Note: Descriptions are shown in the official language in which they were submitted.
8~3
Descri~tion
Improved Somatic Embryogenesis Employing Or~anic Acids
Technical Field
The present discovery relates generally to somatic
embryogenesis in plant cells and, more particularly, to
methods and materials for increasing the quantity and
quality of plant embryos produced from somatic cell
cultures.
Background of the Invention
The process of plant cell cloning can be
accomplished by a variety of methods, one of which is
somatic embryogenesis. This method offers advantages of
scale and efficiency not found in other methods of cell
culture since, in one step, somatic embryos develop both
a root and shoot. This method of cloning also provides
an opportunity for commercial scale production of naked
or encapsulated somatic embryos. These embryos can be
germinated and provide a substitute for true plant seed.
Evans, D.A. et al., "Growth and Behavior of cell
cultures: Embryogenesis and organogenesis in Plant
Tissue Culture," T . A . Thorpe, e.d., Academic Press, New
York, pp. 45-113 (1981).
When practicing cloning by somatic embryogenesis,
one must be mindful of the genotypic background of the
explant source as well as the physiological conditions
of culture. By understanding in detail the
physiological conditions of regeneration, it is
generally believed that the ~enetic background of the
plant material will become a less important factor. For
example, attempts to regenerate soybean through somatic
embryogenesis for several decades were not successful.
Recently, a defined set of conditions have been
described for soybean regeneration. Christianson, M.L.
~L2~ 3Z3
et al~., "A morphogenetically competent soybean
suspension cul~uxe," Science 222:632 (1983). Thus, a
genotype previously impossible to manipulate in vitro
(the cultivated soybean) can now be regenerated as a
result of a breakthrough in cell culture conditions.
There are two major drawbacks to using somatic
embryos as a commercial scale cloning methodO First, in
many crops the yield or number of somatic embryos
produced in plant cell culture is low. Improvements in
the efficiency of embryo yield is always a concern in
production systems. Techniques for somatic
embryogenesis in carrot have been sufficiently developed
so that 90% of the cell culture produces somatic
embryos. Fujimura, T. and A Komamine, "Synchronization
of Somatic Embryogenesis in a Carrot Cell Suspension
Culture," Plant Physiol. 64:162-164 (1979). It would
be desirable to obtain similar rates of embryo formation
in other species as well.
A second obstacle to commercial scale cloning of
plant~ through somatis embryogenesis is the failure of
most embryos to develop into plants upon germination.
Lutz, J.D. et al., "Somatic Embryogenesis for Mass
Cloning of Crop Plants" in Tissue Culture in Forestry
and Aqriculture, R.R. Henke, K.W. Hughes,
M.J. Constantin, and A. Hollaender, eds., Plenum Press,
New York, pp. 105-116 (1985). For example, studies
using naked or encapsulated somatic embryos of carrot
found that evsn when the best known germination
conditions are employed, an extremely low percentage of
somatic embryos ever germinate and develop into
plantlets. Drew, R.K.L., "The Development of Carrot
(Daucus carota L.) Embryoids (derived from cell
suspension culture into plantlets on a sugar-free basal
medium). Hort. Res. 19:79-84 (lg79). Kitto, S.L. and
J. Janick, "Production of Synthetic Seed by
~X~ 23
Encapsulating Asexual Embryos o~ Carrot, 11 J. American
Hort. Society, 110:277-282 (1985). To be commercially
useful, somatic embryos mus~ germina~e as rapidly as
seed, and grow out with a vigorous shoot and root.
Typical subculture schemes for plant cell culture
utilize media formulations composed of inorganic
components, vitamins, phytohormones, and a carbohydrate
source. In general, plant cell culture involves
separate stages: (1) callus growth (maintenance~, ~2~
induction to alter the developmental pathway of callus
cells, t3) regeneration o~ shoots on emhryos from
callus, and ~4) production of complete plants. Sharp,
W.R. et al., "The Physiology of In Vitro Asexual
Embryogenesis," Hort. Rev., 2:268-310 (1980). Tisserat,
B., "Somatic Embryogenesis in Angiosperms," Hort. Rev.,
1:1_78 (1979). deFossard, R.A., "Tissue Culture for
Plant Propagators," University of New England Printery,
Armidale, New South Wales, Australia, p. 409 tl976).
The effect of organic acids upon plant cell
cultures has been studied on a number of occasions. In
general, organic acids have been used as a nitrogen
source or as buffering agents. Cells of soybean, wheat,
and flax can be grown for extended periods when ammonium
is the sole source of nitrogen if citrate, malate,
fumarate, or succinate are included in the medium.
Gamborg, O.L. and J.P. Shyluk, "The Culture of Plant
Cells with Ammonium Salts as the Sole Nitrogen Source,"
Plant Physiology, 45:598 (1~70).
Tobacco c~lls will grow on ammonium as a sole
source of nitrogen only i~ succinate, malate, fumarate,
citrate, ~-ketoglutarate, glutamate, or pyruvate are
included in the cell culture medium. This efect was
attributed to the need for additional carbon skeletons
for amino acid synthesis through the condensation of
ammonium and ~-ketoglutarate. Behrend, J. and R.E.
~L2~8~3
Mateles, "Nitrogen Metabolism in Plant Cell Suspension
Cultures," Plant Physiol., 58:510 (1976).
Cell growth can be stopped in soybean cultures if
the cells are transferred onto urea as a sole nitroyen
source in a medium containing 10mM citrate. Polacco,
J.C., "Nitrogen Metabolism in Soybean Tissue Culture. I.
Assimilation of Urea," Plant Physiol., 5g:350 (1976).
The citrate effect can be reversed by adding ammonium or
nickel sulfate. This effect has been attributed to the
ability of citra~e to chelate trace amounts of nickel --
a co-factor of urease in plants. Polacco, J.D.
"Nitrogen Metabolism in Soybean Tissue Culture. II.
~rea Utilization and Urease Synthesis Require Ni2~,"
Plant Physiol., 5g:827 (1977). In this instance,
citrate is used to stop cell growth.
The effect of citrate and ~-ketoglutarate was
studied in alfalfa, wheat, and tobacco cells grown on
either ammonium, nitrate, or glutamine. The growth of
cultures was more successful on ammonium medium,
especially if ~-ketoglutarate was used. Growth rates
were found to be poor if citrate was used in place of
~-ketoglutarate. Fukanaga, Y. et al., "The Differential
Effects of TCA-cycle Acids on the Growth of Plant Cells
Cultured in Liquid Media Containing Various Nitrogen
Sources, Planta 139:199 (1978).
In carrot cell cultures grown on ammonium as the
sole source of nitrogen, succinate, fumarate, malate,
~-ketoglutarate, glutamate, maleate, malonate, tartarate,
and citrate were all found to support growth of the cell
cultures. Dougall, D.K. and K.W. Weyrauch, "Abilities
of Organic Acids to Support Growth and Anthocyanin
Accumulation by Suspension Cultures of Wild Carrot Using
Ammonium as the Sole Nitrogen Source, In Vitro 16:969
(1980). The observed growth in responsa to these
organic acids was attributed to the pH buffering effect
~9~ 3
--5--
on the culture medium. Apart from cell growth, an
effect of organic acids on the subsequent regeneration
of the cultures was not observed.
In the above studies, organic acid additions to the
cell culture medium were not shown to have an effect on
somatic embryogenesis.
In carrot cell cultures, somatic embryogenesis is
weak or absent if ammonium succinate medium is used for
regeneration, but embryo yields are high if ammonium or
glutamate is used. Wetherell, D.F~ and D.K. Dougall,
"Sources of Nitrogen Supporting Growth and Embryogenesis
in Cultured Wild Carrot Tissue," Physiologia Planturium
32:97 (1976). This result suggests that succinate is
deleterious to embryogenesis of plant cell cultures.
In eggplant, various concentrations of ammonium
ci~rate and potassium nitrate have been used in the
regeneration medium (Gleddie, S., "Somatic Embryogenesis
and Plant Regeneration from Leaf Explants and Cell
Suspensions of Solanum meloqena (eggplant)," Can. J.
Bot~ 6:656 (1~83)). The optimal~ratio of ammonium to
nitrate was found to be 2:1 (20mM NH4+:40mM N03-). If
ammonium citrate concentration was increased above the
determined optimum, embryogenesis was found to be
inhibited. At 20mM citrate, embryogenesis was totally
blocked. The optimization of embryogenesis in this
instance was attributed to the ~avorable level of NH
and N03- in the medium. Citrate was not considered
important in this response.
In rice cell cultures, succinate has been shown to
inhibit callus growth in the presence of NH4+ and N03-
but will stimulate growth if ammonium is the sole
nitrogen source. Regeneration in th~ latter treatments,
however, was poorer than with cells not treated with
succinate, indicating that organic acid pretreatment is,
by itself, deleterious to subsequent regeneration and
12~ 3
embryogenesis in cell cultures when compared to the
other treatments tested. Chaleff, R.S. "Induction,
Maintenance and Differentiation of Rice Callus Cultures
on Ammonium as Sole Ni~rogen Source," Plant Cell Tissue
organ Culture 2:29 (1983).
Finally, studies using soybean cell cultures grown
on 40mM NH4+ (as ammonium citrate) in the absence of
nitrate reported successful embryo development when
cultures were transferred to Murashige and Skoog (MS)
medium. Christianson, M.L., et al., "A
Morphogenetically Competent Soybean Suspension Culture,"
Science 222:632-634 (1983). Christianson, M.L. "An
Embryogenic Culture o~ Soybean. Towards a General
Theory o~ Somatic Embryogenesis" in Tissue Culture in
Forestry and A~riculture, R.R. Henke, K.W. Hughes, M.L.
Constantin, and A. ~ollaender eds., Plenum Press, New
York, pp. 83-103 (1985). Murashige, T. and F. Skoog, "A
Revised Medium for Rapid Growth and Bioassays with
Tobacco Tissue Cultures," Physiologia Plantarum 15:473-
497 ~1962). The author(s) point out that the hallmark
of this procedure of regenerating soybean involves the
"coordinate removal of 2,4-dichlorophenoxyacetic acid
(an auxin) and a change from 40mM ammonium to 20mM
ammonium and 50mM nitrate." According to the authors, a
change in nitrogen source is essential to the technique.
Christianson, M.L. et al., loc. cit., Christianson,
M.L., loc. cit o In so concluding, the authors teach
against using citrate as an additive to cell cultures to
improve the yield and maturation of somatic embryos. In
recent experimenks with soybean cultures it has been
shown that ammonium citrate inhibits the development of
soybean embryos when added to the regeneration medium
whereas potassium citrate fails to inhibit embryo
expression. ~.L. Christianson loc. cit. It was
concluded that 40mM NH4~ has an adverse effect on embryo
~3~23
expression if included in the regeneration medium. It
was also concluded that potassium citrate has no effect
on embryo expression.
The above examples teach that organic acids, such
as citrate or succinate, have a neutral or deleterious
effect on somatic embryogenesis. In all of these
examples, the authors concluded tha~ the main effector
of somatic embryo production is the level of nitrogen,
either as nitrate or ammonium. Thus, organic acid
addition has been thought to have either a neutral role,
as with eggplant or soybean somatic embryogenesis, or
inhibit somatic embryogenesis, as was the case with rice
somatic embryogenesisO
Disclosure of the Invention
This invention provides materials and methods for
improving the yield and quality of somatic embryos from
plant cell culture by treating cell cultures with
organic acids prior to somatic embryo development.
These materials and methods also improve the
gexminability of somatic embryos produced in cell
cultures so treated.
The present invention provides a medium for
subculturing cultured plant cells which i5 comprised of
at least one organic acid in a balanced salt solution
containing the nutritional and growth factors necessary
for plant growth. This medium contains an amount of the
selected organic acid(s) sufficient to produce cells
which possess an improved ability to undergo somatic
embryogenesis when exposed to hormone and nutrient
conditions avorable ~or regeneration.
Another aspect of this invention provides a medium
for subculturing cultured plant cells which is comprised
of at least one organic acid, together with reduced
nitrogen additives, such as amino acids, in a balanced
salt solution containing the nutritional and growth
factors necessary for plant growth. This medium
contains sufficient amounts of the selected organic
acid(s) and reduced nitrogen to produce cells which
possess an improved ability to undergo somatic
embryogenesis when exposed to hormone and nutrient
conditions favorable for regeneration.
A further aspect of ~he present invention provides
a method for culturing cells utilizing the media of this
invention. The somatic embryos resulting Prom such a
pretreatment also show superior quality as measured by
improved germinabili~y and conversion to plants.
Brief Descripkion of the Drawinas
Figure 1 is a graphic representation of a treatment
scheme used to sllbculture, induce and regenerate alfalfa
somatic embryos, and to form plants from the embryos,
including a pretreatment period in accordance with the
present invention, which represents a specialized case
of the maintenance culture of alfalfa cells; and
Figure 2a-e are graphic representations of the
yield o~ somatic embryos produced in response to a
pretreatment of cell cultures of alfalfa with various
organic acids.
Modes of Practicin~ the Invention
This invention provides novel and improved methods
and materials for producing numerous high quality
somatic e~bryos from plant tissue by the addition of
organic acids and reduced nitrogen to cultures prior to
the processes of somatic embryogenesis and plant
formation.
In the practice of the present invention, various
organic acids are employed. These acids are generally
~7~3
dicarboxylic acids wherein ~he carboxyl groups are
separated by a chain of five or less carbon ~toms, e.g.
~OH
c=o
(CH2)n
C=O
\OH
Alternatively, the carbon chain in an organic acid can
include CHOH groups replacing one or more CH~ groups.
Representative organic acids include oxalic, malonic,
succinic, glutaric, maleic, pimelic and tartaric acids,
among others.
Additional benefits of the present invention can be
obtained by the inclusion of a source of reduced
nitrogen in the pretreatment medium. As an example of
known sources of reduced nitrogen, amino acids can be
employed with good effect. Among the preferred amino
acids useful in the present invention are proline,
alanine, glutamine, arginine and asparagine.
Numerous important crop and horticultural species
have been shown to be capable of propagation through
tissue culture and somatic embryogenesis. For a lengthy
but by no ~eans exhaustive list of species capable of
somatic embryogenesis, see Evans, D.A., et al., "Growth
and Behavior of Cell Cultures: Embryogenesis and
Organogenesis" in Plant Tissue Culture: Methods and
APplications in Aariculture, T. ~horpe, ed., Academic
Press, pg. 45 et ~. (1981).
In alfalfa tMedicago sativa L.)~ a representative
species of the plant family Fabaceae, embryogenesis can be
routinely induced in the Regen S line. Saunders, J.W. and E.T.
Bingham, "Production of Alfalfa Plants from Callus Tissue,"
Crop Sci. 12:804-808 (1972). A representative protocol for
alfalfa somatic embryogenesis is provided therein and,
b~- in the practice of the present invention, modifications
--10--
to such procedures included a pretreatment period,
generally as follows:
E~perimental
Definition of Pretreatment Period
According to published procedures, alfalfa cell
cultures can be obtainQd from explants, such as leaf
petioles placed on Schenk-Hildebrandt (SH) bas~d medium
(Schenk, R.V. and A.C. Hildebrandt, Can. J. Bot.,
50:199-204 (197~)) with 3% (w/v) sucrose also containing
25~M ~-Naphthaleneacetic acid (~-NAA) and 10~M kinetin
(maintenance period). Cell cultures can be maintained
by repeated subcultures onto resh medium of the same
kind. Walker, K.~. and S.J. Sato, 'IMorphogenesis in
Callus Tissue of Medicago sativa: The Role of Ammonium
Ion in Somatic Embryogenesis," Plant Cell Tissue Organ
Culture 1:109-121 (1981). As depicted in Figure 1, the
period of cell subculture immediately preceding
induction is herein termed the pretreakment period.
This period is generally 14 to 21 days long.
Cells are subsequently treated with SH containing
3% sucrose, 50~M 2,4~dichlorophenoxyacetic acid (2,4-D)
and 5~M kinetin for 3 to 4 days (Induction). Transfer
of cells to regeneration medium results in the
development of embryos. This regeneration medium is
characterized as having reduced or no hormones but
containing ammonium ion and other supplements such as
amino acids or carbohydrate sources other than sucrose,
in the case of alfalfa. The final step in plant
production is conversion, which occurs on a simple salt
medium with sugar but not hormones (Figure 1). The
effect of pretreatment conditions, therefore, i5
assessed by measuring embryogenesis and plant formation
which occurs after pretreatment, induction and
regeneration phases of culture. Measurements of embryo
~2~8~3
yield, shape, structure, and overall vigor in
germination or conversion assays are used to determine
the effectiveness of the pretreatment conditions on
somatic embryo quantity and quality. Consequently, the
pretreatment period occurs well before any embryos are
initiated or formed.
In other emhryogenesis methods or protocols the
maintenance medium and induction medium, as these terms
are used here, are often one and the same. That is,
other methods utilize nutrient and hormone conditions
which cause plant cell growth and also predispose
cultures to or initiate the process of embryogenesis.
In this sense, the pretreatment medium would be a medium
on which plant cultures have been incubated on prior to
transfer to regeneration or embryo develop~ent medium.
In a representative protocol of the present
invention, the steps include:
Maintenance. Plants of edicago satlva L. Cultivar
Regen S derived from the second cycle recurrent
selection for regeneration from the cross of the
varieties Vernal and Saranac were used. Callus was
initiated by surface sterilizing petioles with 50%
Clorox~ for ~ive minutes, washing with H20 and plating
on Schank-Hildebrandt medium (SH). The medium contained
25~M ~-Naphthyleneacetic acid (~-NAA) and lO~M kinetin
(SH or other cell growth medium so modified is termed
maintenance medium). The ionic and organic chemical
constitution of SH medium is summarized in Table 1.
-12-
Table 1
The Composition of Schenk and ~ildebrand'c Medium
Ma~or Salts mM
KN03 25.0
CaC12 1. a~
MgSO4 1.6
~H4H2P04 2.6
Micronutrient Salts ~
KI 6.0
H3BO3 80.0
MnSO4 60.0
ZnS04 3.5
Na2MoO4 0.4
CUSO4 0.8
CoC12 0.4
Na2-Ethylene diaminetetraacetic acetic 55.0
FeSO4 55.0
2~
Or~anic ComPounds mq/l
Inositol 1000.0
Nicotinic acid ~5.0
Thiamine ~Cl 5.0
25 Note: No hormones added unless specified.
Carbohydrate ~/1
Sucrose 30.0
Gamborg, O.L. et al., "Plant Tissue Culture Media,"
In Vitro 12:473-478 (1976).
Callus which is formed on the explant tissue was
separated from the r~maining uncallused tissue and
repeatedly subcultured on maintenance medium. Callus
was subcultured at three week intervals and grown under
indirect light at 27C.
Pretreatment. Culture pretreatment wa~ given by
varying the components of the maintenance medium to
include filter sterilized organic acids and/or amino
acids. This medium in all instances included 25~M ~-NAA
and 10~M kinetin to promote callus growth. This medium
is referred to herein as pretreatment medium.
-13-
Pretreatment was carri~d out for 21 days (one subculture
cycle) unless otherwise noted.
Induction. Three to nine grams of callus were
collected at 17 to 24 days post-subculture from plates
of maintenance or pre~reatment medium and transferred to
lOOmm x 15mm plates o~ agar solidified containing 50~M
2,4-dichlorophenoxyacetic acid (2,4-D) and 5~M kinetin
for induc~ion. Walker, X.A., et al.,"Organogenesis in
Callus ~issue of Medicago sativa: The Temporal
Separation of Induction Processes from Differentiation
Processes," Plant Sci. Lett. 16:23_30 (1979). This
medium is termed the induction medium. Cells were
cultured for thrae days at 27C under indirect light.
Re~enerat on. Induced callus was squashed with a
spatula and transferred to regeneration medium. For
replicate treatment, 75mg fresh weight of callus was
maasured using a calibrated stainless steel scoop.
Alternatively, induced cells were aseptically sized on a
series of column sieves (Fisher Scientific) under gentle
vacuum. Cell clumps either fell or were forced through
a 35 mesh (480~M) and collected on a 60 mesh (230~M)
stainless steel screen. Cells retained on the 60 mesh
screen were washed with 500ml of SH minus hormone medium
for every three plates of induction culture volume. The
washing medium was removed by vacuum. The fresh weight
of the cell clumps was measured and cells were
resuspended in SH medium without hormones at 150mg fresh
weight per milliliter. Seventy-five mg (0.5ml) of
resuspended cells were pipeted onto approximateiy lOml
of agar solidified medium in 60mm x 15mm petri dishes.
Somatic embryogenesis will also occur in suspension
culture if 300mg (2ml) of resuspended cells are
delivered to 8ml of liquid SH medium contained in a 50ml
erlenmeyer flask.
-14~
Regeneration medium used in many of the examples
contained SH medium with 3% (w/v) sucrose and with no
hormones. The total NH4+ level was lOmM and the L-
proline level was 30mM. These components were added
after autoclaving from a ~ilter sterilized stock
solution. Medium, when solidi~ied, contained 0.8% (w/v)
tissue culture quality agar.
Each treatment was generally replicated ten times.
Dishes were Parafilm~ wrapped and incubated for 21 days.
SuspPnsion flasks were foam plugged, sealed with Saran
Wrap~ and incuba~ed for 14 days on a orbital shaker at
lOOrpm. Incubation was at 27C under 12 hour
illumination from cool white fluorescent tubes at 28cm
away from solidified cultures or 2m from suspension
cultures.
Embryogenesis was visually measured after incubation
by counting green centers of organization on th~ callus
using a stereomicroscope at a magnification of lOX.
Embryo size was measured using a calibrated ocular scale
at lOX magnification. Embryo shape was determined by
visual examination.
Conversion. Conversion of embryos to whole plants
with root and shoot axis with the first primary leaf was
done by aseptically transferring embryos from selected
treatments at 21 days of initial culture to
half-strength SH medium and solidified with 0.8% agar.
The above specification can be altered to contain
less or more of any particular chemical component
without broadly affecting the final embryo number or
quality. Thus, pretreatments involving organic acids
improved not only the yield of somatic embryos but also
the quality o~ somatic embryos as measured in conversion
assays. This result was unexpected as the organic acid
pretreatment of callus tissue incapable of embryogenesis
without further treatment was not expected to have an
~2~78~3
effect on ~mbryo development. Furthermore, th~ effect
of the organic acid in the cell cultures was to reduce
the growth rate siynificantly. This result was
surprising since pretreatment of callus with organic
acid ends 15 days before the first appearance of embryos
in the culture on regeneration medium. (Figure 1).
Such dramatic effects of culture treatment prior to the
addition of the active induction agent have not
previously been described for plant cell culture.
Exam~le 1. Somatic Embryo Formation in Response to
Organic Acid Pretreatment
Citrate was added to the pretreatment medium for 21
days at concentration of 2.5, 10, 15, 20, 25 or 30mM
with 25mM L-glutamine. After pretreatment, the callus
was removed from the medium and processed through the
induction and regeneration media as previously described
(Figure 1). At 20mM citrate, embryo yield (numbers~ at
the end of the regeneration period was increased to 290
as compared to 150 for a pretreatment without citrate
(Figure 2a~.
la. Malate was substituted for citrate at
concentrations of 10, 20, 30, 40, 50, 60, 70 and 100mM.
Embryo yield was increased to between 200 and 300 (60mM
malate produced 300 embryos) when malate was used. The
highest malate concentration of 100mM had a yield equal
to the control (130 embryos) without organic acids
(Figure 2b).
lb. Succinate was substituted for citrate at
concentrations of 10, 20, 30, 40 and 50mM. Embryo
yields were increased only at the highest concentration.
With 60mM succinate the yield was 190 embryos as
compared to the control of 60 embryos tFigure 2c~.
lc. Tartarate was substituted for citrate at
concentrations of 10, 20, 30, 40, 50, 60, 70 and 100mM.
..
-16-
All but the highest concentration improved embryo yield.
At 70mM tartrate, yield was 330 as compared to 180 for
the control (Figure 2d).
ld. Oxalate was substituted for citrate at
concentration of 2.5, 10 and 2OmM. Embryo yield was
increased at 10mM (190 embryos) as compared to the
control at 80 embryos.
As demonstrated by these examples, a number of
organic acids were discovered to be effective over a
broad concentration range in improving embryo quality
and yield (Table 2).
Table 2
Organic Acid Activity on Improved
Regeneration of Alfalfa Somatic Em~ryos
-
Organic Acid Concentration Range Approximate
(mM~ Optimum
(m~)
- -
Citric 10-25 20
L-Malic 10-100 60
Succinic 30-~0 50
25L-(+) Tartaric 10-100 70
Oxalic 5-20 10
Exampls 2. The Response to Citrate and Minus-Nitrate
Pretreatment Conditions
The effect of citrate pretreatment on somatic
embryo yield was investigated as described in Example l
with a variety of additives to the pretreatment medium
(Table 3).
78;2:3
Table 3
Effect of Citrate Pretreatment in the
Presence and Absence of Nitrate on
Subsequent Embryogenesis of Al~al~a Cultures
-
Callus Pretreatmenk Embryo Yield
Maintenance Medium (SH plus 3% sucrose 301 + 17
plus 25~M ~ NAA plus lO~M kinPtin)
Maintenance Medium plus 20mM512 + 33
K2-citrate
Naintenance Medium minus N03-22 + 5
Maintenance Medium plus 20mM249 + 13
K2-citrate minus N03-
Maintenance Medium plus 2smM255 + 13
L-glutamine
Maintenance Medium plus 20mM543 + 25
K-citrate plus N03
Maintenance Nedium plus 25mM533 + 23
L-glutamine plus 2OmM K2-citrate
minus N03-
Addition of 20mM potassium citrate (K-citrate) alone
caused a 70~ increase in embryo yield with no other
medium modifications. Deletion of nitrate from the
pretreatment medium inhibited the embryogenesis
response, and hence, minus nitrate medium alone did not
stimulate embryogenesis. Citric acid, supplied here as
K2-citrake, provided the best embryogenesis from callus
pretreated with this organic acid. By providing only
glutamine to the maintenance medium, which acts as an
alternative nitrogen source for ammonium or nitrate,
lower embryo yields resulted. If citrate was added in
the presence of glutamine an increase in embryogenesis
resulted even if nitrate was present or absent.
~9~78~
-18-
Example 3. _Use of OrgLanic Acid Pretreatments in
Combination with Amino Acids Supplements
The effect of citrate pretreatment on somatic
embryo yield was investigated as described in Example 1
with the addition of amino acids (Table 4: A&B).
The addition of only amino acids as a pretreatment
to cell cultures did not stimulate embryogenesis. If
the amino acids L-glutamine or L-proline were used in
the pretreatment medium with 20mM K2-citrate, however,
embryogenesis was stimulated. In the case of proline
plus citrate pretreatment, the highest embryo yields
were achieved. The positive amino acid response was
seen even when the pretreatment medium with citrate
contained no nitrate (Table 4B).
~2~23
--19--
Table 4
The Effect of Pretreating Cell
Cultures with organic Acids and Amino Acids
. . .
Pretreatment Medium Somatic Embryo Yield
A. ~
Main~enance Medium 486 + 19
Maintenance Medium plus 25mM 355 + 19
L-glutamine only
Maintenance Medium plus 25mM 510 + 18
~-glutamine plus 2OmM K2-citrate
Maintenance medium plus 25mM 428 + 12
L-proline only
Maintenance medium plus 25mM 620 + 33
L-proline plus 20mM K2-citrate
B.
Maintenance Medium 288 + 16
Maintenance Medium plus 25mM L-proline 391 + 18
plus 20 mM K-citrate minus N03-
Maintenance Medium plus 25mM L-alanine 328 + 14
plus 20mM K-citrate minus N03-
As dPmonstrated in these examples, a number of
amino acids, when used in conjunction with organic
acids, were effective over a broad range of
concentrations in improving embryo quality and yield
(Table 5).
Table 5
Effective levels of amino acids used in embryogenesis
pretreatment medium in combination with organic acids.
Amino AcidEffective Concentration Range
.~
L-proline 10 to 300 mM
L-alanine 10 to 200 mM
L-glutamine 5 to 100 mM
L-asparagine 2.5 to 80 mM
L-arginine 2.5 to 80 mM
-20-
Example 4. Effect of Orqanic ACid Pretreatment on
Somatic Embryo Conversion to a Plantlet
The effect of organic acid pretreatment in somatic
embryo quality, measured in terms of conversion-to-
plant frequency, was determined as describe~ in Example
1 (Table 6A-C).
Table 6
The Effect of Organic Acid Pretreatment
on Somatic Embryo Conversion to Plantlets
Pretreatment Conversion Percentage
Maintenance medium 22 + 3
Maintenance medium plU5 25mM 25 + 5
L-glutamine
Maintenance medium plus 25mM 38 + 7
L-glutamine plus 20mM citrate
.
B. -
Maintenance medium 32 + 2
Maintenance medium plus 25mM
L-glutamine plus 20mM citrate 47 + 2
Maintenance medium plus 25mM
L-glutamine plus 50mM malate 50 + 4
Maintenance medium plus 25mM
L-glutamine plus 70mM tartrate 48 + 5
. .
C .
Maintenance medium 5
Naintenance medium plus 25mM 43
L-glutamine plus 20mM citrate
Maintenance medium minus NO3- plus 38
25mM L-glutamine plus 20mM cikrate
The performance of a naked embryo produced in vltro
was measured by evaluating the development of entire
plantlets from a population of somatic embryos. This
~7~23
-21-
value is called the conversion percen~age or frequency,
which is analogous to the germination percentage or
frequency of true seeds. Improvements in conversion
frequency occurred as a result of pretreating callus
with organic acids prior to induction and regeneration
of somatic embryos.
With pretreatment of callus cultures with organic
acids, citrate, malate, succinate and tartrate, embryo
conversion to plantlets was significantly improved by 50
percent or more in each experiment.
Exam~le 5. Effect of Orqanic Acid Pretreatment on
Callus Growth Rate an_d OveralL ~fficiency of Plant
Production.
The effect of citrate pretreatment on somatic
embryo yield and guality was investigated as described
in Example 1 by comparing the overall effect of
maintenance medium with and without organic acids.
One gram of callus when placed for 21 days on
maintenance medium yielded 3.9 + 0.5g. callus. From one
gram of the callus, 8,400 somatic embryos were produced
based on the ~ubsample using the induction and
regeneration conditions previously described. Of these
embryos, 1,800 converted to whole plants, again using a
subsample.
One gram of callus when placed for 21 days on
pretreatment medium consisting of maintenance medium
with 25mM glutamine, 25mM K-citrate, and no nitrate
yielded significantly less callus, 2.2 + 0.lg. From
one gram of this callus, 8,000 somatic embryos were
produced, based on a subsample. Of these embryos 3,400
converted to whole plants, again using a subsample.
one gram of callus when placed for 21 days on
pretreatment medium consisting of maintenance medium
with 25mM glutamine, 25mM K-malate, and no nitrate
z~
yielded significantly less callus, 1 6 + 0.lg, than the
control. From one gram of this callus, 5,600 embryos
were produced, based on a subsample. Of these e-mbryos,
2,200 converted to whole plants, again using a
subsample.
Obviously, many modi~ications and varia~ions of the
present invention are possible after consideration of
the present disclosure. It is therefore to be
understood that within the scope of the appended claims,
the invention may be practiced otherwise than as is
specifically described.
