid medium is
desired.
BM3¨Development Medium BM+25 mg/L abscisic acid + 12% PEG-8000 + 800 mg/L
additional myo-inositol + 0.1% activated charcoal + 1%
glucose, +2.5% Maltose. The following amino acid mixture
is added: L-proline (100 mg/L), L-asparagine (100 mg/L),
L-arginine (50 mg/L), L-alanine (20 mg/L), and L-serine
(20 mg/L). GELRITE (2500 mg/L) is added when a solid
medium is desired.
BM5¨Stratification Medium BM3 modified by omitting abscisic acid, and PEG-
8000.
GELRITE (2500mg/L) is added when a solid medium is
desired.
BM6¨Germination Medium BM modified by replacing maltose with 2% sucrose.
Myo-inositol is reduced to 100.0 mg/L, glutamine and
casamino acids are reduced to 0.0 mg/L. FeSO4.7H20 is
reduced to 13.9 mg/L and Na2EDTA reduced to 18.6mg/L.
Agar at 0.8% and activated charcoal at 0.25% are added.
Induction: Sterile gametophytes with intact embryos are placed on a solid BMI
culture medium and held in an environment at 22 -25 C with a 24 hour dark
photoperiod
for a time of 3-5 weeks. The length of time depends on the particular genotype
being
cultured. At the end of this time a white mucilaginous mass forms in
association with the
original explants. Microscopic examination typically reveals numerous early
stage
embryos associated with the mass. These are generally characterized as having
a long
thin-walled suspensor associated with a small head with dense cytoplasm and
large
nuclei.
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Osmolality of the induction medium may in some instances be as high as
150 mM/kg. Normally it is about 120 mM/kg or even lower (such as 110 mM/kg).
Maintenance and Multiplication: Early stage embryos removed from the masses
generated in the induction stage are first placed on a BM2 gelled maintenance
and
multiplication medium. This differs from the induction medium in that the
growth
hormones (both auxins and cytokinins) are reduced by at least a full order of
magnitude.
Osmolality of this medium 130 mM/kg or higher (typically within the range of
about
120-150 mM/kg for Pinus taeda) The temperature is again 22 -25 C in the dark.
Embryos are cultured 12-14 days on the BM2 solid medium before transferring to
a liquid
medium for further subculturing. This liquid medium has the same composition
as BM2,
but lacks the gellant. The embryos at the end of the solid maintenance stage
are typically
similar in appearance to those from the induction stage. After 5 to 6 weekly
subcultures
on the liquid maintenance medium advanced early stage embryos have formed.
These are
characterized by smooth embryonal heads, estimated to typically have over
100 individual cells, with multiple suspensors.
Embryo Development: embryo development is conducted using the system shown
in FIGURE 1.
The osmotic potential of this development medium may be raised substantially
over that of the maintenance medium. It has been found advantageous to have an
osmolality as high as 350 mM/kg or even higher. Development is preferably
carried out
in complete darkness at a temperature of 22 -25 C until cotyledonary embryos
have
developed. Development time is typically several weeks, such as 7 to 12 weeks.
Stratification: Cotyledonary embryos are singulated and transferred to
stratification medium BM5. This medium is similar to development medium but
lacks
abscisic acid, PEG-8000, and gellan gum. Embryos are cultivated on
stratification
medium at between about 1 C and about 10 C in the dark for between three to
six weeks.
Condidtioning over water: The mature embryos still on their nylon membrane
support are lifted from the pad and placed in a closed container over H20 at a
relative
humidity of 97%, for a period of about three weeks.
Germination: The dried mature embryos were rehydrated by placing them, while
still on the nylon membrane support, for about 24 hours on a pad saturated
with liquid
germination medium. The embryos were then placed individually on solid BM6
medium
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for germination. This is a basal medium lacking growth hormones which has been
modified by reducing sucrose, myo-inositol and organic nitrogen. The embryos
are
incubated on BM6 medium for about 12 weeks under environmental conditions
of 23 -25 C, and a 16 hour light-8 hour dark photoperiod, until the resulting
plantlets
have a well developed radicle and hypocotyl and green cotyledonary structure
and
epicotyl.
Because of the reduced carbohydrate concentration, the osmotic potential of
the
germination medium is further reduced below that of the development medium. It
is
normally below about 150 mM/kg (such as about 100 mM/kg).
EXAMPLE 2
This Example shows that Loblolly pine somatic embryos can be developed on
porous nylon membrane disposed on cellulose pads that are soaked in liquid
development
medium.
Embryo Treatment: Loblolly pine genotypes A, B, and C were bulked in a large
flask. 0.75 mls of cells were applied onto either WhatmanTM No. 4 filter
paper, or onto
nylon membrane (SeFar Co., Product No. 0-100-44 having 100 1.i.m pore size),
disposed
on a double layer of absorbent pads in a Petri plate. Each pad had a diameter
of 2" and
the double layer of pads was soaked with approximately 40 mls of development
medium.
After development treatment, all plates were stratified for four weeks in the
cold.
The embryos were then singulated on dry filter paper and suspended over water
in large
boxes for three weeks in order to condition the embryos. The embryos were then
imbibed
on liquid germination medium for 24 hours, then planted into solid germination
medium.
The germination boxes containing the germinating embryos were moved to the
light after
seven days of dark treatment.
Embryo Yield After Development Treatment: Embryos were counted at the
beginning of the conditioning treatment. Embryo numbers were calculated per
milliliter
of cells plated for each genotype. The embryo yield for embryos developed on
filter
paper (combining all genotypes) was 48 28. The embryo yield for embryos
developed
on nylon membrane (all genotypes combined) was 55 14.
The average percentage germination for embryos (all genotypes combined)
cultured on filter paper was 30 5. The average percent germination for embryos
(all
genotypes combined) cultured on nylon membrane was 32 7.
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CA 02563893 2010-05-25
Thus, there was not a statistically significant difference in the numbers of
embryos obtained after development, or germination, using filter paper
compared to
nylon membrane.
EXAMPLE 3
This Example describes the successful use of a bioreactor to develop Loblolly
pine somatic embryos on a nylon membrane that is intermittently contacted with
development medium.
Loblolly pine genotype A was used. 12 ml per treatment were plated in half
size
CambroTM boxes. Each plate had 0.5 mls of cells.
The bioreactor system shown in FIGURE 1 was used to perform the experiments
described in this example.
Four development treatments were used: Treatment 1 had 10% C.C. (cellulose)
pad with a nylon membrane (100 vim pore size) disposed on the pad; Treatment 2
had 10% C.C. pad with filter paper (Whatman #4 fp.) instead of the nylon
membrane;
Treatment 3 had filter paper disposed on top of the nylon membrane (no pad);
Treatment 4 had only nylon membrane (no pad, no filter paper).
Development medium was pumped into the Cambro boxes until the medium
touched the nylon membrane. The medium started draining 15 minutes after
pumping
stopped. The pump delivered medium once every 24 hours.
The experiment was stopped after 8 weeks. Every treatment produced embryos.
In particular, good quality (zygotic-like) embryos developed on the membrane
that was
not supported by a cellulose pad.
While the preferred embodiment of the invention has been illustrated and
described, it will be appreciated that various changes can be made therein
without
departing from the spirit and scope of the invention.
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