Enhanced regeneration system

Whole scutella are isolated from immature zygotic embryos of cereal plants and cultured, in the absence of the zygotic embryo axis, to produce somatic embryos, which in turn are converted into plantlets. The scutellar cells optionally are transformed with foreign DNA so that at least some of the resulting plantlets are transgenic. The regeneration is much more efficient and rapid than with conventional methods.

BACKGROUND OF THE INVENTION 
1. 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. 
2. 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 contribute about 90% of the total grain production 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 unique 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 
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 establishing 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 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-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. 
SUMMARY OF THE 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 zygotic embryo. Applicants have discovered that 
the embryogenic cells of the zygotic embryo are principally in the 
scutellum, and that the embryo axis may be detached from the scutellum 
without injury to the latter. As a result of the removal of the embryo 
axis, the growth of non-embryogenic callus is inhibited. The culturing of 
isolated scutella promotes the growth of 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 and 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 
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 purpose of large scale 
propagation and genetic engineering of cereal crops. 
The frequency of somatic embryo formation from scutella is very high, 
usually on the order of 85-99%. 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 50-85% (typically about 70%), of these somatic embryos, 
can be converted into complete plantlets in two more weeks. Foreign DNA 
may be targeted into the competent embryogenic cells of scutellum e.g., by 
the particle bombardment method of gene delivery (FIG. 1A), and stably 
transformed somatic embryos and plants regenerated (FIG. 1B) 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 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 into 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.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
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 embryogenic 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 cultured until embryogenic 
callus forms, typically 4-6 weeks post initiation of cultures (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 unusual for 8-10 weeks or more 
to elapse from the initiation of cultures 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 efficiently. 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 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 Angiospermae (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. 
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, 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 isolated scutella. In a 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 my be applied to any cereal 
crop 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 
developmental 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 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. 
5. Nitrilase gene for resistance to bromaxynil. 
B. Insect and disease resistance 
1. Bacillus thuringienses (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. 
5. 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-1-phosphate dehydrogenase gene for stress tolerance. 
3. Genes for improved chilling and cold tolerance. 
D. Improvement of quality and productivity 
1. Bacterial glgC 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. Ribonuclease 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 
phosphotransferase (encodes resistance to kanamycin, geneticin and G418 
sulphate), hygromycin phosphotransferase (resistance to hygromycin), 
phosphinothricin acetyltransferase (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 1D 
promoter. The foreign DNA may include other regulatory sequences as well, 
such as intron sequences. Plant genes frequently contain intron sequences, 
which increase transcriptional efficiency. A preferred intron is one 
derived from a cereal gene, such as the rice actin 1D 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 (Adh1) promoter and/or its intron. 
4. Maize shrunken-1 (Sh1) 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 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 present in isolated scutellar tissue. 
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 scutellum 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 embryogenic 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. 
It is expected that for most cereal plant varieties, the zygotic embryo is 
best harvested from the plant at about 8-14 days post anthesis. With 
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 harvesting time in 
the manner set forth in Example 5. The purpose of harvesting the embryo is 
to permit the isolation of the scutella, and at too early a stage, the 
embryo axis and the scutellum are too 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 scutella are not isolated immediately after harvesting; a 
holding period of about 5-10 days is desirable. Preferably, the embryos 
are refrigerated during this holding period, e.g., at about 5-10 degrees 
C. This refrigeration facilitates the separation. It is not necessary that 
the embryos be cultured prior to isolation of the scutella. 
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 scutella have hard embryogenic callus, 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 
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, U.S. Pat. No. 4,945,050; 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). 
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 rupture 
pressure at the time of impact is 900-1300 lb/in.sup.2, more preferably 
1100 lb/in.sup.2. If the preculture is longer, so that an embryogenic 
callus must be penetrated, the projectile may need to exert a pressure of 
as high as 2000 lb/in.sup.2. Commercially available projectiles are made 
of Tungsten or Gold; Gold particles are superiors possible because of 
their non-toxic nature, uniform size and smooth microsurface. 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 
too many cells are damaged. Typically, 2.5-10 .mu.g DNA is coated onto 
microprojectiles and suspended in 60 ml ethanol in a microfuge tube. 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 culture the transformed cells for a time prior to 
selection. It is also possible to apply 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 .mu.E.m.sup.-2 s.sup.-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 
preferred for conversion of somatic embryos into plants is half-strength 
MS salt formulation. 
Preferably, at least 50%, more preferably at least 85%, 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 50%, 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 40-50%, rather than 85-100%, of isolated transformed scutella 
forming somatic embryos. The frequency of conversion of transformed 
somatic embryos into plants is comparable to that for 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, 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 from 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.degree..+-.2.degree. 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 (2,4-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/l , but about 2 
mg/l 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 whites 
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 tissue. 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 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 
embryogenesis. 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 larger 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 culturing the carefully isolated scutella in an appropriate 
position. Further dissection of scutellum into several segments, as shown 
in FIG. 2A, revealed that the embryogenic cells were concentrated in a 
particular zone of scutellum at the junction of embryo axis and scutellum, 
as shown in FIG. 2B, and Table 1. FIG. 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 embryogenic callus and somatic embryos, but 
promoted vigorous growth of non-embryogenic callus from the cut ends. To 
overcome this problem, a technique was developed for removal of embryo 
axis while avoiding any injury to the scutellum at both ends. 
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-post anthesis from the plants 
grown in a growth chamber under 16 h photoperiod (150 .mu.E.m.sup.-2 
s.sup.-1) at 25.degree. C. day and 20.degree. C. night temperature. To 
facilitate isolation of scutella, it was necessary to store the spikes in 
a refrigerator at, e.g., 5.degree.-7.degree. C., preferably 5.degree. C. 
for at least five days, e.g., 5-10 days, more 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 70% 
ethanol (1 min) and 20% 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). 
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 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/l 
2,4-D and 100 mg/l 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.degree..+-.2.degree. C. for one 
week for induction of embryogenic callus and then transferred to low light 
(e.g., 10 .mu.E.m.sup.-2 s.sup.-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 
cultures to low light (10 .mu.E.m.sup.-2 s.sup.-1) enhanced the 
development of somatic embryos and suppressed the growth of peripheral 
non-embryogenic callus. Within a week after removal of cultures 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-embryogenic callus developed from 
intact immature wheat embryo six weeks after culture. 
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 (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 improvement in embryogenic callus 
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 difference in 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 
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/l was tested. 
All concentrations of 2,4-D were supplemented with 100 mg/l 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 (92.5%) and number of somatic embryo/explant (10.8) was obtained 
with 2 mg/l 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. 
EXAMPLE 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 .mu.E.m.sup.-2 s.sup.-1) on somatic 
embryogenesis. As evident from data 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 .mu.E.m.sup.-2 s.sup.-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 
.mu.E.m.sup.2 s.sup.-1 light intensity. Most explants formed green spots 
instead of developed somatic embryos at 40 and 60 .mu.E.m.sup.2 s.sup.-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 somatic embryos into plants, the mature 
somatic embryos separated from 3 week-old cultures were plated on 
different media. The highest rate (68%) 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 embryos formed per explant. In 
general, the Canada Prairie Spring (CPS) wheat genotypes gave better 
response to somatic embryogenesis than Canada Western Red 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. 
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 Biotechnology, Chapt. 5, pp. 103-127. 
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) 
Mitsui Toatsu Chemicals, Canadian Patent 1,288,713 
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. 
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. Pat. 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 vulgare. 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. 
All references cited in this specification are hereby incorporated by 
reference. No admission is made that any cited reference constitutes prior 
art. 
The appended claims are hereby incorporated by reference as a further 
description of the preferred embodiments. 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. 
TABLE 1 
______________________________________ 
Embryogenic potential of different scutellar 
segments of wheat cv. Fielder.sup.z. 
No. of mature 
% explants somatic 
% explants forming 
forming embryos/ 
Scutellar 
embryogenic callus 
somatic embryos 
explant 
segment 
after 2 weeks after 4 weeks 
after 4 weeks.sup.y 
______________________________________ 
I 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 
______________________________________ 
.sup.z Mean separation within column by Tukey's HSD (P = 0.05) on 
transformed data. Original means are presented. 
.sup.y Total number of somatic embryos/number of explants forming somatic 
embryos. 
TABLE 2 
______________________________________ 
Comparison of embryogenic potential of intact 
immature embryos (conventional method) and 
isolated scutella (new method) for wheat cv. Fielder. 
% explants No. of mature 
forming somatic 
% explants forming 
somatic embryos/ 
Explant embryogenic callus 
embryos explant 
(position) 
after 2 weeks after 4 weeks 
after 4 weeks.sup.z 
______________________________________ 
Conventional 
method 
Immature 12.5 17.5 2.8 
embryo.sup.y 
(embryo axis 
down) 
New method 
Scutellum 
95.0 97.5 10.3 
(cut surface 
down) 
Significance 
*** *** *** 
______________________________________ 
***Significant at P = .001 by paired t test. 
.sup.z Total number of somatic embryos/number of explants forming somatic 
embryos. 
.sup.y The germinated embryo axis was removed within a week after culture 
initiation. 
TABLE 3 
______________________________________ 
Effect of developmental stage (size) of scutellum on 
somatic embryogenesis of wheat cv. Fielder.sup.z. 
% explants No. of mature 
Days post- forming somatic 
anthesis 
% explants forming 
somatic embryos/ 
(size range 
embryogenic callus 
embryos explant 
in mm) after 2 weeks after 4 weeks 
after 4 weeks.sup.y 
______________________________________ 
8 (1.2-1.5) 
87.5a 42.5a 4.8a 
10 (1.7-2.0) 
92.5a 85.0b 15.0b 
12 (2.2-2.5) 
87.5a 82.5b 14.2b 
14 (2.7-3.0) 
29.8b 26.4a 4.7a 
______________________________________ 
.sup.z Mean separation within column by Tukey's HSD (P = 0.05) on 
transformed data. Original means are presented. 
.sup.y 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 followed by the 
same letter are nonsignificant. 
TABLE 4 
______________________________________ 
Effect of hormone concentration on somatic 
embryogenesis of wheat cv. Fielder.sup.z. 
No. of 
% explants % explants mature 
forming forming % explants 
somatic 
embryogenic 
somatic forming embryos/ 
Hormone callus embryos roots explant 
concentration 
after after after after 
(mg/l) 2 weeks 4 weeks 4 weeks 4 weeks.sup.y 
______________________________________ 
0D + 0CA.sup.x 
0.0a 0.0a 0.0a 0.0a 
1D + 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 0.0a 4.4b 
______________________________________ 
.sup.z Mean separation within column by Tukey's HSD (P = 0.05) on 
transformed data. Original means are presented. 
.sup.y Total number of somatic embryos/number of explants forming somatic 
embryos. 
.sup.x D = 2,4dichlorophenoxyacetic acid; CA = casamino acid (vitamin 
free) 
TABLE 5 
______________________________________ 
Effect of light intensity on somatic embryogenesis 
of wheat cv. Fielder.sup.z. 
% explants No. of mature 
Light forming somatic 
intensity 
% explants forming 
somatic embryos/ 
(.mu.E .multidot. 
embryogenic callus 
embryos explant 
m.sup.-2 s.sup.-1) 
after 2 weeks after 4 weeks 
after 4 weeks.sup.y 
______________________________________ 
0 (Dark) 
93.8a 93.8a 7.6a 
10 95.0a 98.8a 9.6a 
20 51.2b 68.8b 3.2b 
40 28.8c 36.2c 2.3b 
60 0.0d 17.5c 0.9c 
______________________________________ 
.sup.z Mean separation within column by Tukey's HSD (P = 0.05) on 
transformed data. Original means are presented. 
.sup.y Total number of somatic embryos/number of explants forming somatic 
embryos. 
TABLE 6 
______________________________________ 
Frequency of conversion of somatic embryos of wheat cv. 
Fielder into plantlets on different media.sup.z. 
% somatic 
embryos 
forming Average Average 
plantlets length length 
Medium.sup.y 
after 2 weeks 
of shoot (cm) 
of root (cm) 
______________________________________ 
Half strength MS 
68.0a 6.7a 2.2a 
Full strength MS 
49.0b 5.0a 2.3a 
MS + .025 mg/l 
38.0b 2.9b 3.9b 
ABA 
MS + .050 mg/l 
42.0b 3.4b 3.7b 
ABA 
______________________________________ 
.sup.z Mean separation within column by Tukey's HSD (P = 0.05) on 
transformed data. Original means are presented. 
.sup.y MS = Murashige and Skoog's mineral salts (1962); ABA = abscisic 
acid 
TABLE 7 
______________________________________ 
Response of different wheat genotypes to somatic 
embryogenesis from isolated scutellum.sup.z. 
% explants No. of mature 
forming somatic 
% explants forming 
somatic embryos/ 
embryogenic callus 
embryos explant 
Genotype 
after 2 weeks after 4 weeks 
after 4 weeks.sup.y 
______________________________________ 
CPS (Canada Prairie Spring Wheat) 
Fielder 92.5 .+-. 4.8.sup.x 
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 Wheat) 
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 
______________________________________ 
.sup.z Tenday post anthesis 
.sup.y Total number of somatic embryos/number of explants forming somatic 
embryos. 
.sup.x Mean .+-. S.E. 
TABLE 8 
______________________________________ 
Response of different barley genotypes to somatic 
embryogenesis from isolated scutellum.sup.z. 
% explants No. of mature 
forming somatic 
% explants forming 
somatic embryos/ 
embryogenic callus 
embryos explant 
Genotype 
after 2 weeks after 4 weeks 
after 4 weeks.sup.y 
______________________________________ 
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 
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 
______________________________________ 
.sup.z Eightday post anthesis (twoday post anther protrusion) 
.sup.y Total number of somatic embryos/number of explants forming somatic 
embryos. 
.sup.x Mean .+-. S.E.