Note: Descriptions are shown in the official language in which they were submitted.
2092588
ENHANCED REGENERATION SYSTEM FOR CEREALS
BACKGROIIND OF THE INVENTION
Field of the Invention
The present invention is directed to an enhanced
regeneration system for cereal plants, especially wheat and
barley, and the use thereof in genetic engineering.
Description of the Background Art
The crop species belonging to family Gramineae (Poaceae),
order Graminale, class Monocotyledoneae, and subdivision
Angiospermae of the Plant Kingdom are known as cereals. The
major cereal grain crops of the world include crops such as
wheat, rice, barley, corn, oats, rye, sorghum, and millets.
Cereal grain crops contribuce about 90% of the total grain
population of the world. Among cereal grain crops wheat, rice,
and corn provide three-fourth of the world's cereal grain
production. Barley, sorghum, rye, oats and millets account for
the remainder (Stoskopf, N.C. 1985, Cereal Grain Crops, Virginia,
U.S.A.). Cereals are regarded as principal source of
carbohydrate and protein in animal and human diet. In addition,
cereals provide fats, minerals and vitamins. The starch stored
in cereal grains can also be fermented into ethanol for use in
beverages and as a fuel source.
Both wheat and barley occupy a uni que position in the global
agricultural economy because of their widespread cultivation and
trade at the international level. The annual world production
of wheat and barley for 1992 is estimated to be 547 and 171
million tons, respectively (Market Commentary, 1991, Agricultural
Canada Publication). Wheat and barley together accounts for more
than 40% of the total world's cereal grain production. Wheat is
the single largest commodity traded in the world. Wheat and
barley together accounts for more than 50% of the total world
2092588
2
grain export. With the constantly increasing world population,
the demand for wheat, barley and other cereals is expected to
rise, resulting in an increase in production of about 2%- annually
(Stoskopf, 1985).
Conventional plant breeding has, thus far, contributed
significantly to the improvement of cereal crops. However, the
advent of genetic engineering techniques now provides an
opportunity for further improvement of wheat, barley, and other
cereals, by incorporating genes for resistance to herbicides,
insect pests and diseases, and better nutritional quality into
elite genotypes.
A major problem with genetic engineering of wheat and barley
is the inability to recover fertile plants from the transformed
cells. Although several procedures have been described in the
literature for plant regeneration from various tissues of wheat
and barley, all of these procedures have drawbacks.
Many different types of explants, such as mature and
~ immature zygotic embryos, immature inflorescence segments,
~. _.
anthers, young leaves and roots have been successfully used for
establishiifg-in vitro cultures of both wheat and barley (Vasil,
--_~---~
1988; Gobel and Lorz, 1988). Plants have also been regenerated
from such cultures via organogenesis and somatic embryogenesis.
(Organogenesis refers to development of adventitious shoots or
roots (unipolar structures) which maintain their link to initial
explant tissue or callus originated from explant tissues.
Somatic embryogenesis refers to development of distinct somatic
embryos, (bipolar structures) with shoot and root apices
integrated into one axis, from somatic cells. The somatic
embryos are not attached to the parental tissue and are capable
of germinating into complete plants.) However, in general, the
regeneration frequency from most explants has been very low.
Moreover, the success with plant regeneration from the most
responsive explants such as immature zygotic embryos and
inflorescence segments (Thomas and Scott, 1985; Redway et al.,
1990) depends on the tedious and subjective process of
2~9~588
3
identification, selection and maintenance of embryogenic callus.
The entire process of shoot regeneration from these explants
often takes more than ten weeks from the initiation of cultures.
This prolonged culture period not only results in loss of
regeneration potential, but also adds to the risk of genetic
instability among regenerants. The major limitation with the use
of immature anther culture for plant regeneration is the genotype
dependence of the process and the occurrence of albino plantlets
at a high frequency among regenerants.
In the recent past, several attempts have been made to
establish cell suspension cultures from embryogenic callus
cultures and subsequent plant regeneration from established cell
suspensions or protoplasts isolated from such cultures, in both
wheat and barley (Vasil, 1990; Jahne et al., 1991; He et al.,
1992). However, in most cases either the procedure was not
reproducible or it resulted in the production of infertile
plants, with the exception of one instance in which normal
fertile plants were recovered from protoplasts of an Australian
genotype of wheat (He et al., 1992). Additionally, the
establishment of cell suspension cultures of wheat and barley is
an extremely difficult and time consuming process. Consequently,
the immature zygotic embryos have been extensively used for
obtaining embryogenic callus and subsequent plant regeneration
in wheat and barley. Since immature zygotic embryos contain
embryo axes, the shoot regeneration from such explants is
sometimes confused with the precocious germination of embryo axis
or axillary shoot proliferation from the remains of embryo axis
removed after germination. The precocious germination of embryo
axis is undesirable for genetic engineering and other
biotechnological applications of tissue culture methods and
therefore should be avoided. The most desirable mode of plant
regeneration from somatic cells is through somatic embryogenesis
as described in this invention.
The process of shoot regeneration from intact immature
zygotic embryos is slow because a large proportion of callus
produced from such explants constitutes an undesirable non-
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embryogenic callus. The non-embryogenic callus grows at a faster
rate than embryogenic callus and thus suppresses the growth of
embryogenic callus by competing for nutrients and other
constituents of the tissue culture medium. The identification,
selection and maintenance of embryogenic callus from the mixture
of different callus types is as mentioned earlier, a tedious and
time consuming process.
Successful genetic engineering of cereal crop plants
primarily depends upon the availability of a high frequency,
genotype-independent regeneration procedure which has the
potential of producing a large number of plants in a short period
of time.
SUbEiARY OF TS73 INVENTION
The present invention is directed to a novel enhanced
regeneration system for high frequency somatic embryogenesis of
wheat, barley and other cereal plants, from isolated scutellar
tissue. This procedure fulfills all of the above-mentioned
requirements of an ideal system for accomplishing genetic
transformation of wheat, barley, and other cereals.
The present invention contemplates regeneration of plants
from the isolated scutellar tissue of the embryo. Applicants
have discovered that the embryonic cells of the embryo are
_.
---
principally in the scutellum, and that the embryo axis may be
detached from the scutellum without injury to the latter. As a
,ti..
-__.~,~.....~.~..
result of the removal of the embryo axis,tihe.__
growth of non--'{
--_ -------____-~. ~
embryogenic callus is inhibi-ted_ .___) The culturing of isolated'-
scutella promotes the 'grow~i- o~ competent embryogenic cells
leading to enhanced regeneration of somatic embryos. The
procedure therefore enriches the growth of embryogenic callus and
speeds up the process of somatic embryo formation andM plant
regeneration. As opposed to 10-12 weeks required for shoot or
somatic embryo formation in traditional immature embryo system,
the regeneration of somatic embryos using the new system of
isolated scutella is typically achieved within 2-3 weeks from
------------
2092588
initiation of cultures. This is a significant advantage as
prolonged culture period often results in loss of regeneration
potential and increases the risk of genetic instability and
sterility among the regenerants, which is undesirable for the
5 purpose of large scale propagation and genetic engineering of
cereal crops.
The frequency of somatic embryo formation from scutella is
highly efficient, usually on the order of 85-99W. The practice
also results in production of a large number of mature somatic
embryos (10-15) from a single scutellum within 2-4 weeks
(average: 3) of the initiation of cultures. A large proportion,
about 50-85W (typically about 70%r), of these somatic embryos, can
be converted into complete plantlets in two more weeks. Foreign
DNA may be targeted into these competent embryogenic cells, e.g.,
by the particle bombardment method of gene delivery, and
transformed plants regenerated by the method set forth herein.
The enhanced regeneration system employing in vitro culture
of scutella reported here for wheat and barley is also applicable
to other Gramineae crop species such as corn, rice, oats, sorghum
and other millets and grasses since in all these crops plant
regeneration is generally accomplished through the use of
conventional intact immature embryo culture.
It is known that in the conventional intact immature embryo
based regeneration system, the scutella contribute to the
production of embryogenic callus. (I.K. Vasil (1987) J. Plant
Physiol. 128:193-218.) However, the isolation of scutella was
discouraged by the fear that any injury to the immature embryo
would result in death of injured cells leading to complete loss
of regeneration potential, as monocots do not show a wound
response responsible for callus induction in most dicot species.
In dicot plants, meristematic activity can be induced by
culturing wounded tissues on a tissue culture medium. This
meristematic or cell division activity originates primarily from
the cambial tissue at the wound site, giving rise to a mass of
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unorganized cells known as callus. The actively dividing cells
of callus can be redifferentiated to form organs such as root
shoot and somatic embryos. In contrast, monocots in general and
cereals in particular do not show this type of wound response,
due to their inherent lack of cambial tissue. As a result, in
cereals, certain types of organs, such as immature zygote embryos
that contain cells predisposed to undergo active cell division
are cultured without causing any injury. The notion is that the
meristematic cells are capable of dividing actively only when the
organs are cultured intact and that any injury to such organs
would lead to loss of regeneration potential by disrupting the
pattern of active cell division.
However, in the present invention, the inventors have
provided a procedure for isolation of scutella without causing
injury to their sensitive parts. In addition, the present
inventors have conclusively established that competent
embryogenic cells are predominantly contained in the scutella. \':~'
-- _-- --__..
These competent embryogenic cells can be rapidly converted irito
a large number of somatic embryos by culturing the isolated
scutella under experimentally defined in vitro culture
conditions. These manipulations have lead to the development of
a novel enhanced regeneration system for both wheat and barley
which has not previously been available. The system is genotype-
independent, very efficient and results in regeneration of a
large number of fertile plants within 5 weeks as opposed to
several months required for very low frequency plant regeneration
in the conventional immature embryo system. The results obtained
by the process of the present invention also demonstrate that the
enhanced regeneration system can be used to provide an efficient
transformation system for wheat and barley.
{~,=.','
BRIEF DESCRIPTION OF THE DRAWINGS
Fig.1. Transient and stable GUS expression in wheat. (A) Scutella
of wheat showing transient GUS activity in competent embryogenic
cells, 48 hour after bombardment. (B) A developing transformed
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7
somatic embryo of wheat expressing GUS activity 4 weeks after
bombardment.
Fig. 2. Regeneration potential of different segments of wheat
scutellum. (A) An isolated scutellum showing different segments
used for culture. (B) Development of callus and somatic embryos
from different segments of scutellum after 3 weeks in culture.
Note a high frequency (Table 1) somatic embryo formation from
segment II representing the point of attachment of embryo axis
to scutellum.
Fig. Somatic embryogenesis and plant regeneration from isolated
scutella of wheat and barley. (A) An immature zygotic embryo of
wheat excised from caryopses 10 day-post anthesis. (B) An
isolated scutellum of wheat showing cut surface after dissection
of embryo axis. (C) Scutellum of wheat cultured with its cut
surface in contact with the medium. (D) Scutella of wheat showing
the development of transparent embryogenic rings 2 days after
culture. Note the development of larger rings in smaller scutella
(top row) and smaller rings in bigger ones (bottom row). (E)
Formation of nodular embryogenic callus surrounded by friable
non-embryogenic callus from isolated scutellum of wheat one week
after culture. (F) A cluster of wheat somatic embryos developed
from embryogenic callus two weeks after culture. (G) Mature
embryos of wheat formed from isolated scutellum 3 weeks after
culture. (H) Poorly organized somatic embryos surrounded by a
mass of non-embryogenic callus developed from intact immature
embryo of wheat 6 weeks after culture. (I) Globular embryogenic
callus developed on isolated scutellum of barley one week after
culture. (J) Proliferation of embryogenic callus and development
of somatic embryos from isolated scutellum of barley two weeks
after culture. (K) Mature somatic embryos of barley formed three
week after culture. (L) Plant regeneration from wheat somatic
embryos on hormone free MS medium. (M) A plantlet developed from
a single somatic embryo of wheat. (N) Fertile flowering plants
from control seeds (left) and scutellar-derived somatic embryos
(right) of wheat.
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DETAILED DESCRIPTION OF THE PREFERRED EbBODIbENTS
The process of the present invention for enhanced
regeneration from isolated scutellum overcomes the problems
associated with the intact immature zygotic embryo system. The
process of the present invention not only enriches the growth of
embryonic callus, but also expedites the process of somatic
embryo development. These attributes are essential for the
successful genetic engineering of cereal crops.
In conventional methods for regeneration of cereal plants
by somatic embryogenesis, the immature zygotic embryo is
cultivated until embryogenic callus forms, typically 4-6 weeks
post anthesis (See, e.g., Jahne, et al., 1991). This embryogenic
callus must then be separated from the nonembryogenic callus and
recultured for several more weeks before somatic embryos appear,
so that it is not usual for 8-10 weeks or more to elapse from
harvesting to somatic embryo formation.
In contrast, with the method of the present invention, the
isolated scutella typically forms embryogenic callus within a
mere 3-5 days after initiation of the culture. The somatic
embryos develop, without any need to subculture the embryogenic
callus a week or two later. Thus, a complete regeneration can
be achieved more quickly and with less labor. The rapid
development of the plant also makes it less susceptible to
developmental abnormalities, such as loss of fertility.
The anthers and ovules of a plant are formed by meiotic
division. When the anthers of a plant mature, they open up
(anthesis) and release (anther dehiscence) pollen grains, which
travel to the stigma. The pollen grains germinate there, forming
a pollen tube, through which the nuclei travel to the embryo sac.
There, they fertilize the ovules, forming a zygotic cell. The
zygote divides mitotically, forming an immature zygotic embryo.
The embryo is contained (after 8-16 days post-anthesis) in a
seed, which initially is light green in color (hence the name
"green grain") but darkens as development proceeds. The
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embryonic cell mass multiplies rapidly until about 30 days post-
anthesis, when the rate of cell division slows down. The seed
also begins to dry out, turning yellow in the process. By 50
days post-anthesis, the seed is dry, and there is little active
cell division. The embryos are now mature, and the seed is ready
for germination.
Plants are unique in their ability to also produce somatic
embryos. Somatic embryos are structurally similar to zygotic
embryos found in seeds, and are able to grow into complete
plants. However, they develop from somatic cells, instead of
zygotes, and they lack certain nutritive and protective tissues
found in seeds.
The Angiosperm.ae (flowering plants) are divided into two
classes, Monocotyledonae ( 'monocots") and Dicotyledonae
("dicots"). In dicots the zygotic embryo or a rudimentary plant
is enclosed with two cotyledons or seed leaves whereas monocots
have only one cotyledon. The single cotyledon or seed leaf of
monocots is botanically known as scutellum. The zygotic embryo
of cereals is present at the base of the seed and is composed of
two major parts, the embryo axis and the scutellum. The embryo
axis, at seed germination, develops into a seedling, and the
scutellum provides nourishment to the germinating embryo axis.
The embryo axis is composed of the shoot apex (plumule) pointing
towards the top of the seed and the root apex (radicle) pointing
towards the base of the seed. The protective sheath covering the
shoot apex is called coleoptile whereas that covering the root
apex is called coleorhiza. Root initials are the initials
present at the base of the primary root or radicle. The root
initials gives rise to secondary roots in a germinating embryo
axis. Attached to the embryo axis, near to the endosperm, is the
shield-shaped cotyledon or scutellum of the embryo.
Callus is an unorganized mass of cells. Embryogenic cells
are cells competent to form plants via somatic embryogenesis or
organogenesis.
2092588
The monocotyledoneae ("monocots") are divided into nine
different orders. The cereals belong to the order Graminale
(Glumiflorae) which is comprised of only one family i.e.
Gramineae (Poaceae). Cereal crop species such as wheat, barley,
5 corn, rice, oats, rye, sorghum, and millets belong to the family
Gramineae. The size of seed, embryo and scutellum may vary among
the species of cereal plants.
The present invention is generally directed to the
regeneration of cereals from cultured plant cells. In a
10 preferred embodiment, it contemplates regeneration of the
aforementioned cereal plants. Its use in regeneration of wheat
and barley is especially preferred. Fielder is the preferred
wheat variety, and Ellice the preferred barley variety.
The regeneration technology disclosed herein may be applied
to any cereal plant cells having a desirable genotype, whether
that genotype has occurred spontaneously in nature, or has arisen
through traditional hybridization techniques, mutation (with
radiation or chemicals) and selection, or genetic engineering.
If only a few seeds of a desirable plant are available, the
most effective means of increasing stock quickly may be to
amplify the embryos in cell culture. Theoretically, a culture
initiated from a single explant can be used to produce an
unlimited number of embryos. Conventional vegetative propagation
systems are limited to the amount of material that can be
harvested from the mother plant. Also, little labor input is
required to regenerate a complete plant from a somatic embryo,
which carries the program to make a complete plant. Vegetative
propagation systems must be manipulated as they require separate
shoot growth and rooting steps to make complete plantlets.
A desirable plant may have been obtained, e.g., by classical
breeding or by protoplast fusion. However, the most important
application for plant regeneration technology is in amplifying
transformed cells. Transformed cells are cells genetically
modified by direct transfer of foreign DNA into the cell by in
11 2092588
vitro manipulations, such as those hereafter described, or their
equivalents.
The DNA may be genomic DNA, complementary DNA, synthetic
DNA, or a combination thereof. DNA may be obtained from a
suitable source and then modified by mutagenesis. DNA from
several sources may be ligated together.
The term "foreign DNA," as used herein, means DNA which
encodes at least one gene product which the recipient cells are
not otherwise capable of producing. The gene product may,
however, be similar to a gene product native to the recipient
cells. The gene product may be one which is produced by at least
some member of the species to which the recipient cells belong,
or it may be entirely foreign to that species, or to the genus,
family, order, class or even a higher taxon to which the cells
belong. For example, the gene product may be one produced by
microbial or animal cells rather than plant cells. If the gene
product is from an organism whose relationship to cereal plants
is remote, it may be desirable to prepare a synthetic or
mutagenized gene whose codons are selected to enhance expression
in cereal plant cells.
By way of example and not limitation, the encoded gene
product may be one which provides insect, disease or herbicide
resistance, stress tolerance, or some form of quality
improvement. Suitable genes are set forth in more detail below:
A. Herbicide resistance
1. Phosphinothricin acetyltransferase (bar)
* ~:
gene for resistance to bialaphos or basta.
2. 5-enolpyruvylskhimate-3-phosphate synthetase
(EPSPS) gene for resistance to glyphosate
(Roundup).
3. Acetolactate synthase (ALS) gene for
resistance to sulfonylurea.
4. 2,4-dichlorophenoxyacetate * monooxygenase
gene for resistance to 2,4-D.
~, * Trademark
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12
5. Nitrilase gene for resistance to bromaxynil.
B. Insect and disease resistance
1. Bacillus thuringiensis (B.T.) endotoxin gene for insect
tolerance.
2. Proteinase inhibitor I and II genes for insect
tolerance.
3. Coat protein genes for viral tolerance.
4. PR (pathogenesis related) proteins for pathogen
resistance.
S. Chitinase genes for pathogen resistance.
6. Ribosome-inactivating proteins (RIP) for disease
resistance.
7. Gene encoding osmotin for disease resistance.
8. Genes for resistance to various fungi, bacteria and
nematodes.
C. Stress tolerance
1. Betaine aldehyde dehydrogenase (BADH) and other genes
for drought and salt tolerance.
2. Mannitol-l-phosphate dehydrogenase gene for stress
tolerance.
3. Genes for improved chilling and cold tolerance.
D. Improvement of quality and productivity
1. Bacterial g1gC gene encoding ADP-glucose
pyrophosphorylase for enhancing starch biosynthesis.
2. Genes for starch modification in seeds.
3. Genes for improved amino acid composition of seeds.
4. Ribonulease genes for induction of male sterility for
hybrid production.
Conveniently, the foreign DNA also comprises one or more
selectable or scorable marker genes whereby transformed cells may
be selected or screened. Suitable selectable markers include the
neomycin phosphotra4sferase (encodes resistance to kanamyciri;
geneticin* and G418* sulphate), hygromycin phosphotransferase
(resistance to hygromycin), phosphinothricin acetyltransferase
Trademark
2092588
13
(see above) and dihydrofolate reductase (resistance to
methotrexate) genes, or other genes conferring resistance to
antibiotics or herbicides. Scorable markers include beta-
glucuronidase bacterial or firefly luciferase, chloramphenicol
acetyltransferase, nopaline synthase and octopine synthase genes.
The coding sequences of the genes carried by the foreign DNA
will be operably linked either to their native promoters, or to
a new promoter. The new promoter may be a promoter native to the
recipient cells, or a promoter derived from another organism, but
functional in the recipient cells. The promoter may be a
constitutive or a regulatory promoter. A preferred promoter is
a cereal gene promoter, such as the rice actin 1B promoter. The
foreign DNA may include other regulatory sequences as well, such
as enhancer sequences. Plant genes frequently contain enhancer
sequences, e.g., in introns, which increase transcriptional
efficiency. A preferred enhancer is one derived from the intron
of a cereal gene, such as the rice actin 1B gene. A sampling of
suitable plant regulatory sequences appears below:
1. Cauliflower mosaic virus 35S promoter.
2. Rice actin promoter and/or its first intron.
3. Maize alcohol dehydrogenase (Adhl) promoter
and/or its intron.
4. Maize shrunken-1 (Shl) promoter and/or its
intron.
5. Emu promoter.
6. Nopaline synthase promoter.
7. Various combinations of above listed
promoters and introns.
8. Other constitutive and tissue specific
promoters and introns isolated from
different organisms.
The foreign DNA is usually cloned into a plasmid or phage
and amplified in bacteria such as E. coli. It may also be
amplified in vitro by PCR. However, the present invention is not
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14
limited to any particular method of amplification, nor is
amplification an absolute requirement. The foreign DNA, or a
fragment thereof, is then introduced into the target cells, which
are isolated scutellar cells.
The scutellum may be isolated by any art-recognized
technique, however it is desirable to remove as much of the
embryo axis as possible, while injuring the scutellum as little
as possible. In a preferred embodiment of this invention, the
scutella of wheat and barley were isolated using a stero
*
microscope, scalpel blade (Fishers size 11) mounted on a handle,
and ordinary forceps. Alternatively, blades of different sizes
or specially modified blades may be used for isolation of
scutella. In an immature embryo of appropriate stage selected
for isolation, the scutellum appears as a transparent to creamy
white shield-shape disk with its concave surface partially
covering the embryo axis. The embryo axis with its pointed shoot
and root apices is attached to the scutellum in the lower half.
The most critical feature of the three step procedure described
in this invention for isolation of scutellum is that it avoids
any injury to the sensitive parts of the scutelluin while removing
the entire embryo axis.
While it is desirable that the embryo axis be entirely
removed, and the scutellum uninjured, it will be appreciated that
an imperfectly isolated scutellum may still offer somatic embryo
regeneration potential superior to that of intact embryos, and
may therefore still be encompassed by the present invention. The
scutellum may be isolated at any stage that will yield enhanced
regeneration on potential relative to intact embryos.
The isolated scutella may be cultured in any medium suitable
for the development of embryonic cells and development of somatic
embryos. However, the isolated scutella are preferably cultured
in agar solidified MS (Murashige and Skoog, 1962) salt
formulation containing 2-3 mg/l phytohormone 2,4-D and 100-200
mg/l vitamin-free casamino acid.
* Trademark
C~L~;,
2092588
It is expected that for most cereal plant varieties, the
scutellum is best isolated from the immature zygotic embryo at
about 8-14 days post anthesis. (It should be noted that this is
prior to formation of embryogenic callus by the embryo.) With
5 wheat, the preferred time is 10-12 days post-anthesis, and with
barley, 8-10 days, but the optimum time differs from one variety
to the next and it is prudent to systematically determine the
optimum isolation time in the manner set forth in Example 5. At
too early a stage, the embryo axis and the scutellum are too
10 fragile, and scutellar damage is likely. At too late a stage,
the embryo axis adheres so tightly to the scutellum that it is
difficult to separate them without injury. Preferably, the
embryos are refrigerated for several days after harvesting, and
before dissecting out the scutella, as this facilitates the
15 separation.
If a genetically engineered plant is desired, the isolated
scutellum must be transformed with the DNA of interest. However,
it is preferable to preculture the scutella, typically for 2-5
days, prior to transformation. After two days, the cells are
actively dividing, as is desirable. By 4-5 days, the embryos has
a hard caryopses, making it more difficult to introduce the
foreign DNA into all embryonic cells. Still later, while
transformation can still occur, the resulting plant is likely to
be chimeric.
For the purpose of the present invention, it is not critical
which transformation technique is used, provided it achieves an
acceptable level of gene transfer. Potentially suitable
approaches include Agrobacterium vectors which infect monocots,
direct DNA uptake (possibly assisted by CaPO4 or PEG), viral
vectors, pollen-mediated transformation, electroporation,
microinjection and bombardment ("biolistics"). The latter
technique is preferred for genetic transformation of wheat,
barley and other cereals. (Sanford, USP4, 945,050; Franks and
Birch, 1991; Kartha et al., 1989; Chibbar et al., 1991; Vasil et
al., 1991, 1992). For a more general survey, see Gobel and Lorz
(1988) and Parrott, et al. (1991).
16 2092588
In "Biolistics", particles are coated with DNA and
accelerated by a mechanical device to a speed high enough to
penetrate the plant cell wall and nucleus. The foreign DNA thus
delivered into plant cells get incorporated into the host DNA,
resulting in production of genetically transformed cells. For
more details regarding the biolistics process and factors
affecting gene delivery and subsequent transformation efficiency,
please refer to Frank and Birch, (1991).
If the scutella have been precultured for two days, the
optimum pressure at the time of impact is 900-1300 lb/in2, more
preferably 1100 lb/in2. If the preculture is longer, so that a
caryopses must be penetrated, the projectile may need to exert
a pressure of as high as 2000 lb/in2. Commercially available
projectiles are made of Tungsten or Gold; Gold particles are
superior, possible because their microsurface is smooth.
Particles with a diameter of one micron work well but other sizes
are commercially available. The DNA concentration on the
particles should also be adjusted empirically; too little, and
too few cells are transformed, too much, and DNA is damaged.
Typically, 2.5-10 mg DNA is added to a 60 ml Eppendorf tube, and
each tube is good for 4-6 bombardments.
As previously mentioned, marker genes may be used to
select or screen for transformed cells. One strategy is to
transform, then select immediately. However, it is better to
cultivate the transformed cells for a time prior to selection.
It is also possible to do a further selection after the
transformed cells have developed into a somatic embryo.
Preferably, after isolation (or, if the cells are to be
transformed, after transformation) the scutellar cells are
initially cultivated in the dark, for a period of 5 to 7 days.
Subsequently, they may be transferred to low light conditions,
i.e., light of less than 10 E.m'2s'1. Since the regeneration
frequency in low light and complete dark is statistically
comparable, the use of low light is not essential but preferred
for fast and uniform development of somatic embryos. The medium
Trademark
A
2092588
17
preferred for conversion of somatic embryos into plants is half-
strength MS salt formulation.
Preferably, at least 50%, more preferably at least 85t, of
the isolated, untransformed scutella form one or more somatic
embryos. Desirably, an average of at least about 10 somatic
embryos is formed per regenerating scutellum. Advantageously,
at least 50k, more preferably, at least 70% of the somatic
embryos are converted into plants.
When cells are transformed by biolistic methods, a certain
amount of cell injury can be expected, which lowers the
regeneration efficiency, with typically 40-50W, rather than 85-
100t, of isolated transformed scutella forming somatic embryos.
The frequency of conversion of transformed somatic embryos into
plants is comparable to that f or nontransgenic embryos. However,
owing to the failure of some cells to be transformed, and of some
transformed cells to integrate the foreign DNA, only 1-3%; of
bombarded wheat scutella can be expected to develop into
transgenic plants. It must be emphasized, however, that this
level of transgenic plant formation is not considered
unsatisfactory; in using intact zygotic cereal plant embryos as
a source of cells for transformation and regeneration, it was not
possible to obtain anY transgenic plants.
Example 1:
Localization of Competent Embryogenic Cells in Scutellum
The culture of intact immature zygotic embryos results in
the production of a mixture of watery, friable non-embryogenic
and nodular embryogenic callus. Plant regeneration from these
cultures is obtained by selection and maintenance of embryogenic
callus from a mixture of different callus types, which becomes
a cumbersome and time consuming process.
In an attempt to localize the cells which were competent to
form embryogenic callus and to enrich the growth of such cells,
various components of immature zygotic embryos such as scutellum,
2092588
18
embryo axis, shoot apex, root apex, coleorhiza, and coleoptile
were dissected and tested separately for callus formation and
regeneration. These explants were placed onto culture medium
with either cut surface in contact with the medium or away form
the medium surface.
For the experiments described below, the immature embryos
were obtained from caryopses (a botanical name for grains or
seeds of cereals) of cultivated varieties (cvs.) Fielder and
HY320 of wheat 10-12 day post anthesis and from cv. Ellice of
barley 8-10 day post anthesis. All cultures were incubated in
the dark at 26 2 C on Murashige and Skoog's (1962) nutrient
medium supplemented with 2 mg/L 2,4-dichlorophenoxyacetic acid
(2,4D) and 100 mg/L vitamin free casamino acids (CA). 2,4-
dichlorophenoxyacetic acid (2e4-D) is a synthetic auxin
(phytohormone) which is desirable for induction of callus and
development of somatic embryos from embryogenic cells contained
in isolated scutella. The acceptable range of 2,4-D for cereals
is about 1-3 mg/i, but about 2 mg/1 is preferred for wheat and
barley scutella culture.
In cereal cell cultures, primarily two different types of
calluses are encountered i.e. non-embryogenic and embryogenic
callus. The non-embryogenic callus is characterized as a fast
growing, soft white, friable callus that sometimes gives watery
appearance. The cells contained in this type of callus do not
form somatic embryos. By contrast, the embryogenic callus is
~. __..
compact, organized and pale yellow in color. The growth of
embryogenic callus is slow but this is the type of callus that
results in formation of somatic embryos.
It was found from the above experiments that non-
embryogenic watery and friable callus originated mainly from
various components of embryo axis, whereas the embryogenic cells
-.----
------- --____. _. .~ -- -----.
were predominantly contained in the _scutellar..._.t1.ssue. The
coleoptile, coleorhiza and root initials in root apex were the
main contributors to the pool of watery and non-embryogenic
callus. In shoot apex, after removal of watery coleoptile
2092588
19
callus, the basal end of plumule sometimes formed compact creamy
type of callus, which grew slowly and rarely differentiated into
shoots after 12-16 weeks of culture. Occasionally, the intact
embryo axis also formed this type of callus when cultured with
the cut surface away from the medium. Among all explants tested,
the embryogenic callus and somatic embryos were obtained from
isolated scutellar tissue.
The position of isolated scutellum was critical for
obtaining somatic emb~yogenesis. In wheat, somatic embryos were
formed when the cut surface of the scutellum was kept in contact
with the medium, whereas only non-embryogenic callus developed
when the cut surface was kept away from the medium. However, in
barley the position of scutellum did not prevent regeneration,
but a large number of explants regenerated when the cut surface
was kept in contact with the medium. These experiments suggested
that growth of embryogenic cells contained in the scutellar
tissue of immature zygotic embryo could be enriched for
development of somatic embryos by cutting the carefully isolated
scutella in an appropriate position. Further dissection of
scutellum into several segments, as shown in Figure 2A, revealed
that the embryogenic cells were concentrated in a parti ular zone
of scutellum at the junction of embryo axis and scutellum, as
shown in Figure 2B, and Table 1. Figure 2B shows development of
callus and somatic embryos from different segments of scutellum
after three weeks in culture. Table 1 shows a high frequency of
somatic embryo formation from segment II, representing the point
of attachment of the embryo axis to the scutellum. However, the
microscopic detachment of embryo axis was essential for promoting
the growth and development of such cells into somatic embryos.
The dissection of scutellum into several segments did not hamper
the development of__embryoqenic-__callus-.and-.s.Qmatic embryous, but__..
promoted vigorous growth of non-embryogenic t~allus from the cut
ends. i'Clo overeome this problem, a technique was developed for
removal of embryo axis while avoiding any injury to the scutellum
at both ends.
2092588
Example 2:
Isolation and culture of scutellum
For isolation of scutella, the spikes of wheat were
harvested 10 day-post anthesis and the spikes of barley 8 day-
5 post anthesis from the plants grown in a growth chamber under 16
h. photoperiod (150 E.m2s'1) at 25 C day and 20 C night
temperature. To facilitate isolation of scutella, it was
necessary to store the spikes in a refrigerator at, e.g., 5-7 C,
preferably 5 C for at least five days, e:g., 5-10 days, more
10 preferably 5-7 days. The immature embryos obtained from spikes
immediately after harvesting were fragile and difficult to
dissect whereas those obtained from spikes stored for longer than
10 days were mature and gave poor response to somatic embryo
formation. The immature caryopses were surface sterilized with
15 70t ethanol (1 min) and 20t javex* (20 min) for wheat and 5%- javex
(3 min) for barley followed by five rinses with sterile distilled
water.
The immature zygotic embryos were excised from caryopses ten
days post anthesis using a stereo dissecting microscope (Fig.3A) .
20 Further separation of the embryo axis from scutellum was a
three step procedure. In first step, a slanting cut was made by
sliding a scalpel blade on the right side of the embryo starting
from the shoot apex to the end of the root apex along the ridge
joining the embryo axis to scutellum, while gently supporting the
left side of embryo with forceps. The immature embryo was then
turned around and a similar cut was made on the left side by
sliding the scalpel blade from the root apex to the shoot apex
along the ridge. Finally, the embryo axis was removed by gently
holding the root apex with forceps and lifting the shoot apex
with the tip of a scalpel blade to obtain isolated scutella.
Fig.3B shows the cut surface after dissection of the embryo axis.
The morphology of all cereal plant immature embryos is
essentially similar. Therefore the technique would not be
different for barley or any other cereal. It should be
emphasized here that any injury caused to the scutella, at either
* Trademark
~
2092588
21
end, during this painstaking operation would promote the
development of non-embryogenic callus and result in the reduction
of somatic embryo regeneration potential of the scutella.
The isolated scutella were cultured on MS medium
supplemented with 2 mg/1 2,4-D and 100 mg/1 vitamin-free casamino
acids. The scutella were placed with their cut surface in
contact with medium (Fig.3C) . (The cut surface may also lie away
from the medium, but this is less effective.) The cultures were
incubated in the dark at 26 2 C for one week for induction of
embryogenic callus and then transferred to low light (e.g., 10
E.mZs-1) for two weeks for development of embryogenic callus into
mature somatic embryos.
Example 3:
Somatic embryogenesis and plant regeneration
A transparent circular ring was observed in the basal
portion of isolated wheat scutella within 2-3 days after
initiating culture (Fig.3D). The size of the ring was dependent
on the developmental stage of scutella; the smaller scutella
(1.7-2.0 mm) produced a larger ring compared to the bigger (2.2-
2.5) ones (Fig.3D). The circular ring further developed into a
mass of nodular compact embryogenic callus surrounded by
peripheral non-embryogenic friable callus within a week from
culture initiation (Fig.3E). The embryogenic callus at this
stage contained several globular to slightly advanced stage
somatic embryos. Although embryogenic callus developed into a
mass of distinct somatic embryos in dark, the transfer of
culture~a_ to-- low light ___(10gE.ni2s-1) enhanced the development of
---- -- ----
----
somatic embryos and suppressed the growth of peripheral non-
_~._.. ..._..._..__ ._...._.._.... _._.__ __w. _..._
_
embryo e~ c.~lus y Wi.thin a week af ter remo o ures to
low light, the whole embryogenic callus turned into a cluster of
distinct somatic embryos (Fig.3F) which further developed into
mature somatic embryos in another week (Fig.3G). Fig.3H shows
poorly organized somatic embryos surrounded by a mass of non-
embryonic callus developed from intact immature wheat embryo six
weeks after culture.
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22
The process of somatic embryogenesis in barley was similar
to wheat except that the formation of a distinct circular ring
was not observed. Instead the entire surface of scutellum turned
transparent and formed globular somatic embryos within a week
5(Fig.3I). These globular somatic embryos further proliferated
(Fig.3J) and developed into mature somatic embryos (Fig.3K)
within 3 weeks from the initiation of cultures. The mature
somatic embryos of both wheat and barley easily germinated on
half strength MS medium (Fig.3L and M) resulting in the rapid
production of a large number of fertile plants (Fig.3N).
Example 4:
Comparison of plant regeneration potential
After establishing the technique for isolation and in vitro
culture of scutella, the regeneration potential of conventional
immature embryo system was compared with the new enhanced
regeneration system. The data presented in Table 2 show a
significant im~r.Qvement in embryogeniccallus induction, somatic
embryo formation and number of somatic embryos per explant with
the new method of culturing isolated scutella. Callusing was
visible in the conventional immature embryo explants within two
weeks, but most of the callus developed at this stage was watery
and non-embryogenic type of callus. However, some explants
exhibited tiny sectors of creamy embryogenic callus, embedded in
the mass of non-embryogenic callus, which formed a few poorly
organized somatic embryos after 4-6 weeks in culture (Fig.3H).
On the other hand, in the new method, distinct nodular
embryogenic callus (Fig.3E) developed on almost all isolated
scutella within a week which eventually developed into a prolific
mass of mature somatic embryos in 3 weeks from culture initiation
(Fig.3G).
Example 5:
Effect of developmental stage of scutellum
The developmental stage of scutellum was found to influence
the regeneration potential. Therefore in this experiment the
immature embryos were obtained after 8, 10, 12 and 14 day-post
anthesis for isolation of scutella. There was no significant
2092588
23
differencein embryogenic callus formation (Table 3) from
scutella ranging from 1.2-2.5 mm in size (8-12 day-post
anthesis). However, the larger scutella (2.7-3.0 mm, 14 day-post
anthesis) gave poor response to embryogenic callus and somatic
embryo formation. Although very young scutella (1.2-1.5 mm, 8
day-post anthesis) formed embryogenic callus at a high frequency,
the growth of calli from such explants was slow and resulted in
lower frequency of somatic embryo formation, as shown in Table
3. The best response for embryogenic callus induction and
somatic embryo formation was obtained from scutella in the range
of 1.7-2.5 mm in size (10-12 day-post anthesis) . Therefore, this
particular stage of scutella is hereby recommended to obtain
enhanced somatic embryogenesis and plant regeneration.
Example 6 s
Effect of hormone concentration
Our experiments indicated that addition of vitamin-free
casamino acids to the culture medium had little effect on
frequency of somatic embryogenesis but helped in uniform
development of somatic embryos. In this experiment the effect
of 2,4-D concentrations in the range of 1-4 mg/1 was tested. All
concentration of 2,4-D were supplemented with 100 mg/1 vitamin-
free casamino acids. Although there was no significant
difference in the frequency of embryogenic callus and somatic
embryo formation at different concentrations of 2,4-D (Table 4),
the highest response to somatic embryo formation (92.5g) and
number of somatic embryo /explant (10.8) was obtained with 2 mg/1
2,4-D concentration. The lower concentration of 2,4-D promoted
root formation as shown in Table 4, and higher concentrations
suppressed the development of somatic embryos.
Examp 1 e 7:
Effect of light intensity
The transfer of cultures to low light after induction of
embryogenic callus in the dark was found to enhance the
development of somatic embryos. Therefore a detailed experiment
was conducted to test the effect of various light intensities (0-
60 E.m'Zs'1) on somatic embryogenesis. As evident from data
------ - - - ---------
2092588
24
presented in Table 5, there was no significant difference in the
frequency of embryogenic callus and somatic embryo formation
between dark and low light (10 E.iri2s'1) . Well developed distinct
somatic embryos were formed in dark and low light but the
development of somatic embryos was more uniform and faster at low
light. The higher light intensity levels were found to interfere
with the development of embryogenic callus and somatic embryos.
Poorly organized and often fused somatic embryos were observed
on some explants at 20 E.m2s'1 light intensity. Most explants
formed green spots instead of developed somatic embryos at 40 and
60 E.mzs'1 light intensity.
Example 8:
Conversion of somatic embryos into plants
The frequency of conversion of somatic embryos into plants
is considered critical for the success of a system based on
somatic embryogenesis. To test the conversion efficiency of
somatic embryos into plants, the mature somatic embryos separated
from 3 week-old cultures were plated on different media. The
highest rate (68t) of conversion was obtained on half strength
MS medium (Table 6). Significantly lower rate of conversion was
obtained on full strength MS medium or on MS medium containing
low levels of abscisic acid (ABA). Somatic embryos developed
into complete plants with long shoots and short roots within 2
weeks on MS medium without growth hormones. The addition of ABA
suppressed the growth of shoot but promoted root growth as shown
in Table 6.
Example 9:
Genotypic response to Somatic Embryogenesis
Ten commercial genotypes of wheat were tested for their
response to somatic embryogenesis from isolated scutella under
the conditions standardized for cv. Fielder. All genotypes
formed embryogenic callus and somatic embryos, cf. Table 7.
However, differences were' observed in frequency of somatic
embryogenesis and number of somatic embryps formed per explant.
In general, the Canada Prairie Spring (CPS)* wheat genotypes gave
better response to somatic embryogenesis than Canada Western Red
* Trademark
,';
25 2092588
Spring (CWRS)* wheat genotypes. In recent experiments designed
to evaluate the interaction between explant size and genotypes,
it was observed that regeneration frequency of some of the
genotypes could be considerably improved by culturing the
scutella of different size groups.
Six barley genotypes were also tested under culture
conditions optimized for wheat. All genotypes responded to
somatic embryo formation, albeit at different frequencies, when
scutella in the range of 1.2 -1.5 mm (8 day-post anthesis) in
size were cultured, cf. Table 8. However, the highest response
to somatic embryogenesis (82.5%) was obtained in cv. Ellice.
The foregoing description of the specific embodiments will
so fully reveal the general nature of the invention that others
can, by applying current knowledge, readily modify and/or adapt
for various applications such specific embodiments without
departing from the generic concept, and, therefore, such
adaptations and modifications should and are intended to be
comprehended within the meaning and range of equivalents of the
disclosed embodiments. It is to be understood that the
phraseology or terminology employed herein is for the purpose of
description and not of limitation.
* Trademark
~
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26
LITERATURE CITED:
Chibbar, R.N., Kartha, K.K., Leung, N., Qureshi, J. and Caswell,
K. 1991. Transient expression of marker genes in immature
zygotic embryos of spring wheat (Triticum aestivum) through
microprojectile bombardment. Genome 34:453-460.
Franks and Birch, 1991. Microprojectile Techniques for Direct
Gene Transfer into Intact Plant Cells, in Murray ed.,
Advanced Methods in Plant Breeding and Biotechnologv, Chapt.
5, pp. 103-127.
Mitsui Toatsu Chemicals, Canadian Patent 1,288,713
Gobel, E. and Lorz, H. 1988. Genetic manipulation of cereals.
Oxford Survey of Plant Molecular and Cell Biology 5:1-22.
He, D.G., Yang, Y.M. and Scott, K.J. 1992. Plant regeneration
from protoplasts of wheat (Triticum aestivum cv. Hartog).
Plant Cell Reports 11:16-19.
Jahne, A., Lazzeri, P.A. and Lorz, H. 1991. Regeneration of
fertile plants from protoplasts derived from embryogenic
cell suspensions of barley (Hordeum vulgare L.). Plant Cell
Reports 10:1-6.
Kartha, K.K., Chibbar, R.N., Georges, F., Leung, N., Caswell,
K., Kendall, E. and Qureshi, J. 1989. Transient expression
of chloramphenicol acetyltransferase (CAT) gene in barley
cell cultures and immature embryos through microprojectile
bombardment. Plant Cell Reports 8:429-432.
Kartha, K.K., Chibbar, R.N., Nehra, N.S., Leung, N., Caswell,
K., Baga, M., Mallard, C.S. and Steinhauer, L. 1992.
Genetic engineering of wheat through microprojectile
bombardment using immature zygotic embryos. J Cellular
Biochem. Supplement 16F:198. (Abstract Y001)
Murashige, T. and Skoog, F. 1962. A revised medium for rapid
growth and bioassays with tobacco tissue cultures. Physiol.
Plant. 15:473-497.
Parrott, et al., Somatic Embryogenesis: Potential in Use in
Propagation and Gene Transfer Systems, in Murray ed.,
Advanced Methods in Plant Breeding and Biotechnology, Chapt.
5, pp. 103-127.
2092588
27
Redway, F.A., Vasil, V., Lu, D., and Vasil I.K. 1990.
Identification of callus types for long-term maintenance and
regeneration from commercial cultivars of wheat (Triticum
aestivum L.). Theoret. Appl. Genet. 79:609-617.
Sanford, J.C., Klein, T.M., Wolf, E.D., and Allen, N. 1987.
Delivery of substances into cells and tissues using a
particle bombardment process. Particulate Science
Technology 5:27-37.
Sanford, et al., U. S. Patent No. 4,945,050 (1990)
Thomas, M.R. and Scott, K.J. 1985. Plant regeneration by somatic
embryogenesis from callus initiated from immature embryos
and immature inflorescences of Hordeum vulaare. J. Plant
Physiol. 121:159-169.
Vasil, I.K. 1988. Progress in the regeneration and genetic
manipulation of cereal crops. Bio/Technology 6:397-402.
Vasil, V., Redway, F.A., and Vasil, I.K. 1990. Regeneration of
plants from embryogenic suspension culture protoplasts of
wheat (Triticum aestivum L.). Bio/Technology 8:429-433.
Vasil, V., Brown, S.M., Re, D., Fromm, M.E. and Vasil, I.K.
1991. Stably transformed callus lines from microprojectile
bombardment of cell suspension - cultures of wheat.
Bio/Technology 9:743-747.
Vasil, V., Castillo, A.M., Fromm, M.E. and Vasil, I.K. 1992.
Herbicide resistant fertile transgenic wheat plants
obtained by microprojectile bombardment of regenerable
embryogenic callus. Bio/Technology 10:667-674.
No admission is made that any cited reference
constitutes prior art.
The specification of any range shall be deemed the
description of all included subranges. Any reference to
a multi-membered class, such as the class of cereal
plants, should be deemed a description not only of that
class, but also all possible subclasses, e.g., cereal
plants other than rice.
~
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28
Table 1. Embryogenic potential of different scutellar segments of wheat cv.
Fielder=.
Scutellar % explants forming % explants forming No.of mature
segment embryogenic callus somatic embryos somatic embryos
after 2 weeks after 4 weeks /explant
after 4 weeks"
20.4a 20.4a 1.9a
II 70.4b 70.4b 4.8b
III 17.6a 18.5a 0.7a
IV 2.8a 5.6a 0.3a
= Mean separation within column by Tukey's HSD (P = 0.05) on transformed data.
Original means are presented.
" Total number of somatic embryos/number of explants forming somatic embryos.
29
Table 2. Comparison of embryogenic potential of intact immature embryos
(conventional method)
and isolated scutella (new method) for wheat cv. Fielder.
Explant % explants forming % explants forming No. of mature
(position) embryogenic callus somatic embryos somatic embryos
after 2 weeks after 4 weeks /explant
after 4 weeksz
Conventional method
Immature embryo'' 12.5 17.5 2.8
(embryo axis down)
New method
Scutellum 95.0 97.5 10.3
(cut surface down)
Significance * * * * * * * *
Significant at P=.001 by paired t test.
= Total number of somatic embryos/number of explants forming somatic embryos.
The germinated embryo axis was removed within a week after culture initiation.
2092588
Table 3. Effect of developmental stage (size) of scutellum on somatic
embryogenesis of wheat cv.
Fielder=.
Days post- % explants forming % explants forming No. of mature
5 anthesis embryogenic callus somatic embryos somatic embryos
(size range after 2 weeks after 4 weeks /explant
in mm) after 4 weeks''
8 (1.2-1.5) 87.5a 42.5a 4.8a
10 10 (1.7-2.0) 92.5a 85.Ob 15.Ob
12 (2.2-2.5) 87.5a 82.5b 14.2b
14 (2.7-3.0) 29.8b 26.4a 4.7a
15 Z Mean separation within column by Tukey's HSD (P = 0.05) on transformed
data. Original means
are presented.
'' Total number of somatic embryos/number of explants forming somatic embryos.
The letters a, b, c and d with each number in Tables 3-6 represents
statistical significance. The
numbers followed by different letters are statistically significant from each
other whereas those
20 followed by the same letter are non-significant.
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31
Table 4. Effect of hormone concentration on somatic embryogenesis of wheat cv.
Fielderz.
Hormone % explants % explants % explants No. of
concentration forming forming forming mature
(mg/I) embryogenic somatic roots somatic
callus embryos after embryos/
after after 4 weeks explant
2 weeks 4 weeks after
4 weeksY
OD + OCAx O.Oa O.Oa O.Oa O.Oa
1 D+ 100CA 67.5b 81.2b 81.2b 5.3b
2D + 100CA 76.2b 92.5b 27.5c 10.8c
3D + 100CA 62.5b 88.8b 7.5d 6.4b
4D + 100CA 53.8b 76.2b O.Oa 4.4b
Z Mean separation within column by Tukey's HSD (P = 0.05) on transformed data.
Original means
are presented.
Y Total number of somatic embryos/number of explants forming somatic embryos.
" D = 2,4-dichlorophenoxyacetic acid; CA = casamino acid (vitamin free)
2092588
32
Table 5. Effect of light intensity on somatic embryogenesis of wheat cv.
Fielderz.
Light intensity % explants forming % explants forming No. of mature
(ErE.m-zs'') embryogenic callus somatic embryos somatic embryos
after 2 weeks after 4 weeks /explant
after 4 weeks"
0 (Dark) 93.8a 93.8a 7.6a
95.Oa 98.8a 9.6a
10 20 51.2b 68.8b 3.2b
40 28.8c 36.2c 2.3b
60 O.Od 17.5c 0.9c
z Mean separation within column by Tukey's HSD (P = 0.05) on transformed data.
Original means
are presented.
" Total number of somatic embryos/number of explants forming somatic embryos.
2092588
33
Table 6. Frequency of conversion of somatic embryos of wheat cv. Fielder into
plantlets on
different media'.
Medium'' % somatic embryos Average length Average laudi
forming plantlets of shoot (cm) of root (cm)
after 2 weeks
Half strength MS 68.Oa 6.7a 2.2a
Full strength MS 49.Ob 5.Oa 2.3a
MS + .025 mg/I ABA 38.Ob 2.9b 3.9b
MS + .050 mg/I ABA 42.Ob 3.4b 3.7b
= Mean separation within column by Tukey's HSD (P = 0.05) on transformed data.
Original means
are presented.
'' MS = Murashige and Skoog's mineral salts (1962); ABA = abscisic acid
2092588
34
Table 7. Response of different wheat genotypes to somatic embryogenesis from
isolated
scutellum=.
Genotype % explants forming % explants forming No. of mature
embryogenic callus somatic embryos somatic embryos
after 2 weeks after 4 weeks /explant
after 4 weeksY
CPS (Canada Prairie Spring Wheat)
i---,
Fielder 92.5 4.8x 85.0 6.4 15.0 0.7
Taber 87.5 + 4.8 52.5 11.1 3.4 0.5
Genesis 77.5 12.5 65.0 13.2 3.9 0.4
Biggar 55.0 5.0 45.0 9.6 3.5 0.2
HY320 52.5 4.8 35.5 4.8 4.0 0.7
CWRS (Canada Western Red Spring Whe t)
Minto 50.8 + 11.0 ~ 39.4 + 9.8 4.4 +
_ _ _ 0.8
Laura 30.0 9.1 27.5 8.5 2.8 0.2
Pasqua 20.0 0.0 12.5 4.8 2.9 1.0
Katepwa 18.3 6.9 10.5 4.5 2.0 0.7
Makwa 17.5 6.3 17.5 6.3 2.0 0.8
= Ten-day post anthesis
'' Total number of somatic embryos/number of explants forming somatic embryos.
" Mean + S.E.
2092588
Table 8. Response of different barley genotypes to somatic embryogenesis from
isolated scutellum=.
Genotype % explants forming % explants forming No. of mature
embryogenic callus somatic embryos somatic embryos
5 after 2 weeks after 4 weeks /explant
after 4 weeks''
Ellice 72.5 6.3 82.5 6.2 3.4 0.3
Manley 92.5 4.8 67.5 4.8 2.5 0.1
10 Bridge 67.5 2.5 47.5 6.3 2.8 0.2
Guardian 85.0 2.9 35.0 2.9 3.0 0.2
Harrington 45.0 9.6 47.5 16.0 2.4 0.4
TR-941 40.0 7.1 35.0 8.7 1.7 0.3
15 = Eight-day post anthesis (two-day post anther protrusion)
'' Total number of somatic embryos/number of explants forming somatic embryos.
" Mean S.E.
