U.S. patent application number 11/590071 was filed with the patent office on 2007-02-22 for genetically engineered duckweed.
Invention is credited to Nirmala Rajbhandari, Anne-Marie Stomp.
Application Number | 20070044177 11/590071 |
Document ID | / |
Family ID | 46281374 |
Filed Date | 2007-02-22 |
United States Patent
Application |
20070044177 |
Kind Code |
A1 |
Stomp; Anne-Marie ; et
al. |
February 22, 2007 |
Genetically engineered duckweed
Abstract
Methods and compositions for the efficient transformation of
duckweed are provided. Preferably, the methods involve
transformation by either ballistic bombardment or Agrobacterium. In
this manner, any gene or nucleic acid of interest can be introduced
and expressed in duckweed plants. Transformed duckweed plants,
cells, tissues are also provided. Transformed duckweed plant tissue
culture and methods of producing recombinant proteins and peptides
from transformed duckweed plants are also disclosed.
Inventors: |
Stomp; Anne-Marie; (Raleigh,
NC) ; Rajbhandari; Nirmala; (Raleigh, NC) |
Correspondence
Address: |
MYERS BIGEL SIBLEY & SAJOVEC
PO BOX 37428
RALEIGH
NC
27627
US
|
Family ID: |
46281374 |
Appl. No.: |
11/590071 |
Filed: |
October 31, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10273974 |
Oct 18, 2002 |
7161064 |
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11590071 |
Oct 31, 2006 |
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09448105 |
Nov 23, 1999 |
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10273974 |
Oct 18, 2002 |
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09132536 |
Aug 11, 1998 |
6040498 |
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09448105 |
Nov 23, 1999 |
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60055474 |
Aug 12, 1997 |
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Current U.S.
Class: |
800/278 ;
435/419 |
Current CPC
Class: |
C12N 15/8205 20130101;
A01H 13/00 20130101; C12N 15/8257 20130101; C12N 15/8207 20130101;
A01H 4/005 20130101 |
Class at
Publication: |
800/278 ;
435/419 |
International
Class: |
A01H 1/00 20060101
A01H001/00; C12N 15/82 20060101 C12N015/82; C12N 5/04 20060101
C12N005/04 |
Goverment Interests
[0002] This invention was made with Government support under grant
number R823570-01-1 from the United States Environmental Protection
Agency. The government has certain rights in this invention.
Claims
1. A stably transformed duckweed tissue culture produced according
to a method comprising the steps of: (a) providing a duckweed
tissue target, the cells of the duckweed tissue culture including
cell walls; and (b) propelling a heterologous nucleotide sequence
of interest at the duckweed tissue target at a velocity sufficient
to pierce the cell walls and deposit the heterologous nucleotide
sequence within a cell of the tissue, wherein the heterologous
nucleotide sequence is carried by a microprojectile; and wherein
the heterologous nucleotide sequence is propelled at the tissue by
propelling the microprojectile at the tissue; and (c) producing a
stably transformed duckweed tissue culture.
2. The stably transformed duckweed tissue culture according to
claim 1, wherein the nucleotide sequence comprises at least one
expression cassette comprising a gene which confers resistance to a
selection agent.
3. The stably transformed tissue culture according to claim 2
further comprising the step of culturing the stably transformed
tissue culture with the selection agent.
4. The stably transformed duckweed tissue culture according to
claim 2, wherein the gene which confers resistance to a selection
agent is selected from the group consisting of neo, bar, pat, ALS,
HPH, HYG, EPSP and Hml.
5. The stably transformed duckweed tissue culture according to
claim 1, wherein the nucleotide sequence comprises two genes of
interest.
6. The stably transformed duckweed tissue culture according to
claim 1, wherein the nucleotide sequence encodes a protein or
peptide selected from the group consisting of insulin, growth
hormone, .alpha.-interferon, .beta.-glucocerebrosidase,
retinoblastoma protein, p53 protein, angiostatin, leptin, and serum
albumin.
7. The stably transformed duckweed tissue culture according to
claim 1, wherein the nucleotide sequence encodes at least one
protein or peptide subunit of a multimeric protein selected from
the group consisting of hemoglobin, collagen, P450 oxidase, and a
monoclonal antibody.
8. The stably transformed duckweed tissue culture according to
claim 1, wherein the nucleotide sequence encodes a secreted protein
or peptide.
9. The stably transformed duckweed tissue culture according to
claim 1, wherein the duckweed tissue culture is a callus tissue
culture.
10. The stably transformed duckweed callus tissue culture according
to claim 9, wherein the duckweed callus tissue culture is a Type I
callus tissue culture.
11. The stably transformed duckweed tissue culture according to
claim 1, wherein the duckweed tissue culture is a Spirodela,
Wolffia, Wolfiella, or Lemna tissue culture.
12. The stably transformed duckweed tissue culture according to
claim 11, wherein the duckweed tissue culture is a Lemna tissue
culture.
13. The stably transformed duckweed tissue culture according to
claim 12, wherein the duckweed tissue culture is a Lemna minor,
Lemna miniscula, or Lemna gibba tissue culture.
14. The stably transformed duckweed tissue culture according to
claim 13, wherein the duckweed tissue culture is a Lemna minor
tissue culture.
15. A stably transformed duckweed tissue culture comprising a
chimeric nucleotide sequence of interest, said chimeric nucleotide
sequence comprising a coding sequence operably linked to a
transcription initiation region that is heterologous to said coding
sequence, the method comprising the steps of: (a) providing a
duckweed tissue culture target, the cells of the duckweed tissue
culture including cell walls; and (b) propelling a chimeric
nucleotide sequence of interest at the duckweed tissue culture
target at a velocity sufficient to pierce the cell walls and
deposit the chimeric nucleotide sequence within a cell of the
tissue, wherein the chimeric nucleotide sequence is carried by a
microprojectile; and wherein the chimeric nucleotide sequence is
propelled at the tissue by propelling the microprojectile at the
tissue; and (c) producing a stably transformed duckweed tissue
culture.
16. The stably transformed duckweed tissue culture according to
claim 15, wherein the chimeric nucleotide sequence comprises a
duckweed coding sequence operably linked to a transcription
initiation region that is heterologous to the coding sequence.
17. The stably transformed duckweed tissue culture according to
claim 15, wherein the chimeric nucleotide sequence of interest
comprises at least one expression cassette comprising a gene which
confers resistance to a selection agent.
18. The stably transformed tissue culture according to claim 17
further comprising the step of culturing the stably transformed
tissue culture with the selection agent.
19. The stably transformed duckweed tissue culture according to
claim 17, wherein the gene which confers resistance to a selection
agent is selected from the group consisting of neo, bar, pat, ALS,
HPH, HYG, EPSP and Hml.
20. The stably transformed duckweed tissue culture according to
claim 15, wherein the chimeric nucleotide sequence comprises two
genes of interest.
21. The stably transformed duckweed tissue culture according to
claim 15, wherein the chimeric nucleotide sequence encodes a
protein or peptide selected from the group consisting of insulin,
growth hormone, .alpha.-interferon, .beta.-glucocerebrosidase,
retinoblastoma protein, p53 protein, angiostatin, leptin, and serum
albumin.
22. The stably transformed duckweed tissue culture according to
claim 15, wherein the chimeric nucleotide sequence encodes at least
one protein or peptide subunit of a multimeric protein selected
from the group consisting of hemoglobin, collagen, P450 oxidase,
and a monoclonal antibody.
23. The stably transformed duckweed tissue culture according to
claim 15, wherein the chimeric nucleotide sequence encodes a
secreted protein or peptide.
24. The stably transformed duckweed tissue culture according to
claim 15, wherein the duckweed tissue culture is a callus tissue
culture.
25. The stably transformed duckweed callus tissue culture according
to claim 24, wherein the duckweed callus tissue culture is a Type I
callus tissue culture.
26. The stably transformed duckweed tissue culture according to
claim 15, wherein the duckweed tissue culture is a Spirodela,
Wolffia, Wolfiella, or Lemna tissue culture.
27. The stably transformed duckweed tissue culture according to
claim 26, wherein the duckweed tissue culture is a Lemna tissue
culture.
28. The stably transformed duckweed tissue culture according to
claim 27, wherein the duckweed tissue culture is a Lemna minor,
Lemna miniscula, or Lemna gibba tissue culture.
29. The stably transformed duckweed tissue culture according to
claim 28, wherein the duckweed tissue culture is a Lemna minor
tissue culture.
Description
RELATED APPLICATION INFORMATION
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/273,974, filed Oct. 18, 2002, which is a
continuation of U.S. patent application Ser. No. 09/448,105 filed
23 Nov. 1999, which is a divisional of U.S. patent application Ser.
No. 09/132,536 filed 11 Aug. 1998, which claims the benefit of U.S.
Provisional Application No. 60/055,474 filed 12 Aug. 1997, the
disclosures of which are incorporated by reference herein in their
entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to methods and compositions
for the transformation of duckweed, particularly to methods for
transformation utilizing ballistic bombardment and
Agrobacterium.
BACKGROUND OF THE INVENTION
[0004] The duckweeds are the sole members of the monocotyledonous
family, Lemnaceae. The four genera and 34 species are all small,
free-floating, fresh-water plants whose geographical range spans
the entire globe. Landolt, Biosystematic Investigation on the
Family of Duckweeds: The family of Lemnaceae--A Monograph Study.
Geobatanischen Institut ETH, Stiftung Rubel, Zurich (1986).
Although the most morphologically reduced plants known, most
duckweed species have all the tissues and organs of much larger
plants, including roots, stems, flowers, seeds and fronds. Duckweed
species have been studied extensively and a substantial literature
exists detailing their ecology, systematics, life-cycle,
metabolism, disease and pest susceptibility, their reproductive
biology, genetic structure, and cell biology. Hillman, Bot. Review
27, 221 (1961); Landolt, Biosystematic Investigation on the Family
of Duckweeds: The family of Lemnaceae--A Monograph Study.
Geobatanischen Institut ETH, Stiftung Rubel, Zurich (1986).
[0005] The growth habit of the duckweeds is ideal for microbial
culturing methods. The plant rapidly proliferates through
vegetative budding of new fronds, in a macroscopic manner analogous
to asexual propagation in yeast. Duckweed proliferates by
vegetative budding from meristematic cells. The meristematic region
is small and is found on the ventral surface of the frond.
Meristematic cells lie in two pockets, one on each side of the
frond midvein. The small midvein region is also the site from which
the root originates and the stem arises that connects each frond to
its mother frond. The meristematic pocket is protected by a tissue
flap. Fronds bud alternately from these pockets. Doubling times
vary by species and are as short as 20-24 hours. Landolt, Ber.
Schweiz. Bot. Ges. 67, 271 (1957); Chang et al., Bull. Inst. Chem.
Acad. Sin. 24, 19 (1977); Datko and Mudd., Plant Physiol. 65,16
(1980); Venkataraman et al., Z. Pflanzenphysiol. 62, 316
(1970).
[0006] Intensive culture of duckweed results in the highest rates
of biomass accumulation per unit time (Landolt and Kandeler, The
family of Lemnaceae--A Monographic Study. Vol. 2: Phytochemistry,
Physiology, Application, Bibliography, Veroffentlichungen des
Geobotanischen Institutes ETH, Stiftung Rubel, Zurich (1987)), with
dry weight accumulation ranging from 6-10% of fresh weight
(Tillberg et al., Physiol. Plant. 46, 5 (1979); Landolt, Ber.
Schweiz. Bot. Ges. 67, 271 (1957); Stomp, unpublished data).
Protein content of a number of duckweed species grown under varying
conditions has been reported to range from 15-45% dry weight (Chang
et al, Bull. Inst. Chem. Acad. Sin. 24, 19 (1977); Chang and Chui,
Z. Pflanzenphysiol. 89, 91 (1978); Porath et al., Aquatic Botany 7,
272 (1979); Appenroth et al., Biochem. Physiol. Pflanz. 177, 251
(1982)). Using these values, the level of protein production per
liter of medium in duckweed is on the same order of magnitude as
yeast gene expression systems. Prior to now, the systematic
optimization of medium components and culturing conditions for
maximal growth and protein content for specific duckweed strains
has not been done.
[0007] Sexual reproduction in duckweed is controlled by medium
components and culturing conditions, including photoperiod and
culture density. Flower induction is a routine laboratory procedure
with some species. Plants normally self-pollinate and selfing can
be accomplished in the laboratory by gently shaking cultures. By
this method, inbred lines of Lemna gibba have been developed.
Spontaneous mutations have been identified (Slovin and Cohen, Plant
Physiol. 86, 522 (1988)), and chemical and gamma ray mutagenesis
(using EMS or NMU) have been used to produce mutants with defined
characteristics. Outcrossing of L. gibba is tedious but can be done
by controlled, hand pollination. The genome size of the duckweeds
varies from 0.25-1.63 pg DNA/2C with chromosome counts ranging from
20 to 80 and averaging about 40 across the Lemnaceae (Landolt,
Biosystematic Investigation on the Family of Duckweeds: The family
of Lemnaceae--A Monograph Study. Geobatanischen Institut ETH,
Stiftung Rubel, Zurich (1986)). Ploidy levels are estimated to
range from 2-12 C. Id. Genetic diversity within the Lemnaceae has
been investigated using secondary products, isozymes, and DNA
sequences. McClure and Alston, Nature 4916, 311 (1964); McClure and
Alston, Amer. J. Bot. 53, 849 (1966); Vasseur et al., Pl. Syst.
Evol. 177, 139 (1991); Crawford and Landolt, Syst. Bot. 10, 389
(1993).
[0008] Accordingly, the characteristics described above make
duckweed an ideal choice to develop as an efficient, plant-based,
gene expression system.
SUMMARY OF THE INVENTION
[0009] The present invention is drawn to methods and compositions
for the efficient transformation of duckweed. The methods involve
the use of ballistic bombardment, Agrobacterium, or electroporation
to stably introduce and express a nucleotide sequence of interest
in transformed duckweed plants. In this manner, any gene(s) or
nucleic acid(s) of interest can be introduced into the duckweed
plant. Transformed duckweed cells, tissues, plants and seed are
also provided.
[0010] As a first aspect, the present invention provides a method
for transforming duckweed with a nucleotide sequence of interest,
wherein said nucleotide sequence comprises at least an expression
cassette containing a gene which confers resistance to a selection
agent, the method comprising the steps of: (a) providing a duckweed
tissue target, the cells of the duckweed tissue including cell
walls; and (b) propelling the nucleotide sequence at the duckweed
tissue target at a velocity sufficient to pierce the cell walls and
deposit the nucleotide sequence within a cell of the tissue to
thereby produce a transformed tissue, wherein the nucleotide
sequence is carried by a microprojectile; and wherein the
nucleotide sequence is propelled at the tissue by propelling the
microprojectile at the tissue.
[0011] As a second aspect, the present invention provides a method
for transforming duckweed with a nucleotide sequence of interest,
the method comprising the steps of: (a) inoculating a duckweed
plant tissue with an Agrobacterium comprising a vector which
comprises the nucleotide sequence, wherein the nucleotide sequence
comprises at least an expression cassette containing a gene which
confers resistance to a selection agent; and (b) co-cultivating the
tissue with the Agrobacterium to produce transformed tissue.
[0012] As a third aspect, the present invention provides a method
of transforming duckweed by electroporation.
[0013] As a fourth aspect, the present invention provides
transformed duckweed plants and transformed duckweed tissue culture
produced by the methods described above.
[0014] As a fifth aspect, the present invention provides a
transformed duckweed plant and methods of using transformed
duckweed plants to produce a recombinant protein or peptide.
[0015] Duckweed offers an ideal plant-based gene expression system.
A duckweed gene expression system provides the pivotal technology
that would be useful for a number of research and commercial
applications. For plant molecular biology research as a whole, a
differentiated plant system which can be manipulated with the
laboratory convenience of yeast provides a very fast system in
which to analyze the developmental and physiological roles of
isolated genes. Model plants such as tobacco and Arabidopsis are
currently used for this purpose by plant molecular biologists.
These plants require greenhouse or field facilities for growth
(often difficult for plant molecular biologists to obtain).
Alternative gene expression systems are based on microbial or cell
cultures where tissue and developmentally regulated gene expression
effects are lost. Heterologous gene expression systems also require
restructuring of the gene of interest prior to insertion, an
expensive and time-consuming process. A duckweed system overcomes
both of these problems and is far easier to grow and maintain in a
laboratory setting. If it is desirable to harvest the expressed
proteins or peptides (or molecules produced thereby), this can be
accomplished by any suitable technique known in the art, such as
mechanical grinding or lysing of cells.
[0016] For commercial production of valuable proteins, a
duckweed-based system has a number of advantages over existing
microbial or cell culture systems. In the area of mammalian protein
production, plants show post-translational processing that is
similar to mammalian cells, overcoming one major problem associated
with microbial cell production of mammalian proteins. Duckweed is
also far cheaper to produce than mammalian cell cultures. It has
already been shown by others (Hiatt, Nature 334, 469 (1990)) that
plant systems have the ability to assemble multi-subunit proteins,
an ability often lacking in microbial systems. Plant production of
therapeutic proteins also limits the risk from contaminating
substances, including animal viruses, produced in mammalian cell
cultures and in microbial systems. Contaminating substances are a
major concern in therapeutic protein production. Unlike other
suggested plant production systems, e.g., soybeans and tobacco,
duckweed can be grown in fermentor/bioreactor vessels, making the
system's integration into existing protein production industrial
infrastructure far easier.
[0017] As a manufacturing platform for lower cost industrial
enzymes and small molecules, duckweed offers the advantage that
production is readily scaleable to almost any quantity because it
can be grown under field conditions using nutrient-rich wastewater.
A genetically engineered duckweed system growing on wastewater
could produce a valuable product while simultaneously cleaning up
wastewater for reuse. Such a system would turn a net capital loss
(remediation of wastewater from discharge) into a chemical or
enzyme production system with a positive economic balance.
Duckweeds' advantage over chemical syntheses in field crops is that
production does not require arable crop land or irrigation water
necessary to increase food production for the world's increasing
population.
[0018] These and other aspects of the present invention are
disclosed in more detail in the description of the invention given
below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 presents an autoradiograph produced by Southern
hybridization of untransformed duckweed DNA and transformed
duckweed DNA from line D with a radioactively labeled 3.2 kb
fragment from pBI121 containing the GUS gene. Channels: 1)
Isolated, undigested pBI121 DNA. The expected major band is at 12.8
kb. The lower molecular weight band is probably represents the
supercoiled plasmid. 2) HindIII digested, pBI121 DNA. This
digestion linearizes the plasmid and shows the expected 12.8 kb
band. The lower molecular weight band indicates incomplete
digestion. 3) pBI121 DNA digested with both HindIII and EcoR1.
Digestion was incomplete but yielded the expected bands: 12.8 kb
(left from incomplete digestion), the approximately 9 kb band, and
a faint supercoiled band. The 3.2 kb band did not give visible
hybridization in this exposure. 4) DNA from untransformed duckweed
with the equivalent of 1 copy of doubly-digested, pBI121 DNA giving
the expected 9 and 3.2 kb bands. 5) Untransformed duckweed DNA. 6)
Undigested DNA from transformed duckweed line D. 7) HindIII
digested DNA from transformed duckweed line D. 8) HindIII and EcoR1
digested DNA from transformed duckweed-line D.
DETAILED DESCRIPTION OF THE INVENTION
[0020] The present invention is directed to methods for
transforming duckweed. In preferred embodiments, the methods
utilize ballistic bombardment or Agrobacterium to stably transform
the duckweed cells. Alternately, the methods use electroporation to
transform duckweed. The methods and transformed plants of the
present invention find use as a plant-based gene expression system
possessing many of the advantages of yeast.
[0021] As far as the inventors are aware, there are no previous
reports of stable gene transfer in duckweed nor of regeneration of
transformed duckweed plants. In the present investigations two
strategies have been utilized for the production of transgenic
duckweed plants: (1) By directly transferring and inserting foreign
DNA into meristematic frond cells followed by asexual propagation
and selfing to produce transgenic duckweed (a plant-to-plant
system), and (2) Transformation of undifferentiated callus cells,
followed by selection of proliferating callus, and frond
regeneration (a callus-to-plant system). Limited tissue culture
systems for callus production from L. gibba and L. minor have
previously been reported by Chang's group (Chang and Chui, Bot.
Bull. Academia Sinica 17, 106 (1976); Chang and Chui, Z.
Pflanzenphysiol. Bd. 89.S, 91 (1978)) and Frick (Frick, J. Plant
Physiology 137, 397 (1991)), respectively. The present
investigations have significantly extended the work in this area by
developing an organized callus system that regenerates fronds.
[0022] Preferably, the present invention utilizes one of two
systems to stably transform duckweed: ballistic transformation
using microprojectile bombardment or Agrobacterium-mediated
transformation. Although duckweeds would be expected to be
refractory to Agrobacterium transformation because they are
monocotyledonous plants, it has unexpectedly been found that
duckweed can be transformed using Agrobacterium. Transformed
duckweed plants according to the present invention may also be
generated by electroporation. See, e.g., Dekeyser et al., Plant
Cell 2, 591 (1990); D'Halluin et al., Plant Cell 4, 1495 (1992);
U.S. Pat. No. 5,712,135 to D'Halluin et al. One advantage of
electroporation is that large pieces of DNA, including artificial
chromosomes, can be transformed into duckweed by this method. Any
suitable duckweed cell or tissue type can be transformed according
to the present invention. For example, nucleic acids can be
introduced into duckweed cells in tissue culture. Alternately, the
small size and aquatic growth habit of duckweed plants allows for
nucleic acids to be introduced into duckweed cells of intact
embryos, fronds, roots, and other organized tissues, such as
meristematic tissue. As a further alternative, nucleic acids can be
introduced into duckweed callus.
[0023] It is preferred that the transformed duckweed plants
produced by the claimed methods exhibit normal morphology and are
fertile by sexual reproduction. Preferably, transformed plants of
the present invention contain a single copy of the transferred
nucleic acid, and the transferred nucleic acid has no notable
rearrangements therein. Also preferred are duckweed plants in which
the transferred nucleic acid is present in low copy numbers (i.e.,
no more than five copies, alternately, no more than three copies,
as a further alternative, fewer than three copies of the nucleic
acid per transformed cell).
[0024] The term "duckweed", as used herein, refers to members of
the family Lemnaceae. There are known four genera and 34 species of
duckweed as follows: genus Lemna (L. aequinoctialis, L. disperma,
L. ecuadoriensis, L. gibba, L. japonica, L. minor, L. miniscula, L.
obscura, L. perpusilla, L. tenera, L. trisulca, L. turionifera, L.
valdiviana); genus Spirodela (S. intermedia, S. polyrrhiza, S.
punctata); genus Wolffia (Wa. angusta, Wa. arrhiza, Wa. australina,
Wa. borealis, Wa. brasiliensis, Wa. columbiana, Wa. elongata, Wa.
globosa, Wa. microscopica, Wa. neglecta) and genus Wolfiella (Wl.
caudata, Wl. denticulata, Wl. gladiata, Wl. hyalina, Wl. lingulata,
Wl. repunda, Wl. rotunda, and Wl. neotropica). Any other genera or
species of Lemnaceae, if they exist, are also aspects of the
present invention. Lemna gibba, Lemna minor, and Lemna miniscula
are preferred, with Lemna minor and Lemna miniscula being most
preferred. Lemna species can be classified using the taxonomic
scheme described by Landolt, Biosystematic Investigation on the
Family of Duckweeds: The family of Lemnaceae--A Monograph Study.
Geobatanischen Institut ETH, Stiftung Rubel, Zurich (1986)).
[0025] As will be evident to one of skill in the art, now that a
method has been provided for the efficient transformation of
duckweed, any nucleic acid of interest can be used in the methods
of the invention. For example, a duckweed plant can be engineered
to express disease and insect resistance genes, genes conferring
nutritional value, antifungal, antibacterial or antiviral genes,
and the like. Alternatively, therapeutic (e.g., for veterinary or
medical uses) or immunogenic (e.g., for vaccination) peptides and
proteins can be expressed using transformed duckweed according to
the present invention.
[0026] Likewise, the method can be used to transfer any nucleic
acid for controlling gene expression. For example, the nucleic acid
to be transferred can encode an antisense oligonucleotide.
Alternately, duckweed can be transformed with one or more genes to
reproduce enzymatic pathways for chemical synthesis (e.g., for the
synthesis of plastics) or other industrial processes (e.g.,
keratinase). The nucleic acid may be from duckweed or from another
organism (i.e., heterologous). Moreover, nucleic acids of interest
can be obtained from prokaryotes or eukaryotes (e.g., bacteria,
fungi, yeast, viruses, plants, mammals) or the nucleic acid
sequence can be synthesized in whole or in part. In particular
preferred embodiments, the nucleic acid encodes a secreted protein
or peptide.
[0027] Preferably, the transferred nucleic acid to be expressed in
the transformed duckweed encodes a protein hormone, growth factor,
or cytokine, more preferably, insulin, growth hormone (in
particular, human growth hormone), and .alpha.-interferon.
Alternatively, it is also preferred that the nucleic acid expresses
.beta.-glucocerebrosidase.
[0028] Also preferred are nucleic acids that encode peptides or
proteins that cannot effectively be commercially-produced by
existing gene expression systems, because of cost or logistical
constraints, or both. For example, some proteins cannot be
expressed in mammalian systems because the protein interferes with
cell viability, cell proliferation, cellular differentiation, or
protein assembly in mammalian cells. Such proteins include, but are
not limited to, retinoblastoma protein, p53, angiostatin and
leptin. The present invention can be advantageously employed to
produce mammalian regulatory proteins; it is unlikely given the
large evolutionary distance between higher plants and mammals that
these proteins will interfere with regulatory processes in
duckweed. Transgenic duckweed can also be used to produce large
quantities of proteins such as serum albumin (in particular, human
serum albumin), hemoglobin and collagen, which challenge the
production capabilities of existing expression systems.
[0029] Finally, as described in more detail below, higher plant
systems can be engineered to produce (i.e., synthesize, express,
assemble) biologically-active multimeric proteins (e.g., monoclonal
antibodies, hemoglobin, P450 oxidase, and collagen, and the like)
far more easily than can mammalian systems. Those skilled in the
art will appreciate that the term "biologically active" includes
multimeric proteins in which the biological activity is altered as
compared with the native protein (e.g, suppressed or enhanced), as
long as the protein has sufficient activity to be of interest for
use in industrial or chemical processes or as a therapeutic,
vaccine, or diagnostics reagent.
[0030] One exemplary approach for producing biologically-active
multimeric proteins in duckweed uses an expression vector
containing the genes encoding all of the polypeptide subunits. See,
e.g., During et al. (1990) Plant Molecular Biology 15:281; van
Engelen et al., (1994) Plant Molecular Biology 26:1701. This vector
is then introduced into duckweed cells using any known
transformation method, such as a gene gun or Agrobacterium-mediated
transformation. This method results in clonal cell lines that
express all of the polypeptides necessary to assemble the
multimeric protein. As one alternate method, independent vector
constructs are made that encode each polypeptide subunit. Each of
these vectors is used to generate separate clonal lines of
transgenic plants expressing only one of the necessary
polypeptides. These transgenic plants are then crossed to create
progeny that express all of the necessary polypeptides within a
single plant. See Hiatt et al., (1989) Nature 342:76; U.S. Pat.
Nos. 5,202,422 and 5,639,947 to Hiatt et al. A variation on this
approach is to make single gene constructs, mix DNA from these
constructs together, then deliver this mixture of DNAs into plant
cells using ballistic bombardment or Agrobacterium-mediated
transformation, more preferably ballistic bombardment. As a further
variation, some or all of the vectors may encode more than one
subunit of the multimeric protein (i.e., so that there are fewer
duckweed clones to be crossed than the number of subunits in the
multimeric protein). Finally, in some instances, it may be
desirable to produce less than all of the subunits of a multimeric
protein, or even a single protein subunit, in a transformed
duckweed plant, e.g., for industrial or chemical processes or for
diagnostic, therapeutic or vaccination purposes.
A. Expression Cassettes.
[0031] According to the present invention, the nucleic acid to be
transferred is contained within an expression cassette. The
expression cassette comprises a transcriptional initiation region
linked to the nucleic acid or gene of interest. Such an expression
cassette is provided with a plurality of restriction sites for
insertion of the gene or genes of interest (e.g., one gene of
interest, two genes of interest, etc.) to be under the
transcriptional regulation of the regulatory regions. Preferably,
the expression cassette encodes a single gene of interest. In
particular embodiments of the invention, the nucleic acid to be
transferred contains two or more expression cassettes, each of
which encodes at least one gene of interest (preferably one gene of
interest).
[0032] The transcriptional initiation region, (e.g., a promoter)
may be native or homologous or foreign or heterologous to the host,
or could be the natural sequence or a synthetic sequence. By
foreign, it is intended that the transcriptional initiation region
is not found in the wild-type host into which the transcriptional
initiation region is introduced. As used herein a chimeric gene
comprises a coding sequence operably linked to a transcription
initiation region that is heterologous to the coding sequence.
[0033] Any suitable promoter known in the art can be employed
according to the present invention (including bacterial, yeast,
fungal, insect, mammalian, and plant promoters). Plant promoters
are preferred, with duckweed promoters being most preferred.
Exemplary promoters include, but are not limited to, the
Cauliflower Mosaic Virus 35S promoter, the opine synthetase
promoters (e.g., nos, mas, ocs, etc.), the ubiquitin promoter, the
actin promoter, the ribulose bisphosphate (RubP) carboxylase small
subunit promoter, and the alcohol dehydrogenase promoter. The
duckweed RubP carboxylase small subunit promoter is known in the
art. Silverthrone et al., (1990) Plant Mol. Biol. 15:49. Other
promoters from viruses that infect plants, preferably duckweed, are
also suitable including, but not limited to, promoters isolated
from Dasheen mosaic virus, Chlorella virus (e.g., the Chlorella
virus adenine methyltransferase promoter; Mitra et al., (1994)
Plant Molecular Biology 26:85), tomato spotted wilt virus, tobacco
rattle virus, tobacco necrosis virus, tobacco ring spot virus,
tomato ring spot virus, cucumber mosaic virus, peanut stump virus,
alfalfa mosaic virus, and the like.
[0034] Finally, promoters can be chosen to give a desired level of
regulation. For example, in some instances, it may be advantageous
to use a promoter that confers constitutive expression (e.g. the
ubiquitin promoter, the RubP carboxylase gene family promoters, and
the actin gene family promoters). Alternatively, it other
situations, it may be advantageous to use promoters that are
activated in response to specific environmental stimuli (e.g., heat
shock gene promoters, drought-inducible gene promoters,
pathogen-inducible gene promoters, wound-inducible gene promoters,
and light/dark-inducible gene promoters) or plant growth regulators
(e.g., promoters from genes induced by abscissic acid, auxins,
cytokinins, and gibberellic acid). As a further alternative,
promoters can be chosen that give tissue-specific expression (e.g.,
root, leaf and floral-specific promoters).
[0035] The transcriptional cassette includes in the 5'-3' direction
of transcription, a transcriptional and translational initiation
region, a nucleotide sequence of interest, and a transcriptional
and translational termination region functional in plants. Any
suitable termination sequence known in the art may be used in
accordance with the present invention. The termination region may
be native with the transcriptional initiation region, may be native
with the nucleotide sequence of interest, or may be derived from
another source. Convenient termination regions are available from
the Ti-plasmid of A. tumefaciens, such as the octopine synthetase
and nopaline synthetase termination regions. See also, Guerineau et
al., Mol. Gen. Genet. 262, 141 (1991); Proudfoot, Cell 64, 671
(1991); Sanfacon et al., Genes Dev. 5,141 (1991); Mogen et al.,
Plant Cell 2, 1261 (1990); Munroe et al., Gene 91, 151 (1990);
Ballas et al., Nucleic Acids Res. 17, 7891 (1989); and Joshi et
al., Nucleic Acids Res. 15, 9627 (1987). Additional exemplary
termination sequences are the pea RubP carboxylase small subunit
termination sequence and the Cauliflower Mosaic Virus 35S
termination sequence. Other suitable termination sequences will be
apparent to those skilled in the art.
[0036] Alternatively, the gene(s) of interest can be provided on
any other suitable expression cassette known in the art. Where
appropriate, the gene(s) may be optimized for increased expression
in the transformed plant. Where mammalian, yeast or bacterial or
plant dicot genes are used in the invention, they can be
synthesized using monocot or duckweed preferred codons for improved
expression. Methods are available in the art for synthesizing plant
preferred genes. See, e.g., U.S. Pat. Nos. 5,380,831; 5,436,391;
and Murray et al., Nucleic Acids. Res. 17, 477 (1989); herein
incorporated by reference.
[0037] The expression cassettes may additionally contain 5' leader
sequences. Such leader sequences can act to enhance translation.
Translation leaders are known in the art and include: picornavirus
leaders, e.g., EMCV leader (Encephalomyocarditis 5' noncoding
region; Elroy-Stein et al., Proc. Natl. Acad. Sci USA, 86, 6126
(1989)).; potyvirus leaders, e.g., TEV leader (Tobacco Etch Virus;
Allison et al., Virology, 154, 9 (1986)); human immunoglobulin
heavy-chain binding protein (BiP; Macajak and Sarnow, Nature 353,
90 (1991)); untranslated leader from the coat protein mRNA of
alfalfa mosaic virus (AMV RNA 4; Jobling and Gehrke, Nature 325,
622 (1987)); tobacco mosaic virus leader (TMV; Gallie, MOLECULAR
BIOLOGY OF RNA, 237-56 (1989)); and maize chlorotic mottle virus
leader (MCMV; Lommel et al., Virology 81, 382 (1991)). See also,
Della-Cioppa et al., Plant Physiology 84, 965 (1987). Other methods
known to enhance translation can also be utilized, e.g., introns
and the like.
[0038] The exogenous nucleic acid of interest may additionally be
operably associated with a nucleic acid sequence that encodes a
transit peptide that directs expression of the encoded peptide or
protein of interest to a particular cellular compartment. Transit
peptides that target protein accumulation in higher plants cells to
the chloroplast, mitochondrion, vacuole, nucleus, and the
endoplasmic reticulum (for secretion outside of the cell) are known
in the art. Preferably, the transit peptide targets the protein
expressed from the exogenous nucleic acid to the chloroplast or the
endoplasmic reticulum. Transit peptides that target proteins to the
endoplasmic reticulum are desirable for correct processing of
secreted proteins. Targeting protein expression to the chloroplast
(for example, using the transit peptide from the RubP carboxylase
small subunit gene) has been shown to result in the accumulation of
very high concentrations of recombinant protein in this organelle.
A duckweed nucleic acid encoding an RubP carboxylase transit
peptide has already been cloned. Stiekma et al., (1983) Nucl. Acids
Res. 11:8051-61; see also U.S. Pat. Nos. 5,717,084 and 5,728,925 to
Herrera-Estrella et al. The pea RubP carboxylase small subunit
transit peptide sequence has been used to express and target
mammalian genes in plants. U.S. Pat. Nos. 5,717,084 and 5,728,925
to Herrera-Estrella et al. Alternatively, mammalian transit
peptides can be used to target recombinant protein expression, for
example, to the mitochondrion and endoplasmic reticulum. It has
been demonstrated that plant cells recognize mammalian transit
peptides that target endoplasmic reticulum. U.S. Pat. Nos.
5,202,422 and 5,639,947 to Hiatt et al.
[0039] The expression cassettes may contain more than one gene or
nucleic acid sequence to be transferred and expressed in the
transformed plant. Thus, each nucleic acid sequence will be
operably linked to 5' and 3' regulatory sequences. Alternatively,
multiple expression cassettes may be provided.
[0040] Generally, the expression cassette will comprise a
selectable marker gene for the selection of transformed cells.
Selectable marker genes are utilized for the selection of
transformed cells or tissues. Selectable marker genes include genes
encoding antibiotic resistance, such as those encoding neomycin
phosphotransferase II (NEO) and hygromycin phosphotransferase
(HPT), as well as genes conferring resistance to herbicidal
compounds. Herbicide resistance genes generally code for a modified
target protein insensitive to the herbicide or for an enzyme that
degrades or detoxifies the herbicide in the plant before it can
act. See, DeBlock et al., EMBO J. 6, 2513 (1987); DeBlock et al.,
Plant Physiol. 91, 691 (1989); Fromm et al., BioTechnology 8, 833
(1990); Gordon-Kamm et al., Plant Cell 2, 603 (1990). For example,
resistance to glyphosphate or sulfonylurea herbicides has been
obtained using genes coding for the mutant target enzymes,
5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and
acetolactate synthase (ALS). Resistance to glufosinate ammonium,
boromoxynil, and 2,4-dichlorophenoxyacetate (2,4-D) have been
obtained by using bacterial genes encoding phosphinothricin
acetyltransferase, a nitrilase, or a 2,4-dichlorophenoxyacetate
monooxygenase, which detoxify the respective herbicides.
[0041] For purposes of the present invention, selectable marker
genes include, but are not limited to, genes encoding: neomycin
phosphotransferase II (Fraley et al., CRC Critical Reviews in Plant
Science 4, 1 (1986)); cyanamide hydratase (Maier-Greiner et al.,
Proc. Natl. Acad. Sci. USA 88, 4250 (1991)); aspartate kinase;
dihydrodipicolinate synthase (Perl et al., BioTechnology 11, 715
(1993)); bar gene (Toki et al., Plant Physiol. 100, 1503 (1992);
Meagher et al., Crop Sci. 36, 1367 (1996)); tryptophane
decarboxylase (Goddijn et al., Plant Mol. Biol. 22, 907 (1993));
neomycin phosphotransferase (NEO; Southern et al., J. Mol. Appl.
Gen. 1, 327 (1982)); hygromycin phosphotransferase (HPT or HYG;
Shimizu et al., Mol. Cell. Biol. 6, 1074 (1986)); dihydrofolate
reductase (DHFR; Kwok et al., Proc. Natl. Acad. Sci. USA vol, 4552
(1986)); phosphinothricin acetyltransferase (DeBlock et al., EMBO
J. 6, 2513 (1987)); 2,2-dichloropropionic acid dehalogenase
(Buchanan-Wollatron et al., J. Cell. Biochem. 13D, 330 (1989));
acetohydroxyacid synthase (U.S. Pat. No. 4,761,373 to Anderson et
al.; Haughn et al., Mol. Gen. Genet. 221, 266 (1988));
5-enolpyruvyl-shikimate-phosphate synthase (aroA; Comai et al.,
Nature 317, 741 (1985)); haloarylnitrilase (WO 87/04181 to Stalker
et al.); acetyl-coenzyme A carboxylase (Parker et al., Plant
Physiol. 92, 1220 (1990)); dihydropteroate synthase (sulI;
Guerineau et al., Plant Mol. Biol. 15, 127 (1990)); and 32 kDa
photosystem II polypeptide (psbA; Hirschberg et al., Science 222,
1346 (1983)).
[0042] Also included are genes encoding resistance to:
chloramphenicol (Herrera-Estrella et al., EMBO J. 2, 987 (1983));
methotrexate (Herrera-Estrella et al., Nature 303, 209 (1983);
Meijer et al., Plant Mol. Biol. 16, 807 (1991)); hygromycin
(Waldron et al., Plant Mol. Biol. 5, 103 (1985); Zhijian et al.,
Plant Science 108, 219 (1995); Meijer et al., Plant Mol. Bio. 16,
807 (1991)); streptomycin (Jones et al., Mol. Gen. Genet. 210, 86
(1987)); spectinomycin (Bretagne-Sagnard et al., Transgenic Res. 5,
131 (1996)); bleomycin (Hille et al., Plant Mol. Biol. 7, 171
(1986)); sulfonanide (Guerineau et al., Plant Mol. Bio. 15, 127
(1990); bromoxynil (Stalker et al., Science 242, 419 (1988)); 2,4-D
(Streber et al., Bio/Technology 7, 811 (1989)); phosphinothricin
(DeBlock et al., EMBO J. 6, 2513 (1987)); spectinomycin
(Bretagne-Sagnard and Chupeau, Transgenic Research 5, 131
(1996)).
[0043] The bar gene confers herbicide resistance to
glufosinate-type herbicides, such as phosphinothricin (PPT) or
bialaphos, and the like. As noted above, other selectable markers
that could be used in the vector constructs include, but are not
limited to, the pat gene, also for bialaphos and phosphinothricin
resistance, the ALS gene for imidazolinone resistance, the HPH or
HYG gene for hygromycin resistance, the EPSP synthase gene for
glyphosate resistance, the Hml gene for resistance to the Hc-toxin,
and other selective agents used routinely and known to one of
ordinary skill in the art. See generally, Yarranton, Curr. Opin.
Biotech. 3, 506 (1992); Chistopherson et al., Proc. Natl. Acad.
Sci. USA 89, 6314 (1992); Yao et al., Cell 71, 63 (1992);
Reznikoff, Mol. Microbiol. 6, 2419 (1992); BARKLEY ET AL., THE
OPERON 177-220 (1980); Hu et al., Cell 48, 555 (1987); Brown et
al., Cell 49, 603 (1987); Figge et al., Cell 52, 713 (1988);
Deuschle et al., Proc. Natl. Acad. Sci. USA 86, 5400 (1989); Fuerst
et al., Proc. Natl. Acad. Sci. USA 86, 2549 (1989); Deuschle et
al., Science 248, 480 (1990); Labow et al., Mol. Cell. Biol. 10,
3343 (1990); Zambretti et al., Proc. Natl. Acad. Sci. USA 89, 3952
(1992); Baim et al., Proc. Natl. Acad. Sci. USA 88, 5072 (1991);
Wyborski et al., Nuc. Acids Res. 19, 4647 (1991);
Hillenand-Wissmah, Topics in Mol. And Struc. Biol. 10, 143 (1989);
Degenkolb et al., Antimicrob. Agents Chemother. 35, 1591 (1991);
Kleinschnidt et al., Biochemistry 27, 1094 (1988); Gatz et al.,
Plant J. 2, 397 (1992); Gossen et al., Proc. Natl. Acad. Sci. USA
89, 5547 (1992); Oliva et al., Antimicrob. Agents Chemother. 36,
913 (1992); HLAVKA ET AL., HANDBOOK OF EXPERIMENTAL PHARMACOLOGY 78
(1985); and Gill et al., Nature 334, 721 (1988). Such disclosures
are herein incorporated by reference.
[0044] The above list of selectable marker genes are not meant to
be limiting. Any selectable marker gene can be used in the present
invention.
[0045] Where appropriate, the selectable marker genes and other
gene(s) and nucleic acids of interest to be transferred can be
synthesized for optimal expression in duckweed. That is, the coding
sequence of the genes can be modified to enhance expression in
duckweed. The synthetic nucleic acid is designed to be expressed in
the transformed tissues and plants at a higher level. The use of
optimized selectable marker genes may result in higher
transformation efficiency.
[0046] Methods for synthetic optimization of genes are available in
the art. The nucleotide sequence can be optimized for expression in
duckweed or alternatively can be modified for optimal expression in
monocots. The plant preferred codons may be determined from the
codons of highest frequency in the proteins expressed in duckweed.
It is recognized that genes which have been optimized for
expression in duckweed and other monocots can be used in the
methods of the invention. See, e.g., EP 0 359 472, EP 0 385 962, WO
91/16432; Perlak et al., Proc. Natl. Acad. Sci. USA 88, 3324
(1991), and Murray et al., Nuc. Acids Res. 17, 477 (1989), and the
like, herein incorporated by reference. It is further recognized
that all or any part of the gene sequence may be optimized or
synthetic. In other words, fully optimized or partially optimized
sequences may also be used.
[0047] Additional sequence modifications are known to enhance gene
expression in a cellular host. These include elimination of
sequences encoding spurious polyadenylation signals, exon-intron
splice site signals, transposon-like repeats, and other such
well-characterized sequences which may be deleterious to gene
expression. The G-C content of the sequence may be adjusted to
levels average for a given cellular host, as calculated by
reference to known genes expressed in the host cell. When possible,
the sequence is modified to avoid predicted hairpin secondary mRNA
structures.
B. Target Tissues and Callus.
[0048] The methods of the invention are useful for transforming
duckweed plant cells, preferably frond and meristematic cells. Such
cells also include callus, which can be originated from any tissue
of duckweed plants. Preferably, the tissue utilized in initiating
callus is meristematic tissue. Alternatively, the callus can be
originated from any other frond cells, or in principal from any
other duckweed tissue capable of forming callus. Alternatively
stated, any tissue capable of subsequent clonal propagation,
whether by organogenesis or embryogenesis, may be employed to
transform duckweed according to the present invention. The term
"organogenesis", as used herein, means a process whereby fronds and
roots are developed sequentially from meristematic centers. The
term "embryogenesis", as used herein, means a process whereby
fronds and roots develop together in a concerted fashion (not
sequentially), whether from somatic cells or gametes.
[0049] The method can also be used to transform cell suspensions.
Such cell suspensions can be formed from any duckweed tissue.
[0050] The duckweeds make three kinds of callus: (a) a compact,
semi-organized callus (designated Type I); (b) a friable, white,
undifferentiated callus (designated Type II); and (c) a green,
differentiated callus (designated Type III). In tissue culture,
callus can only regenerate plants two ways: via embryos and via
shoot formation (in duckweed the frond is the shoot). Methods of
callus induction are known in the art, and the particular
conditions to be employed can be optimized for each duckweed
species and for the type of callus desired, as demonstrated in the
Examples below. Preferably, Type I or Type III callus, more
preferably Type I callus, is used to transform duckweed according
to the present invention.
[0051] Callus can be induced by cultivating duckweed tissue in
medium containing plant growth regulators, i.e., cytokinins and
auxins. Preferred auxins for callus induction from duckweed tissue
include 2,4-dichlorophenoxyacetic acid (2,4-D) and
naphthaleneacetic acid (NAA). Preferred auxin concentrations are
1-30 .mu.M, more preferably 5-20 .mu.M, yet more preferably 5-10
.mu.M. The preferred cytokinin is benzyladenine (BA) or thidiazuron
(TDZ). Preferred cytokinin concentrations are 0.1-10 .mu.M, more
preferably 0.5-5 .mu.M, yet more preferably 0.5-1 .mu.M. In other
more preferred embodiments, callus is induced by cultivating
duckweed tissue in medium containing both BA or TDZ and either
2,4-D or NAA. In general, low concentrations of auxin or "weak"
auxins (e.g., indoleacetic acid) promote frond proliferation rather
than callus formation, and high concentrations of auxin or "strong"
auxins (e.g., 2,4-D) promote callus formation. Preferred basal
media for callus formation include N6 medium (Chu et al., Scientia
Sinica 18, 659 (1975)) and Murashige and Skoog medium (Murashige
and Skoog, Physiol. Plant. 15, 473 (1962)), with Murashige and
Skoog medium being more preferred. In general, callus induction
frequency is variable. In these species, callus may not be visible
for two to three weeks in culture, and it may take four to eight
weeks of cultivation before calli are of sufficient size for
transformation. Preferably, callus induction is carried out for a
period of 1-10 weeks, more preferably 2-8 weeks, yet more
preferably 3-5 weeks. For callus growth, the preferred media are as
for callus induction, but the auxin concentration is reduced.
C. Transformation of Duckweed by Ballistic Bombardment.
[0052] One embodiment of the invention is a method of transforming
duckweed with a nucleotide sequence of interest, wherein the
nucleotide sequence contains at least an expression cassette
carrying a gene that confers resistance to a selection agent. The
nucleotide sequence is carried by a microprojectile. As far as the
inventors are aware, there are no previous reports of producing
stably transformed duckweed by means of ballistic
transformation.
[0053] According to preferred embodiments of the present invention,
the ballistic transformation method comprises the steps of: (a)
providing a duckweed tissue as a target; (b) propelling the
microprojectile carrying the nucleotide sequence at the duckweed
tissue at a velocity sufficient to pierce the walls of the cells
within the tissue and to deposit the nucleotide sequence within a
cell of the tissue to thereby provide a transformed tissue. In
particular embodiments of the invention, the method further
includes the step of culturing the transformed tissue with a
selection agent, as described below. In a further alternate
embodiment, the selection step is followed by the step of
regenerating transformed duckweed plants from the transformed
tissue. As noted below, the technique could be carried out with the
nucleotide sequence as a precipitate (wet or freeze-dried) alone,
in place of the aqueous solution containing the nucleotide
sequence.
[0054] Any ballistic cell transformation apparatus can be used in
practicing the present invention. Exemplary apparatus are disclosed
by Sandford et al. (Particulate Science and Technology 5, 27
(1988)), Klein et al. (Nature 327, 70 (1987)), and in EP 0 270 356.
Such apparatus have been used to transform maize cells (Klein et
al., Proc. Natl. Acad. Sci. USA 85, 4305 (1988)), soybean callus
(Christou et al., Plant Physiol. 87, 671 (1988)), McCabe et al.,
BioTechnology 6, 923 (1988), yeast mitochondria (Johnston et al.,
Science 240, 1538 (1988)), and Chlamydomonas chloroplasts (Boynton
et al., Science 240, 1534 (1988)).
[0055] In the investigations presented herein, a
commercially-available helium gene gun (PDS-1000/He) manufactured
by DuPont was employed. Alternately, an apparatus configured as
described by Klein et al. (Nature 70, 327 (1987)) may be utilized.
This apparatus comprises a bombardment chamber, which is divided
into two separate compartments by an adjustable-height stopping
plate. An acceleration tube is mounted on top of the bombardment
chamber. A macroprojectile is propelled down the acceleration tube
at the stopping plate by a gunpowder charge. The stopping plate has
a bore hole formed therein, which is smaller in diameter than the
microprojectile. The macroprojectile carries the
microprojectile(s), and the macroprojectile is aimed and fired at
the bore hole. When the macroprojectile is stopped by the stopping
plate, the microprojectile(s) is propelled through the bore hole.
The target tissue is positioned in the bombardment chamber so that
a microprojectile(s) propelled through the bore hole penetrates the
cell walls of the cells in the target tissue and deposit the
nucleotide sequence of interest carried thereon in the cells of the
target tissue. The bombardment chamber is partially evacuated prior
to use to prevent atmospheric drag from unduly slowing the
microprojectiles. The chamber is only partially evacuated so that
the target tissue is not desiccated during bombardment. A vacuum of
between about 400 to about 800 millimeters of mercury is
suitable.
[0056] In alternate embodiments, ballistic transformation is
achieved without use of microprojectiles. For example, an aqueous
solution containing the nucleotide sequence of interest as a
precipitate could be carried by the macroprojectile (e.g., by
placing the aqueous solution directly on the plate-contact end of
the macroprojectile without a microprojectile, where it is held by
surface tension), and the solution alone propelled at the plant
tissue target (e.g., by propelling the macroprojectile down the
acceleration tube in the same manner as described above). Other
approaches include placing the nucleic acid precipitate itself
("wet" precipitate) or a freeze-dried nucleotide precipitate
directly on the plate-contact end of the macroprojectile without a
microprojectile. In the absence of a microprojectile, it is
believed that the nucleotide sequence must either be propelled at
the tissue target at a greater velocity than that needed if carried
by a microprojectile, or the nucleotide sequenced caused to travel
a shorter distance to the target tissue (or both).
[0057] It is currently preferred to carry the nucleotide sequence
on a microprojectile. The microprojectile may be formed from any
material having sufficient density and cohesiveness to be propelled
through the cell wall, given the particle's velocity and the
distance the particle must travel. Non-limiting examples of
materials for making microprojectiles include metal, glass, silica,
ice, polyethylene, polypropylene, polycarbonate, and carbon
compounds (e.g., graphite, diamond). Metallic particles are
currently preferred. Non-limiting examples of suitable metals
include tungsten, gold, and iridium. The particles should be of a
size sufficiently small to avoid excessive disruption of the cells
they contact in the target tissue, and sufficiently large to
provide the inertia required to penetrate to the cell of interest
in the target tissue. Particles ranging in diameter from about
one-half micrometer to about three micrometers are suitable.
Particles need not be spherical, as surface irregularities on the
particles may enhance their DNA carrying capacity.
[0058] The nucleotide sequence may be immobilized on the particle
by precipitation. The precise precipitation parameters employed
will vary depending upon factors such as the particle acceleration
procedure employed, as is known in the art. The carrier particles
may optionally be coated with an encapsulating agents such as
polylysine to improve the stability of nucleotide sequences
immobilized thereon, as discussed in EP 0 270 356 (column 8).
[0059] After ballistic bombardment of the target tissue,
transformants may be selected and transformed duckweed plants
regenerated as described below in Section E.
D. Agrobacterium-Mediated Transformation.
[0060] In one embodiment of the present invention, duckweed is
transformed using Agrobacterium tumefaciens or Agrobacterium
rhizogenes, preferably Agrobacterium tumefaciens.
Agrobacterium-mediated gene transfer exploits the natural ability
of A. tumefaciens and A. rhizogenes to transfer DNA into plant
chromosomes. Agrobacterium is a plant pathogen that transfers a set
of genes encoded in a region called T-DNA of the Ti and Ri plasmids
of A. tumefaciens and A. rhizogenes, respectively, into plant
cells. The typical result of transfer of the Ti plasmid is a
tumorous growth called a crown gall in which the T-DNA is stably
integrated into a host chromosome. Integration of the Ri plasmid
into the host chromosomal DNA results in a condition known as
"hairy root disease". The ability to cause disease in the host
plant can be removed by deletion of the genes in the T-DNA without
loss of DNA transfer and integration. The DNA to be transferred is
attached to border sequences that define the end points of an
integrated T-DNA.
[0061] Gene transfer by means of engineered Agrobacterium strains
has become routine for many dicotyledonous crop plants.
Considerable difficulty has been experienced, however, in using
Agrobacterium to transform monocotyledonous plants, in particular,
cereal plants. As far as the inventors are aware, there are no
reports to date of producing stably transformed duckweed by means
of Agrobacterium-mediated transformation.
[0062] While the following discussion will focus on using A.
tumefaciens to achieve gene transfer in duckweed, those skilled in
the art will appreciate that this discussion applies equally well
to A. rhizogenes. Transformation using A. rhizogenes has developed
analogously to that of A. tumefaciens and has been successfully
utilized to transform, for example, alfalfa, Solanum nigrum L., and
poplar. U.S. Pat. No. 5,777,200 to Ryals et al. As described by
U.S. Pat. No. 5,773,693 to Burgess et al., it is preferable to use
a disarmed A. tumefaciens strain (as described below), however, the
wild-type A. rhizogenes may be employed. An illustrative strain of
A. rhizogenes is strain 15834.
[0063] The Agrobacterium strain utilized in the methods of the
present invention is modified to contain a gene or genes of
interest, or a nucleic acid to be expressed in the transformed
cells. The nucleic acid to be transferred is incorporated into the
T-region and is flanked by T-DNA border sequences. A variety of
Agrobacterium strains are known in the art particularly for
dicotyledon transformation. Such Agrobacterium can be used in the
methods of the invention. See, e.g., Hooykaas, Plant Mol. Biol. 13,
327 (1989); Smith et al., Crop Science 35, 301 (1995); Chilton,
Proc. Natl. Acad. Sci. USA 90, 3119 (1993); Mollony et al.,
Monograph Theor. Appl. Genet NY 19, 148 (1993); Ishida et al.,
Nature Biotechnol. 14, 745 (1996); and Komari et al., The Plant
Journal 10, 165 (1996), the disclosures of which are incorporated
herein by reference.
[0064] In addition to the T-region, the Ti (or Ri) plasmid contains
a vir region. The vir region is important for efficient
transformation, and appears to be species-specific. Binary vector
systems have been developed where the manipulated disarmed T-DNA
carrying foreign DNA and the vir functions are present on separate
plasmids. In this manner, a modified T-DNA region comprising
foreign DNA (the nucleic acid to be transferred) is constructed in
a small plasmid which replicates in E. coli. This plasmid is
transferred conjugatively in a tri-parental mating or via
electroporation into A. tumefaciens that contains a compatible
plasmid with virulence gene sequences. The vir functions are
supplied in trans to transfer the T-DNA into the plant genome. Such
binary vectors are useful in the practice of the present
invention.
[0065] In preferred embodiments of the invention C58-derived
vectors are employed to transform A. tumefaciens. Alternately, in
other embodiments, super-binary vectors are employed. See, e.g.,
U.S. Pat. No. 5,591,615 and EP 0 604 662, herein incorporated by
reference. Such a super-binary vector has been constructed
containing a DNA region originating from the hypervirulence region
of the Ti plasmid pTiBoS42 (Jin et al., J. Bacteriol. 169, 4417
(1987)) contained in a super-virulent A. tumefaciens A281
exhibiting extremely high transformation efficiency (Hood et al.,
Biotechnol. 2, 702 (1984); Hood et al., J. Bacteriol. 168, 1283
(1986); Komari et al., J. Bacteriol. 166, 88 (1986); Jin et al., J.
Bacteriol. 169, 4417 (1987); Komari, Plant Science 60, 223 (1987);
ATCC Accession No. 37394.
[0066] Exemplary super-binary vectors known to those skilled in the
art include pTOK162 (Japanese patent Appl. (Kokai) No. 4-222527, EP
504,869, EP 604,662, and U.S. Pat. No. 5,591,616, herein
incorporated by reference) and pTOK233 (Komari, Plant Cell Reports
9,303 (1990); Ishida et al., Nature Biotechnology 14, 745 (1996);
herein incorporated by reference). Other super-binary vectors may
be constructed by the methods set forth in the above references.
Super-binary vector pTOK162 is capable of replication in both E.
coli and in A. tumefaciens. Additionally, the vector contains the
virB, virC and virG genes from the virulence region of pTiBo542.
The plasmid also contains an antibiotic resistance gene, a
selectable marker gene, and the nucleic acid of interest to be
transformed into the plant. The nucleic acid to be inserted into
the duckweed genome is located between the two border sequences of
the T region. Super-binary vectors of the invention can be
constructed having the features described above for pTOK162. The
T-region of the super-binary vectors and other vectors for use in
the invention are constructed to have restriction sites for the
insertion of the genes to be delivered. Alternatively, the DNA to
be transformed can be inserted in the T-DNA region of the vector by
utilizing in vivo homologous recombination. See, Herrera-Esterella
et al., EMBO J. 2, 987 (1983); Horch et al., Science 223, 496
(1984). Such homologous recombination relies on the fact that the
super-binary vector has a region homologous with a region of pBR322
or other similar plasmids. Thus, when the two plasmids are brought
together, a desired gene is inserted into the super-binary vector
by genetic recombination via the homologous regions.
[0067] Any suitable vector for transforming duckweed may be
employed according to the present invention. For example, the
heterologous nucleic acid sequence of interest and the flanking
T-DNA can be carried by a binary vector lacking the vir region. The
vir region is then provided on a disarmed Ti-plasmid or on a second
binary plasmid. As another alternative, the heterologous nucleic
acid sequence and the T-DNA border sequences can be put into the
T-DNA site on the Ti-plasmid through a double recombination event
by which the new T-DNA replaces the original Ti-plasmid T-DNA. The
vir region can be supplied by the Ti-plasmid or on a binary
plasmid. As yet a further alternative, the heterologous nucleic
acid sequence and flanking T-DNA can be integrated into the
bacterial chromosome as described by U.S. Pat. No. 4,940,838 to
Schilperoort et al., and the vir region can then be supplied on a
Ti-plasmid or on a binary plasmid.
[0068] The Agrobacterium-mediated transformation process of the
present invention can be thought of as comprising several steps.
The basic steps include an infection step and a co-cultivation
step. In some embodiments, these steps are followed by a selection
step, and in other embodiments by a selection and a regeneration
step.
[0069] An optional pre-culture step may be included prior to the
infection step. The pre-culture step involves culturing the callus,
frond, or other target tissue prior to the infection step on a
suitable medium. The pre-culture period may vary from about 1 to 21
days, preferably 7 to 14 days. Such a pre-culture step was found to
prevent transformation of maize cultures. See, e.g., EP 0 672
752.
[0070] In the infection step, the cells to be transformed are
exposed to Agrobacterium. The cells are brought into contact with
the Agrobacterium, typically in a liquid medium. As noted above,
the Agrobacterium has been modified to contain a gene or nucleic
acid of interest. The nucleic acid is inserted into the T-DNA
region of the vector. General molecular biology techniques used in
the invention are well-known by those of skill in the art. See,
e.g., SAMBROOK ET AL., MOLECULAR CLONING: A LABORATORY MANUAL
(1989).
[0071] Agrobacterium containing the plasmid of interest are
preferably maintained on Agrobacterium master plates with stock
frozen at about -80.degree. C. Master plates can be used to
inoculate agar plates to obtain Agrobacterium that is then
resuspended in medium for use in the infection process.
Alternatively, bacteria from the master plate can be used to
inoculate broth cultures that are grown to logarithmic phase prior
to transformation.
[0072] The concentration of Agrobacterium used in the infection
step and co-cultivation step can affect the transformation
frequency. Likewise, very high concentrations of Agrobacterium may
damage the tissue to be transformed and result in a reduced callus
response. Thus, the concentration of Agrobacterium useful in the
methods of the invention may vary depending on the Agrobacterium
strain utilized, the tissue being transformed, the duckweed species
being transformed, and the like. To optimize the transformation
protocol for a particular duckweed species or tissue, the tissue to
be transformed can be incubated with various concentrations of
Agrobacterium. Likewise, the level of marker gene expression and
the transformation efficiency can be assessed for various
Agrobacterium concentrations. While the concentration of
Agrobacterium may vary, generally a concentration range of about
1.times.10.sup.3 cfu/ml to about 1.times.10.sup.10 cfu/ml is
employed, preferably within the range of about 1.times.10.sup.3
cfu/ml to about 1.times.10.sup.9 cfu/ml, and still more preferably
at about 1.times.10.sup.8 cfu/ml to about 1.times.10.sup.9 cfu/ml
will be utilized.
[0073] The tissue to be transformed is generally added to the
Agrobacterium suspension in a liquid contact phase containing a
concentration of Agrobacterium to optimize transformation
efficiencies. The contact phase facilitates maximum contact of the
tissue to be transformed with the suspension of Agrobacterium.
Infection is generally allowed to proceed for 1 to 30 minutes,
preferably 1 to 20 minutes, more preferably 2 to 10 minutes, yet
more preferably 3 to 5 minutes prior to the co-cultivation
step.
[0074] Those skilled in the art will appreciate that the conditions
can be optimized to achieve the highest level of infection and
transformation by Agrobacterium. For example, in preferred
embodiments of the invention the cells are subjected to osmotic
pressure (e.g., 0.6 M mannitol) during the infection and
co-cultivation steps. Additionally, to enhance transformation
efficiency, tissue may be cultured in medium containing an auxin,
such as IAA, to promote cell proliferation (i.e., it is believed
that Agrobacterium integrates into the genome during mitosis). As
further alternatives, tissue wounding, vacuum pressure, or
cultivation in medium containing acetosyringone can be employed to
promote the transformation efficiency.
[0075] In the co-cultivation step, the cells to be transformed are
co-cultivated with Agrobacterium. Typically, the co-cultivation
takes place on a solid medium. Any suitable medium, such as Schenk
and Hildebrandt medium (Schenk and Hildebrandt, Can. J. Bot. 50,
199 (1972)) containing 1% sucrose and 0.6% agar, may be utilized.
The optimal co-cultivation time varies with the particular tissue.
Fronds are co-cultivated with the Agrobacterium for about 2 to 7
days, preferably 2 to 5 days, more preferably 3 to 5 days, and more
preferably 4 days. In contrast, callus is co-cultivated with the
Agrobacterium for 0.5 to 4 days, more preferably 1 to 3 days, more
preferably 2 days. Co-cultivation may be carried out in the dark or
under subdued light conditions to enhance the transformation
efficiency. Additionally, as described above for the inoculation
step, co-culturing can be done on medium containing IAA or
acetosyringone to promote transformation efficiency. Finally, the
co-culturing step may be performed in the presence of cytokinins,
which act to enhance cell proliferation.
[0076] Following the co-cultivation step, the transformed tissue
may be subjected to an optional resting and decontamination step.
For the resting/decontamination step, the transformed cells are
transferred to a second medium containing an antibiotic capable of
inhibiting the growth of Agrobacterium. This resting phase is
performed in the absence of any selective pressures to permit
recovery and proliferation of transformed cells containing the
heterologous nucleic acid. An antibiotic is added to inhibit
Agrobacterium growth. Such antibiotics are known in the art which
inhibit Agrobacterium and include cefotaxime, timetin, vancomycin,
carbenicillin, and the like. Concentrations of the antibiotic will
vary according to what is standard for each antibiotic. For
example, concentrations of carbenicillin will range from about 50
mg/l to about 250 mg/l carbenicillin in solid media, preferably
about 75 mg/l to about 200 mg/l, more preferably about 100-125
mg/l. Those of ordinary skill in the art of monocot transformation
will recognize that the concentration of antibiotic can be
optimized for a particular transformation protocol without undue
experimentation.
[0077] The resting phase cultures are preferably allowed to rest in
the dark or under subdued light, preferably in subdued light. Any
of the media known in the art can be utilized for the resting step.
The resting/decontamination step may be carried out for as long as
is necessary to inhibit the growth of Agrobacterium and to increase
the number of transformed cells prior to selection. Typically, the
resting/decontamination step may be carried out for 1 to 6 weeks,
preferably 2 to 4 weeks, more preferably 2 to 3 weeks prior to the
selection step. In more preferred embodiments, the selection period
is started within 3 weeks following co-cultivation. Some strains of
Agrobacterium are more antibiotic resistant than are others. For
less resistant strains, decontamination is typically performed by
adding fresh decontamination medium to the calli every five days or
so. For more resistant strains, a stronger antibiotic (e.g.,
vancomycin) may be added to the calli every other day.
[0078] Following the co-cultivation step, or
resting/decontamination step, transformants may be selected and
duckweed plants regenerated as described below in Section E.
E. Selection of Transformants and Regeneration of Transformed
Duckweed Plants.
[0079] Duckweed tissue or callus is transformed according to the
present invention, for example by ballistic bombardment or
Agrobacterium-mediated transformation, each of which is described
in more detail above in Sections C and D, respectively. After the
transformation step, the transformed tissue is exposed to selective
pressure to select for those cells that have received and are
expressing the polypeptide from the heterologous nucleic acid
introduced by the expression cassette. The agent used to select for
transformants will select for preferential growth of cells
containing at least one selectable marker insert positioned within
the expression cassette and delivered by ballistic bombardment or
by the Agrobacterium.
[0080] The conditions under which selection for transformants (from
any tissue type or callus) is performed are generally the most
critical aspect of the methods disclosed herein. The transformation
process subjects the cells to stress, and the selection process can
be toxic even to transformants. Typically, in response to this
concern, the transformed tissue is initially subject to weak
selection, utilizing low concentrations of the selection agent and
subdued light (e.g., 1-5 .mu.mol/m.sup.2sec, with a gradual
increase in the applied selection gradient by increasing the
concentration of the selection agent and/or increasing the light
intensity. Selection pressure may be removed altogether for a time
and then reapplied if the tissue looks stressed. Additionally, the
transformed tissue may be given a "resting" period, as described
above in Section D, before any selection pressure is applied at
all. The selection medium generally contains a simple carbohydrate,
such as 1% to 3% sucrose, so that the cells do not carry out
photosynthesis. In addition, the selection is initially performed
under subdued light conditions, or even in complete darkness, so as
to keep the metabolic activity of the cells at a relatively low
level. Those skilled in the art will appreciate that the specific
conditions under which selection is performed can be optimized for
every species or strain of duckweed and for every tissue type being
transformed without undue experimentation.
[0081] There is no particular time limit for the selection step. In
general, selection will be carried out long enough to kill
non-transformants and to allow transformed cells to proliferate at
a similar rate to non-transformed cells in order to generate a
sufficient callal mass prior to the regeneration step. Thus, the
selection period will be longer with cells that proliferate at a
slower rate. Type I duckweed callus, for example, proliferates
relatively slowly and selection may be carried out for 8-10 weeks
prior to regeneration.
[0082] Methods of regenerating certain plants from transformed
cells and callus are known in the art. See, e.g., Kamo et al., Bot.
Gaz. 146, 327 (1985); West et al., The Plant Cell 5, 1361 (1993);
and Duncan et al., Planta 165, 322 (1985). Several refinements to
these methods are recommended for regenerating duckweed. Frond
regeneration following transformation and selection can be achieved
most reliably with Type I and Type III callus. Regeneration in Type
I calli, for example, can be identified by green centers (sites
where fronds are organizing) appearing on the pale yellow callus
surface. Typically, duckweed regeneration does not occur under the
same medium conditions that support callus proliferation. A lean
solid medium (e.g., water-agar or half-strength Schenk and
Hildebrandt medium contain 0.5% sucrose and 0.8% agar) is
preferred. It is usually necessary, however, to intermittently
culture the regenerating duckweed callus for short periods on
full-strength medium to maintain nutrient balance in the
regenerating cells. In some instances, with slowly regenerating
strains or species, this process may have to repeated several times
before fronds are regenerated. Typically, plant growth regulators
are not added to the frond regeneration medium (because they
inhibit the organization of fronds), however, cytokinins, such as
BA and adenine sulfate, can increase frond regeneration with some
species. Callus cultures do not loose their ability to regenerate
fronds over prolonged periods of callus culture.
[0083] During the regeneration process, any method known in the art
may be utilized to verify that the regenerating plants are, in
fact, transformed with the transferred nucleic acid of interest.
For example, histochemical staining, ELISA assay, Southern
hybridization, Northern hybridization, Western hybridization, PCR,
and the like can be used to detect the transferred nucleic acids or
protein in the callal tissue and regenerating plants.
[0084] Now that it has been demonstrated that duckweed can be
transformed utilizing ballistic bombardment and Agrobacterium,
alterations to the general methods described herein can be used to
increase efficiency or to transform strains that may exhibit some
recalcitrance to transformation. Factors that affect the efficiency
of transformation include the species of duckweed, the tissue
infected, composition of the media for tissue culture, selectable
marker genes, the length of any of the above-described step, kinds
of vectors, and light/dark conditions. Specifically for
Agrobacterium-mediated transformation, the concentration and strain
of A. tumefaciens or A. rhizogenes must also be considered.
Therefore, these and other factors may be varied to determine what
is an optimal transformation protocol for any particular duckweed
species or strain. It is recognized that not every species and
strain will react the same to the transformation conditions and may
require a slightly different modification of the protocols
disclosed herein. However, by altering each of the variables, an
optimum protocol can be derived for any duckweed line.
[0085] The following Examples are offered by way of illustration
and not be way of limitation. As used in the Examples, "hr" means
hour, "sec" means second, "g" means gram, "mg" means milligram, "l"
means liter, "ml" means milliliter, "mmol" means millimole, "mM"
means millimolar, ".mu.M" means micromolar, "m" means meter, "mm"
means millimeter, "BA" means benzyladenine, "2,4-D" means
2,4-dichlorophenoxyacetic acid, "NAA" means naphthaleneacetic acid,
and "IAA" means indoleacetic acid.
EXAMPLES
Tissue Culture:
[0086] This section presents experiments pertaining to methods of
making duckweed callus. A number of examples use Lemna gibba G3 as
the duckweed strain, the strain used to optimize culturing
parameters: (1) basal medium formulation, (2) type and
concentration of plant growth regulators, and (3) transfer
schedule. As knowledge of callus formation increased, it was
applied to other duckweed species. The duckweeds make three kinds
of callus: (a) a compact, semi-organized callus (designated Type
I); (b) a friable, white, undifferentiated callus (designated Type
II); and (c) a green, differentiated callus (designated Type III).
In tissue culture, callus can only regenerate plants two ways: via
embryos and via shoot formation (in duckweed the frond is the
shoot). The data presented below demonstrate that transformed
duckweed plants can be regenerated from all known pathways of
callus regeneration of fronds.
Example 1
[0087] Eighteen combinations of an auxin, 2,4-dichlorophenoxyacetic
acid (2,4-D), and a cytokinin, benzyladenine (BA), were tested for
their effects on callus induction in a duckweed species, Lemna
gibba G3.
[0088] Duckweed fronds were grown in liquid Hoagland's medium
(Hoagland and Snyder, Proc. Amer. Soc. Hort. Sci. 30, 288 (1933))
containing 3% sucrose for two weeks at 23.degree. C. under a 16 hr
light/8 hr dark photoperiod with light intensity of approximately
40 .mu.mol/m.sup.2sec prior to experimentation. For callus
induction, eighteen, 100 ml portions of Murashige and Skoog basal
medium (Murashige and Skoog, Physiol. Plant. 15, 473 (1962))
containing 3% sucrose, 0.15% Gelrite and 0.4% Difco Bacto-agar were
prepared with 2,4-D concentrations of 10, 20 and 50 .mu.M and BA
concentrations of 0, 0.01, 0.1, 1.0, 2.0, and 10.0 .mu.M. All media
were pH adjusted to 5.8, autoclaved at 121.degree. C. for 20
minutes, cooled, and each 100 ml was poured into 4, 100 mm.times.15
mm petri dishes.
[0089] A three 2,4-D concentrations.times.six BA concentrations,
full-factorial experimental design (18 treatments in total) with
four replications, with one petri dish per replication and 5 fronds
per petri dish was used. For callus induction, 5 individual
duckweed fronds were placed abaxial side down on each plate of
medium. The 72 plates were incubated at 23.degree. C., for 27 days
under a 16 hr light/8 hr dark photoperiod with light intensity of
approximately 40 .mu.mol/m.sup.2sec. After 27 days the duckweed
tissue was transferred to fresh media of the same type and
incubation was continued another 35 days under the same temperature
and light culturing conditions.
[0090] The results, measured as the frequency of callus induction
(#fronds making any callus/total # fronds) showed three types of
callus proliferation after the 62 days of incubation. (1) A
compact, white-yellow callus was identified and designated as "Type
I". A low frequency of fronds, approximately 5%, proliferated this
type of callus. (2) A friable white callus was identified and
designated as "Type II". Between 20 and 40% of fronds proliferated
this type of callus. (3) A green callus ranging in its degree of
cellular organization was identified and designated as "Type III".
This callus type was produced by greater than 50% of all fronds
proliferated during the incubation time. All three types of callus
demonstrated proliferation at all 18 2,4-D and BA combinations in
varying frequencies. Callus proliferation was the most vigorous in
a broad range of 2,4-D concentrations, from 20-50 .mu.M, and BA
concentrations between 0.01 and 0.1 .mu.M.
Example 2
[0091] Forty concentrations of an auxin, 2,4-dichlorophenoxyaectic
acid (2,4-D), and a cytokinin, benzyladenine (BA) were tested to
better optimize the auxin and cytokinin concentrations for callus
induction from duckweed fronds of Lemna gibba G3.
[0092] Duckweed fronds were grown in liquid Hoagland's medium
containing 3% sucrose for two weeks at 23.degree. C. under a 16 hr
light/8 hr dark photoperiod with light intensity of approximately
40 .mu.mol/m.sup.2sec. prior to experimentation. For callus
induction, forty 100 ml portions of Murashige and Skoog medium with
3% sucrose, 0.15% Gelrite, and 0.4% Difco Bacto-agar were prepared
with 2,4-D concentrations of 20, 30, 40, 50, 60, 70, 80, 100 .mu.M
and BA concentrations of 0.01, 0.05, 0.1, 0.5, and 1.0 .mu.M. All
media were pH adjusted to 5.8, autoclaved at 121.degree. C. for 20
minutes, cooled, and each 100 ml was poured into 4, 100 mm.times.15
mm petri dishes.
[0093] An eight 2,4-D concentrations.times.five BA concentrations,
full-factorial experimental design (40 treatments in total) with
four replications, with one petri dish per replication and 5 fronds
per petri dish was used. For callus induction, 5 individual
duckweed fronds were placed abaxial side down on each plate of
medium. The plates were incubated at 23.degree. C., for 27 days
under a 16 hr light/8 hr dark photoperiod with light intensity of
approximately 40 .mu.mol/m.sup.2sec. After 27 days, the duckweed
tissue was transferred to fresh media of the same type and
incubation was continued another 35 days under the same temperature
and light culturing conditions.
[0094] Results taken after 63 days of incubation showed that three
types of callus had proliferated: (1) Type I, (2) Type II, and (3)
a Type III callus. Regression analysis (quadratic response surface)
of the numerical frequency data (#fronds making any callus/total #
fronds) revealed differences in frond response for callus induction
of the different types. The frequencies of Type II and Type III
callus types were significantly affected by the concentrations of
both 2,4-D and BA, however, the frequency of Type I callus was
significantly affected by the 2,4-D concentration only. No specific
concentration of 2,4-D or BA proved optimal, callus induction
occurred over a broad range of both plant growth regulators.
Approximately 50% of the fronds produced Type III callus,
approximately 25% produced Type II callus, and less than 5%
produced Type I callus.
Example 3
[0095] Forty combinations of an auxin, dicamba, and a cytokinin,
benzyladenine (BA), were tested to compare the relative efficacy of
dicamba versus 2,4-D for callus induction in a duckweed species,
Lemna gibba G3.
[0096] Duckweed fronds were grown in liquid Hoagland's medium
containing 3% sucrose for two weeks at 23.degree. C. under a 16 hr
light/8 hr dark photoperiod with light intensity of approximately
40 .mu.mol/m.sup.2sec. prior to experimentation. For callus
induction, forty 100 ml portions of Murashige and Skoog medium with
3% sucrose, 0.15% Gelrite, and 0.4% Difco Bacto-agar were prepared
with dicamba concentrations of 10, 20, 30, 40, 50, 60, 80, 100
.mu.M and BA concentrations of 0.01, 0.05, 0.1, 0.5, and 1.0 .mu.M.
All media were pH adjusted to 5.8, autoclaved at 121.degree. C. for
20 minutes, cooled, and each 100 ml was poured into 4, 100
mm.times.15 mm petri dishes.
[0097] An eight dicamba concentrations.times.five BA
concentrations, full-factorial experimental design (40 treatments
in total) with four replications, with one petri dish per
replication and 5 fronds per petri dish was used. For callus
induction, 5 individual duckweed fronds were placed abaxial side
down on each plate of medium. The plates were incubated at
23.degree. C., for 27 days under a 16 hr light/8 hr dark
photoperiod with light intensity of approximately 40
.mu.mol/m.sup.2sec. After 28 days the duckweed tissue was
transferred to fresh media of the same type and incubation was
continued another 45 days under the same temperature and light
culturing conditions.
[0098] After 73 days of incubation three types of callus
proliferation were observed:
[0099] (1) Type I, (2) Type II, and (3) a Type III callus. Overall,
callus proliferation was poor and occurred on dicamba
concentrations of 10 and 20 .mu.M; above 30 .mu.M callus
proliferation did not occur. Type II and Type III callus
proliferated in response to dicamba; Type I callus proliferation
was rare.
Example 4
[0100] Two concentrations of 2,4-D in combination with BA were used
determine if callus growth could be maintained and callus lines
established from the three types observed with Lemna gibba G3.
[0101] Duckweed fronds were grown in liquid Schenk and Hildebrandt
medium (Schenk and Hildebrandt, Can. J. Bot. 50, 199 (1972))
containing 1% sucrose for two weeks at 23.degree. C. under a 16 hr
light/8 hr dark photoperiod with light intensity of approximately
40 .mu.mol/m.sup.2sec. prior to experimentation. For callus
induction, 400 ml of Murashige and Skoog medium with 3% sucrose,
0.15% Gelrite, 0.4% Difco Bacto-agar, 0.01 .mu.M BA were prepared
with 2,4-D concentrations of 10 and 40 .mu.M. All media were pH
adjusted to 5.8, autoclaved at 121.degree. C. for 20 minutes,
cooled, and each 200 ml portion was poured into 8, 100 mm.times.15
mm petri dishes.
[0102] A two treatment, random block experimental design with four
replications, with one petri dish per replication and 5 fronds per
petri dish was used. For callus induction, 5 individual duckweed
fronds were placed abaxial side down on each plate of medium. The
plates were incubated at 23.degree. C., for 27 days under a 16 hr
light/8 hr dark photoperiod with light intensity of approximately
40 .mu.mol/m.sup.2sec. After 4 weeks, the duckweed tissue was
transferred to fresh media of the same type and incubation was
continued another 4 weeks under the same temperature and light
culturing conditions.
[0103] After 8 weeks of incubation three types of callus
proliferation were observed: (1) Type I, (2) Type II, and (3) a
Type III callus. All three callus types were transferred to fresh
medium of identical composition from that they had been on, and
incubation on identical culturing conditions was continued with
four week subcultures. After two more months of culture, Type I and
Type III callus on 10 .mu.M 2,4-D and 0.01 .mu.M BA established
healthy, proliferating callus cultures. Type II callus did not
proliferate. Although callus proliferation could be maintained on a
four-week subculture schedule, callus decline was noted during the
third and fourth weeks of the subculture period.
Example 5
[0104] The subculture schedule to maintain callus proliferation was
tested with Lemna gibba G3. Duckweed fronds were grown in liquid
Schenk and Hildebrandt medium containing 1% sucrose for two weeks
at 23.degree. C. under a 16 hr light/8 hr dark photoperiod with
light intensity of approximately 40 .mu.mol/m.sup.2sec prior to
experimentation. For callus induction, 500 ml of Murashige and
Skoog medium with 3% sucrose, 0.15% Gelrite, and 0.4% Difco
Bacto-agar, 30 .mu.M 2,4-D and 0.02 .mu.M BA was prepared, the pH
adjusted to 5.8, autoclaved at 121.degree. C. for 30 minutes,
cooled, and poured into 20, 100 mm.times.15 mm petri dishes.
[0105] A two treatment, random block experimental design with two
replications, with five petri dish per replication and 5 fronds per
petri dish was used. For callus induction, 5 individual duckweed
fronds were placed abaxial side down on each plate of medium. The
plates were incubated at 23.degree. C., for 2 weeks under a 16 hr
light/8 hr dark photoperiod with light intensity of approximately
40 .mu.mol/m.sup.2sec. After 2 weeks, the duckweed tissue on half
the plates (10 plates) was transferred to fresh medium of the same
composition and incubation was continued under the same conditions
as those of the non-transferred tissue. After 4 weeks the tissue
was assessed for callus proliferation. Three types of callus
proliferated: Type I, Type II, and Type III. No difference in
callus type or proliferation was observed between duckweed tissue
transferred at 2 weeks as compared with duckweed tissue incubated
for 4 weeks without transfer.
[0106] Type I and Type III callus were subcultured away from the
original fronds and continued in culture on Murashige and Skoog
medium with 3% sucrose, 0.15% Gelrite, 0.4% Difco Bacto-agar, 10
.mu.M 2,4-D and 0.01 .mu.M BA. Proliferating callus was continually
subcultured to fresh medium of the same composition at 2 week
intervals. Longer intervals between transfer resulted in an abrupt
decline in callus health between 2 and 3 weeks. Callus
proliferation continued without loss of vigor when a two-week
subculture schedule was maintained.
Example 6
[0107] Two different basal media, Murashige and Skoog and Nitsch
and Nitsch (Science 163, 85 (1969)), were tested to compare their
relative efficacy for callus induction of Lemna gibba G3.
[0108] Duckweed fronds were grown in liquid Schenk and Hildebrandt
medium containing 1% sucrose for two weeks at 23.degree. C. under a
16 hr light/8 hr dark photoperiod with light intensity of
approximately 40 .mu.mol/m.sup.2sec prior to experimentation. For
callus induction, 500 ml, each, of Murashige and Skoog and Nitsch
and Nitsch media with 3% sucrose, 0.15% Gelrite, 0.4% Difco
Bacto-agar, 30 .mu.M 2,4-D, and 0.01 .mu.M BA were prepared, the pH
adjusted to 5.8, autoclaved at 121.degree. C. for 30 minutes,
cooled, and each used to pour 20, 100 mm.times.15 mm petri
dishes.
[0109] A two treatment, random block experimental design with two
replications, with five petri dishes per replication and 5 fronds
per petri dish was used. For callus induction, 5 individual
duckweed fronds were placed abaxial side down on each plate of
medium. The plates were incubated at 23.degree. C., for 2 weeks
under a 16 hr light/8 hr dark photoperiod with light intensity of
approximately 40 .mu.mol/m.sup.2sec. After 2 weeks, the duckweed
tissue was transferred to fresh medium of the same composition and
incubation was continued under the same conditions.
[0110] After 4 weeks the tissue on all the plates was assessed for
callus proliferation. Fronds cultured on Nitsch and Nitsch medium
failed to proliferate significant amounts of callus. Duckweed
tissue on this medium was pale and had begun to yellow. Duckweed
fronds cultured on Murashige and Skoog medium proliferated the
usual three types of callus: Type I, Type II, and Type III
callus.
[0111] Type I and Type III callus were subcultured away from the
original fronds and continued in culture on Murashige and Skoog
medium with 3% sucrose, 0.15% Gelrite, 0.4% Difco Bacto-agar, 10
.mu.M 2,4-D and 0.01 .mu.M BA. Proliferating callus was continually
subcultured to fresh medium of the same composition at two-week
intervals. Longer intervals between transfer resulted in an abrupt
decline in callus health between 2 and 3 weeks. Callus
proliferation continued without loss of vigor.
Example 7
[0112] Three different basal media, Murashige and Skoog, Schenk and
Hildebrandt, and Gamborg's B5 (Gamborg et al., In Vitro 12, 473
(1976)) were tested to compare their relative efficacy for callus
induction and growth of Lemna gibba G3.
[0113] Duckweed fronds were grown in liquid Schenk and Hildebrandt
medium containing 1% sucrose for two weeks at 23.degree. C. under a
16 hr light/8 hr dark photoperiod with light intensity of
approximately 40 .mu.mol/m.sup.2sec prior to experimentation. For
callus induction, 500 ml, each, of the three media were prepared
with 3% sucrose, 0.15% Gelrite, 0.4% Difco Bacto-agar, 30 .mu.M
2,4-D and 0.02 .mu.M BA, the pH adjusted to 5.8, and autoclaved at
121.degree. C. for 30 minutes, cooled, and each portion was used to
pour 20, 100 mm.times.15 mm petri dishes.
[0114] A three treatment, random block experimental design with two
replications, with five petri dishes per replication and 5 fronds
per petri dish was used. For callus induction, 5 individual
duckweed fronds were placed abaxial side down on each plate of
medium. The plates were incubated at 23.degree. C., for 2 weeks
under a 16 hr light/8 hr dark photoperiod with light intensity of
approximately 40 .mu.mol/m.sup.2sec. After 2 weeks, the duckweed
tissue was transferred to fresh medium of the same composition and
incubation was continued under the same conditions.
[0115] After 4 weeks the tissue on all the plates was assessed for
callus proliferation. Fronds cultured on Gamborg's B5 medium were
pale, and yellow senescent fronds were present. No appreciable
callus proliferation had occurred. Fronds cultured on Schenk and
Hildebrandt medium were dark green and proliferated aberrant
fronds, and no appreciable callus proliferation had occurred.
Duckweed fronds cultured on Murashige and Skoog medium proliferated
the three usual types of callus: Type I, Type II and Type III
callus.
[0116] Type I and Type III callus were subcultured away from the
original fronds and continued in culture on Murashige and Skoog
medium with 3% sucrose, 0.15% Gelrite, 0.4% Difco Bacto-agar, 10
.mu.M 2,4-D and 0.01 .mu.M BA. Proliferating callus was continually
subcultured to fresh medium of the same composition at two-week
intervals. Longer intervals between transfer resulted in an abrupt
decline in callus health between 2 and 3 weeks. Callus
proliferation continued without loss of vigor.
Example 8
[0117] Four basal media: Murashige and Skoog (MS), Schenk and
Hildebrandt (SH), Nitsch and Nitsch (NN), and Gamborg's B5 (B5),
were used to compare their efficacy to support Lemna gibba G3 Type
II callus proliferation in liquid medium.
[0118] Duckweed fronds were grown in liquid Schenk and Hildebrandt
medium containing 1% sucrose for two weeks at 23.degree. C. under a
16 hr light/8 hr dark photoperiod with light intensity of
approximately 40 .mu.mol/m.sup.2sec prior to experimentation. For
callus induction, 500 ml of Murashige and Skoog medium was prepared
with 3% sucrose, 0.15% Gelrite, 0.4% Difco Bacto-agar, 30 .mu.M
2,4-D and 0.02 .mu.M BA, the pH adjusted to 5.8, and autoclaved at
121.degree. C. for 30 minutes, cooled, and poured into 20, 100
mm.times.15 mm petri dishes.
[0119] For callus induction, 5 individual duckweed fronds were
placed abaxial side down on each plate of medium. The 20 plates
were incubated at 23.degree. C., for 2 weeks under a 16 hr light/8
hr dark photoperiod with light intensity of approximately 40
.mu.mol/m.sup.2 sec. After 2 weeks, the duckweed tissue was
transferred to fresh medium of the same composition and incubation
was continued under the same conditions.
[0120] After 4 weeks Type II callus tissue was used to inoculate
liquid medium for callus suspension cultures. For suspension callus
establishment, 100 ml, each, of the four basal media, MS, SH, NN,
and B5, were prepared with 3% sucrose, 10 .mu.M 2,4-D, and 0.01
.mu.M BA. The media were adjusted to pH 5.8, four 25 ml aliquots
were placed in 125 ml flasks, and all 16 flasks of media were
autoclaved at 121.degree. C. for 18 minutes. After cooling, each
flask was inoculated with 1-2 small pieces of Type II, friable
white callus. The flasks were wrapped with aluminum foil and
incubated 23.degree. C., for 2 weeks, with constant shaking at 100
rpm, in the dark.
[0121] After two weeks the flasks were assessed for callus
proliferation. A slight amount of growth was noted with Murashige
and Skoog medium and with Nitsch and Nitsch medium. The flasks were
incubated for another 2 weeks without change of medium and no
further callus proliferation was noted.
Example 9
[0122] Thirty-two duckweed strains across 15 species, broadly
representative of the genetic diversity of the Lemnaceae, were used
to determine the degree to which the methods and media for callus
induction developed with Lemna gibba G3 will extrapolate across the
entire family. Table I lists the strains tested.
[0123] Duckweed fronds were grown in liquid Schenk and Hildebrandt
medium containing 1% sucrose for two weeks at 23.degree. C. under a
16 hr light/8 hr dark photoperiod with light intensity of
approximately 40 .mu.mol/m.sup.2sec prior to experimentation. For
callus induction, six basal media were used: Murashige and Skoog,
Schenk and Hildebrand (Schenk and Hildebrandt, Can. J. Bot. 50, 199
(1972)), Nitsch and Nitsch, N6 (Chu et al., Scientia Sinica 18, 659
(1975), Gamborg's B5, and Hoagland's. The two plant growth
regulator combinations known to elicit callus proliferation in L.
gibba G3 were used: 30 .mu.M 2,4-D and 0.02 .mu.M BA, and 5 .mu.M
2,4-D and 2 .mu.M BA. For each strain, 200 ml of each basal medium
was prepared with 3% sucrose, 0.15% Gelrite, and 0.4% Difco
Bacto-agar. The 200 ml was divided into 2, 100 ml portions, each to
be used to prepare the two plant growth regulator concentrations.
The pH of all media was adjusted to 5.8, the media were autoclaved
for 30 minutes at 121.degree. C., cooled and 4, 100 mm.times.15 mm
petri dishes were poured from each 100 ml portion.
[0124] A 6 media.times.2 plant growth regulator combinations, 12
treatment, random block experimental design was used for each
duckweed strain tested. The design was replicated four times, with
one petri dishes per replication and 6 fronds per petri dish. For
callus induction, 6 individual duckweed fronds were placed abaxial
side down on each plate of medium for the larger fronds of Lemna,
Spirodela and Wolfiella species. For strains within Wolffia, the
small fronds technically prohibited plating of individual fronds,
rather, small clumps of fronds were used as the experimental unit.
The plates were incubated at 23.degree. C., for 4-5 weeks under a
16 hr light/8 hr dark photoperiod with light intensity of
approximately 40 .mu.mol/m.sup.2sec. At this time the fronds were
evaluated for general health (judged by color: green to yellow, and
vigor of proliferation) and the frequency of callus initiation of
the three types: Type I, Type II, and Type III.
[0125] The results showed a variation in responsiveness of the
different duckweed species to callus induction medium. In general,
species and strains in the genera Lemna and Wolffia were the most
responsive. All five Lemna gibba strains showed callus induction to
varying degrees on MS, B5, and N6 medium containing 5 .mu.M 2,4-D
and 2 .mu.M BA. Both Lemna minor strains followed the same pattern,
with a greater degree of callus induction relative to the Lemna
gibba strains. Both Lemna miniscula strains showed a high frequency
of callus induction, with proliferation of a white callus somewhat
dissimilar to Lemna minor or Lemna gibba. Lemna aequinoctialis
showed frond curling and swelling at the highest auxin
concentrations, but the proliferation of a true callus culture was
not observed, indicating that the auxin concentrations used were
not high enough. Lemna valdiviana did not show callus induction. In
the Wolffia species, Wolffia arrhiza showed a small amount of
callus proliferation on B5 medium with 5 .mu.M 2,4-D and 2 .mu.M
BA. Wolffia brasiliensis and Wolffia columbinana showed callus
induction on Hoaglands medium supplemented with 5 .mu.M 2,4-D and 2
.mu.M BA. The remaining Wolffia species, Wolffia australiana, did
not show callus induction, although fronds showed swelling and
somewhat abnormal growth. The Wolffiella and Spirodela species did
not show callus induction. Fronds of the Spirodela species did not
survive on the higher concentration of 2,4-D and did not grow well
at the lower concentration. This pattern of response is consistent
with the interpretation that Spirodela is more sensitive to auxin
than the Lemna and Wolffia species and that lower auxin
concentrations should be used in subsequent experiments to induce
callus formation. TABLE-US-00001 TABLE I Genus Species Strain
Designation Country of Origin Spirodela polyrrhiza 7970 USA 4240
China 8652 China 8683 Kenya Spirodela punctata 7488 USA 7776
Australia Spirodela intermedia 7178 Wolffia arrhiza 7246 S. Africa
9006 Japan Wolffia australiana 7267 Tasmania 7317 Australia Wolffia
brasiliensis 7397 Venezuela 7581 Venezuela 8919 Venezuela Wolffia
columbiana 7153 USA 7918 USA Wolffiella lingulata 8742 Argentina
9137 Brazil Wolfiella neotropica 7279 Brazil 8848 Brazil Wolfiella
oblongata 8031 USA 8751 Argentina Lemna aequinoctialis 7558 USA
Lemna gibba G3 USA 6861 Italy 7784 8405 France 8678 Kashmir Lemna
minor 8744 Albania 8627 Denmark Lemna miniscula 6600 California
6747 California Lemna valdiviana 8821 Argentina 8829 Argentina
Example 10
[0126] Four auxins, napthaleneacetic acid (NAA), 2,4-D,
indolebutyric acid (IBA), and dicamba were tested for their ability
to induce callus formation from L. gibba G3 fronds on three
different basal media: SH, MS and N6.
[0127] Duckweed fronds were grown in liquid Schenk and Hildebrandt
medium containing 1% sucrose for two weeks at 23.degree. C. under a
16 hr light/8 hr dark photoperiod with light intensity of
approximately 40 .mu.mol/m.sup.2sec prior to experimentation. For
callus induction, three basal media were tested: Murashige and
Skoog, Schenk and Hildebrandt, and N6. Benzyladenine was used as
the cytokinin at a concentration of 1 .mu.M. The auxin
concentrations varied with auxin type. For the relatively strong
auxins, 2,4-D and dicamba, concentrations were 0, 1, 5, 10 and 20
.mu.M. For weak auxins, NAA and IBA, the concentrations were 0, 5,
10, 20 and 50 .mu.M. For each medium dose-response experiment, 2
liters of basal medium were prepared with BA and the pH adjusted to
5.8. The volume was aliquoted as 20, 100 ml portions. To each of
these portions, the appropriate amount of auxin was added and the
medium was adjusted to 0.15% Gelrite, and 0.4% Difco Bacto-agar.
The media were autoclaved for 30 minutes at 121.degree. C., cooled
and 4, 100 mm.times.15 mm petri dishes were poured from each 100 ml
portion.
[0128] A 3 media.times.4 auxin.times.5 concentration combinations,
60 treatment, randomized dose-response experimental design was
used. The design was replicated two times, with one petri dish per
replication and 5 fronds per petri dish. For callus induction, 5
individual duckweed fronds were placed abaxial side down on each
plate of medium. The plates were incubated for five weeks at
23.degree. C. under a 16 hr light/8 hr dark photoperiod with light
intensity of approximately 40 .mu.mol/m.sup.2sec. After five weeks,
the fresh weight of duckweed tissue arising from each original
frond was measured and these tissue populations were visually
examined for the number of calli induced and the type of callus
produced.
[0129] A number of trends were seen in the results. First, low
auxin concentrations and weak auxins promote frond proliferation.
This proliferation is greater than that seen without auxin present.
When fronds are proliferating, callus induction frequency is low.
At high auxin concentration or with stronger auxins, frond curling
and greatly reduced proliferation was observed. Callus formation
was associated with frond curling. The auxin types ranked (from
most curling to least curling) as follows: 2,4-D, dicamba, NAA and
IBA. Both N6 and MS supported callus formation, SH did not. N6
supported greater proliferation than MS. Higher concentrations of
auxin were required on N6 to elicit callus formation than on MS
medium. For compact, Type I callus induction, 2,4-D, dicamba, and
NAA all showed some degree of callus induction on MS medium, on N6
medium only 2,4-D and dicamba produced callus. The greatest callus
induction was seen on MS medium containing 10 .mu.M NAA.
Example 11
[0130] Four cytokinins: benzyladenine (BA), kinetin, thidiazuron
(TDZ), and 2-iP were tested for their ability to induce callus
formation from L. gibba G3 fronds on three different basal media:
SH, MS and N6.
[0131] Duckweed fronds were grown in liquid Schenk and Hildebrandt
medium containing 1% sucrose for two weeks at 23.degree. C. under a
16 hr light/8 hr dark photoperiod with light intensity of
approximately 40 .mu.mol/m.sup.2sec prior to experimentation. For
callus induction, three basal media were tested: Murashige and
Skoog, Schenk and Hildebrandt, and N6. 2,4-D was used as the auxin
at a concentration of 20 .mu.M. The cytokinin concentrations used
were 0, 0.05, 0.1, 0.5, 1 and 5 .mu.M. For each medium
dose-response experiment, 2400 ml of basal medium were prepared
with 2,4-D and the pH adjusted to 5.8. The volume was aliquoted as
24, 100 ml portions. To each of these portions, the appropriate
amount of cytokinin was added and the medium was adjusted to 0.15%
Gelrite, and 0.4% Difco Bacto-agar. The media were autoclaved for
30 minutes at 121.degree. C., cooled and 4, 100 mm.times.15 mm
petri dishes were poured from each 100 ml portion.
[0132] A 3 media.times.4 cytokinin types.times.6 cytokinin
concentrations combinations, 72 treatment, randomized dose-response
experimental design was used. The design was replicated two times,
with one petri dish per replication and 5 fronds per petri dish.
For callus induction, 5 individual duckweed fronds were placed
abaxial side down on each plate of medium. The plates were
incubated for five weeks at 23.degree. C. under a 16 hr light/8 hr
dark photoperiod with light intensity of approximately 40
.mu.mol/m.sup.2sec. After five weeks, the fresh weight of duckweed
tissue arising from each original frond was measured and these
tissue populations were visually examined for the number of calli
induced and the type of callus produced.
[0133] A number of trends were seen in the results. Frond
proliferation did not occur across all treatments due to the 2,4-D
concentration of 20 .mu.M being too high. Frond curling was evident
across all treatments. MS and N6 showed callus induction with MS
clearly superior. Callus induction did not occur on SH medium. TDZ
gave the greatest frequency of callus induction on MS medium across
a broad range of concentrations. A trade-off exists between
induction of Type I and Type II callus. When Type I callus
induction is high, Type II callus induction is low.
Example 12
[0134] As Lemna minor strains 8744 and 8627 showed greater callus
induction and more rapid callus proliferation than L. gibba strains
(see Example 9 and Table I), further optimization of culturing
conditions was done for L. minor. Variables tested for callus
induction included: a) screening of basal medium composition, b)
auxin type and concentration screening, and c) cytokinin type and
concentration screening.
[0135] In the basal medium screen, three media were tested: Schenck
and Hildebrandt, Murashige and Skoog, and F medium as developed by
Frick (Frick, (1991) J. Plant Physiol. 137:397-401). Stock fronds
used for these experiments were grown on F-medium supplemented with
24 .mu.M 2,4-D and 2 .mu.M 2iP for two weeks prior to use. Callus
induction media were prepared as in Example 8. Fronds were
separated, the roots cut off, and half were forced through a
strainer (following the method of Frick) prior to placement on
callus induction media, the remaining half of the fronds were
plated whole. The fronds were incubated under conditions given in
Example 8 for 6 weeks at which time cultures were evaluated for the
presence or absence of callus induction, the degree to which the
callus proliferated, and the basic morphology of the callus.
[0136] Murashige and Skoog medium showed the best callus induction
with both L. minor strains. Schenk and Hildebrandt medium failed to
produce callus, and callus induction was minimal on F medium.
Forcing fronds through a sieve prior to plating had no effect on
callus induction.
[0137] In the auxin type and concentration experiment, four auxins:
2,4-D, NAA, IBA and dicamba were tested, each at four
concentrations: 2, 5, 10 and 20 .mu.M, for their ability to induce
callus formation from L. minor strains 8744 and 8627. The basal
medium used was MS and media and experimental protocol was
basically that followed in Example 10. Fronds used in this
experiment were grown for 2 weeks prior to plating on callus
induction medium under 3 different culture conditions: 1) SH medium
without plant growth regulators, 2) F medium with 24 .mu.M 2,4-D
and 2 .mu.M 2-iP, and 3) SH medium with 24 .mu.M 2,4-D and 2 .mu.M
2-iP. Fronds were separated, the roots cut off and then plated on
induction medium. The fronds were incubated under conditions given
in Example 8 for 6 weeks at which time cultures were evaluated for
the presence or absence of callus induction, the degree to which
the callus proliferated, and the basic morphology of the callus
present.
[0138] Callus induction was not observed on any treatment in which
the inducing auxin was either NAA or IBA. For strain 8744, prolific
callus induction was observed in 2,4-D treatments of either 5 or 10
.mu.M concentrations, with 5 .mu.M 2,4-D giving the best induction.
Callus induction was also observed at the highest dicamba
concentration, 20 .mu.M. For L. minor strain 8627, callus induction
was also observed on 2,4-D and dicamba, but at lower
concentrations. For 2,4-D, the most prolific callus induction was
observed at 1 and 5 .mu.M, with 5 .mu.M giving the best induction.
Useful concentrations of dicamba for callus induction were 5 and 10
.mu.M. Regardless of callus induction treatment, callus formation
came only from fronds previously grown on Schenk and Hildebrandt
medium without plant growth regulators.
[0139] In the cytokinin type and concentration experiment, four
cytokinins: BA, kinetin, 2-iP, and thidiazuron were tested, each at
five concentrations: 0.05, 0.1, 0.5, 1 and 5 .mu.M, for their
ability to induce callus formation from L. minor strains 8744 and
8627. The basal medium used was MS and the media and experimental
protocol were basically as described in Example 11. Fronds used in
this experiment were grown for 2 weeks prior to plating on callus
induction medium under 3 different culture conditions: 1) SH medium
without plant growth regulators, 2) F medium with 24 .mu.M 2,4-D
and 2 .mu.M 2-iP, and 3) SH medium with 24 .mu.M 2,4-D and 2 .mu.M
2-iP. Fronds were separated, the roots cut off and then plated on
induction medium. The fronds were incubated under conditions given
in Example 8 for 6 weeks at which time cultures were evaluated for
the presence or absence of callus induction, the degree to which
the callus proliferated, and the basic morphology of the
callus.
[0140] For strain 8744, prolific callus induction was observed with
either 2-iP or thidiazuron, each at either 0.5 or 1 .mu.M. Callus
induction was only observed with fronds grown on F-medium prior to
plating on callus induction medium. For L. minor strain 8627,
callus induction was also observed with either 2-iP or thidiazuron
but at lower concentrations: either 0.1 or 0.5 .mu.M. In this
strain, callus induction was also observed using BA at 0.5 and at 1
.mu.M.
Example 13
[0141] Basal medium composition was tested for its effect on callus
proliferation and long term establishment using L. minor strains
8627 and 8644.
[0142] Three basal medium compositions were tested for their
ability to maintain healthy callus growth: MS, F-medium and
half-strength SH. All media contained 3% sucrose and were gelled
with 0.4% Difco Bacto-agar and 0.15% Gelrite. The MS medium was
supplemented with 1 .mu.M 2,4-D, 2 .mu.M BA; the half-strength SH
medium was supplemented with 1 .mu.M BA; and the F-medium was
supplemented with 9 .mu.M 2,4-D and 1 .mu.M 2-iP. Callus cultures
from both strain 8744 and strain 8627 proliferated in a previous
callus induction medium as in Example 12 were used for this
experiment. Callus was grown for a two-week subculture period and
scored for growth, color and general health.
[0143] For L. minor strain 8744, half-strength SH supplemented with
1 .mu.M BA proved the best for maintaining callus growth and
health, with the resulting callus showing areas of organizations
and aberrant frond regeneration. Sectors of color, ranging from
green to pale yellow were also present on this medium. Culturing
callus on MS or F-medium resulted in very fast proliferation, with
fresh weight doubling every 6 days. Callus proliferated on these
two media showed much less organization and frond regeneration. For
strain 8627, there was little effect of basal media, callus
proliferation was equally good on all 3 media. As with strain 8744,
callus showed more organization when grown on half-strength SH
supplemented with 1 .mu.M BA.
Example 14
[0144] As Lemna minor showed greater callus induction than Lemna
gibba, an additional screening of three more L. minor strains, all
exceptional in frond growth rate and protein content, was done to
determine if the protocol for callus induction from L. minor 8744
and 8627 would extrapolate to these new strains. The strains are
were designated as L. minor 7501, 8626, and 8745.
[0145] The callus induction system developed in the previous
Examples was followed: Murashige and Skoog basal medium
supplemented with 3% sucrose, 5 .mu.M 2,4-D and 2 .mu.M BA, and
gelled with 0.4% Difco Bacto-agar and 0.15% Gelrite was used for
callus induction. Fronds were grown on liquid SH medium devoid of
plant growth regulators and supplemented with 1% sucrose prior to
plating on callus induction medium. Fronds were plated onto callus
induction medium and scored 5 weeks later for relative frequencies
of callus induction and relative rates of callus proliferation.
[0146] For strains 8626 and 8745 callus induction did not occur
during the 5-week induction period, however subsequent culture did
yield a low frequency of callus proliferation. The morphology and
color of callus from strains 8626 and 8745 was quite similar to
that proliferated from 8744 and 8627 and proliferated quite well
when transferred to callus maintenance medium. Strain 7501 showed a
low frequency of callus induction, with callus similar in
morphology to that produced from strains 8626 and 8745.
Example 15
[0147] As Lemna minuscula showed significant callus induction on
the first screening (see Example 9), callus induction was repeated
with Lemna miniscula strains 6600 and 6747. Callus induction medium
was prepared and fronds cultured as described in Example 14.
[0148] Both Lemna miniscula strains, 6600 and 6747, showed very
high frequencies of callus induction, with callus proliferating
from virtually every frond. Callus initiation occurred quickly in
these strains with callus first observed 2-3 weeks after plating.
Callus was pale in color and proliferated more slowly than that
produced from Lemna minor strains 8744 or 8627 (see Example
14).
Example 16
[0149] Based on the investigations described in the previous
Examples, the preferred methods for callus induction and growth in
Lemna are as follows.
[0150] Callus induction, growth and frond regeneration from
duckweed plants is accomplished through incubation on the
appropriate medium and manipulation of the plant growth regulator
types and concentrations at specific developmental stages to
promote callus formation, growth and reorganization to fully
differentiated plants. Typically, for species within the genus
Lemna, the preferred media for callus induction are N6 and MS, most
preferred is MS. Fronds are incubated in the presence of both an
auxin and a cytokinin, the preferred auxins are NAA and 2,4-D and
the preferred cytokinins are BA and TDZ. The concentrations of
these plant growth regulators vary over a broad range. For the
auxins, the preferred concentrations are 5-20 .mu.M, the most
preferred are 5-10 .mu.M, and for the cytokinins, the preferred
concentrations are 0.5-5 .mu.M, the most preferred are 0.5-1 .mu.M.
The fronds are incubated for an induction period of 3-5 weeks on
medium containing both plant growth regulators with callus
proliferating during this time.
[0151] For callus growth, the preferred media are as for callus
induction, but the auxin concentration is reduced. For auxins, the
preferred concentrations are 1-5 .mu.M, and for cytokinins the
preferred concentrations are 0.5-1 .mu.M. The subculture period is
also reduced from 4-5 weeks, for callus induction, to 2 weeks for
long-term callus growth. Callus growth can be maintained on either
solid medium gelled with agar, Gelrite, or a combination of the
two, with the preferred combination of 0.4% Difco Bacto-agar and
0.15% Gelrite, or on liquid medium. Callus cultures can be
maintained in a healthy state for indefinite periods of time using
this method.
Example 17
[0152] Strains within Wolffia respond to callus-inducing plant
growth regulator concentrations in a manner similar to that for
strains within Lemna. Therefore, select Wolffia strains were
further investigated for their ability to proliferate callus.
[0153] Four Wolffia arrhiza strains: 7246, 8853, 9000, 9006 and
four Wolffia brasiliensis strains: 7393, 7581, 7591, and 8319 were
tested for their ability to proliferate callus in response to plant
growth regulators. The basal medium used was MS supplemented with
3% sucrose, 5 .mu.M 2,4-D, 5 .mu.M, each BA and kinetin, and 65
.mu.M phenylboric acid. Cultures were plated and incubated on
callus induction medium for 5 weeks then scored for callus
proliferation.
[0154] Callus proliferation was not obtained during the 5-week
incubation period from any of the strains tested. However,
pre-callus induction morphology was readily apparent in several
strains, including Wolffia arrhiza 8853, 9000, 9006 and Wolffia
brasilensis 7581. With these strains, frond thickening was
apparent, a response frequently seen in fronds before callus
formation becomes apparent and indicates that the auxin
concentrations used was insufficient to support callus
proliferation.
[0155] Transformation: This section covers experiments pertaining
to the methods used for actual gene transfer. There are three
sections: (1) Transformation of fronds using the gene gun, (2)
Agrobacterium-mediated transformation using duckweed fronds, and
(3) Agrobacterium-mediated transformation using duckweed callus.
The transformation of fronds experiments were used to optimize the
parameters affecting actual gene transfer: (a) bacterial growth,
(b) inclusion of acetosyringone, (c) bacterial concentration, (d)
solution for resuspending bacteria and the effect of osmotic shock,
(e) co-cultivation medium for fronds and callus, (f) duration of
the time of inoculation, (g) co-cultivation time for fronds and
callus, and (h) light conditions during co-cultivation. The
protocol developed with fronds was applied to transform the callus
cultures obtained using the optimized tissue culture procedure. It
is this transformed callus that is taken on to selection and then
through regeneration to obtain transformed fronds.
Gene Gun Mediated Transformation:
Example 18
[0156] Fronds of Lemna gibba G3 were subjected to microcarrier
bombardment to test their ability to express foreign gene
constructs.
[0157] For frond proliferation, 60 ml of high salt medium (De
Fossard, TISSUE CULTURE FOR PLANT PROPAGATORS 132-52 (1976))
supplemented with 3% sucrose and 0.8% agar was prepared, the pH
adjusted to 5.8, autoclaved for 20 minutes at 121.degree. C.,
cooled, and used to pour 6, 60 mm.times.15 mm petri dishes. One
frond was inoculated to each petri dish. The fronds were grown for
two weeks at 23.degree. C. under a 16 hr light/8 hr dark
photoperiod with light intensity of approximately 40
.mu.mol/m.sup.2sec.
[0158] For bombardment, 1.6 .mu.m gold microcarriers were prepared
and DNA from plasmid pRT99 was precipitated on the microcarriers
following the manufacturer's (Bio-Rad) gene gun protocols. The
plasmid, pRT99 (Topfer et al., Nucleic Acid Res. 16, 8725 (1988))
encodes the neomycin phosphotransferase gene and the
.beta.-glucuronidase gene (GUS; Jefferson et al., EMBO J. 6, 3901
(1987)), both under the control of CaMV35S promoters.
[0159] Duckweed fronds were turned abaxial side up and bombarded
with the DNA coated microcarriers at four pressure levels of
helium: 800, 600, and 400 lbs/sq. inch. Histochemical staining for
GUS activity using 5-bromo-4-chloro-3-indolyl-.beta.-D-glucuronic
acid (X-gluc) as the substrate following the method of Stomp
(Histochemical localization of beta-glucoronidase, in GUS PROTOCOLS
103-114 (S. R. Gallagher ed. 1991)) was done 24 hours after
bombardment. The frequency of GUS positive staining centers was
directly proportional to the pressure used for bombardment, with
the greatest number of GUS expressing cells found in the 800 psi
treatment, with frequency ranging from 4-20 staining cells/frond.
In all treatments, bombardment resulted in the destruction of more
than half the fronds.
Example 19
[0160] Fronds of Lemna gibba G3 were subjected to microprojectile
bombardment to test the effect of microcarrier size on the
frequency of foreign gene expression.
[0161] For frond proliferation, 200 ml of high salt medium
supplemented with 3% sucrose and 0.8% agar was prepared, the pH
adjusted to 5.8, autoclaved for 20 minutes at 121.degree. C.,
cooled, and used to pour 20, 60 mm.times.15 mm petri dishes. One
frond was inoculated to each petri dish. All fronds were grown for
two weeks at 23.degree. C. under a 16 hr light/8 hr dark
photoperiod with light intensity of approximately 40
.mu.mol/m.sup.2sec. Two microcarrier size 1.0 and 1.6 .mu.m were
tested at 3 helium pressure levels: 400, 800, and 1200 psi, using a
PDS-1000/He gene gun manufactured by DuPont. Gold microcarriers
were prepared and pRT99 DNA was precipitated onto the microcarriers
following methods supplied by the manufacturer (Bio-Rad).
[0162] Bombarded duckweed fronds were assayed for GUS expression 24
hours after bombardment using histochemical staining methods of
Stomp (Histochemical localization of beta-glucoronidase, in GUS
PROTOCOLS 103-114 (S. R. Gallagher ed. 1991)). The greatest
frequency of GUS expression was found in fronds bombarded with 1.6
.mu.m microcarriers and a helium pressure of 800 psi. The number of
GUS positive events ranged from 1-21 per frond.
Example 20
[0163] Transgenic duckweed plants are regenerated from duckweed
callus transformed by ballistic bombardment. Type I callus cultures
are grown as described in Example 42 below. Typically, 20-30
duckweed callus pieces, approximately 2-4 mm in diameter, are
spread evenly across the bombardment area on MS medium (MS medium
described in Example 42). Gold particles (1.6 .mu.M in diameter)
and bombardment (helium pressure of 800 psi) as described in
Example 18 and Example 19 are used. The DNA for bombardment
consists of an expression plasmid containing the gene of interest
(e.g., GUS, another marker gene, a gene encoding a mammalian
protein, or a gene encoding a bacterial, fungal, plant or mammalian
enzyme) and a gene encoding a selectable marker gene, e.g., nptII
(kanamycin resistance), hptII (hygromycin resistance), sh ble
(zoecin resistance), and bar (phosphinotricin resistance), as well
as other sequences necessary for gene expression (e.g., promoter
sequences, termination sequences). After bombardment at 800 lbs/sq.
inch, the callus is incubated in the dark for two days (or longer
if necessary), followed by incubation under a light intensity of
3-5 .mu.mol/m.sup.2sec for 4-6 weeks. Callus is transferred to
fresh medium every two weeks, with the selectable agent added to
the medium 2-4 weeks post-bombardment. Selection of resistant
callus is continued for 8-16 weeks, until fully resistant callus is
produced. Regeneration of transgenic fronds and plants is carried
out as described in Example 42.
Transformation with Agrobacterium Using Duckweed Fronds:
Example 21
[0164] Duckweed fronds of Lemna gibba G3 were used to test the
susceptibility of duckweed to Agrobacterium tumefaciens using two
different media for co-cultivation, Schenk and Hildebrandt and
Murashige and Skoog.
[0165] Agrobacterium tumefaciens strain AT656 and non-virulent A.
tumefaciens strain A136 were used to inoculate the duckweed fronds.
Strain AT656 is constructed from strain EHA105 (Hood et al.,
Transgenic Res. 2, 208 (1993)) which contains the pTiBo542 vir
region on a disarmed pTiBo542 plasmid. The T-DNA is carried on a
binary plasmid, pCNL56 (Li et al., Pl. Mol. Biol. 20, 1037 (1992)).
This binary plasmid is derived from pBIN19, and as modified carries
a neomycin phosphotransferase gene under the control of the
nopaline synthetase promoter and a nopaline synthetase terminator,
and a .beta.-glucuronidase (GUS) gene (Janssen and Gardner, Plant
Mol. Biol. 14, 61 (1989)) under the control of the mas2'-CaMV35S
promoter and an octopine synthetase terminator. The GUS coding
region contains an intron within the coding sequence of the gene to
prevent bacterial expression of GUS (Vancanneyt et al., Mol. Gen.
Genet. 220, 245 (1990)). Strain A136 is derived from the broad host
range strain, C58. When C58 is grown at temperatures above
30.degree. C. it loses its Ti-plasmid becoming avirulent A136.
These two strains, AT656 and A136, were grown overnight on AB
minimal medium (Chilton et al., Proc. Nat. Acad. Sci. USA 71, 3672
(1974)) solidified with 1.6% agar and supplemented with 100 .mu.M
acetosyringone at 28.degree. C.
[0166] Duckweed fronds were grown in liquid Hoagland's medium
containing 3% sucrose for two weeks at 23.degree. C. under a 16 hr
light/8 hr dark photoperiod with light intensity of approximately
40 .mu.mol/m.sup.2sec prior to experimentation. For co-cultivation,
500 ml of Schenk and Hildebrandt medium containing 1% sucrose and
0.6% agar was prepared, the pH adjusted to 5.6, autoclaved at
121.degree. C. for 30 minutes, and cooled. Five-hundred ml of
Murashige and Skoog medium containing 3% sucrose and 0.6% agar were
also prepared, the pH adjusted to 5.8, autoclaved at 121.degree. C.
for 30 minutes, and cooled. To both media, a filter-sterilized
solution of acetosyringone was added to a final, medium
concentration of 20 mg/L. Twenty, 100 mm.times.15 mm petri dishes
were poured from each cooled medium. For each bacterial strain, the
bacteria from one, 100 mm.times.15 mm petri dish were resuspended
for at least one hour prior to use in 100 ml of the following
solution (Hiei et al., The Plant J. 6, 271 (1994)): Gamborg's B5
salts, Murashige and Skoog vitamins, glycine (8 mg/L), aspartic
acid (266 mg/L), arginine (174 mg/L), glutamine (876 mg/L),
casamino acids (500 mg/L), sucrose (6.85%), glucose (3.6%), and
acetosyringone (20 mg/L). The solution was prepared, the pH
adjusted to 5.8, and filter sterilized before the addition of the
bacteria.
[0167] A 2 bacterial strains.times.2 co-cultivation media,
full-factorial experimental design (4 treatments in total) with 5
replications, with 2 petri dish per replication and 20 fronds per
petri dish was used. For inoculation, duckweed fronds were floated
in the bacterial solution for several minutes. For co-cultivation,
the fronds were transferred to either Schenk and Hildebrandt or
Murashige and Skoog medium as described above. The fronds were
incubated at 23.degree. C. under a 16 hr light/8 hr dark
photoperiod with light intensity of approximately 40
.mu.mol/m.sup.2sec for four days. The fronds were then transferred
to fresh medium of the same composition except that acetosyringone
was absent and 500 mg/L of timentin and 50 mg/L kanamycin sulfate
were added to the medium.
[0168] Histochemical staining for GUS activity following the method
of Stomp et al. (Histochemical localization of beta-glucoronidase,
in GUS PROTOCOLS 103-114 (S. R. Gallagher ed. 1991)) was used to
confirm gene transfer in fronds. Staining of fronds inoculated with
A136 was done as a control to test bacterially inoculated fronds
for endogenous GUS activity. Staining done 10 days after
inoculation showed no GUS staining in A136 inoculated controls and
high frequencies of staining in fronds inoculated with AT656,
regardless of what basal medium, MS or SH, was used for
co-cultivation. Transformation frequencies of greater than 70% of
the original inoculated fronds were observed, showing GUS positive
cells somewhere within the fronds.
Example 22
[0169] Fronds of Lemna gibba G3 were used to determine the effect
of wounding on the frequency of GUS expression after
co-cultivation.
[0170] Duckweed fronds were grown in liquid Schenk and Hildebrandt
medium containing 1% sucrose for two weeks at 23.degree. C. under a
16 hr light/8 hr dark photoperiod with light intensity of
approximately 40 .mu.mol/m.sup.2sec prior to experimentation. For
co-cultivation, one liter of Murashige and Skoog medium containing
3% sucrose, 0.6% agar, 20 .mu.M 2,4-D, 2 .mu.M BA, and 20 mg/L
acetosyringone was prepared, the pH adjusted to 5.8, autoclaved at
121.degree. C. for 30 minutes, and cooled. A filter-sterilized
solution of acetosyringone was added to a final, medium
concentration of 20 mg/L. Forty, 100 mm.times.15 mm petri dishes
were poured from the cooled medium.
[0171] For inoculation, Agrobacterium tumefaciens strain AT656 was
used and was grown overnight at 28.degree. C. on AB minimal medium
(Chilton et al., Proc. Nat. Acad. Sci USA 71, 3672 (1974))
containing 50 mg/L kanamycin sulfate and 20 mg/L acetosyringone.
For inoculation, the bacteria from one 100 mm.times.15 mm petri
dish were resuspended as described in Example 21.
[0172] A 2 wounding treatments.times.2 bacterial inoculations,
full-factorial experimental design (four treatments in total) with
5 replications, with 2 petri dish per replication and 20 fronds per
petri dish was used. For wounding treatments, clumps of duckweed
fronds were removed from SH medium onto moist, sterile filter
paper. The clumps were separated into individual fronds, the fronds
were turned abaxial side up, and fronds were wounded one of two
ways: 1) cut transversely across the frond centrum, thus cutting
through the adjacent meristematic regions from left to right, or 2)
cut on each side of the centrum, thus cutting longitudinally
through each meristematic region. For bacterial treatments, both
classes of wounded fronds were floated on: 1) resuspended AT656 or
2) in the resuspension fluid without the bacteria. For inoculation,
fronds were left floating for 10-30 minutes.
[0173] For co-cultivation, fronds were transferred to Murashige and
Skoog medium as described above with 3% sucrose, 20 .mu.M 2,4-D, 2
.mu.M BA, 100 .mu.M acetosyringone, and 0.6% agar. The fronds were
incubated at 23.degree. C. under a 16 hr light/8 hr dark
photoperiod with light intensity of approximately 40
.mu.mol/m.sup.2sec for four days. A frond subsample was stained for
GUS following the procedure of Stomp (Histochemical localization of
beta-glucoronidase, in GUS PROTOCOLS 103-114 (S. R. Gallagher ed.
1991). Staining of co-cultivated fronds four days after inoculation
showed that the direction of wounding did not affect the frequency
of fronds with GUS staining, which averaged approximately 70%.
Control, wounded fronds inoculated with bacterial resuspension
solution without bacteria showed no GUS staining. The number of
fronds with staining within the meristematic regions averaged
approximately 40%.
Example 23
[0174] Fronds of Lemna gibba G3 were used to determine the effect
of inoculation time for wounded fronds in bacterial resuspension
medium on the frequency of GUS expression after co-cultivation.
[0175] Duckweed fronds were grown in liquid Hoagland's medium
containing 1% sucrose to a density of approximately 120 fronds per
25 ml of medium in a 125 ml flask at 23.degree. C. under a 16 hr
light/8 hr dark photoperiod with light intensity of approximately
40 .mu.mol/m.sup.2 sec prior to experimentation. For
co-cultivation, 1500 ml of Schenk and Hildebrandt medium with 1%
sucrose and 0.6% agar was prepared, the pH adjusted to 5.6,
autoclaved at 121.degree. C. for 30 minutes, and cooled. A
filter-sterilized solution of acetosyringone was added to a final,
medium concentration of 20 mg/L. Sixty, 100 mm.times.15 mm petri
dishes were poured from the cooled medium.
[0176] A randomized block experimental design with 4 inoculation
time treatments, with 3 replications, with 5 petri dish per
replication, and 25 fronds per petri dish was used. For
inoculation, Agrobacterium tumefaciens strain AT656 was used and
was grown overnight at 28.degree. C. on AB minimal medium
containing 50 mg/L kanamycin sulfate and 20 mg/L acetosyringone.
For inoculation, the bacteria from one 100 mm.times.15 mm petri
dish were resuspended as described in Example 21.
[0177] For inoculation, individual fronds were separated from
clumps, each turned abaxial side up and wounded with a sterile
scalpel in the meristematic regions, then transferred to bacterial
suspensions and incubated for 15, 30, 45, or 60 minutes. For
co-cultivation, fronds were transferred to Schenk and Hildebrandt
co-cultivation medium as described above. All 60 petri dishes were
incubated at 23.degree. C. under a 16 hr light/8 hr dark
photoperiod with light intensity of approximately 40
.mu.mol/m.sup.2sec for six days. The three replicates were done
over a four-day period. Subsamples of co-cultivated fronds from
each incubation time (15, 30, 45, or 60 minutes) were taken after
2, 3 and 6 days of co-cultivation. Subsampled fronds were stained
for GUS expression following the procedure of Stomp (Histochemical
localization of beta-glucoronidase, in GUS PROTOCOLS 103-114 (S. R.
Gallagher ed. 1991)) The results are presented in Table II.
TABLE-US-00002 TABLE II Incubation Co-cultivation time time
Replicate Total # fronds # staining 2 days 15 1 27 27 30 1 27 27 45
1 28 26 60 1 28 26 3 days 15 2 30 29 30 2 28 28 45 2 25 24 60 2 27
26 6 days 15 3 27 24 30 3 26 22 45 3 23 21 60 3 30 28
[0178] Although GUS staining on wounded stem ends was evident at 2
days, GUS staining within the meristematic regions was not evident
at 2 days of co-cultivation. Meristematic staining was greatest at
3 days co-cultivation and decreased by 6 days of co-cultivation.
The time of incubation of duckweed fronds in the bacterial
suspension solution did not have a significant effect on the
frequency of overall GUS expression after co-cultivation.
Example 24
[0179] Fronds of Lemna gibba G3 were used to determine the effect
of Agrobacterium strain and foreign gene construct on the frequency
of GUS expression after co-cultivation.
[0180] Two Agrobacterium tumefaciens strains were used: AT656 and
C58sZ707pBI121. C58sZ707pBI121 is a disarmed, broad host range
C.sub.5-8 strain (Hepburn et al., J. Gen. Microbiol. 131, 2961
(1985)) into which pBI121 has been transferred. The binary plasmid,
pBI121 is derived from pBIN19 and its T-DNA encodes a neomycin
phosphotransferase gene under the control of the nopaline
synthetase promoter and a nopaline synthetase terminator, and a
.beta.-glucuronidase (GUS) gene under the control of a CaMV35S
promoter and an octopine synthetase terminator. AT656 was streaked
on AB minimal medium containing kanamycin sulfate at 50 mg/L and
C58sZ707pBI121 was streaked on AB minimal medium containing
streptomycin at 500 mg/L, spectinomycin at 50 mg/L and kanamycin
sulfate at 50 mg/L. Both bacterial strains were grown overnight at
28.degree. C.
[0181] Duckweed fronds were grown in liquid Hoagland's medium
containing 1% sucrose for four weeks at 23.degree. C. under a 16 hr
light/8 hr dark photoperiod with light intensity of approximately
40 .mu.mol/m.sup.2sec prior to experimentation. For co-cultivation,
500 ml of Schenk and Hildebrandt medium with 1% sucrose and 0.6%
agar was prepared, the pH adjusted to 5.6, autoclaved at
121.degree. C. for 30 minutes, and cooled. A filter-sterilized
solution of acetosyringone was added to a final, medium
concentration of 20 mg/L. Twenty, 100 mm.times.15 mm petri dishes
were poured from the cooled medium.
[0182] A randomized block, experimental design with 2 bacterial
strain treatments, with 2 replications, with 5 petri dish per
replication, and 25 fronds per petri dish was used. For
inoculation, bacteria from one AB plate of each strain were
resuspended as described in Example 21.
[0183] For inoculation, individual fronds were separated from
clumps, each turned abaxial side up and wounded with a sterile
scalpel in the meristematic regions, then transferred to bacterial
suspensions and incubated for 15-30 minutes. For co-cultivation,
fronds were transferred to Schenk and Hildebrandt co-cultivation
medium as described above. All 20 petri dishes were incubated at
23.degree. C. under a 16 hr light/8 hr dark photoperiod with light
intensity of approximately 40 .mu.mol/m.sup.2sec for six days. A
subsample of fronds was taken at 6 days of co-cultivation and
stained for GUS expression. With AT656, 12 of the 13 duckweed frond
clumps sampled showed GUS staining, however none was seen in the
meristematic region. With C58sZ707pBI121, all the duckweed frond
clumps showed extensive staining.
[0184] Incubation was continued for all remaining fronds for one
week after transfer to fresh medium containing kanamycin sulfate.
For transfer after co-cultivation, 1500 ml of Schenk and
Hildebrandt medium containing 1% sucrose and 0.6% agar was
prepared, the pH adjusted to 5.6, autoclaved at 121.degree. C. for
30 minutes, and cooled. Two antibiotics, timentin and kanamycin
sulfate were added as filter-sterilized solutions to the cooled
medium to a final medium concentration of 500 mg/L and 2 mg/L,
respectively. The cooled medium was used to pour 60, 100
mm.times.15 mm petri dishes.
[0185] After one week the fronds were scored for growth on
kanamycin and GUS expression. The proliferating fronds showed 3
categories of response to kanamycin: (1) approximately 20% of the
fronds arising from those originally co-cultivated with bacterial
strain AT656 showed vigorous growth in the presence of kanamycin
and approximately 30% of fronds arising from those originally
co-cultivated with bacterial strain C58sZ707pBI121 showed vigorous
growth in the presence of kanamycin, (2) another group of fronds
clearly had not proliferated and were bleached of chlorophyll and
were dying, (3) an intermediate group of fronds showed some
proliferation in the presence of kanamycin but the fronds were half
bleached, indicating sensitivity to kanamycin. Results of GUS
staining indicated that active enzyme was still present at high
frequency in the originally co-cultivated fronds.
Example 25
[0186] Fronds of Lemna gibba G3 were used to determine the effect
of Agrobacterium strain, foreign gene construct, and frond
pre-treatment on the frequency of GUS expression after
co-cultivation.
[0187] Two Agrobacterium tumefaciens strains were used: AT656 and
EHA101pJR1. EHA101pJR1 is a binary Agrobacterium tumefaciens strain
containing a disarmed pTiBo542 plasmid harboring the hypervirulence
region of wild-type strain, Bo542, and a small binary plasmid
harboring a hygromycin-phosphotransferase gene under the control of
an alcohol dehydrogenase 1 enhanced, CaMV35S promoter and a
.beta.-glucuronidase gene constructed as in AT656. These two
strains were streaked on potato dextrose agar with 50 mg/L
kanamycin and grown overnight at 28.degree. C.
[0188] Duckweed fronds were grown on liquid Schenk and Hildebrandt
medium containing 1% sucrose with and without 10 .mu.M indoleacetic
acid (IAA), a concentration sufficient to increase proliferation
rate. Fronds were grown in 25 ml aliquots of medium in 125 ml
flasks, at 23.degree. C. under a 16 hr light/8 hr dark photoperiod
with light intensity of approximately 40 .mu.mol/m.sup.2sec. For
co-cultivation, 500 ml of Schenk and Hildebrandt medium containing
1% sucrose, 0.8% agar, 20 mg/L acetosyringone, and with and without
10 .mu.M indoleacetic acid was prepared, pH adjusted to 5.6,
autoclaved at 121.degree. C. for 30 minutes, and cooled.
Filter-sterilized solutions of acetosyringone, and acetosyringone
and indoleacetic acid were added to the cooled medium, to the
final, appropriate concentrations. Twenty, 100 mm.times.15 mm petri
dishes were poured from the cooled medium.
[0189] A randomized block, experimental design with 2 bacterial
strain treatments.times.2 frond growth media, with 5 replications,
with one petri dish per replication, and 20 fronds per petri dish
was used. For inoculation, bacteria of each strain were separately
resuspended as described in Example 21. For inoculation, individual
fronds were separated from clumps, each turned abaxial side up and
wounded with a sterile scalpel in the meristematic regions, then
transferred to bacterial suspensions of either AT656 or EHA101pJR1,
and incubated for 10-15 minutes. For co-cultivation, fronds were
transferred to solid Schenk and Hildebrandt medium with 1% sucrose,
0.8% agar and 100 .mu.M acetosyringone with and without 10 .mu.M
indoleacetic acid as described above, abaxial side down.
[0190] The fronds were co-cultivated for 4 days at 23.degree. C.
under a 16 hr light/8 hr dark photoperiod with light intensity of
approximately 40 .mu.mol/m.sup.2 sec. The fronds from two plates
from each of the four treatments were stained for GUS expression.
Table III presents the results of GUS staining. TABLE-US-00003
TABLE III Media Strain Total # fronds Total # Stained SH AT656 61
12 SH EHA101pJR1 62 4 SH + IAA AT656 66 32 SH + IAA EHA101pJR1 68
2
[0191] Regardless of the presence or absence of IAA, fronds
co-cultivated with EHA101pJR1 had much lower frequencies of fronds
showing GUS expression. An effect of IAA in the incubation medium
was detected with medium containing IAA giving 48% of co-cultivated
fronds showing GUS expression compared to 20% of fronds
co-cultivated on medium without IAA.
Example 26
[0192] Fronds of Lemna gibba G3 were co-cultivated for five
different times: 12.5, 18.5, 40.5, 82, and 112 hours, with
bacterial strain AT656 to test the effect of co-cultivation time on
GUS expression after co-cultivation.
[0193] Duckweed fronds were grown for two weeks on liquid Schenk
and Hildebrandt medium containing 1% sucrose and 10 .mu.M
indoleacetic acid at 23.degree. C. under a 16 hr light/8 hr dark
photoperiod with light intensity of approximately 40
.mu.mol/m.sup.2sec prior to experimentation. For co-cultivation,
750 ml of Schenk and Hildebrandt medium with 1% sucrose, 0.8% agar,
10 .mu.M indoleacetic acid, and 20 mg/L acetosyringone was
prepared, the pH was adjusted to 5.6, the medium autoclaved at
121.degree. C. for 30 minutes, and cooled. A filter-sterilized
solution of acetosyringone and indoleacetic acid was added to the
final medium concentration. Thirty, 100 mm.times.15 mm petri dishes
were poured from the cooled medium. Bacterial strain AT656 was
streaked on potato dextrose agar with 50 mg/L kanamycin sulfate and
grown overnight at 28.degree. C.
[0194] A randomized block, experimental design with 5 incubation
time treatments, with 6 replications, with one petri dish per
replication, and 60 fronds per petri dish was used. For
inoculation, bacteria were resuspended as described in Example 21.
For inoculation, individual fronds were separated from clumps and
each turned abaxial side up and wounded with a sterile scalpel in
the meristematic regions. Fronds were then transferred to the
bacterial resuspension solution and incubated for approximately
10-15 minutes. For co-cultivation, fronds were transferred to solid
Schenk and Hildebrandt medium as described above, abaxial side
down. The fronds were co-cultivated under a 16 hr light/8 hr dark
photoperiod with light intensity of approximately 40
.mu.mol/m.sup.2sec.
[0195] At the appropriate times, 10 fronds were removed from each
petri dish (6 samples) and histochemically stained for GUS
expression. Table IV gives the results of GUS staining:
TABLE-US-00004 TABLE IV Time (hr) Total # Fronds Total # Staining
12.5 61 0 18.5 61 0 40 61 0 82 75 24 112 67 25
[0196] Co-cultivation time had a significant effect on the
frequency of fronds with GUS expression. Before 40 hours, no GUS
expression was detectable. By 3.5 days (82 hours) GUS expression
was readily detectable. Longer co-cultivation did not significantly
increase the frequency, intensity, or tissue association pattern of
GUS expression in duckweed fronds. It was concluded that 3.5-4 days
is the shortest co-cultivation time that will give the maximum
frequency of gene transfer in duckweed fronds.
Example 27
[0197] Bacteria of strain AT656, grown on three different bacterial
media: AB minimal, potato dextrose, and mannitol glutamine Luria
broth, were used to co-cultivate Lemna gibba G3 fronds, that had
been grown with and without indoleacetic acid prior to
co-cultivation, in light and in the dark to test the effects of
these treatments on GUS expression following co-cultivation.
[0198] Lemna gibba G3 fronds were grown for two weeks on liquid
Schenk and Hildebrandt medium containing 1% sucrose and with or
without 10 .mu.M indoleacetic acid in 25 ml aliquots in 125 ml
flasks, at 23.degree. C. under a 16 hr light/8 hr dark photoperiod
with light intensity of approximately 40 .mu.mol/m.sup.2sec prior
to experimentation. For co-cultivation, 900 ml of Schenk and
Hildebrandt medium containing 1% sucrose, 0.8% agar, with and
without 10 .mu.M indoleacetic acid, and 20 mg/L acetosyringone was
prepared, the pH was adjusted to 5.6, the medium autoclaved at
121.degree. C. for 30 minutes, and cooled. Filter-sterilized
solutions of acetosyringone and indoleacetic acid were added to the
appropriate, final medium concentrations. Thirty-six, 100
mm.times.15 mm petri dishes were poured from the cooled medium.
Three bacterial media: 1) AB minimal containing 1.6% agar (AB),
Difco potato dextrose medium with 1.6% agar (PDA), and mannitol
glutamine (Roberts and Kerr, Physiol. Plant Path. 4, 81 (1974)
Luria broth medium with 1.6% agar (MGL; Miller, EXPERIMENTS IN
MOLECULAR GENETICS 433 (1972)) were prepared, autoclaved at
121.degree. C. for 20 minutes, and cooled. A filter-sterilized
solution of kanamycin sulfate and acetosyringone was added to the
cooled media to final medium concentrations of 50 mg/L and 20 mg/L,
respectively. AT656 was streaked on these three media and incubated
overnight at 28.degree. C.
[0199] A full-factorial experimental design with 3 bacterial
media.times.2 plant media.times.2 light condition treatments (12
treatments in total), with 3 replications, with one petri dish per
replication, and 20-25 fronds per petri dish was used. For
inoculation, bacteria from each medium were separately resuspended
as described in Example 21.
[0200] For inoculation, individual fronds were separated from
clumps, each turned abaxial side up and wounded with a sterile
scalpel in the meristematic regions. Fronds were then transferred
to the bacterial resuspension solution and incubated for
approximately 10-15 minutes. After inoculation, fronds were
transferred to solid Schenk and Hildebrandt co-cultivation medium
as described above. The fronds were co-cultivated for 4 days under
a 16 hr light/8 hr dark photoperiod with light intensity of
approximately 40 .mu.mol/m.sup.2sec for the light treatment or
placed in total darkness for the dark treatment. After
co-cultivation, all fronds were stained for GUS expression
following the procedure of Stomp et al. (Histochemical localization
of beta-glucoronidase, in GUS PROTOCOLS 103-114 (S. R. Gallagher
ed. 1991)). Table V gives the results of GUS staining:
TABLE-US-00005 TABLE V Bacterial Plant Light Total Total # #
Stained Medium Medium or Dark # Fronds Stained Meristem PDA SH D 63
60 5 PDA SH L 67 66 8 MGL SH D 66 65 7 MGL SH L 70 65 9 AB SH D 58
58 7 AB SH L 62 60 6 PDA SH + IAA D 61 61 14 PDA SH + IAA L 68 63
14 MGL SH + IAA D 62 61 11 MGL SH + IAA L 46 39 2 AB SH + IAA D 62
61 6 AB SH + IAA L 58 53 3
[0201] Bacterial medium has a significant effect on the frequency
of GUS expression after 4 days of co-cultivation. AB medium gave
the lowest frequency of GUS expression and PDA the highest. Growing
fronds on indoleacetic acid prior to inoculation increased the
frequency of GUS expression after co-cultivation. The presence of
light during co-cultivation did not significantly affect the
frequency of GUS expression after co-cultivation in treatments
using fronds grown without indoleacetic acid, however,
co-cultivation in the dark did increase the frequency of GUS
expression in treatments that used fronds grown in the presence of
indoleacetic acid. Averaging frequencies from PDA and MGL across
the duckweed fronds grown on Schenk and Hildebrandt medium with
indoleacetic acid gives a frequency of GUS expression in
meristematic tissue of approximately 17%.
Example 28
[0202] Six co-cultivation times and the presence or absence of
light during co-cultivation were examined for their effect on GUS
expression following co-cultivation.
[0203] Lemna gibba G3 fronds were grown for 17 days on Schenk and
Hildebrandt medium containing 1% sucrose and 10 .mu.M indoleacetic
acid at 23.degree. C. under a 16 hr light/8 hr dark photoperiod
with light intensity of approximately 40 .mu.mol/m.sup.2sec. For
co-cultivation, 150 ml of Schenk and Hildebrandt medium containing
1% sucrose, 1% agar, 10 .mu.M indoleacetic acid, and 20 mg/L
acetosyringone was prepared, the pH adjusted to 5.6, autoclaved for
30 minutes, and cooled. Filter-sterilized solutions of
acetosyringone and indoleacetic acid were added to the final medium
concentrations to the cooled medium. The medium was used to pour 6,
100 mm.times.15 mm petri dishes. Agrobacterium tumefaciens strain
AT656 was streaked on AB minimal medium containing kanamycin
sulfate at 50 mg/L and 20 mg/L acetosyringone and grown overnight
at 23.degree. C.
[0204] A randomized block, experimental design with 6
co-cultivation time treatments, with 6 replications, with one petri
dish per replication, and 30 fronds per petri dish was used. For
inoculation, the bacteria from one petri dish were resuspended as
described in Example 21.
[0205] For inoculation, individual fronds were separated from
clumps, each turned abaxial side up and wounded with a sterile
scalpel in the meristematic regions. Fronds were transferred to
bacterial suspensions and incubated for 10 minutes. For
co-cultivation, fronds were transferred to Schenk and Hildebrandt
co-cultivation medium as described above. Three plates were wrapped
in aluminum foil to effect complete darkness and all plates were
incubated at 23.degree. C. under a 16 hr light/8 hr dark
photoperiod with light intensity of approximately 40
.mu.mol/m.sup.2sec for six time points: 13, 23, 36, 49, 73.5, and
93 hours. After co-cultivation for the appropriate time, 5 fronds
were removed from each of 6 plates (3 samples dark and 3 samples
light) and stained for GUS expression following the procedure of
Stomp et al. (Histochemical localization of beta-glucoronidase, in
GUS PROTOCOLS 103-114 (S. R. Gallagher ed. 1991)).
[0206] The results showed that GUS expression became evident by 23
hours after co-cultivation, with expression detected only at the
broken end of stems. By 36 hours, staining was detected in cells
surrounding wounds and at the broken ends of stems. Staining was
more intense overall, however the level of staining intensity was
greater in the fronds incubated in the dark. By 49 hours, the
difference in staining intensity and staining pattern were evident
in the dark versus light treatments. Staining was more extensive in
fronds incubated in the dark, however the frequency of fronds
showing GUS expression and the frequency of GUS expressing
meristematic regions was not significantly different between light
and dark treatments. By 73.5 hours the staining pattern and the
frequency of staining did not differ significantly between dark and
light treatment except that wounded tissue staining was more
prevalent in the dark treatment. By 93 hours (approximately 4 days)
the greatest number of GUS expressing, meristematic regions was
detected, with the dark treatment definitely superior to the light
treatment. Intense staining was still present in wounded cells.
Example 29
[0207] Fronds of Lemna gibba G3 were used to determine the effect
of bacterial resuspension solutions, the osmotic potential of these
solutions, and frond wounding on the frequency of GUS expression
following co-cultivation.
[0208] Fronds were grown on liquid Schenk and Hildebrandt medium
containing 1% sucrose at 23.degree. C. under a 16 hr light/8 hr
dark photoperiod with light intensity of approximately 40
.mu.mol/m.sup.2sec. For co-cultivation, 1800 ml of Schenk and
Hilderbrandt (SH) medium with 1% sucrose, 0.8% of unwashed agar,
and 20 mg/L acetosyringone was prepared, the pH adjusted to 5.6,
autoclaved for 30 minutes at 121.degree. C. for 30 minutes, and
cooled. Heat labile acetosyringone was added to autoclaved, cooled
medium as a filter-sterilized solution. The cooled medium was used
to pour 72, 100 mm.times.15 mm petri dishes. Agrobacterium strain
AT656 was streaked onto AB minimal medium containing 20 mg/L
acetosyringone and 50 mg/L kanamycin sulfate and grown overnight at
28.degree. C.
[0209] A full-factorial experimental design with 12 bacterial
resuspension solution.times.2 wounding treatments (24 treatments in
total), with 3 replications, with one petri dish per replication,
and 20 fronds per petri dish was used. Ten combinations of two
different bacterial resuspension solutions: 1) Gamborg's B5 salts,
Murashige and Skoog vitamins, glycine (8 mg/L), aspartic acid (266
mg/L), arginine (174 mg/L), glutamine (876 mg/L), casamino acids
(500 mg/L), sucrose (6.85%), glucose (3.6%), and acetosyringone (20
mg/L), and 2) Schenk and Hildebrandt medium with 1% sucrose, each
at 5 different mannitol concentrations: 0, 0.2, 0.4, 0.6 and 0.8 M,
were tested for their efficacy in gene transfer. In addition, two
other solutions were tested: 3) Gamborg's B5 salts, Murashige and
Skoog vitamins, glycine (8 mg/L), aspartic acid (266 mg/L),
arginine (174 mg/L), glutamine (876 mg/L), casamino acids (500
mg/L), and acetosyringone (20 mg/L), and 4) Schenk and Hildebrandt
medium with sucrose (6.85%), glucose (3.6%), and acetosyringone (20
mg/L). All bacterial resuspension solutions were filter-sterilized
prior to use. For inoculation, bacteria from one AB plate were
resuspended in 100 ml of each of the 12 resuspension solutions at
least one hour prior to use.
[0210] The importance of wounding fronds prior to inoculation was
also tested. For either wounded or unwounded fronds, individual
fronds were first separated from clumps. For wounding, fronds were
turned abaxial side up and stabbed with a sterile scalpel in the
meristematic regions. Unwounded fronds received no further
treatment after separation into individual fronds.
[0211] For inoculation, 120 fronds, 60 wounded and 60 unwounded,
were floated on each of the 12 bacterial resuspension media for 10
minutes, with wounded fronds inoculated separately from unwounded
fronds. For co-cultivation, the fronds were transferred to solid
Schenk and Hildebrandt medium as described above. All treatments
were co-cultivated for 4 days in the dark. After four days of
co-cultivation, two plates from each treatment were randomly picked
and stained for GUS expression.
[0212] The results indicated that regardless of media, 0.6M of
mannitol gave the highest frequencies of GUS expression after
co-cultivation. The simpler, Schenk and Hildebrandt medium
formulation worked as well as the more complex medium formulation
using Gamborg's B5 salts. Wounding gave a measurable, but not
statistically significant, increase in the frequency of fronds
showing GUS expression and did not increase the frequency of
staining in the meristematic region.
Example 30
[0213] Fronds of Lemna gibba G3 were used to test the effect of
bacterial concentrations during inoculation on the frequency of GUS
expression following co-cultivation.
[0214] Duckweed fronds grown on liquid Schenk and Hildebrandt
medium containing 1% sucrose and 10 .mu.M indoleacetic acid in 25
ml aliquots in 125 ml flasks at 23.degree. C. under a 16 hr light/8
hr dark photoperiod with light intensity of approximately 40
.mu.mol/m.sup.2sec for two weeks prior to use. For co-cultivation,
750 ml of Schenk and Hildebrandt medium with 1% sucrose, 1% agar,
20 mg/L acetosyringone, and 10 .mu.M indoleacetic acid was
prepared, the pH adjusted to 5.6 autoclaved at 121.degree. C. for
30 minutes, and cooled. Filter-sterilized solutions of
acetosyringone and indoleacetic acid were added to the cooled
medium to obtain the final medium concentrations. The cooled medium
was used to pour 30, 100 mm.times.15 mm petri dishes. Agrobacterium
strain AT656 was streaked on half-strength potato dextrose
agar-mannitol glutamine Luria broth medium with 1.6% Difco
Bacto-agar, 20 mg/L acetosyringone, and 50 mg/L kanamycin sulfate
and grown overnight at 28.degree. C.
[0215] A randomized block experimental design with 10 bacterial
concentration treatments, with 3 replications, with one petri dish
per replication and 20 individual fronds or frond clumps per petri
dish was used. For inoculation, bacteria from one petri dish were
resuspended as described in Example 21. This bacterial solution
constituted the "undiluted" sample and was the beginning of a
serial dilution series for the following dilutions: 1/3, 10.sup.-1,
1/33, 10.sup.-2, 1/333, 10.sup.-3, 1/3333, 10.sup.-4, 10.sup.-5.
The 1/3 dilution had an OD540 nm of 1.006, which corresponded to
approximately 1.6.times.10.sup.9 bacteria/ml.
[0216] For inoculation, individual fronds were separated from
clumps, each turned abaxial side up and wounded with a sterile
scalpel in the meristematic regions. Fronds were then transferred
to each of the ten different bacterial resuspension solution
concentrations and incubated for approximately 10-15 minutes. For
co-cultivation, fronds were transferred to solid Schenk and
Hildebrandt co-cultivation medium described above, abaxial side
down. The fronds were co-cultivated for 4 days at 23.degree. C. in
the dark. After co-cultivation, all fronds were stained for GUS
expression following the procedure of Stomp et al. (Histochemical
localization of beta-glucoronidase, in GUS PROTOCOLS 103-114 (S. R.
Gallagher ed. 1991)).
[0217] The results showed that the frequencies of GUS expression
varied ten-fold across bacterial concentration. The greatest
frequency of GUS expression was observed at the highest bacterial
concentration tested. At dilutions greater than 10.sup.-3 no GUS
expression was detected.
Example 31
[0218] Fronds of Lemna gibba G3 were used to test the effect of
four co-cultivation media on GUS expression using an optimized
transformation protocol.
[0219] Fronds were grown on liquid Schenk and Hildebrandt medium
containing 1% sucrose and 10 .mu.M indoleacetic acid at 23.degree.
C. under a 16 hr light/8 hr dark photoperiod with light intensity
of approximately 40 .mu.mol/m.sup.2sec. For co-cultivation, four
media were used: 1) Murashige and Skoog medium (MS) with 20 .mu.M
2,4-D and 0.1 .mu.M BA (MS1), 2) MS medium with 20 .mu.M 2,4-D and
1 .mu.M BA (MS2), 3) MS medium with 1 .mu.M 2,4-D and 2 .mu.M BA
(MS 3), and 4) Schenk and Hilderbrandt medium (S H). For each
medium, 100 ml containing the appropriate plant growth regulators
containing 3% sucrose, 0.15% Gelrite and 0.4% Difco Bacto-agar was
prepared, the pH adjusted to 5.6, autoclaved at 121.degree. C. for
20 minutes, and cooled. A filter-sterilized acetosyringone solution
was added to each cooled medium to a final concentration of 20
mg/L. Each medium was used to pour 4, 100 mm.times.15 mm petri
dishes. Bacterial strain AT656 was streaked on potato dextrose agar
with 20 mg/L acetosyringone and 50 mg/L kanamycin sulfate and grown
overnight at 28.degree. C.
[0220] A randomized block experimental design with 4 media
treatments with four replications, with one petri dish per
replication and 20 fronds per petri dish was used. For inoculation,
the bacteria from one petri dish were resuspended for one hour
prior to use in 100 ml of filter-sterilized SH medium with 0.6M
mannitol and 20 mg/L acetosyringone at pH 5.6. For inoculation,
individual fronds were separated from clumps and floated in the
resuspended bacteria for 8-10 minutes. For co-cultivation, the
fronds were transferred to co-cultivation medium described above
(MS1, MS2, MS3, SH). Fronds were co-cultivated at 23.degree. C. in
the dark for four days. After four days of co-cultivation, all
fronds were stained for GUS expression.
[0221] The frequency of fronds showing GUS expression ranged from
80-90% across all treatments. Co-cultivation medium did not have a
significant effect on this frequency. The intensity of GUS staining
ranged from light to intense. Staining was associated with root
tips, stems, broken ends of stems and wounds, meristematic regions,
and the frond margins.
Example 32
[0222] Frond transformation using Agrobacterium is accomplished
through manipulation of the cell division rate of the fronds prior
to inoculation, the medium on which the Agrobacteria are grown,
optimization of co-cultivation parameters including secondary
metabolites such as acetosyringone, the concentration of the
Agrobacteria, the osmolarity of the inoculation fluid, the duration
of the co-cultivation period, and the light intensity of the
co-cultivation period.
[0223] Based on the studies described in the previous Examples, a
preferred method of frond transformation and selection is as
follows. Typically, fronds are grown on medium containing an auxin
that increases the proliferation rate of the fronds, with NAA, IBA
and IAA being the preferred auxins and the preferred concentrations
ranging from 0.2-1 .mu.M. Agrobacteria are grown on a medium
without rich nutrient supplements and including such secondary
metabolites as acetosyringone, with potato dextrose agar and
mannitol glutamine Luria broth as preferred media. The frequency of
transformation is determined by the composition of the inoculating
fluid, with the preferred fluid being MS or SH basal salts
supplemented with 0.6 M mannitol and 100 .mu.M acetosyringone. The
concentration of Agrobacteria resuspended in this inoculating fluid
also affects the frequency of transformation, with the preferred
concentration on the order of 1.times.10.sup.9 bacteria per ml.
Inoculation time can vary with the preferred time ranging from 2-20
minutes. Co-cultivation time also affects the frequency of
transformation, with a time of 3-4 days being preferred.
Co-cultivation can be carried on under light or dark conditions,
with darkness (e.g., subdued light) being preferred.
[0224] Growth of transformed fronds is also dependent on preferred
conditions. MS and SH are the preferred media. Decontamination of
the fronds from infecting Agrobacteria is done using the
appropriate antibiotics at high concentrations, typically 100-500
mg/L, with frequent transfer of infected tissue, the preferred
method being transfer to fresh medium with antibiotic every 2-4
days. Incubation under low light intensity, the preferred range
being 1-5 .mu.mol/m.sup.2.sec, for an initial resting/recovery
period of 3-6 weeks is preferred.
[0225] Selection by growth in the presence of the selection agent
can be initiated at variable times, with the preferred time being
1-3 weeks after inoculation. Initial selection under reduced light
levels and low selection agent concentration is also preferred,
with light levels of 1-5 .mu.mol/m.sup.2.sec and low concentration
ranges appropriate for the selection agent as determined from
toxicity studies for the specific agent. For kanamycin sulfate, the
typical range is 2-10 mg/L.
Example 33
[0226] Fronds from strains within 10 species of duckweed: Lemna
trisulca 7315, Lemna minor 7101, Lemna japonica 7427, Lemna
turionifera 6601, Lemna gibba G3, Lemna valdiviana 7002, Lemna
aequinocitalis 7001, Lemna miniscula 6711, Lemna obscura 7325, and
Spirodela punctata 7273, were tested for their ability to give GUS
expression following co-cultivation using the transformation
protocol developed with Lemna gibba G3.
[0227] All duckweed strains except L. gibba G3 were grown on liquid
Schenk and Hildebrandt medium with 1% sucrose in 25 ml aliquots in
125 ml flasks. Lemna gibba G3 was grown on Schenk and Hildebrandt
medium with 1% sucrose and 10 .mu.M indoleacetic acid. All duckweed
cultures were incubated at 23.degree. C. under a 16 hr light/8 hr
dark photoperiod with light intensity of approximately 40
.mu.mol/m.sup.2sec. For co-cultivation, 400 ml of Schenk and
Hildebrandt medium containing 1% sucrose, 0.9% agar, and 20 mg/L
acetosyringone was prepared, the pH adjusted to 5.6, autoclaved at
121.degree. C. for 30 minutes and cooled. A filter-sterilized
solution of acetosyringone was added to the cooled medium to obtain
the final medium concentration. The cooled medium was used to pour
10, 100 mm.times.15 mm petri dishes. Agrobacterium tumefaciens
strain AT656 was streaked on half-strength potato dextrose agar
mixed with half-strength mannitol glutamine Luria broth medium
containing 20 mg/L acetosyringone and 50 mg/L kanamycin sulfate
overnight at 28.degree. C.
[0228] A randomized block experimental design with 10 duckweed
strain treatments, with one replication, with one petri dish per
replication and 20-25 fronds per petri dish was used. For
inoculation, bacteria from one petri dish were resuspended as
described in Example 21.
[0229] For inoculation, individual fronds were separated from
clumps, each turned abaxial side up and wounded with a sterile
scalpel in the meristematic regions. Fronds were transferred to
bacterial suspensions and incubated for approximately 10 minutes.
For co-cultivation, fronds were transferred to Schenk and
Hildebrandt co-cultivation medium described above and incubated at
23.degree. C. in the dark for four days.
[0230] After co-cultivation the fronds were stained for GUS
expression. Of the 10 strains tested, 8 showed GUS expression in a
pattern identical to L. gibba G3 and at frequencies ranging from
14% to 80%.
Example 34
[0231] Twenty strains of duckweed from the 4 genera of the
Lemnaceae were tested for their ability to give GUS expression
following co-cultivation with Agrobacterium strain AT656 using the
transformation protocol developed with L. gibba G3. The twenty
strains were: Wolffiella lingulata strains 8742 and 9137, Wl.
neotropica strains 7279 and 8848, Wl. oblongata strains 8031 and
8751, Wolffia arrhiza strains 7246 and 9006, Wa. australiana 7317,
Wa. brasiliensis strains 7397, 7581, and 8919, Wa. columbiana
strains 7121 and 7918, Spirodela intermedia 7178, S. polyrrhiza
strains 7960 and 8652, S. punctata strains 7488 and 7776, and L.
gibba G3.
[0232] All strains were grown on liquid Schenk and Hildebrandt
medium with 1% sucrose, pH of 5.6 for two weeks prior to
experimentation. For co-cultivation, 1500 ml of Schenk and
Hildebrandt medium containing 1% sucrose, 0.8% agar, and 20 mg/L
acetosyringone was prepared, the pH adjusted to 5.6, autoclaved at
121.degree. C. for 30 minutes, and cooled. A filter-sterilized
solution of acetosyringone was added to the cooled medium to obtain
the final medium concentration. The cooled medium was used to pour
60, 100 mm.times.15 mm petri dishes. The bacterial strain, AT656
was streaked on potato dextrose agar with 20 mg/L acetosyringone
and 50 mg/L kanamycin sulfate and grown overnight at 28.degree. C.
For inoculation, the bacteria from one petri dish were resuspended
for at least one hour prior to use in Schenk and Hildebrandt medium
with 0.6M mannitol, 20 mg/L acetosyringone, pH of 5.6 that was
filter-sterilized before use.
[0233] A randomized block experimental design with 20 duckweed
strain treatments, with 3 replications, with one petri dish per
replication and 20 individual fronds or frond clumps per petri dish
was used. For inoculation, individual fronds of Spirodela and
Wolfiella strains and L. gibba G3 were separated from clumps. For
Wolffia strains, fronds were inoculated as clumps because their
small size made individual frond separation difficult. For
inoculation, fronds of each duckweed strain were floated in the
bacterial suspension solution for 2-5 minutes. For co-cultivation,
the fronds were transferred from bacterial solution to solid Schenk
and Hildebrandt co-cultivation medium described above. For
Spirodela and Wolfiella strains and for L. gibba G3, 20 individual
fronds were transferred to each of 3 replicate dishes; for Wolffia
strains, 20 small frond clumps were transferred to each of 3
replicate plates. All strains were co-cultivated in darkness at
23.degree. C. for four days.
[0234] After co-cultivation, 2 plates from each strain were stained
for GUS expression. Staining results showed that all but one
species tested and the majority of duckweed strains within species
gave some GUS expression 4 days after co-cultivation. Of the 4
Wolffia species tested, all showed varying frequencies of GUS
expression. The three strains of Wolffia brasiliensis showed the
highest frequencies of GUS expression, ranging from 50-75%. Across
the 6 strains within the genus Wolfiella, the frequency of GUS
expression was lower, ranging from 5-12%. Two of the three
Spirodela species gave GUS expression of 10 and 35%; the third gave
no indication of GUS expression. Lemna gibba G3, serving as the
positive control had a GUS expression frequency of approximately
50%.
Transformation by Agrobacteria Using Callus Cultures:
Example 35
[0235] Type I callus produced from Lemna gibba G3 fronds was used
to test its ability to give GUS expression using the optimized
transformation protocol developed with L. gibba G3 fronds and to
test the effect of vacuum infiltration.
[0236] Type I callus was produced by growing fronds on solid
Murashige and Skoog medium containing 3% sucrose, 0.15% Gelrite,
0.4% Difco Bacto-agar, 5 .mu.M 2,4-dichlorophenoxyacetic acid
(2,4-D), and 2 .mu.M benzyladenine (BA). Callus induction and all
subsequent culture was at 23.degree. C. and under a 16 hr light/8
hr dark photoperiod with light intensity of approximately 40
.mu.mol/m.sup.2sec. After 4 weeks of callus induction, Type I
callus clumps were separately cultured on the same medium with the
2,4-D concentration reduced to 1 .mu.M. The callus was subcultured
to fresh medium every two weeks until sufficient callus was
proliferated for experimentation.
[0237] For co-cultivation, 400 ml of solid Murashige and Skoog
medium (MS) with 3% sucrose, 0.15% Gelrite, 0.4% Difco Bacto-agar,
1 .mu.M 2,4-D, and 2 .mu.M BA was prepared, the pH adjusted to 5.6,
autoclaved at 121.degree. C. for 20 minutes, and cooled. A
filter-sterilized solution of acetosyringone was added to a final,
medium concentration of 20 mg/L. The cooled medium was used to pour
16, 100 mm.times.15 mm petri dishes. Agrobacterium strain AT656 was
streaked on potato dextrose agar with 20 mg/L acetosyringone and 50
mg/L kanamycin sulfate and grown overnight at 28.degree. C.
[0238] A randomized block experimental design with two vacuum
infiltration treatments with four replications with two petri
dishes per replication and ten callus pieces per petri dish was
used. For inoculation, the bacteria were resuspended in
filter-sterilized Schenk and Hildebrandt medium containing 0.6M
mannitol and 20 mg/L of acetosyringone at pH 5.6 for at least one
hour before use. Inoculation with bacteria was done with and
without vacuum infiltration. Without vacuum infiltration, small
pieces of Type I callus were placed in the bacterial solution for
10 minutes, then blotted and transferred to MS co-cultivation
medium as described above. With vacuum infiltration, the callus was
placed in bacterial solution, a vacuum of 10 inches of mercury
applied for 10 minutes, then the callus was blotted and transferred
to MS co-cultivation medium. All dishes were co-cultivated in the
dark at 23.degree. C.
[0239] After four, six and nine days of co-cultivation,
approximately 40, 20 and 20 callus pieces, respectively, were
stained for GUS expression. Results showed that the frequencies of
callus pieces showing GUS expression did not vary with respect to
vacuum infiltration treatment nor did the frequencies vary with the
time of co-cultivation. Without vacuum infiltration across all time
points, GUS staining ranged from 25-78% and with vacuum
infiltration the frequencies ranged from 25-74%. The intensity of
GUS staining varied from dark to light blue and had no correlation
with treatment.
Example 36
[0240] Four different co-cultivation media were tested for their
effect on the frequency of GUS expression following co-cultivation
of Type I callus with Agrobacterium strain AT656.
[0241] Type I callus was produced by growing Lemna gibba G3 fronds
on solid Murashige and Skoog medium containing 3% sucrose, 0.15%
Gelrite, 0.4% Difco Bacto-agar, 5 .mu.M 2,4-D, and 2 .mu.M BA.
Callus induction and all subsequent culture was at 23.degree. C.
and under a 16 hr light/8 hr dark photoperiod with light intensity
of approximately 40 .mu.mol/m.sup.2sec. After 4 weeks of callus
induction, Type I callus clumps were separately cultured on the
same medium with the 2,4-D concentration reduced to 1 .mu.M. The
callus was subcultured to fresh medium every two weeks until
sufficient callus was proliferated for experimentation.
[0242] For co-cultivation, four media were tested: Murashige and
Skoog medium (MS) with 20 .mu.M 2,4-D and 0.1 .mu.M BA (MS1), MS
medium with 20 .mu.M 2,4-D and 1 .mu.M BA (MS2), MS medium with 1
.mu.M 2,4-D and 2 .mu.M BA (MS3), and Schenk and Hilderbrandt
medium (SH) without plant growth regulators. Fifty milliliters of
each media were prepared containing 3% sucrose, 0.15% Gelrite and
0.4% Difco Bacto-agar, the pH adjusted to 5.6, autoclaved at
121.degree. C. for 20 minutes, cooled and a filter-sterilized
solution of acetosyringone added to the cooled medium to a final
concentration of 20 mg/L. The media were used to pour 24, 100
mm.times.15 mm petri dishes. Agrobacterium strain AT656 was
streaked on potato dextrose agar containing 20 mg/L acetosyringone
and 50 mg/L kanamycin sulfate and grown overnight at 28.degree.
C.
[0243] A randomized block experimental design with 4 co-cultivation
media treatments with two replications with one petri dish per
replication and 20 callus pieces per petri dish was used. For
inoculation, the bacteria from one petri dish were resuspended in
filter sterilized SH medium containing 0.6M mannitol and 20 mg/L
acetosyringone at pH 5.6 for at least one hour prior to use. For
inoculation, Type I callus pieces were placed in bacterial solution
for 8 minutes, blotted and then transferred to the four different
co-cultivation media. All plates were co-cultivated at 23.degree.
C. in the dark for four days. After co-cultivation, all callus was
stained for GUS expression following the procedure of Stomp et al.
(Histochemical localization of beta-glucoronidase, in GUS PROTOCOLS
103-114 (S. R. Gallagher ed. 1991)).
[0244] The co-cultivation medium did not have a significant effect
on the frequency of callus pieces showing GUS expression. Across
all treatments, the frequency of GUS expression ranged from 70-85%.
The intensity of GUS expression varied, with staining ranging from
dark to light blue.
Example 37
[0245] Two different co-cultivation times, two and four days, were
tested for their effect on the frequency of GUS expression
following co-cultivation of Type I callus with Agrobacterium strain
AT656.
[0246] Type I callus was produced by growing Lemna gibba G3 fronds
on solid Murashige and Skoog medium containing 3% sucrose, 0.15%
Gelrite, 0.4% Difco Bacto-agar, 5 .mu.M 2,4-D, and 2 .mu.M BA.
Callus induction and all subsequent culture was at 23.degree. C.
and under a 16 hr light/8 hr dark photoperiod with light intensity
of approximately 40 .mu.mol/m.sup.2sec. After 4 weeks of callus
induction, Type I callus clumps were separately cultured on the
same medium with the 2,4-D concentration reduced to 1 .mu.M. The
callus was subcultured to fresh medium every two weeks until
sufficient callus was proliferated for experimentation.
[0247] For co-cultivation, 400 ml of solid Murashige and Skoog
medium (MS) with 3% sucrose, 0.15% Gelrite, 0.4% Difco Bacto-agar,
1 .mu.M 2,4-D and 2 .mu.M BA was prepared, the pH adjusted to 5.6,
autoclaved at 121.degree. C. for 20 minutes, and cooled. A
filter-sterilized solution of acetosyringone was added to the
cooled medium to a final concentration of 20 mg/L. The cooled
medium was used to pour 16, 100 mm.times.15 mm petri dishes.
Agrobacterium strain AT656 was streaked on potato dextrose agar
with 20 mg/L acetosyringone and 50 mg/L kanamycin sulfate and grown
overnight at 28.degree. C.
[0248] A randomized block experimental design with two
co-cultivation time treatments with two replications with four
petri dishes per replication and 10 callus pieces per petri dish
was used. For inoculation, the bacteria were resuspended in
filter-sterilized Schenk and Hildebrandt medium containing 0.6M
mannitol and 20 mg/L acetosyringone at pH 5.6 for at least one hour
before use. For inoculation, Type I callus pieces were placed in
bacterial solution. For co-cultivation, the pieces were blotted,
then transferred to MS co-cultivation medium described above. All
plates were co-cultivated in the dark at 23.degree. C. for either
two or four days. After either two or four days of co-cultivation,
all callus was stained for GUS expression.
[0249] The results showed that the frequencies of GUS expression
did not vary with respect to co-cultivation time. Across all
treatments, the frequencies of GUS expression ranged from 50-70%.
The intensity of GUS staining ranged from dark to light blue.
However, heavy bacterial overgrowth was present after four days of
co-cultivation and this bacterial coating was found to inhibit GUS
staining.
Example 38
[0250] A different gene construct was used to test the efficacy of
the Type I callus co-cultivation protocol with another
Agrobacterium strain, C58sZ707pBI121.
[0251] Type I callus was produced by growing Lemna gibba G3 fronds
on solid Murashige and Skoog medium containing 3% sucrose, 0.15%
Gelrite, 0.4% Difco Bacto-agar, 5 .mu.M 2,4-D, and 2 .mu.M BA.
Callus induction and all subsequent culture was at 23.degree. C.
and under a 16 hr light/8 hr dark photoperiod with light intensity
of approximately 40 .mu.mol/m.sup.2sec. After 4 weeks of callus
induction, Type I callus clumps were separately cultured on the
same medium with the 2,4-D concentration reduced to 1 .mu.M. The
callus was subcultured to fresh medium every two weeks until
sufficient callus was proliferated for experimentation.
[0252] For co-cultivation, 400 ml of solid Murashige and Skoog
medium (MS) with 3% sucrose, 0.15% Gelrite, 0.4% Difco Bacto-agar,
1 .mu.M 2,4-D and 2 .mu.M BA was prepared, the pH adjusted to 5.6,
autoclaved at 121.degree. C. for 20 minutes, and cooled. A
filter-sterilized solution of acetosyringone was added to the
cooled medium to a final concentration of 20 mg/L. The cooled
medium was used to pour 16, 100 mm.times.15 mm petri dishes.
Agrobacterium strain C58sZ707pBI121 was streaked on potato dextrose
agar with 20 mg/L acetosyringone, 500 mg/L streptomycin sulfate, 50
mg/L spectinomycin, and 50 mg/L kanamycin sulfate and grown
overnight at 28.degree. C.
[0253] A randomized block experimental design with one bacterial
strain treatment with four replications with four petri dishes per
replication and 10 callus pieces per petri dish was used. For
inoculation, the bacteria from one petri dish were resuspended in
filter-sterilized Schenk and Hildebrandt medium containing 0.6M
mannitol and 20 mg/L of acetosyringone at pH 5.6 for at least one
hour before use. For inoculation, Type I callus pieces were placed
in bacterial solution for 8-10 minutes. For co-cultivation, the
pieces were blotted and then transferred to MS co-cultivation
medium described above. All callus was co-cultivated in the dark at
23.degree. C. for two days. After co-cultivation, two callus pieces
were selected from one plate per replication (8 pieces in total)
and stained for GUS expression.
[0254] All callus pieces showed GUS expression ranging from dark to
pale blue. The remaining callus was transferred from MS
co-cultivation medium to identical MS medium that contained 500
mg/L of cefotaxime for the first two weeks and 500 mg/L, each,
cefotaxime and carbenecillin, thereafter, to rid the tissue of the
bacterial contaminant. All callus tissue was transferred to fresh
MS medium containing cefotaxime and carbenecillin at two-week
intervals. At each transfer, a subsample of callus pieces were
stained for GUS expression. The frequencies of GUS expression
decreased slightly but remained high with 70-95% of pieces showing
some GUS expression. Visual inspection of callus on antibiotic
medium showed no indication of bacterial contamination after 4
weeks of culture.
Example 39
[0255] Type II callus and Type III callus were tested for their
ability to give GUS expression following co-cultivation in the
presence of Agrobacterium strain AT656.
[0256] Both callus types were induced by culturing Lemna gibba G3
fronds on solid Murashige and Skoog medium containing 3% sucrose,
0.15% Gelrite, 0.4% Difco Bacto-agar, 30 .mu.M 2,4-D and 0.02 .mu.M
BA at 23.degree. C. under a 16 hr light/8 hr dark photoperiod with
light intensity of approximately 40 .mu.mol/m.sup.2sec. After four
weeks, Type II callus and Type III callus were separated from the
original fronds and transferred to solid Murashige and Skoog medium
containing 3% sucrose, 0.15% Gelrite, 0.4% Difco Bacto-agar, 10
.mu.M 2,4-D, and 0.01 .mu.M BA for callus maintenance under the
same temperature and light conditions. The callus was subcultured
to fresh medium every two weeks until sufficient callus was
proliferated for experimentation.
[0257] Agrobacterium strain AT656 was streaked on potato dextrose
agar containing 20 mg/L acetosyringone and 50 mg/L kanamycin
sulfate and grown overnight at 28.degree. C. For co-cultivation,
200 ml of Murashige and Skoog medium (MS) with 3% sucrose, 0.15%
Gelrite and 0.4% Difco Bacto-agar, 10 .mu.M 2,4-D and 0.02 .mu.M BA
was prepared, the pH adjusted to 5.6, autoclaved at 121.degree. C.
for 20 minutes, and cooled. A filter-sterilized solution of
acetosyringone was added to the cooled medium to a final
concentration of 20 mg/L. The cooled medium was used to pour 8, 100
mm.times.15 mm petri dishes.
[0258] A randomized block experimental design with two callus type
treatments was used. Forty clumps of green callus, transferred
evenly to 4 petri dishes, and 9 clumps of white callus, transferred
evenly to 4 petri dishes, were inoculated. For inoculation,
bacteria were resuspended in filter-sterilized Schenk and
Hildebrandt medium containing 0.6M mannitol and 20 mg/L
acetosyringone at pH 5.6 for at least one hour before inoculation.
For inoculation, pieces of green callus and white callus were
dipped in the bacterial solution for 2-5 minutes. For
co-cultivation, callus pieces were blotted then transferred as
clumps to MS co-cultivation medium described above. All callus was
incubated at 23.degree. C. in the dark for two days.
[0259] After co-cultivation, all white callus and 3 pieces of green
callus per plate were randomly picked and stained for GUS
expression. Out of nine clumps of white callus, 7 clumps showed GUS
expression of varying intensity. Out of 12 pieces of green callus,
6 showed GUS expression of varying intensity.
Example 40
[0260] Type I callus established from two different fast-growing
strains of Lemna gibba (strain 6861 and 7784) and one strain of
Lemna minor were co-cultivated with AT656 to determine the
frequency of transformation with the protocol established using
Lemna gibba G3.
[0261] Agrobacterium strain AT656 was streaked on potato dextrose
agar containing 20 mg/L acetosyringone and 50 mg/L kanamycin
sulfate and grown overnight at 28.degree. C. For co-cultivation,
200 ml of Murashige and Skoog medium (MS) with 3% sucrose, 0.15%
Gelrite and 0.4% Difco Bacto-agar, 10 .mu.M 2,4-D and 0.02 .mu.M BA
was prepared, the pH adjusted to 5.6, autoclaved at 121.degree. C.
for 20 minutes, and cooled. A filter-sterilized solution of
acetosyringone was added to the cooled medium to a final
concentration of 20 mg/L. The cooled medium was used to pour 8, 100
mm.times.15 mm petri dishes.
[0262] For inoculation, bacteria were resuspended in
filter-sterilized Schenk and Hildebrandt medium containing 0.6 M
mannitol and 20 mg/L acetosyringone at pH 5.6 for at least one hour
before inoculation. For inoculation, approximately 10-15 pieces of
Type I callus from the 3 different duckweed strains and from L.
gibba G3 (positive control) were dipped in the bacterial solution
for 2-5 minutes. For co-cultivation, callus pieces were blotted,
then transferred as clumps to two plates (for each duckweed strain)
of co-cultivation medium, as described above. All callus was
incubated at 23.degree. C. in the dark for two days.
[0263] After co-cultivation, all callus pieces of the two Lemna
gibba strains and 3 pieces of callus from the Lemna minor strain
were randomly picked and stained for GUS expression. All callus
pieces showed multiple small spots of GUS staining cells two weeks
after co-cultivation, consistent with successful
transformation.
Selection Using Fronds:
Example 41
[0264] Lemna gibba G3 fronds were used to test the effect of three
co-cultivation media on rescue of fronds expressing GUS and growing
on kanamycin selection medium.
[0265] Fronds were grown for 3 days on liquid Schenk and
Hildebrandt medium containing 1% sucrose, and 10 .mu.M indoleacetic
acid prior to use. The bacterial strain, AT656, was grown overnight
on potato dextrose agar containing 20 mg/L acetosyringone and 50
mg/L kanamycin sulfate at 28.degree. C. Three solid media were used
for co-cultivation: 1) Schenk and Hildebrandt medium (SH)
containing 1% sucrose, 1% agar, 20 mg/L acetosyringone, and 10
.mu.M indoleacetic acid, 2) Murashige and Skoog medium (MS)
containing 3% sucrose, 1% agar, 20 mg/L acetosyringone, and 50
.mu.M 2,4-dichlorophenoxyacetic acid (2,4-D), and 3) Murashige and
Skoog medium containing 3% sucrose, 1% agar, 20 mg/L
acetosyringone, 5 .mu.M 2,4-D, 10 .mu.M naphthaleneacetic acid, 10
.mu.M giberrellic acid G3, and 2 .mu.M benzyladenine. The media
were prepared, the pH adjusted to 5.6 (SH) or 5.8 (both MS types),
autoclaved, cooled, heat labile components acetosyringone,
indoleacetic acid and giberrellic acid added as filter-sterilized
solutions, and the medium poured into 100 mm.times.15 mm petri
dishes. For each medium, 20 petri dishes (500 ml) were
prepared.
[0266] A randomized block experimental design with three
co-cultivation media treatments with 4 replication with 5 petri
dishes per replication and 20 fronds per petri dish was used. For
inoculation, the bacteria on one petri dish were resuspended as
described in Example 21.
[0267] For inoculation, individual fronds were separated from
clumps, each turned abaxial side up and wounded with a sterile
scalpel in the meristematic regions. Fronds were transferred to
bacterial suspensions and incubated for 10 minutes. For
co-cultivation, fronds were transferred to the three co-cultivation
media described above. All plates were incubated for 5.5 days in
the dark at 23.degree. C.
[0268] After 5.5 days of co-cultivation, the fronds from two petri
dishes per medium were stained for GUS expression. The results
showed that GUS expression was present to a large extent on fronds
co-cultivated on Schenk and Hildebrandt medium, less staining was
seen with fronds on Murashige and Skoog media. The remaining fronds
from the other 18 plates of each medium (18 plates.times.3 media=54
plates) were transferred to both solid and liquid media of the same
composition without acetosyringone, and with 500 mg/L of timentin.
The fronds from 3 plates were transferred into 3 flasks with 25 ml
of liquid media and were grown under 23.degree. C. under a 16 hr
light/8 hr dark photoperiod with light intensity of approximately
40 .mu.mol/m.sup.2sec. The fronds on 15 plates of solid media were
divided into 2 groups: 1) the fronds from 10 original plates were
transferred to 12 new plates and incubated in the dark (12 plates),
2) the fronds from 5 original plates were transferred to 6 new
plates and incubated under subdued light conditions of less than 5
.mu.mol/m.sup.2. sec.
[0269] After 11 days of growth, subsamples of fronds were taken to
stain for GUS expression. The results showed that regardless of
light treatment or medium treatment, GUS expression was still
present. All fronds, regardless of media, incubated under subdued
light showed the highest intensity of GUS expression. Fronds
incubated in the dark showed an intermediate level of GUS
expression and fronds incubated in the light showed very low
levels. Fronds incubated on Shenk and Hildebrandt medium showed the
highest frequencies of GUS positive tissue, however no GUS
expression was associated with newly expanding fronds.
[0270] On Murashige and Skoog media formulated to induce callus,
the staining pattern was restricted to single cells and very small
regions. Fronds on MS medium containing 2,4-D, NAA, GA3 and BA
showed more intense staining than those incubated on MS medium
containing only 2,4-D. Callus formation on both MS based media,
with plant growth regulators adjusted to induce callus, did not
occur in the dark, but had started on MS medium with 2,4-D, NAA,
GA3 and BA under subdued light. Based on these results, fronds on
Schenk and Hildebrandt medium were dropped from the experiment. All
remaining fronds on MS media in darkness were transferred to
subdued light conditions to continue incubation. All tissue was
kept on the same medium formation but transferred to fresh medium
with timentin and incubation was continued under subdued light
conditions for about 5 weeks.
[0271] Seven weeks after co-cultivation all remaining tissue was
again transferred to fresh medium and kanamycin sulfate at either
10 mg/L (about 25% of the tissue) or 2 mg/L (about 75% of the
remaining tissue) was included. One week later, a subsample of
tissue from both kanamycin treatments was stained for GUS
expression. Three types of staining was present: 1) staining
associated with the original, co-cultivated fronds, 2) staining
associated with Type I callus, and 3) staining associated with Type
III callus. The frequency of callus staining was not high,
estimated at about 5-8 fronds giving rise to a kanamycin resistant
culture per hundred fronds co-cultivated. Incubation and
subculturing of the tissue was continued for another 5 weeks under
subdued light.
[0272] At twelve weeks, all tissue remaining was from cultures on
MS medium containing 2,4-D, NAA, GA3 and BA. The tissue was
transferred to Murashige and Skoog medium with 1 .mu.M 2,4-D, 2
.mu.M BA, 0.15 g/L Gelrite, 0.4 g/L Difco Bacto-agar, 500 mg/L
timentin and 10 mg/L kanamycin sulfate. Heat labile components were
filter-sterilized and added to autoclaved, cooled medium. Healthy
tissue that had proliferated from each originally co-cultivated
frond was transferred to an individual petri dish. All tissue was
incubated at 23.degree. C. and shifted from subdued to full light
intensity of approximately 40 .mu.mol/m.sup.2sec and a 16 hr
light/8 hr dark photoperiod. At this time a small subsample of
tissue was stained for GUS expression and the results showed a low
frequency of GUS staining associated with Type III callus. After
two weeks it became obvious from visual observation that transfer
to full light had enhanced the segregation of kanamycin resistant
callus from kanamycin sensitive tissue. Growth of callus on
kanamycin was continued for another 4 weeks (to 16 weeks total) by
transfer of all living tissue to fresh medium.
[0273] Between sixteen and twenty weeks after co-cultivation,
kanamycin resistant callus lines became established. These compact
Type I callus and Type III callus cultures were characterized by
growth on 10 mg/L kanamycin in the light. Eight kanamycin resistant
callus cultures were proliferated from 360 original co-cultivated
fronds. As these eight lines developed, subsamples of the callus
were transferred to half-strength Schenk and Hildebrandt medium
containing 0.5% sucrose to regenerate fronds. Of these eight, three
regenerated fronds in the absence of kanamycin, frond regeneration
would not occur in the presence of kanamycin. None of these fronds
showed GUS expression when stained.
Selection of Callus Cultures and Regeneration of Transformed
Fronds:
Example 42
[0274] Type I callus was tested for its ability to give GUS
expression and kanamycin sulfate resistant cultures following
co-cultivation in the presence of Agrobacterium strain
C58sZ707pBI121.
[0275] Type I callus was produced by growing Lemna gibba G3 fronds
on solid Murashige and Skoog medium containing 3% sucrose, 0.15%
Gelrite, 0.4% Difco Bacto-agar, 5 .mu.M 2,4-D, and 2 .mu.M BA.
Callus induction and all subsequent culture was at 23.degree. C.
and under a 16 hr light/8 hr dark photoperiod with light intensity
of approximately 40 .mu.mol/m.sup.2sec. After 4 weeks of callus
induction, Type I callus clumps were separately cultured on the
same medium with the 2,4-D concentration reduced to 1 .mu.M. The
callus was subcultured to fresh medium every two weeks until
sufficient callus was proliferated for experimentation.
[0276] For co-cultivation, 400 ml of solid Murashige and Skoog
medium (MS) with 3% sucrose, 0.15% Gelrite, 0.4% Difco Bacto-agar,
1 .mu.M 2,4-D and 2 .mu.M BA was prepared, the pH adjusted to 5.6,
autoclaved at 121.degree. C. for 20 minutes, and cooled. A
filter-sterilized solution of acetosyringone was added to the
cooled medium to a final concentration of 20 mg/L. The cooled
medium was used to pour 20, 100 mm.times.15 mm petri dishes.
Agrobacterium strain C58sZ707pBI121 was streaked on potato dextrose
agar with 20 mg/L acetosyringone, 500 mg/L streptomycin, 50 mg/L
spectinomycin, and 50 mg/L kanamycin sulfate and grown overnight at
28.degree. C.
[0277] A randomized block experimental design with one bacterial
strain treatment with one replication with 20 petri dishes per
replication and approximately 10 callus pieces per petri dish was
used. For inoculation, the bacteria were resuspended in
filter-sterilized Schenk and Hildebrandt medium containing 0.6M
mannitol and 20 mg/L acetosyringone at pH 5.6 for at least one hour
before use. For inoculation, Type I callus pieces were placed in
bacterial solution. For co-cultivation, the pieces were blotted
then transferred to MS co-cultivation medium described above. All
callus pieces were co-cultivated for two days at 23.degree. C. in
the dark. After co-cultivation, a subsample of callus pieces were
histochemically stained for GUS expression. The results showed a
high frequency of GUS expression of varying intensity.
[0278] The approximately 200 remaining callus pieces were
transferred to decontamination medium. For decontamination, 500 ml
of solid MS medium containing 3% sucrose, 0.15% Gelrite, 0.4% Difco
Bacto-agar, 1 .mu.M 2,4-D, and 2 .mu.M BA was prepared, the pH
adjusted to 5.6, autoclaved for 20 minutes at 121.degree. C., and
cooled. A filter-sterilized solution containing cefotaxime was
added to the cooled medium to a final medium concentration of 500
mg/L. The cooled medium was used to pour 20 plates. Approximately
10 callus pieces, each, were transferred to the 20 petri dishes of
decontamination medium. All petri dishes were incubated at
23.degree. C. in the dark. Weekly subcultures of the callus pieces
to identical fresh medium were done and the callus was incubated
under the same conditions. At week 5, a small subsample of callus
tissue was stained for GUS expression. Expression was present at
high frequency and at varying intensity.
[0279] On week 5, the remaining callus pieces were transferred to
selection medium. For selection, 500 ml of MS with 3% sucrose,
0.15% Gelrite and 0.4% Difco Bacto-agar, supplemented with 1 .mu.M
2,4-D and 2 .mu.M BA was prepared, the pH adjusted to 5.6,
autoclaved for 20 minutes at 121.degree. C., and cooled. A
filter-sterilized solution containing cefotaxime, carbenicillin,
and kanamycin sulfate was added to the cooled medium to a final
medium concentration of 500, 500 and 2 mg/L, respectively. The
cooled medium was used to pour 20 plates. Approximately 9-10 callus
pieces were transferred to the 20 petri dishes of selection medium.
All callus was incubated at 23.degree. C. under a 16 hr light/8 hr
dark photoperiod of subdued light intensity of approximately 3-5
.mu.mol/m.sup.2sec. After one week of incubation, the callus was
transferred to the same medium except that the kanamycin
concentration was increased to 10 mg/L. Callus culture was
continued under the same incubation conditions for another week,
subcultured once to fresh medium of identical composition. At the
end of this two-week, kanamycin selection period, approximately 25%
of the original callus pieces showed healthy callus growth with the
remainder in decline.
[0280] On week seven, 64 of the healthiest callus pieces were
transferred to solid MS medium with 3% sucrose, 0.15% Gelrite, 0.4%
Difco Bacto-agar, 1 .mu.M 2,4-D, 2 .mu.M BA, 500 mg/L, each,
carbenecillin and cefotaxime, and four different concentrations of
kanamycin sulfate: 10, 20, 40, and 80 mg/L. 160 ml of the medium
was prepared, the pH adjusted to 5.6, autoclaved, and cooled.
Filter-sterilized solutions of the heat labile antibiotics were
added to the appropriate concentrations. The cooled media were then
used to pour 16, 60 mm.times.15 mm petri dishes. Approximately 4
callus pieces were transferred to each plate and 4 plates were
prepared of each kanamycin concentration (16 callus pieces per
kanamycin concentration). Incubation of the callus continued at
23.degree. C. under subdued light. At weekly intervals, 4 plates,
one from each of the kanamycin concentrations, were transferred to
a higher light intensity of 40 .mu.mol/m.sup.2.sec. On week nine,
regardless of light conditions, all callus was transferred to fresh
medium of identical composition as the previous subculture. By week
12, all callus was under the higher light intensity of 40
.mu.mol/m.sup.2.sec. Callus culture was continued for another four
weeks (to week 16), with subculture to fresh medium at two-week
intervals.
[0281] On week 16, a small subsample of the remaining healthy
callus was stained for GUS expression. All healthy callus pieces
showed GUS expression with whole callus pieces showing uniform
staining indicating segregation of GUS expressing callus from
non-expressing callus. Most of the callus had died by this time,
but greater than 10% showed varying degrees of healthy callus
proliferation. Three callus lines, A, B, and C were identified and
transferred to medium to promote frond regeneration. Upon further
subculture of growing callus pieces on selection medium, 6 more
callus lines, D-I, were identified and transferred to regeneration
medium. Eight of the 9 lines were found on medium containing 10
mg/L kanamycin. The exception was line D which showed good growth
on 40 mg/L kanamycin. Upon subsequent subculture, six callus lines
continued to grow: A, B, D, F, H, and I.
[0282] Two of the nine identified lines went on to regenerate
fronds that were positive for GUS expression when stained and that
would proliferate readily in the presence or absence of kanamycin.
For regeneration, water agar was prepared from 100 ml of distilled
water with 0.4% Difco Bacto-agar and 0.15% Gelrite, the pH was
adjusted to 5.6, and the medium autoclaved for 18 minutes at
121.degree. C. This medium was used to pour 10, 60 mm.times.15 mm
petri dishes. Small pieces of callus from lines A, D, F, H, and I
were transferred to two petri dishes, each, of medium. The callus
was incubated at 23.degree. C. under a 16 hr light/8 hr dark
photoperiod with light intensity of approximately 40
.mu.mol/m.sup.2sec. Callus culture on water agar was continued for
six weeks, with subculture to fresh water agar at two-week
intervals. By week six, the callus from all lines had turned
yellowish and brown. The callus was transferred at the end of week
6 to either solid or liquid, half-strength Schenk and Hildebrandt
medium containing 0.5% sucrose and 0.8% Difco Bacto-agar (solid
medium only). After 4-6 weeks the callus had organized green
nodules that differentiated into thickened, frond like structures.
As fronds could be detached from the callus clumps they were
transferred to full-strength Schenk and Hildebrandt medium
containing 1% sucrose, with incubation at 23.degree. C. under a 16
hr light/8 hr dark photoperiod with light intensity of
approximately 40 .mu.mol/m.sup.2sec. These fronds proliferated in
liquid SH medium indefinitely. The fronds proliferated equally well
on SH medium with or without kanamycin. Bleaching of fronds was not
seen in the presence of kanamycin. Frond subsamples were taken
periodically and stained for GUS expression. All fronds showed GUS
expression.
[0283] To confirm transformation, Southern hybridization analysis
was done on DNA isolated from lines A and D. Duckweed DNA
preparations were prepared from untransformed L. gibba G3 and from
transformed lines A and D using the CTAB procedure of Doyle and
Doyle (Amer. J. of Botany 75, 1238 (1988)). Isolated DNA was
digested with restriction enzymes EcoRI and Hind III, and with both
enzymes, and fragments were electrophoretically separated on a 0.7%
agarose gel. The gel was blotted to a nylon membrane following the
methods of Sambrook. SAMBROOK ET AL., MOLECULAR CLONING: A
LABORATORY MANUAL (1989). For probe, plasmid DNA from pBI121 was
isolated using an alkali SDS procedure of Sambrook. Id. The 12.8 kb
plasmid DNA was digested with restriction enzymes EcoR1 and Hind
III to produce a 3.2 kb fragment consisting of the
.beta.-glucuronidase gene and an approximately 9 kb fragment
containing the neomycin phosphotransferase gene. Both fragments
were isolated from the agarose gel and radioactively labeled by
random priming using the Prime-a-Gene kit (Promega). Using these
probes, hybridization was done with blots carrying untransformed
duckweed DNA and either DNA from transformed line A or transformed
line D. The hybridization reaction was carried out at 65.degree. C.
overnight in a hybridization oven. The membrane was washed under
stringent conditions of 0.1.times.SSC, 0.1% SDS. The blot was then
placed in contact with BIOMAX MS film (Kodak), and the
autoradiograph exposed for 2 days at -70.degree. C.
[0284] The results of the hybridization experiments showed that GUS
and NPTII hybridizing DNA was present in duckweed lines A and D,
but not in DNA from untransformed duckweed (results for line D
shown in FIG. 1). Double digestion of transformed duckweed DNA gave
hybridization at the expected molecular weight. Single digestion
showed that hybridization was associated with DNA fragments of
unexpected molecular weight, indicating that the hybridizing DNA
was not of bacterial origin and was integrated into plant DNA.
Probing the same blots with labeled virulence region probe showed
the absence of hybridization, indicating that the positive GUS and
NPTII signals came from plant, not bacterial origin.
Example 43
[0285] Type I callus was tested for its ability to give GUS
expression and kanamycin sulfate resistant cultures following
co-cultivation in the presence of Agrobacterium strain
C58sZ707pBI121.
[0286] Type I callus was produced by growing Lemna gibba G3 fronds
on solid Murashige and Skoog medium containing 3% sucrose, 0.15%
Gelrite, 0.4% Difco Bacto-agar, 5 .mu.M 2,4-D, and 2 .mu.M BA.
Callus induction and all subsequent culture was at 23.degree. C.
under a 16 hr light/8 hr dark photoperiod with light intensity of
approximately 40 .mu.mol/m.sup.2sec. After 4 weeks of callus
induction, Type I callus clumps were separately cultured on the
same medium with the 2,4-D concentration reduced to 1 .mu.M. The
callus was subcultured to fresh medium every two weeks until
sufficient callus was proliferated for experimentation.
[0287] For co-cultivation, 750 ml of solid Murashige and Skoog
medium (MS) with 3% sucrose, 0.15% Gelrite, 0.4% Difco Bacto-agar,
1 .mu.M 2,4-D and 2 .mu.M BA was prepared, the pH adjusted to 5.6,
autoclaved at 121.degree. C. for 20 minutes, and cooled. A
filter-sterilized solution of acetosyringone was added to the
cooled medium to a final concentration of 20 mg/L. The cooled
medium was used to pour 30, 100 mm.times.15 mm petri dishes.
Agrobacterium strain C58sZ707pBI121 was streaked on potato dextrose
agar with 20 mg/L acetosyringone, 500 mg/L streptomycin, 50 mg/L
spectinomycin, and 50 mg/L kanamycin sulfate and grown overnight at
28.degree. C.
[0288] A randomized block experimental design with one bacterial
strain treatment with one replication with 30 petri dishes per
replication and approximately 5 callus pieces per petri dish was
used. For inoculation, the bacteria were resuspended in
filter-sterilized MS medium containing 0.6M mannitol and 20 mg/L
acetosyringone at pH 5.8 for at least one hour before use. For
inoculation, Type I callus pieces were placed in bacterial
solution. For co-cultivation, the pieces were blotted then
transferred to MS co-cultivation medium described above. All callus
pieces were co-cultivated for two days at 23.degree. C. in the
dark. After co-cultivation, a subsample of callus pieces were
histochemically stained for GUS expression. The results showed a
high frequency of GUS expression.
[0289] The approximately 150 remaining callus pieces were
transferred to decontamination medium. For decontamination, 750 ml
of solid MS medium containing 3% sucrose, 0.15% Gelrite, 0.4% Difco
Bacto-agar, 1 .mu.M 2,4-D, and 2 .mu.M BA was prepared, the pH
adjusted to 5.8, autoclaved for 20 minutes at 121.degree. C., and
cooled. A filter-sterilized solution containing cefotaxime and
carbenicillin was added to the cooled medium to a final medium
concentrations of 500 mg/L, each. The cooled medium was used to
pour 30 plates. Approximately 5 callus pieces were transferred to
each of the 30 petri dishes of decontamination medium. The callus
was then incubated at 23.degree. C. in the dark. Weekly subcultures
of the callus pieces to identical fresh medium were done and the
callus was incubated under the same conditions.
[0290] Selection for kanamycin resistant callus lines was begun on
week 5. For selection, 750 ml of solid MS medium containing 3%
sucrose, 0.15% Gelrite, 0.4% Difco Bacto-agar, 1 .mu.M 2,4-D, and 2
.mu.M BA was prepared, the pH adjusted to 5.8, autoclaved for 20
minutes at 121.degree. C., and cooled. A filter-sterilized solution
containing cefotaxime, carbenicillin, and kanamycin was added to a
final medium concentration of 500 mg/L, 500 mg/L, and 2 mg/L,
respectively. The cooled medium was used to pour 30 plates.
Approximately 5 callus pieces were transferred to each of the 30
petri dishes of selection medium. The callus was then incubated at
23.degree. C. in the dark.
[0291] For weeks 6 and 7, the callus was transferred to identical
fresh medium with the kanamycin concentration increased to 10 mg/L.
Incubation was continued in the dark at 23.degree. C. At the
beginning of week 8 the kanamycin concentration was increased.
Murashige and Skoog medium with composition identical to that of
previous subcultures was prepared with half the medium containing
kanamycin at 10 mg/L and the other half with kanamycin at 40 mg/L.
Approximately half of the remaining callus was transferred to each
kanamycin concentration. The light conditions of incubation were
changed as well. All callus was incubated at 23.degree. C. under a
16 hr light/8 hr dark photoperiod with light intensity of
approximately 3-5 .mu.mol/m.sup.2sec. The callus was maintained
under these medium and incubation conditions for two weeks. After
two weeks at the subdued light level, the callus was transferred to
fresh medium of identical composition containing kanamycin at
either 10 or 40 mg/L, and the light intensity was increased to 40
.mu.mol/m.sup.2sec. The callus was maintained on a two-week
subculture regime on identical medium and incubation
conditions.
[0292] By week 12, approximately 10% of the callus remained healthy
and growing. Of the 15 proliferating callus lines remaining, half
were growing on 10 mg/L kanamycin and the remainder on 40 mg/L.
Histochemical staining of small subsamples of callus from 6 lines
showed GUS expression in sectors of the callus pieces.
[0293] Fronds were regenerated following the procedure of Example
42. Frond proliferation was normal when grown in the presence or
absence of kanamycin sulfate, and no bleaching of the fronds was
observed. Fronds also showed intense GUS histochemical staining.
Southern hybridization analysis showed the presence of DNA
sequences of the expected fragment size for both the GUS and
neomycin phosphotransferase genes, and the absence of DNA sequences
from the vir region. Appropriate restriction enzyme analysis was
carried out as in Example 42, and the results were consistent with
a finding of integration of foreign genes into the plant
genome.
Example 44
[0294] Based on the previous Examples, the following method is
preferred for transforming duckweed callus with Agrobacterium,
followed by selection and regeneration of transformed plants.
Overall, Lemna minor has a particularly vigorous callus system,
which makes it easier to regenerate transformed plants from this
species.
[0295] Typically, callus transformation, selection, and frond
regeneration is dependent upon a well-established callus system and
a number of parameters optimized for each step of the process. A
vigorously growing callus culture is maintained as described in
Example 16. Agrobacteria are grown (on Potato Dextrose Agar with
appropriate antibiotics and 100 .mu.M acetosyringone) and
resuspended as in Example 32, except that the preferred
resuspension medium is MS rather than SH. Callus pieces are
inoculated by immersing in the solution of resuspended bacteria for
a minimum of 3-5 minutes, blotted to remove excess fluid, and
plated on co-cultivation medium consisting of MS supplemented with
auxin and cytokinin optimized to promote callus growth and 100
.mu.M acetosyringone. Inoculated callus is incubated in darkness
for 2 days.
[0296] After co-cultivation, callus is transferred to fresh media
containing antibiotics to decontaminate the cultures from infecting
Agrobacteria. The preferred medium is MS with 3% sucrose, 1 .mu.M
2,4-D, 2 .mu.M BA, gelled with 0.15% Gelrite and 0.4% Difco
Bacto-agar and antibiotic(s). The callus is incubated under subdued
light of 3-5 .mu.mol/m.sup.2.sec. The callus is transferred every
2-5 days, 3 days is preferred, to fresh medium of the same
composition. The total recovery period lasts for 2-3 weeks, 3-6
subcultures.
[0297] Callus selection follows after the recovery period. Callus
is transferred to MS medium supplemented with 1 .mu.M 2,4-D, 2
.mu.M BA, 3% sucrose, 0.4% Difco Bacto-agar, 0.15% Gelrite, and 10
mg/L kanamycin sulfate. The callus is incubated under subdued light
of 3-5 .mu.mol/m.sup.2.sec, with transfer to fresh medium of the
same composition every 2 weeks. The callus is maintained in this
way for 4-6 weeks. Then the callus is incubated under full light of
40 .mu.mol/m.sup.2.sec on the same medium. Selection is considered
complete when the callus shows vigorous growth on the selection
agent.
[0298] Callus showing vigorous growth on callus maintenance medium
in the presence of the selection agent is transferred to
regeneration medium to organize and produce plants. In general,
duckweed regenerates on lean media. For L. minor it is
half-strength SH medium with 1% sucrose; for L. gibba it is water
agar. Typically, the selection agent is not present in the
regeneration medium. The callus is incubated, under full light, on
regeneration medium for 2-4 weeks until fronds appear. Fully
organized fronds are transferred to liquid SH medium with 1-3%
sucrose and no plant growth regulators and incubated under full
light for further clonal proliferation.
Example 45
[0299] The effect of light intensity and kanamycin sulfate
concentration were tested for its effect on the frequency of
transformation of Lemna minor callus cultures.
[0300] Lemna minor fronds were grown in liquid Schenk and
Hildebrandt medium containing 1% sucrose for two weeks at
23.degree. C. under a 16 hr light/8 hr dark photoperiod with light
intensity of approximately 40 .mu.mol/m.sup.2.sec prior to callus
induction. Callus induction was accomplished as in Example 14 using
fronds from Lemna minor strain 8744. Callus was maintained on MS
medium containing 3% sucrose, 1 .mu.M 2,4-D, 2 .mu.M BA, 0.4%
Bacto-agar and 0.15% Gelrite for 13 weeks prior to co-cultivation.
Callus was subcultured to fresh medium every 2 weeks during this
13-week period.
[0301] Agrobacterium strain C58sz707 harboring the T-DNA containing
binary plasmid from strain AT656, as described in Example 21, was
grown on PDA containing 50 mg/L kanamycin sulfate, 50 mg/L
spectinomycin, and 500 mg/L streptomycin for 2 days at 28.degree.
C. For co-cultivation, solid MS medium with 3% sucrose, 1 .mu.M
2,4-D, 2 .mu.M BA, 0.4% Bacto-Agar, and 0.15% Gelrite was prepared,
the pH was adjusted to 5.6, the medium was autoclaved at
121.degree. C. for 20 minutes, and cooled. A filter-sterilized
solution of acetosyringone was added to the cooled medium to a
final concentration of 100 .mu.M. The cooled medium was used to
pour 8, 100 mm.times.15 mm petri dishes.
[0302] For inoculation, the Agrobacteria were resuspended in
filter-sterilized, MS medium containing 0.6 M mannitol and 100
.mu.M acetosyringone at pH 5.6 for at least one hour before
inoculation. For inoculation, approximately 160 pieces of Type I
callus were dipped in the bacterial solution for 2-5 minutes in
batches of 20 callus pieces. For co-cultivation, callus pieces were
blotted, then transferred as clumps to co-cultivation medium, 20
callus clumps per 100 mm.times.15 mm petri dish. All inoculated
callus was incubated at 23.degree. C. in the dark for 2 days.
[0303] For selection, 200 ml of MS medium containing 1 .mu.M 2,4-D,
1 .mu.M BA, 3% sucrose, 500 mg/L carbenicillin, 500 mg/L
cefotaxime, 10 mg/L kanamycin sulfate, 0.4% Bacto-Agar and 0.15%
Gelrite was prepared, the pH was adjusted to 5.6, the medium was
autoclaved at 121.degree. C. for 20 minutes and 8, 100 mm.times.15
mm petri dishes were poured. The antibiotics were added to cooled,
autoclaved medium as a filter-sterilized solution just prior to
pouring. Co-cultivated callus clumps were transferred to the fresh
selection medium, 20 callus clumps per petri dish. Eighty callus
clumps (4 plates) were incubated under subdued light of less than 5
.mu.mol/m.sup.2.sec and the other 80 callus clumps (4 plates) were
transferred to a higher light intensity of 40 .mu.mol/m.sup.2.sec.
For 3 weeks, callus was subcultured to fresh, antibiotic-containing
medium every week. On week 4, half (40 callus clumps) of the callus
from each light treatment was transferred to fresh medium in which
the kanamycin concentration was increased from 10 mg/L to 40 mg/L.
The remaining 40 callus clumps were transferred to fresh medium
maintaining the original kanamycin concentration of 10 mg/L.
Incubation under identical subdued or full light conditions and low
or high kanamycin concentrations was continued for 2 more weeks,
with weekly subcultures. At 6 weeks post-inoculation, all samples
were transferred to fresh medium and incubated under full light
intensity. From this point onward, subculture was at 2-week
intervals.
[0304] After 12 weeks of culture on kanamycin, vigorously growing
callus was transferred to fresh, regeneration medium. Frond
regeneration medium consisted of half-strength Schenk and
Hildebrandt medium containing 1% sucrose, 0.4% Bacto-agar, and
0.15% Gelrite. Callus clumps were transferred to fresh medium of
the same composition every 2 weeks. Fronds regenerated from callus
clumps 3-6 weeks after transfer to regeneration medium.
[0305] Two transformed, clonal frond lines were regenerated from
this experiment. Both lines showed GUS histochemical staining, had
different levels of GUS enzyme activity (0.31% and 0.14% of
extractable protein) as measured in a soluble assay using
methylumbelliferone-glucuronic acid (MUG) as the substrate, and had
detectable levels of neomycin phosphotransferase enzyme as measured
using and ELISA assay. Southern hybridization analysis confirmed
the presence of foreign DNA sequences in high molecular weight DNA,
which when digested with the appropriate restriction enzymes gave
the expected fragment sizes. Re-probing of the stripped blot with
DNA sequences representing the virulence region of the original
Agrobacterium failed to give detectable hybridization.
Example 46
[0306] The effect of Lemna minor genotype of the frequency of
rescue of transformed fronds was tested using Lemna minor callus
cultures from strain 8627.
[0307] Callus maintenance prior to inoculation, bacterial strain,
bacterial growth for inoculation, bacterial resuspension, callus
inoculation procedure, and co-cultivation for 2 days in darkness
were performed as in Example 45.
[0308] For kanamycin selection, following co-cultivation, 180
callus clumps were transferred to MS medium containing 1 .mu.M
2,4-D, 2 .mu.M BA, 500 mg/L carbenicillin, 500 mg/L cefotaxime and
10 mg/L kanamycin sulfate. All callus was incubated under subdued
light levels of less than 5 .mu.mol/m.sup.2.sec. On the second week
after inoculation, half the callus pieces were transferred to fresh
selection medium in which the kanamycin sulfate concentration was
increased from 10 mg/L to 40 mg/L, the rest were transferred to
fresh selection medium containing 10 mg/L of kanamycin sulfate.
Weekly subculture was continued through week 5, post-inoculation at
which time subcultures were done every two weeks.
[0309] To regenerate transformed fronds, callus lines growing
vigorously on kanamycin and showing GUS expression using
histochemical staining after 12 weeks were transferred to frond
regeneration medium containing half-strength Schenk and Hildebrandt
medium with 1% sucrose, 0.4% Bacto-agar and 0.15% Gelrite. Fronds
regenerated after 3-4 weeks on regeneration medium. Regenerated
fronds were maintained on SH medium with 1% sucrose.
[0310] Three transformed, clonal frond lines were regenerated in
this experiment. All 3 lines showed GUS histochemical staining,
variable levels of GUS activity as measured by the MUG assay
(0.2-0.3% of extractable protein), and detectable levels of
neomycin phosphotransferase protein as measured in an ELISA assay.
Southern hybridization was used to confirm transformation and
integration of foreign DNA sequences into duckweed DNA.
Example 47
[0311] The effect of medium composition on frond regeneration from
L. minor callus cultures was also tested. Seven media formulations
were tested: (1) water agar, (2) water agar with 100 .mu.M adenine
sulfate, (3) water agar with 10 .mu.M BA, (4) water agar with 10
.mu.M BA and 1 .mu.M IBA, (5) half-strength SH, (6) half-strength
SH with 10 .mu.M BA, and (7) half-strength SH with 10 .mu.M BA and
1 .mu.M IBA. Callus cultures from both strain 8744 and strain 8627
proliferated in a previous callus induction medium as in Example 12
were used for this experiment. Callus was incubated on the seven
different media for 8 weeks, with continual observation for the
development of fronds.
[0312] Frond regeneration was only achieved on the half-strength SH
treatments. When half-strength SH was supplemented with 10 .mu.M BA
only, callus growth was faster than that plated on half-strength SH
without plant growth regulators, however, regeneration took longer
than on half-strength SH. The addition of IBA to the medium had no
effect on the timing or ability of callus to regenerate fronds.
Example 48
[0313] The efficiency of the duckweed system for mammalian gene
expression was tested using a human .beta.-hemoglobin gene
construct and a P450 oxidase construct.
[0314] Two Agrobacterium strains were used to inoculate Type I
callus of Lemna minor strain 8627. For .beta.-hemoglobin
transformations, strain C58 C1, harboring 3 plasmids: pGV3850,
pTVK291, pSLD34 was used. pTVK291 contains the supervirulence G
gene from pTiBo542. pSLD34 is an Agrobacterium binary plasmid,
derived from pBIN19, consisting of a neomycin phosphotransferase
gene under the control of CaMV35S promoter, and a human
.beta.-hemoglobin gene driven by the super-mac promoter.
[0315] For P450 oxidase transformations, strain C58 C1, harboring 3
plasmids: pGV3850, pTVK291 and pSLD35 were used. The T-DNA is
carried on the binary plasmid, pSLD35, which is similar in
structure to pSLD34, with the exception that pS:D35 does not
contain the .beta.-hemoglobin gene and instead contains DNA
sequences encoding 3 proteins: a human P450 oxidase, an
oxidoreductase, and a cytochrome B5. Each gene is driven by a
super-mac promoter. The pSLD35 plasmid contains both hygromycin and
kanamycin selectable marker genes.
[0316] Several experiments with the basic experimental design of 2
bacterial strains.times.2 light intensities during early
selection.times.2 kanamycin concentrations during selection
experimental design (8 treatments in total) with 3 replications,
with 2 petri dishes per replications and 10 callus pieces per petri
dish was used. Callus cultures produced from Lemna minor strains
8627 and 8744 and Lemna gibba strain G3 were used in these
experiments. Callus maintenance prior to inoculation, bacterial
strain, bacterial growth for inoculation, bacterial resuspension,
callus inoculation procedure, and co-cultivation for 2 days in
darkness were performed as in Example 45, with the exception that
bacteria were grown on PDA containing 50 mg/L kanamycin, 50 mg/L
gentamycin, 100 mg/L carbenicillin, and 100 .mu.M acetosyringone
prior to inoculation.
[0317] For kanamycin selection, following co-cultivation, callus
clumps were transferred to MS medium containing 1 .mu.M 2,4-D, 2
.mu.M BA, 500 mg/L carbenicillin, 500 mg/L cefotaxime and two
concentrations of kanamycin: 10 mg/L and 40 mg/L. The callus
cultures were further divided during incubation with half of the
callus pieces on each kanamycin concentration going to subdued
light and the other half being incubated under full light. Callus
was subcultured to fresh medium of the same composition at weekly
intervals for the first four weeks after co-cultivation. At week 5,
all cultures were incubated under full light intensity for another
6 weeks, with subculture to fresh medium every two weeks.
[0318] Frond regeneration was accomplished using the appropriate
media for frond regeneration from L. gibba G3 or L. minor strains
as described in Example 42 and Example 47. Fronds regenerated after
3-4 weeks on regeneration medium. Regenerated fronds were
maintained on SH medium with 1% sucrose.
[0319] Across all experiments, more than 20 transformed clonal
frond lines were rescued. More lines were found using 10 mg/L
kanamycin as the selection concentration, as opposed to 40 mg/L.
Subdued light intensity during selection proved advantageous. All
lines showed vigorous callus growth on kanamycin, had detectable
and variable levels of neomycin phosphotransferase protein as
measured by an ELISA test. The presence of the P450 oxidase and
.beta.-hemoglobin DNA, RNA and/or protein is detected in stably
transformed duckweed plants by any method known in the art, e.g.,
Southern, Northern and Western hybridizations, respectively.
[0320] All publications and patent applications mentioned in the
specification are indicative of the level of those skilled 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 to be incorporated by
reference.
[0321] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims.
* * * * *