U.S. patent application number 12/977715 was filed with the patent office on 2011-07-07 for direct and continuous root alone or root/shoot production from transgenic events derived from green regenerative tissues and its applications.
This patent application is currently assigned to Pioneer Hi-Bred International, Inc.. Invention is credited to David C. Cerf, Myeong-Je Cho, Deping Xu, Zuo-Yu Zhao.
Application Number | 20110165561 12/977715 |
Document ID | / |
Family ID | 43663622 |
Filed Date | 2011-07-07 |
United States Patent
Application |
20110165561 |
Kind Code |
A1 |
Cho; Myeong-Je ; et
al. |
July 7, 2011 |
DIRECT AND CONTINUOUS ROOT ALONE OR ROOT/SHOOT PRODUCTION FROM
TRANSGENIC EVENTS DERIVED FROM GREEN REGENERATIVE TISSUES AND ITS
APPLICATIONS
Abstract
The present invention provides assays and methods for
efficiently testing a polynucleotide of interest for a phenotype in
a root. In some embodiments, the assays and methods include
regenerating green tissue that is transgenic for at least one
polynucleotide of interest into one or more transgenic plantlets
that have at least one transgenic root. Further provided are
methods of making a root assay by contacting green tissue with a
first rooting medium to produce a plantlet and a plurality of
roots. Additionally provided are methods of assaying for
insecticidal activity on a live root. Accordingly provided herein
is a substantially contamination-free, root bioassay. Further
provided are methods of identifying a promoter having activity in a
root.
Inventors: |
Cho; Myeong-Je; (Alameda,
CA) ; Cerf; David C.; (Palo Alto, CA) ; Xu;
Deping; (Johnston, IA) ; Zhao; Zuo-Yu;
(Johnston, IA) |
Assignee: |
Pioneer Hi-Bred International,
Inc.
Johnston
IA
|
Family ID: |
43663622 |
Appl. No.: |
12/977715 |
Filed: |
December 23, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61291704 |
Dec 31, 2009 |
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Current U.S.
Class: |
435/6.1 ; 435/29;
435/32; 47/58.1R |
Current CPC
Class: |
G01N 2333/415 20130101;
G01N 33/5097 20130101; C12N 15/8201 20130101; Y02A 40/146 20180101;
C12N 15/8286 20130101; Y02A 40/162 20180101; G01N 2333/43552
20130101; C12N 15/821 20130101; C12N 15/8227 20130101; A01G 22/00
20180201 |
Class at
Publication: |
435/6.1 ; 435/32;
435/29; 47/58.1R |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12Q 1/18 20060101 C12Q001/18; C12Q 1/02 20060101
C12Q001/02; A01G 1/00 20060101 A01G001/00 |
Claims
1. A method for efficiently testing a polynucleotide of interest
for a phenotype in roots comprising: a) regenerating green tissue
that is transgenic for at least one polynucleotide of interest into
one or more transgenic plantlets, wherein the one or more
transgenic plantlets comprise at least one transgenic root; and b)
determining at least one phenotype of the transgenic root.
2. The method of claim 1, further comprising subjecting the
transgenic root to a pest or pathogen.
3. The method of claim 1, wherein the phenotype is increased root
size, increased overall root mass, altered root architecture,
increased expression level of mRNA or protein, increased
biochemical content, increased tolerance to stress, increased
resistance to a pest, increased resistance to an insecticide,
increased yield, or increased nitrogen use efficiency as compared
to the corresponding phenotype of a control, wherein the
polynucleotide of interest has not been introduced into the
control.
4. The method of claim 1, wherein the at least one transgenic root
is isolated from the plantlet prior to determining the at least one
phenotype.
5. The method of claim 1, further comprising producing a transgenic
plant from the green tissue.
6. The method of claim 1, further comprising growing the transgenic
plantlet into a transgenic plant.
7. The method of claim 1, further comprising rooting the plantlet
on medium.
8. The method of claim 7, wherein the medium is medium gelled with
agar or an agar substitute.
9. The method of claim 1, wherein the plantlet is a monocot
plantlet.
10. The method of claim 1, wherein the green tissue is obtained by
transforming an explant from a monocot and subjecting the explant
to green tissue initiation medium for a time and under conditions
sufficient to initiate growth from the explant, thereby producing
green tissue.
11. The method of claim 10, wherein the explant comprises an
embryo, green tissue, callus, leaf, meristem, seedling, seed, stem,
shoot, node, leaf base, or root.
12. The method of claim 1, further comprising regenerating
transgenic green tissue into one or more transgenic plantlets by
contacting the green tissue with a rooting medium that induces root
formation for a time and under conditions sufficient to initiate
root growth from the green tissue, thereby producing a
plantlet.
13. The method of claim 1, wherein determining at least one
phenotype of the transgenic root occurs under sterile
conditions.
14. A method of making a root assay comprising: a) contacting green
tissue with a first rooting medium gelled with agar or an agar
substitute to produce a plantlet having at least one transgenic
root; b) removing the root from the medium; and c) contacting the
root with a second rooting medium in the absence of agar or an agar
substitute to produce a plurality of roots.
15. The method of claim 14, further comprising regenerating a
plurality of transgenic plantlets from the green tissue.
16. The method of claim 14, placing the at least one transgenic
root of the plantlet in a culture dish.
17. The method of claim 14, contacting the roots with the second
rooting medium, wherein the second rooting medium comprises a
biocide.
18. The method of claim 14, contacting the roots with the second
rooting medium using an absorbent material.
19. The method of claim 14, wherein the green tissue is obtained by
transforming an explant from a monocot and subjecting the explant
to green tissue initiation medium for a time and under conditions
sufficient to initiate growth from the explant, thereby producing
green tissue.
20. The method of claim 19, wherein the explant comprises an
embryo, green tissue, callus, leaf, meristem, seedling, seed, stem,
shoot, node, leaf base, or root.
21. The method of claim 14, further comprising regenerating
transgenic green tissue into one or more transgenic plantlets by
contacting the green tissue with a first rooting medium that
induces root formation for a time and under conditions sufficient
to initiate root growth from the green tissue, thereby producing a
plantlet.
22. The method of claim 14, wherein the roots are prepared under
substantially sterile conditions.
23. The method of claim 14, further comprising subjecting the root
to a chemical, pest, or pathogen.
24. The method of claim 23, further comprising sterilizing the pest
prior to contacting the root.
25. The method of claim 23, further comprising feeding the pest
prior to infesting the pest on the transgenic roots.
26. The method of claim 14, wherein the assay is produced within
about 4 to 14 days.
27. The method of claim 14, further comprising growing the
transgenic plantlets into plants.
28. The method of claim 27, further comprising obtaining transgenic
seeds from the transgenic plants.
29. A method of assaying for insecticidal activity on a live root
comprising: a) regenerating green tissue into one or more plantlets
comprising at least one live root; b) contacting the at least one
root of the plantlet with a rooting medium; c) exposing the root to
one or more pests to infest the medium for infestation; wherein the
medium and pest are substantially contamination-free; and d)
determining a phenotype of the root.
30. The method of claim 29, further comprising regenerating green
tissue transgenic for a polynucleotide of interest into one or more
transgenic plantlets comprising at least one live transgenic
root.
31. The method of claim 29, wherein the live root is assayed for
endogenous resistance to root damage by the pest.
32. The method of claim 29, further comprising contacting the green
tissue with rooting medium to induce root formation.
33. The method of claim 29, wherein the rooting medium comprises a
biocide.
34. The method of claim 29, contacting the root with the rooting
medium using an absorbent material.
35. The method of claim 29, determining the phenotype of the root
by scoring the root for damage as compared to the phenotype of the
control.
36. The method of claim 29, further comprising sterilizing the pest
prior to contacting the root.
37. The method of claim 29, further comprising feeding the pest
prior to infesting the pests on the transgenic roots.
38. The method of claim 29, wherein the pest is from the order of
lepidoptera, homoptera, heteroptera, or coleoptera.
39. The method of claim 29, wherein the pest is of a developmental
stage comprising an egg, larva, instar, or adult.
40. The method of claim 29, further comprising determining a
phenotype of the pest.
41. The method of claim 40, wherein the phenotype is growth or
mortality of the pest.
42. The method of claim 41, further comprising determining the
phenotype by scoring the pest for mortality or stunted growth.
43. The method of claim 42, wherein more than half of the pests are
dead or have stunted growth indicates the polynucleotide of
interest is effective for controlling pest infestation or damage to
the root or combinations thereof.
44. The method of claim 29, comprising observing the stem area
above a crown, wherein the stem area darkens from pest damage as
compared to a control indicates that the root is not effective in
controlling pest infestation.
45. The method of claim 29, wherein the green tissue is obtained by
transforming an explant from a monocot and subjecting the explant
to green tissue initiation medium for a time and under conditions
sufficient to initiate growth from the explant, thereby producing
green tissue.
46. The method of claim 45, wherein the explant comprises an
embryo, green tissue, callus, leaf, meristem, seedling, seed, stem,
shoot, node, leaf base, or root.
47. The method of claim 29, further comprising regenerating
transgenic green tissue into one or more transgenic plantlets by
contacting the green tissue with a first rooting medium that
induces root formation for a time and under conditions sufficient
to initiate root growth from the green tissue, thereby producing a
plantlet.
48. The method of claim 29, further comprising producing a
plurality of transgenic plantlets from the green tissue.
49. The method of claim 29, further comprising producing a
plurality of roots from the green tissue.
50. The method of claim 29, further comprising placing the at least
one root of the plantlet in a culture dish.
51. The method of claim 29, wherein the plantlet has had its leaves
removed.
52. The method of claim 29, wherein the root is intact.
53. The method of claim 29, wherein the assay is completed within
about 4 to 14 days.
54. The method of claim 29, further comprising regenerating
transgenic plantlets from the green tissue from which a root has
been assayed for its phenotype.
55. A substantially contamination-free, root bioassay comprising: a
monocot plantlet, wherein the plantlet comprises at least one live
root in a culture dish, and wherein the dish comprises a rooting
medium in contact with the root.
56. The assay of claim 55, wherein the plantlet and root are
transgenic for a polynucleotide of interest.
57. The assay of claim 55, further comprising a plantlet having a
plurality of roots.
58. The assay of claim 55, further comprising a plurality of
plantlets obtained from the green tissue.
59. The assay of claim 55, wherein the rooting medium comprises a
biocide.
60. The assay of claim 55, wherein the assay comprises a means for
bringing the root into contact with the rooting medium.
61. The assay of claim 55, wherein the culture dish comprises a
substantially sterile absorbent material that contacts the rooting
medium and the roots.
62. The assay of claim 55, wherein the assay comprises a sterilized
pest, pathogen, or chemical.
63. The assay of claim 55, wherein the pest is from the order of
lepidoptera, homoptera, heteroptera, or coleoptera.
64. The assay of claim 55, wherein the pest is of a developmental
stage comprising an egg, larva, instar, or adult.
65. The assay of claim 62, wherein the chemical is a pesticide,
insecticide, fungicide, or bactericide.
66. The assay of claim 62, wherein the root is transgenic for a Bt
gene and wherein the pest is western corn root worm (WCRW).
67. A method of identifying a promoter having activity in the root
comprising: a) regenerating green tissue transgenic for a promoter
of interest operably linked to a polynucleotide into one or more
stably transformed transgenic plantlets, wherein the plantlets
comprise at least one transgenic root; and b) determining whether
the polynucleotide is expressed in root cells of the plantlet.
68. The method of claim 67, determining whether the polynucleotide
is expressed preferentially in root cells of the plantlet.
69. The method of claim 67, comprising determining whether the
polynucleotide is expressed preferentially in root cells of the
plantlet as compared to expression in cells of non-root tissues of
the plantlet; wherein increased expression of the polynucleotide in
root cells in comparison to expression of the polynucleotide in
non-root cells indicates that the promoter is preferentially
expressed in root cells.
70. The method of claim 67, wherein the at least one transgenic
root is isolated from the plantlet prior to determining the
expression of the polynucleotide.
71. The method of claim 67, further comprising producing a
transgenic plant from the green tissue.
72. The method of claim 67, further comprising growing the
transgenic plantlet into a transgenic plant
73. The method of claim 67, further comprising rooting the plantlet
on medium.
74. The method of claim 73, further comprising rooting the plantlet
on medium gelled with agar or an agar substitute.
75. The method of claim 67, wherein the plantlet is a monocot
plantlet.
76. The method of claim 67, wherein the green tissue is obtained by
transforming an explant from a monocot and subjecting the explant
to green tissue initiation medium for a time and under conditions
sufficient to initiate growth from the explant, thereby producing
green tissue.
77. The method of claim 76, wherein the explant comprises an
embryo, green tissue, callus, leaf, meristem, seedling, seed, stem,
shoot, node, leaf base, or root.
78. The method of claim 67, regenerating transgenic green tissue
into one or more transgenic plantlets by contacting the green
tissue with a rooting medium that induces root formation for a time
and under conditions sufficient to initiate root growth from the
green tissue, thereby producing a plantlet.
79. The method of claim 67, wherein the polynucleotide encodes a
marker polypeptide.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/291,704, filed Dec. 31, 2009, which is herein
incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The invention relates to the field of genetic manipulation
of plants; in particular, the invention provides assays and methods
for efficiently testing a polynucleotide of interest for a
phenotype in a plant tissue such as a plantlet, a root or a
leaf.
BACKGROUND OF THE INVENTION
[0003] The economic value of roots arise not only from harvested
roots, but also from the ability of roots to alter the soil in
which they grow and to funnel nutrients to support growth and
increase vegetative material, seeds, fruits, etc.
[0004] Roots have four main functions. First, they anchor the plant
in the soil. Second, they facilitate and regulate the molecular
signals and molecular traffic between the plant, soil and soil
fauna. Third, the root provides a plant with nutrients gained from
the soil or growth medium. Fourth, they condition local soil
chemical and physical properties. Roots arise from meristems cells
that are protected by a root cap during root elongation, but as the
root grows out, the cap cells abscise and the remaining cells
differentiate to the tip. Depending on the plant species, some
surface cells of roots can develop into root hairs. Some roots
persist for the life of the plant, others gradually shorten as the
ends slowly die back and some may cease to function altogether due
to external influences.
[0005] Because plants are sessile organisms, their survival is
critically dependent on rapid adaptation to environmental changes.
In the soil, change can arise from alteration of the concentration
of oxygen or carbon dioxide, nutrient availability, the presence
(or absence) of microorganisms and overall soil humidity. For
example, oxygen levels in the rhizosphere decrease rapidly during
flooding. Hypoxic or anoxic conditions occur in submerged plant
tissues and can have lasting effects on the subsequent growth
and/or development of the plant.
[0006] Roots are also the sites of intense chemical and biological
activities and as a result can strongly modify the soil they
contact. For example, roots secrete a wide variety of high and low
molecular weight molecules into the rhizosphere in response to
biotic and abiotic stresses. They are also capable of absorbing
toxic substances from the soil and then storing or modifying the
toxins, resulting in soil improvement.
[0007] Roots coat themselves with surfactants and mucilage to
facilitate these types of activities. Specifically, roots attract
and interact with beneficial microfauna and flora that help to
mitigate the effects of toxic chemicals, pathogens and stress in
addition to facilitating water and nutrient assimilation and
mobilization. Nutrients can take the form of ions and organic and
inorganic compounds. Uptake of nutrients by roots produces a
"source-sink" effect in a plant. The greater the source of
nutrients, the larger "sinks" (such as stems, leaves, flowers,
seeds, fruits, etc.) can grow.
[0008] Currently, transient gene expression has been applied to
dicot species using the hairy root system to do a quick gene
testing in roots, but establishing the hairy root system for maize
and delivering transgenes in roots using Agrobacterium rhizogenes
has been difficult. Generating transgenic maize plants with a
callus tissue system by standard protocols uses a whole cycle of
the transformation process which is a time-consuming process.
[0009] To date, there is only limited ability to efficiently and
quickly test genes and root promoters in vivo in a root, for
example, to assess the strength of a promoter in a root, to assess
a gene's effect on the tolerance of roots to pests that attack
roots (e.g., insects, fungi, bacteria, viruses, or nematodes) or to
assess a gene's effect on the nutritional composition of roots for
human food or animal feed applications. Thus a need exists for a
highly efficient way to test polynucleotides in the root of a plant
and generate plants expressing them in the root.
SUMMARY OF THE INVENTION
[0010] Compositions and methods are provided for efficiently
testing a polynucleotide of interest for a phenotype in a plant
tissue. While the invention is primarily discussed with respect to
the root, it is recognized that the leaf, the plantlet, or other
tissues may be used in the methods of the invention. More
specifically, the embodiments of the present invention relate to
assays and methods of regenerating green tissue into one or more
plantlets that have at least one root. In some examples, the green
tissue is transgenic for at least one polynucleotide of interest
and the green tissue is regenerated into one or more transgenic
plantlets having at least one transgenic root. The root, plantlet,
or leaf, non-transgenic or transgenic, may be optionally subjected
to a biotic stress, pest, or pathogen. The plant tissue may be
assayed for one or more phenotypes. Such root phenotypes include
but are not limited to increased root size, increased overall root
mass, altered root architecture, increased expression level of mRNA
or protein, increased biochemical content, increased tolerance or
resistance to a pest or pathogen, modulation in biotic mass of the
root, modulated yield, such as increased yield, as compared to the
corresponding phenotype of a control. Similar phenotypes can be
assessed for the leaf and plantlet.
[0011] Also provided herein are methods of making a root assay by
contacting green tissue with a first rooting medium to produce a
plantlet. The rooting medium may be a liquid, gel, or solid medium,
including, for example, a medium gelled with agar or an agar
substitute. The plantlet has at least one root that is removed from
the medium and is contacted with a second rooting medium to produce
a plurality of roots. In some embodiments, the rooting medium lacks
agar or an agar substitute
[0012] Additionally, a method of assaying for insecticidal activity
on a live root is provided herein. The method includes regenerating
green tissue into one or more plantlets comprising at least one
live root. In some embodiments, the green tissue is transgenic for
a polynucleotide of interest. The at least one root of the plantlet
is contacted with a rooting medium. The root is exposed to one or
more pests to infest the medium for infestation. In some
embodiments, the medium and pest are substantially free of
contamination. A phenotype of the root and/or pest is
determined.
[0013] Accordingly, one of the embodiments includes a substantially
contamination-free, root bioassay. The bioassay includes a live
monocot plantlet with at least one live root. In some examples, the
plantlet has at least one live root that is transgenic for a
polynucleotide of interest. The root is placed in culture dish. The
dish includes a rooting medium that contacts the root of the
plantlet.
[0014] Methods of identifying a promoter having activity in plant
tissue, particularly the root, are also provided. The methods
relate to regenerating green tissue transgenic for a promoter of
interest operably linked to a polynucleotide into one or more
stably transformed transgenic plantlets. The plantlets have at
least one live transgenic root. Further encompassed by the methods
is determining whether the polynucleotide is expressed in root
cells of the plantlet. The relative strength of a promoter in a
root cell, the spatial expression of a promoter in the root, or
whether the promoter is a root-preferred promoter may also be
evaluated if desired. The methods may include determining the
expression level of the polynucleotide and/or polypeptide encoded
by the polynucleotide in root cells of the plantlet.
[0015] Other objects, features, advantages and aspects of the
present invention will become apparent to those of skill from the
following description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 shows a flow chart demonstrating an efficient
screening scheme using in vitro bioassay plantlets.
DETAILED DESCRIPTION OF THE INVENTION
[0017] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Unless
mentioned otherwise, the techniques employed or contemplated herein
are standard methodologies well known to one of ordinary skill in
the art. The materials, methods and examples are illustrative only
and not limiting. The following is presented by way of illustration
and is not intended to limit the scope of the invention.
[0018] Indeed, the invention may be embodied in many different
forms and should not be construed as limited to the embodiments set
forth herein; rather, these embodiments are provided so that this
disclosure will satisfy applicable legal requirements.
[0019] Many modifications and other embodiments of the invention
set forth herein will come to mind to one skilled in the art to
which these inventions pertain having the benefit of the teachings
presented in the foregoing descriptions. Therefore, it is to be
understood that the invention are not to be limited to the specific
embodiments disclosed and that modifications and other embodiments
are intended to be included within the scope of the appended
claims. Although specific terms are employed herein, they are used
in a generic and descriptive sense only and not for purposes of
limitation. The articles "a" and "an" are used herein to refer to
one or more than one (i.e., to at least one) of the grammatical
object of the article. By way of example, "an element" means one or
more than one element.
[0020] As used herein, the term "transgenic" means a plant or plant
cell or plant part (e.g., a plant tissue or a plant organ) that
comprises genetic material additional to the naturally occurring
nucleic acid within the plant, cell or part. For example, the
genome of a transgenic plant or plant cell or plant part may
comprise nucleic acid from a different organism such as an animal,
insect, bacterium, fungus or different plant species or variety.
Alternatively, the genome of a transgenic plant or plant cell or
plant part may comprise one or more additional copies of nucleic
acid that occur naturally in the same plant species or variety.
Alternatively, the genome of a transgenic plant or plant cell or
plant part may comprise nucleic acid that does not occur in nature
e.g., RNAi. The genome of a transgenic plant or plant cell or plant
part may also contain a deletion relative to the genome of an
isogenic or near-isogenic naturally-occurring plant e.g., as a
result of homologous recombination or recombinase-induced
recombination.
[0021] As used herein, the term "green tissue" refers to green
regenerative tissue or green callus tissue which is green, shiny,
nodular and compact as compared to monocot plant callus tissue.
Green tissues are organogenic and have meristem-like structures.
See U.S. Pat. No. 7,102,056, incorporated by reference in its
entirety.
[0022] The term "root-preferred" is intended to mean that
expression of the heterologous polynucleotide sequence is most
abundant in the root. While some level of expression of the
heterologous nucleotide sequence may occur in other plant tissue
types, expression occurs most abundantly in a cell of the root or
in a type of root, which may include, but is not limited to
primary, lateral, and adventitious roots.
[0023] The term "root" is intended to mean any part of the root
structure, including but not limited to, the root cap, apical
meristem, protoderm, ground meristem, procambium, endodermis,
cortex, vascular cortex, epidermis, and the like.
[0024] "Promoter" refers to a nucleotide sequence capable of
controlling the expression of a coding sequence or functional RNA.
In general, a coding sequence is located 3' to a promoter sequence.
The promoter sequence consists of proximal and more distal upstream
elements, the latter elements often referred to as enhancers.
[0025] Accordingly, an "enhancer" is a nucleotide sequence which
can stimulate promoter activity and may be an innate element of the
promoter or a heterologous element inserted to enhance the level or
tissue-specificity of a promoter. Promoters may be derived in their
entirety from a native gene, or be composed of different elements
derived from different promoters found in nature, or even comprise
synthetic nucleotide segments. It is understood by those skilled in
the art that different promoters may direct the expression of a
gene in different tissues or cell types, or at different stages of
development, or in response to different environmental conditions.
Promoters which cause a nucleic acid fragment to be expressed in
most cell types at most times are commonly referred to as
"constitutive promoters".
[0026] The term "dim light" refers to light that is approximately 5
to 50 .mu.E m.sup.-2s.sup.-1.
[0027] The term "operably linked" refers to the association of two
or more nucleic acid fragments on a single nucleic acid fragment so
that the function of one is affected by the other. For example, a
promoter is operably linked with a coding sequence when it is
capable of affecting the expression of that coding sequence (i.e.,
that the coding sequence is under the transcriptional control of
the promoter). Coding sequences can be operably linked to
regulatory sequences in sense or antisense orientation.
[0028] The term "expression", as used herein, refers to the
transcription and stable accumulation of sense (mRNA) or antisense
RNA derived from the nucleic acid fragment of the invention.
Expression may also refer to translation of mRNA into a
polypeptide. "Antisense inhibition" refers to the production of
antisense RNA transcripts capable of suppressing the expression of
the target protein. "Overexpression" refers to the production of a
gene product in transgenic organisms that exceeds levels of
production in normal or non-transformed organisms. "Co-suppression"
refers to the production of sense RNA transcripts capable of
suppressing the expression of identical or substantially similar
foreign or endogenous genes (U.S. Pat. No. 5,231,020, incorporated
herein by reference).
[0029] "Altered levels" refers to the production of gene product(s)
in transgenic organisms in amounts or proportions that differ from
that of normal or non-transformed organisms.
[0030] "Transformation" refers to the transfer of a nucleic acid
fragment into the genome of a host organism, resulting in
genetically stable inheritance. Host organisms containing the
transformed nucleic acid fragments are referred to as "transgenic"
organisms. Examples of methods of plant transformation include
Agrobacterium-mediated transformation (De Blaere et al. (1987)
Meth. Enzymol. 143:277) and particle-accelerated or "gene gun"
transformation technology (Klein et al. (1987) Nature (London)
327:70-73; U.S. Pat. No. 4,945,050, incorporated herein by
reference).
[0031] Previously a whole cycle of the transformation process was
used to generate transgenic maize plants with a callus tissue
system, but this is a time-consuming process. Transient gene
expression has been applied to dicot species using the hairy root
system to do a quick gene testing in roots, but establishing the
hairy root system for maize and expressing transgenes using
Agrobacterium rhizogenes have been difficult. The current invention
utilizes a highly regenerative tissue system which can produce
transgenic organogenic tissues ready for shoot regeneration and
root formation. For example, using a visible marker, transgenic
sectors can be easily identified under a fluorescence microscope
and used for fast and continuous root production as well as shoot
regeneration from transgenic green tissues by placing them directly
on the rooting medium. Green tissues are more organogenic than
callus tissues, and advantageously these green tissues can be
maintained for long periods with a minimal loss of regenerability.
This is in contrast to the rapid loss of regenerability that occurs
when using a standard callus tissue system. A further advantage
from practicing the methods and bioassays described herein is that,
because green tissue is used, multiple plants can be produced from
the same transgenic event during an extended time period.
[0032] Accordingly, provided herein are methods and assays for
efficiently identifying the affects of expression of one or more
polynucleotides of interest on plant tissues, particularly on the
roots, transgenic for the one or more polynucleotides. The plant
tissues are then analyzed for expression of the polynucleotides.
Such affects or phenotypes for roots include, but are not limited
to, modulated root size, overall root mass, root architecture,
expression level of mRNA or protein, biochemical content of the
root, tolerance to a biotic stress, tolerance or resistance to a
pest, tolerance or resistance to a pathogen, yield, agronomic
traits, increased disease resistance, nutritional enhancement, and
the like. Also provided herein are methods and assays for
efficiently determining whether a live plantlet or root has
endogenous resistance or susceptibility to a biotic stress, such as
a pest or pathogen. This would be of interest when screening
germplasm using non-transgenic germinating plantlets/roots. In one
example, polynucleotides effective for preventing corn root worm
infestation or damage to roots associated with corn root worms may
be identified using the provided methods and assays. In another
aspect, the provided methods and assays may be used to determine
whether a promoter is functional in a root cell, the relative
strength of a promoter in a root cell, the spatial expression of a
promoter in the root, or whether the promoter is a root-preferred
promoter. In addition, the methods and assays described herein can
be applied for rapid production of non-transgenic monocot plants or
transgenic monocot plants. Efficient regeneration of plants would
facilitate the study of plants with improved traits or
phenotypes.
[0033] In one aspect, the methods include regenerating green tissue
into one or more plantlets having at least one root. In some
examples, the green tissue is transgenic for at least one
polynucleotide of interest and gives rise to a transgenic plantlet
having at least one transgenic root. In other applications, the
green tissue can be used to produce root cultures, for example,
transgenic root cultures. The polynucleotide of interest may be any
suitable polynucleotide and may be either endogenous or
heterologous to the plant cell being transformed. Polynucleotides
encompass all forms of nucleic acid sequences including, but not
limited to, single-stranded, double-stranded, triplexes, linear,
circular, branched, hairpins, stem-loop structures, branched
structures, and the like. In some instances, the polynucleotide of
interest may encode a polypeptide of interest which is expressed in
the cell. The polynucleotide of interest may confer a particular
trait of interest to the plant, for example, such as, but not
limited to disease resistant traits, insect resistant traits,
nutritional enhancements, agronomic traits, firmness, acidity
content, sugar content, texture, oil, starch, carbohydrate, or
nutrient metabolism, increased oil production, increased protein
production, unique oil and protein production, increased
fermentable starch production, increased content of essential amino
acids, increased content of fatty acids and the like. The
polynucleotide of interest may be thioredoxin (Cho et al. 1999,
Proc Natl Acad Sci USA 96: 14641-14646), lactoferrin, or lysozyme
(Humphrey et al. 2002, J of Nutrition 32(6): 1214-1218). In one
example, the polynucleotide of interest is a selectable or
screenable marker gene. Exemplary marker genes are described
elsewhere herein. In some instances, the polynucleotide of interest
may suppress the expression of a target molecule in the plant cell,
for example, Ca.sup.2+-dependent protein kinase1 (CDPK1), Plant
Cell 17:2911-2921 (2005); Arabidopsis Ran binding protein,
AtRanBP1c, Plant Cell, 13: 2619-2630 (2001). The inhibitory
polynucleotide may any suitable polynucleotide including but not
limited to miRNA, a siRNA, dsRNA, an antisense polynucleotide and
the like.
[0034] In one embodiment, recombinant vectors including one or more
polynucleotides of interest suitable for the transformation of
plant cells are prepared. These may be used to construct a
recombinant expression cassette which can be introduced into the
desired plant cell. In one example, an expression cassette will
typically comprise a polynucleotide of interest operably linked to
a promoter sequence and other transcriptional and translational
initiation regulatory sequences which are sufficient to direct the
transcription of the polynucleotide sequence in the intended
tissues (e.g., entire plant, leaves, roots, etc.).
[0035] A number of promoters can be used in the practice of the
present invention. The promoters can be selected based on the
desired outcome. That is, the nucleic acids can be combined with
constitutive, inducible, tissue-preferred, root-preferred promoters
or other promoters for expression in the explant, green tissue,
root, or regenerated plant.
[0036] Constitutive promoters include, for example, the core
promoter of the Rsyn7 promoter and other constitutive promoters
disclosed in WO 99/43838 and U.S. Pat. No. 6,072,050; the core CaMV
35S promoter (Odell et al. (1985) Nature 313:810-812); rice actin
(McElroy et al. (1990) Plant Cell 2:163-171); ubiquitin
(Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and
Christensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last
et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten et al.
(1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No.
5,659,026), and the like. Other constitutive promoters include, for
example, those disclosed in U.S. Pat. Nos. 5,608,149; 5,608,144;
5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142;
and 6,177,611.
[0037] Chemical-regulated promoters can be used to modulate the
expression of a gene in a plant through the application of an
exogenous chemical regulator. Depending upon the objective, the
promoter may be a chemical-inducible promoter, where application of
the chemical induces gene expression, or a chemical-repressible
promoter, where application of the chemical represses gene
expression. Chemical-inducible promoters are known in the art and
include, but are not limited to, the maize 1n2-2 promoter, which is
activated by benzene sulfonamide herbicide safeners; the maize GST
promoter, which is activated by hydrophobic electrophilic compounds
that are used as pre-emergent herbicides; and the tobacco PR-1a
promoter, which is activated by salicylic acid. Other
chemical-regulated promoters of interest include steroid-responsive
promoters. See, for example, the glucocorticoid-inducible promoter
in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425
and McNellis et al. (1998) Plant J. 14(2):247-257 and the
tetracycline-inducible and tetracycline-repressible promoters for
example, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S.
Pat. Nos. 5,814,618 and 5,789,156, herein incorporated by
reference.
[0038] Root-preferred promoters are known and can be selected from
the many available from the literature or isolated de novo from
various compatible species. See, for example, Hire et al. (1992)
Plant Mol. Biol. 20(2):207-218 (soybean root-specific glutamine
synthetase gene); Keller et al. (1991) Plant Cell 3(10):1051-1061
(root-specific control element in the GRP 1.8 gene of French bean);
Sanger et al. (1990) Plant Mol. Biol. 14(3):433-443 (root-specific
promoter of the mannopine synthase (MAS) gene of Agrobacterium
tumefaciens); and Miao et al. (1991) Plant Cell 3(1): 11-22
(full-length cDNA clone encoding cytosolic glutamine synthetase
(GS), which is expressed in roots and root nodules of soybean). See
also Bogusz et al. (1990) Plant Cell 2(7):633-641, which discloses
two root-specific promoters isolated from hemoglobin genes from the
nitrogen-fixing nonlegume Parasponia andersonii and the related
non-nitrogen-fixing nonlegume Trema tomentosa. The promoters of
these genes were linked to a beta-glucuronidase reporter gene and
introduced into both the nonlegume Nicotiana tabacum and the legume
Lotus corniculatus, and in both instances root-specific promoter
activity was preserved. Leach et al. (1991) describe their analysis
of the promoters of the highly expressed rolC and rolD
root-inducing genes of Agrobacterium rhizogenes (see Plant Science
(Limerick) 79(1):69-76). They concluded that enhancer and
tissue-preferred DNA determinants are dissociated in those
promoters. Teeri et al. (1989) EMBO J. 8(2):343-350 used gene
fusion to lacZ to show that the Agrobacterium T-DNA gene encoding
octopine synthase is especially active in the epidermis of the root
tip and that the TR2' gene is root specific in the intact plant and
stimulated by wounding in leaf tissue, which is an especially
desirable combination of characteristics for use with an
insecticidal or larvicidal gene. The TR1' gene, fused to nptII
(neomycin phosphotransferase II), showed similar characteristics.
Additional root-preferred promoters include the VfENOD-GRP3 gene
promoter (Kuster et al. (1995) Plant Mol. Biol. 29(4):759-772); the
ZRP2 promoter (U.S. Pat. No. 5,633,636); the IFS1 promoter (U.S.
patent application Ser. No. 10/104,706) and the rolB promoter
(Capana et al. (1994) Plant Mol. Biol. 25(4):681-691). See also
U.S. Pat. Nos. 5,837,876; 5,750,386; 5,459,252; 5,401,836;
5,110,732; and 5,023,179.
[0039] A strongly or weakly constitutive plant promoter that
directs expression of a polynucleotide of interest nucleic acid in
all tissues of a plant can be employed. Such promoters are active
under most environmental conditions and states of development or
cell differentiation. In addition to the promoters mentioned above
examples of constitutive promoters include the 1'- or 2'-promoter
of Agrobacterium tumefaciens, and other transcription initiation
regions from various plant genes known to those of skill. Where
over expression of a polypeptide of interest is detrimental to the
plant, one of skill will recognize that weak constitutive promoters
can be used for low-levels of expression. Generally, by "weak
promoter" a promoter that drives expression of a coding sequence at
a low level is intended. By "low level" levels from about 1/1000
transcripts to about 1/100,000 transcripts, to about as low as
1/500,000 transcripts per cell are intended. Alternatively, it is
recognized that weak promoters also include promoters that are
expressed in only a few cells and not in others to give a total low
level of expression. Where a promoter is expressed at unacceptably
high levels, portions of the promoter sequence can be deleted or
modified to decrease expression levels. In those cases where high
levels of expression is not harmful to the plant, a strong
promoter, e.g., a t-RNA, or other pol III promoter, or a strong pol
II promoter, e.g., the cauliflower mosaic virus promoter, CaMV, 35S
promoter can be used.
[0040] Alternatively, a plant promoter can be under environmental
control. Such promoters are referred to as "inducible" promoters.
Examples of environmental conditions that may alter transcription
by inducible promoters include pathogen attack, anaerobic
conditions, or the presence of light. In some cases, it is
desirable to use promoters that are "tissue-specific" and/or are
under developmental control such that the polynucleotide of
interest is expressed only in certain tissues or stages of
development, e.g., leaves, roots, shoots, etc. Promoters of genes
related to pesticide resistance and related phenotypes may also be
used.
[0041] Tissue specific promoters can also be used to direct
expression of heterologous structural genes, including
polynucleotides of interest. Thus, the promoters can be used in
recombinant expression cassettes to drive expression of any gene
whose expression is desirable in the transgenic plantlets.
Similarly, enhancer elements, e.g., derived from the 5' regulatory
sequences or intron of a heterologous gene, can also be used to
improve expression of a heterologous structural gene.
[0042] In general, the particular promoter used in the expression
cassette in plants depends on the intended application. Any of a
number of promoters which direct transcription in plant cells can
be suitable. In addition to the promoters noted above, promoters of
bacterial origin which operate in plants include the octopine
synthase promoter, the nopaline synthase promoter and other
promoters derived from T1 plasmids. See, Herrera-Estrella et al.
(1983) Nature 303:209. Viral promoters include the .sup.35S and 19S
RNA promoters of CaMV. See, Odell et al. (1985) Nature 313:810.
Other plant promoters include the ribulose-1,3-bisphospha- the
carboxylase small subunit promoter and the phaseolin promoter. The
promoter sequence from the E8 gene (see, Deikman and Fischer (1988)
EMBO J. 7:3315) and other genes are also favorably used. Promoters
specific for monocotyledonous species are also considered (McElroy
and Brettell (1994) "Foreign gene expression in transgenic cereals"
Trends Biotech. 12:62-68.) Alternatively, novel promoters with
useful characteristics can be identified from any viral, bacterial,
or plant source by methods, including sequence analysis, enhancer
or promoter trapping, and the like, known in the art.
[0043] In preparing expression vectors, sequences other than the
native promoter of the polynucleotide of interest may also be used.
If proper polypeptide expression is desired, a polyadenylation
region can be derived from the native gene, from a variety of other
plant genes, or from T-DNA. Signal/localization peptides, which,
e.g., facilitate translocation of the expressed polypeptide to
internal organelles (e.g., chloroplasts) or extracellular
secretion, can also be employed.
[0044] The vector can include a selectable or screenable marker
gene as, or in addition to, a particular polynucleotide of interest
to provide or enhance the ability to identify transformants by
conferring a selectable phenotype on the transformed plant cells.
"Marker genes" are genes that impart a distinct phenotype to cells
expressing the marker gene and thus allow such transformed cells to
be distinguished from cells that do not have the marker. Such genes
may encode either a selectable or screenable marker, depending on
whether the marker confers a trait which one can "select" for by
chemical means, i.e., through the use of a selective agent (e.g., a
herbicide, antibiotic, or the like), or whether it is simply a
trait that one can identify through observation or testing, i.e.,
by "screening", e.g., bar, pat, GAT, PMI, hpt, nptII, DS-RED, GFP,
YFP, GUS. Of course, many examples of suitable marker genes are
known to the art and can be employed in the methods and assays.
Marker genes may also be used to monitor gene expression and
protein localization in plant cells, such as root cells via
visualizable reaction products or by direct visualization of the
gene product itself. Accordingly, many selectable marker coding
regions may be used in connection with a promoter. Examples of
selectable markers include nptII. (Potrykus et al., 1985), which
provides kanamycin resistance and can be selected for using
kanamycin, G418, paromomycin, etc.; bar, which confers bialaphos or
phosphinothricin resistance; a nitrilase such as bxn from
Klebsiella ozaenae which confers resistance to bromoxynil (Stalker
et al., 1988) and a mutant acetolactate synthase (ALS) which
confers resistance to imidazolinone, sulfonylurea or other ALS
inhibiting chemicals (European Patent Application 154,204, 1985)
and a methotrexate resistant DHFR (Thillet et al., 1988). Such
vectors also generally include one or more dominant selectable
marker genes, including genes encoding antibiotic resistance (e.g.,
resistance to hygromycin, kanamycin, bleomycin, G418, streptomycin,
paromomycin, or spectinomycin) and herbicide-resistance genes
(e.g., resistance to phosphinothricin acetyltransferase or
glyphosate) to facilitate manipulation in bacterial systems and to
select for transformed plant cells.
[0045] A number of techniques and protocols may be used to produce
green tissue. For example, when it is desired that green tissue be
transgenic for a polynucleotide of interest, the green tissue
itself may be transformed using conventional methods, for example,
particle bombardment or Agrobacterium. See Example 1. Alternately,
green tissue can be made transgenic for a polynucleotide of
interest by transforming an explant that can give rise to green
tissue when the explant is cultured for a time and under conditions
sufficient for the initiation and growth of green tissue to occur.
As described, green tissue induction is carried out under dim
light. The length of exposure of the plant cells to dim conditions
may vary based in part on the type of plant species and genotype
being transformed.
[0046] Any suitable explant that can give rise to green tissue may
be used in the methods described herein. The explant can be from a
monocot. It will be understood by one skilled in the art that the
explant may comprise a plant cell, a tissue or an organ. Exemplary
explants for use with the methods include but are not limited to
embryos, green tissue, callus such as Type I or II, cell
suspensions, cotyledons, including scutella, meristems, seedlings,
mature and immature seeds, leaves, stems, shoots, scutella, nodes,
leaf bases, or roots. See U.S. patent application publication no.
20080280361, U.S. Pat. Nos. 5,569,834; 5,416,011; 5,824,877;
7,064,248. When the explant is an embryo from a maize plant, the
method may include pollinating ears from the treated maize plant,
harvesting the ears so that the ears or embryos may be prepared for
transformation. See, for example, Green and Phillips (Crop Sci.
15:417-421, 1976). Maize immature embryos can be isolated from
pollinated plants, as another example, using the methods of Neuffer
et al. ("Growing Maize for genetic purposes." In: Maize for
Biological Research W. F. Sheridan, Ed., University Press,
University of North Dakota, Grand Forks, N. Dak. 1982.). The
explant may be prepared using any suitable technique and may
include, for example, isolating the explant from the plant,
excising plant cell, tissue, or organ from the explant, sterilizing
the plant cell, tissue, organ, or explant or combinations thereof.
In some cases, the explant is an embryo, such as an immature embryo
from a monocot such as corn. In one example, the methods include
transforming one or more immature embryos from the monocot using
conventional methods such as Agrobacterium-mediated transformation
or particle bombardment. See Example 1. As described, green tissue
induction is carried out under dim light for a length of time
sufficient for the initiation and growth of green tissue to occur.
The length of exposure of the plant cells to dim conditions may
vary based in part on the type of plant species and genotype being
transformed.
[0047] In cases where the explant is other than green tissue, the
explant can be used to generate green tissue using commonly known
techniques. For example, green tissue can be obtained by culturing
immature embryos under appropriate conditions to initiate the
formation of green tissue. See, for example, U.S. Pat. Nos.
6,541,257, 6,235,529, 7,102,056. When Agrobacterium is used to
transform cells of the explant to generate transgenic green tissue,
typically the explant such as immature embryos are co-cultivated
for about 1-3 days in the dark and rested for an additional 1-3
days in resting medium, typically without selection. The explant is
contacted with green tissue induction medium under dim light to
produce green tissue. The green tissue induction medium may
optionally contain a selective agent, e.g. bialaphos and
carbinicellin, when producing transgenic green tissue. Usually, the
green tissue-induction media used in the methods contains different
combinations of an auxin, cytokinin, and copper in amounts
effective to initiate the formation of green tissue. In one example
of green tissue-induction medium the auxin is
2,4-dichlorophenoxyacetic acid (2,4-D), the cytokinin is
6-benzylaminopurine (BAP) and copper is CuSO.sub.4. This culturing
step usually takes about 2-3 weeks, preferably at about 24.degree.
C.-28.degree. C. under dim light.
[0048] In some circumstances, it may be desirable to break the
green tissue into one or more pieces to facilitate proliferation
and more stringent selection. The method may also include
subculturing the broken pieces of green tissue in the presence of
the selection agent for about 2-3 weeks. About 3 to 5 rounds of
subculturing with a selective agent is typically considered
sufficient to select for transformed tissue.
[0049] Transgenic green tissue can also be obtained by bombarding
immature embryos and culturing them under appropriate conditions to
initiate the formation of green tissue. Immature embryos are
isolated using any suitable technique and placed scutellum-side up
in an osmotic medium. The embryos are bombarded with solid
particles, such as gold particles, coated with the polynucleotide
of interest. The embryos are contacted with green tissue induction
medium that typically lacks a selective agent and cultured under
dim light for about 3-7 days, usually at about 24.degree.
C.-28.degree. C. to produce transgenic green tissue. In some
circumstances, it may be desirable to break the green tissue into
one or more pieces to facilitate proliferation and more stringent
selection. The method may also include subculturing the broken
pieces of green tissue in the presence of the selection agent for
about 2-3 weeks. About 3 to 5 rounds of subculturing with a
selective agent is typically considered sufficient to select for
transformed tissue. Optionally, the putative transgenic tissues are
maintained and proliferated on green tissue induction or
maintenance medium containing a selective agent. Once a sufficient
amount of green tissues are obtained, the green tissue may be
plated on solid regeneration/rooting medium optionally containing a
selective agent and exposed to a higher light intensity,
approximately 45 to 100 .mu.E m.sup.-2s.sup.-1, on a 16-h light
cycle. After about 4 to 6 weeks, regenerated plantlets may be
transferred to soil.
[0050] When green tissues are used as targets for bombardment, the
green tissue may be pretreated with an osmotic solution. See
Example 1. After about 4 hours, the green tissue is bombarded using
any suitable particle. One transformed, transgenic green tissues
are selected and cultured in a similar manner as that used for
green tissue obtained by particle bombardment of immature embryos.
See Example 1.
[0051] Transgenic regions of the green tissue obtained by any
method may be confirmed or identified using any suitable gene such
as a maker gene. For convenience, visible marker genes such as RFP,
GFP, EGFP, lucieferase or YFP are normally utilized to identify
transgenic regions in the green tissue using standard techniques
and instruments such as a fluorescence microscope. The transgenic
regions of the green tissue may be isolated by cutting the
transgenic regions from the non-transgenic regions of the green
tissue. The transgenic regions are typically cut into several
pieces and placed on maintenance medium for further proliferation.
In some examples, the non-transgenic green tissue may be cut into
several pieces and placed on maintenance medium for further
proliferation. In some cases, the maintenance medium and green
tissue initiation medium are the same.
[0052] The green tissues are transferred directly onto rooting
medium so that one or more roots are produced. Any suitable rooting
medium may be used, including but not limited to phytohormone-free
medium. For example, MS basal medium supplemented with IBA (e.g.,
0.5 mg/L) can be used to induce root formation, if necessary.
Depending upon the genotype, different levels of an auxin and
cytokinin (i.e., a different auxin/cytokinin ratio) provide optimal
results. The medium may be of any suitable form such as solid,
liquid or gel, for example, medium gelled with agar or an agar
substitute gelling agent such as PHYTAGEL.TM. (Sigma-Aldrich, St.
Louis, Mo., USA). Normally, when the green tissue is transgenic the
rooting medium contains a selective agent or one is added to the
medium. The green tissue is contacted with rooting medium that
induces root formation for a time and under conditions sufficient
to initiate root growth from the green tissue, thereby producing a
plantlet.
[0053] If desired, the green tissue may be incubated on
regeneration medium prior to placing tissues on rooting medium. Any
suitable regeneration medium can be used including without
limitation to phytohormone-free medium and others. One skilled in
the art will be familiar with such media. Exposing the green tissue
to regeneration medium can facilitate more efficient root
production. The length of incubation is often for a short period of
time such as 1-3 weeks.
[0054] In one embodiment, the methods of the invention may use
plant tissues selected from, but not limited to, whole plantlets,
plantlet parts, plantlet leaves or plantlet roots.
[0055] The following discussion is directed to assay of roots but
can be adapted for the assay of other plant tissues including
plantlets and leaves. Roots can be produced with shoot regeneration
or without any shoot regeneration. If shoot regeneration is
desired, the green tissue is contacted with shoot regeneration
medium for a sufficient length of time and under conditions to
generate shoots. In some instances, the green tissue may be
contacted with shoot regeneration medium prior to, concurrent with,
or subsequent to contacting the green tissue with rooting medium in
order to produce a plantlet.
[0056] Plantlets having one or more roots may be removed from the
solidified agar medium and the agar rinsed off the roots. In one
aspect, the roots are placed onto suitable assay dishes or
containers such as a culture dish, e.g. Phyta trays or Petri
dishes.
[0057] When doing so, it may prove advantageous to continue to keep
the roots growing and alive to more closely mimic real life
infestations, infections or stresses of plants. Accordingly, the
roots are placed in a rooting medium such as MSA and MSB in the
culture dish so that root production is continuous. Typically, MSA
includes MS salts and vitamins, 2% sucrose, 0.35% Phytagel and 3
mg/L bialaphos and MSB includes MS salts and vitamins, 2% sucrose,
0.25% Phytagel, 0.5 mg/L IBA and 3 mg/L bialaphos. Additional
exemplary rooting media are described above and in the Examples
herein. In some examples, the rooting medium lacks agar or an
agar-substitute.
[0058] In another aspect, steps are taken to prevent or inhibit the
growth of unwanted contaminants e.g. microbes, such as bacteria,
mold, or fungi in the assay. For example, a biocide such as Plant
Preservative Mixture (PPM-0.1350%
5-chloro-2-methyl-3(2H)-isothiazolone, 0.0412%
2-methyl-3(2H)-isothiasolone, 99.8238% inert ingredients, Plant
Cell Biotechnology, Inc., Washington, D.C.) may be added to the
rooting medium to prevent or inhibit fungal and bacterial
development. Additionally, the dish may be sterile or substantially
sterile. Further, the use of sterile filter papers rather than agar
in the dish can be used to facilitate the transfer of rooting
solution to plant roots and reduce fungal and bacterial
development. Any means, technique or object, such as filter paper,
that facilitates the contact between the rooting medium and roots
may be used so long as it does not break off or kill the roots.
Contact between the medium and the roots may be facilitated by
placing an object on top of the roots to force the roots downward
into the dish. The object may be a screen or grid.
[0059] Use of the methods and assays described herein serve as an
efficient means for testing endogenous genes or a polynucleotide of
interest for various phenotypes in non-transgenic roots or roots
transgenic for the polynucleotide respectively. As described
elsewhere herein, the polynucleotide may be any suitable
polynucleotide. Polynucleotides suitable for use in the methods and
assays described herein may be either endogenous or heterologous to
the plant cell of the explant to be transformed. The polynucleotide
may be RNA, DNA or both. Polynucleotides encompass all forms of
nucleic acid sequences including, but not limited to,
single-stranded, double-stranded, triplexes, linear, circular,
branched, hairpins, stem-loop structures, branched structures, and
the like. The polynucleotide may be a ds RNA molecule, such as a
dsRNA molecule that upon consumption by a pest decreases pest
infestation. See U.S. Publication No. 20060021087. In some
instances, the polynucleotide of interest may encode a polypeptide
of interest which is already expressed in the native root cell. The
polynucleotide of interest may confer or modulate one or more
particular phenotypes of interest to the root, for example, such
as, but not limited to increased root size, increased overall root
mass, altered root architecture, increased expression level of mRNA
or protein, increased biochemical content, increased tolerance to
stress, increased tolerance or resistance to a pest, increased
tolerance or resistance to a pathogen, increased yield desirable
agronomic traits, increased disease resistance, nutritional
enhancements, and the like. As will be appreciated by one skilled
in the art, there may be overlap or correlations between the
observed phenotypes.
[0060] Exemplary promoters to drive expression of the
polynucleotide of interest include without limitation constitutive,
inducible, or root-preferred promoters and are described elsewhere
herein and can be selected from the many available from the
literature. Known or novel promoters may be tested for
functionality in a root cell, the relative strength of the promoter
in a root cell, the spatial expression of the promoter in the root,
or whether the promoter is a root-preferred promoter using the
methods and assays described herein.
[0061] The plant tissue is evaluated for expression levels of
endogenous polynucleotides of interest or heterologous
polynucleotides of interest. Expression at the RNA level can be
determined to identify and/or quantitate expression of a
polynucleotide of interest. Standard techniques for RNA analysis
can be employed and include PCR amplification assays using
oligonucleotides primers designed to amplify only the heterologous
RNA templates and solution hybridization assays using heterologous
nucleic acid-specific probes. The transgenic tissue may be
evaluated for the polynucleotide of interest's impact on resistance
to diseases, pests, pathogens, stresses, nutrients, or chemicals
and the like. The chemical may be a pesticide or bactericide and
the like.
[0062] Roots may be evaluated for expression levels of endogenous
polynucleotides of interest or heterologous polynucleotides of
interest, for example, in a transgenic root. Expression in the
roots at the RNA level can be determined to identify and/or
quantitative expression for a polynucleotide of interest. When the
polynucleotide of interest is a root-preferred promoter, as will be
understood by one skilled in the art, the transgenic root may be
evaluated for expression of the polynucleotide operably linked to
the root-preferred promoter. Standard techniques for RNA analysis
can be employed and include PCR amplification assays using
oligonucleotides primers designed to amplify only the heterologous
RNA templates and solution hybridization assays using heterologous
nucleic acid-specific probes. If desired, the roots can be analyzed
for protein expression by fluorescent microscopy, FACS, or Western
immunoblot analysis using the specifically reactive cognate
antibodies. In addition, in situ hybridization and
immunocytochemistry according to standard protocols can be done
using heterologous nucleic acid specific polynucleotide probes and
antibodies, respectively, to localize sites of expression within
transgenic root tissue. Generally, a number of transgenic roots are
usually screened for the polynucleotide of interest to identify and
select plantlets with the most appropriate expression profiles, for
example, in some examples, those that comparatively express the
polynucleotide at the highest level or as compared to a control
null for the polynucleotide of interest. In some cases, it may be
desirable to have low levels of expression of an endogenous gene in
the root.
[0063] Roots, such as roots transgenic for the polynucleotide of
interest, may be evaluated for root size. Root size includes but is
not limited to root biomass, root strength, root thickness, the
formation of aerial roots, the number of aerial roots, length of
roots, and the number of lateral and/or adventitious roots and the
like and combinations thereof as compared as to a control. See U.S.
Pat. No. 7,259,296. In one aspect, expression of the polynucleotide
of interest increases root biomass, produces thicker roots,
produces stronger roots, increases the formation of aerial roots,
increases the number of aerial roots, increases the length of
roots, and increases the number of lateral and/or adventitious
roots or combinations thereof.
[0064] In another aspect, the roots may be evaluated for the
endogenous or heterologous polynucleotide of interest's impact on
root architecture. Aspects of root architecture that may be
evaluated include without limitation root depth, root angle, root
branching, number of root tips, nodal root diameter, nodal root
volume, and root metabolic activity and the like or combinations
thereof. See U.S. Pat. No. 7,557,266. One skilled in the art will
be familiar with techniques for determining such aspects.
[0065] Expression of the endogenous or heterologous polynucleotide
of interest may also affect the biochemical content of the root.
See, for example, J. Exp. Bot. (2003) 54: 203-211 describing the
effect of pmt gene overexpression on tropane alkaloid production in
transformed root cultures of Datura metel and Hyoscyamus
muticus.
[0066] The transgenic roots may be evaluated for the polynucleotide
of interest's impact on resistance to diseases, pests, pathogens,
stresses, nutrients, or chemicals and the like. The chemical may be
a pesticide or bactericide and the like.
[0067] Accordingly, the embodiments encompass methods that are
directed to protecting plants against root pathogens or biotic
stresses such as fungal pathogens, bacteria, viruses, nematodes,
pests, and the like. By "disease resistance" or "insect resistance"
is intended that the plants avoid the harmful symptoms that are the
outcome of the plant-pathogen interactions. U.S. Pat. No.
7,456,334. Pathogens of the embodiments include, but are not
limited to, viruses or viroids, bacteria, insects, nematodes,
fungi, and the like. Viruses include tobacco or cucumber mosaic
virus, ringspot virus, necrosis virus, maize dwarf mosaic virus,
etc. Nematodes include parasitic nematodes such as root knot, cyst,
and lesion nematodes, etc. As described elsewhere herein, various
changes in phenotype may be determined in the plantlet root, e.g.
altering a plant's pathogen or insect defense mechanism or
increasing the plant's tolerance to herbicides
[0068] Advantageously, the present methods and assays use an
intact, live root that takes place in a dish and allows for the
continuous generation of plants from roots obtained from the green
tissue. With respect to the continuous generation of transgenic
plants from transgenic green tissue, advantageously these may be
obtained from the same transgenic event. This is in contrast to
other rootworm bioassay techniques that employ ground-up
transformed roots or use seedlings in soil which are infested with
either eggs or neonates. The former destroys the plant and requires
new plants to be recreated with the same genomic character. The
latter requires an assay of at least 14 days of duration and is
often destructive in nature. It is challenging to determine the
activity of the plant on the larvae as it is difficult to find the
larvae in the soil. Advantageously, the status of the pests and
root are easily observable using the assays and methods described
herein since they do not require that the roots be immersed or
buried in soil.
[0069] The assays and methods described herein are also economical
from a time and space standpoint as they have a duration of about 1
to 14 days or less and extensive greenhouse space is not needed to
perform the methods and assays, rather they can be performed in an
incubator with lights. In one embodiment of the invention, the
effect of insect application on plantlet, root or leaf damage may
be assayed within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14 or more days after infestation with one or more pests.
[0070] Pests that may be used in the methods and assays include
without limitation those insects belonging to the order of
Lepidoptera, which would feed on the stalk and leaves, e.g.
European corn borer (Ostrinia nubilalis), Corn earworm (Helicoverpa
zea), Fall armyworm (Spodoptera frugiperda), Western bean cutworm
(Richia albicosta), Black cutworm (Agrotis ipsilon), Lesser
cornstalk borer (Elasmopalpus lignosellus), Southwest corn borer
(Diatraea grandiosella), Sugarcane borer (Diatraea saccharalis),
Homoptera, e.g. Aphid (leaf feeding and root feeding), Leafhoppers,
Coleoptera, e.g. Corn rootworms, Wireworms, or White grubs
(Scarabs) or Heteroptera. In another aspect, steps are taken to
prevent or inhibit the growth of unwanted contaminants e.g.
microbes, such as bacteria, mold, or fungi in the assay. For
example, a biocide such as PPM may be added to the medium to
prevent or inhibit fungal and bacterial development. Further, the
use of an absorbent material such as filter papers, rather than
agar, in the Phytatray or Petri dish can be used to facilitate the
transfer of nutrient solution to plant roots, while reducing fungal
and bacterial development. If further contact is desired between
the medium and the roots, a wire grid or screen may be placed on
top of the roots.
[0071] Pests may be sterilized prior to contact with the root. In
some examples, the eggs, larvae, instars or adults of the pests are
treated to remove or kill bacterial or fungal spores which may
include washing with once or multiple times with a solution such as
ethanol or CHLOROX.RTM. bleach (The Chlorox company, Oakland,
Calif.). The pests may be feed prior to infesting the roots. For
example, neonates may be placed on artificial diet for 24 hours
prior to being placed on the test roots. This eliminates any larvae
that will have died in the initial 24 hours as well as allowing for
the selection of uniform-sized and healthy test subjects. Allowing
the neonates to feed for 24 hours provides the further benefit of
causing the evacuation of any fungal or bacterial spores in the gut
with the elimination of frass. In another aspect, the pest in any
developmental stage such as eggs, larvae, instars, or adults may be
sprayed with LYSOL.RTM. disinfectant, e.g. EPA Reg No 777-72, in
particular Professional LYSOL.RTM. disinfectant spray, EPA Reg No
777-72-625, or another disinfectant prior to infestation to help
kill fungi and bacteria (LYSOL.RTM. disinfectant Reckitt Benckiser
Inc, Parsippany, N.J.).
[0072] Subsequent to pest infestation or exposure to the stress or
pathogen, the root is incubated under appropriate conditions, for
example, incubating the dish plus roots and pests at about
24.degree. C.-28.degree. C. under light or dark conditions. The
roots may be subjected to pests of the appropriate developmental
stage, for example, larvae, and appropriate number. Generally the
duration of the assay is about 4-14 days. The phenotypes of the
roots or pests or both may be observed at any suitable time point
but are typically performed at completion. As understood by one
skilled in the art, the damage to roots and pests can be determined
in various ways, including objective and subjective techniques. For
example, the roots may be scored for their damage by the pests on a
scale of 0 to 5 with 0 indicating little or no observable damage to
severe root damage. In addition, leaf damage can also be scored for
direct feeding by the insects, or by color changes and wilting due
to damage to the roots or stem. In some instances, color change in
various plantlet parts, such as the leaves, stems, and/or roots,
may be observed as a result of pest damage. Color change may occur
in none, some or all of these parts. Pests may be scored to count
"live" versus "dead" or "stunted" larvae and tabulating the results
to express as a percentage of mortality. Any result of dead or
stunted or combinations thereof over 50% is considered a positive
result. In another aspect, the roots may be evaluated for
resistance to any rootworm, for example, resistance to Southern
corn rootworm (Diabrotica undecimpuncata), Western corn rootworm
(Diabrotica virgifera), and/or Northern corn rootworm (Diabrotica
barber), and the like. In some cases, the transgenic roots may be
evaluated for the polynucleotide of interest's impact on resistance
to a pest, such as any rootworm.
[0073] As another example, the plantlet may be scored for damage by
the pests by observing the color change of the stem area above the
crown of the roots. With respect to a normal plantlet of maize
exposed to a pest, when the stem area above the crown darkens from
pest damage, e.g. from WCRW damage; this color change indicates
that the polynucleotide of interest is not effective for
controlling pest infestation and/or damage to the stem. However,
when little or no color change of the stem area above the crown is
observed when the plantlet is exposed to a pest, this indicates
that the polynucleotide of interest is effective for controlling
pest infestations and/or damage to the plantlet. As another
example, the crown of the plantlet may be scored for damage by the
pests by determining the existence of holes in the crown. The
number of holes in the crown can be translated into a numerical
value which can be used to determine the overall activity of the
polynucleotide of interest in protecting the plantlet.
[0074] Transgenic plants may be regenerated from green tissue that
has a root testing positive for a desirable phenotype. A plant
having the desired phenotype may be produced by regenerating the
plant from the green tissue and the resultant plant entered into a
plant breeding program. After 3-4 weeks, the regenerated transgenic
plantlets may be transferred to soil and grown into a transgenic
plant in a greenhouse. Accordingly, in one aspect, the methods may
include growing the transgenic plantlet into a transgenic plant.
Transgenic seed may also be obtained from the plant if desired.
[0075] Any well-known regeneration medium may be used for the
practice of the provided methods. "Regeneration medium" (RM)
promotes differentiation of totipotent plant tissues into shoots,
roots, and other organized structures and eventually into plantlets
that can be transferred to soil. Auxin levels in regeneration
medium are reduced relative to MPM or, preferably, auxins are
eliminated. It is also preferable that copper levels are reduced,
e.g., to levels common in basal plant culture media such as MS
medium. It is preferable to include a cytokinin in RM, as
cytokinins have been found to promote regenerability of the
transformed tissue. However, regeneration can occur without a
cytokinin in the medium. Typically, cytokinin levels in RM are from
about 0 mg/L to about 4 mg/L. RM also preferably includes a carbon
source, preferably about 20 g/L to about 30 g/L, e.g., either
sucrose or maltose.
[0076] Transformed plant cells which are derived by any of the
above transformation techniques can be cultured to regenerate a
whole plant which possesses the transformed genotype. Such
regeneration techniques often rely on manipulation of certain
phytohormones in a tissue culture growth medium, typically relying
on a biocide and/or herbicide marker which has been introduced
together with a polynucleotide of interest. For transformation and
regeneration of maize see, Gordon-Kamm, et al., (1990) The Plant
Cell 2:603-618.
[0077] Regeneration can also be obtained from explants, green
tissue, roots, plantlets, or parts thereof. Such regeneration
techniques are described generally in Klee, et al., (1987) Ann.
Rev. of Plant Phys. 38:467-486. The regeneration of plants from
either single plant protoplasts or various explants is well known
in the art. See, for example, Methods for Plant Molecular Biology,
Weissbach and Weissbach, eds., Academic Press, Inc., San Diego,
Calif. (1988). This regeneration and growth process includes the
steps of selection of transformant cells and shoots, rooting the
transformant shoots and growth of the plantlets in soil. For maize
cell culture and regeneration see generally, The Maize Handbook,
Freeling and Walbot, Eds., Springer, New York (1994); Corn and Corn
Improvement, 3.sup.rd edition, Sprague and Dudley Eds., American
Society of Agronomy, Madison, Wis. (1988).
[0078] Plants to be transferred to the growth chamber are removed
from sterile containers and the solidified agar medium is rinsed
off the roots. The plantlets are placed in a commercial potting mix
in a growth chamber equipped with a misting device which maintains
the relative humidity near 100% without excessively wetting the
plant roots. Approximately three to four weeks are required in the
misting chamber before the plants are robust enough for
transplantation into pots or into field conditions. At this point,
many plantlets, especially those regenerated from short-term callus
cultures will grow at a rate and to a size similar to seed-derived
plants. Plants regenerated from long-term callus, from suspension
cultures, and from in vitro-selected callus will sometimes show
phenotypic abnormalities, such as reduced plant size, leaf striping
and delayed maturation. Care must be taken to assure controlled
pollination with such plants. Ten to fourteen days after
pollination, the plants are checked for seed set. If there is seed,
the plants are then placed in a holding area in the greenhouse to
mature and dry down. Harvesting is typically performed six to eight
weeks after pollination.
[0079] One of skill will recognize that after the recombinant
expression cassette comprising the polynucleotide of interest is
stably incorporated in transgenic plants and confirmed to be
operable, it can be introduced into other plants by sexual
crossing. Any of a number of standard breeding techniques can be
used, depending upon the species to be crossed.
[0080] This invention can be better understood by reference to the
following non-limiting examples. It will be appreciated by those
skilled in the art that other embodiments of the invention may be
practiced without departing from the spirit and the scope of the
invention as herein disclosed and claimed.
EXAMPLES
[0081] The present invention is further defined in the following
Examples, in which parts and percentages are by weight and degrees
are Celsius, unless otherwise stated. The disclosure of each
reference set forth herein is incorporated herein by reference in
its entirety.
Example 1
Production of Transgenic Maize Events Via Bombardment
Immature Embryos as a Bombardment Target
[0082] Ears of a maize (Zea mays L.) cultivar, PHR03, were
surface-sterilized for 15-20 min in 20% (v/v) bleach (5.25% sodium
hypochlorite) plus 1 drop of Tween 20 followed by 3 washes in
sterile water. Immature embryos (IEs), typically 9 to 12 days after
pollination, were isolated from ears and were placed scutellum-side
up in an osmotic medium containing equimolar amounts of mannitol
and sorbitol to give a final concentration of 0.4 M. The embryos
were bombarded with gold particles coated with DNA containing
bar/moPAT or another selectable marker using a PDS-1000 He
biolistic device (Bio-Rad, Inc., Hercules, Calif.) at 650-1300 psi.
Between 16 hr and 18 hr after bombardment, the bombarded embryos
were placed on green tissue induction medium without osmoticum and
grown at 26.degree. C..+-.2.degree. C. under dim light (10-50 uE
m.sup.-2 s.sup.-1). Following the initial 4 to 10 day culturing
period, each green tissue was broken into 1 to 3 pieces depending
on tissue size and transferred to green tissue induction medium
supplemented with bialaphos or another selective agent. Three weeks
after the first round of selection, cultures were transferred to
fresh green tissue induction medium containing a selective agent at
3 to 4 week intervals. Following identification of sufficient sized
green, regenerative structures, tissues were then transferred
directly onto 2 different shoot and root regeneration culturing
schemes: (1) 7-14 days of incubation on 289F shoot regeneration
medium prior to placing tissues on rooting medium and (2) directly
onto rooting medium. Two rooting media were also tested: (1) MSA
containing MS salts and vitamins, 2% sucrose, 0.35% Phytagel and 3
mg/L bialaphos and (2) MSB containing MS salts and vitamins, 2%
sucrose, 0.25% Phytagel, 0.5 mg/L IBA and 3 mg/L bialaphos. MSB was
more efficient in root formation than MSA.
Green Tissues as a Bombardment Target
[0083] Ears of PHR03 were surface-sterilized as described above.
Green tissues were induced and proliferated by culturing IEs on
green tissue induction medium and used for bombardment. Green
tissues, approximately two to three months old, were used as
targets for bombardment. Tissues (4 to 6 mm) were transferred for
osmotic pretreatment to green tissue induction medium containing
0.2 M mannitol and 0.2 M sorbitol. After 4 hr, tissues were
bombarded as described above. Sixteen to 18 hr after bombardment,
the bombarded tissues were placed on green tissue induction medium
without osmoticum and grown at 26.degree. C..+-.2.degree. C. under
dim light (10-50 uE m.sup.-2 s.sup.-1). Following the initial 4 to
10 day culturing period, each green tissue was broken into 1 to 3
pieces depending on tissue size and transferred to green tissue
induction medium supplemented with bialaphos or another selective
agent. Three weeks after the first round of selection, cultures
were transferred to fresh green tissue induction medium containing
a selective agent at 3 to 4 week intervals. Once transformed,
transgenic green tissues are selected and cultured in a similar
manner as that used for green tissue obtained by particle
bombardment of immature embryos.
Example 2
Production of Transgenic Maize Events Via Agrobacterium
Preparation of Agrobacterium Suspension:
[0084] Agrobacterium tumefaciens harboring a binary vector
containing DS-RED (RFP) reporter gene and a selectable marker (moPA
7) with or without a Bt gene was streaked out from a -80.degree.
frozen aliquot onto solid PHI-L medium and cultured at 28.degree.
C. in the dark for 2-3 days. PHI-L media comprised 25 ml/L stock
solution A, 25 ml/L stock solution B, 450.9 ml/L stock solution C
and spectinomycin added to a concentration of 50 mg/L in sterile
ddH.sub.2O (stock solution A: K.sub.2HPO.sub.4 60.0 g/L,
NaH.sub.2PO.sub.4 20.0 g/L, adjust pH to 7.0 with KOH and
autoclave; stock solution B: NH.sub.4Cl 20.0 g/L,
MgSO.sub.4.7H.sub.2O 6.0 g/L, KCl 3.0 g/L, CaCl.sub.2 0.20 g/L,
FeSO.sub.4.7H.sub.2O 50.0 mg/L, autoclave; stock solution C:
glucose 5.56 g/L, agar 16.67 g/L and autoclave). Two ways to grow
Agrobacterium were used for transformation.
[0085] 1. Growing Agrobacterium on Solid Medium
[0086] A single colony or multiple colonies were picked from the
master plate and streaked onto a plate containing PHI-M medium and
incubated at 28.degree. C. in the dark for 1-2 days.
[0087] Five mL Agrobacterium infection medium and 5 .mu.L of 100 mM
3'-5'-Dimethoxy-4'-hydroxyacetophenone (acetosyringone) were added
to a 14 mL Falcon tube in a hood. About 3 full loops of
Agrobacterium were suspended in the tube and the tube was then
vortexed to make an even suspension. One mL of the suspension was
transferred to a spectrophotometer tube and the OD of the
suspension was adjusted to 0.35 at 550 nm. The Agrobacterium
concentration was approximately 0.5.times.10.sup.9 cfu/mL. The
final Agrobacterium suspension was aliquoted into 2 mL
microcentrifuge tubes, each containing 1 mL of the suspension. The
suspensions were then used as soon as possible.
[0088] 2. Growing Agrobacterium on Liquid Medium
[0089] One day before infection, a 125 mL flask was set up with 30
mLs of 557A and 30 uL spectinomycin (50 mg/mL) and 30 uL
acetosyringone (20 mg/mL). A half loopful of Agrobacterium was
suspended into the flasks and placed on a 200 rpm shaker at
28.degree. C. overnight. The Agrobacterium culture was centrifuged
at 5000 rpm for 10 min. The supernatant was removed and the
Agrobacterium infection medium+acetosyringone solution was added.
The bacteria were resuspended by vortex and the OD of Agrobacterium
suspension was adjusted to 0.35 at 550 nm.
Maize Transformation:
[0090] Ears of maize (Zea mays L.) cultivars, PHR03 and PH4CN, were
surface-sterilized for 15-20 min in 20% (v/v) bleach (5.25% sodium
hypochlorite) plus 1 drop of Tween 20 followed by 3 washes in
sterile water. Immature embryos (IEs) were isolated from ears and
were placed in 2 mL of the Agrobacterium infection medium plus
acetosyringone solution. The optimal size of the embryos was
1.5-1.8 mm and 1.3-2.1 mm for PHR03 and PH4CN, respectively. The
solution was drawn off and 1 mL of Agrobacterium suspension was
added to the embryos and the tube vortexed for 5-10 sec.
[0091] The microfuge tube was allowed to stand for 5 min in the
hood. The suspension of Agrobacterium and embryos were poured onto
co-cultivation medium. Any embryos left in the tube were
transferred to the plate using a sterile spatula. The Agrobacterium
suspension was drawn off and the embryos placed axis side down on
the media. The plate was sealed with Parafilm tape and incubated in
the dark at 21.degree. C. for 1-3 days of co-cultivation.
[0092] Embryos were transferred to resting medium without
selection. Three to 7 days later, they were transferred to green
tissue induction medium containing 3-5 mg/L bialaphos (Meiji Seika
K.K., Tokyo, Japan) plus 100 mg/L carbenicillin (ICN, Costa Mesa,
Calif.). The plate was sealed with Parafilm and incubated at
26.degree. C..+-.2.degree. C. under dim light. At 2-3 weeks after
the first round selection, each callusing piece, broken into 1 to 3
pieces, depending on initial size, was transferred to fresh medium
supplemented with a selective agent. Tissues were subcultured on
fresh medium containing bialaphos at 2-3 week intervals. At
3.sup.rd round selection, transgenic sectors were identified by
visible markers (e.g. RFP) under a fluorescence microscope and
chopped into small pieces to place on maintenance medium for
further proliferation. Transgenic green tissues were proliferated
until sufficient amount of tissues was obtained. Table 1 below
shows transgenic events produced from PHR03 and PH4CN using
different Bt gene constructs.
TABLE-US-00001 TABLE 1 Transgenic maize events transformed with
different Bt gene constructs #transgenic Construct Inbred Bt gene
events/ PHP26650 PH4CN Control 37 PHR03 44 PHP36779 PH4CN Shuffled
Bt variant 12 2A12-V1 PHR03 2 PHP36782 PH4CN Shuffled Bt variant 14
2A12-V2 PHR03 38 34651- Bt PH4CN Bt variant V6 2 Variant V6 PHR03
169 34651- Shuffled Bt PH4CN Shuffled Bt variant 3 variant 2A12-V5
PHR03 2A12-V5 86 34651- Shuffled Bt PH4CN Shuffled Bt variant --
variant 2A12-V3 PHR03 2A12-V3 48 34651- Shuffled Bt PH4CN Shuffled
Bt variant -- variant 2A12-V4 PHR03 2A12-V4 49
Example 3
Continuous Root or Root/Shoot Production from Green Regenerative
Tissues of Transgenic Maize Events
[0093] Highly regenerative, green tissues of monocot crops species
contain multiple, light to dark green, shoot meristem-like
structures. These tissues regenerate multiple shoots without loss
or with minimum loss of regenerability for more than a year. These
green tissues are organogenic, rather than embryogenic, and are
likely to have meristem-like tissues which are ready for shoot
regeneration and root formation. Transgenic sectors were identified
by visible markers under a fluorescence microscope and chopped into
small pieces to place on maintenance medium for minimal
proliferation. Tissues were then transferred directly onto 2
different shoot and root regeneration culturing schemes: (1) 7-14
days of incubation on 289F shoot regeneration medium prior to
placing tissues on rooting medium and (2) directly onto 2 rooting
media, MSA and MSB. MSB was more efficient in root formation than
MSA. When green tissues were incubated directly on MSB, both shoot
and root formation or root formation only was observed. The use of
289F shoot regeneration medium could facilitate shoot production
more efficiently. Transgenic roots could be produced without any
shoot regeneration when green tissues with no shoot regeneration on
289F were transferred onto MSB Regenerated shoot and root tissues
showed uniform expression of RFP. This system can be used to do
quick gene testing in roots such as corn root worm assay and
functionality test of root-specific promoters using stably
transformed tissues.
Example 4
Screening of Transgenic Maize Events with Bt Gene Expression by
Western Blot Analysis
[0094] In order to screen transgenic events with Bt gene
expression, western blot hybridization analysis was carried out.
Forty to 100 mg of green tissues or leaf or root tissues from each
transgenic event were mixed with 0.1 to 0.25 mL CCLR protein
extraction buffer (100 mM phosphate buffer, 1 mM EDTA, 1% Triton,
10% Glycerol, 7 mM BME, pH 7.8) (Cat. #E1531, Promega Corp.,
Madison, Wis.) in a 2 mL microfuge tube. After adding two steel
balls in the tube, the samples were ground two to three times with
the GenoGrinder2000 (1.times. rate, 2 min 30 sec/run; 250
strokes/min). After centrifugation (10,000.times.g for 2 min), 30
.mu.L of the supernatant and 10 .mu.L of 4.times. loading dye were
mixed in a fresh tube and heated for 5 min at 95.degree. C. Twenty
.mu.L of total soluble protein (total 6000 .mu.g wet tissue
equivalent) from each event and purified Bt protein as a positive
control were separated on SDS-PAGE using NuPAGE 4-12% Bis Tris gel
(Invitrogen Corp, Carlsbad, Calif.) and transferred to
nitrocellulose membrane. After transfer, the membrane was blocked
in TBS-T (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% Tween 20)+5%
nonfat dried milk overnight. After washing (2.times.5 min) in
TBS-T, primary rabbit polyclonal Bt antibody was added at 1:1000
dilution and incubated for 1.5 to 2 hr at room temperature. After
washing in 1% BSA/TBS-T, the membrane was incubated in goat
anti-rabbit alkaline phosphatase (AP) antibody at 1:1000 dilution
for 1-2 hr at room temperature and washed as indicated above.
Labeling was monitored by using Bio-Rad (Hercules, Calif.) AP
conjugate substrate kit for detection according to manufacturer's
instructions. Expression signal was quantified using UN-SCAN-IT gel
(Gel & Graph Digitizing Software 6.1, Silk Scientific, Inc.,
Orem, Utah).
[0095] Bt expression in transgenic events was determined by western
analysis using green tissues and leaf and root tissues from each
event. Out of 6 Bt variants, shuffled Bt variant 2A12-V5 had the
highest expression of Bt (60 ppm) in green tissues while shuffled
Bt variant 2A12-V2 was lowest in expression (7 ppm). Shuffled Bt
variants 2A12-V1, 2A12-V3, 2A12-V4, and 2A12-V6 showed 26, 36, 28
and 38 ppm, respectively. In general, root and leaf tissues had
much lower levels of Bt expression, compared with that of green
tissues derived from the same event, e.g., root (7 ppm) and leaf
tissues (6 ppm) vs. green tissues (23 ppm) from shuffled Bt variant
2A12-V4 event #2. For each construct 3 to 8 highest expressors were
selected for shoot and/or root regeneration for Western corn
rootworm (WCRW) bioassay.
Example 5
Establishment and Optimization of a Contamination-Free In Vitro
Insect Bioassay System Using Live Plantlets or Root Tissues
Preparation of Plantlets or Root Tissues:
[0096] Plantlets used for western corn rootworm bioassay were
selected from high expression events containing Bt genes. Plantlet
selections were based on root thickness and root mass, preferably
with a single shoot and either thin young roots or highly branched
young roots. A mixture of MSA with 0, 1, 1.5, 2 and 5% Plant
Preservative Mixture (PPM--0.1350%
5-chloro-2-methyl-3(2H)-isothiazolone, 0.0412%
2-methyl-3(2H)-isothiasolone, 99.8238% inert ingredients, Plant
Cell Biotechnology, Inc.) was prepared for plantlet soak and as a
nutrient solution. Two mL MSA-PPM mixture on two 70 mm circular
Whatman (Schleicher & Schuell) filter papers in Petri dishes
(100.times.15 mm, VWR) were set-up for plantlet/root containment.
Individual plantlets were extracted from MSB media and roots were
removed from agar by forceps. Extraneous yellow and browning
tissue, leaves and roots were cut away or excised. Each plantlet
was soaked in 50 mL MSA containing different levels of PPM for 3 to
5 minutes. After soaking, stray residual agar and extra nutrient
solution were removed from each plantlet, and the roots then
arranged for maximum contact with the filter paper within the
prepared Petri dishes, one plantlet per Petri dish. Plant materials
were submitted for 1 day old WCRW infestation Root tissues alone
without leaves also could be prepared for the assay using the same
protocol described above.
Preparation of Western Corn Root Worms:
[0097] Non-diapausing WCRW eggs were cleaned and surface sterilized
in successive steps using a modification of the procedure described
by Marrone et al. (1985) (J of Economic Entomology 78: 290-293),
with 1% Clorox bleach, 0.25% peracetic acid solution, 70% ethanol,
and then de-ionized water. The final step utilizes washing with
deionized water four times. Eggs were suspended in a 0.22% agarose
solution and transferred to agar Petri plates covered with two 9 cm
filter paper disks. Once dry, the eggs were sprayed with a thin
layer of Professional Lysol Disinfectant Spray (28.1% a.i.). After
they were dry, the plates were covered and four dishes were placed
in 32 ounce Nalgene containers that had been sprayed with Lysol,
wiped down, and allowed to dry. The Nalgene containers were closed
and put in incubators kept at 28.degree. C. in the dark for two
days until egg hatch. Neonates were transferred to single-well
plates with artificial WCRW diet, sealed with heat-sealable Mylar
with pin holes, and kept at 25.degree. C. in the dark for 24
hr.
Infestation of Plantlets with Western Corn Root Worms:
[0098] Under a laminar flow hood, larvae were tapped into a
heat-sterilized #80 sieve. Larvae were sprayed with or without
Professional Lysol Disinfectant Spray (28.10% a.i.) and allowed to
soak for 2 min. The larvae were then washed with autoclaved water
and transferred to a 50 mL centrifuge tube. Additional autoclaved
water was added to the tube and the larvae swirled for 1 minute.
The water was decanted, leaving the larvae and a small amount of
water in the bottom of the tube. A sterile disposable inoculating
loop was used to transfer .about.25 larvae to the Petri dishes
containing the plantlet. The Petri dish/plantlet/larvae are placed
in an incubator at 25.degree. C. in a 16-hr light phase for 5-7
days prior to checking contamination of the plates and plants.
[0099] No fungal or bacterial contamination was observed in the
plates with MSA solution plus 5% PPM when non-sterilized rootworms
were infested (Table 2). In contrast, fungal contamination was
detected with 1% or 2% PPM; the higher the level of PPM, the less
the contamination. Rootworms, however, were severely stunted at 5%
PPM.
TABLE-US-00002 TABLE 2 Number of contaminated plates treated with
non-sterilized WCRWs at different PPM concentrations in liquid MSA
medium. PPM level 0% PPM 1% PPM 2% PPM 5% PPM # plates 2/2 1/2 1/2
0/2 contaminated/# (severe mold (severe mold (minor mold (No mold)
plates tested contamination) contamination) contamination) The
MSA-PPM mixtures on 2 Whatman filter papers in Petri dishes were
set for plantlet/root containment.
[0100] Another set of experiments was conducted with rootworms
sterilized with Lysol. Fungal contamination was controlled in the
plates with both 1% and 1.5% PPM treatments (Table 3), but mild
bacterial contamination was occasionally observed at 1% PPM. When
sterilized rootworms were placed onto agar medium, severe bacterial
contamination was detected (data not shown). Based on these
results, a filter paper system supplemented with MSA liquid medium
containing 1.5% PPM was employed using rootworms sterilized with
Lysol for further WCRW bioassay.
TABLE-US-00003 TABLE 3 Number of contaminated plates treated with
sterilized WCRWs 1% PPM 1.5% PPM Non-sterilized 1/2 1/2 rootworm
(severe mold (minor mold contamination contamination Sterilized 0/2
0/2 Rootworm w/Lysol (No Contamination) (No Contamination)
Example 6
Western Corn Rootworm Bioassay Using Transgenic Maize Events
Expressing Bt Genes
[0101] Transgenic plant materials were produced as described in
Example 1-3. Plantlets/root tissues were prepared as described in
Example 3. One to 3 plantlets per event were extracted from MSB
media and roots were removed of agar by forceps. After soaking in
MSA containing 1.5% PPM, stray residual agar and extra nutrient
solution were removed from each plantlet, and the roots were then
arranged for maximum contact with 3 filter papers within the
prepared Sigma Phytatrays, one plantlet per Phyatray vessel. Roots
not in contact with the filter paper were weighed down by single
2''.times.2'' stainless steel mesh screens until it was accessible
to the nutrient solution. About 20 plantlets were prepared for WCRW
bioassay, as well as 1 to 3 control plantlets per inbred line, and
used for WCRW infestation.
[0102] Under a laminar flow hood, the one-day-old larvae were
transferred to a heat-sterilized #80 sieve and sprayed until
drenched with Professional Lysol Disinfectant Spray and allowed to
soak for 2 min. The larvae were then washed with autoclaved water
and transferred to a 50 mL centrifuge tube and swirled for 1 min.
The water was decanted, leaving the larvae in a small amount of
water in the bottom of the tube. A sterile disposable inoculating
loop was used to transfer .about.25 larvae to the Phytatray
containing the transformed plantlet. After infestation, the
Phytatrays were then placed in an incubator at 25.degree. C. in a
16-hr light phase for 7 days prior to scoring the plants and larvae
for plant damage and larval development.
[0103] As shown, 24 to 36% of larvae developed to the 2.sup.nd
instars (Table 4) and root tissues had severe damage on control
plantlets of both PH4CN and PHR03 1 wk after infestation while the
growth of rootworms was severely stunted and little root damage was
detected on Herculex.RTM. plantlets (Table 4). Herculex.RTM. RW
contains Cry 34/Cry35 toxins. (Pioneer and Dow Agro). The
expression level of 6 Bt variants was 7 to 60 ppm in green tissue.
In general the higher the Bt expression of the same Bt variant in
green tissue, the more resistant the plantlets and roots to
rootworms (data not shown). A few events with high Bt expression in
green tissue were susceptible to rootworms; these events did not
show Bt expression in roots possibly due to production of multiple
events derived from the same embryo or transgene silencing as
tissue reaches stages of plantlets or roots. All Bt variants
tested, except variant 2A12-V2, were slightly to moderately
resistant to rootworms. (Table 4). Event #3 transformed with Bt
variant V6 (at 21 ppm) had little root damage and stunted root
worms 4 days after infestation (data not shown). Event #23
transformed with shuffled variant 2A12-V3 (at 9 ppm) had severe
root damage and bigger root worms 4 days after infestation. (data
not shown). Event #23 transformed with variant 2A12-V3 (Table 4)
and 3 events (#s 3, 5 and 7) with variant 2A12-V2 were low in
efficacy, possibly due to low expression (Table 4). The number of
the 2.sup.nd instars and root damage appeared to be the best
indicators to evaluate the efficacy of Bt genes and transgenic
events (Table 4).
TABLE-US-00004 TABLE 4 WCRW bioassay results using T.sub.0 PH4CN
and PHR03 plantlets Crown Root Leaf Expt Inbred Construct Event #
Rep # II instars Damage Damage Damage Plant Bt expression in
tissue, ppm #1 PH4CN 26650 6 0 #1 6 ++ ++ none live (-control)
Shuffled 5 12 #1 2 + + none live Bt variant 11 12 #1 2 none + none
live 2A12-V1 Shuffled 3 7 #1 4 ++ ++ none live Bt variant 5 6 #1 5
+++ ++ none live 2A12-V2 7 5 #1 8 +++ +++ none live Herculex .RTM.
(+control) n.a. #1 0 none (+) none live RW Bt expression in GT, ppm
#2 PHR03 26650 6 0 #1 9 +++ +++ some dying (-control) 0 #2 6 +++
+++ moderate dead Shuffled 22 27 #1 3 + ++ none healthy Bt variant
27 #2 1 + ++ some dying 2A12-V3 23 9 #1 12 +++ +++ some dying
Shuffled 3 21 #1 0 none + none dying Bt variant 21 #2 0 + ++ none
dying 2A12-V6 21 #3 0 none none none healthy 10 23 #1 7 ++ ++ some
dying 23 #2 0 ++ + none healthy Herculex .RTM. (+control) n.a. #1 0
none + some healthy RW
Example 7
Application of the Current WCRW Bioassay System to Other
Insects
[0104] This example illustrates significant pest inhibition
obtained by feeding lepidopteran larvae on corn tissue transformed
with Bt genes.
[0105] Corn earworm, fall armyworm, black cutworm and sugarcane
borer eggs were received from Benzon Research (Carlisle, Pa.).
European corn borer eggs were received from Pioneer (Johnston,
Iowa). Soybean looper eggs were received from DuPont (Wilmington
Experiment Station, DE). Eggs were kept at 28.degree. C. and
allowed to hatch. Neonates were placed on a multi-species
lepidopteran diet (Southland Products, Lake Village, Ak.) and kept
at 28.degree. C. for 24 hr. Under a laminar flow hood, 10
one-day-old larvae were transferred to the Phytatray containing the
transgenic plantlet using a sterile disposable inoculating loop.
Prior to infestation, a minimal amount of plant nutrients and 1.5%
PPM was applied to the 3 sterile filter papers on which the
plantlet was placed, as described in Examples 4, 5 and 6. Metal
mesh screen was placed on the roots of the plantlet to insure good
contact between the roots and the plant nutrients on the filter
paper if necessary. The Phytatrays were then placed in an incubator
at 26.degree. C. in a 16-hr light phase for 3-7 days prior to
scoring the plants and larvae for plant damage and larval
development. Plants were observed to determine those expressing Bt
to kill or stunt the lepidopteran pests compared to the control
which were not expressing toxin.
[0106] Table 5 demonstrates the effect of Bt 1 expression in corn
plantlets on resistance to soybean looper. All transgenic events
(#s 2, 16, 17, and 33) showing the Bt1 gene presence had good
resistance to soybean looper while negative control (#583) and
Herculex.RTM.RW without Bt1 were susceptible to soybean looper
(Table 5)
TABLE-US-00005 TABLE 5 In vitro soybean looper bioassay results
using T.sub.0 PHR03 plantlets Construct Bt1 gene Insect # Insect #
Condition of leaves (event #) presence alive dead (0% to 100%)
Condition of insects 24600 (#583) - 20 0 50% eaten Several third
instars Bt1 (#40) - 7 13 20% eaten All seven still 1st instars
(stunted) Bt1 (#33) + 0 20 0% eaten All dead neonates Bt1 (#17) + 0
20 0% eaten All dead neonates Bt1 (#16) + 0 20 0% eaten All dead
neonates Bt1 (#2) + 0 20 2% eaten, slight All dead neonates holes
in leaves Herculex .RTM.RW - 14 6 80% eaten Various instars up to
3rds Twenty neonates of soybean looper were infested onto corn
plantlets. Five days after infestation plant damage and insect
growth, stunting and survival were scored.
[0107] Table 6 demonstrates the effect of Bt 1 expression in corn
plantlets on resistance to black cutworm. Transgenic event #54
showing the Bt1 gene presence had good resistance to black cutworm
while event #40 without Bt1 was susceptible to black cutworm.
TABLE-US-00006 TABLE 6 In vitro black cutworm bioassay results
using T.sub.0 PHR03 plantlets Bt1 gene Insect # Insect # Plant
presence alive dead Leaf damage health Bt1 (#54) + 3 7 10%, stem
& leaves pocked Alive Bt1 (#40) - 10 0 95%, stem completely
shredded, Dead everything eaten Herculex .RTM.RW - 5 5 80%, stem
shredded; leaves Dead shredded Ten neonates of black cutworm were
infested onto corn plantlets. Seven days after infestation plant
damage and insect growth, stunting and survival were scored.
[0108] Table 7 demonstrates the effect of Bt 1 expression in corn
plantlets on resistance to corn earworm. Transgenic event #s 25, 42
and 82 showing the Bt1 gene presence had good resistance to corn
earworm while event #40 and Herculex.RTM.RW without Bt1 were
susceptible to corn earworm.
TABLE-US-00007 TABLE 7 In vitro corn earworm bioassay results using
T.sub.0 PHR03 plantlets Bt1 gene Insect # Insect # presence alive
dead Leaf damage Plant health Bt1 (#42) + 0 10 0%, no penetration
Healthy Bt1 (#82) + 0 10 0%, no penetration Healthy Bt1 (#25) + 0
10 0%, one penetration hole, Healthy but did not go anywhere Bt1
(#40) - 10 0 95%, stem completely Dead shredded, everything eaten
Herculex .RTM.RW - 5 5 80%, stem shredded; leaves Dead shredded Ten
neonates of corn earworm were infested onto corn plantlets. Seven
days after infestation plant damage and insect growth, stunting and
survival were scored.
[0109] Table 8 demonstrates the effect of Bt 1 expression in corn
plantlets on resistance to fall armyworm and sugarcane borer. All
transgenic events except event #20 showing the Bt1 gene presence
had good resistance to both fall armyworm and sugarcane borer while
negative control (#5) and Herculex.RTM.RW without Bt1 were
susceptible to these two lepidopteras (Table 8). All events were
consistent in tolerance to both fall armyworm and sugarcane (Table
8). Transgenic event #20 was susceptible to both lepidopteras
possibly due to lack of Bt1 expression even with the presence of
Bt1 gene.
TABLE-US-00008 TABLE 8 In vitro fall armyworm and sugarcane borer
bioassay results using T.sub.0 PHR03 plantlets Condition of
Construct Bt1 gene Insect Insect leaves (0% Insect event # presence
# alive # dead to 100%) Condition of insects Fall 26500 (#5) - 14 1
70% eaten Survivors premolt to III armyworm Bt1 (#8) + 0 15 2%
eaten All dead neonates Bt1 (#51) + 0 15 4% eaten All dead neonates
Bt1 (#12) + 4 11 8% eaten Survivors still 1sts Bt1 (#20) + 11 4 65%
eaten Survivors premolt to III Herculex .RTM. RW - 13 2 85% eaten
Survivors premolt to III Construct Bt1 gene Stalk Insect event #
presence holes Condition of leaves (0% to 100%) Sugarcane 26650
(#5) - 10 Plant dead, stalk collapsed borer Bt1 (#8) + 2 Plant
healthy Bt1 (#51) + 6 Plant alive Bt1 (#12) + 6 Plant dying Bt1
(#20) + 8 Plant dying, stalk collapsed Herculex .RTM. RW - ? Plant
dead, stalk completely collapsed unable to count *Some feeding on
leaves, but neonates of sugarcane borer borrowed into stalks within
an hour and survivors could not be scored live/dead/.
Example 8
In Vitro Bioassay Using Multiple Insects
[0110] This innovative in vitro bioassay using plantlets provides a
novel method to analyze effectiveness of gene constructs using both
damages to the plant as well as development of insect. Until
recently, we have only performed this bioassay using one insect at
a time; however, a multiple insect test has been successfully
implemented in this example. To prepare plantlets for this system,
the whole plantlet was carefully extracted from the MSB rooting
medium with roots intact and with as much agar off roots as
possible and soaked for 5 minutes in a MSA+1.5% PPM solution. A
wire grid might be used to weigh down the plantlet in the solution.
After 5 min, the plantlet was carefully taken out and placed in a
phytatray container containing 3 pieces of filter paper with 3 mL
of MSA+1.5% PPM solution. The roots were spread out and the
root/leaf was cut down if necessary so that all plantlets were
equivalent. If only using one type of insect for infestation, the
plantlets were infested and scored according to protocol about one
week later. Multiple insects could also be used. If the feeding
source of the insects was different, i.e. one type fed on roots
whereas the other fed on leaves, they could be infested together on
the same day. When using multiple insects, they could also be
infested separately depending on how soon results showed, i.e.
leaf-eating insect (lepidoptera) on one day and root-eating insect
three days later.
[0111] Table 9 demonstrates in vitro bioassay results using
multiple insects in T.sub.o quality event corn plantlets; all
quality events contain Bt1 gene for lepidopteran resistance and Bt2
and Bt3 genes for rootworm resistance. All transgenic events showed
resistance to both FAW and WCRW while negative control plantlets
were susceptible to both insects (Table 9). Infestation of both FAW
and WCRW together was very efficient in screening of transgenic
events resistant to both insects compared with infestation of each
insect separately. The surviving plantlets were transplanted to
soil for additional insect bioassay, further molecular assay and
grown to maturity to harvest seed. This bioassay scheme provides an
efficient and time-saving pre-screening system for transgenic
events (FIG. 1).
TABLE-US-00009 TABLE 9 In vitro bioassay using multiple insects in
T.sub.0 PHR03 plantlets Construct Insect treatment (event #)
Condition of leaves Condition of insects WCRW + FAW* - control
plant dead; entire stalk and roots 5 FAW alive (III instars),
(#623) destroyed (unclear if FAW ate roots as no WCRW found well)
QE #8 no stalk damage - healthy plant; tiny no live FAW, no growth
pinhole damage (very slight), no root of WCRW damage QE #76 no
stalk damage - healthy plant; 5% 2 FAW alive (II instars), damage
to leaves by FAW, small WCRW alive but stunted damage to roots
FAW-> WCRW** - control plant dying; stalk damage, 50% leaf 8 FAW
alive (III instars), (#623) eaten, no roots eaten by FAW one
rootworm visible QE #8 healthy plant; no leaf damage no live FAW,
WCRW not on roots (repellency?) QE #76 healthy plant; no leaf
damage no live FAW, WCRW on roots but stunted FAW-> WCRW* -
control plant dead; nothing left 4 FAW alive (III instars) (#623)
QE #76 healthy plant; 5% leaf damage from 1 FAW (I), little WCRW
FAW, no root damage yet mortality QE #105 plant collapsing,
browning; 10% leaf 3 FAW (II), some WCRW damage, minor root damage
mortality QE #119 plant alive, green; 15% leaf damage, no 3 FAW
(II), no WCRW root damage mortality FAW + WCRW** - control plant
dead; stalk left, no roots, 70% leaf 3 FAW (III) +' 1 eaten, 1
(#623) damage WCRW QE #76 healthy plant; no leaf damage, some 0
FAW, some WCRW root damage mortality QE #105 alive but collapsed;
20% leaf damage, 4 FAW (II), some WCRW no root damage mortality QE
#119 plant alive; 2 FAW (II), ~50% 30% leaf damage, no root damage
WCRW mortality WCRW->FAW*** - control plant dead; stalk only
left, major root 6 FAW (3 III, 3 II), 3 (#623) damage, 70% leaf
damage WCRW (II) QE #76 Alive; 20% leaf damage, minor root 3 FAW
(I), 0 WCRW feeding QE #105 alive but collapsed; 5% leaf damage, no
6 FAW (I), 0 WCRW root damage QE #119 plant alive; pinhole leaf
damage, no 2 FAW (I), 70% WCRW root damage mortality *Both 25
neonates of western corn rootworm (WCRW) and 10 neonates of fall
armyworm (FAW) were infested together at the same time. **10
neonates of FAW were infested first and 4 days later 25 neonates of
WCRW were infested. ***25 neonates of WCRW were infested first and
6 days later 10 neonates of FAW were infested.
Example 9
In Vitro Insect Bioassay for Promoter Testing
[0112] This example illustrates the application of the in vitro
lepidopteran insect bioassay system to test different promoters do
drive the expression of the Bt gene. Two constructs were used for
corn transformation: one containing Bt1 driven by the maize
ubiquitin promoter (Ubi1-Bt1) and another driven by the banana
streak virus promoter (BSV TR-Bt1). Plantlets were regenerated from
transgenic events and leaf punches were harvested for copy # assay
by qPCR. Single copy events were used for in vitro insect
bioassay.
[0113] Under a laminar flow hood, 10 one-day-old larvae of fall
armyworm (FAW) were transferred to the Phytatray containing the
transgenic plantlet using a sterile disposable inoculating loop.
Prior to infestation, a minimal amount of plant nutrients and 1.5%
PPM was applied to the 3 sterile filter papers on which the
plantlet was placed. Metal mesh screen was placed on the roots of
the plantlet to insure good contact between the roots and the plant
nutrients on the filter paper if necessary. The Phytatrays were
then placed in an incubator at 26.degree. C. in a 16-hr light phase
for 3-7 days prior to scoring the plants and larvae for plant
damage and larval development. Plants were observed to determine
those expressing Bt to kill or stunt FAW compared to the control
which were not expressing toxin.
[0114] Table 10 demonstrates the effect of Bt 1 expression driven
by 2 different promoters in corn plantlets on resistance to FAW.
All transgenic events showed good resistance to fall armyworm, but
the degree of insect resistance was higher with the maize ubiquitin
promoter than banana streak virus promoter (Table 10). Bt1
expression of each event will be measured by ELISA or western blot
analysis.
TABLE-US-00010 TABLE 10 Promoter test in T.sub.0 PHR03 plantlets
using in vitro insect bioassay Condition of leaves Promoter
Construct event # (0% to 100%) Condition of insects -Control 26500
(#5) 70-100% eaten Survivors premolt to III Maize UbiI-Bt1 (#8) 0%
eaten All dead neonates ubiquitin UbiI-Bt1 (#12) 5% pinhole damage
All dead neonates UbiI-Bt1 (#18) 2% eaten All dead neonates
UbiI-Bt1 (#47) 0% eaten All dead neonates UbiI-Bt1 (#51) 4% eaten
All dead neonates Banana streak BSV TR-Bt1 (#12) 8% eaten Survivors
still 1sts virus BSV TR-Bt1 (#76) 5% eaten Survivors still 1sts BSV
TR-Bt1 (#82) 0% eaten, stalk All dead neonates damage BSV TR-Bt1
(#105) 10% eaten 3 survivors to II BSV TR-Bt1 (#116) Pinhole damage
All dead neonates BSV TR-Bt1 (#119) 15% eaten 3 survivors to II
Example 10
In Vitro Insect Bioassay Using Transgenic Green Regenerative Tissue
Events
[0115] Our green tissue bioassay system also can provide a method
for pre-screening of genes/promoters. The process is similar to the
in vitro bioassay system that was described in Example 5, but
instead of using maize plantlets, green regenerative tissues were
used. Using all sterile materials, two pieces of filter paper were
placed into a Petri dish and about 1.5 mL of MSA+1.5% PPM solution
were pipetted onto the filter paper. If there was excess solution,
or not enough solution, solution was removed/added until the filter
papers were evenly soaked but not dripping. Five good pieces of
green tissue were selected and placed onto the filter paper; good
callus tissue is defined as a piece about 5 mm, and regenerable,
compact, and green. Also, for all events being screened, there
should be the same amount of green tissues and they should all be
equivalent in quality and size. The Petri dish was infested with
the insect which the gene that was being tested was resistant
against and the plate was sealed with parafilm to prevent
contamination. Two days after infestation, additional solution was
added to keep the tissue healthy. After 6 to 12 days, tissue damage
and insect growth/stunting/death were scored per scoring
protocol.
[0116] Transgenic maize green tissue events transformed with 4
different shuffled Bt variants were tested for WCRW resistance. As
expected, negative control events were susceptible to WCRW and
tissues became brown and some neonates grew to the healthy 2.sup.nd
instars (Table 11). Event #s 48 and 50 transformed with the
shuffled Bt variant 14 showed good efficacy to rootworms while
event #95 transformed with the shuffled Bt variant 1, event #36
transformed with the shuffled Bt variant 2 and event #10 with the
shuffled Bt variant 3 showed slightly to moderately resistant to
rootworms. Clearer results could be obtained when the tissues were
maintained for longer than 2 weeks. Thus, this green tissue
bioassay system can be used for early screening of transgenic
events.
TABLE-US-00011 TABLE 11 WCRW bioassay using T.sub.0 PHR03 green
regenerative tissues Construct Event # Tissue Damage Insect
growth/healthiness 24600 (- control) 6 +++, brown healthy 26650 (-
control) 1 +++, brown healthy Shuffled Bt variant 1 34 ++(+), brown
healthy 59 +++, brown healthy 95 +, most of them green several
dead/stunted Shuffled Bt variant 2 36 +(+), most of them green
several stunted Shuffled Bt variant 3 9 ++(+), brown healthy 10
+(+), some green some stunting Shuffled Bt variant 14 4 ++(+),
brown healthy 48 0, green mortality and stunting 50 (+), green
mortality but some healthy Tissue damage and insect
growth/stunting/death were scored 6 days after WCRW
infestation.
Example 11
In Vitro Bioassay Using Different Explants for Insect
Infestation
[0117] Different types of tissue were used for in vitro insect
bioassay following the similar sterilization and culture protocol
used for the in vitro insect bioassay protocol described above.
Roots, leaves, callus- and green tissue-derived plantlets, and
mature seed- and immature embryo-derived seedlings were used as
target explants for insect infestation. Plantlets and germinating
seedlings were, in general, best for in vitro insect bioassay using
both rootworms and lepidopteras (Table 12).
TABLE-US-00012 TABLE 12 In vitro insect bioassay efficiency using
different in vitro-derived tissue types Germinating Germinating
Green seedling from seedling from Tissue type regenerative
Regenerated mature seed w/ immature seed Insect Root Leaf tissue
Callus plantlet w/ root root w/ root Rootworm +++ + ++(+) n.t. +++
+++ +++ Lepidoptera ++ +++ +++ n.t. +++ +++ +++ *n.t.: not
tested
[0118] All publications and patent applications in this
specification are indicative of the level of ordinary skill in the
art to which this invention pertains. All publications and patent
applications are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated by reference.
[0119] The invention has been described with reference to various
specific and preferred embodiments and techniques. However, it
should be understood that many variations and modifications may be
made while remaining within the spirit and scope of the
invention.
* * * * *