Note: Descriptions are shown in the official language in which they were submitted.
~9Z9 ~ ;9
Technical Field
This invention relates generally to the culturing
of embryonic plant cells and tissue and more specifically
to an improved medium particularly adapted for sustaining
embryos produced by induction from somatic tissue in vitro
and a method of using the medium.
Background Art
It has been recognized that recent progress in
genetic engineering offers plant breeders the ability to
avoid the delay in crop improvement inherent in classical
breeding techniques. However, there remain difficulties
in the application of these techniques, for unicellular
and multicellular organisms require different procedures
to change the entire genetic message. In microorganisms,
one attempts to e~fect change at the cellular level with
confidence that this will be reproduced through succeeding
generations of cells.
In multicellular organisms, such as plants, it is
advantageous to perform genetic manipulations at the
cellular level, then regenerate and raise a mature plant
expressing the new characteristic. Foreign genetic
material can be incorporated into the host cell by, e.g.,
plasmid insertion to provide for specific changes or
protoplast fusion to provide wholesale genetiG
manipulation. Further genetic manipulation using the
techniques of traditional plant breeding with mature
plants can be used to incorporate the new trait into
agriculturally useful varieties. Allard, R.W., Princip~es
of Pl~t Br~in~, John Wiley and Sons, New York (1960);
Simmons, N.W., Principles of Crop Improvement, Langman
Group, Ltd., London, (1972).
In vitro cultivation of plant cells and tissue
requires that the cultures be maintained in a medium which
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provides nutrition and sustains viability. Examples of
commonly used tissue culture media have been reviewed in
Huang, L. and T. Murashige, "Plant Tissue Culture Media:
Major constituents, their preparation, and some
applications" Tissue Culture Association Manual 3:539-
548. Tissue Culture Association, Rockville, M.D., (1977)
and de Fossard, R.A. Tissue Culture for Plant Propaaators,
University of New England Printery, Armidale, N.S.W.,
Australia (1976~. This culture maintenance can be
promoted in plant organ, tissue, or cell cultures.
In somatic embryogenesis culture, somatic plant
cells are typically induced to undergo repeated cell
divisions on a nutritive culture medium substrate,
producing an amorphous cell mass known as callus. The
callus can be maintained through subculture to allow mass
proliferation. The callus may also be induced to undergo
di~erentiation, which produces the organized tissues and
organs of the mature plant. Somatic embryos may also form
in culture from other pre-existing embryos. The parent
embryos may range from the immature globular stage to
mature, germinating embryos. Lupotto, E., "Propagation of
an embryonic culture of Medicaqo sativa L.", Zeit.
Pflanzenphysiol. 111:95-104 (1983). Thus, somatic embryos
may arise from undifferentiated callus or from pre-
existing embryos in plant tissue culture.
In this manner, genetic changes may be affected
on a cellular or embryo level and then maintained through
subsequent development to produce an entire crop with
identical genetic characteristics. This allows the plant
breeder to bypass the normal genetic barriers in plant
reproduction, and obtain a more uniform and advantageous
field crop.
Using current technology, it is possible to
produce thousands of plants from one gram of cells using
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the process of somatic embryogenesis. Evans, D.A., W.R.
Sharp, and C.E. Flick, "Growth and behavior of cell
cultures: embryogenesis and organogenesis", in Plant
Tissue Culture -_1981, T.A. Thorpe, ed., Academic Press,
pp. 45-113 (1981). These embryos could be germinated and
transferred into the greenhouse or field where mature
plants can develop. A plant breeder would use these
plants to recover useful genetic variation or to clonally
propagate varieties for use in a plant breeding program.
Certain techniques are known for determining the
quantity and quality of the embryonic tissue obtained
through culture of somatic plant parts. The quantity of
somatic embryos can be measured by determining the yield
of structures associated with the stages of development of
the embryo. Fujimura, T., and A. Komamine,
"Synchronization of somatic embryogenesis in a carrot cell
suspension culture" Plant Physiology, 64:162-164 (1979);
Verma, D.C. and D.K. Dougall, "Influence of carbohydrates
on quantitative aspects of growth and embryo formation in
wild carrot suspension cultures" Plant Physiology 59:81-85
(1977). This measurement is generally done by counting
structures using a dissecting microscope.
Somatic embryo quality can be assessed by various
methods. Embryo development is typically determined
visually by searching for globular, heart, torpedo and
plantlet stages. Ammirato, P.V., "The effects of abscisic
acid on the development of somatic embryos from cells of
caraway (Carum carri L.) "Botanical Gazette 135:328-337
(1974). Embryo development or quality can also be
determined from the yield of plantlets obtained from
individual somatic embryos. Drew, R.L.W., "The
development cf carrot (Daucus carota L.) embryoids
tderived from cell suspension culture) into plantlets on a
sugar-free basal medium" Horticultural Research 19:79
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(1979). However, plantlet formation is rarely measured
despite its importance in determining the yield of
functionally useful embryos for field use.
Techniques have been described for improving the
quantity or yield of embryos. For example, formation of
somatic embryos is known to require a source of ammonium
in the culture medium for embryogenesis to occur.
Halperin, W. and D.F. Wetherell, "Ammonium requirement for
embryogenesis in vitro", Nature 205:519-520 (1965).
Walker, K.A., and S.J. Sato, "Morphogenesis in callus
tissue of Medicago sativa: the role of ammonium ion in
somatic embryogenesis", Plant Cell Tissue Organ Culture
1:109-121 (1981). Since most plant cell culture media
contain some ammonium, it has been considered important to
adjust the ammonium concentration to an appropriate level
to achieve high embryo yield and quality. See Walker and
Sato, su~ra, and Wetherell, D,F. and D.X. Dougall,
"Sources o~ nitrogen supporting growth and embryogenesis
in cultured wild carrot tissue", Physiologia Plantarum
37:97-103 (1976).
In plant cell culture and in mature plants,
ammonium and glutamine or glutamate can be readily
interconverted by the cells. Ojima, K. and K. Ohira,
"Nutritional requirements of callus and cell suspension
cultures" in FrontieEs of Plant Tissue Culture-1978, T.A.
Thorpe, ed., University of Calgary Offset Printing
Services, pg. 265-275 (1978); Miflin, 8.J. and P.L. Lea,
"Ammonium Assimilation" in The Biochemistry of Plants, P.
Stumpf and E. Conn eds., Academic Press., Vol. 6, pg. 169-
201 1980; Dougall, D.K. "Current problems in the
regulation of nitrogen metabolism in plant cell cultures",
in Plant Tissue Culture and its Biotechnoloaical
Applicat on. W. Barz, E. Reinhard, and M.H. Zenk, eds.,
Springer-Verlag, Berlin, pg. 76-81 (1977).
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The perceived proximity of ammonium to glutamine
and glutamate in plant metabolism is reflected by the
protocols for systematic studies of the effects of amino
acids on somatic embryogenesis. Studies using carrot
cells have removed ammonium from the plant cell culture
medium and tested single amino acids for their effect on
somatic embryogenesis. Wetherell and Dougall, supra;
Xamada, H. and H. Harada, "Studies on the organogenesis in
carrot tissue cultures II. Effects of amino acids and
inorganic nitrogenous compounds on somatic embryogenesis,"
Zeit. Pflanzenphysiologie 91:453-463 (1979). Wetherell
and Dougall found that amino acid additions did not
improve embryogenesis compared to ammonium ion. Kamada
and Harada, on the other hand, found that alanine and
glutamine act as good reduced nitrogen sources for
embryogenesis in the absence of ammonium or in the absence
of ammonium and nitrate. ~ne report haa recommended
against the use o~ amino acid additions which were 20 mM
or higher (Reinert, J. and M. Tazawa, "Wirkung von
Stickstoffverbindungen und von Auxin auf die Embryogenese
in Gewebekulturen," Planta 87:239 ~1969), (Text in
German).
The above studies indicate that there is an
equivalence among sources of reduced nitrogen, such as
ammonium and amino acids. Wetherell and Doug~ll, supra;
Kamada and Harada, supra. The metabolic equivalence of
ammonium and amino acids is further shown by the studies
of Tazawa and Reinert (Tazawa, M. and Reinert, J.,
"Extracellular and intracellular chemical environments in
relation to embryogenesis in vitro", Protoplasma 68:157-
173 (1969)). In an investigation o~ the internal levels
of ammonium in carrot cultures undergoing somatic
embryogenesis, it was found that the level of ammonium
correlated with the amount of embryogenesis in the culture
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regardless of whether cultures were fed ammonium, amino
acid or nitrate. It was concluded that the internal level
of NH4' is the critical factor in stimulating somatic
embryogenesis. The internal NH4' level is derived from
either externally supplied NH~ or amino acids, or by the
biological reduction of nitrate to NH4~. Internally, NH
can be converted to organic nitrogen compounds to supply
amino acids for normal cell requirements, Tazawa and
Reinert, supra. Hence amino acids are believed to act by
releasing ammonium, which stimulates embryogenesis.
Factors which have been noted to improve embryo
development are abscisic acid, zeatin, gibberellic acid,
high sucrose concentrations and light. Ammirato, P.V. and
F.C. Steward, "Some effects of environment on the
development of embryos from cultured free cells",
Botanical Gazette 132:149-158 ~1971); Ammirato, P.V.,
"Hormonal control of somatic embryo development from
cultured cells of caraway. Interactions of abscisic acid,
zeatin and gibberellic acid", Plant Physiology 59:579-586
(1977). The effect of ammonium and amino acids on embryo
quality is not known to have been recognized. Ammirato,
P.V., "The regulation of somatic embryo development in
plant cell cultures: Suspension culture techniques and
hormone requirements", Bio/Technology 1:68_74 (1983).
In addition, conversion frequency, as a measure
of embryo quality, has heretofore not been recognized or
used systematically to improve embryo development. Embryo
maturation has been determined by visual assessment of
embryo morphology, but this method does not measure the
frequency of plant formation from individual embryos.
Therefore it is an object of this invention to
provide method3 and materials to increase the quantity and
quality of somatic embryos produced from plant tissue.
It is a further ob;ect of this invention to
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provide optimized sources of reduced nitrogen for somatic
embryogenesis.
It is yet another object of this invention to
provide methods and materials allowing mass propagation of
numerous species of plants through somatic embryogenesis.
It is a still further object of this invention to
provide methods and materials for a generation of numerous
viable somatic embryos with identical genetic and
phenotypic traits.
Other objects, advantages, and features of the
present invention will become apparent from the following
description and the accompanying examples.
~isclosure of the Invention
This invention provides novel and improved
methods and materials for producing numerous high qualit~
somatic embryos from plant tisue by the addition of
optimal amounts of amino acids and sources of reduced
nitrogen. One aspect of the present invention provides a
method for producing embryonic tissue from Gramineae,
Umbelliferae or Malvaceae somatic tissue, wherein somatic
tissue is regenerated from induced cells in a nutritive
plant cell culture medium to form embryonic tissue, said
method comprising:
providing a nutritive plant cell cul~ure medium
comprising:
a medium having all mineral salts, vitamins and
nutrients required to maintain tissue viability, together
with a source of ammonium ion; and
an addition to said medium of at least one amino
acid component selected from the group consisting of L-
proline, L-arqinine, L-lysine, L-asparagine, L-serine, L-
ornithine and the amide, alkyl ester and dipeptidyl
derivatives thereof, upon which somatic embryos can
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differentiate from induced cells, the ammonium and amino
acid components of said medium provided in amounts
sufficient to increase the quantity or improve the quality
of somatic tissue to be produced;
exposing undifferentiated plant somatic tissue
selected from the group consisting of Gramineae,
Umbelliferae and Malvaceae somatic tissue to said culture
medium to cause embryogenesis in said plant somatic tissue
to form somatic embryos; and
sustaining said somatic embryos on said culture
medium to permit embryo development into plantlets.
Another aspect of the present invention provides
a method of producing embryonic tissue from Gramineae,
Umbelliferae or Malvaceae somatic tissue, wherein somatic
tissue is regenerated from induced cells in a nutritive
plant cell culture medium to ~orm embryonic tissue, said
method comprising:
providing a nutritiVe plant cell culture medium
comprising all mineral salts, vitamins and nutrients
required to maintain tissue viability, which medium is
substantially free of ammonium ion, together with an
addition to said medium of L-glutamine and at least one
amino acid component selected from the group consisting of
L-proline, L-arginine, L-lysine, L-asparagine, L-serine,
L-ornithine, the amide, alkyl ester and dipep~idyl
derivatives thereof, upon which somatic embryos
differentiate from induced cells, the amino acid
components of said medium provided in amounts sufficient
to increase the quantity or improve the quality of somatic
tissue to be produced;
exposing undifferentiat~d plant somatic tissue
selected from the group consisting of Gr~mineae,
Umbelliferae and:Malvaceae somatic tissue to said culture
medium to cause embryogenesis in said plant somatic tissue
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to form somatic embryos; and
sustaining said somatic embryos on said provided
nutritive plant cell culture medium to thereby permit
embryo development into plantlets.
Brief Description of the Drawinq
The single drawing is a graphic representation of
the increase in number of somatic embryos produced as a
function of the concentration of amino acids added to the
medium.
Best Mode for Carryinq Out the Invention
The present invention provides methods for
enhanced quantity and quality of embryos produced from
plant somatic tissue by providing a medium for culturing
said cells and tissue which contains a suf~icient amount
of ~elected amino acids to stimulate somatic
embryogenesis.
The present invention also provides for such
enhanced quantity and quality by providing a medium for
culturing such cells and tissue which contains selected
amino acids together with sources of ammonium ion in
amounts sufficient to stimulate the quantity and quality
of somatic embryos. Also provided is a method for using
such plant tissue culture medium.
While it has previously been believed that amino
acids served as simple equivalents to the desired ammonium
media component, it has surprisingly been found that amino
acids can serve as replacement for ammonium ion which
enhance the production of somatic embryos over the
equivalent concentrations of ammonium. It has also been
surprisingly found that selected amino acids together with
an additional source of ammonium ion can provide
substantially increased benefits which would not be
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predicted from a simple additive effect of increased
ammonium ion concentration.
It has been found that a medium which contained
an amino acid selected from the group consisting of
proline, arginine, lysine, asparagine, ornithine, and the
amides, alkyl esters and dipeptidyl derivatives of these
amino acids, which medium is substantially free of
ammonium ion, provides enhanced quantity and quality of
somatic embryos derived from the cultured somatic tissue.
It has also been found that a medium containing
ammonium ion and at least one amino acid selected from the
group consisting of proline, alanine, arginine, glutamine,
lysine, asparagine, serine, ornithine, glutamate and the
am~des, alkyl esthers and dipeptidyl derivatives of these
amino acids in an amount sufficient to stimulate
embryogenesi~ or embryo conversion can provide similar
embryo enhancements.
It has been surprisingly discovered that the
medium of the present invention provides increased yield
of somatic embryos from callus tissue over the typical
media used heretofore in the induction, regeneration and
maintenance of embryonic tissue. In each stage o~ these
embryogenic procedures, a far greater yield of embryonic
tissue can be attained using the media of the present
invention than the results previously provided.
Furthermore, the advantages obtained through the practice
of the present invention have been achieved in a variety
of useful plant species including alfalfa, celery, cotton,
corn and rice.
Plant cell culture media which can be improved by
the practice of the present invention include previously
known plant tissue culture media such as those reviewed in
Huang, L. and T. Murashige, supra, and in Clonina
agricultural plan~s ~ in vitro techniques, B.V. Conger,
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ed., CRC Press, Inc. pp. 172 et seq. In general, plant
culture media provide plant nutrients, sources of energy
such as sugar, plant hormones and buffered salts to
control the pH and osmotic balance of an aqueous solution.
Representative of such plant cell culture media
is the medium known as Schenk and Hildebrandt (SH) medium,
Schenk, R.U. and A.C. Hildebrandt, "Medium and techniques
for induction and growth of monocotyledonous and
dicotyledonous plant cell cultures", Can. J. Bot., 50:199
(1972). As used hereinafter, hormone-free SH medium is
the medium disclosed therein including the major salts,
vitamins and sucrose, but without the 2,4-D, pCPA and
kinetin.
Alternatively, the medium known as Murashige and
Skoog (MS), Murashige, T. and F, Skoog, "A revised medium
for rapid growth and bioassays with Tobacco tissue
cultures", Physiologia Planta,, 15:473-4g7 (1962) can be
employed in place of SH medium. As used hereinafter,
hormone-free MS medium is the medium disclosed therein
including the major salts, vitamins and sucrose, but
without the indole-3-acetic acid and kinetin.
The selection of the basic plant cell culture
medium to be utilized in the practice of the present
invention will be dictated, in part, by the species of
plant somatic tissue selected, and is conside~ed to be
within the ordinary skill of one experienced in the tissue
culture of plant cells and the practice of somatic
embryogenesis.
Numerous important crop and horticultural species
have been shown to be capable of propagation through
tissue culture and somatic embryogenesis. These varieties
include, but are not limited to:
Table 1
Veaetable crops Fruit and nut trees
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12
alfalfa almond
asparagus apple
beet banana
brussels sprouts coffee
carrot date
cauliflower grapefruit
eggplant lemon
onion ol ive
spinach orange
sweet potato peach
tomato Bulbs
Fruit and berries lily
blackberry daylily
grape Easter lily
pineapple hyacinth
strawberry Flowers
Foliaae African violet
silver vase anthurium
begonia chrysanthemum
crytanthus gerbera daisy
dieffenbachia gloxinia
dracaena petunia
eiddleleaf rose
pointsettia orchid
weeping fig Pharmaceutical
rubber plant atropa
ginseng
pyrethium
Ferns Silviculture (forestry)
Australia tree fern douglas fir
Boston fern pine
Maidenhair fern quaking aspen
rabbitsfoot fern redwood
staghorn fern rubber tree
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13
sword f ern
Cereal Grains
barley
corn
millet
pennisetum
wheat
For a more exhaustive list of species capable of
somatic embryogenesis, see Evans, D.A. et al., "Growth and
Behavior of Cell Cùltures: Embryogenesis and
Organogenesis" in Plant Tissue Culture: Methods and
Appli ations in Aariculture, Thorpe, ed., Academic Press,
page 45 et seq. ~1981).
Numerous amino acids are known in the prior art
which, with certain exceptions, have the common feature of
a ~ree carboxyl group and a free unsubstituted amino group
in the ~-carbon atom. Proline is a notable exception,
since the ~-amino group o~ proline i8 substituted so that
it is in reality an ~-imino acid.
Amino acids can be divided generally into protein
and nonprotein amino acids wherein protein amino acids
include the 20 most commonly recognized. These amino
acids include ~our subgroups: Those with nonpolar or
hyrophobic substitutions, including alanine, leucine,
isoleucine, valine, proline, phenylalanine, tr~ptophan and
methionine; amino acids with uncharged polar R groups
including serine/ threonine, tyrosine, asparagine,
glutamine, cysteine and, presumably, glycine; amino acids
with negatively charged R groups including aspartic acid
and glutamic acid; and amino acids with positively charged
R groups including lysine, arginine, and, presumably,
histidine.
In addition to the 20 common amino acids, there
are numerous others which appear rarely or not at all in
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14
are numerous others which appear rarely or not at all in
proteins. Hydroxylysine and hydroxyproline are rarely
found and only in fibrous proteins. Also, over 150 other
amino acids are known to occur in different cells and
tissues, in free or combined from, which include most
commonly citrulline and ornithine, which are intermediates
in the synthesis of arginine, as well as numerous ~, y and
h forms of the common amino acids.
In addition to the basic amino acid structures
discussed above, amino acids can be modified in numerous
ways without altering their ability to function in the
present invention. Among these alterations include the
formation of amino acid amides and amino acid alkyl esters
by the addition of amino and carboxy groups respectively.
In addition, dipeptidyl derivatives of the amino acids can
be ~ormed by linking two amino acids throuyh the ~-carboxy
group and ~-amino group. It will be readily appreciated
that each pair o~ amino acids will have two potential
dipeptidyl derivatives.
Also of importance to the present invention is
the provision of a source of ammonium ion (NH4~) to
supplement the amino acid-containing media of the present
invention. Sources of ammonium ion are also well known in
the art of plant tissue culture. Typically, such ammonium
ion is provided by way of the inclusion in the medium of a
quantity of non-toxic salt of ammonium, formed with an
anion which balances the ammonium ion charge, e.g.,
ammonium chloride, ammonium phosphate or ammonium
sulphate. Other sources o~ ammonium ion are disclosed in
Walker, K.A. and S.J. Sato, "Plant Cell Tissue Organ
Culture" 1:109-121 (1981).
The following examples are provided in order to
illustrate various aspects o~ the present invention The
examples should not be taken as implying any limitation to
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exclusively the claims appended hereto.
xperimental
In general, the methods utilized to practice
somatic embryogenesis with plant cell and tissue cultures
are well known and require only slight modification for
adaptation to a selected plant species. See, for example,
Plant Tissue Culture: Methods and Applications in
Aqriculture, Thorpe, ed., supra (1981).
For example, alfalfa, embryogenesis can be
routinely induced in the Regen S line of Saunders and
Bingham, "Production of Alfalfa Plants from Callus
Tissue," Crop Sci., 12:804-808 (1972).
Plants of Medica~o sativa 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 five minutes,
washing with H20 and plating on hormone-free SH medium,
containing the salts, vitamins and sucrose of Schenk-
Hildebrandt medium (Schenk, R.U. and A.C. Hildebrandt,
supra, (1972)). The medium contained 25 ~M ~-
naphthyleneacetic acid and 10 ~M kinetin and 0.8% (w/v)
agar (termed maintenance medium). Callus which formed on
the explant tissue was separated from the remaining
uncallused tissue and repeatedly subcultured on
maintenance medium. Callus was subcultured at 3 week
intervals and grown under indirect light at 27C.
Three to nine grams of callus was collected at 17
to 24 days post-subculture from plates of maintenance
medium and transferred to 100 ml of liquid SH containing
50 ~M 2,4-dichlorophenoxyacetic acid (2,4-D) and 5 ~M
kinetin (B) for induction. Walker, K.A., M.~. Wendeln and
E.G. Jaworski, Plant Sci. Lett. 16:23_30 (1979). Cells
were cultured in 500 ml flasks for 3 days at 27C on an
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16
orbital shaker at 100 R.P.M. under indirect light.
Induced cells were asceptically 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) through
stainless steel screen. Cells retained on the 60 mesh
screen were washed with 500 ml of SH minus hormone medium
for every 100 ml of induction culture volume. The washing
medium was removed by vacuum. The fresh weight of the
cell clumps was taken and cells were resuspended in SH
medium without hormones at 150mg fresh weight per ml.
Seventy-five mg (0.5ml) of resuspended cells were pipeted
onto approximately 10 ml of agar solidified medium in 60mm
x 15 mm petri dishes.
Alternatively, somatic embryogenesis in
suspension culture will occur if 300 mg (2ml) of
resuspended cells are delivered to 8 ml of hormone-free
liquid SH medium contained in a 50 ml erlenmeyer flask.
The embryogenesis media contained SH medium (NH~+ equal to
2.6mM with 3% (w/v) sucrose without hormones. Ammonium
ion free medium was made by substituting an equivalent
amount of NaH2PO4 for the NH~2PO~ of SH. The 25mM NH
control medium consisted of ammonium free medium
supplemented with 12.5mM (NH4)2SO4. All organic and
inorganic sources of reduced nitrogen were sterilized by
0.2 ~m filtration and subsequently added to freshly
autoclaved medium.
Each treatment was generally plated in 10
replicates. Dishes were parafilm wrapped and incubated
for 21 days. Suspension flasks were foam plugged, sealed
with Saran Wrap~ and incubated for 14 days on an orbital
shaker at 100 rpm. Incubation was at 27C under 12 hour
illumination from cool white fluorescent tubes at 28 cm
from solidified cultures or 200 cm from suspension
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cultures.
Embryogenesis was visually measured after
incubation by counting green centers of organization on
the callus using a stereo microscope 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 of embryos to whole
plants with root and shoot axis (first primary leaf) was
done by aseptically transferring embryos from amino acid
treatments at 21 days of initial culture to half-strength
hormone-free SH medium supplemented with 25 ~M gibberelic
acid and 0.25 ~M ~-naphthyleneacetic acid solidified with
0.8% agar.
1. Somatic Embryoaenesis in Media Containing Ammonium
A. Leguminosae somatic cell culture
As a representative of the Leauminosae family,
alfalfa tissue, Medicao sativa, Regen S was cultured as
outlined above and assayed for somatic embryogenesis in
culture medium containing 2.6 mM ammonium in accordance
with the present invention. All protein amino acids were
tested at between 1 and 100 mM concentrations. Two
response types emerged from this initial screen and based
on these results further tests with sieved cells were
performed. Table 2 excludes-amino acids of the first
response type, those which were found to be to~ic to
growth or inhibitory to embryogenesis compared to the SH-
medium (2.6mM NH4~) control. These amino acids included
the sulfur and aromatic and most of the branched chain
family. None of these amino acids stimulated
embryogenesis over the SH medium control and all were
toxic, in that they inhibited growth or caused browning of
the callus either at 1 or 10 mM.
The second response type from the initial screen
either stimulated embryogenesis or caused an increase in
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18
embryo size when compared to the SH control. See Table 2.
Detailed concentration dependence studies were performed
on these amino acids and the results are shown in Figure
1. The amino acid most effective in stimulating somatic
embryo formation was proline, which yielded nearly 3-fold
more embryos than the 2.6 mM NH4+ control and was twice as
effective as 25 mM NH4~, the optimal ammonium concentration
in alfalfa (D). (Walker, et al. supra). Alanine,
arginine, glutamine and lysine were all less effective but
stimulated embryo formation to approximately the level of
25 mM NH4~. Serine and asparagine showed less stimulation
of embryogenesis compared to the SH control, but increased
embryo size.
Table 2 summarizes the amino acids and other
nitrogen sources which have been found to be stimulatory
to somatic embryogenesis in alfalfa. It is important to
note that the ester and amide forms of proline are highly
active in stimulating embryo numbers and quality as is the
dipeptide, prolyl alanine. It is interesting to note that
the nonprotein amino acid ornithine is also active.
Table 2
The Effect of Redùced Nitrogen Sources on Somatic
Embryoqenesis in Alfalfa
Stimulatorv Sources
Ammonium
Proline
Alanine
Glutamine
Arginine
Asparagine
Ornithine
Serine
Lysine
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19
L-proline amide
L-prolyl-L-alanine
L-proline methyl ester
Using the techniques described above, embryo
quality was measured by visual inspection of the embryo
size. The data is presented in Table 3.
Table 3
Effect of Reduced Nitrogen Treatment on Somatic
Embryo Size
Treatment Length Width
Control (25mM NH4+) 0.805 + O.084 0.383 + O.019
lOOmM L-Proline 1.143 + 0.0810.867 + 0.046
30mM L-Alanine 1.163 + 0.0900.744 + 0.037
lOOmM L-Alanine 1.192 + 0.1020.833 + 0.049
30mM L-Arginine 1.521 i 0.1420.663 i 0.048
30mM L-Glutamine 1.342 i 0.1220.713 i 0.051
3mM L-Lys~ne 1.163 + 0.0960.652 + 0.039
3mM L-Asparagine 0.699 + 0.0620.446 + 0.027
lOmM L-Asparagine 1.239 + 0.1020.610 + 0.034
Based on the data of Table 2 and Table 3, the
effectiveness of the amino acid additives in improving
embryo size can be ranked in the following order:
Arginine > glutamine > alanine ~ proline > NH4~.
Using the techniques described above, the
conversion of embryos to whole plants with root and shoot
axis (first primary leaf) was observed and tabulated. The
results are as follows:
Table 4
Conversion of Somatic Embryos to Alfalfa Plantlets
Initial Treatment % Plants with First primary Leaf
25 mM NH4t 33.3% + 4.2
100 mM L-Proline 54.0% + 6.4
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50 mM L-Alanine 63.5% + 4.4
30 mM L-Arginine 59.0% + 6.2
30 mM L-Glutamine 67.0% + 3.4
Based on the data of Table 4, the effectiveness
of an additive on the conversion of embryos to plantlets
is as follows:
Glutamine > alanine > arginine > proline > NH4'
From the correlation between these sets of data
it is shown that embryo size is a good indicator of embryo
conversion to plantlets and thus a good indicator of the
quality of embryos produced by a given technique.
The optimal amounts of added amino acids were
determined for the stimulation of somatic embryogenesis on
both agar solidified cultures and liquid suspension
cultures. These data are presented in Tables 5 and 6 as
follows:
Table 5
Stimulation o~ Al~alfa Somatic Embryogenesis
by Amino Acid Additions to Agar Solidified
Cultu~e i~ the _~esence of 2.6 mM NH~
~ MaximalConcentration Concentration
SourceStimulationRange in ~M Optimum in mM
Control (2.6 100 --- ---
mM NH4f)
L-Proline 330 10 to 300 (100)
L-Alanine 314 20 to 150 (75-100)
L-Glutamine 175 20 to 50 (30-40)
L-Arginine 244 5 to 50 (30-40)
L-Asparagine 155 0.5 to 3 (1)
L-Ornithine 156 1 to 3 (1-3)
L-Serine 160 0.5 to 2 (1)
L-Lysine 233 1 to 10 (3)
L-Proline amide 240 30 to 200 (50-100)
L-Proline
methyl ester 241 5 to 25 (10)
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L-Prolyl-L-
alanine 210 30 to 200 (50-100)
Table 6
Stimulation of Alfalfa somatic Embryogenesis by
Amino Acid Additions in Liquid
suspension Cultures in the Presence of 2.6 mM NH~
% Maximal Concentration Concentration
Source StimulationRange in mM Optimum in mM
Control (2.6
mM NH4~) 100
L-Proline 528 10 to 300 (100)
L-Alanine 240 25 to 200 (50)
L-Glutamine 243 5 to 75 (50)
L-Arginine 168 5 to 75 (50)
L-Lysine 180 1 to 10 (3)
B. Umbelli~erae somatic cell culture
As a representative o~ the Umbelliferae family,
seeds o~ celery, Apium qraveolens (variety Calmario) were
germinated ~or one to two weeks. The resulting seedlings
were sterilized with a solution of 10% CloroxR for 20
minutes. Cotyledons or hypocotyls were removed and
explants were placed on 0.8% agar solidified hormone-free
SH medium containing 25 ~M 2,4-D and 5 ~M benzyladenine.
After initiation of callus (3-4 weeks), callus was
transferred to SH medium with 2.5 ~M 2,4-D and 0.5 ~M
~inetin. Heat labile additives were filter sterilized and
added to warm medium. When required, specific amounts of
tissue for innoculation were obtained using a modified
spatula device and filling this to uniform volume.
Subsequent subcultures of callus were on SH medium plus 1
~M picloram and 0.5 ~M benzyladenine. For somatic embryo
production 75 mg of callus cells was transferred to 0.8%
agar solidified hormone-free SH medium containing filter
sterilized additives and incubated for 18 to 30 days at
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22
24OC under the same conditions as alfalfa.
Amino acids proline, alanine and glutamine were
compared against NH4+ control-treated embryos. Treating
cultures with 50 mM alanine resulted in higher frequency
embryogenesis than all other treatments, as well as
embryos which had better cotyledons, root and primary leaf
development than other cultures. The following order of
total embryo numbers formed was observed:
20-100 mM alanine > 50 mM proline >
25 mM glutamine-N > 25 mM NH4~
Although proline stimulated embryogenesis better
than glutamine, the latter resulted in better development
of seedling-like embryos. Ammonium treated cultures
developed smaller and fewer embryos than all other
treatments Glutamic acid, when added singly to celery
regeneration medium at 30 mM, stimulates embryo number in
celery compared to 25 mM NHI~ - treated material. Alanine,
proline, glutamine and glutamate at the above
concentrations improve celery embryo conversion to
plantlets compared to NH4~ treated embryos.
C. Gramineae Somatic Cell Culture
As a species representative of the Gramineae
family, Zea mays somatic embryogenesis was performed
employing media in accordance with the present invention.
Ears of corn at ten days post fertilization were
harvested and immature embryos were dissected from these
aseptically. Embryos were placed onto N-6 mineral salt
medium (Chu, C.C., Wang, C.C., Sun, C.S., Hsu, C., Yin,
K.C. and Chu, C.Y., 1975. Establishment of an efficient
medium for anther culture of rice throuqh comparative
experiments on the nitroqen sources. Sci. Sin. 16, 659-
688) plus 3% sucrose and 5 ~M 2,4-D fox 21 days. After
încubation callus was scored for formation of embryo
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23
masses on each callus formed while in the presence or
absence of L-proline. The results were as indicated in
Table 7, where, the percent response is the average
frequency of embryo formation of between 287 and 1165
replicate embryo explants.
Table 7. Effect of L-Proline on Embryo Callus Formation
in Corn.
Proline Conc. (mM) % Embryo Callus Formation
o 15.8
6 20.6
12 20.8
24 20.2
As a further example of somatic embryogenesis
involving the Gramineae, the rice species Qxyza sativa was
regenerated in accordance with the practice of the present
invention.
Seeds of Oryza sativa were dehusked, surface
sterilized and placed on Murashige and Skoog ~MS) salts
(Murashige, T. and F. Skoog. (1962), supra) plus 4%
sucrose, 0 26 mM tryptophan, 5 ~M 2,4-D, 1 ~M kinetin, pH
6.2 with 2.5g/1 Gelrite as a gelling agent. After 15 to
21 days individual seeds were scored for embryo formation
on the scutellar region of the seed with a dissecting
microscope. Treatments treated with and without L-proline
are noted in Table 8.
Table 8. Effect of L-Proline on Embryo Formation in Rice
Callus Cultures
.... . . .. _ _ . _
Proline Conc. (mM) % Embryo Formation
0 57.8
3 59.0
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24
lo 77.8
64.9
70.1
100 68.4
D. Malvaceae Somatic Cell Culture
As an example of the practice of the present
invention in plant somatic tissue of the Nalvaceae family,
cultures of two cotton species were regenerated in media
prepared in accordance with the present disclosure.
Gossvpium hirsutum. Cultures initiated from
surface sterilized seed of Gossvpium hirsutum were
subcultured on Murashige and Skoog salts plus 3% sucrose
and 0.5 ~M NAA, 5 ~M 2-isopentanyladenine with o.8~ agar
medium for four week subcultures. Cultures were induced
for 10 days to form embryos on medium containing either
0.5 ~M 2,4-D plus 0.2 ~M kinetin or 1 ~M NAA and 0.5 ~M
kinetin with or without proli~e in liquid suspension
culture. Cells were then transferred to hormone-free SH
medium with 3% sucrose and 10 mM L-glutamine for
regeneration. After four weeks cultures were evaluated
for formation of embryos with mature cotyledons. The
results were as indicated in Table 9.
:
Table 9. Effect of Added Proline on the Regeneration of
Cotyledonary Cotton (Gossy~ium hirsutum) Embryos in
Suspension Culture
30Proline Conc (mM) Embrvos with Cotvledons
5.5
24 12.5
~,~...
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Gossypium klotzschianum. Cultures initiated from
surface sterilized seed were subcultured on Murashige and
Skoog salts, 3% sucrose, 0.5 ~M NAA and 5 ~M 2-
i,sopentanyladenine. Callus was suspended for 10 days in
MS salts, 3% sucrose and 0.2 ~M picloram prior to
regeneration for 21 days on hormone-free medium with added
L-glutamine. The results were as indicated in Table 10.
Table 10. Effect of L-Glutamine on Embryo For~ation in
Goss~pium klotzschianum
L-Glutamine Conc. (mM) Cotyladonar~ Embryos
5 0
10 2.0
20 3.5
2. ,Amino Acid Interaction With Sources of Ammonium Ion
Cells were induced, sieved and plated as in the
above experiments. The concentrations of proline or
arginine and NH4+ were varied to determine if the optimum
concentration for any additive alone was influenced by the
presence of the additional additive.
1. Proline: Proline was tested over a range of
30 mM to 300 mM where the amount of added NH4~ varied
between 0 and 25 mM. The results are indicated in Table
11 .
2. Arginine: A similar experiment where the
concentration of arginine was varied in addition to the
concentration of NH~ added to the medium. The results are
shown in Table 11.
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26
Table 11
Effect of Amino Acid Interaction with Ammonium
Ion in Alfalfa (mean number of embryos produced
in at least seven trials~
proline Concentration (mM) 30 100 300
Concentration (mM~
0 326 470 133
1.0 502 747 541
2.6 753 731 825
10.0 887 811 572
25.0 744 1,0472 844
Arainine Concentration (mM) O 10 30 lOO
_~ concentration (mM)
0 12 147 126 99
1.0 70 252 246 157
2.6 207 298 306 264
10.0 340 408 411 3~1
25.0 335 297 233 148
It is seen in each case that a synergistic effect
resulted when the optimum amounts of arginine or proline
and optimum amounts of NH4~ were added.
In a repeat of a portion of the Example portrayed
in Table 11, various concentrations of NH4~ and L-proline
were tested for their effect on somatic embry0 quantity
and conversion to plantlets.
Table 12
Effect of Ammonium and Proline on Somatic Embryo
Ouantitv and Ouality
%
Proline (mM~ NH,~ lmM? SE Ouantity Conversio~
O 0 4 + 1 3 + 3
0 2.6 36 + 5 4 ~ 3
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27
0 25 60 + 6 9 + 5
2.6 95 + 12 25 + 5
2.6 134 + 17 42 + 7
100 2.6 175 i 19 42 + 4
187 + 24 42 + 6
It is seen that proline plus ammonium media
improve embryo quantity and that proline improves embryo
quality in the presence of high or low ammonium.
3. Amino Acid Additions to Media
Substantially Free of Ammonium Ion
Alfalfa cells were induced and plated as in the
above examples, except that NH4' was deleted from the media
formulation. A range of amino acid concentrations was
tested ~or their e~ect on somatic embryogenesis and the
result~ are summarized in Table 13.
Table 13
Stimulation of Alfalfa Somatic Embryogenesis
by Amino Acid Additions to Agar Solidified
Cultures in Media Substantially Free of NH~~
% MaximalConcentration Concen,tration
Addition StimulationRanae in mM Optimum in mM
Control 100
without NH4~)
L-Proline 525 6 to 300 (10-30)
L-Arginine 1,200 1 to 100 (20-50)
L-Asparagine 750 1 to 100 (2-10)
L-Ornithine goo 0.3 to 3 (1)
L-Lysine 500 1 to 10 (3)
In the substantial absence of NH4~, the above
amino acids are shown to stimulate somatic embryogenesis
as the sole reduced nitrogen source in culture media. In
addition, with the exception of ornithine, substantial
,,~,,
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28
improvements in the quality of the produced embryos, as
determined by size, shape and degree of maturation, were
obtained by the above additions throughout the stated
concentration ranges.
4. Combinations of Amino Acids
The following Table shows the effect of adding
combinations of amino acids to alfalfa cultures in the
absence of NH4'. Combinations of amino acids have a
synergistic effect on embryo numbers.
Table 14
EmbrYo Number
Expt. 1 50 mM L-Proline 80
30 mM L-Glutamine 50
50 mM Proline and 30 mM Glutamine 248
Expt. 2 100 mM L-Proline 27
100 mM L-Alanine 74
100 mM L-Proline+50mM L-Alanine 215
30 mM L-Arginine 135
100 mM Proline ~ 30 mM Arginine 215
- The largest and highest quality embryos were
observed in treatments containing proline combined with
other amino acids.
Although the foregoing invention has been
described in some detail by way of illustration and
example for purposes of clarity of understanding, it will
be obvious to one skilled in the art that certain changes
and modifications may be practiced within the scope of the
appended claims.
. . .
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