Note: Descriptions are shown in the official language in which they were submitted.
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5-17094/+/CGC 1352
Method of Controlling Plant Pests
The present invention relates to a method of controlling pests on plants.
More particularly, it relates to a method of controlling pests on plants
by exposing the pests to proteinase inhibitors which are biologically
synthesized by the plants. The invention also relates to plants capable
of exhibiting such control.
A. Proteinases and their Inhibitors
A proteinase is conventionally regarded as an enzyme which hydrolyzes
peptide bonds, thereby destroying molecules, such as proteins, which
contain such bonds. If the proteinase severs a peptide bond of a
terminal amino acid, it is designated an "exoproteinase". If the enzyme
hydrolyzes a non-terminal~peptide bond, it is designated an "endo-
proteinase". However, for purposes of the present invention, the term is
also considered applicable to materials which sever peptide bonds by
mechanisms other than hydrolysis.
Both classes of proteinase exhibit different modes of action. Exoprotein-
ases may cleave at either the N-terminus or the C-terminus of the
peptide. Aminopeptidases are ones acting at the N-terminus while carboxy-
peptidases are ones attacking the C-terminus.
Endoproteinases are more specific in their activities. By convention
these enzymes are categorized in four principal classes reflecting the
nature of the hydrolyzed bonds and/or the cavity surrounding the
hydrolysis site and/or the requirement of a characteristic moiety in the
proteinase.
Serine endoproteinases are characterized by the participation of a serine
hydroxyl group in the hydrolysis reaction of the protein being de-
activated. As a group these proteinases are probably the best charac-
terized. However, they appear to be far more prevalent in microbial
tissues and animal tissues than in plant tissues.
Thiol proteinases, or sulfhydryl proteinases, appear to be the most
prevalent kind of plant tissue proteinases. They are characterized by the
apparent involvement of sulfur in some form in the hydrolysis reaction. A
free sulfhydryl group on cysteine has been identified in the active site
of a number of these proteinases.
The acidic proteinases, also known as carboxylic proteinases, are
widespread throughout the plant and animal kingdoms. Active sites appear
to contain two aspartic acid side chains which share a common proton and
a hydroxyl group from a tyrosine residue.
The metalloproteinases, for purposes of the present discussion, are
generically defined as those proteinases which do not fall into one of
the above three groups and which require at least one metal ion for their
hydrolytic activities. Calcium, zinc and iron are among the more common
metals found in such proteinases.
Proteinase inhibitors are materials which destroy or limit the activity
of proteinases. A variety of classification schemes has been developed to
correlate the activities of particular inhibitors with their target
proteinases and/or other inhibitors. The predominant one correlates the
activity of the inhibitor with that of the proteinase. Thus, the four
principal classes of proteinase inhibitors are serine proteinase
inhibitors, thiol proteinase inhibitors, acidic proteinase inhibitors and
metalloproteinase inhibitors.
Serine proteinase inhibitors are found naturally in several kinds of
tissues in many plants. They appear to be active against a wide variety
of serine proteinases from both insects and microorganisms. Evidence
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suggests that the target proteinase is deactivated by the formation of a
stable complex between proteinase and inhibitor, a complex which is
incapable of hydrolyzing peptides.
Inhibitors of thiol proteinases, acidic proteinases and metalloprotein-
ases are less well understood from a mechanistic point of view. They are
found throughout the plant kingdom, although they appear to be less
ubiquitous than the serine proteinase inhibitors. It is thought that the
inhibitors of thiol proteinases act primarily through a mechanism in
which the characteristic sulfur moiety is blocked.
Proteinase inhibitors may also be classified according to structural
considerations. A variety of "low molecular weight" proteinase inhibitors
are known, largely of non-natural synthetic origin, and are useful as
laboratory reagents. A number of naturally occuring low molecular weight
inhibitors have been isolated from bacterial and fungal sources and
characterized; this group includes such inhibitors as the leupeptins,
antipains and pepstatins. A large number of naturally occurring protein-
ase inhibitors are actual proteins which are produced directly from gene
expression in the cell, or synthesized in a series of chemical reactions
which are controlled by enzymes that are produced directly from gene
expression in the cell.
B. Proteinase Inhibitors in Plants
Plant proteinase inhibitors are commonly found in plant tissues,
apparently as a part of a biological control mechanism to protect the
plant against attack by a variety of pests, particularly insects or
microorganisms. In response to such attack, it is thought that the plant
releases one or more proteinase inhibitors which, when ingested by the
attacker, deactivates) proteinases within the body of the attacker. Such
deactivation probably interferes with the metabolic processes of the
attacker, particularly its digestive processes, thus slowing or termina-
ting its metabolism.
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It has been shown that the addition of proteinase inhibitors to the diets
of some insects inhibits growth of certain insects. For example, Gate-
house and Boulter (1983) have shown that cowpea (Yi,yna ungulculatal
trypsin inhibitor decreases the development of cowpea weevil (Callo-
sobruchus maculatus~ larvae; however, soybean (Clycine maxJ and lima bean
(Phaseolus Iunatus~ inhibitors are much less effective. Murdock et al.
(1987) have demonstrated that some Coleopteran insects experience growth
retardation when exposed to inhibitors of thiolproteinases.
These results indicate that naturally occurring proteinase inhibitors
provide a potent means to control insect or microbial pests on plants.
However, large scale use of naturally occurring inhibitors suffers from a
number of disadvantages. Such compounds are typically proteinaceous and,
therefore, are expensive to isolate (or otherwise produce) in large
quantities, to purify and to formulate. Because many are readily degraded
in the environment, activity is short-lived, requiring multiple applica-
tions. Ln addition to degradation, many such proteinase inhibitors are
water soluble and thus wash away after the first rain storm or watering.
However, of perhaps even greater concern is the non-specificity of many
proteinase inhibitors. That is, many inhibitors exhibit activity against
a variety of insects and/or microorganisms. Accordingly, use of a
particular inhibitor against a particular pest may very well have an
adverse impact upon beneficial insects and/or microorganisms.
C. In Vivo Synthesis of Proteinase Inhibitors in Plants
As the field of molecular biology has developed, control of the genetic
processes leading to biological production of proteinaceous materials in
a wide variety of biological organisms is now routine in many instances.
Of particular interest in the development of genetic engineering, or
recombination DNA technology, has been the transplanting of genes from
one organism into another that is quite different in its properties, thus
endowing the receiving organism with phenotypes not characteristic of
that organism.
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The transformation of plants has developed more slowly than trans-
formation of other eukaryotic organisms. However, in recent years
techniques to transform plant tissue and regenerate whole, fertile plants
have become more numerous and reliable. Therefore, the possibility of
transforming plants to express particular proteins is now an objective
that can be realized effectively and in a reasonably efficient manner.
In recognition of the advances of recombinant DNA technology, and of the
disadvantages associated with the use of proteinase inhibitors in a pure,
or substantially pure form, it now becomes possible to develop transgenic
plants which are capable of biologically synthesizing proteinase in-
hibitors which provide the plants with a new, or an additional, mechanism
to protect themselves from attack by insects and/or microorganisms making
it unnecessary to apply exogenous chemicals.
The disadvantages outlined previously for the use of substantially pure
proteinaceous proteinase inhibitor are entirely or substantially overcome
by such an approach. The inhibitor is produced in pure form in sufficient
amount, and there is no need to apply or formulate it. Environmental
degradation and loss become minimal if not irrelevant. And the biological
synthesis of a particular proteinase inhibitor permits some control over
the specificity of the inhibitor used in the plant of interest.
The possibility of endowing plants at the genetic level with the ability
to express a foreign proteinase inhibitor, that is, a proteinase in-
hibitor which it does not normally express, has recently been
demonstrated (Hilder e,t al., 1987). In this work tobacco plants were
transformed with the gene which encodes cowpea trypsin inhibitor, thus
imparting to tobacco resistance to fyelioth~s virescens.
Clearly this demonstration is quite informative. However, tobacco is a
model system which is easily transformed, and is therefore widely used
for introductory experiments in plant transformation. The present
invention is directed to the transformation of a variety of plants, both
for purposes of model studies and commercial utility, with genes for a
variety of proteinase inhibitors.
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The variety of plants and inhibitors embraced by the present invention is
extensive. The transformation of tobacco with genes encoding non-trypsin
proteinase inhibitors is of specific interest for its value as a model.
However, the transformation of a variety of monocot and dicot plants with
widespread utility and substantial commercial benefit represents a
significant advance, the benefits of which span the entire realm of
agriculture. That is, the development of plants with increased resistance
to pests has obvious advantages to the growers and users of plants useful
as food, forage, ornamental, fiber, energy and pharmaceutical resources.
Summary of the Invention
The present invention relates to a method of controlling a plant pest
which comprises exposing that pest to a pesticidally effective amount of
a proteinase inhibitor wherein the inhibitor is biologically synthesized
as a result of genetic manipulation in the plant and the plant is a
monocot or dicot. It also relates to transgenic plants containing genes
which encode such proteinase inhibitors, or genes which encode protein
precursors of the desired proteinase inhibitors and preferably such
plants which express these genes.
A prinicipal object of this invention is a transgenic plant which
exhibits as a phenotype the ability of in vivo synthesis of a foreign
proteinase inhibitor, preferably in an amount effective to control a
particular pest, thus imparting to the plant new or improved resistance
to that pest.
A corollary object is the development of new transgenic monocot plants
showing such a phenotype.
Another corollary object is the development of new transgenic dicot
plants showing such a phenotype.
Another principal object of this invention is a method of controlling
plant pests by exposing them to such a transgenic plant.
6a
According to one aspect of the present invention,
there is provided a method of controlling a pest which is
attacking a target plant, which method comprises exposing
the pest to a pesticidally effective amount of a non-plant
proteinase inhibitor in or on the plant, wherein: (a) said
inhibitor is biologically synthesized in the plant as a
result of expression of a foreign gene which encodes the
proteinase inhibitor, or as a result of expression of one or
more foreign genes encoding one or more precursors of the
proteinase inhibitor; and (b) said plant is: (i) a monocot
selected from the group of plant types consisting of
Cereals, Vegetables and Tubers, Oil Crops, Sugar Crops,
Forage and Turf Grasses, Fiber Plants and Woods and Spices
and Flavorings; or (ii) a dicot selected from the group of
plant types consisting of Cereals, Protein Crops, Fruit
Crops, Vegetables and Tubers, Nuts, Oil Crops, Sugar Crops,
Forage Legumes, Fiber Plants and Woods and Spices and
Flavorings.
The invention likewise provides a method of
controlling a pest which is attacking a target plant, which
method comprises exposing the pest to a pesticidally
effective amount of a non-plant proteinase inhibitor in or
on the plant, wherein said inhibitor: a) is biologically
synthesized in the plant as a result of expression of a
foreign gene which encodes the proteinase inhibitor, or as a
result of expression of one or more foreign genes encoding
one or more precursors of the proteinase inhibitor; and (b)
is selected from the group of proteinase inhibitors
consisting of inhibitors of thiolproteinases,
metalloproteinases, acidic proteinases and non-trypsin
serine proteinases.
According to another aspect of the present
invention, there is provided a plant cell containing a
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6b
foreign gene capable of expressing a non-plant proteinase
inhibitor, or a foreign gene or gene group capable of
expressing one or more precursors of a non-plant proteinase
inhibitor, wherein: (a) said gene or gene group comprises
at least one coding section which encodes a proteinase
inhibitor, or a precursor for a proteinase inhibitor, the
proteinase inhibitor being selected from the group
consisting of inhibitors of serine proteinases,
thiolproteinases, metalloproteinases and acidic proteinases;
and (b) said plant cell is: (i) a monocot cell selected
from the group of plant types consisting of Cereals,
Vegetables and Tubers, Oil Crops, Sugar Crops, Forage and
Turf Grasses, Fiber Plants and Woods and Spices and
Flavorings; or (ii) a dicot cell selected from the group of
plant types consisting of Cereals, Protein Crops, Fruit
Crops, Vegetables and Tubers, Nuts, Oil Crops, Sugar Crops,
Forage Legumes, Fiber Plants and Woods and Spices and
Flavorings.
According to still another aspect of the present
invention, there is provided a DNA sequence coding for
chicken egg white cystatin according to FIG. 9, wherein the
codons are those preferred in corn.
According to yet another aspect of the present
invention, there is provided a vector comprising a DNA
sequence coding for a proteinase inhibitor, which vector can
be used or described herein.
According to a further aspect of the present
invention, there is provided use of a transgenic plant
containing a gene capable of expressing a non-plant
proteinase inhibitor, or a gene or gene group capable of
expressing one or more precursors of a non-plant proteinase
inhibitor, for controlling a plant pest.
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6c
According to yet a further aspect of the present
invention, there is provided a process for generating a
transgenic plant comprising crossing a transgenic first
parent plant with a second parent plant wherein the first
parent plant contains a gene capable of expressing a non-
plant proteinase inhibitor.
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Brief Description of the Figures
Fig. 1 describes the construction of pRK252/Tn903/BglII.
Fig. 2 shows the construction of pCIB5.
Figs. 3 and 4 illustrate the construction of pCIB4.
Fig. 5 discloses the construction of pCIB2.
Fig. 6 contains the construction of pCIBlO, a broad host range plasmid
containing T-DNA borders and gene for plant selection.
Fig. 7 describes the construction of pCIB710.
Fig. 8 reports the construction of pCIBlO/710.
Fig. 9 contains the sequence of a cystatin gene which is incorporated
into a transgenic plant according to this invention.
Fig. 10 discloses the synthetic fragments which are prepared and then
assembled to produce the cystatin gene of Figure 9.
Fig. 11 contains the synthetic fragments which are prepared and then
assembled for the soybean Kunitz trypsin inhibitor gene.
Brief Description of Tables A and B
Tables A contains a listing of plants according to their uses. It has
been extracted from Christie (1987).
Table B illustrates representative transgenic plants containing genes for
proteinase inhibitors, or precursors for proteinase inhibitors, prepared
in accordance with this invention and representative insects and other
pests which may be controlled by these plants. It is intended in no way
to limit the scope of the invention as described and claimed herein.
DETAILED DESCRIPTION OF THE INVENTION
A. Generic Asvects of the Invention
In a generic aspect the present invention is directed to a method of
controlling a pest which is attacking a target plant, which method
comprises exposing the pest to a pesticidally effective amount of a
non-plant proteinase inhibitor in or on the plant, wherein:
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(a) said inhibitor is biologically synthesized in the plant as a result
of expression of a foreign gene which encodes the proteinase inhibitor,
or as a result of expression of one or more foreign genes encoding one or
more precursors of the proteinase inhibitor; and
(b) said plant is:
(i) a monocot selected from the group of plant types consisting of
Cereals, Vegetables and Tubers, 0i1 Crops, Sugar Crops, Forage and Turf
Grasses, Fiber Plants and Woods and Spices and Flavorings; or
(ii) a dicot selected from the group of plant types consisting of
Cereals, Protein Crops, Fruit Crops, Vegetables and Tubers, Nuts, 011
Crops, Sugar Crops, Forage Legumes, Fiber Plants and Woods and Spices and
Flavorings.
In a second generic aspect the present invention is also directed to a
method of controlling a pest which is attacking a target plant, which
method,cQ~nprises exposing the pest to a pesticidally effective amount of a
non-plant proteinase inhibitor in or on the plant, wherein said inhibitor:
(a) is biologically synthesized in the plant as a result of expression of
a foreign gene which encodes the proteinase inhibitor, or as a result of
expression of one or more foreign genes encoding one or more precursors
of the proteinase inhibitor; and
(b) is selected from the group of non-trypsin proteinase inhibitors
consisting of inhibitors of thiolproteinases, metalloproteinases, acidic
proteinasea and serine proteinases.
More specifically, the present invention includes a method of controlling
a pest which is attacking a target plant, which method_comprises exposing
the pest to a pesticidally effective amount of a nori-plant prOtelnase
inhibitor in or on the plant, wherein said inhibitors
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(a) is biologically synthesized in the plant as a result of expression of
a foreign gene which encodes the proteinase inhibitor, or as a result of
expression of one or more foreign genes encoding one or more precursors
of the proteinase inhibitor; and
(b) is selected from the group of proteinase inhibitors consisting of
inhibitors of thiolproteinases, metalloproteinases, acidic proteinases
and non-trypsin serine proteinases.
In a further aspect the present invention embraces a transgenic plant
containing a foreign gene capable of expressing a non-plant proteiriase
inhibitor, or a foreign gene or gene group capable of expressing one or more
precursors of a proteinase inhibitor, wherein:
(a) said gene or gene group comprises at least one coding section which
encodes a proteinase inhibitor, or one or more precursors for a protein-
ase inhibitor, the proteinase inhibitor being selected from the group
consisting of inhibitors of serine proteinases, thiolproteinases,
metalloproteinases and acidic proteinases; and
(b) said plant is:
(i) a monocot selected from the group of plant types consisting of
Cereals, Vegetables and Tubers, Oil Crops, Sugar Crops, Forage and Turf
Grasses, Fiber Plants and Woods and Spices and Flavorings; or
(ii) a dicot selected,from the group of plant types consisting of
Cereals, Protein Crops, Fruit Crops, Vegetables and Tubers, Nuts, 011
Crops, Sugar Crops, Forage Legumes, Fiber Plants and Woods and Spices and
Flavorings.
In another aspect the present invention embraces a transgenic plant
containing a foreign gene capable of expressing a riori-plant protelriase
inhibitor, or a foreign gene or gene group capable of expressing one or more
precursors of a proteinase inhibitor, wherein:
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(a) said gene or gene group comprises at least one coding section which
encodes a proteinase inhibitor, or one or more precursors for a protein-
ase inhibitor, the proteinase inhibitor being selected from the group
consisting of inhibitors of thiolproteinases, metalioproteinases, acidic
proteinases and non-trypsin serine proteinases; and
(b) said plant is:
(1) a monocot selected from the group of plant types consisting of
Cereals, Vegetables and Tubers, Oil Crops, Sugar Crops, Forage and Turf
Grasses, Fiber Plants and Woods and Spices and Flavorings; or
(ii) a dicot selected from the group of plant types consisting of
Cereals, Protein Crops, Fruit Crops, Vegetables and Tubers; Nuts, Oil
Crops, Sugar Crops, Forage Legumes, Fiber Plants and Woods, Drug Crops
and Spices and Flavorings.
Furthermore the present invention is directed to transgenic plants
expressing a non-plant proteinase inhibitor useful in the mentioned method
of controlling a pest which is attacking a target plant.
B. Definitions
In order to provide a clear and concise understanding of the specifica-
tion and the claims, including the scope given to the language in each,
the following definitions are provided:
Biological Synthesis of a Proteinase Inhibitor: Synthesis of a protein-
ase inhibitor in a host cell. This term is intended to embrace: (1)
processes in which an active proteinaceous proteinase inhibitor is
produced as a result of expression of a gene encoding that proteinase
inhibitor; (2) processes in which a translation product is modified by
enzymatic action post-translationally; and (3) processes in which one or
more proteins are produced as a result of expression of one or more
genes, the proteins participating in a reaction scheme which converts one
or more proteinaceous or non-proteinaceous precursors to an active
proteinase inhibitor. This last process specifically embraces, but is not
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limited to, the situations in which a material exogenous or endogenous to
the plant is converted by such a reaction scheme to an active proteinase
inhibitor.
Biological Synthesis as a Result of Gene Expression: Synthesis in a host
cell of a product that results directly from gene expression (that is, as
a product of translation) or synthesis of a product that results
indirectly from gene expression (that is, as a product of chemical
reactions carried out by translation products).
- Control of a Pest: The killing of a pest or the inhibition of activity
deleterious to a host by that pest.
Foreign Gene or Gene Group: A gene or group of genes which originates
from a host different from that which ultimately exhibits the desired
phenotype; or which originates from the host but which has been modified
in some fashion. Modification in this latter situation specifically
includes, but is not limited to, any change in the DNA sequence of the
gene or any addition of one or more coding sections which encode an
additional phenotype (such as an antibiotic marker trait).
Gene: A DNA sequence containing any segments necessary to effect trans-
lation of a protein. The necessary segments include at least a promoter
sequence, a coding sequence and a terminator signal. However, those
segments do not necessarily originate from the same source; that is, the
term "gene" as used herein embraces DNA segments which originate from the
same or different sources.
Non-trypsin proteinase: A proteinase whose mode of action differs
substantially from that of a trypsin according to conventional classifi-
cation schemes or the understanding of one skilled in the art.
The term specifically embraces, but is not limited to, proteinases having
two or more modes of action, the most significant of which differs
substantially from that of a trypsin, although a less or the least
significant of which may be like that of a trypsin. For purposes of this
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invention, an inhibitor of this kind of non-trypsin proteinase is one
which interferes with at least the primary mode of action, irrespective
of any interference with the trypsin mode of action.
Pesticidallv effective amount: An amount sufficient to achieve control of
a pest.
Plant: A plant in the conventional sense of the term as well as plant
tissue in a plant or grown, for example, in a tissue culture medium.
Precursor of a proteinase inhibitor: A material, proteinaceous or
non-proteinaceous, which is converted enzymatically to an active protein-
ase inhibitor in the host cell, or which participates (either as a
reactant or as an enzyme) in the synthesis of a proteinase inhibitor. The
term specifically embraces, but is not limited to, a single compound or a
group of compounds which is or are endogenous or exogenous to a host
cell. The term also specifically embraces one or more enzymes participat-
ing in a pathway to synthesize a proteinase inhibitor from cellular
substrates; in this situation both the substrate and the enzyme are
considered as precursors of a proteinase inhibitor.
Proteinase: A material, either a unique compound or a group of compounds
operating in concert, which deactivates a protein. Although the term
"proteinase" is used interchangeably with "protease" herein, "proteinase"
is the term of predominant usage.
Proteinase inhibitor: A material, either a unique compound or a group of
compounds operating in concert, which deactivates a proteinase.
Transgenic plant: A plant containing at least one DNA sequence, which may
or may not be a gene, which is different from that occurring in the
analogous wild-type plant. As used herein the term embraces any plant in
which any DNA has been modified relative to the DNA of the wild-type
plant. However, there is no requirement that the modified DNA endow a new
phenotype relative to the wild-type plant. The term also embraces plants
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containing an increased number of gene copies relative to the wildtype
and plants containing any modified DNA sequences not necessarily
restricted to those which encode proteins.
Functional Similarity of Inhibitors: Inhibitors have functional
similarity if they can be exchanged by each other without a significant
loss of action, i.e. if they show an action of the same kind and if their
effectiveness is in the same order of magnitude.
C. TransQenic Plants and Method of Pest Control
(1) Transgenic Plants Containing, and Use of,--Proteinase Inhibitors of
Ail Four Classes
The present invention provides a means of controlling pests on a number
of transgenic target monocot and dicot plants using proteinase inhibitors
which are representative of all four conventional classes of those
inhibitors and which are biologically synthesized in those plants. Pests
that can be controlled in this manner are predominantly insects, acari,
fungi or bacteria.
Plants of particular interest to this aspect of the present invention
include: (a) monocots selected from the group of plant types consisting
of Ornamentals and those listed in Table A as Cereals, Vegetables and
Tubers, Oil Crops, Sugar Crops, Forage and Turf Grasses, Fiber Plants and
Woods, and Spices and Flavorings; and (b) dicots selected from the group
of plant types consisting of Ornamentals and those listed in Table A as
Cereals, Proteins Crops, Fruit Crops, Vegetables and Tubers, Nuts, Oil
Crops, Sugar Crops, Forage Legumes, Fiber Plants and Woods and Spices and
Flavorings. That is, dicots of the Drug Crops are not included in this
aspect of the invention.
Preferred monocot plant types include those of the Cereals, Vegetables
and Tubers, Sugar Crops and Forage and Turf Grasses. Particularly
preferred are plants of the genera A versa (oats), Nordeum (barleys),
Oryza (rice), Sorghum (sorghum), Trtt~cum (wheats), Dactylls (cocksfoot,
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orchard grass) and Saccharum (sugar cane), and Zea ways (maize). Most
preferred are plants of the genus Dactylic and Zea mays, in particular
Zea mays.
Preferred dicot plant types include those of the Fruit Crops, Vegetables
and Tubers, Oii Crops, Sugar Crops, Forage Legumes and Fiber Plants and
Woods. Particularly preferred are plants of the genera lycoperslcon
(tomatoes), Solarium (potatoes), Pisum (peas), Beta (beets), Glyci»e
(soybean), Brassica (rapes and kales) and Gossypium (cottons). Most
preferred are plants of the genera Lycvpersicon, Solarium and Gossypium.
Another group of preferred plants consists of potato, rape, tomato,
soybean, pea and cotton.
Inhibition may be observed in transgenic plants containing a foreign gene
or gene group encoding an inhibitor of a serine proteinase. Preferably
that inhibitor is an inhibitor of at least one serine proteinase selected
from the group consisting of thrombin, plasmin, elastase, kallikrein,
subtilisin, cathepsin G, chymase, acrosin, plasminogen activator,
C1-esterase, enterokinase, tryptase, post-proline cleaving enzyme
(prolyl endoproteinase), ATP-dependent protease, thermitase, mast cell
proteinase I and II, Streptomyces griseus proteinase A, Staphylococcus
aureus V8 proteinase, Tenebrio a-proteinase, urokinase, the blood
clotting factors, the complement-activating factors, and the serine
carboxypeptidases, or of a proteinase which has substantial structural or
functional similarity to any of these.
In addition, the inhibitor may be an inhibitor of trypsin or chymotryp-
sin. When the proteinase is chymotrypsin, the inhibitor is preferably a
potato I, potato II, tomato I or tomato II inhibitor.
Inhibition is also observed as a result of in vivo synthesis of other
inhibitors of serine proteinases when one or more of such inhibitors is a
member of the Bowman-Birk inhibitor family, the soybean Kunitz inhibitor
family, the bovine pancreatic trypsin (Kunitz) inhibitor family, the
Kazal trypsin inhibitor family, the Streptomyces subtilisin inhibitor
family, the potato inhibitor I family, the potato inhibitor II family,
the al-proteinase inhibitor family, the hirudin family, the bdellin
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family, the eglin family, the inter-al trypsin inhibitor family, the
serpin superfamily, the C1-inhibitor family, the Ascaris inhibitor
family, the leupeptins, the antipains, elastinal and chymostatin, or an
inhibitor which has substantial structural of functional similarity to
any of these.
Particularly preferred serine protease inhibitors are soybean Kunitz
trypsin inhibitor, al-antitrypsin (an example of the al-proteinase
inhibitor family), eglin C and eglin C mutants, especially eglin C
(Arg 45).
A preferred group of inhibitors biologically synthesized in a transgenic
plant and useful in controlling pests, particularly insect pests,
contains an inhibitor of thiolproteinase. Preferred examples of such
inhibitors include inhibitors of papain, bromelain, ficin, calpain,
cathepsin B, cathepsin C, cathepsin L, cathepsin H, cathepsin S,
chymopapain, clostripain, asclepain, prolyl endopeptidase, pyroglutamyl
peptidase, dipeptidyl proteinase I, yeast proteinase B, Streptococcus
proteinaae, Staphylococcus thiol proteinase or actinidin, or an inhibitor
of a proteinase which has substantial structural or functional similarity
to any of these.
Particularly preferred is an inhibitor which is, or has substantial
structural or functional similarity to, a cystatin, calpastatin,
bromelain inhibitor, antipain, leupeptin, chymostatin or E64 or a
derivative thereof. E64 is a simple name used to designate the compound
[N-(L-3-transcarboxyoxiran-2-carbonyl)-L-leucyl]-amido(4-guanido)-butane.
Derivatives of E64 include such compounds as those in which the group
-NH-(CHz)4-NH-C(=NH)-NHz is replaced by various alkyl groups or by such
groups as -NH-(CHz)4-NHz, -NH-(CHz)z-CH3, -NH-(CHz)7-NHz or -OzCCH=CH-
CO-NHCH(i-propyl)-CO-NH-(CHz)z-CH-(CH3)z~
Preferred cystatin type inhibitors are selected from the group consisting
of egg white cystatin, human cystatin A, human cystatin B, human cystatin
C, human cystatin S, rat cystatin a, rat cystatin S and kininogen, e.g.
L-kininogen and H-kininogen.
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A particularly preferred thiolproteinase inhibitor is egg white cystatin.
In addition, transgenic plants may show resistance to pests when in vivo
synthesis produces an inhibitor of a metalloproteinase. Preferred in-
hibitors are those which inhibit carboxypeptidase A, carboxypeptidase B,
aminopeptidase, collagenase, calcium-dependent neutral proteinase,
thermolysin, angiotensin-converting enzyme, renal dipeptidase,
enkephalinase, gelatinase or keratinase or inhibitors of proteinases
which have substantial structural or functional similarity to any to
these.
Particularly preferred is an inhibitor which is, or has substantial
structural or functional similarity to, a potato carboxypeptidase
inhibitor, mammalian collagenase inhibitor, az-macroglobulin, tissue
bradykinin-potentiating peptide, phosphoramidon, bestatin or amastatin.
Finally, inhibition is observed when the inhibitor is an inhibitor of an
acidic proteinase. Preferred inhibitors are those which inhibit pepsin,
rennin, cathepsin D, chymosin, Penicillinopepsin or Scytalidium acid
protease B, or inhibitors of proteinases which have substantial
structural or functional similarity to any of these.
Particularly preferred as such an inhibitor is one which is, or has
substantial structural or functional similarity to, pepstatin, Ascar~s
carboxyl proteinase inhibitor, Bauhinia pepsin inhibitor, Scopolia
,japonica proteinase inhibitor or potato cathepsin D inhibitor.
While typical pests of these preferred plants are of insect, acarid,
fungal or bacterial origin, insect pests are of special significance.
Table B provides some preferred examples of target plants genetically
manipulated to produce the indicated proteinase inhibitors) which
imparts) control to the indicated target insect. These results are
intended to be no way limiting of the invention.
Primary target pests on monocots, for example maize, are members of the
order Coleoptera or Lep.tdoptera, in particular of the genera D~abrot~ca,
Diatraea, Ostr~n~a and Hel~othis, for example corn root worm or corn
13 4 ~a ~ ~
borer. Primary target pests on dicots, for example potato, rape, tomato,
soybean, pea or cotton are also members of the order Coleoptera or
Lepidoptera, in particular of the genera D.iabrotica, Diatraea, Ostrin~a,
Helioth~s, Spodoptera and Anthonomus, for example potato beetle.
The proteinase inhibitor may be expressed in any part of the plant, for
example in the roots, stalks, leaves, seed or pollen of the plant.
Preferably the proteinase inhibitor is expressed in that plant part which
is the primary attack point of the pest to be controlled.
- (2) Transgenic Plants Containing, and Use of, Inhibitors of Non-trypsin
Proteinase
The present invention also provides a means of controlling pests on an
extensive number of transgenic target monocot and dicot plants using
proteinase inhibitors which are representative of all four conventional
classes and which are biologically synthesized in those plants. More
specifically the invention relates to using inhibitors of non-trypsin
proteinases of all four classes, preferably to using inhibitors of
non-trypsin serine proteinases. Pests that can be controlled in this
manner are predominantly of insect, acarid, fungal or bacterial origin.
Plants of particular interest to this aspect of the present invention
include monocots and dicots in Table A as mentioned above selected from
the group of plant types consisting of Cereals, Protein Crops, Fruit
Crops, Vegetables and Tubers, Nuts, Oil Crops, Sugar Crops, Forage and
Turf Grasses, Forage Legumes, Fiber Plants and Woods, and Spices and
Flavorings, and further to these plant types also Drug Crops.
Preferred monocot plant types include those of the Cereals, Vegetables
and Tubers, Sugar Crops and Forage and Turf Grasses. Particularly
preferred are plants of the genera Avena (oats), fi'orderrm (baileys),
Oryza (rice), Sorghum (sorghum), Triticum (wheats), Dacty I~s (cocksfoot,
orchard grass) and Saccharum (sugar cane), and Zea ways (maize). Most
preferred are plants of the genus Dactylis and Zea mays.
i3 4 1 ~ ~ Z
Preferred dicot plant types include those of the Fruit Crops, Vegetables
and Tubers, Oil Crops, Sugar Crops, Forage Legumes, Fiber Plants and
Woods and Drug Crops. Particularly preferred are plants of the genera
Lycopersicon (tomatoes), Solarium (potatoes), Pisum (peas), Beta (beets),
Glycine (soybean), Brasslca (rapes and kales), Gossypium (cottons) and
Nicotiana (tobaccos). Most preferred are plants of the genera
Lvcopersicon, Solarium, Gossypium and Nicotjana.
Inhibition may be observed in transgenic plants containing a foreign gene
or gene group encoding an inhibitor of a non-trypsin serine proteinase.
Preferably that is an inhibitor of at least one non-trypsin serine
proteinase selected from the group consisting of thrombin, plasmin,
elastase, kallikrein, subtilisin, cathepsin G, chymase, acrosin,
plasminogen activator, C1-esterase, enterokinase, tryptase; post-
proline cleaving enzyme (prolyl endoproteinase), ATP-dependent protease,
thermitase, mast cell proteinase 1 and II, Streptomyces griseus
proteinase A, Staphylococcus aureus V8 proteinase, Tenebrio a-proteinase,
urokinase, the blood clotting factors, the complement-activating factors,
and the serine carboxypeptidases, or of a proteinase which has
substantial structural or functional similarity to any of these. In
addition, the inhibitor may be an inhibitor of chymotrypsin. When the
proteinase is chymotrypsin, the inhibitor is preferably a potato I,
potato II, tomato I or tomato II inhibitor.
Inhibition is also observed as a result of in vivo synthesis of other
inhibitors of non-trypsin serine proteinases when one or more of such
inhibitors is a member of the Bowman-Birk inhibitor family, the
Streptomyces subtilisin inhibitor family, the potato inhibitor I family,
the potato inhibitor II family, the al-proteinase inhibitor family, the
hirudin family, the bdellin family, the eglin family, the inter-ai
trypsin inhibitor family, the serpin superfamily, the C1-inhibitor
family, the Ascaris inhibitor family, the leupeptins, the antipains,
elastinal and chymostatin, or an inhibitor which has substantial
structural of functional similarity to any of these.
13 41~ ~ 1
- 19 -
Particularly preferred non-trypsin serine protease inhibitors of this
aspect of the invention are eglin C and eglin C mutants, especially
eglin C (Arg 45).
A preferred group of inhibitors biologically synthesized in the trans-
genic plants of this aspect of the invention and useful in controlling
pests, particularly insect pests, contains an inhibitor of a thiol-
proteinase mentioned above. Preferred and particularly preferred
examples of such inhibitors are listed in the preceding sections and are
also preferred in this aspect of the invention.
In addition, transgenic plants may show resistance to pests when in vivo
synthesis produces an inhibitor of a metalloproteinase. Preferred and
particularly preferred inhibitors of this aspect of the invention are
those which are mentioned as preferred in the preceding sections.
Finally, inhibition is observed when the inhibitor is an inhibitor of an
acidic proteinase. Again preferred and particularly preferred inhibitors
of this aspect of the invention are those which are mentioned as
preferred in the preceding sections.
While typical pests of these preferred plants are of insect, acarid,
fungal or bacterial origin, insect pests are of special significance.
Table B provides some preferred examples of target plants genetically
manipulated to produce the indicated proteinase inhibitors) which
imparts) control to the indicated target insect. These results are
intended to be no way.limiting of the invention. Typical pests to be
treated are mentioned in the preceding sections.
D. DNA Sequences
In order to carry out the transformation of plants that is needed to
generate the transgenic plants of this invention and to practice the
method of this invention, DNA sequences which code for the proteinase
inhibitors, or for precursors of the proteinase inhibitors, must be
1341511
-ZO-
available for manipulation. Accordingly this invention also embraces the
DNA sequences so utilized. Techniques for carrying out these trans-
formations are described in detail subsequently.
Generically, the invention is directed to a substantially pure DNA
sequence which comprises a coding sequence for a proteinase inhibitor
selected from the group consisting of inhibitors of thiolproteinases,
metalloproteinases, acidic proteinases and non-trypsin serine protein-
ases, or a coding sequence for one or more precursors participating in
the biologica2 synthesis of a proteinase inhibitor, preferably s non-
trypsin proteinase inhibitor, selected from the group consisting of
thiolproteinases, metalloproteinases, acidic proteinases and serine
proteinases. In addition, the invention is directed to a substantially
pure DNA sequence coding for a proteinase inhibitor selected from the
group consisting of inhibitors of thiolproteinases, metalloproteinases,
acidic proteinases and non-trypsin serine proteinases.
The invention is also intended to embrace vectors which carry sequences
encoding proteinase inhibitors, or precursors of proteinase inhibitors,
and such sequences isolated in substantially pure form.
Preferred are those sequences coding for inhibitors which are effective
against insect, acarid, fungal or bacterial pests. Of particular
importance are those sequences coding for inhibitors, or precursors of
inhibitors, which are effective against insects.
Typically the sequence will be one wherein the inhibitor gene originates
from, or has substantial sequence homology to a proteinase inhibitor gene
originating from, an animal, a bacterium or a fungus or from a plant of
a species different from that of the target plant.
When the invention is directed to an inhibitor of a non-trypsin serine
proteinase, it may be selected from the group consisting of inhibitors of
chymotrypsin, thrombin, plasmin, elastase, kallikrein, subtilisin,
cathepsin G, chymase, acrosin, plasminogen activator, C1-esterase,
enterokinase, tryptase, post-proline cleaving enzyme (prolyl endo-
proteinase), ATP-dependent protease, thermitase, mast cell proteinase I
13 ~+15 ~ Z
- 2I -
and II, Streptomyces griseus proteinase A, Staphylococcus aureus V8
proteinase, Tenebrio a-proteinase, urokinase, the blood clotting factors,
the complement-activating factors, and the serine carboxypeptidases, or
an inhibitor of a proteinase which has substantial structural or
functional similarity to any of these.
As mentioned previously, the invention embraces DNA sequences which
encode one or more precursors participating in the biological synthesis
of a proteinase inhibitor wherein said inhibitor is selected from the
group consisting of thiolproteinases, metalloproteinases, acidic protein-
ases and serine proteinases. In this instance the serine proteinase is
selected from the group consisting of trypsin, chymotrypsin, thrombin,
plasmin, elastase, kallikrein, subtilisin, cathepsin G, chymase, acrosin,
plasminogen activator, C1-esterase, enterokinase, tryptase, post-
proline cleaving enzyme (prolyl endoproteinase), ATP-dependent protease,
thermitase, mast cell proteinase I and II, Streptomyces griseus
proteinase A, Staphy~IococcUS aureus V8 proteinase, Tenebrio a-proteinase,
urokinaee, the blood clotting factors, the complement-activating factors,
and the serine carboxypeptidases, and a proteinase which has substantial
structural or functional similarity to any of these.
More specifically, the sequences) may be one or more which code for
precursors leading to an inhibitor that is a member of the Bowman-Birk
inhibitor family, the soybean Kunitz inhibitor family, the bovine
pancreatic trypsin (Kunitz) inhibitor family, the Kazal trypsin inhibitor
family, the Streptomyces subtilisin inhibitor family, the potato in-
hibitor I family, the. potato inhibitor II family, the al-proteinase
inhibitor family, the hirudin family, the bdellin family, the eglin
family, the inter-al trypsin inhibitor family, the serpin superfamily,
the C1-inhibitor family, the Ascaris inhibitor family, the leupeptins,
the antipains, elastinal and chymostatin, or an inhibitor of chymotrypsin
or an inhibitor having substantial structural or functional similarity to
an inhibitor of chymotrypsin. In this last case the sequence may be one
encoding an inhibitor wherein this inhibitor is a potato I, potato II,
tomato I or tomato II inhibitor.
13 4?~' ~
- 22 -
In another embodiment the DNA sequence may encode a proteinase inhibitor,
or one or more precursors of a proteinase inhibitor, belonging to the
class of inhibitors of thiolproteinases. In this instance the inhibitor
may be an inhibitor of papain, bromelain, ficin, calpain, cathepsin B,
cathepsin C, cathepsin L, cathepsin H, cathepsin S, chymopapain,
clostripain, asclepain, prolyl endopeptidase, pyroglutamyl peptidase,
dipeptidyl proteinase I, yeast proteinase B, Streptococcus proteinase,
Staphylococcus thiol proteinase or actinidin, or an inhibitor which has
substantial structural or functional similarity to any of these.
Particularly preferred in this situation is an inhibitor which is, or has
substantial structural or functional similarity to, a cystatin, cal-
pastatin, bromelain inhibitor, antipain, leupeptin, chymostatin or E64 or
a derivative thereof. If the inhibitor is a cystatin, it may be selected
from the group consisting of egg white cystatin, human cystatin A, human
cystatin B, human cystatin C, human cystatin S, rat cystatin a, rat
cystatin s and kininogen, e.g. L-kininogen and H-kininogen.
Furthermore, the sequence may encode a proteinase inhibitor, or one or
more precursors of a proteinase inhibitor, which is an inhibitor of a
metalloproteinase. For example, the inhibitor may be an inhibitor of
carboxypeptidase A, carboxypeptidase B, aminopeptidase, collagenase,
calcium-dependent neutral proteinase, thermolysin, angiotensin-converting
enzyme, renal dipeptidase, enkephalinase, gelatinase or keratinase, or an
inhibitor of a proteinase having substantial structural or functional
similarity to any of these.
Particularly preferred in this instance are DNA sequences which encode
inhibitors, or one or more precursors of inhibitors, wherein the in-
hibitors are, or have substantial structural or functional similarity to,
a potato carboxypeptidase inhibitor, mammalian collagenase inhibitor,
az-macroglobulin, tissue bradykinin-potentiating peptide, phosphoramidon,
bestatin or amastatin.
Finally, the invention embraces DNA sequences encoding a proteinase
inhibitor, or one or more precursors of a proteinase inhibitor, which is
an inhibitor of an acidic proteinase. Of particular interest are those
134~~1~
- 23 -
cases wherein the inhibitor is an inhibitor of pepsin, rennin, cathepsin
D, chymosin, penicillinopepsin or Scytali dium acid protease B, or an
inhibitor of a proteinase which has substantial structural or functional
similarity to any of these.
Particularly preferred are those situations wherein the inhibitor is, or
has substantial structural or functional similarity to, pepstatin,
Ascaris carboxyl proteinase inhibitor, Bauhinja pepsin inhibitor,
Scopolia japonica proteinase inhibitor or potato cathepsin D inhibitor.
E. Vectors
Vectors, produced by standard techniques and comprising the DNA sequences
described above, represent an additional feature of the invention.
Vectors are recombinant DNA sequences which may be used for isolation and
multiplication purposes of the mentioned DNA sequences and for the
transformation of suitable hosts with these sequences. Preferred vectors
for isolation and multiplication are plasmids which can be propagated in
a suitable host microorganism, for example in B'. coli. Preferred vectors
for transformation are those useful for transformation of plant cells or
of Agrobacterium. More particularly, if plant cells other than proto-
plasts are being transformed, then the preferred vector is a Ti-plasmid
derived vector. For the direct DNA transfer into protoplasts, any of the
mentioned vectors may be used. Appropriate vectors which can be utilized
as starting materials are known in the art. Suitable vectors for trans-
forming plant tissue and protoplasts have been described by
deFramond et al. (1983); An et al. (1985); Potrykus et al. (1985);
Rothstein et al. (1987). In addition to these, many other vectors have
been described in the art which are suitable for use as starting
materials in the present invention.
The construction and multiplication of the vectors can be performed in a
suitable host, for example, in B'. cold. Suitable ~'. coli strains include
HB101, JM83, DH1, DHSa, LE392 and the like. The vectors of the invention
may be used as such in a direct gene transfer or a micro-infection
technique. In certain instances it may be preferable to linearize the
vector before use. Alternatively the vectors may be transferred to an
- 24 -
Agrobacter~um host. This transfer is accomplished by conventional
techniques including biparental mating (Simon et al., 1983b), triparental
mating (Ditta et al., 198Uj or transformation (Holsters et al.,1978j.
Suitable strains of Agrobacterium include but are not limited to
A. tumefaciens LBA44U4, CIB542 and C58Z7U7.
Preferred vectors are those comprising the preferred DNA sequences
mentioned above. Furthermore a preferred vector is one that is functional
in plant cells or in Agrobacterium. Particularly preferred are the
vectors described in the Examples.
F. Processes of Preparation
The invention further embraces a process for the preparation of the
mentioned DNA sequences wherein the DNA sequence is isolated from a
naturally occurring source, optionally mutated, or synthesized chemically
or enzymatically.
The invention also embraces a process for the preparation of the
mentioned vectors comprising a DNA sequence as defined above wherein said
DNA sequence is ligated into a vector functional in plant cells or in
Agrobacterium.
The invention further embraces a process for the preparation of trans-
genic plants as defined above wherein a plant cell is transformed with a
vector comprising a DNA sequence of the invention or is co-cultivated
with Agrobacterium containing such a vector, and the plant cell is
regenerated into a plant.
Preferred processes are those which lead to the preferred DNA sequences,
vectors and transgenic plants of the invention. Particularly preferred
are the processes described in the Examples.
13 41511
- 25 -
EXAMPLES
The following examples are provided in order to demonstrate both general
and specific aspects of the invention. Accordingly, they are not intended
to limit the scope of the disclosure or claims in any way.
ABBREVIATIONS
b~ base pair.
2,4-D: 2,4-dichlorophenoxyacetic acid.
Dicamba: 3,6-dichloro-2-methoxy benzoic acid.
EDTA: 1-ethylendiamine N,N,N',N'-tetraacetic acid
kb: kilo base pair.
MES: 2-(N-morpholino)ethane sulfonic acid.
MW: molecular weight.
NAA: a-naphthaleneacetic acid.
PEG: polyethylene glycol.
rpm: rounds per minute
SDS: sodium dodecyl sulfate.
Tris-HC1: tris(hydroxymethyl)methylamine hydrochloride.
- 26 -
MEDIA
SH-0 medium: Medium of Schenk and Hildebrandt (1972); without hormones.
SH medium can be liquid or solidified with U.8 % agar or with 0.5
GelRite~'. The medium is normally sterilized by heat in an autoclave at
110° to 121°C for 15-20 min.
SH-30 medium: SH-0 medium containing 30 uM Dicamba.
SH-45 medium: SH-O medium containing 45 pM Dicamba.
MS Medium and OMS medium: Media of Murashige and Skoog (1962). The media
can be solidified with 0.8 ,,°a agar or agarose or with 0.5 %
GelRite'~'.
Beasley and Ting Medium (embryo germination medium): Medium of Beasley
and Ting (1973).
KM-8p Medium: This medium comprises macroelements, microelements and
Fe-EDTA as described by Kao and Michayluk (1975), and the following
organic compounds: Biotin (0.01 mg/liter), pyridoxine-HC1 (1 mg/liter),
thiamine-HC1 (10 mg/liter), nicotinamide (1 mg/liter), nicotinic acid
(0.1 mg/liter), folic acid (0.4 mg/liter), D-calcium-pantothenate
(1 mg/liter), p-aminobenzoic acid (0.02 mg/liter), choline chloride
(1 mg/liter), riboflavine (0.2 mg/liter), vitamin B12 (0.02 mg/liter),
glycine (0.1 mg/liter), sucrose (0.25 g/liter), glucose (68.4 g/liter),
mannitol (0.25 g/liter), sorbitol (0.25 g/liter), cellobiose
(0.25 g/liter), fructpse (0.25 g/liter), mannose (0.25 g/liter), rhamnose
(0.25 g/liter), ribose (0.25 g/liter), xylose (0.25 g/liter), myo-
inositol (O.1 g/liter), citric acid (40 mg/liter), fumaric acid
(40 mg/liter), malic acid (40 mg/liter), sodium pyruvate (20 mg/liter),
adenine (0.1 mg/liter), guanine (0.03 mg/liter), thymidine
(0.03 mg/liter), uracil (0.03 mg/liter), hypoxanthine (0.03 mg/liter),
cytosine (0.03 mg/liter), glutamine (5.6 mg/liter), alanine
(0.6 mg/liter), glutamic acid (0.6 mg/liter), cysteine (0.2 mg/liter),
asparagine, aspartic acid, cystine, histidine, isoleucine, leucine,
134~51~
- 27 -
lysine, methionine, phenylalanine, proline, serine, threonine,
tryptophan, tyrosine and valine (each O.1 mg/liter). The solution is
filter sterilized. The final pH is 5.8.
Macroelements are made up as a 1U X concentrated stock solution, and
microelements as a 1000 X concentrated stock solution. Citric, fumaric
and malic acid and sodium pyruvate are prepared as a 10U X concentrated
stock solution adjusted to pH 6.5 with NHaOH. Adenine, guanine,
thymidine, uracil, hypoxanthine and cytosine are prepared as a 1000 X
concentrated stock solution, adjusted to pH 6.5 with NH40H. The amino
acids are added using a 10 X stock solution (pH 6.5 with NH40H) to yield
the given final concentrations. Vitamin stock solution is normally
prepared 100 X concentrated.
N6 Medium: This medium comprises macroelements, microelements and Fe-EDTA
as described by Chu et al. (1975), and the following organic compounds:
Pyridoxine-HC1 (0.5 mg/liter), thiamine-HC1 (0.1 mg/liter), nicotinic
acid (0.5 mg/liter), glycine (2.0 mg/liter), and sucrose (30.0 g/liter).
The solution is autoclaved. The final pH is 5.6.
Macroelements are made up as a 1U X concentrated stock solution, and
microelements as a 1000 X concentrated stock solution. Vitamin stock
solution is normally prepared 100 X concentrated.
YEB Medium: 5 g/liter beef extract, 1 g/liter yeast extract, 5 g/liter
peptone, 5 g/liter sucrose, adjusted to pH 7.2 with NaOH, 2 mM MgCl2
added after autoclaving.
Agarose: Preparation and purification of agarose are described, for
example by Guiseley and Renn (1975). Agarose is one of the constituents
of agar. Commercially available agar normally consists of a mixture of
neutral agarose and ionic agaropectin with a large number of side groups.
Usually a certain number of side chains remains intact and determines the
physicochemical properties of the agarose such as gel formation and
melting temperature. Low-melting agarose, especially SeaPlaque~' agarose
is a preferred solidifying agent within the process described herein-
of ter.
_ 28 _
Casein hydrolysate: Casein Hydrolysate - Enzymatic Hydrolysate from
bovine mills, Type 1, Sigma Co., St. Louis, M0, USA.
Cellulase RS and R-10: Yakult Honsha Co. Ltd., Tokyo, Japan.
GelRite~': GelRite Gellan Gum, Scott Laboratories Ins., Fiskersville,
R.I., USA.
Nalgene'g'filter: Nalge Co., Division of Sybron Corp., Rochester,
New York, USA.
Pectolvase Y-23~': Seishin Pharmaceutical Co. Ltd., Tokyo, Japan.
Parafilm~': Parafilm~' laboratory film - American Can Co., Greenwich, CT,
USA.
GENERAL RECOMBINANT DNA TECHNIQUES
Since many of the recombinant DNA techniques used in this invention are
routine for those skilled in the art, a brief description of these
commonly used techniques is included here rather than at each instance
where they appear below. Except where noted, all of these routine pro-
cedures are described in the reference by Maniatis et al. (1982).
A. Restriction Endonuclease Digestions. Typically, DNA is present in the
reaction mixture at approximately 50 to 500 ug/ml in the buffer solution
recommended by the manufacturer, New England Biolabs, Beverly, MA. 2 to
S units of restriction endonucleases are added for each ug of DNA, and
the reaction mixture incubated at the temperature recommended by the
manufacturer for one to three hours. The reaction is terminated by
heating to 65°C for ten minutes or by extraction with phenol, followed
by precipitation of the DNA with ethanol. This technique is also
described on pages 104-106 of the Maniatis et al. reference.
B. Treatment of DNA with Polymerase to Create Flush Ends. DNA fragments
are added to a reaction mixture at 50 to 500 ug/ml in the buffer
recommended by the manufacturer, New England Biolabs. The reaction
4 '~ ~ '~
- 29 -
mixture contains all four deoxynucleotide triphosphates at a concentra-
tion of 0.2 mM. The reaction is incubated at 15°C for 30 minutes, and
then terminated by heating to 65°C for ten minutes. For fragments
produced by digestion with restriction endonucleases that create 5'-pro-
truding ends, such as EcoRI and BamHI, the large fragment, or Klenow
fragment, of DNA polymerase is used. For fragments produced by endo-
nucleases that produce 3'-protruding ends, such as PstI and SacI, T4 DNA
polymerase is used. Use of these two enzymes is described on
pages 113-121 of the Maniatis et al. reference.
C. Agarose Gel Electrophoresis and Purification of DNA Fragments from
Gels. Agarose gel electrophoresis is performed in a horizontal apparatus
as described on pages 150-163 of the Maniatis et al. reference. The
buffer used is the Tris-borate buffer described therein. DNA fragments
are visualized by staining with 0.5 ug/ml ethidium bromide, which is
either present in the gel and tank buffer during electrophoresis or added
following electrophoresis. DNA is visualized by illumination with
short-wavelength or long-wavelength ultraviolet light. idhen the fragments
are to be isolated from the gel, the agarose used is the low temperature
gelling variety, obtained from Sigma Chemical, St. Louis, Missouri. After
electrophoresis, the desired fragment is excised, placed in a plastic
tube, heated to 65°C for approximately 15 minutes, then extracted with
phenol three times and precipitated with ethanol twice. This procedure is
slightly modified from that described in the Maniatis et al. reference at
page 170.
D. Addition of Synthetic Linker Fragments to DNA Ends. When it is desired
to add a new restriction endonuclease site to the end of a DNA molecule,
that molecule is first treated with DNA polymerase to create flush ends,
if necessary, as described in section B above. Approximately U.1 to
1.0 ug of this fragment is added to approximately 10U ng of phosphoryl-
ated linker DNA, obtained from New England Biolabs, in a volume of 20 to
30 u1 containing 2 u1 of T4 DNA ligase, from New England Biolabs, and
1 mM ATP in the buffer recommended by the manufacturer. After incubation
overnight at 15°C, the reaction is terminated by heating the mixture at
65°C for ten minutes. The reaction mixture is then diluted to
approximately 100 u1 in a buffer suitable for the restriction endo-
~3 4 ~ 5 ~ ~
- 3U -
nuclease that cleaves at the synthetic linker sequence, and approxi-
mately 50 to 200 units of this endonuclease are added. The mixture is
incubated at the appropriate temperature for 2 to 6 hours, then the
fragment is subjected to agarose gel electrophoresis and the fragment
purified as described in C above. The resulting fragment will now have
ends with termini produced by digestion with the restriction endo-
nuclease. These termini are usually cohesive, so that the resulting
fragment is now easily ligated to other fragments having the same
cohesive termini.
-~ E. Removal of 5'-Terminal Phosphates from DNA Fragments. During plasmid
cloning steps, treatment of the vector plasmid with phosphatase reduces
recircularization of the vector (discussed on page 13 of the
Maniatis et al. reference). After digestion of the DNA with the
appropriate restriction endonuclease, one unit of calf intestine alkaline
phosphatase, obtained from Boehringer-Mannheim, Indianapolis, IN, is
added. The DNA is incubated at 37°C for one hour, then extracted twice
with phenol and precipitated with ethanol.
F. Ligation of DNA Fragments. When fragments having complementary
cohesive termini are to be joined, approximately 100 ng of each fragment
are incubated in a reaction mixture of 20 to 40 u1 containing approxi-
mately 0.2 units of T4 DNA ligase from New England Biolabs in the buffer
recommended by the manufacturer. The incubation is conducted for 1 to
2U hours at 15°C. When DNA fragments having flush ends are to be
joined,
they are incubated as above, except the amount of T4 DNA ligase is
increased to 2 to 4 units.
G. Transformation of DNA into L~'. coli. ~'. coli strain HB101 is used for
most experiments. DNA is introduced into E. coli using the calcium
chloride procedure described by Maniatis et al. on pages 250-251.
Transformed bacteria are selectively able to grow on medium containing
appropriate antibiotics. This selective ability allows the desired
bacteria to be distinguished from host bacteria not receiving trans-
forming DNA. Determining what antibiotic is appropriate is routine, given
knowledge of the drug resistance genes present on incoming transforming
DNA and the drug sensitivity of the host bacteria. For example, where the
13 4 ~ 5 ~ 1
- 31 -
host bacterium is known to be sensitive to ampicillin and there is a
functional drug resistance gene for ampicillin on the incoming trans-
forming DNA, ampicillin is an appropriate antibiotic for selection of
transformants.
H. Screening E. coli for Plasmids. Following transformation, the
resulting colonies of ~'. coli are screened for the presence of the
desired plasmid by a quick plasmid isolation procedure. Two convenient
procedures are described on pages 366-369 of the Maniatis et al.
reference.
I. Large Scale Isolation of Plasmid DNA. Procedures for isolating large
amounts of plasmids in F,. cold are found on pages 88-94 of the
Maniatis et al. reference.
J. Cloning into M13 Phage Vectors. In the following description, it is
understood that the double-stranded replicative form of the phage M13
derivatives is used for routine procedures such as restriction endo-
nuclease digestions, ligations, etc.
I. Identification of Proteinase Inhibitors of Interest
Example 1: In Vitro Assay for Selection of Protease Inhibitors)
Effective against Target Insects
A. Assay Procedure. Protease Inhibitors are initially screened for
activity against the desired target insect by measuring the ability of
particular inhibitors to inhibit proteolysis by homogenates of the insect
gut. Guts are dissected from CO~-anesthetized or frozen second or third
instar larvae; guts are frozen on dry ice immediately after dissection.
Guts are homogenized in 1U0 mM Tris, 10 mM EDTA, pH 8.5 (Wolfson and
Murdock, 1987) using 5 to 10 u1 buffer per gut. The homogenate is
centrifuged at 5000 rpm for 5 min at 4°C to remove particulates and the
supernatant is stored in 1 ml aliquots at -20°C. Protease activity is
measured essentially using the method of Wolfson and Murdock (1987) using
1''C-BSA (New England Nuclear) as the substrate and measuring the rate of
~3 41a ~ ~
- 32 -
soluhilization of radioactivity with time over a 32 min time period. The
pH optimum for protease activity is determined by measuring the amount of
hydrolysis in each of the following buffers:
~H Buffer
2.0 2U0 mM glycine/HC1
3.0 2UU mM glycine/HC1
4.0 2U0 mM S-alanine/HC1
5.0 2UU m.'~t sodium acetate
6.0 10U mM sodium phosphate/biphosphate
- 7.0 100 mM sodium phosphate/biphosphate
8.0 100 mM Tris/HC1
9.0 200 mM glycine/NaOH
Subsequent experiments are performed at the pH of maximum activity.
Inhibitor is incubated with the homogenate and assay buffer for 5 min at
25°C. The substrate, 1''C-methylated bovine serum albumin (NEN
Products,
Boston) adjusted to a specific activity of ~ U.5 uCi/mg with unlabelled
BSA, is added and 2U u1 aliquots are removed at 0, 2, 4, 8, lfi and
32 min. These aliquots are added to 2U0 girl ice-cold 10 % trichloroacetic
acid solution and stored on ice for 3U min. Samples are centrifuged for
min at 5000 rpm at 4°C to precipitate insoluble protein. Two 75 u1
aliquots of each supernatant are added to 8 to 10 ml of Scintiverse II
scintillation cocktail to count the radioactive decay events. The rate of
solubilization of radioactivity is determined and the rate for each
inhibitor is compared with that of the control.
B. Screening with Gut Homogenates from Southern Corn Rootworm (Diabrotica
undecimpunctataJ. In a typical experiment using gut homogenates from
Diabrotica undecympunctata, the pH of optimum hydrolysis is found to be
pH 4. A number of inhibitors are tested in a standard assay reaction
containing 10 u1 homogenate, 8U,U00 cpm 1''C-BSA in a final volume of
2UU u1 U.2 M S-alanine HC1, pH 4Ø Pepstatin activity is assayed in
U.2 M glycine HC1, pH 2Ø Typical data are presented in Table 1.
~~ ~' S 1 ~
- 33 -
Table 1: Effect of Proteinase Inhibitors on Proteinase Activity of
Homogenates of D. undec_impunctata Guts
Class Inhibitor Conc. (ug/ul) % of Control
Thiol E-64 0.25 7.2
Antipain 0.1 19.8
Cystatin 0.072 30.9
Thiol + Leupeptin 0.1 21.6
Serine
Serine Aprotinin (Bovine 0.1 85.4
pancreatic trypsin
inhibitor)
Chick trypsin inh. 0.1 62
Potato I chymo- U.i 55.4
trypsin inhibitor
Potato II chymo- 0.1 42.8
trypsin inhibitor
Soybean I (Kunitz) 0.1 69.7
trypsin inhibitor
Soybean II (Bowman-
Birk) 0.1 72.5
Lima bean trypsin 0.1 161
inhibitor
Acidic Pepstatin O.U2 2.1
Proteolysis by gut homogenates of D. Wrgifera and D. balteata show
similar strong inhibition by inhibitors of thiol proteases.
C. Inhibition of Proteolysis in Gut Homogenates from Western Corn Root
Worm (Diabrotica virgiferal and European Corn Borer (Ostrinia nubilalis~.
Proteinase inhibitors are tested for their ability to inhibit proteolytic
activity in homogenates of guts from both Diabrotlca virgi fern and
Ostrinia nubllalis using the methods described above. The results shown
in Table 2 below are consistent with data obtained with other inhibitors
which show that gut proteases in Ostrlnia are inhibited by serine
protease inhibitors while those in Diabrotica are not. In particular
eglin C (Arg45) is shown to be highly effective in inhibiting proteolysis
by O. nubilalis gut homogenates; eglin C (Arg45) is significantly more
effective than eglin C and another trypsin inhibitor, cowpea trypsin
inhibitor (Hilder et al., 1987).
13 415 ~ ~
- 34 -
Table 2: Inhibition of Proteolysis in Insect Gut Homogenates
Inhibitor Conc. (ug/ul) Percent of control activity
D. vir~ifera O, nubzlalis
Eglin C 0.5 107.6 % 29.9
Eglin C (Arg45) 0.5 110.8 % 3.4
Leupeptin 0.002 6.2 % 24.9
Cowpea trypsin inhibitor 0.5 99.2 % 29.5 °o
Table 3: Concentration of Inhibitors Required for 70 % Inhibition of
Proteolysis by O. nubilalis Gut Homogenates
Inhibitor IC70
Eglin C (Arg45) 12 uM
Eglin C 62 ~.tM
Cowpea trypsin inhibitor 50 uM
Example 2: Feeding Assa1- for Selection of Protease Inhibitors Effective
against Target Insects
An alternative method of determining the efficacy of inhibitors against a
particular target insect is to incorporate the inhibitor into the diet.
Inhibitor solution is applied to the surface of the normal diet (e. g.
young corn leaves for Ostrinia nubi1a11s and Hel~othis zea, tobacco leaf
disks for H. virescens, corn roots or cultured corn tissue for Dia-
brotica sp.) and allowed to dry. Newly hatched larvae are exposed to this
diet and the survival rate and weight measured after 5 to 6 days.
Alternatively, second or third instar larvae may be weighed before and
after three days exposure to the treated diet.
A. Effect of Proteinase Inhibitors of Growth of Neliothis zea. In a
typical experiment corn leaf strips (1 x 1 cm) are coated with 12 u1
aliquots of solutions of 10 mg/ml test material and placed in individual
diet cups with one newly hatched N. zea larva. The weight of the larvae
is measured after 5 days. Control proteins such as bovine serum albumin
or thaumatin cause a significant (P<0.05) increase in weight compared to
untreated leaves. Leaves with serine protease inhibitors such as Kunitz
soybean trypsin inhibitor or lima bean trypsin inhibitor cause a
significant decrease in larval weight compared to the control (Table 4).
1~ 4~5~ ~
- 35 -
Table 4: Effect of Proteinase Inhibitions on Growth of Newly Hatched H.
zea
Treatment Mean wt (mg) n
untreated 3.41 15
BSA 6.65 13 O.OUS
thaumatin 4.57 15 0.1
soybean trypsin inhibitor1.59 19 0.005
I
lima bean trypsin inhibitor2.38 13 0.1
B. Effect of Proteinase Inhibitors on Growth of Diabrotica. Feeding
assays with Diabrotica sp. corroborate the in vitro assay described in
Example 1. In a representative experiment cells from a corn cell
suspension culture are soaked in 2.5 % sucrose containing desired amounts
of inhibitors and fed to second instar D. undecimpunctata larvae. Weight
gain after 5 days is significantly (t test p<0.05) lower than controls
when cells are treated with leupeptin, antipain, or pepstatin at
concentrations exceeding 0.3 mg/ml. In similar experiments D. virgifera
larval growth is inhibited by doses of leupeptin as low as U.1 mg/ml
(Table 5).
13 4 1 ~ ~ 1
- 36 -
Table 5: Effect of Protease Inhibitors on Growth of Diabrotica Larvae
Species Inhibitor Dose (mg/mlj Mean wt (mg) p
D. undecimpunctata None 7.54
Leupeptin 1.0 5.53 0.05
3.0 5.02 0.05
10.0 3.55 0.05
Antipain 1.0 5.44 0.05
3.0 5.79 0.05
10.0 3.69 U.US
Pepstatin 0.1 7.42 NS
0.3 6.18 0.05
1.0 5.99 0.05
3.0 6.2 0.05
D, virgifera None 3.67
Leupeptin 0.1 2.51 0.05
0.3 1.91 O.US
1.0 1.56 U.05
3.0 1.28 U.05
C. Effect of Proteinase Inhibitors on Growth of Ustrinla. The effect of
eglin C (Arg45j on the growth of newly hatched Ostrinia nub~1a11s larvae
is determined using the asssay described above. Aliquots of inhibitor
solution are applied to 1 x 1 cm pieces of corn leaf; each leaf piece is
placed in a separate diet cup along with one newly hatched larva. Twenty
larvae are tested at each dose. The cups are incubated at 29°C for six
days after which survival rate, final insect weight and leaf area eaten
are scored. Applfcation of 125 ug of eglin C (Arg45j to each leaf piece
results in a significAnt decrease in final weight of the larvae (p)0.05).
Typically exposure to eglin C (Arg45) results in a decrease of about 30
in the average final larval weight when compared to untreated controls.
II. Genes and Vectors
A protease inhibitor gene is isolated from the source in which it exists
naturally or synthetically and, if necessary, characterized by
conventional methods. If the gene is inducable, it is activated by the
appropriate regulator. RNA resulting from that activation is isolated and
is used to construct a cDNA library. This library is used for differenti-
~3 ~f~~ ~ ~
- 37 -
al screening using radiolabelled cDNA generated_from (1) RNA isolated
from the activated system and (Z) RNA isolated from a second system not
so activated. cDNA clones corresponding to induced clones are then
isolated and sequenced.
If the desired gene is not known to be inducable, it m~~= be isolated
using immunological procedures. The protease inhibitor protein is used as
antigen to produce antibody, which is then used to screen a recombinant
phage cDNA library in a-GT11 according to the procedure of Young and
Davis (1983). Positive clones are screened with radioiabelied mixed
oligonucleotides (Wood et al., 1985; generated according to the known
amino acid seauence of the protein.
Example 3: Proteinase Inhibitcr Genee
A. Purification of Proteinase Inhibitors. Many proteinase inhibitors are
available commercially in purified or partially purified form and the
purification protocols fcr numerous others have beer. published jsee
Barrett and Salvesen (1986; fcr extensive referencesj. Further purifica-
tion of material can be effectee using affinity chremato~traphv~ on columns
of the appropriate inactivated enzyme.
In a typical purification with pineapple bromeiain inhibitor, the enzyme
column is prepared with bromelain essentially as described by
Anastasi et al. (19$3) for carboxymeth_ylpapain. The bromelain is reacted
with iodoacetamide to inactive it. The carboxymethyl-bromelain is then
coupled to CNBr-activated Sepharcse*~Pharmacia) using the protocol
recommended by the manufacturer. After extensive washing the column is
equilibrated in 0.05 M NaP04, pH 4.0, 0.5 M NaCl, U.1 % Brig*~5 and
treated with one column volume of 50 mg/ml bovine serum albumin in the
same buffer to block non-specific binding sites. The column is thoroughly
washed and the partially purified bromelain (Sigma) dissolved in pH 4.0
buffer containing 10 % glycerol instead of Brij 35 is applied to the
column. After thorough washing with pH 4.0 buffer the column is eluted
with 0.05 M NaPO~" pH 11.5, 0.5 M NaCl, 10 iv glycerol. Activity is
monitored by testing for the ability to inhibit the degradation of
3'' C-BSA by bromelain.
*Trade-mark
E
1311511
- 38 -
When isolating inhibitors which have a high degree of activity on the
protease of a particular insect the efficiency of purification can be
tested using the assay described in Example 1. To increase selectivity,
the insect enzyme can be purified by affinity chromatography on an
inhibitor column and then used to make an affinity column for purifi-
cation of new inhibitors.
If necessary further purification of inhibitors for protein sequencing
can be effected using reverse phase HPLC.
B. Amino Acid Sequencing of Proteinase Inhibitors. In the case of
proteins whose amino acid sequence is not already available in the
literature, the amino acid sequence is determined by automatic Edman
degradation using a Model 470A Protein Sequencer (Applied Biosystems,
Foster City, CA) equipped with an on-line reverse phase HPLC for analysis
of the phenylthiohydantoin derivatives of the amino acids and a Model 900
data analysis system. Peptides are produced by enzymatic digestion with
trypsin, Lys-C (lysine endopeptidase), Arg-C (arginine endopeptidase) or
Glu-C (StaphylococcUS aureus protease V8) and separated by reverse phase
HPLC prior to sequencing.
C. Synthesis of a Gene Encoding Proteinase Inhibitor: Chicken Egg White
Cystatin. For proteins of less than 150 amino acids whose amino acid
sequence is entirely known, a gene may be constructed by DNA synthesis.
In the case of chicken 'egg white cystatin, the amino acid sequence
(Schwabe et al., 1984; Turk et al., 1983) is back-translated using the
genetic code with the codon frequency calculated from all available corn
proteins in the GenBank data bank using the computer programs of the
University of Wisconsin Genetics Computer Group. Translational stop and
start signals are added along with BamHI linkers at both ends for
convenience in subsequent manipulations. This process yields the sequence
shown in Figure 9.
Oligonucleotides corresponding to the regions A-K (Figure 10) are
synthesized using a Model 380A DNA synthesizer (Applied Biosystems,
Foster City, CA) with s-cyanoethyl chemistry.
13 41511
- 39 -
The gene is assembled in three steps:
(1) Addition of 5'-phosphate: A 5'-phosphate is added to the 5' ends of
all fragments except fragments A and K by mixing 4U pmol of each fragment
D, C, U, E, F, G, H, I and J with T4 polynucleotide kinase according to
the method described in Maniatis et al. (1982).
(:.') Annealing: After removal of excess reagent by phenol/chloroform
extraction, chloroform extraction, and ethanol precipitation, the
precipitate containing the phosphorylated fragments is dissolved in T4
- liease buffer. 40 pmol each of fragment A and K are added, the mixture is
heated to 85°C, then slowly cooled to 15°C and maintained at
15°C for at
least 4 h tv allow the fragments to anneal.
(3) Ligation: ATP is added to 1 mM along with T4 lipase and incubation is
continued for 4 h. Reagents are removed by extraction and precipitation
follows as in step (1). To check for the efficacy of the reaction, an
aliquot of the products is analyzed on a 10 to 15 % acrylamide gel. A
363 by band is visible. If necessary the corresponding DNA fragments may
be purified by preparative gel electrophoresis before proceeding to
ligate it into the vector.
D. Synthesis of a Gene Encoding a Proteinase Inhibitor: Eglin C and
El;lin C Mutants.
(1) Eglin C Gene: The preparation of the plasmid pMLl47 containing the
e~lin C gene is described in the Carladlan Patent Application 468,119. The
strain F. cola HlllU1/phIL147 harboring this plasmid was deposited on
January 28, 1988 at the Ueutsche Sammlung von Mikroorganismen (DSM),
Mascheroder Weg lb, D-33UU $raunschweig, Federal Republic of Germany,
under the number DSM 4380. The 23U by EcoAI-Bamiil Fragment containing the
complete eglin C gene i.s isolated from lU Ng of plasmid pML147 by means
of digestion with the restriction endonucleases EcoRI and $amllI and
subsequent electrophoresis on 1.5 % low melting point agarose. The EcoRI
end is converted to a $amilI end by ligating with the appropriate linker
chosen so that the coding sequence will be in the correct reading frame.
After isolation, the modified fragment is inserted into the $amHl site of
the CaMV35S promoter in the pCI$71U vector as described in Example 5.
s
r.
~ 3 ~+ ' S 1 l
40 -
(2) M13 Cloning of the Eglin C Gene: Approximately 0.5 up of the 230 by
EcoRI-BamHI fragment containing the complete eglin C gene is isolated
from 1U up of plasmid pMLl47 as above. This DNA fragment (1U ng) is mixed
with 40 ng of M13mp8 pre-digested with EcoRI and BamHI and incubated in
50 mM Tris-HCl pH i.4, 10 mM MgCl2, 10 mM ATP, IO mM dithiothreitol in
the presence of 6.125 units of T4 DNA lipase in a volume of 15 u1
(Zoller et al., 198.:;. The resulting solution is used to transform the
~'. roll strain JM101 (Zoller et al., 1983). The transformation mixture
is coated cntc X-Gal (IPTG-indictor agar) plates (teller et al., 1983).
40 blue (wild type) plaques and 650 colorless plaques are obtained.
(3) Prcducticn of Mi~~mp8 tingle-stranded DNA: ~ ml of 2 culture of
..r, col~~ JM10~~ grown in l. medium (10 g/liter Bactc tryptone, 5 g/liter
Bacte yeast extract, 5 g/liter NaCi, 5 g/liter glucose, 0.1 g/liter
ampicillin) up to an ODe?~ of approximately 0.5 are inoculated with a
colorless plaque taken from the agar plate (see above) and maintained for
approximately- 4 tc 5 hrs at 37°C and 180 rpm. subsequently the grown
culture i= ce,~,trifu~ec for 5 min in a Eppenecri cer:trifuge. The
supernatant ~.. transferred intc s frost-: cent:~._uge tube and centrifuged
again. 200 ui cf 2C ~~ PEG, 1.5 M haCl are added, and the mixture is
maintainer at room temperature for 2G min and then centrifuged again. The
supernatant is discarded and the pellet is dissclver ir: 100 u1 50 mM
Tris-HCi, pH 1.~, 2 mM EDTA (TE buffer). The mixture is mixed with 5U ui
phenol/TE buffer, kept for 15 min at room temperature and then
centrifuged for 5 min in a Eppendorf centrifuge. 1U u1 sodium acetate
buffer, pH 6. and 25U u1 absolute ethanol are added to 100 u1 of
supernatant, and the mixture is maintained at -20°C overnight and then
centrifuged as described above for 10 min. The pellet is washed with 1 rol
80 lo ethanol and again centrifuged. The pellet is dried at room
temperature for 10 min and then dissolved in 50 u1 of TE buffer. The
solution contains approximately 5 ~g of M13mp8 single-stranded DNA.
(4) Production of the Gene Coding for Eglin C (Arg45): A method known as
"site directed mutagenesis" according to Zoller et al., (1983) is used.
The following nucleotide is produced by chemical synthesis for the
mutagenesis of the eglin C gene:
*Trad e-m ark
1~ x:15 ~ 1
- 41 -
5'-CT CCT GTT ACT CGG GAC C-3'
The starred base differs from the corresponding base in the coding strand
of the eglin C gene (a T) and will finally cause the mutation from Leu45
to Arg45. 10 u1 of the oligonucleotide (1 OD/ml = 50U ng) are kinased in
20 u1 of 0.07 M Tris-HC1 pH 7.6, O.O1 M MgClz, 50 mM dithiothreitol with
r-32P ATP and T4 polynucleotide kinase (Boehringer) according to the
method of Maniatis et al. (1982) p. 125. The kinased oligonucleotide is
dissolved in 1U u1 TE buffer (50 ng/ul).
1 ug of the M13mp8 single-stranded DNA and 50 ng of the kinased oligo-
nucleotide primer in 10 u1 50 mM Tris-HC1 pH 7.8, 100 m."1 MgClz are
maintained at 45°C for 30 min and then at room temperature for 5 min
for
annealing. 1 u1 each of 10 mM dATP, dGTP, dCTP and dTTP, 1 u1 T4 DNA-
ligase, 2 u1 50 mM dithiothreitol, 1 u1 10 mM ATP, 1 u1 gelatine
(5 mg/ml), 1 u1 of 10 x hlenow buffer (0.66 M Tris-HC1 pH 7.6, 50 m.'~!
MgCl~, 50 mM dithiothreitol), and 1 u1 (2.5 units) DNA polymerase (Klenow
fragment) are added. The mixture is maintained at 22°C for 5 min and
then
at 15°C for 16 hrs and finally separated electrophoretically on 1
agarose. The resulting circular, double-stranded DNA is made visible with
ethidium bromide and isolated from the gel by electroelution (approxi-
mately 1U ng in 15 u1 TE buffer). 5 u1 (approximately 3.5 ng) of the DNA
obtained in this manner are transformed into ~. toll strain JM101 and
coated onto X-Gal/IPTG indicator plates (see above). Approximately 100
colorless plaques are obtained.
40 of these plaques are used to inoculate each a 2 ml ~'. toll JM101
culture (see paragraph (3)). After cultivation, the ~'. toll cells are
centrifuged off from the supernatants containing phages and single-
stranded DNA. The cell pellets already contain the corresponding mutated
double-stranded DNA. 50 u1 of each of the 4U phage supernatants are
filtered over nitrocellulose, washed twice with TE buffer, maintained
under vacuum for 2 hrs at 80°C and examined according to Southern
(1975) for the presence of the mutated DNA sequence using the oligo-
nucleotide primer as a radioactive probe in hybridization. Twelve phage
supernatants potentially containing the eglin C (Arg45) gene are
identified. Four of these positive phage supernatants are diluted
13 451 ~
4~0 _
approximately 1:10', mixed with B. coli strain JM101 and coated onto
indicator agar. Phages of three each of the resulting plaques are
isolated. The single-stranded DNA is isolated therefrom in the manner
described above. These 12 single-stranded DNAs are sequenced in
accordance with Sanger (1977 and 1981). All 12 single-stranded DNAs
exhibit the desired mutated eglin C sequence. In a minipreparation, the
respective mutated double-stranded DNA (eglin C (arg45) gene in plasmid
M13mp8) is then prepared from the corresponding ~'. toll cell pellets (see
above). By restriction digestion with the endonucleases EcoRI and BamHI,
the EcoRI-BamHI insert containing the mutated gene is cut out from the
vector, isolated and cloned in the vector pHRi148/EcoRI/BamHI (European
Patent Application 146 785). The plasmid pJPGl8 resulting therefrom is
isolated and transformed into ~'. coli strain HB101.
The 230 by EcoRI-BamHI fragment containing the mutated eglin C (arg45)
gene is again cut out of the vector pJPGlB and adapted as described for
the non-mutated eglin C gene in paragraph (1) above.
(5) Production of the Gene Coding for Eglin C (Pro44): The Thr44 to Pro44
mutation is carried out in a manner analogous to that descsribed in
paragraph (4) above. The mutagenic oligonucleotide used has the following
structure:
5'-CT CCT GTT CCT CTG GAC-3'
The starred base differs from the corresponding base in the coding strand
of the eglin C gene (an A) and will finally cause the mutation from Thr44
to Pro44. On working up the mutation mixture 18 potential eglin C (Pro44)
mutants are obtained. By cloning the eglin C (Pro44) DNA into the vector
pHRi148/EcoRI/BamHI, plasmid pJB591 is obtained and used as above.
Example 4: Construction of a Ti Plasmid-derived Vector.
The vector pCIBlO (Rothstein et al., 1987) is a Ti-plasmid-derived vector
useful for transfer of the chimeric gene to plants via Agrobacterium
tumefaciens. The vector is derived from the broad host range plasmid
pRK252, which may be obtained from Dr. W. Barnes, Washington University,
St. Louis, Mo. The vector also contains a gene for kanamycin resistance
-43- i3 4 ~ 5 1 1
in Agrobacterium, from 'Tn903, and left and right T-DNA border sequences
from the Ti plasmid pTi'T37. Between the border sequences are the poly-
linker region from the plasmid pUCl8 and a chimeric gene that confers
kanamycin resistance in plants.
First, plasmid pRK252 is modified to replace the gene conferring tetra-
cycline-resistance with one conferring resistance to kanamycin from the
transposon Tn903 (Oka et al., 1981), and is also modified by replacing
the unique EcoRI site in pRK252 with a BglII site (see Figure 6 for a
summary of these modifications). Plasmid pRK252 is first digested with
endonucleases SalI and SmaI, then treated with the large fragment of DNA
polymerase I to create flush ends, and the large vector fragment purified
by agarose gel electrophoresis. Next, plasmid p368 is digested with
endonuclease BamHI, treated with the large fragment of DNA polymerase,
and an approximately 1050 by fragment is isolated after agarose gel
electrophoresis; this fragment contains the gene from transposon Tn903
which confers resistance to the antibiotic kanamycin (Oka et al., 1981).
Plasmid p368 has been deposited with ATCC, accession number 67700. Both
fragments are then treated with the large fragment of DNA polymerase to
create flush ends. Both fragments are mixed and incubated with T4 DNA
ligase overnight at 15°C. After transformation into ~. coli strain
HB101
and selection for kanamycin resistant colonies, plasmid pRK252/Tn903 is
obtained.
Plasmid pRK252/Tn903 is digested at its unique EcoRI site, followed by
treatment with the large fragment of ~'. coll DNA polymerase to create
flush ends. This fragment is added to synthetic BglII restriction site
linkers, and incubated overnight with T4 DNA ligase. The resulting DNA is
digested with an excess of BglII restriction endonuclease and the larger
vector fragment purified by agarose gel electrophoresis. The resulting
fragment is again incubated with T4 DNA ligase to recircularize the
fragment via its newly added BglII cohesive ends. Following trans-
formation into ~'. coli strain HB101, plasmid pRK252/Tn903/BglII is
obtained (see Figure 1).
13 415t1
- 44 -
A derivative of plasmid pBR322 is constructed which contains the Ti
plasmid T-DNA borders, the polylinker region of plasmid pUCl9, and the
selectable gene for kanamycin resistance in plants (see Figure 2).
Plasmid pBR325/Eco29 contains the 1.5 kbp EcoRI fragment from the
nopaline Ti plasmid pTi'r37. This fragment contains the T-DNA left border
sequence (Yadav et al., 1982). To replace the EcoRI ends of this fragment
with HindIII ends, plasmid pBR325/Eco29 DNA is digested with EcoRI, then
incubated with nuclease S1, followed by incubation with the large
fragment of DNA polymerase to create flush ends, then mixed with
synthetic HindIII linkers and incubated with T4 DNA ligase. The resulting
DNA is digested with endonucleases ClaI and an excess of HindIII, and the
resulting 1.1 kbp fragment containing the T-DNA left border is purified
by gel electrophoresis. Next, the polylinker region of plasmid pUCl9 is
isolated by digestion of the plasmid DNA with endonucleases EcoRI and
HindIII and the smaller fragment (approx. 53 bp) is isolated by agarose
gel electrophoresis. Next, plasmid pBk322 is digested with endonucleases
EcoRI and ClaI, mixed with the other two isolated fragments, incubated
with T4 DNA ligase and transformed into ~'. coli strain HB101. The
resulting plasmid, pCIBS, contains the polylinker and T-DNA left border
in a derivative of plasmid pBR322 (see Figure 2).
A plasmid containing the gene for expression of kanamycin resistance in
plants is constructed (see Figure 4). Plasmid Bin6 is obtained from
Dr. M. Bevan, Plant Breeding Institute, Cambridge, UK. This plasmid is
described in the reference by Bevan (1984). Plasmid Bin6 DNA is digested
with EcoRI and HindIII and the fragment approximately 1.5 kbp in size
containing the chimer~c neomycin phosphotransferase (NPT) gene is
isolated and purified following agarose gel electrophoresis. This
fragment is then mixed with plasmid pUCl8 DNA which has been cleaved with
endonucleases EcoRI and HindIII. Following incubation with T4 DNA ligase,
the resulting DNA is transformed into ~'. coli strain HB101. The resulting
plasmid is called pUCl8/neo. This plasmid DNA contains an unwanted BamHI
recognition sequence between the neomycin phosphotransferase gene and the
terminator sequence of the nopaline synthase gene (see Bevan, 1984). To
remove this recognition sequence, plasmid pUCl8/neo is digested with
endonuclease BamHI, followed by treatment with the large fragment of DNA
polymerase to create flush ends. The fragment is then incubated with T4
13 41511
- 4.5 -
DNA ligase to recircularize the fragment, and is transformed into
coli strain HB101. The resulting plasmid, pUCl8/neo (Bam) has lost the
BamHI recognition sequence.
The T-DNA right border sequence is then added next to the chimeric NPT
gene (see Figure 4). Plasmid pBR325/Hind23 contains the 3.4 kbp HindIII
fragment of plasmid pTiT37. This fragment contains the right T-DNA border
sequence (Bevan et al., 1983). Plasmid pBR325/Hind23 DNA is cleaved with
endonucleases SacII and HindIII and a 1.9 kbp fragment containing the
right border is isolated and purified following agarose gel electro-
phoresis. Plasmid pUCl8/neo(Bam) DNA is digested with endonucleases SacII
and HindIII and the 4.0 kbp vector fragment is isolated by agarose gel
electrophoresis. The two fragments are mixed, incubated with T4 DNA
ligase and transformed into ~', coli strain HB101. The resulting plasmid,
pCIB4 (Figure 4), contains the T-DNA right border and the plant-
selectable marker for kanamycin resistance in a derivative of plasmid
pUClB.
Next, a plasmid is constructed which contains both the T-DNA left and
right borders, with the plant selectable kanamycin-resistance gene and
the polylinker of pUCl8 between the borders (see Figure 5). Plasmid
pCIB4 DNA is digested with endonuclease HindIII, followed by treatment
with the large fragment of DNA polymerise to create flush ends, followed
by digestion with endonuclease EcoRI. The 2.6 kbp fragment containing the
chimeric kanamycin resistance gene and the right border of T-DNA is
isolated by agarose gel electrophoresis. Plasmid pCIBS DNA is digested
with endonuclease AatII, treated with T4 DNA polymerise to create flush
ends, then cleaved with endonuclease EcoRI. The larger vector fragment is
purified by agarose gel electrophoresis, mixed with the pCIB4 fragment,
incubated with T4 DNA ligase, and transformed into E. coli strain HB101.
The resulting plasmid, pCIB2 (Figure 5) is a derivative of plasmid pBR322
containing the desired sequences between the two T-DNA borders.
The following steps complete the construction of the vector pCIBlO, and
are shown in Figure 6. Plasmid pCIB2 DNA is digested with endonuclease
EcoRV, and synthetic linkers containing BglII recognition sites are added
as described above. After digestion with an excess of BgiII endonuclease,
~3 ~'~511
- 46 -
the approximately 2.6 kbp fragment is isolated after agarose gel electro-
phoresis. Plasmid pRK252/Tn903/BglII, described above, is digested with
endonuciease BglII and then treated with phosphatase to prevent re-
circularization. These two DNA fragments are mixed, incubated with T4 DNA
ligase and transformed into ~', cvli strain HB101. The resulting plasmid
is the completed vector, pCIBlO.
Example 5: Construction of a chimeric gene with the CaMV 35S promoter
(A) Construction of a CaMV 35S Promoter Cassette Plasmid. pCIB710 is
- constructed as shown in Figure 7. This plasmid contains CaMV promoter and
transcription termination sequences for the 35S RNA transcript
(Covey et al., 1981). A 1149 by BglII restriction fragment of CaMV DNA
(bp 6494-7643 in Hohn et al., 1982, 194-220 and Appendices A to G) is
isolated from plasmid p1.W111 which has been deposited with the American
Type Culture Collection on May 14, 1986 and assigned the number
ATCC 40~~35. Alternatively, the fragment can be isolated directly from
CaMV DNA by preparative agarose gel electrophoresis. It is mixed with
BamHI-cleaved plasmid pUCl9 DNA, treated with T4 DNA ligase, and trans-
formed into ~'. coli. (Note the BamHI restriction site in the resulting
plasmid has been destroyed by ligation of the BglII cohesive ends to the
BamHI cohesive ends.) The resulting plasmid, called pUCl9/35S, is then
used in oligonucleotide-directed in vitro mutagenesis to insert the BamHI
recognition sequence GGATCC immediately following CaMV nucleotide 7483
(as designated in Hohn et al., 1982). The resulting plasmid, pCIB710,
contains the CaMV 35S promoter region and transcription termination
region separated by a BamHI restriction site. DNA sequences inserted into
this BamHI site will be expressed in plants by these CaMV transcription
regulation sequences. (Also note that pCIB710 does not contain any ATG
translation initiation codons between the start of transcription and the
BamHI site.)
(B) Insertion of the CaMV 35S Promoter/Terminator Cassette into pCIBlO.
The following steps are outlined in Figure 8. Plasmid pCIBlO and pCIB710
DNAs are digested with EcoRI and SalI, mixed and ligated. The resulting
plasmid, pCIBlO/710 has the CaMV 35S promoter/terminator cassette
- 47 -
inserted into the plant transformation vector pCIBlO. The CaMV 35S
sequences are between the T-DNA borders in pCIBlO, and thus will be
inserted into the plant genome in plant transformation experiments.
C. Transfer to Agrobacterlum. In order to transfer the binary pCIBlO
derived plasmid from ~'. cola HB101 to Agrobacterium, an intermediate
cola host strain S17-1 is used. This strain, obtainable from
Agrigenetics Research Corp., Boulder, Co., is described in Simon et al.
(1983a). It contains mobilization functions that can transfer plasmid
pCIBlO directly to Agrobacterium via conjugation, thus avoiding the
necessity to transform naked plasmid DNA directly into Agrobacterium.
First, plasmid pCIBlO DNA is introduced into calcium chloride-treated
S17-1 cells. Next, cultures of transformed S17-1 cells and
A. tumefaciens strain LBA 4404 (Ooms et al., 1981) are mixed and mated on
an N agar (Difco) plate overnight at room temperature. A loopful of the
resulting bacteria are streaked onto AB minimal media (pitta et al.,
1980), plated with 5U ug/ml kanamycin and incubated at 28°C. Colonies
are
restreaked onto the same media, then restreaked onto N agar plates.
Slow-growing colonies are picked, restreaked onto AB minimal media with
kanamycin and single colonies isolated. This procedure selects for
Agrobacteria containing the pCIBlO plasmid.
III. Transformation and Regeneration
Example 6: Transformation of Tobacco
Protoplasts of Nicotiana tabacum cv. "Coker 176" are prepared as follows:
Four to five week old shoot cultures are grown aseptically in MS medium
without hormones at 16°C with a 16 hour light/8 hour dark photoperiod.
Approximately 1.5 g of leaf tissue are removed from the plant and
distributed equally among 8 to 10 Petri dishes (100 x 25 mm, Lab-Tek),
each containing 10 ml of enzyme solution. The enzyme solution contains
1 % cellulase R-10, U.25 % macerase, from Calbiochem Co., 1 % pectolyase
Y-23, from Seishin Pharmaceutical Co., 0.45 M mannitol and 0.1 x K3 salts
(Nagy and Malaga, 1976). Tobacco leaves are cut into thin strips with a
scalpel, the dishes are sealed, placed on a gyrotory shaker at 35 rpm and
incubated with the enzymes for 4 to 5 hours at room temperature.
-48_ 1~ ~ ~ ~ l '1
Next, contents of the dishes are filtered through a cheesecloth-lined
funnel and collected in a flask. The filtrate is pipetted into Babcock
flasks containing 35 ml each of rinse solution. The rinse solution
contains 0.45 M sucrose, 0.1 % MES and 0.1 x K3 salts. The bottles are
centrifuged at 80 x g fcr ten minutes, after which the protoplasts will
have floated to the tep cf the bcttle. The protoplasts are removed with a
1 ml pipet, combined into one bettie, and rinsed twice more. The
resulting protoplasts are suspended in K~ medium in a 15 ml disposable
centrifuge tube. The concer:tration of protoplasts is determined by
counting in a Fuchs-Rosenthal hemocytometer. The protoplasts are then
plated at a densitv~ of 100.000/mi in 6 ml of liquid K~ medium per
x
100 }: 20 mm Fetr-~ dis't: (Cert~-;ng'. The dishes containing the protoplasts
arE incubated at 2~'C it: the cart: for twc days, during c,°hich time
cell
wall regeneration will occur.
After the two-day incubation, 5 uI of a stationary culture of A. tume-
faciens containing the desired piasmids (grown it YEF medium containing
50 ug kanamvcin per ml at 28°C until the stationar~: phase is reached)
arE
added to the dish cf Frctoplasts. After incubation fcr threE mere days at
?b'G, cefotoxime (Calbiochem; is added tc make a final concentration of
500 ug/mi to kill the Agrcbacteria. The following dG°, cells are
diluted
»'lth .~ ml fresh ~_ W EoiL:ru: pEr ClSfi, anG CefCtaz;lmE cGQEG a~cln (flnal
ccncentration 50G ug/ml,. Cells are then grown. at 2G'C fcr ? tc 3 weeks
and then screened on selective medium as described b~~ de Block et al.
(1984).
Example l: Regeneration of Cotton Plants Starting from Cotyledon
Explants
A. Cotton Variety Acala SJ2 in Regular Callus Inducing Medium. Seeds of
Gossypium hirsvtum variety Acala SJ2 (cotton) are sterilized by contact
with 95 % alcohol for three minutes, then twice rinsed with sterile water
and immersed with a 15 % solution of sodium hypochlorite for 15 minutes,
then rinsed in sterile water. Sterilized seeds are germinated on a basal
agar medium in the dark for approximately 14 days to produce a seedling.
ThE cotyledons ef the seedlings are cut into segments which are trans-
* Trad e-m ark
1~ X1511
- 49 -
ferred aseptically to a callus inducing medium consisting of Murashige
and Skoog (MS) major and minor salts supplemented with 0.4 mg/liter
thiamine-HC1, 30 g/liter glucose, 2.0 mg/liter NAA, 1 mg/liter kinetin,
100 mg/liter myo-inositol, and agar (0.8 %). The cultures are incubated
at about 30°C under conditions of 16 hours light and 8 hours darkness
in
a Percival incubator with fluorescent lights (cool daylight) providing a
light intensity of about 2000 to 4000 lx. Calls are formed on the
cultured tissue segments within 3 to 4 weeks and are white to gray-
greenish in color. The calls formed are subcultured every three to four
weeks onto a callus growth medium comprising MS medium containing
100 mg/liter myo-inositol, 2.0 g/liter sucrose, 2 mg/liter NAA and agar.
Somatic embryos form four to six months after first placing the tissue
explants on the callus inducing medium. The callus and embryos are
maintained on callus growth medium by subculturing onto fresh callus
growth medium every three to four weeks.
Somatic embryos which formed on tissue pieces are explanted either to
fresh callus growth medium, or to Beasley and Ting (1973)'s medium
(embryo germination medium). The somatic plantlets which are formed from
somatic embryos are transferred onto Beasley and Ting's medium which
contains 15 mg/liter ammonium nitrate and 15 mg/liter casein hydrolysate
as an organic nitrogen source. The medium is solidified by a solidifying
agent (Gelrite) and plantlets are placed in Magenta boxes. The somatic
embryos develop into plantlets within about three months. The plantlets
are rooted at the six to eight leaf stage (about 7.5 to 10 cm tall), and
are transferred to soil and maintained in an incubator under high
humidity for three to four weeks, after which they are transferred to the
greenhouse. After hardening, plants are transferred to open tilled soil.
B. Cotton Variety Acala SJ2 in Half-strength Callus Inducing Medium.
The procedure of A (above) is repeated using instead half-strength MS
medium in which all medium components have been reduced to one-half the
specified concentration. Essentially the same results are obtained.
1341511
- 50 -
C. Different Cotton Varieties. The procedures of A and B (above) are
repeated with Acala cotton varieties SJ4, SJ2C-1, GC510, B1644, B2724,
B1810, the picker variety Siokra and the stripper variety FC2U17. All are
successfully regenerated.
D. Regeneration of Cotton Plants from Cotyledon Explants with Suspension
Cell Culture as Intermediate Step. The procedure of A (above) is repeated
to the extent of obtaining callus capable of forming somatic embryos.
Pieces of about 750 to 1000 mg of actively growing embryogenic callus are
suspended in 8 ml units of liquid suspension culture medium comprised of
MS major and minor salts, supplemented with 0.4 mg/liter thiamine HC1,
20 g/liter sucrose, 100 mg/liter myo-inositol and NAA (2 mg/liter) in
T-tubes and placed on a roller drum rotating at 1.5 rpm under 16 hours
light/8 hours dark regime. Light intensity of about 2000 to 4500 lx is
again provided by fluorescent lights (cool daylight). After four weeks,
the suspension is filtered through an 840 um size nylon mesh to remove
larger cell clumps. The fraction smaller than 840 um is allowed to
settle, washed once with about 20 to 25 ml of fresh suspension culture
medium. This cell suspension is transferred to T-tubes (2 ml per tube)
and each tube diluted with 6 ml of fresh suspension culture medium. The
cultures are maintained by repeating the above at 10 to 12 day intervals.
At each subculture, the suspension is filtered and only the fraction
containing cell aggregates smaller than 840 um is transferred to fresh
suspension culture medium. In all instances, the fraction containing cell
clumps larger than 840 um is placed onto the callus growth medium to
obtain mature somatic embryos. The somatic embryos that are formed on
callus growth medium are removed and transferred to embryo germination
medium. Using the protocol of A (above), these are germinated, developed
into plantlets and then field grown plants.
E. Alternative Intermediate Suspension Culture. The procedure of D
(above) is repeated except that suspension cultures are formed by
transferring 750 to 1000 mg of embryogenic calls to a DeLong flask
containing 15 to 20 ml of liquid MS medium containing 2 mg/liter NAA. The
culture containing flask is placed on a gyrotory shaker and shaken at 100
to 110 strokes/minute. After three weeks the suspension is filtered
through an 840 yam nylon mesh to remove the large cell clumps for plant
1~ 4151 ~
- 51 -
growth, as in D (above), The less than 840 um suspension is allowed to
settle, washed once in the liquid MS medium and resuspended in 2 to 5 ml
of liquid MS medium. The suspension is subcultured by transfer to fresh
medium in a DeLong flask containing 1 to 2 ml of suspension and 15 ml of
fresh liquid MS medium. The cultures are maintained by repeating this
procedure at seven to ten day intervals. At each subculture only the less
than 840 um suspension is subcultured and the large clumps (840 um or
greater) are used for plant growth.
F. Production of Plants from Large Clumps of Suspension Cultured Cells.
After three or four subcultures using the suspension growth procedure of
D and E (above), 1.5 ml to 2.0 ml of cell suspension from the T-tube and
DeLong flask are in each instance plated onto agar-solidified MS medium
containing 2 mg/liter NAA and Beasley and Ting medium containing
500 mg/liter casein hydrolysate. Within three to four weeks embryogenic
calli with developing embryos become visible. Again, the 840 um or
greater cell clumps are plated on the callus growth medium, giving rise
to embryogenic clumps with developing embryos, which ultimately grow into
plants.
Example 8: Transformation of Cotton Suspension Culture Cells to
Tumorous Phenotype by Ag robacter~um LBA 4434.
A. Growth of the Plant Suspension Culture. An Acala cotton suspension
culture is subcultured into "T" tubes with the medium (MS medium
containing 2 g/liter NAA) being changed every seven to ten days. After a
medium change, the "T" tube is rotated 90° and the cells allowed to
settle out. The supernatant is removed by pipeting prior to trans-
formation and the resulting cells treated as described below.
B. Description of the Agrobacterium Vector. The Agrobacterium strain
LBA 4434 (Hoekema et al., 1983) contains a Ti plasmid-derived binary
plant transformation system. In such binary systems, one plasmid contains
the T-DNA of a Ti-plasmid, the second plasmid contains the vir-region of
a Ti-plasmid, and together the two plasmids function to effect plant
transformation. In the Agrobacterium strain LBA 4434, the T-DNA plasmid
pAL1050 contains T of pTiAchS, an octopine Ti-plasmid. The vir plasmid
L
~~ X1511
- 52 -
in strain LBA 4434, pAL4404, contains the intact virulence regions of
pTiAchS (Uoms et al., 1982). Strain LBA 4434 is available from
Dr. Robert Schilperoort of the Department of Biochemistry, University of
Leiden, the Netherlands.
C. Growth of Agrobacteria. The transforming Agrobacterium strain is taken
from a glycerol stock, inoculated in a small overnight culture, from
which a 50 ml culture is inoculated the following day. Agrobacteria are
grown on YEB medium to which antibiotics as appropriate have been added.
The absorbance at 600 nm of the 50 ml overnight culture is read, the
- culture is centrifuged and the pellet resuspended in the plant cell
growth medium (MS medium containing 2 mg/ml NAA) to a final absorbance
at 60U nm of 0.5. 8 ml of this bacterial suspension is added to each "T"
tube containing the plant cells from part A above.
D. Infection. The "T" tube containing the plant and bacteria cells is
agitated to resuspend all cells and returned to a roller drum for three
hours to allow the Agrobacteria to attach to the plant cells. The cells
are then allowed to settle and the residual supernatant removed. A fresh
aliquot of growth medium is added to the "T" tube and this allowed to
incubate on a roller drum for a period of 18 to 20 hours-in the presence
of any residual Agrobacteria which remained. After this time, the cells
are again allowed to settle, the supernatant is removed and the cells are
washed twice with a solution of growth medium containing cefotaxime
(200 ug/ml). After washing, the cells from each T-tube are resuspended in
ml growth medium containing cefotaxime (200 ug/ml in all cases) and
1 ml aliquots of this plated on petri dishes.
E. Growth of Transformed Tissue. The cells infected with Agrobacteria
grow on the growth medium which has no added phytohormones, indicating
the tissue has received the wild-type phytohormone genes in T-DNA.
These cells develop into tumors, further indicating transformation of the
cultures.
~~ X1511
_ 53 _
Example 9: Transformation of Cotton Suspension Culture Cells to a
Kanamycin-resistant Non-tumorous Phenotype.
The same procedure as in Example 8 is followed except that different
transforming Agrobacteria are used and that the plant selection medium
contains an antibiotic for the selection of transformed plant tissue.
A. Growth of Plant Tissue. As in Example 8, part A.
B. Description of the Agrobacterium Vector. The transforming Agro-
- bacteria contain the T-DNA containing binary vector pCIBIU
(Rothstein et al., 1987) as well as the pAL4404 vir plasmid. The T-DNA of
pCIBlO contains a chimeric gene composed of the promoter from the
nopaline synthase gene, the coding region from Tn5 (encoding the enzyme
neomycin phosphotransferase), and the terminator from the nopaline
synthase gene.
C. Growth of Agrobacteria. Agrobacteria containing pCIBlO are grown on
YEB containing kanamycin (50 ug/ml). Otherwise, conditions are as in
Example 8, part C.
D. Infection. Transformation is accomplished as detailed in Example 8
with the change that the 1 ml aliquots resulting in part C are plated
immediately on medium containing selective antibiotics. Selection medium
contains either kanamycin (50 ug/ml) or 6418 (2.5 ug/ml). Expression of
the nos/neo/nos chimeric gene in transformed plant tissue allows the
selection of this tissue on either of these antibiotics.
E. Growth of Transformed Tissue. Plant growth media in this and all
following examples contain phytohormones as indicated in Example 7.
In 2 to 4 weeks, transformed tissue becomes apparent on the selection
plates. Uninfected tissue or control tissue shows no signs of growth,
turns brown and dies. Transformed tissue grows very well in the presence
of kanamycin or 6418. At this time, tissue pieces which are growing well
are subcultured to fresh selection medium.
~34~5~1
- 54 -
F. Growth of Somatic Embryos. Somatic embryos form on these tissue
pieces. Somatic embryos are explanted to fresh medium (non selective).
G. Germination. When the embryos begin to differentiate and germinate,
1.e. at the point where they are beginning to form roots and have two or
three leaves, they are transferred to boxes containing growth medium.
Growth is allowed to proceed until the plantlet has 6 to 8 leaves, at
which time it is removed from the agar medium.
H. Growth of Plantlet. The plantlet is placed in potting soil, covered
with a beaker to maintain humidity and placed in an incubator for 4 to
8 weeks. At this time, the beaker is removed and the plant is transferred
to the greenhouse. The plants grow in the greenhouse, flower and set
seed.
Example 10: Transformation of Cotton Suspension Culture Cells to a
Hygromvcin-resistant Non-tumorous Phenotype.
The same procedure as in Example 9 is followed except where noted.
Different transforming Agrobacteria are used and the plant selection
medium contains an antibiotic appropriate for the selection of trans-
formed plant tissue.
A. Growth of Plant Tissue. As in Example 8, part A.
B. Description of the Agrobacterium Vector. The transforming Agro-
bacteria contain the T-DNA containing binary vector pCIB715
(Rothstein et al., 1987) as well as the vir plasmid. The T-DNA of pCIB715
contains a chimeric gene composed of the promoter and terminator from the
CaMV 35S transcript (Odeli et al., 1985) and the coding sequence for
hygroroycin B phosphotransferase (Gritz and Davies, 1983).
C. Growth of Agrobacteria. Agrobacteria containing pCIB715 are grown on
YEB containing kanamycin (50 ug/ml).
-
D. Infection. Transformation is accomplished as detailed in Example 8
with the change that the 1 ml aliquots resulting in part (c) are plated
immediately on medium containing selective antibiotics. Selection medium
contains 50 ug/ml hygromycin. Expression of the chimeric hygromycin gene
in transformed plant tissue allows the selection of this tissue on medium
containing hygromycin.
E. Growth of Transformed Tissue. As in Example 9, part E except that the
antibiotic hygromycin is used in the plant selection growth medium.
w Example 11: Transformation of Plant Cells by Microprojectile Bombardment
Another method to introduce foreign DNA sequences into plant cells
comprises the attachment of said DNA to particles which are then forced
into plant cells by means of a shooting device as described by
Klein et al. (1988). Any plant tissue or plant organ may be used as the
target for this procedure, including but not limited to embryos, apical
and other meristems, buds, somatic and sexual tissues in vivo and in
vitro. Transgenic cells and callus are selected following established
procedures known in the art. Targeted tissues are induced to form somatic
embryos or regenerate shoots to give transgenic plants according to
established procedures known in the art. The appropriate procedure may be
chosen in accordance with the plant species used.
The regenerated plant may be chimeric with respect to the incorporated
foreign DNA. If the cells containing the foreign DNA develop into either
micro- or macrospores, the integrated foreign DNA will be transmitted to
sexual progeny. If the cells containing the foreign DNA are somatic cells
of the plant, non-chimeric transgenic plants are produced by conventional
methods of vegetative propagation either in vivo, i.e. from buds or stem
cuttings, or in vitro following established procedures known in the art.
Such procedures may be chosen in accordance with the plant species used.
1~ ~~1511
- 56 -
Example 12: Transformation of Plant Cells by Injection.
Transfer of DNA into pl<~nt cells is also achieved by injection into
isolated protoplasts, cultured cells and tissues as described by
Reich et al. (1986 a and b) and injection into meristematic tissues of
seedlings and plants as described by de la Pena et al. (1987), Graves
and Goldman (1986), Hooykaas-Van Slogteren et al. (1984) and
Grimsley et al. (1987 and 1988). Transgenic plants and progeny therefrom
are obtained by conventional methods known in the art.
- Example 13: Preparation of a Special Type of Callus of Zea maws, Elite
Inbred Line Funk 2717
Zea mays plants of the :inbred line Funk 2717 are grown to flowering in
the greenhouse, and self pollinated. Immature ears containing embryos
approximately 2 to 2.5 mm in length are removed from the plants and
sterilized in 10 °o Cloro x' solution for 20 minutes. Embryos are
aseptically removed from the kernels and placed with the embryo axis
downwards on OMS medium containing 0.1 mg/liter 2,4-D, 6 % (w/v) sucrose
and 25 mM L-proline solidified with 0.24 % (w/v) Gelrite~' (initiation
medium). After two weeks' culture in the dark at 27°C, the callus
developing on the scute:llum is removed from the embryo and placed on B5
medium (Gamborg et al., 1968), containing 0.5 mg/liter 2,4-D and solidi-
fied with 0.24 % (w/v) Gelrite'~. The callus is subcultured every 2 weeks
to fresh medium. After a total of 8 weeks after placing the embryos on
the initiation medium, the special type of callus is identified by its
characteristic morphology. This callus is subcultured further on the same
medium. After a further period of 2 months, the callus is transferred to,
and serially subcultured on, N6 medium containing 2 mg/liter 2,4-D and
solidified with Gelrite~'.
Example 14: Preparation of a Suspension Culture of Zea ways Elite Inbred
Line Funk 2717
The callus described in this example is subcultured for a total of at
least 6 months. The type of callus chosen for subculture is relatively
non-mucilaginous, granular and very friable, such that it separates into
~341~~~
- 57 -
small individual cell aggregates upon placing into liquid medium.
Cultures containing aggregates with large, expanded cells are not
retained. Approximately 500 mg aliquots of the special callus of Zea
mays elite inbred line Funk 2717 are placed into 3U ml of N6 medium
containing 2 mg/liter 2,4-D in 125 ml Delong flasks. After 1 week of
culture at 26°C in the dark on a gyrotory shaker (130 rpm, 2.5 cm
throw)
the medium is replaced with fresh medium. The suspensions are again
subcultured in this way after another week. At that time, the cultures
are inspected, and those which do not show large numbers of expanded
cells are retained. Suspension cultures containing aggregates with large,
expanded cells are discarded. The preferred tissue consists of densely
cytoplasmic dividing cell aggregates which have a characteristically
smoother surface than the usual type of cell aggregates. The cultures
retained have at least 5U % of the cells represented in these small
aggregates. This is the desired morphology. These suspensions also have a
rapid growth rate, with a doubling time of less than 1 week. The
suspension cultures are subcultured weekly by transferring U.5 ml packed
cell volume (PCV: settled cell volume in a pipette) into 25 ml of fresh
medium. After 4 to 6 weeks of subculture in this fashion, the cultures
increase 2 to 3 fold per weekly subculture. Cultures in which more than
75 % of the cells are of the desired morphology are retained for further
subculture. The lines are maintained by always choosing for subculture
the flask whose contents exhibit the best morphology. Periodic filtration
through 630 um pore size stainless steel sieves every 2 weeks is used in
some cases to increase the dispersion of the cultures, but is not
necessary.
A Zea ways suspension culture is deposited with the American Type Culture
Collection (ATCC) accession number 40326. This deposition was made in
accordance with the Budapest Treaty (date of deposit: May 2U, 1987).
Example 15: Preparation of Protoplasts from Suspension-Cultures of Zea
ma ys
1 to 1.5 ml PCV of the suspension culture cells prepared as in the
preceding example are incubated in 10 to 15 ml of a filter-sterilized
mixture consisting of 4 % (w/v) cellulase R5 with 1 % (w/v) Rhozyme in
~3~15~1
- 58 -
KMC (8.65 g/liter KC1, 16.47 g/liter MgCl~.6 H20 and 12.5 g/liter
CaC12.2 HBO, 5 g/liter MES, pH 5.6) salt solution. Digestion is carried
out at 30°C on a slow rocking table, for a period of 3 to 4 hours. The
preparation is monitored under an inverted microscope for protoplast
release. The protoplasts which are released are collected as follows: The
preparation is filtered through a 100 um mesh sieve, followed by a 5U um
mesh sieve. The protoplasts are washed through the sieves with a volume
of KMC salt solution equal to the original volume of enzyme solution.
ml of the protoplast preparation is placed in each of several plastic
disposable centrifuge tubes, and 1.5 to 2 ml of 0.6 M sucrose solution
(buffered to pH 5.6 with 0.1 % (w/v) MES and KOH) layered underneath. The
tube is centrifuged at 60 to 100 x g for 10 minutes, and the protoplasts
banding at the interfaces are collected using a pipette and placed in a
fresh tube. The protoplast preparation is resuspended in 10 ml of fresh
KMC salt solution, and centrifuged for 5 minutes at 60 to 100 x g. The
supernatant is removed and discarded, and the protoplasts resuspended
gently in the drop remaining, and then 10 ml of a 13/14 strength KMC
solution gradually added. After centrifuging again for 5 minutes, the
supernatant is again removed and the protoplasts resuspended in a 6/7
strength KMC solution. An aliquot is taken for counting, and the proto-
plasts again sedimented by centrifugation. The protoplasts are re-
suspended at 107 (ten million) per ml in KM-8p medium in U.5 M
mannitol containing 6 mM MgCl~ or in any other appropriate medium for
use in transformation as described in the following examples.
Example 16: Transformation of Zea ma vs Protoplasts by Electroporation
(A) All steps except the heat shock are carried out at room temperature
(22° to 28°C). The protoplasts from the last step of the example
immedi-
ately above are resuspended in U.5 M mannitol containing 0.1 % (w/v) MES
and 6 mM MgCl2. The resistance of this suspension is measured in the
chamber of a Dialog Electroporator~' (DIA-LOG GmbH, D-4000 Dusseldorf 13,
Federal Republic of Germany) and adjusted to 1 to 1.2 kS2 using a 300 mM
MgCl2 solution. The protoplasts are heat-shocked by immersing the tube
containing the sample in a water bath at 45°C for 5 minutes, followed
by
cooling to room temperature on ice. 4 ug of linearized plasmid containing
a plant-selectable hygromycin resistance gene such as described by Roth-
1 ~ ~~ 1 5 1 ~
- 59 -
stein et al. (1987) or c:himeric gene constructs as described in
Examples 32, 36, 41 and 46, and 20 ug of calf thymus carrier DNA are
added to aliquots of 0.25 ml of this suspension. 0.125 ml of a 24 % (w/v)
polyethylene glycol (PECJ) solution (MW of PEG 8000) in 0.5 M mannitol
containing 30 mM MgClz are added to the protoplasts. The mixture is mixed
well but gently, and incubated for 10 minutes. The sample is transferred
to the chamber of the electroporator and samples pulsed 3 times at
second intervals at initial voltages of 1500, 1800, 2300 or
2800 Vcm 1, and an exponential decay time of 10 us.
The protoplasts are cultured as follows: The samples are plated in 6 cm
Petri dishes at room temperature. After a further 5 to 15 minutes, 3 ml
of KM-8p medium containing 1.2 % (w/v) SeaPlaque~ agarose and 1 mg/liter
2,4-D are added. The agarose and protoplasts are mixed well, and the
medium allowed to gel.
B. The procedure of A 1:o repeated with one or more of the following
modifications:
(1) The resistance of the protoplast preparation is adjusted to 0.5 to
0. 7 kS~.
(2) The PEG used is PEG with a molecular weight of 4000.
(3) No PEG is added, or one half volume of 12 % (w/v) PEG is added.
(4) The pulses are applied at intervals of 3 seconds.
(5) The protoplasts are plated after the electroporation in dishes placed
on a plate cooled to a temperature of 16°C.
(6) The protoplasts are placed in tubes after the electroporation step,
washed with 10 ml of ~/7 strength KMC solution or with W5 solution
(comprised of 380 mg/liter KC1; 18.375 g/liter CaCl~~2H~0; 9 g/liter
NaCl; 9 g/liter glucose; pH 6.0), then collected by centrifugation at
60 x g for 10 minutes, resuspended in 0.3 ml of KM medium, and plated as
in A.
(7) The calf thymus carrier DNA is not added.
1a X1511
- 60 -
Example 17: Transformation of Zea mars Protoplasts by Treatment with
Polyethylene Glycol (PEG)
A. The protoplasts are resuspended at the last step of example 15 in a
0.5 M mannitol solution containing 12 to 30 mM MgCl2. A heat shock of
45°C for 5 minutes is given as described in Example 16. The protoplasts
are distributed in aliquots for transformation in centrifuge tubes,
0.3 ml of suspended prot=oplasts per tube. During the next 10 minutes the
following are added: DNA (as in Example 16A) and PEG solution (40 % (w/v)
MW 6000; containing 0.1 M Ca(N03)2 and 0.4 M mannitol; pH 8 - 9 with KOH)
to give a final concentration of 20 % PEG. The aliquots are incubated for
3U minutes with occasional gentle shaking and then the protoplasts are
placed in petri dishes (0.3 ml original protoplast suspension per 6 cm
diameter dish) and cultured as described in Example 16A.
B. The procedure of A above is repeated, but the protoplasts are washed
after 30 minutes of incubation in the PEG solution by adding 0.3 ml of
W5 solution 5 times at 2 to 3 minutes intervals. The protoplast
suspension is centrifuged, the supernatant removed, and the protoplasts
are cultured as for Example 16 (A).
C. The procedures of A and B above are repeated with the modification
that the final concentration of PEG is between 13 and 25 % (w/v).
Example 18: Regeneration of Callus from Protoplasts
The plates containing_the protoplasts in agarose are placed in the dark
at 26°C. After 14 days, cell colonies arise from the protoplasts. The
agarose containing the colonies is transferred to the surface of a 9 cm
diameter petri dish containing 30 ml of N6 medium containing 2 mg/liter
2,4-D, solidified with 0.24 % (w/v) Gelrite~'. This medium is referred to
as 2N6. The callus is cultured further in the dark at 26°C and callus
pieces subcultured every 2 weeks onto fresh solid 2N6 medium.
13 415??
- 61 -
Example 19: Selection of Transformed Callus of Zea ma
Example 18 is repeated with the modification that 100 mg/liter or
200 mg/liter hygromycin B is added to the 2N6 medium in order to select
for transformed cells.
Example 20: Regeneration of Corn Plants
A. Callus is placed on 2N6 medium for maintenance and on ON6 (comprising
N6 medium lacking 2,4-D) and N61 medium (comprising N6 medium containing
0.25 mg/liter 2,4-D and 10 mg/liter kinetin) to initiate regeneration.
Callus growing on O'~6 and N61 media is grown in the light (I6 hrs/day
light of 800 to 8000 lx from white fluorescent lamps). Callus growing on
N61 medium is transferred to ON6 medium after 2 weeks, as prolonged time
on the N61 medium is detrimental. The callus is subcultured every 2 weeks
even if the callus is to be transferred again on the same medium
formulation. Plantlets appear in about 4 to 8 weeks. Once the plantlets
are at least 2 cm tall, they are transferred to ON6 medium in GA7
containers. Roots form i.n 2 to 4 weeks, and when the roots look well-
formed enough to support: growth, the plantlets are transferred to soil in
peat pots, under a light: shading for the first 4 to 7 days. It is often
helpful to invert a clear plastic cup over the transplants for two to
three days to assist hardening off. Once the plants are established, they
are treated as normal corn plants and grown to maturity in the green-
house. In order to obtain progeny plants are self pollinated or crossed
with wild type.
B. The procedure of A above is repeated with the modification that
100 mg/liter or 200 mg/liter hygromycin B is added to the medium used to
maintain the callus.
Example 21: Preparation of Embryogenic Suspensions from Tissue of
Dactylis glomerata L. (orchard gras)
A. Embryogenic callus is initiated from basal sections of the youngest
leaves of greenhouse-grown Dactylis glomerata L. as described by Harming
and Conger (1982). The leaves are surface sterilized by immersion in a
~~ 4~51~ ._
- 62 -
1:10 dilution of Clorox~~ solution (a solution of 5.25 % (w/v) sodium
hypochlorite; The Clorox: Company, Oakland, CA 94623, USA) for about
minutes and then cut aseptically into small segments of 1 to 5 mm in
length or in diameter. These segments are placed on sterile SH-30 medium
containing 0.8 % (w/v) agarose as a gelling agent. Callus and/or embryo-
genic structures appear within 2 to 6 weeks after plating, upon culture
at about 25°C. Embryogenic callus is maintained by subculturing onto
fresh SH-30 medium every 2 to 4 weeks and culturing in the dark at
25°C.
B. Embryogenic suspension cultures are initiated by placing approximate-
ly 0.5 g fresh weight of embryogenic callus into 50 ml of liquid medium
described by Gray and Conger (1985) containing 45 pM dicamba and
4 g/liter casein hydrolysate. The suspension cultures are grown at 27°C
under a 16 hours light (3300 lx), 8 hours dark photoperiod on a gyrotory
shaker at about 130 rpm in 125 ml Delong flasks sealed with a metal cap
and parafilm'~'. After approximately four weeks the large clumps are
allowed to settle for about 30 seconds and 10 ml aliquots of the
supernatant medium cont:~ining small cell clusters are removed and
transferred to 5U ml of fresh medium. This proceess is repeated every 3
to 4 weeks using the most successful cultures as judged by smaller clump
size and better quality based on the presence of small, cytoplasmic
cells. After 5 to 8 transfers the suspensions are essentially free of non
embryogenic cells and the majority of the embryogenic cell clusters are
quite small (150 to 2000 um).
Example 22: Isolation and Purification of Dactt~Iis glomerata L.
Protoplasts
Protoplast are prepared from embryogenic suspension cultures of the
preceding example by aseptically filtering the cells on a Nalgene~
0.2 um filter unit and then adding 0.5 g cells (fresh weight) to each
12.5 ml protoplast enzyme mixture in a petri dish. The enzyme mixture
consists of 2 % (w/v) Cellulase RS, 7 mM CaCl~ ~ H20, U.7 mM
NaHzPOw ~ H20, 3 mM MES (pH 5.6), glucose (550 mOs/kg H20 of pH 5.6), and
is filter sterilized. The mixture is swirled on an orbital shaker at
about 50 rpm in dim (< 400 lx) light for about 4 to 5 hours. The digest
is then sieved through a stainless steel sieve (100 um mesh size) and
1341511
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distributed into 12 ml centrifuge tubes which are centrifuged at about 60
to 100 x g for about 5 minutes. The protoplast-containing sediment is
then washed three times with protoplast culture medium KM-8p adjusted to
550 mOs/kg H~0 with glucose. At this point a flotation step may be
included for further purification of the protoplasts. In this case, the
washed protoplasts are layered atop 10 ml of KM-8p culture medium
adjusted to 7U0 mOs/kg H20 with sucrose. After centrifugation at 6U to
100 x g for about 10 minutes, protoplasts banding at the interface are
collected using a fine pipette. Finally, the protoplasts are resuspended
in 1 to 2 ml KM-8p culture medium and sieved through a stainless mesh
screen (20 um mesh size). The protoplasts released are collected and
washed and resuspended in KM-8p medium for culture or in osmotically
adjusted medium suitable for transformation.
Example 23: Dactvl~s glnmerata L. Protoplast Culture and Growth of
~.,i ~..~
A. The purified protoplasts are plated at a density of about 5 x 105
protoplasts/ml in KM-8p culture medium containing 1.3 % (w/v) SeaPlaque~'
agarose (FMC Corp., Marine Colloids Division, Rockland, Maine, USA) and
30 to 40 % (v/v) of conditioned medium (obtained from 3 to 4 week-old
D. glomerata L. embryogenic suspension cultures by filtering the medium
through a sterile Nalgene~' 0.2 um filter, making the medium 550 mOs/kg
H20 by addition of glucose, and again filter sterilizing). The plates are
then placed in the dark at a constant temperature of 28°C. After 10 to
14 days the agarose is cut into wedges and placed into 'bead culture' as
described by Shillito.ei= al. (1983) using 2U ml SH-45 suspension culture
medium with 3 % (w/v) sucrose per 3 ml original agarose embedded culture.
The plates are put on a platform shaker and agitated at about 50 rpm in
light at 670 lx. New suspension cultures are formed as the colonies grow
out of the agarose and release cells into the liquid medium. The
resultant suspension cultured cells are plated onto agar-solidified SH-30
medium and placed in the dark at 25°C until callus is formed.
B. Protoplasts are cultured as described in A above except that the
culture medium contains no conditioned medium.
~~ ~r1511
- 64 -
Example 24: Transformation of Dactylic glomerata L. Protoplasts by Means
of Electroporation
A. Immediately after purification of the protoplasts, electroporation is
performed according to Shillito et al. (1985) using linearized plasmid as
described in Example 16. The protoplasts are resuspended after the last
wash at a density of about 7 x 106 protopiasts/ml in the electroporation
buffer (0.4 M mannitol, 6 mM MgCl2). The protoplasts are placed in 0.7 ml
aliquots in 10 ml plastic centrifuge tubes. Plasmid DNA to give a final
concentration of 10 ug/ml and sonicated calf thymus DNA (Sigma) to give
50 ug/ml are added to the tubes. Then 0.38 ml PEG solution (24 % (w/v)
PEG 6000 in 0.4 M mannit:ol, 30 mM MgCl2, 0.1 % (w/v) MES (pH 5.6)~ is
added and the solution gently mixed. The protoplast suspension is
transferred into the chamber of a Dialo g' Electroporator and 10 pulses of
3250 V/cm initial voltage and exponential decay constant of IO us applied
at 30 s intervals. The sample is removed from the chamber, and placed in
a 10 cm diameter petri clash. 10 ml of KM-8p medium containing 1.2 % (w/v)
SeaPlaque~ agarose is added, the protoplasts distributed evently through-
out the medium, and the agarose allowed to gel.
B. Example 24A is repeated except that the initial voltage used is
'1.500 Vcm 1, 3000 Vcm 1, 3500 Vcm 1, 4000 Vcm 1 or 5000 Vcm 1.
C. Examples 24 A and B are repeated except that PEG of MW 4000 or PEG of
MW 8000 is used.
D. Examples 24 A to C are repeated except that the final PEG concentra-
tion is between 10 % and 30 % (w/v).
Example 25: Transformation of Dactyl.f~: glomerata L. Protoplasts b
Treatment with Polyethylene Glycol (PEG)
A. PEG mediated direct gene transfer is performed according to
Negrutiu et al. (1987). The protoplasts are suspended following the last
wash in 0.5 M mannitol containing 15 mM MgCl2 at a density of about
2 x 106 per ml. The protoplast suspension is distributed as 1 ml
aliquots into 10 ml plastic centrifuge tubes. The DNA is added as
~3 41511
- 65 -
described in Example 24, and then 0.5 ml of the PEG solution added (40
(w/v) PEG 4000 in 0.4 M mannitol, 0.1 M Ca(N03)z, pH 7.0j. The solutions
are mixed gently and incubated for 30 minutes at room temperature (about
24°C) for 30 minutes with occasional shaking. 1.4 ml of the wash
solution
is then added, and the contents of the tube gently mixed. The wash
solution consists of 87 mM mannitol, 115 mM CaCl~, 27 mM MgCl2, 39 mM
KC1, 7 mM Tris/HCl and 1..7 g/liter myo-inositol, pH 9Ø Four further
1.4 ml aliquots of wash solution are added at 4 minute intervals, with
mixing after each addition. The tube is then centrifuged at about 60 x g
for about 1U minutes, and the supernatant discarded. The sedimented
protoplasts are taken up in 1 ml KM-8p culture medium, and placed in a
cm Petri dish. 10 ml of KM-8p medium containing 1.2 % (w/v) SeaPlaque~'
agarose is added. The protoplasts are evenly distributed throughout the
medium, and the agarose allowed to gel.
13. The procedure of A above is repeated with one or more of the following
modifications:
(1) The pH of the wash solution is adjusted to 5.6 or 7Ø
(2) The PEG used is PEG of MW 2000, 6000 or 8000.
(3) The wash medium consists of (a) 154 mM NaCl, 125 mM CaCl2, 5 mM KC1,
5 mM glucose, pH 6.0 with KOH, of (b) 0.2 M CaClz, 0.1 %a (w/v) MES,
pH 6.U with KOH, or of (c) 0.2 M CaCl~, 7 mM Tris/HC1, pH 9.0 with KOH.
Example 26: Transformation of Dactvl~s glomerata L. Protoplasts after
Heat Shock Treatment
Transformation is carried out as described in Examples 24 or 25, except
that the protoplasts are treated at 45°C for about 5 minutes prior to
distribution of the aliquots into tubes for transformation or after
distribution of the aliquots, and before addition of the PEG.
Example 27: Selection of Transformed Colonies
A. The culture plates (Petri dishes) containing the protoplasts from
examples 24 to 26 are incubated for 10 days in the dark at about 25°C
and
then cut into 5 equal slices for 'bead cultures' (Shillito et al., 1983).
4 of. the slices are placed each into 20 ml SH-45 culture medium with
1~ 4 X511
- 6_6 -
4 g/liter casein hydrolysate and 20 ug/ml hygromycin B. The fifth slice
is put into 20 ml of the same medium but without hygromycin B as a
non-selected control. After 4 to 5 weeks the putative transformed
protoplast-derived cell colonies growing in hygromycin B are cut out of
the agarose and placed into a 19 mm Petri dish with 2 ml of liquid SH-45
medium containing 20 ug/ml hygromycin B, which is agitated at about
50 rpm on an orbital shaker. After another 4 to 5 weeks all colonies
which grow to make new suspensions are transferred into 125 ml Erlenmeyer
flasks and grown in a manner similar to the parent suspension culture,
except that 20 ug/ml hygromycin B is included in the medium.
The new suspensions are subcultured every 1 to 3 weeks using SH-45 medium
containing 4 g/liter casein hydrolysate and 20 ug/ml hygromycin B. Cells
from these suspensions are also plated on solidified SH-30 medium
containing 2U pg/ml hygromycin B and incubated at about 25°C in the
dark.
Calii grown from the plated cells are subcultures every two weeks onto
fresh medium. The cells which grow in the presence of hygromycin B are
presumed to be transformants.
B. Selection is carried out as described in Example 27 (A) except that
the protoplast-derived cell colonies growing in hygromycin B - containing
medium are placed on agar plates of SH-30 medium containing 20 ug/ml
hygromycin B and incubated at about 25°C in the dark.
Example 28: Regeneration of Transformed Dactylis ~Iomerata L. Plants
A. D. glomerata L, callus (obtained as described in Example 27) derived
from protoplasts is grown on solidified SH-30 medium, and subcultures
every two weeks. Any embryos which form are removed and plated on
germination medium (SH-0) and placed in the light (3700 to 4600 lx).
Germination of these embryos occurs in 1 to 4 weeks and the resultant
plantlets are placed on SH-O medium in the light to form root systems.
They are moved into the greenhouse at the six to twelve leaf stage, and
hardened off gradually.
~~ ~~511
_ 6_7 _
B. Callus (obtained as described in Example 27) derived from protoplasts
is grown on SH-0 medium solidified with 0.24 % (w/vj GelRite~ in the
light (3700 to 4600 lx), and subcultured every two weeks. The resultant
plantlets are placed on a 1:1 mixture of SH-0 and OMS media solidified
with a combination of 0.12 % (w/v) GelRite~' and 0.4 % (w/v) agar in the
light to form root systems. They are moved to the greenhouse at the six
to twelve leaf stage, and hardened off gradually.
C. Small plantlets are obtained as described in A and B above, and are
placed on OMS medium solidified with 0.8 % (w/v) agar in the light to
form root systems. They are moved to the greenhouse at the six to twelve
leaf stage, and hardened off gradually.
D. Small plantlets are obtained as described in A above and are placed on
a 1:1 mixture of SH-0 and OMS media solidified with a combination of
U.12 ~o (w/v) GelRite'~ and 0.4 iv (w/v) agar in the light to form root
systems. They are moved to the greenhouse at the six to twelve leaf
stage, and hardened off gradually.
Example 29: Transformation and Regeneration of Tomato
Tomatoes are transformed (Nelson et al., 1988) with engineered A.
tumefaciens bearing a plant selectable marker (eg. kanamycin-resistance)
and the chimeric gene encoding the protease inhibitor of interest. Leaf
disk transformation of tomato with A. tumefaciens is according to
McCormick et al. (1986)..
Example 30: Transformation and Regeneration of Potato
Potatoes are transformed (Stockhaus et al., 1987) with engineered
A. tumefaciens bearing a plant selectable marker (eg. kanamycin-
resistance) and the chimeric gene encoding the protease inhibitor of
interest.
1~4~5~'
_ 6g _
IV. Results
Example 31: Bioassay of Transformed Cotton
Neliothis virescens eggs laid on sheets of cheesecloth are obtained from
the Tobacco Insect Control Laboratory at North Carolina State University,
Raleigh, North Carolina. The cheesecloth sheets are transferred to a
large covered glass beaker and incubated at 29°C with wet paper towels
to
maintain humidity. The eggs hatch within three days. As soon as possible
after hatching, the larvae (one larva per cup) are transferred to covered
small plastic cups. Each cup contains cotton leaf discs. Larvae are
transferred using a fine bristle paint brush.
Leaf discs one cm in diameter are punched from leaves of cotton plants
and placed on a circle of wet filter paper in the cup with the larva. At
least 6 to 10 leaf discs, representing both young and old leaves, are
tested from each plant. Leaf discs are replaced at two day intervals, or
as necessary to feed the larvae. Growth rates (size or combined weight of
all replica worms) and mortality of larvae feeding on leaves of trans-
formed plants are compared with those of larvae feeding on untransformed
cotton leaves.
Larvae feeding on discs of cotton transformed with a gene encoding a
protease inhibitor show a large decrease in growth rate and 100
mortality compared with controls.
Example 32: Maize Resistant to Damage by Diabrotica (Corn Root Worm
through Expression of Egg White Cystatin
A. Construction of the pCIB715/710-Cystatin Vector. The cystatin gene is
synthesized as described in Example 3 C. The 363 by DNA sequence is
purified and ligated into the BamHI site of the pCIB710 vector as
described in Example 5. The resulting vector is denoted as pCIB710-
cystatin. This vector is cut with EcoRI and the resulting DNA is treated
with alkaline phophatase. Plasmid pCIB715 (Rothstein et al., 1987) is
cut with EcoRI and ligated with the pCIB710-cystatin from above. The
resulting vector is named pCIB715/710-cystatin.
~3 41511
- 69 -
B. Transformation and Regeneration of Maize. Maize tissue is transformed
with the pCIB710 vector or, preferably, the pCIB715/710 vector carrying
the cystatin gene insert and plants are regenerated as described in
Examples 16 to 20. For controls, plants transformed with the pCIB7IU
vector or the pCIB715 vector without the gene insert are prepared in the
same way. Transformed plant tissue is selected on the appropriate
antibiotic. The initial plants are self-fertilized and seed (T1 seed) is
obtained.
C. Testing Plants for Cystatin Expression. Plants grown from the T1 seed
are analyzed for the presence and expression of the cystatin gene using
several tests.
(1) DNA is isolated and digested with BamHI; the digest is electro-
phoresed on a 1.5 is agarose gel. The DNA fragments are transferred to
nitrocellulose and hybridized with the cystatin gene labelled with 3zP by
nick translation (see Maniatis et al. (1982) for technique s . The
presence of the cystatin gene is detected by a band of approximately 363
by which hybridizes to the probe.
(2) RNA is detected by the Northern blot procedure (Maniatis et al.,
1982) as a band of appraximately 380 bases which hybridizes with the
szP-cystatin gene described above.
(3) Cystatin protein is detected using standard immunological techniques
with a polyclonal rabbit antibody raised against commercially (Sigma)
available cystatin.
(4) Cystatin activity is detected by immunopurifying material from the
plant extracts using the rabbit polyclonal anti-cystatin antibody and
protein A Sepharose and assaying the isolated material for the ability to
inhibit proteolysis of 1''C-BSA by papain and by corn root worm gut
homogenates as described in Example 1.
~,~ 4111
D. Resistance of Transformed Corn Plants to Diabrotica Damage. T1
seedlings are planted in coarse vermiculite in 100 mm Petri dishes (5 per
dish, 5 dishes). When the second leaves on the seedlings emerge, each
dish is infested with 20 second instar IJiabrotica larvae. After seven
days, the number and weight of the survivors is measured along with the
weight of the washed roots of the corn plant. Resistance of transformed
plants is detected by a statistically significant (Student's t test
p<0.05) decrease in larval weight gain, decrease in larval survival rate,
or decrease in loss of root weight relative to insect-free plants when
plants expressing the cystatin gene are compared to control plants
- transformed with the vector without gene insert or untransformed plants.
Example 33: Potato and Tomato Resistant to Damage by Leptinotarsa
decemlineata through Expression of Cystatin
A. Construction of the pCIBlO-Cystatin Vector. The cystatin gene is
synthesized and ligated into vector pCIB710 as described in Example 32 A.
The cystatin gene plus t:he 355 CaMV promoter are subcloned out of the
pCIB710 vector into the pCIBlO vector by digesting with XbaI and EcoRI,
isolating the 1593 by fragment and ligating this fragment with XbaI,
EcoRI digested pCIBlO to make the pCIBlO-cystatin vector.
B. Transformation and Regeneration of Plants. The pCIBlO-cystatin vector
or, preferably, the pCIB715/710-cystatin vector is introduced into
A, tumefaciens carrying a virulence plasmid such as LBA 4404 (Example 5)
or pCIB542. pCIB542 is an engineered A. tumefaciens vir plasmid derived
from pTiBo542 (Hood et al., 1986). pCIB542 has the bacterial kanamycin
resistence gene replaced by a bacterial streptomycin/spectinomycin
resistence gene. The strain carrying both pCIBlO-cystatin or pCIB715/710
cystatin and pCIB542 is used to produce transformed tomato plants
according to the method of Fischhoff et al. (1987), or as in Example 29.
Potato plants containing pCIBlO-cystatin or pCIB715/710-cystatin are
obtained by the method of Stockhaus et al. (1987), Example 30.
C. Testing Plants of Cystatin Expression. Testing of transformants for
cystatin expression is carried out as described above for maize.
~3 ~ ~ ~ ~ i
- ~.l _
D. Resistance of Transformed Plants to Damage by L. decemlineata. Ten
four week old plants are each infested with five second instar
L. decemlineata larvae. Larvae are allowed to feed for four days at which
time insect mortality, insect weight gain and amount of damage to the
plant are scored. Resistance of transformed plants is detected by a
statistically significant (Student's t test p<0,05) decrease in larval
weight gain, decrease in larval survival rate, or decrease in plant
damage when plants expressing the cystatin gene are compared to control
plants transformed with the vector without the gene insert or untrans-
formed plants.
Example 34: Dactyls glomerata (Orchard Grass) Resistant to Damage
Coleopterans through Expression of Cystatin
A. Construction of Vector. pCIB710-cystatin and pCIB715/710 cystatin are
prepared as described in Example 32A.
B. Transformation and Regeneration of Plants. Transformed plants are
obtained as described in Examples 24 to 28 using pCIB710-cystatin and
pCIB715/710-cystatin.
C. Testing Plants for Cystatin Expression. Testing of transformants for
cystatin expression is carried out as described in Example 32C.
D. Resistance of Transformed Plants to Damge by Diabrotica undecim-
punctata. T1 seedlings a re planted in fine soil in 100 mm Petri dishes
(10/dish, 5 dishes). When the second leaves on the seedlings emerge, each
of five dishes is infested with 20 second instar D. undecimpunctata
larvae. After seven days, the number and weight of the survivors is
measured along with the weight of the washed roots of the plant.
Resistance of transformed plants is detected by a statistically
significant (Student's t test p<0.05) decrease in larval weight gain,
decrease in larval survival rate, or decrease in loss of root mass
relative to insect-free plants when plants expressing the cystatin gene
are compared to control plants transformed with the vector without the
gene insert or untransformed plants.
t3 4 15 t t
_ 7,2 -
Example 35: Cotton Resistant to Damage by ArJthonomus grandis (Boll
Weevil) through Expression of Cystatin
A. Construction of Vector. pCIB715/710 cystatin and pCIBlO-cystatin are
prepared as described in Examples 32A and 33A.
B. Transformation and Regeneration of Plants. Transformed plants are
obtained using pCIB715/710-cystatin or pCIBlO-cystatin in Agrobacterium
as described in Examples 7 to 10.
C. Testing Plants for Cystatin Expression. Testing of transformants for
cystatin expression is carried out as described in Example 32C.
D. Resistance of Transformed Plants to Damage by A. grandis. Ten
transformed plants are grown until bolls start to form. Each plant is
infested with three adu:Lt female A. grandis. Damage to plants is rated
after one week and surviving adults are removed. Damage by larva, larval
numbers and weight per plant are measured at weekly intervals for four
weeks. Resistance of transformed plants is detected by a statistically
significant (Student's t test p<U.US) decrease in damage ratings,
decrease in larval number, or decrease in larval weight when plants
expressing the cystatin gene are compared to control plants transformed
with the vector without the gene insert or untransformed plants.
Example 36: Maize Resistant to Damage by Lepidopterous Larvae through
Expression of Soybean Kunitz Trypsin Inhibitor
A. Construction of the pCIB715/710-KTI Vector. The soybean trypsin
inhibitor cDNA is obtained by the method of Hoffman et al. (1984). The
652 by fragment is modified so that the sequence begins at the methionine
start codon (44 in the original fragment) and codons for the missing
three amino acids plus a stop codon are added to the 3'end. BamHI linkers
are added to both ends. This construction is accomplished by digesting
the 652 by fragment with Eco57I and BstXI and isolating the resulting 517
by fragment following agarose gel electrophoresis. The oligonucleotides
shown in Figure 11 are synthesized. Phosphates are added to the 5' ends
_ i
of the 517 by fragment and oligonucleotides 3M.and 5C and all fragments
are ligated as described above. The resulting 630 by fragment is isolated
and ligated into the BamHI site of the pCIB710 vector as described above.
The resulting vector is denoted pCIB710-KTI.
B. Transformation and Regeneration of Maize. Maize tissue is transformed
with the pCIBIIO vector or, preferably, the pCIB715/710 vector carrying
the soybean Kunit~ try~psin inhibitor gene insert and plants are
regenerated as described in Examples i6-20. For controls, plants trans-
formed with the pCIBllO vector or the pCIB715 vector without the gene
insert are prepared in the same way. transformed plant tissue is selected
or. the appropriate antibiotic. The initisl pints arE self-fertilized and
seed (T1 seed) is obtained.
C. Testing Plants for Soybean hunitz Trypsin Inhibitor Expression. Plants
grown from the T1 seeds are analyzed for the presence and expression of
the soybean Kunitz try~psin inhibitor gene using the following tests.
~ 1 J DIVA 1S iSOlatEd ETC digEste(7 t,'i tn I'cT!kHi ; the ClgESt iE EleCtrC-
proresed on a i.~ i~ agarose gel. The Dr:fi. fragments are transferred to
nitrocellulose and hybridized with the soybean Kunitz trypsin inhibitor
gene labelled kite '~F' by nic't: transition (sEe Maniatis Et al. (1982)
for technioues~. ThE presence of the soy>bEan Kunitz try-psin inhibitor
gene is detectee by a band Of appraximstelv 630 by which hybridizes to
the probe.
(2) RNA is detected by the Northern blot procedure as a band of about
650 bases which hybridizes with the ='P-soybean Kunitz trypsin inhibitor
gene described above.
(3) Soybean Kunitz trypsin inhibitor protein is detected using standard
immunological techniques with a polyclonal rabbit antibody raised against
commercially (Sigma) available soybean Kunit~ trypsin inhibitor which has
been repurified by reverse phase HPLC on a Vydac phenyl column.
*Trade-mark
.3 !~~5~1
(4) Soybean Kunitz trypsin inhibitor activity is detected by immuno-
purifying material from the plant extracts using the rabbit polyclonal
anti-soybean Kunitz trypsin inhibitor antibody and protein A Sepharose
and assaying the isolated material for the ability to inhibit proteolysis
of 1''C-BSA by trypsin using the assay described in Example 1.
D. Resistance of Transformed Plants to Damage by Lepidopterous Larvae. T1
seeds are germinated and leaf pieces obtained from seedlings at the four
leaf stage are used to feed neonatal Ostrinia nubilalis or Neliothis zea
larvae. Neonatal larvae are placed in individual diet cups with a 1 cm2
piece of leaf. Fifty insects are tested per group. After 5 days insect
weight, insect survival, and amount of leaf eaten are rated. Resistance
of transformed plants is detected by a statistically significant
(Student's t test p<0.05) decrease in larval survival, larval weight or
amount of leaf consumed when leaves from expressing the soybean Kunitz
trypsin inhibitor gene are compared to those of control plants trans-
formed with the vector without the gene insert or those of untransformed
plants.
Example 37: Cotton Resistant to Damage by Lepidopterous Larvae through
Expression of Soybean Kunitz Trypsin Inhibitor
A. Construction of the pCIBlO-KTI Vector. The soybean Kunitz trypsin
inhibitor gene together with the 35S CaMV promoter is removed from
pCIB710-KTI (Example 36A) using appropriate restriction enzymes and
ligated into pCIBlO. This vector is denoted as pCIBlO-KTI.
B. Transformation and Regeneration of Plants. Transformed plants are
obtained using pCIBlO-KTI or pCIB715/71UKTI (Example 36A) in Agro-
bacterium as described in Examples 7 to 10.
C. Testing Plants for Trypsin Inhibitor Expression. Testing of trans-
formants for soybean Kunitz trypsin inhibitor expression is carried out
as described in Example 36C.
-75- 13 41511
D. Resistance of Transformed Plants to Damage from Lepidopterous
Insects. Leaf disks from four week old transformed plants are fed to
neonatal Helioth~s virescens, H. zea or Pectinophora gossvpiella.
Neonatal larvae are placed in individual diet cups with a 1 cmz piece of
leaf. Fifty insects are tested per group. After 5 days insect weight,
insect survival, and amount of leaf eaten are rated. Resistance of
transformed plants is detected by a statistically significant (Student's
t test p<0.05) decrease in larval survival, larval weight or amount of
leaf consumed when leaves from plants expressing the soybean Kunitz
trypsin inhibitor gene are compared to those of control plants trans-
formed with the vector without gene insert or to those of untransformed
plants.
Example 38: Tomato Resistant to Damage by Lepidopterous Larvae through
Expression of Soybean Kunitz Trypsin Inhibitor
A. Construction of Vector. pCIB715/710-KTI and pCIBlO-KTI are prepared as
described in Example 36A and 37A.
B. Transformation and Regeneration of Plants. Transformed plants are
obtained as described in Example 33B using the pCIBlO-KTI vector or the
pCIB715/710-KTI vector.
C. Testing Plants for Trypsin Inhibitor Expression. Testing of trans-
formants for soybean Kunitz trypsin inhibitor expression is carried out
as described in Example~36C.
D. Resistance of Transformed Plants to Damage from Lepidopterous Insects.
Leaf disks from four week old transformed plants are fed to neonatal
Neliothis zea or Manduca sexta. Neonatal larvae are placed in individual
diet cups with a 1 cm2 piece of leaf. Fifty insects are tested per group.
After 5 days insect weight, insect survival, and amount of leaf eaten are
rated. Resistance of transformed plants is detected by a statistically
significant (Student's t test p<0.05) decrease in larval survival, larval
weight or amount of leaf consumed when leaves from plants expressing the
13 41511
soybean Kunitz trypsin inhibitor gene are compared to those of control
plants transformed with the vector without the gene insert or to those of
untransformed plants.
Example 39: Tobacco Resistant to Damage by Lepidopterous Larvae through
Expression of Soybean Kunitz Trypsin Inhibitor
A. Construction of Vector. The pCIBlO-KTI vector is prepared as
described in Example 37A.
B. Transformation and Regeneration of Plants. Transformed plants are
obtained as described in Example 6 using the pCIBlO-KTI vector or the
pCIB715/710-KTI vector (Example 36A) in Agrobacterium.
C. Testing Plants for Trypsin Inhibitor Expression. Testing of trans-
formants for soybean Kunitz trypsin inhibitor expression is carried out
as described in Example 36C.
D. Resistance of Transformed Plants to Damage from Lepidopterous Insects.
Leaf disks from four week old transformed plants are fed to neonatal
Neljoth~s zea or Manduca sexta. Neonatal larvae are placed in individual
diet cups with a 1 cmZ piece of leaf. Fifty insects are tested per group.
After 5 days insect weight, insect survival, and amount of leaf eaten are
rated. Resistance of transformed plants is detected by a statistically
significant (Student's t test p<0.05) decrease in larval survival, larval
weight or amount of leaf consumed when leaves from plants expressing the
soybean Kunitz trypsin inhibitor gene are compared to those of control
plants transformed with the vector without the gene insert or to those of
untransformed plants.
Example 40: Orchard Grass Resistant to Damage by Lepidopterous Larvae
Through Expression of Soybean Kunitz Trypsin Inhibitor
A. Construction of Vector. pCIB710-KTI and pCIB715/710-KTI are prepared
as described in Example 36A.
13 41511
_"-
B. Transformation and Regeneration of Plants. Transformed plants are
obtained as described in Examples 24 to 28 using pCIB710-KTI and
pCIB715/710-KTI.
C. Testing Plants for Trypsin Inhibitor Expression. Testing of trans-
formants for soybean Kunitz trypsin inhibitor expression is carried out
as described in Example 36C.
D. Resistance of Transformed Plants to Damage by Lepidopterous Larvae.
T1 seedlings are planted in fine soil in 100 mm Petri dishes (10 per
dish, 5 dishes). When the second leaves on the seedlings emerge, each
dish is infested with 2U second instar larvae of Crambus caliginosellus.
After seven days, the number and weight of the survivors is measured
along with the weight of the washed grass roots. Resistance of trans-
formed plants is detected by a statistically significant (Student's t
test p<0.05) decrease in larval weight gain, decrease in larval survival
rate, or decrease in loss of root weight relative to insect-free plants
when plants expressing the soybean Kunitz trypsin inhibitor gene are
compared to control plants transformed with the vector without gene
insert or untransformed plants.
Example 41: Maize Resistant to Damage by Lepidopterous Larvae through
Expression of al-Antitrypsin
(A) Construction of the pCIB715/710-AATI Vector. The DNA sequences
encoding the aI-antitry~sin are obtained by the method of
Rosenberg et al. (1984). For insertion into the BamHI site in the pCIB710
vector, the DNA fragment is digested with appropriate enzymes and the
synthetic oligonucleotides necessary to create compatible ends are
ligated together with BamHI digested pCIB710 using the methodology
described above. The resultant vector is denoted pCIB710-AATI. This
vector is cut with EcoRI and the resulting DNA is treated with alkaline
phophatase. Plasmid pCIB715 (Rothstein et al., 1987) is cut with EcoRI
and Iigated with the pCIB710-AATI from above. The resulting vector is
named pCIB715/710-AATI.
13 4. X511
B. Transformation and Re>generation of Maize. Maize tissue is transformed
with the pCIB710 vector or, preferably, the pCIB715/710 vector carrying
the al-antitrypsin gene insert and plants are regenerated as described in
Examples 16-20. For controls, plants transformed with the pCIB7IU vector
or the pCIB715 vector without the gene insert are prepared in the same
way. Transformed plant tissue is selected on the appropriate antibiotic.
The initial plants are self-fertilized and seed (T1 seed) is obtained.
C. Testing Plants for a~.-Antitrypsin Expression. Plants grown from the
T1 seed are analyzed for the presence and expression of the al-anti-
trypsin gene using the following tests.
(1) DNA is isolated and digested with BamHI; the digest is electro-
phoresed on a 1.5 % agarose gel. The DNA fragments are transferred to
nitrocellulose and hybridized with the al-antitrypsin gene labelled with
j~P by nick translation., The presence of the al-antitrypsin gene is
detected by a band of approximately 1200 by which hybridizes to the
probe.
(2) RNA is detected by t:he Northern blot procedure as a band of
approximately 1200 bases which hybridizes with the 3zP-al-antitrypsin
gene described above.
(3) a1-antitrypsin protein is detected using standard immunological
techniques with a polyclonal rabbit antibody raised against commercially
(Sigma) available al-anf:itrypsin.
(4) al-antitrypsin activity is detected by assaying material
immunopurified from the plant extracts using the rabbit polyclonal
anti-al-antitrypsin antibody and protein A Sepharose and assaying for the
ability of the isolated material to inhibit proteolysis of 1''C-BSA by
trypsin using the assay described above.
D. Resistance of Transformed Plants to Damage by Lepidopterous Larvae.
T1 seeds are germinated and leaf pieces obtained from seedlings at the
four leaf stage are used to feed neonatal Ostrinia nubilalis or lYel~othis
zea larvae. Neonatal larvae are placed in individual diet cups with a
.3 4151 ~
_ ,y _
1 cmz piece of leaf. Fifty insects are tested per group. After 5 days
insect weight, insect survival, and amount of leaf eaten are rated.
Resistance of transformed plants is detected by a statistically signifi-
cant (Student's t test p<0.05) decrease in larval survival, larval weight
or amount of leaf consumed when leaves from plants expressing the
al-antitrypsin gene are compared to those of control plants transformed
with the vector without gene insert or to those of untransformed plants.
Example 42: Cotton Resistant to Damage by Lepidopterous Larvae through
Expression of al-Antitrypsin
A. Construction of the pCIBlO-AATI Vector. The al-antitrypsin gene plus
the 35S CaMV promoter is removed from pCIB710-AATI (Example 41A) and
ligated into pCIBIU usin g appropriate enzymes. This vector is denoted as
pCIBlO-AATI.
B. Transformation and Regeneration of Plants. Transformed plants are
obtained using pCIBlO-AATI or pCIB715/710-AATI (Example 41A) in Agro-
bacterium as described an Examples 7 to 10.
C. Testing Plants for ai-Antitrypsin Expression. Testing of transformants
for a~-antitrypsin expression is carried out as described in Example 41C.
D. Resistance of Transformed Plants to Damage from Lepidopterous
Insects. Leaf disks from four week old transformed plants are fed to
neonatal Heliothls virescens, H. zea or Pectinophora gossypiella.
Neonatal larvae are placed in individual diet cups with a 1 cmz piece of
leaf. Fifty insects are tested per group. After 5 days insect weight,
insect survival, and amount of leaf eaten are rated. Resistance of
transformed plants is detected by a statistically significant (Student's
t test p<0.05) decrease in larval survival, larval weight or amount of
leaf consumed when leaves from plants expressing the al-antitrypsin gene
are compared to those of control plants transformed with the vector
without the gene insert or to those of untransformed plants.
13 41511
-80-
Example 43: Tomato Resistant to Damage by Lepidopterous Larvae through
Expression of al-Antitrypsin
A. Construction of Vector. pCIB7I5/710-AATI and pCIBlO-AATI are prepared
as described in Examples 41A and 42A.
B. Transformation and Regeneration of Plants. Transformed plants are
obtained as described in Example 33B using the pCIBlO-AATI vector and the
pCIB715/710-AATI vector.
C. Testing Plants for a1-Antitrypsin Expression. Testing of transformants
for al-antitrypsin expression is carried out as described in Example 41C.
D. Resistance of Transformed Plants to Damage from Lepidopterous Insects.
Leaf disks from four week old transformed plants are fed to neonatal
fleliothis zea or ~landuca se.tta. Neonatal larvae are placed in individual
diet cups with a 1 cmz piece of leaf. Fifty insects are tested per group.
After 5 days insect weight, insect survival, and amount of leaf eaten are
rated. Resistance of transformed plants is detected by a statistically
significant (Student's 't test p<0.05) decrease in larval survival, larval
weight or amount of leaf consumed when leaves from plants expressing the
al-antitrypsin gene are compared to those of control plants transformed
with the vector without the gene insert or to those of untransformed
plants.
Example 44: Tobacco Resistant to Damage by Lepidopterous Larvae through
Expression of a~-Antitrypsin
A. Construction of Vector. pCIB715/710-AATI and pCIBlO-AATI are prepared
as described in Examples 41A and 42A.
B. Transformation and Regeneration of Plants. Transformed plants are
obtained as described in Example 6 using the pCIBlO-AATI vector or the
pCIB715/710-AATI vector (Example 41A) in Agrobacterjum.
~34~5~~
- 81 -
C. Testing Plants for a1-Antitrypsin Expression. Testing of transformants
for al-antitrypsin expression is carried out as described in Example 41C.
D. Resistance of Transformed Plants to Damage from Lepidopterous Insects.
Leaf disks from four week old transformed plants are fed to neonatal
Helioth~s v~rescens or MandUCa sexta. Neonatal larvae are placed in
individual diet cups with a 1 cmZ piece of leaf. Fifty insects are tested
per group. After 5 days insect weight, insect survival, and amount of
leaf eaten are rated. Resistance of transformed plants is detected by a
statistically significant (Student's t test p<O.US) decrease in larval
survival, larval weight or amount of leaf consumed when leaves from
plants expressing the al-antitrypsin gene are compared to those of
control plants transformed with the vector without the gene insert or to
those of untransformed plants.
Example 45: Orchard Grass Resistant to Damage by Lepidopterous Larvae
Through Expression of ai-Antitrypsin
A. Construction of Vector. pCIB710-AATI and pCIB715/710-AATI are prepared
as described in Example 41A.
B. Transformation and Regeneration of Plants. Transformed plants are
obtained as described in Examples 24 to 28 using pCIB710-AATI and
pCIB715/710-AATI.
C. Testing Plants for ai-Antitrypsin Expression. Testing of transformants
for ai-antitrypsin expression is carried out as described in Example 41C.
D. Resistance of Transformed Plants to Damage by Lepidopterous Larvae.
T1 seedlings are planted in fine soil in 100 mm Petri dishes (10/dish,
S dishes). When the second leaves on the seedlings emerge, each dish is
infested with 20 second instar larvae of Crambus caliginosellus. After
seven days, the number and weight of the survivors is measured along with
the weight of the washed grass roots. Resistance of transformed plants
is detected by a statistically significant (Student's t test p<0.05)
decrease in larval weight gain, decrease in larval survival rate, or
decrease in loss of root mass relative to insect-free plants when plants
~~ 41511
- 82 -
expressing the al-antitrypsin gene are compared to control plants
transformed with the vector without the gene insert or untransformed
plants.
Example 46: Maize Resistant to Damage by Lepidopterous Larvae through
Expression of Eglin
A. Construction of the pCIB715/71U-eglin Vector. The DNA sequences
encoding eglin C and eglin C mutants are obtained as described in
Example 3 D. For insertion into the BamHI site of the pCIB7IU vector, the
DNA fragment is digested with appropriate enzymes and the synthetic
oligonucleotides necessary to create compatible ends are ligated together
with BamHI digested pCIB7IU using the methodology described above. The
resultant vector derived from the eglin C (Arg45) mutant is denoted
pCIB710-eglin. This vector is cut with EcoRI and the resulting DNA is
treated with alkaline phophatase. Plasmid pCIB715 (Rothstein et al.,
1987) is cut with EcoRI and ligated with the pCIB7IU-eglin from above.
The resulting vector is named pCIB715/71U-eglin.
B. Transformation and Regeneration of Maize. Maize tissue is transformed
with the pCIB710 vector or, preferably, the pCIB715/710 vector carrying
the eglin C (Arg45j gene insert and plants are regenerated as described
in Examples 16-2U. for controls, plants transformed with the pCIB710
vector or the pCIB715 vector without the gene insert are prepared in the
same way. Transformed plant tissue is selected on the appropriate
antibiotic. The initial plants are self-fertilized and seed (T1 seed] is
obtained.
C. Testing Plants for Eglin Expression. Plants grown from the Tl seed are
analyzed for the presence and expression of the eglin C (Arg45j gene
using the following tests.
(1) DNA is isolated and digested with BamHI; the digest is electro-
phoresed on a 1.5 % agarose gel. The DNA fragments are transferred to
nitrocellulose and hybridized with the eglin C (Arg45j gene labeled with
3zP by nick translation. The presence of the eglin gene is detected by a
band of approximately 23U by which hybridizes to the probe.
1341~~1
(2) RNA is detected by the Northern blot procedure as a band of approxi-
mately 230 bases which hybridizes with the 3zP-eglin C (Arg45) gene
described above.
(3) Eglin C (Arg45) mutant protein is detected using standard immuno-
logical techniques with a polyclonal rabbit antibody raised against
eglin C (Arg45).
(4) Eglin activity is detected by assaying material immunopurified from
the plant extracts using the rabbit polyclonal anti-eglin C (Arg45)
antibody and protein A Sepharose and assaying for the ability of the
isolated material to inhibit proteolysis of '''C-BSA by trypsin using the
assay described above.
D. Resistance of Transformed Plants to Damage by Lepidopterous Larvae. T1
seeds are germinated, and leaf pieces obtained from seedlings at the four
leaf stage are used to feed neonatal European corn borer (Ostrinia
nubilalis~ or corn earworm (Helioth~s zeal larvae. Neonatal larvae are
placed in individual diet cups with a 1 x 1 cm piece of leaf. Fifty
insects are tested per group. After 5 days insect weight, insect
survival, and amount of leaf eaten are rated. Resistance of transformed
plants is detected by a statistically significant (Student's t test
p>0.05) decrease in larval survival, larval weight or amount of leaf
consumed when leaves frnm plants expressing the eglin C (Arg45) gene are
compared to those of control plants transformed with the vector without
gene insert or those of untransformed plants.
Example 47: Cotton Resistant to Damage by Lepidopterous Larvae through
Expression of Eglin
A. Construction of the pCIBlO-eglin Vector. The eglin C (Arg45) gene plus
the 35S CaMV promoter are removed from pCIB710-eglin (Example 46A) and
ligated into pCIBlO using appropriate enzymes. This vector is denoted
pCIBlO-eglin.
13 41511
- 8.4 -
B. Transformation and Regeneration of Plants. Transformed plants are
obtained using pCIBlO-eglin or pCIB715/710-eglin (Example 46A) in Agro-
bacterium as described in Examples 7 to 10.
C. Testing Plants for Eglin Expression. Testing of transformants for
eglin expression is carried out as described in Example 46C.
D. Resistance of Transformed Plants to Damage from Lepidopterous Insects.
Leaf disks from four week old transformed plants are fed to neonatal
fireliothis c-irescens (tobacco budworm), h'eliothis zea (cotton bollworm) or
Pectinophora goss3pze11a (pink bollworm). Neonatal larvae are placed in
individual diet cups with a 1 x 1 cm piece of leaf. Fifty insects are
tested per group. After 5 days insect weight, insect survival, and amount
of leaf eaten are rated. Resistance of transformed plants is detected by
a statistically significant (Student's t test p>0.05) decrease in larval
survival, larval weight or amount of leaf consumed when leaves from
plants expressing the eglin C (Arg45) gene are compared to those of
control plants transformed with the vector without the gene insert or
those of untransformed plants.
Example 48: Tomato Resistant to Damage by Lepidopterous Larvae through
Expression of Eglin
A. Construction of Vector. pCIB715/710-eglin and pCIBlO-eglin are
prepared as described in Examples 46A and 47A.
B. Transformation and Regeneration of Plants. Transformed plants are
obtained as described in Example 33B using the pCIBlO-eglin vector and
the pCIB715/710-eglin vector.
C. Testing Plants for Eglin Expression. Testing of transformants for
eglin expression is carried out as described in Example 46C.
D. Resistance of Transformed Plants to Damage from Lepidopterous Insects.
Leaf disks from four week old transformed plants are fed to neonatal
Heljothls zea (cotton bollworm) or ~fandvca sexta (tomato/tobacco horn-
worm). Neonatal larvae are placed in individual diet cups with a 1 x 1 cm
~~ ~ r ~ i i
_ g.5 -
piece of leaf. Fifty insects are tested per group. After 5 days insect
weight, insect survival, and amount of leaf eaten are rated. Resistance
of transformed plants is detected by a statistically significant
(Student's t test p<0.05) decrease in larval survival, larval weight or
amount of leaf consumed when leaves from plants expressing the eglin C
(Arg 45j gene are compared to those of control plants transformed with
the vector without the gene insert or those of untransformed plants.
Example 49: Tobacco Resistant to Damage by Lepidopterous Larvae through
Expression of Eglin
A. Construction of Vector. pCIB715/710-eglin and pCIBlO-eglin are
prepared as described in Examples 46A and 47A.
B. Transformation and Regeneration of Plants. Transformed plants are
obtained as described in Example 6 using the pCIBIO-eglin vector or the
pCIB715/7I0-eglin vector (Example 46A) in Agrobacterium.
C. Testing Plants for Eglin Expression. Testing of transformants for
eglin expression is carried out as described in Example 46C.
D. Resistance of Transformed Plants to Damage from Lepidopterous Insects.
Leaf disks from four week old transformed plants are fed to neonatal
Hel.tothis Wrescens (tobacco budworm) or Manduca sexta (tomato/tobacco
hornworm). Neonatal larvae are placed in individual diet cups with a
1 x 1 cm piece of leaf. Fifty insects are tested per group. After 5 days
insect weight, insect survival, and amount of leaf eaten are rated.
Resistance of transformed plants is detected by a statistically signifi-
cant (Student's t test p<0.05) decrease in larval survival, larval weight
or amount of leaf consumed when leaves from plants expressing the eglin C
(Arg45) gene are compared to those of control plants transformed with the
vector without the gene insert or those of untransformed plants.
1~ ~1~11
- 86 -
Example 50: Orchard Grass Resistant to Damage by Lepidopterous Larvae
through Expression of Eglin
A. Construction of Vector. pCIB710-eglin and pCIB715/710-eglin are
prepared as described in Example 46A.
B. Transformation and Regeneration of Plants. Transformed plants are
obtained as described in Examples 24 to 28 using pCIB710-eglin and
pCIB715/710-eglin.
C. Testing Plants for Eglin Expression. Testing of transformants for
eglin expression is carried out as described in Example 46C.
D. Resistance of Transformed Plants to Damage by Lepidopterous Larvae. T1
seedlings are planted in fine soil in 100 mm Petri dishes (1U per dish,
dishes). When the second leaves on the seedling emerge, each dish is
infested with 20 second instar larvae of corn root webworm (Crambus
caliginosellusJ. After seven days, the number and weight of the survivors
is measured along with the weight of the washed grass roots. Resistance
of transformed plants is detected by a statistically significant
(Student's t test p>0.05) decrease in larval weight gain, decrease in
larval survival rate, or decrease in loss of root mass relative to
insect-free plants when plants expressing the eglin C (Arg45) gene are
compared to control plants transformed with the vector without the gene
insert or untransformed plants.
Depositions:
(1) E. cola HB101/pML147 (DSM 4380) was deposited on ,3anuary 28, 1988
with the Deutsche Sammlung von Mikroorganismen and Zellkulturen (DSM)
in Braunschweig FRG.
The following microorganisms were deposited with the American Type
Culture Collection (ATCC) in Rockville, Maryland:
(2) Plasmid pLWlli (ATCC-40235) deposited on May 14, 1986.
(3) Zea m3ys suspension culture (ATCC-40326) deposited on May 20, 1987.
(4) Plasmid p368 (ATCC-67700) deposited on May 19, 1988.
1341511
Table A
PLANT CLASSIFICATION ACCORDING TO USE
CEREALS
Monocot
Dicot
Avena nuda (chirrensis,~Chinese naked oat
A. sati va Common oats
fleusine coracan African millet
Bragrostis tef Tef grass
fordeum d~stichum Two-row barley
H, vulgate Barley
Oryza sate va Rice
Panicum italicium Italian millet
P. miliaceum Broomcorn millet
Pennisetum glaucum Spiked millet
P. spicatum (americanum~Pearl millet
Secale cereale Rye
Sorghum vulgate Grain sorghums
X Triticosecale Triticale
Triticum aestivum Common wheat
T. dicvccum Emmer
T. durum Abyssinian hard wheat
T. monococcum Einkorn wheat
Zea mat's Starch, sweet corn
Amaranthus paniculatus Grain amaranth
Fagopyrum esculentum Buckwheat
F. tataricum Rye buckwheat
PROTEIN CROPS
Dicot
Araehis hypogea Groundnut, peanut
Cajanus indfcus Pigeon pea
Cicer arlet.znum Chickpea
Dolichos Iablab Hyacinth bean
Glycine gracilis Manchurian Soya
G. max Soyabean
G. ussuri'erasis Wild soya
Lathyrus sati vus Grass pea
Lens culinaris Lentil
Mucuna pruriens Cowitch, Florida
velvet bean
Phaseolus acuti folios Tepary bean
P. aureus Mung, green gram
P. lunatus Lima bean, Sieva
P. cvccineus Scarlet runner bean
(foul tifl orusJ
P. mungo Black gram
P. vulgaris French, common, kidney
or dwarf bean
13 415 ~ 1
_88_
hicia faba Horse bean, broad bean
higna angularis Adzuki bean
Y sesquipedali~> Asparagus (yard-long
bean)
V. sinensis Cowpea
FRUIT CROPS
Dicots
Amf gdalus communis Almond
Ananas cvmosus Pineapple
Artocarpus communis Breadfruit
Carica papaya Papaya
Citrullus vulgaris Watermelon
Citrus grandis Pummelo
C. medica Citron, lemon
C. nobilis Tangerine
C. reticulata Mandarin
C. sinensis Orange
Cydonia oblonga Quince
Diospyros kaki Japanese persimmon
Ficus carica Fig
Fragaria chiloe.ns~s Wild strawberry
F. virginiana Strawberry
L1 tCh1 ChlnenSl.S LltCh1
Males asiatica Chinese apple
M pumila Apple
Mangifera indices Mango
Mores rubra Red mulberry
Mesa cavendishii Banana
M. paradisiaca Banana
Passi flora edulis Passion fruit, purple
granadilla
P. ligularis Passion flower
Persea americana Avocado pear
Phoenix dactyli fera Date palm
Prunes armeniaca Apricot
P. a vium Sweet cherry, mazzard
P. cerasifera Cherry plum
(divarica tai
P. cerasus Cherry
P. domestica European plum or prune
P. maheleb Maheleb cherry
P. persica Peach and nectarine
P. pseudocerasus Cherry
P. salicinia Japanese peach
P. serotina Wild black cherry
Psidium guajava Guava
Punica granatum Pomegranate
Pyres communis Pear
P. ussuriensis Chinese pear
Ribes grossularia Gooseberry
R. nigrum Black currant
R. rubrum Red and white currant
Rebus idaeus European raspberry
R. strigosus American raspberry
Tamarindus indices Tamarind
134~5~~
- 89 -
haccinium Sugarberry
angus t~fo1 l um
V, ashei Rabbiteye blueberry
1: cvr3~nbosum Highbush blueberry
h. myrtillus Canada blueberry
1: oxvcoccos Cranberry
li~burnum tr~lobrrmAmerican cranberry bush
l'itis Iabrusca Fox grape
h vinifera Grape
VEGETABLES AND TUBERS
Monocot
Dicot
AII~um ascalvn.tcum Shallot, green onion
A, ceps Onion
A. chinense Onion
A. fistulosum Welsh onion
A. porrum Leek
A. sate vum Garlic
A. schoenoprasum Chives
Asparagus offic.inalisAsparagus (var. attilis)
Zea ways Sweet corn
Amvracia IapathifoliaHorseradish
Apium graveolens Celery
Arabidopsis thaliana Common wall cress
Beta vulgaris Sugar, mangold or
garden beet
Brassica alboglabra Chinese kale
B. campestris Turnip rape
B. carinata Abyssinian mustard
B. cernua Karashina
B. chinensis Chinese mustard or
pak-choi
B. hirta White mustard
B. juncea Pai, brown mustard,
Indian mustard
B. kaber Charlock
B. napobrassica Swede or rutabaga
B. napes Rape, oil rape, kale
B. nigra Black mustard
B. oleracea Cole, kale, collards
brussels sprouts,
cauliflower, cabbage,
kohlrabi, broccoli
B. pekinensis Chinese cabbage or
celery cabbage
B. rape Turnip
Cajanus cajan ('indices)Pigeon pea
Canavalia ensiformis Jack bean
Canna edul~s Edible canna
Capsicum annuum Common cultivated pepper
C. chinense Pepper
C. frutescens Cayenne pepper
C. pendulum Pepper
C. pubescens Pepper
15 41511
- 90 -
Cichorium endi via Endive
C. intylbus Chicory
Colocasia aritiquorum Taro
Crambe maritima Sea kale
Cucumls melo Melon, cantaloupe
C. sati vus Cucumber
Cucurbita fici folia Malabar gourd
C. foetidissima Calabazilla, buffalo
gourd
C. marima Pumpkin
C. moschata Winter pumpkin
C. pepo Summer squash,
vegetable marrow
Cynara scolymus Globe artichoke
Daucus carota Carrot
DlO.s'COrea alata Yam
D. batatas Chinese yam
D. cayennensis Attoto yam
fruca sati va Mill. Rocket salad, rocket
or
roquette
Ipomea batatas Sweet potato
Lactuca sativa Lettuce
Lepidium sativum Garden cress
Lycopersicon cerasiformeCherry tomato
L. esculentum Tomato
Mahihot utilissima Manioc, cassava
Nasturtium officinale Water cress
Pastinaca sati va Parsnip
Petroselinum cr~spurn Parsley
(sa ti vumJ
Physalis peruviana Ground cherry
Pisum sativum Pea
Raphanus sati vus Radish
Rheum offtcinale Rhubarb
R. rhaponticum English rhubarb
Scorzonera hispariica Black salsify
Sechium edule Chayote
Solarium andigenum Andean potato
S. melongena Eggplant
S. muricatum Pepino
S. phureja Potato
S. tuberosum Common potato
Spinaeia oleracea Spinach
NUTS
Dicot
Anacardium occidentaleCashew
Arachis hypogaea Peanut
Carva illinoinensjs Pecan
C. ovata Shagbark hickory
Castariea sari va Chestnut
Cocos nucifera Coconut palm
Corylus americana American hazel, filbert
C. avellana European hazel, cobnut
Juglans nigra Black walnut
J. regia English walnut
141511
- 91 -
J. sinensis Walnut
LItCh1 ChlnenslS LltCh1
Macadamia integrifolia Queensland nut
Pistacia vera Pistachio nut
Prunes amvgdalu~~ Almond
OIL CROPS (COOKING OR VEGETABLE OILS)
Monocot
n; r~nt
Zea ways Corn
Aleurites cordata Tung, China wood oil
A. molLCCana (triloba~Candlenut
Arachis hypogea Ground nut, peanut
Brasslca campestrss Rapeseed oil, canola
oil
B. napes Rapeseed oil, canola
oil
Cannabis sati va Hempseed oil
Carthamus tinctorius Safflower oil
Corns nucifera Coconut palm
Blaeis guineensis Oil palm
Glycine gracilus Manch, soya
G. max Soybean
C. ussuriensis Wild soya
Gossypium hirsutum Cottonseed oil
Belianthus annus Sunflower
Linum usitatissimum Flax
Olea europaea Olive
Papaver somni ferum Poppy seed
Ricinus communis Castor bean
Sesamum indicum Sesame
SUGAR CROPS
Monocot
Dicot
Saccharum offic.inarumSugarcane
(offi c.toarum x
spontaneum~
S. robustum
S. sinense Sugarcane
S. spontaneum Kans grass
Sorghum dochna Sorgo syrup, sugar
sorghum
Acer saccharum Sugar maple
Beta vulgaris Sugar or mangold beet
FORAGE AND TURF GRASSES
Monocot
Agropyron Crested wheatgrass
rristatum
A.desertorum Crested wheatgrass
A.elongatum Tall wheatgrass
A..intermediurn Intermediate wheatgrass
A.smzthii Western wheatgrass
A,spicatum Blue bunch wheatgrass
A.trachycaulum Slender wheatgrass
A.trichophorum Pubescent wheatgrass
13~1~11
- 92 -
Alopecurus pratensis Meadow foxtail
Andropogon gerardi Big bluestem
Arrhenatherum elatiusTall oat grass
Bothriochloa barbinodisCane bluestem
B. ischaemum King ranch bluestem
B. saccharoides Silver bluestem
Bouteloua curipendulaSide oats grama
B. eriopoda Black grama
B. gracilis Blue grama
Bromus erectus Upright brome
B. inermis Smooth brome
B. riparius Meadow brome
Cenchrus ciliar:%s Buffel grass
Chloris gayana Rhodes grass
Cymbopogon nardus Citronella grass
Cynodon dact~yIon Bermuda grass
Dactylis glomera to Cocksfoot, orchard
grass
Dlchanthium annulatumKleberg bluestem
D. aristatum Angleton bluestem
D. sericeum Silky bluestem
Digitaria decumbens Pangola grass
D. smutsii
L'lf~nus angustus Altai wild rye
E. junceus Russian wild rye
Eragrostis curvula Weeping love grass
Festuca arundinacea Tall fescue
F. ovine Sheeps fescue
F, pratensis Meadow fescue
F. rubra Red fescue
Lolium multi florum Italian ryegrass
L. perenne Perennial ryegrass
Panicum maximum Guinea grass
P. purpurascens Para grass
P. virgatum Switchgrass
Paspalum dilatatum Dallis grass, large
water grass
P. notatum Bahia grass
Pennisetum clandestinumKikuyu grass
P. purpureum Dry napier grass
Phalaris arundinacea Reed canary grass
Phleum bertolin_ii Timothy
P. pretense Timothy
Poa fendleriana Mutton grass
P. nemoralis Wood meadow grass
P. pratensis Kentucky bluegrass
Setaria sphacel.~ta Rhodesian timothy
Sorghastrum nutans Indian grass
Sorghum halepense Johnson grass
S. sudanense Sudan grass
Sorghum vulgare Great millet
FORAGE LEGUI~S
Dicot
Coronilia varia Crown vetch
Crotalaria juncea Sunn hemp
Lespedeza stipu.lacea Korean lespedeza
13 41511
- 93 -
L.striata Common lespedeza
L.sericea
Lotus corniculatus Birdsfoot trefoil
L.uliginosus
Lupinus Wolf bean, white lupin
albus
L.angusti folius Blue lupin
L.luteus European yellow lupin
L.mutabilis South American lupin
Medicago Spotted burr-clover
arabica
M.arborea Tree alfalfa
M falcata Yellow lucerne
~lhispida California burr-clover
M,sativa Alfalfa
~Itribuloides Barrel medic
Melilotus albus White sweet clover
M officinalis Yellow sweet clover
Onohrvchis viciifoliaSainfoin
Ornithopus Serradella
sativus
Pueraria Kudzu vine
thunbergiana
Trifolium Egyptian clover
alexandrinum
T.augusttfolium Fineleaf clover
T,diffusum Rose clover
T.t~ybridum Alsike clover
T.incarnatum Cromson clover
T.ingrescens Ball clover
T.pratense Red clover
T.repens White clover
T.resupinatum Persian clover
T.subterraneum Subterranean clover
Trigonella foenum- Fenugreek
graecum
Yicia Common vetch
sativa
Y vt11vsa Hairy vetch
h atropurpurea Purple vetch
Irangusti folic Narrowleaf vetch
l:dasycarpa Woolly pod vetch
Y.ervilia Monantha (bitter) vetch
V pannonica Hungarian vetch
l!calcarata Bard vetch
FI$ER PLANTS AND MOODS
Monocot
Bambusa vrrlgaris Bamboo
Dicot
Agave sisalana Sisal hemp
Boehmeria hives Rhea fiber, ramie
Cannabis indices Hemp
C. sati va Hemp
Ceiba pentandra Silk cotton tree,
kapok tree
Corchorus mucronata Hemp
(striates)
Gossypium arboreum Tree cotton
G. barbadense Egyptian cotton
G. herbaceum Cotton
i3 4511
- 94 -
G. hirsutum Upland cotton
G. nanking Oriental cotton
Linum angustifo.fium Wild flax
L. uSltatlsslmum Flax
Musa tea-tiles Manila hemp, abaca
DRUG CROPS
Dicot
Angelica archangelicaAngelica
Chrysanthemum Palm pyrethrum
cinerariifoli um
Camellia sznensis Chinese tea
C. coecineum Pyrethrum
Coffees arabica Coffee
C. canephora Quillow coffee
Cola acuminata Kola nut
Nicotiana rustica Tobacco
N. tabacum Tobacco
Papa ver dubium Poppy
P. somni forum Opium poppy
Theobroma cacao Cocoa
SPICES AND FLAVORINGS
Monocot
Dicot
Vanilla fragrans Vanilla
Artem~sa dracunculus Tarragon
Cinnamomum zeylanicumCinnamon tree
Hibiscus esculentus Okra
Salvia offlcinal~s Sage
Thymus vulgari s Thyme
Pimpinella anisum Anise
Menthes arvensis Menthol
M. piperita Peppermint
M. viridis Spearmint
Coriandrum sati vum Coriander
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