Note: Descriptions are shown in the official language in which they were submitted.
~ WO 95118862 ~ ~ 7 9 9 8 ~ P~
ffdA GENE SELECTAl~LE MAR~R~ IN PLANTS
AND THE USE THEREOF
Cross Reference to Related ,4T~plicnt~or
This is a in-part of U.S. Application Serial No.
08/179,667, filed Jamuary 11, 1994, the contents of which are fully
ill~,Ul~l~ ' ' by refeRnce herein.
Field of the Invention
The present mvention relates, in general, to transgenic plant cells and
plants. In particular, the present invention relates to 1) a method of selectinga transgenic plant cell , ,, ll~rullllill~ one or more plant cells with
a tfdA gene; 2) a plant cell comprising a tfdA gene wherein said plant cell is
free of other foreign marker genes; and 3) a sweetgum plant cell
a tfdA gene.
Ba~.h~,, rl of the Invention
2,4-Dichluluyl~llu~ y.~ ,ii., acid (2,4-D) is a herbicide used to control
broadleaf weeds. The gene which encodes the first enzyme in the 2,~D
. . pathway (Figure 1) is tfdA. This gene encodes a IllUllU~
which catalyzes the conversion of 2,4-D to 2,4-di~,lllulu~ ,llol (DCP).
Transgenic tobacco and cotton plants containing the tfdA gene have been
shown to have increased tolerance to 2,4-D (Streber et aL, Bio/te~.'.,.ol~;y
7:811-816 (1989) and Bayley et al., Theor. Appl. Genet. 83:645-649 (1992),
ly)~ Until now, attempts to use the tfdA gene as a selectable marker
to identify ~r l~r~ -i plants have failed. Streber et al., Bio/technGlogy
7:811-816 (1989) theorizes that "in a chimeric tissue ~ r l~ ~ ~l cells cannot
develop into shoots because they are overgrown or otherwise inhibited by
1",~l",. c " 1 .. ~ callus that is rapidly deYeloping in the presence of 2,4-D. "
wo 95/18862 r~ 34
~1 ~3~85 i:
--2--
S ~y of the I~vent~on
The mvention provides a method of selecting for a transgenic plant
cell
The invention further provides a method of selecting for a transgenic
S plant cell ~ V a) u.~IlDrullllillg one or more plant cells With a tfdA geneexpressible in the plant cell, b) culturmg the Il~llDr~ .d plant cell with an
amount of 2,4-D which inhibits a.l~ iu~D shoot formation or l~ i",~
from non-~ r.~ plant cells, and c) selecting a plant cell exhibiting
growth.
The invention also provides a method of selecting for a transgenic plant
C~ .v a) ~ r....,.;,.v one or more plant cells with a tfdA gene
expressible in the plant cell, b) culturing the 1, ~ ~r"",.. A plant cell with an
amount of 2,4-D which inhibits a~ iu~LD shoot formation or .I ~5~ .,~ ."I;
from non-LI~ Dru.l.l~,d plant cells, c) selecting a plant cell exhibiting growth,
and d) ,. ~ lYV the plant cell into a plant.
The invention further provides a plant cell ~ a tf&4 gene
expressible in the plant cell wherein the plant cell is free of other foreign
marker genes; a plant l~,g ' from the plant cell; progeny or a pro~agule
of the plant; and seed produced by the progeny.
The invention further provides a sweetgum plant cell ~ a tfdA
gene expressible m the plant cell; a plant Icv ' from the sweetgum plant
cell; progeny of the plant; a propagule of the plant; and seed produced by the
progeny. Herbicide resistance (and more specifically, 2,4-D resistance) in
sweetgum would greatly reduce site plC~ iUII costs, and possibly make
growth of sweetgum (r~ . styraciflua L.) in plantations ~",.. ",.;. ~l
AlKhough transgenic tobaccû and coKon plants containing the tfdA gene have
been shown to have mcreased tolerance to 2,4-D (Streber et al.,
Bio/technology 7:811-816 (1989) and Bayley e~ al., ~f~eor. Appl. Genet.
83:645-649 (1992), I~,D~ ), it was urlknown whether tolerance could be
WO 95118862 r ~ ,c ~ ~4
~17~9~5
--3--
conferred into sweetgum. The present inYention provides transgenic
sweetgum plant cells contau~ing the ~d~ gene.
The invention also relates to a method of obtaining and a method-of
managing a hardwood plantation .
S (a) L-~rul-----.v one or more plant cells with a pV~ llU~,~W~id~.
Cu~ lV a herbicide resistance gene expressible in said plant cell;
(b) ~ said plant cell into a plant;
(c) culturing said ~ ,u~ L~d plant with an amount of herbicide
which will kill non-L-~.~rv-l.l~l plants;
(d) selecting a plant exhibiting growth;
(e) ~IU~ Lillg said plant to produce many plants;
(f) inducing root formation in said plants;
(g) growing said rooted plants to planting stock size;
(h) planting the planting stock srze plants in a sheared, defoliated
lS site, and
(i) applying herbicide over the entrre site to suppress ~
growth until the planting stock can thrive without ~ . growth control.
The invention also relates to a method of obtaining and a method of
managrng a hardwood plantation
(a) 1~. r"",;"~ one or more plant cells with a pvl~ u~ ,uLill~
c~ g a herbicide resistance gene and at least one second gene encoding
a foreign selectable marker expressible in said plant cell;
(b) culturing said plant cell with an amount of a chemical which
will rnhibit ad~ . shoot 1~ ;.. of plarlt cells not 1 .... ~f' ~- - - '-1 wivh
the foreign selectable marker gene;
(c) selecting a plant cell exhibiting a,l~. shoot
(d) I~ , said plant cell into a plant;
(e) ~.ul _ ~ said plant to produce many plants;
(f) inducrng root formation rn said plants;
(g) grûwing said rooted plants to planting stock size;
WO 95~18862 PCTIUS9S/0(!284
21~99~S 1~
~ `,.
(h) planting the planting stock size plants in a sheared, defoliated
site, and
(i) applying herbicide over the entire site to suppress f..".l.. ~il;.,,
growth until the planting stock can thrive without r- ~ "1' i I ;- ,- growth control.
The invention also relates to a method for obtaining and a method for
managing a hardwood plantation ~
(a) L~ r.",.l;"2~ one or more plant cells with a pGlyllu~ id~,
comprising a herbicide resistance gene and at least one second gene encoding
a foreign selectable marker expressible in said plant cell;
(b) culturing said plant cell with an amount of a chemical which
will inhibit adventitious shoot .. g. ,,li(.l~ of plant cells not Ll~ orullll~l with
the foreign selectable marker gene;
(c) selecting a plant cell exhibiting adv. .lLiLiU~ shoot l'L~
(d) I~ r, said plant cell into a plant;
(e) culturing said ~ ' plant with an amount of herbicide
which will kill non-i ~.I.,.,d plants;
(f) selecting a plant exhibiting growth;
(g) ~;,-l;"~ said plant cell mto a plant;
(h) ~lu~ ,dLillg said plant to produce many plants;
(i) mducmg root formation in said plants;
(j) growing said rooted plants to planting stock size;
(k) planting the planting stock size plants in a sheared, defoliated
site, and
(I) applying herbicide over the entire site to suppress (~ :il;.",
growth umtil the planting stock can thrive without ~ growth control.
Preferably, the herbicide resistance gene is the ~dA gene and said
herbicide is 2,~diul~1GIu~ ul~y~ , acid, the herbicide resistance gene is
the mutant ' ~u~y acid synthase gene from Ara~idopis, or the herbicide
resistance gene is the 5 e.lol~yluvy' ' 3-phosphate synthase gene and
said herbicide is glyl'
WO 95/18862 ~ 34
21 79~8S
The invention also relates to a method of obtaining and a method of
managimg a hardwood plantation C~
planting stock size plamts in a sheared,- defoliated site, and
applying herbicide over the entire site to suppress ~ grow~
until the planting stock can thrive without c - ~ - growth control,
wherein said plant is a hardwood comprisrng a herbicide resistance
gene.
The rnvention also relates to a plantation of hardwood trees c. ~
hardwood trees comprising a herbicide resistant gene. Preferably, the
herbicide resistance gene is the tfdA gene, the mutant ~c~tull~d~uAy ~cid
synthase gene from ATabidopis, or the 5 ~llul~ ' -3-phosph2te
synthase gene.
Further objects and advantages of the present imvention will be clear
from the description that follows.
Brief Description of the Figures
FIGURE 1. The 2,4-D ~,~:lddaiivc pathway. Gene~ are
shown in ~J~ICIILh~C:~ for each enzyme.
FIGURE 2. C~ UC~iUII of pUCW101.
FIGURE 3. Construction of pUCW200.
20FIGURE 4. Cullailu.,~iull of pBI121.
FIGURE 5. ELISA of sweetgum 2040 selected on kanamycm.
FIGURE 6. ~LISA of sweetgum 2027 selected on 2,4-D or
kanamycin.
FIGURE 7. Sequence of a tfdA gene from Figure 3 of Streber e~ aL,
25J. of BacteTiolo,~y 169:2950-2955 (1987).
WO 95/18862 ~ ; F~
~1~9985
-6 -
Defini~ions
Plant should be understood as referring to a mlllti~ rf~
organism capable of IJhJ10~ mcluding A~ (monocots and
dicots) and ~
Plant cell should be understood as referring to the structural arld
pllya;ùlur~i~ AI unit of plants. The term "plant cell" refers to any cell which is
either part of or deriYed from a plant. Some examples of cells
by the present invention include dirrt,, ~ cells that are part of a living
plant; dirl~ cells in culture; uliJir~-~ cells in culture; the cells
of ul-~irr~;ll ' tissue such as callus or tumors.
Plant cell progeny should be understood as referring to any cell or
tissue derived from plant cells including callus; plant parts such as stems,
roots, fruits, leaves or flowers; plants; plant seed; pollen; amd plant embryos.Propagu~es should be understood as referring to any plant material
capable of being sexually or asexually ~ ,, 1, or being ,UI~ r 1,, ' ' in vivo
or in vitro. Such propagules preferably consist of the protoplasts, cells, calli,
tissues, embryos or seeds of the 1~ g~ dL~d plants.
Trarsgenic plant should be understood as referring to a plant~aving
stably ;~ exogenous DNA in its genetic material. The term also
includes exogenous DNA which may be introduced into a cell or protoplast
in various forms, including, for eA-~mple, naked DNA in circular, linear or
u;ll`J form, DNA contained m ..~ v~ ~ or ~,Lull~v~ulll~.a or nuc~ei
or parts thereof, DNA complexed or associated with other molecules, DNA
enclosed m liposomes, ,~lD IU~Jlaa~, cells or protoplasts.
Detailed Description of the Invention
lrhe present invention relates to a method of selecting for a transgenic
plant cell.
. .
WO 95/18862 P~,lm.,.5:5- 1
~7~98~
-7-
In one ~.. l,n~l:.. l, the present invention relates to a method of
selecting for a transgenic plant cell C~ Y, a) ~ r~ " " ~ one or more
plant cells with a tfdA gene expressible in the plant cell, b) culturing the
l,.", r""" ;l plant cell with an amount of 2,4-D which irlhibits formation of
dJ~ LiLiuu~ shoots from non-~ r " " d plant cells, and c) selecting a plant
cell exhibiting growth.
In ano&er l ~.hû~l; ~.- ..: the present invention relates to a method of
selecting for a transgenic plant r.~ a) l ~ r(~ g one or more plant
cells with a tfdA gene expressible in said plant cell, b) culturing said
lû ~"~ r""~ l plant cell with an amount of 2,4-D which inhibits formation of
a.l~..LiLi,Ju~ shoots from non-L~ r~Jl~ d plant cells, c) selecting a plant cellexhibiting growth, and d) ~ r, ~ g~ said plant cell into a plant.
All plants which can be I ., . . r " " ,.~. l are intended to be included withinthe scope of the invention (preferably, r~ y' ' plants). Such plants
include, for example, species from the genera Fragaria, Lotus, Medicago,
Onobrychis, Trifolium, Trigonella, Vgna, Ci~rus, Linum, Geranium, Manihot,
Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum,
Da ura, Ilyv~ , Ly,~v~ icv,., Nicotiana, Solanum, Petunia, Digitalis,
Majorana, Cichorium, A'o~;~ ' , Lactuca, Bromus, Asparagus, Ar~
IIel c, v, allis, Nemesia, relu,~ Panicum, r R, ,
Sencia, Su~ v~, Cucumis, Browalia, Glycine, Lolium, Zea, Triticum,
Sorghum, Malus, Apium, and Datura.
Suitable woody di, .J~y' ~( forest tree species (hardwoods) include,
for e cample, species from the genera Acacia, Acer, Actinidia, Albizzia, Alnus,
A~ - " Atriplex, Betula (Birch), Blu~hy ~, Broussonetia, Camellia,
Carya, Castanea (Chestnut), Catalpa, Cinchona, Corylus (Hazelnut),
Diospyrus, Eucalyptus, Fagus (Beech), Ficus, Fraxinus (Ash), Gleditsia,
A', ~';~r, Hedera, llex, Kalmia, ~ (Sweetgum), LiliOdel~ vl~
Moghania, Morus, Paulownia, Populus, Prunus, Quercus (Oalc),
~hnf~ v", Robinia, Salix, Santalum, Sapium, .~ , Tectona,
Tl~,m ' ' . Ulmus (Elm), and Vaccinium.
WO 95/1886~ 1 ~.l/U."._. ~
2~99&~ --
--8--
Plant LL~I~ru~ lLiull techniques are well known im the art and include
direct ~ r...,. -~; - (which includes, but is not limited to: Illi~lU Ij~iU~I
(Crossway, Mol. Gen. Genetics 202:179-185 (1g85)), polyethylene glycol
Lld~rull~l~,iull (Krens et al., Nature 296:72-74 (1982)), high velocity ballistic
I~ ~ 1, ,.I;r.. ~ (Klem et al., Nature 327:70-73 (1987)), fusion of protoplasts with
other entities, either minicells, cells, Iysosomes, or other fusible lipid-surfaced
bodies (Fraley et al., Proc. Natl. Acad. Sci. USA 79:1859-1863 (1982~,
cl~ u~Olrl~iull (Fromm et al., Proc. Natl. Acad. Sci. USA 82:5824 (1985~)
and techniques set forth in U.S. Patent No. 5,231,019)) and Agrobacteriu~
10 r~nof~;o~ mediated 1,,.,,~ r~.. ", ~;.. " (Hoekema etal., Nature 303:179 (1983),
de Framond et al., Bio/tec~mology 1:262 (1983), Fraley et al. WO84/02913,
WO84/02919 and W084/02920, Zambryski et al. EP 116,718, Jordan et ai.,
Plant Cell Reports 7:281-284 (1988), Leple et al. Plant Cell ~eports 11:137-
141 (1992), Stomp et al., Plant Physiol. 92 læ6-1232 (1990), amd Knauf et
al., Plasmid 8:45-54 (1982)). One preferred metbod of 1, ,. "~r, .. " . , ;".. is the
leaf disc tl~ rull~ iull technique as described by Horsch ot al., Scienoe
æ7:1æ9-1230 (1985).
The above-described ~"- ,~r", ~ techniques can utili~e a tfdA gene
or fragment thereof expressible in plants. Included within the scope of-a tfd,4
gene are functional derivatives of the tfdA gene, as well as variant, analog,
species, allelic and mutational derivatives.
A "fragment" of a molecule is meant to refer to any portion of the
amino acid or nucleotide genetic sequence.
A "functional derivative" of a sequence is a molecule that possesses a
biological activity (either functional or st~uctural) that is substantially similar
to a biological activity of the protein or nucleic acid sequence.
A "variant" of a nucleic acid is meamt to refer to a molecule
cl.hr~ fi:.lly similar in structure and biological activity to the nucleic acid, or
to a fragment thereof. Thus, provided that two molecules possess a comrnon
activity and may substitute for each other, they are considered variants as thatterm is used herein even if the nucleotide sequence is not identical.
WO 95118862 r~ 'C
~ 9~85
g
An "analog" of a protein or genetic sequence is meant to refer to a
protein or genetic sequence Cllhc~nt~ y similar in fimction to a protein or
genetic sequence described herein.
An "allele" is an alternative form of a gene occupying a given locus
S on the ~.IUUIUU~VIn~.
A "mutationr is any detectable change in the genetic material which
rnay be transmitted to daughter cells and possibly even to succeeding
giving rise to mutant cells or mutant individuals. If the
of a mutant cell give rise only to somatic cells in mlllfi~
organisms, a mutant spot or area of cells arises. Mutations in the germ line
of sexually lC~/lUdU,illg organisms may be ll~iLlcd by the gametes to the
next generation resulting m an individual with the new mutant conditio~ in
both its somatic and germ cells. A mutation may be any (or a c~....l.: -~;....
ofl detectable, unnatural change affecting the che_ical or physical
~ , mutability, n-r1jr~tinn, phenotypic fimction, or l~. ' of
one or more ~U~l ~ may be added, deleted,
substituted for, inverted, or transposed to new positions with and without
inversion. Mutations may occur ~r~ y amd can be induced
,, ;"" .~ .lly by application of mutagens. A mutant variation of a nucleic
acid molecule results from a mutation. A mutant pol~ may result from
a mutant nucleic acid molecule.
A "species" is a group of actually or potentially b.~li~ natural
Fnp~ tinnc A species variation within a nucleic acid molecule or protein is
a change m the nucleic acid or amino acid sequence that occurs among species
and may be determined by DNA sequencimg of the molecule in question.
The ~d4 gene depicted in Figure 7 (SEQ ID NO: 1) can be altered by
~- ,~li~. :i....~ additions or deletions that provide for .~ ~y equivalent
molecules. Due to the J~ ,.y of rlucleotide codimg sequences, other DNA
sequences which encode substantially the same amino acid sequence as
depicted in Figure 7 (SEQ ID NO:2) may be used m the practice of the
present invention. These include but are not limited to nucleotide sequences
WO95/1886~ ~IIL)~
~1~9~8~ --
-10-
comprising all or portions of the ~d4 gene depicted in Figure 7 (SEQ ID
NO:1) which are altered by the ~ of different codons that encode a
functionally equivalent amino acid residue withm the sequence, thus producing
a silent change.
Such functional alterations of a given nucleic acid sequence afford an
UIJ~VlL~L..liLy to promote secretion and/or processing of l~t~ lo~,Ju~ proteins
encoded by foreign nucleic acid sequences fused thereto. All variations of the
nucleotide sequence of the ~fdA gene and fragments thereof permitted by the
genetic code are, therefore, included in this invention.
lû In addition, the ~ gene may comprise a nucleotide sequence which
results from the addition, deletion or ~ of at least one nucleotide to
the 5'-end and/or the 3'-end of the nucleic acid formula shown in SEQ ID
NO:1 or a derivative thereof. Any nucleotide or polyl.ul,l~uli.le may be used
in this regard, provided that its addition, deletion or i ~. ';1.~1;..~. does not alter
~5 the amino acid sequence of SEQ ID NO:2 which is encoded by the nucleotidesequence. The ~U gene may, as necessary, have restriction L..~1...,... 1. ~_
~, sites added to its 5'-end and/or 3'-end.
Further, it is possible to delete codons or to substitute one or more
codons by codons other than degeneMte codons to produce a ~llu~,~ul~llly
modified pol~ lid~, but one which has subst~mtially the same utility or
activity of the poly~ l., produced by the ~ 1; ri~ d nucleic acid molecule.
As recognized in the art, the two polyl,.,~ .,s are functionally equivalent, as
are the two nucleic acid molecules which give rise to their production, even
though the differences between the nucleic acid molecules are not related to
degeneracy of the genetic code.
The ~.~4 gene is preferably operably linked to a promoter region
functional in plants, a i . initiation site, and a 1 ~ ,"
t .... ~ sequence. The particular promoter used in the expression cassette
is a noncritical aspect of the invention. Any of a number of promoters which
3û direct 11,.. ~. . il,l;.. , in a plant cell is suitable. The promoter can be either
v~ or inducible. Some exarnples of promoters functional in plants
~ WO 9!5/18862 ~17 g 9 8 5 r
include the nopalihe synthase promoter and other promoters derived from
native Ti plasmids, viral promoters mcluding the 35S and l9S RNA promoters
of ~ulillu.._l mosaic virl~ (Odell et al., Nature 313:810-812 (1985)), and
numerous plant promoters.
General methods for selecting transgenic plant cells containing a
selectable marker are well known and taught, for example, by Herrera-
Estrella, L. and Simpson, J., rForeign Gene Expression m Plants, " in Plar~
Molecular Biology, A Practical Approach, C.H Shaw, ed., IRL Press,
Oxford, England (1988), pp. 131-160. For use of the tfdA gene as a
selectable marker, the amount of 2,4-D which inhibits a.l;.. ~iliuuD shoot
formation from non-(~A ~r- ~ ~Sd plant cells and allows al~ LiliuuD shoot
formation from ~ r ~ plant cells can be determined by 1) platmg non-
~.A..~r~.". ;l cells on media containing various ~ of 2,4-D and 2)
by .l.'..,,,;.- c, the lowest ~ of 2,4-D which will inhibit
ad~ iliu D shoot formation by the plant cells. This lowest Wll~, can
then be used to select ~ r~ J plant cells. In general, solubilized 2,4-D
should be present in an amount ranging from about 0.001 to 5 mg/l culture
medium. With regard to the sweetgum plant cells ~ r,ll l ~l with the tfdA
gene, 2,4-D should preferably be present in an amount ranging frorn about
0.01 to 0.5 mg/l culture medium. A preferred amount of 2,4-D is about 0.01
to 0.2 mg/l culture medium. The amount of 2,4-D to be used for selection
of l~ rl~ shoot cultures is determined by identifying the minimum
c., ., -. 1.,-~;.. - of 2,4-D which will inhibit ~d~,llliliuuD shoot formation.
Expanding leaves from a selected sweetgum clone are surface sterilized and
cut into small pieces. The leaf pieces are then placed on WPM 0.1 mg/l
NAA, 2.5 mg/l BA containing 2,4-D m l from 0.0 to 5.0 mg/l.
The leaf pieces are mcubated until the control (no 2,4-D) pieces regenerate
shoots. The ideal .~ of 2,4-D for selection of I A~r~ is the
lowest of 2,4-D which did not allow 1~
In another ~ - t, the present invention relates to a plant cell
Uulll~liDill~ a tfdA gene ~A~ ,DDlbl~, in the plant cell wherein said plant cell is
WO 95/18862 X ~ 7 g ~ ~ 5 PCTIUS9~/00284
-12- s .
free of other foreign marker genes (preferably, other foreign selectable rnarkergenes); a plant ~ from the plant cell; progeny or a propagule of the
plant; and seed produced by the progeny.
Plant ~ ..., techniques are well known in the art and include
those set for~h in the Handbook of Plant Cell Culture, Volumes 1-3, Evans
et al., eds., Macmillan Publishing Co., New York, NY (1983, 1984, 1984,
ic~c,li~ ); Predieri and Malavasi, Plant Cell, Tissue, and Organ Culture
I7:133-142 (1989); James, D.J., et al., J. Plant Plrysiol. 132:148-154 (1988);
Fasolo, F., et al., Plant Cell, Tissue, and Organ Cu7ture 16:75-87 (1989);
Valobra and James, Plant Cell, Tissue, and Organ Culture 21:51-54 (1990);
Srivastava, P.S., et al., Plcnt Science 42:209-214 (1985); Rowland and
Ogden, Hort. Science 27:1127-1129 (1992); Park and Son, Plant Cell, Tissue,
and Organ Culture 15:95-105 (1988); Noh and Minocha, Plant Cen Reports
5:464-467 (1986); Brand and Lineberger, Plant Science 57:173-179 (1988);
Bozhkov, P.V., et al., Plant Cell Reports 11:386-389 (1992); Kvaalen and
von Arnold, P~sant Cell, Tissue, ar~d Organ Culture 27:49-57 (1991);
Tremblay and Tremblay, Plant Cell, Tissue, and Organ Culture 27:95-103
(1991); Gupta and Puls'man, U.S. Patent No. 5,036,007; Michler and Bauer,
Plant Science 77:111-118 (1991); Wetzstein, H.Y., etal., Plant Science
64:193-201 (1989); Mr~`.r~nql~qn G.H., etal., Bio/Technology 6:800-804
(1988); Gingas, V.M., Hort. Science 26:1217-1218 (1991); Chalupa, V.,
Plant Cell Reports 9:398-401 (1990); Gingas and Lineberger, Plant Cell,
Tissue, and Organ Culture 17:191-203 (1989); Bureno, M.A., et al., Phys.
Plant. 85:30-34 (1992); and Roberts, D.R., et al., Can. J. Bot. 68:1086-1090
(1990).
The herbicidal resistant plant enables the farmer to plant a herbicidal
tolerant crop and then treat the field for weeds without adversely affecting thecrop. Further, the herbicidal tolerant plant enables the farmer to grow crops
in fields t~at have been treated with herbicides. These herbicidally treated
fields will contain a certain amount of the herbicide in the soil and thus a
"herbicide carryover" is seen (U.S. Patent No. 4,975,374).
~ WO 95/18862 21~ 9 9 8 r~ ,llU_. '
-13-
Other foreign marker genes (i.e., UAU,,~I.VU~IY introduced genes)
typically used include selectable markers such as a neo gene (Potrykus et al.,
Mol. Gen. Genet 199:183-188 (1985)) which codes for kanamycm resistance;
a bar gene which codes for bialaphos resistance; a mutant EPSP synthase gene
(Hinchee et al., Bio/~ 6:915-922 (1988)) which encodes glyphosate
resistance; a nitrilase gene which confers resistance tû IJlVl.lVAYlUI (Stalker et
al., J. Biol. Chem. 263:6310-6314 (1988)); a mutant '~ synthase
gene (ALS) which confers ;~ ,r or DUIlUllUllylUl~ resistance (EP
s~rpljr~-inn number 154,204); a ll~;fiULI ' resistant DHFR gene (Thillet et
al., J. Biol. Chem. 263:12500-12508) and screenable markers which mclude
ul uluv~lar~ (Gus) or an R-locus gene~ alone or in ~ ;rl~ with a c-
locus gene (Ludwig et al., Proc. Natl. Acad. Sci. USA 86:7092 (1989); Paz-
Ares et al., EMBO J. 6:3553 (1987)).
Plants which contain ~fdA gene and no other foreign marker gene are
avv ~ in that removal of the foreign marker gene, once inserted into
the plant, may be impossible without also removing the tfdA gene. Absence
of the foreign marker gene is desired so as to minimrze the number of foreign
genes eApressed.
A plasmid which contains only tfdA between the Ti-plasmid bnarders
can be c .. ~ l in several ways. One method is to frst partially digest
plasmid pUCW200 with EcoRI such that only the EcoPI site on the right side
of the NOS; (Figure 4) is cut. The ends are then made blumt by
using standard molecular methods (Maniatis, T. e~ al., Molecular Cloning:
A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,
NY (1982)). This linear plasmid is digested with the restriction l~
Hindm. The fragment containing tfdA unde} control of the CaMV 35S
promoter is purified by resolvmg and cutting it out of a low melting
t~,ul,u~ agarose gel. This Hindm-EcoRI fragment is ligated into the
binary vector pBlN19 (Bevan, M., Nllcleic Acids Research 12:8711-8721
(1984)) which has first been digested with the restriction ~ SsdI
and the ends made blunt, and then digested with HindlJI. The resultant
w095/18862 P~-l~... C
21~-998S
-14-
plasmid is then i Cvl....,.l rnto E. coli S17-1 with selection for kanamycrn
resistance conferred by the kanamycrn resistance gene located on plasmid
pBINl9, but outside of the left and right boundaries of the Ti-plasmid. This
plasmid is then mobilized into A~ c, . ", t~/mofnrio)7~ LBA 4404 and used
to h ansforln the target plant tissue
In another ....l.f~.l;,.,...: the present invention relates to a sweetgum
plant cell comprising a tfdA gene expressible in the plant cell; a plant
lcc,~ ,ldlcd from the sweetgum plant cell; progeny of the plant; a propagule
of the plant; and seed produced by the progeny.
~istorically, ~ -1 of bardwood plantations on cut-over forest
land, old a~ri~ lh~r:~l fields, and river bottomland have only been successful
when very intensive site l~lC~UalflLiUII has been employed prior to planting
(Hunt, ~ardwood Short Course, North Carolina State University (1973),
pp. 63-71). An example of the site ~Jlc~JalaLiull methods required is described
in "Hardwood Plantation '`"- l" " (~alac, B. F. and Heeren, R.D.,
Souther,~ Jo~rnal of Applied Forestry 3:3-6 (1979)). Typically, site
,ulclJalaliull requires a regiment of shearing, raking, and disking or beddr~g.
The prepared planting site must be clear and level so tbat directed arpli~ ~tif)n
of herbicide, for ~.. I.~lili.~ conhrol, is possible.
Directed application of herbicide is necessary to avoid killing the
hardwood plants. Not only is this intensive site LJIC~alaliull expensive, it also
results in site ~lr~ - due to soil ~ by heavy machinery and loss
of top soil due to raking. Tbis intensive site flrfj,,..l-l;"" results in lower
yields.
Site ,UII, '' Ir'l-' ` ' I~` will be greatly reduced if hardwoods,
which have been genetically engineered to be herbicide resistant, are planted.
The herbicide may be applied by aerial spraying. Therefore, the site would
not have to be level. This would eliminate the need for raking and disking.
Also, the amount of mowing and cultivation required for ( ~ . control
would be reduced. Less site pl~alaLiull would have the added benefit of less
W0 95/18862 ~ a. .lr - ~4
2179985
-15-
soil ~ and loss of topsoil, thus, less site ~ l. and greater
yields will result.
Planting of herbicide resistant hardwoods would also have several
advantages over planting uuu~. ' ' seedlings or rooted cuttings. These
S advantages incl~lde hand planting, and an u~ for increasing the
number of stems per acre. Hand planting would be possible because, with
aerial herbicide arplir~rinn, straight rows are not needed for tractors. If
restrictions on spacing required to operate tractors between rows are no longer
necessary, more stems per acre could be planted, which would result in
increased yields.
Tolerance to the herbicide 2,4-D can be conferred to sweetgum by
h~LIudu~.liuu of a ~dA gene expressible in sweetgum into the sweetgum
genome. The ~dA gene may be intrc~1uced into sweetgum by the
l"",.r""" :j" techl~iques outlined above or more preferably as set forth in
15 Chen, Z. and Stomp, A. (l991) ~T.. - ~r ~ of l iq~ c~ styraciflua
L. (Sweetgum) using Agrobacterium T~ 1 In:plu~ dll,~ 215t
Southern Forest Tree l~ululu .~ L Conference. Jume 17-20, l99l Knoxville,
TN. A ~dA gene is preferably contained on a plasmid wherein the l~dA gene
is ûperably linked tû a promotrr region functional in plants, a ~
20 initiation site, and a i , ~ ... -';.. sequence (examples ûf which are
provided above). In one preferred ~ - )-1; . ..~ the ~dA gene is linked to a
foreign marker gene (described above).
Thus, the invention also relates to a method of managing a hardwood
plantation c~ 'L
(a) j r ,, one or more plant cells with a p~l~-.. ,l~u~id~.
c-.--.~ a herbicide resistance gene expressible in said plant cell;
(b) I~ ~.. ,-~ .. ~, said plant cell intû a plant;
(c) culturing said " ' plant with an amount of herbicide
which will kill non-i r ~ plants;
(d) selecting a plant exhibiting growth;
(e) ~/lU,U..e,~iill~; said plant to produce many plants;
wo 95/18862 r~l,u.,,s.~ 1
21~8~ ` --
-16-
(f) inducing root formation in said plants;
(g) growing said rooted plants to planting stock sr~e;
(h) planting the plamting stock size plants in a sheared, defoliated
site, and
(i) applying herbicide over the entire site to suppress r~",l.. :;1;
growth until the planting stock can thrive without c- ~ ll l growth control.
The invention also relates to a method of managing a hardwood
plantation ,UIII~ iUI~.
(a) Lldlli~fbllllillg one or more plant cells with a polyllu~
comprising a herbicide resistance gene and at least one second gene encoding
a foreign selectable marker expressible in said plant cell;
(b) culturing said plant cell with an amount of a chemical which
will inhibit ad~ iuu~ shoot ~ 1;.. of plant cells not ~ d with
the foreign selectable marker gene;
(c) selecting a plant cell exhibiting a.l~ iO,.O shoot ~,~...... Al;~l
(d) 1~.~.. ,.1;.. ~ said plant cell into a plant;
(e) ~JlU~ 5d~iUlg said plant to produce many plants;
(3) inducing root formation in said plants;
(g) growing said rooted plants to planting stock size;
(h) plantmg the planting stock srze plants in a sheared, defoliated
site, and
(i) applying herbicide over the entire site to suppress,
growth until the planting stock can tbrive without ~ growth control
The invention also relates to a method for managing a hardwood
plantation ~
(a) I,,.,~r,,.,,,i,~O one or more plant cells with a p~ly~lu~ ide
comprising a herbicide resistance gene and at least one second gene encoding
a foreign selectable marker expressible in said plant cell;
(b) culturing said plant cell with an amount of a chemical which
willinhibita.l~. shoot.~ of plantcellsnotll,~r.. l.. l dwith
the foreign selectable marker gene;
wo 95/18862 r~l"J~ c
2~79~8~
-17-
(c) selecting a plant cell exhibiting a~ iLiuu~ shoot .
ld) ~ said plant cell into a plant;
(e) culturing said O ' plant with an amount of herbicide
which will kill non-~ f ~ l plants;
(f) selecting a plant exhibiting growth;
(g) ,c~,-l;,.~ said plant cell into a plant;
(h) ~JlU~ illg said plant to produce many plants;
(i) inducing root formation m said plants;
(j) growing said rooted plants to planting stock size;
(Ic) plantmg the planting stock size plants in a sheared, defoliated
site, and
(I) applying herbicide oYer the entire site to suppress ~
growth until the plantmg stock can thrive without ~ growth control.
The invention also relates to a method of managmg a hardwood
plantation ~
planting stock size plants m a sheared, defoliated site, and
applying herbicide over the entire site to suppress ~ growth
until the planting stock cam thrive without ~ growth control,
wherein said plant is a hardwood .,ullllni~illO a herbicide resistance
gene.
The invention also relates to a plantation of hardwood trees c, , _
hardwood trees comprising a herbicide resistant gene. Preferably, the
hardwood trees are sweetgum.
In general, herbicide resistant hardwoods can be ~UI~LIu~,t~ by
i ' _ genes which encode proteins which detoxify the herbicide, such
as the ~dA gene, or which encode proteins that are not sensitive to the action
of the herbicide, such as the mutant ~tul~u-~y acid synthase gene from
Arabidopis (~uropean Patent ~pplication Number 91119254.0, by American
Cyanamid Company). Another example of a gene which can confer herbicide
resistance is the 5 ~ !~luv~ 3-phosphate synthase gene (IJnited
States Patent Number 4,940,835, Monsanto Company), which is tolerant to
wo gs/lss62 ~1 79 ~ 8$ . r~
-18-
the herbicide glyphosate. Selection for ~.~"rU..."lLiull of these genes into thetarget plant can be done by selecting for the presence of a selectable marker
gene, such as the kanamycin resistance gene, which is linked to the herbicide
resistance gene. This is done by mcubating the target tissue on media
containing levels of kanamycin which irlhibit .r~ A ~ of non-l .... ~r~., ., .. J
cells. Alternatively, selection can be done by selecting for l~ ;in the
presence of the herbicide at ~:~lllf ~l 'l;-l - which irlhibit l~ of non-
,. d cells, or allowing .. ~ l;, to take place in the absence of
selection and then screening the l~ dLtd plants for presence of the
herbicide resistance gene by growing the putative l~ rul~"l~L~ on media
containing levels of the herbicide which irlhibit growth of non-L-h."rv-.~
plants. The preferred method of selecting for l~a~ru~ iull of herbicide
resistance genes, other than the tfdA gene, is tû first select for ~ ;. . m
the presence of kanamycin and then screen the l.,l, ' plants by
mcubating them on media containing levels of the herbicide that are toxic to
non-l-.~, r~ l plants.
Plantations are a group of a large number of trees umder cultivation.
Preferably, nutrients such as triple ! . '1, ' A I ' or l~ phosphate,
are added to the soil of the site. See, Davey, C.B., Hardwood Short Cf)urse,
North Carolina State University (1973), pp. 72-75), and "Hardwood Plantation
M - L~ " (Malac, B. F. and Heeren, R.D., Southern Journal of Appiied
Forestr~v 3:3-6 (1979)). In addition, the site may be raked prior to planting
the planting stock.
The rnvention also relates to a method of producing or managing a
hardwood plantation, comprising planting stock size plants in a sheared,
defoliated site, and applying herbicide over the entire site to suppress
'""'l' l;l;l-~- growth until the planting stock can thrive without c~....l.~:;l;....
growth control, wherein the plant is a hardwood comprising a tfdA gene.
Preferably, the plant is sweetgum.
The present inveùtion is described in further detail in the following
non-limiting examples.
Wo 95/18862 ~ 1 7 9 9 8 5
-19-
~ , Ics
The followmg protocols and ~ details are referenced in the
exa~nples that follow.
a. Strain Sources and G~owth ~ "'il
The bacterial strains and plasmids used herein are listed in Table 1,
and media formulas are shown in Table 2 and 3. r ~ aeruginosa
PAOlc containing plasmid pRO101 or plasmid pRO1727 were grown on TNA
plates containing 50 ~g/ml tetracycline (TC50) at 37C, P. aeruginosa
PAOlc (pUCW101; Figure 2) was grown on TNA containing 500 ~g/ml
r-~rb~nirillin (Cb500) at 37C. P. pu~ida PPO300 (pUCW200; Figure 3),
Ax,uL,aL~c,~.w.. r ~7r;Pn~ LBA4404 (pUCW200; Figure 3), Escherichia coli
HB101 (pBI121, Figure 4), and E. coli S17-1 (pUCW200; Figure 3) were
grown on TNA contar~ing 50 ~g/ml kanamycm (KmS). The P. p~tida and
A. r ~' strains were grown at 30C, and the E coli strains were
grown at 37C.
Growth of P. aeruginosa PAOlc (pUCW101), P. putida P~0300
(pUCW200), and A. l ~' ~ LBA4404 (pUCW200) for analysis of 2,4-D
conversion to DCP was done by inoculating 50 ml of Burk's/CAA media
containing 1 mM 2,4-D with a loop of culture from an overnight TNA plate
containing the .~ UIJlidt~, antibiotic. These liquid cultures were sbaken at
30C for 4 hours, and then filter sterilr~ed. The sterile filtrate was analyzed
by HPLC as described below.
Wo 95/18862 r~
21~9~8~ ~ ~
-20-
TABLE 1
BAC~EUAL STRUNS A~;D PL~S
Reference or
Strain or Pla~mid Relevant Markersa Source
Ps 1 aeruginosa Prototroph Hollowayb
5 PAOlc
Ps ~ " punQ'a Prototroph ATCCC
PPO300
Escherichia coli thi, pro, recA ~ UIIIU~UIII~IIIY SimOnd
S17-1 integrated RP4
Ax,ubQ~t~,iu,,l tumefaciens Sm Clonteche
LBA~404
PlQsmids
pRO1727 Cb, Tc Cuskeyf
pRO101 2,4-D+, Tc Harkerg
pB1121 Km, Gus+ CloMechd
pUCW101 Cb, ~Q'A+ Figure 2
pUCW200 Km, tfQ'A+ Figure 3
a: Al~ Sm: ~LI~-U.. ~_;I-. Cb: ~ Tc: t~
E~n: karlamycin. Gus: ~ ~,' ''
b: Holloway et al., Microbiol. Rev. 43:73-102 (1979).
c: ATCC 17514, American Type Culture Collection, Rockville, MD
d: Simon, R. et al., Bio/lechnology 1.784-791 (1983)
e: Clontech 1~ C, Inc., Palo Alto, CA.
f: Cuskey et al., J. Bacteriûl. 169:2398-2404 (1987).
~k~ e~ol., ~ /lact~n~l ~n 3~13~0 (1989~
~7~98~
WO95/1886~ P~
-21-
T~BI F 2
TNA
Tryptone 5.0 g/l
Yeast Extract 2.5 g/l
NaCI 8.5 g/l
Glucose 1.0 g/l
Agar 20.0 g/l
AutoclaYe and temper to 50C. Add antibiotic if required and pour plates.
LB
.10 Luria BroLh Base 15.5 gA
Agar 20.0 gA
Autoclave and temper to 50C. Add antibiotic if required and pour plates.
Burk's Salts
Stock Solutions:
a. MgSO4-7H2O 39.90 gA
b. FeSO4-7H2O 0.01 g/l
c. NaMoO4-2H2O 0.05 g/l
d. (NH4)2SO4 100.00 g/l
e. I M Potassium Phosphate
buffer, pH 7.1
Autoclave stock solution and store at room temperature.
Burk's/CAA
To I L of sterile distilled water containing 0.3% casamino acids add:
5 rnl of stock solutions a, b, and c;
10 ml of stock solutions d and e.
r ~ plates
To I L of distilled water containing 0.2% succinate and 2% noble agar, which
has been autoclaved and tempered to 50C add:
5 ml of stock solutions a, b, and c;
10 ml of stock solutions d and e.
Add appropriate antibiotic if desired and pour plates
Page et aL, J. Bacteriol. 125:1080-1087 (1975)
wo 95/18862 ~ ~ 7 ~ 3 8 5 P~111J,.,5
-22-
TAB~ 3
WPM 0.1 mg/l NAA, per liter
2.~ mg/l BA
100 ml
WPM-macro lO ml
5WPM-micro 10 m.
WPM-Ca 10 ml
Inositol (10 mbg/ml) 10 ml
Chelated Iron I ml
WPM Vitamin 20 ml
lOSucrose 2 5 ml
NAA (01 mg/ml)C
BA (01 mg/rnl)d
Bring volume to one liter with distilleo H2O, pH to 5 8, add 7 g agar, and
autoclave
ISWPM-macro g/l WPM-micro g/l
NH4NO3 4 0 H3BO3 0.67
K2SO4 9 9 ZnSO4-7H2O 0 86
KHzPO4 17 MnSO4-H2O 169
MgSO4-7H2O 3 7 Na2MO4-2H2o 0 025
CuSO4-5H2O 0 025
20 WPM-Ca g/100 m- WPM-Vitamin g/100 m.
Ca(NO3)2-4H2O 5.56 Thiamine HCI o l
CaCI2-H2O 0.96 Nicotinic acid 0 05
Pyridoxine HCI 0 05
Glycine 0 2
a Lloyd et al., Comb. Proc. Inter. Plant. Prop. Soc. 30:421-427 (1980)
b. Chelated I}on = Na2 EDTA 3 73 g/l; FeS04 - 7H2O 2 73 g/l
c NAA = N~ acid
d. BA = li-,l~yl~.~lo purme
b. Molecular Biology Methods.
Plasmids were isolated by harvesting the bacterial growth of lO TNA
plates containing the .~,uL,Iul antibiotic by suspending the growth from
each plate in 5 m'l of TE buffer (50 mM Tris-HCI, 20 mM EDTA, pH 8.0),
poolmg the solutions in a 250 ml centrifuge bottle and pelleting the cells by
~ wo 95118862 ~ 3 8 ~ r~.,.~
-23-
... 1 . ir, . ~l ;.... at 10,000 x g for five minutes. The pellet was ~ - 1 m
20 ml of Iysis buffer (50 mM Tris-HCI, pH 8.0, 20 mM EDTA, 50 mM
glucose, 2 mg/ml Iysozyme), and incubated at room ~rll.lf;,-~l lr for five
minutes. Freshly prepared (40 ml) alkaline-SDS solution (0.2M NaOH, 1%
SDS) was added. The cells were Iysed by gentle mversion and incubated in
an ice water bath for 10 minutes. Potassium acetate (30 ml of a 5 M solution)
was added, the solution was mixed by gentle inversion and imcubated in an ice
water bath for 10 minutes. This solution was ~ ~ .r. ~ for 10 minutes at
10,000 x g, 4C. The ~ was decamted to a clean centrifuge bottle
and the DNA was ~., , ' by the addition of two volumes of 95 % ethanol
and incubation in an ice water bath for 1 hour. The precipitate was collected
by Cr .~II;r~ at 10,000 x g for 30 minutes at 4C. The resultmg pellet
was ~ 1 in 10 ml of ice cold TE buffer by slowly passing the mixture
through a pipet. After the pellet was ~ 1 5 ml of 7.5 M: --
acetate was mixed in by gentle inversion. This solution was incubated in an
ice water bath for 20 minutes and then c~n~nfi~fd for 10 minutes at 10,000
X g and 4C. The , was decanted to a 50 ml centrifuse tube and
0.313 volumes of 42 % P~ J I~ glycol (MW 6000-8000) was mixed in by
gentle inversion. The DNA was allowed to precipitate from 4 hours to
overnight at 4C. The DNA was collected by c,~ irl ~, at 10,000 x g for
10 mmutes at 4C. The pellet was . ' ' in 8 ml of ice cold TE buffer
and then added to 8 g of cesium chloride. After the cesium chloride was in
solution, 0.6 ml of a 10 mg/ml solution (m distilled water) of ethidium
bromide was added. The solution was c~nhifil~d m an ~ r..~.. at
40,000 rpm for 42 hours at 20C, usmg a TiS0 rotor. The plasmid band from
this cesium chloride-ethidium bromide gradient was drawn off using a pasteur
pipet. The ethidium bromide was removed by several extractions with water
- saturated n-butanol and then dialyzed for 24 hours, with two buffer changes,
m a TE buffer solution. The purified DNA was stored at -20C.
Routine analysis of strains for the desired plasmid was done by mini-
prep. A loop of culture talcen from a TNA antibiotic plate was suspended in
WO 95/18862 r~
217~98~i
-24-
100 111 of Iysis buffer by vortexing. After 5 minutes of incubation at room
t~ ,Id~UI~, 200 ~LI of alkaline-SDS solution was mrxed in by gentle inversion
and the Iysed cells were incubated in an ice water bath for 10 minutes.
Potassium acetate (150 ,ILI of a 5M solution) was mrb~ed in by gentle inversion
and incubation in the ice water bath was continued for 5 minutes. The Iysate
was cleared by Ill;~,luruS~l.iull at 4C for 5 minutes, and the ~ was
decanted to a fresh tube. The DNA was ~ by adding 1 ml of 95%
etbanol and incubating the mixture at -70C for 30 minutes, followed by
l~iwuru~dliull for 30 minutes. The pellet was 1,~ 1rd in 100 ~1 of ice
cold, sterile distilled water, and 50 ILI of 7.5 M ~ nn~ m acetate was mixed
in by gentle inversion of the tube. This mixture was incubated in an ice water
bath for 10 minutes, ~ ,luru~_d for 2 minutes, and the ~ was
decanted to a fresh tube. The DNA was ~,, , ' by addition of 300 ~1 of
95% ethanol, incubation at -70C for 30 minutes, and ~u;~.luru~;~liull for 30
minutes. The DNA pellet was dried by vacuum desiccation for 10 mmutes,
and l,.-,~l.. .~'1..1 in 40 ~1 of TE buffer. Analysis was done by agarose gel
el~ u~Jllulc~ia as described below.
Restriction ~.. 1.. 1. -~. digestion was done by incubating the purifled
plasmid DNA in the dlJ,UI~, ' ' Boebringer Mannherm buffer with 1-2 ,ul of
the required Boehringer Mannheim restriction .. 1".. 1. -~-, at 37C for 1
hour. The reaction was inactivated by incubation at 70C for 10 minutes,
followed by incubation m an ice water bath for 10 minutes.
DNA ligation was performed by mixing the two restriction
~",1. " ~ digested DNA fragments to be ligated, adding 1/10 volume 7.5
M - ~ acetate, and two volumes of 95 % ethanol. The DNA in this
solution was ~ . ' by incubation at -70C for 30 minutes and then
for 30 minutes at 4C rn the microfuge. The pellet was
. .1 in 100 ~1 of ice cold sterile distilled water by vortexing for 15
seconds. The .~ lrA DNA was ~ t. .1 by the r acetate-
ethanol method described above. After the second u~ J;~l;ull, the DNA
pellet was dried by vacuum desiccation for 10 minutes, I~ in 16 ,ul
~ WO 95/18862 217 9 ~ ~ ~ F~~
-25-
of ice cold sterile distilled water and 4 ~1 of 5X Gibco-BRL ligase buffer was
added. Gibco-BRL T4 ligase was added to 1 Wiess unit. The ligation
mixture was incubated at room t~ tUI~ for 2 hours, and stopped by
addition of 30 ~1 of ice cold, sterile distilled water.
Analysis of plasmids and DNA fragments was done by agarose gel
CI~IIU~IIUI~D;~ The gel is made by adding agarose to a fmal c.,.. ".. I;~,
of 0.7% in TAE buffer (40 mM Tris-acetate, 0.1 mM EDTA). A 15 cm2 gel
had a total volume of 100 ml and a mini-gel had a total volume of 25 ml.
The agarose buffer solution was melted in the microwave, tempered to 50C
and then poured into the gel mold and allowed to solidify for 20 minutes. The
cast gel was then placed in the gel box, and submerged in TAE buffer. The
DNA was loaded into the wells, and ~ ,IIu~l~ul~ .d for 2.5 hours at 100
volts when 15 cm2 gels were r~m, and 45 minutes at 130 volts when mini-gels
were run. The DNA was visualized by staining the gel in 300 ml of water
containing 40 ~1 of 10 mglml etbidium bromide solution for 20 minutes, and
then exposing the gel to W light at 305 nm. The gel was r~ using
a Fisher Brand ~ stem and Polaroid 660 film.
Low meltmg ~ a~ ~se gels were run as described above,
except the amount of low melting agarose was 1% and the gels were run at
4C. The DNA was visualized by ethidium bromide staining, and the desired
fragment was cut out of the gel. The excised fragment was eluted from the
gel matrix by adding lOû ~LI of TE buffer and incubatmg at 70C for 10
minutes. An equal volume of TE saturated phenol was added and mixed in
by gentle inversion. The phases were separated by ~ uru~,i..~ the sample
for 3 minutes at 4C. The top (aqueous) layer was collected, and the phenol
Iayer was extracted twice more with an equal volume of TE buffer. The
aqueous phases were pooled and extracted once with a 1:1 mixture of
rh~nf~ vrullll amd once with chloroform. The DNA was ~ by
addmg 1/10 volume of 7.5 M acetate, 2 volumes of 95% ethanol,
incubation at -70C for 30 minutes, and lll;.,l~ for 30 minutes. The
WO 95/18862 r~ r.'l
-26-
pellet was vacuum dried for 10 minutes and then ~ l in 100 ~1 of TE
buffer.
c. T~ ~, and (~ " " ` Metllods.
TlGll~rVllllGiiVII of P. aeruginosa PAOlc was as described by Mercer
S et aL (Mercer, A.A. and Loutit, J.S., .~. Bactenol. 140:3~42 (1979)).
E. coli S17-1 was IlA ~r'~ as described by Maniatis et al. (Maniatis, T.
et al., Molecular cloning: a laboratory manual, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N~ (1982).
Transfer of plasmid pUCW200 from 1~ coli S17-1 to P putida
PPO300 or A. tllmofn~ ion~ LBA4404 by ; ., was done by growrng the
strains at 30C in 1~3 media or LB containing 50 ILg/ml kanamycin (Æ. coli
S17-1) and then mixing equal volumes of each culture, filtering the mixture
through a sterile 0.22 Ibm filter and placing the filter on an LB plate. The
plates were incubated overnight at 30C, followed by washing the filter with
5 rnl of sterile distilled water and plating dilutions of the cell suspension onBurk's salts containing 0.2% succinate and 50 ~g/rnl kanamycin These plates
were incubated at 30C for 48 hours and the 1~ were purified by
restreaking to identical media.
d. npLc Analysis for 2,4D and DCP.
HPLC analysis was done using a Supelco C8 colurnn, a mobile phase
of 70:30 I~ GIIVI~ - at 1 ml/min., and detection at 280 nm of a 20 ~LI
injection. Peaks were identified by c~ of retention times to those of
known standards.
wo 95/18862 T ~ ~ 5.~ 1
~ 21~98~
-27-
Example 1. ~ . of PlasmidpUCW101.
To construct pUCW101 (Figure 2), plasmid pRO101, which encodes
all the enzymes for the ~lr~ ;., of 2,4-D to chlJlu~lldk.Jla~.ciil, acid, was
digested with restriction ~ r - I-'s ~ BamHI and Hind ,s. The DNA
fragment containing the tfdA gene was isolated from a low melting
1 l l r. agarose gel and ligated into vector plasmid pRO1727 which had
been digested with the saSne restriction ~ Ir -~- ~ The ligated DNA was
r~ d into P. aeruginosa pAolc and llA~l~r~ which contained the
desired insert were selected by plating for growth on TNA Cb500, followed
by replica plating to DNA Tc50 and TNA Cb500. Strains with the correct
phenotype of Tc sensitivity (due to insertional iuac~i~aiiull) and Cb resistancewere, I~ S~ further by isolation of the plasmid DNA and digestion with
BamHI and Hind.~. The digestcd plasmid DNA was analyzed by agarose gel
clC~.Llu~llul~ , to confirm that the desired fragment had been cloned. This
plasmid was designated pUCW101, Figure 2.
Expression of tfdA on plasmid pUCW101 in P. aen~giiAosa PAOlc was
conflrmed by growth of this strain in the presence of 2,4-D and detection of
DCP by HPLC.
Example 2. ~ . ' of PlasmidpUCW200.
The A. ~ binary vector pBI121 (Clontech T ~ Inc.,
Palo Alto, CA.; shown in Figure 4) was used to transfer and stably express
tfdA in sweetgum. pBI121 contains the ~ and l . A~ l IAI start
sequences of the "aulillu.. _l mosaic virus 35S promotor, and the
' t. . ".;., ~ , and pulyaLIl~laLiull sites and the ~ IIA' stop
codons from the nopaline synthase (NOS) gene. pBI121 also contains left and
right T-DNA borders (LB and RB) which are sequences used for
l, ~- ,- r .. ~ ;. ,.. of a plant cell (See Zambryski et al., Cell 56:193-201 (1989)
and Zambryski et al., AniA,u. Rev. Plant Physiol. & Plant Mol. Biol. 43:465-
WO 9!i/18862 2 ~ ~ g 9 8 ~ .. 'C - ~4 ~
-28-,
490 (1992)). DNA between these boundaries will be inseroed and tben
replicated with the plant's LIUUIII~:~VII~I DNA.
Subcloning tfdA into plasmid pBI121 creating plasmid pUCW200 was
~r~ as diagramed in Figure 3. Plasmid pUCW101 was digested
with restriction i ' ' Xbal and SacI. The DNA fragment containing
the tfdA gene was isolated from a low melting t~ Lul~ agarose gel and
ligaoed mto plasmid pBI121 which had been cut with the same enzymes. This
mixture was ~ r~" " ~ d into E. coli S17- 1, and L~ rvlll~u~ were selected
for growth on LB Km50 at 37C. T,,,,,~r,,..,.A,.I~ were picked, grown
overnight on identical media and analyzed for inserts by mini-prep analysis.
One strain which seemed to contain the proper insert was further . .l . ,.. I. .;, . ~l
by purifying the plasmid DNA as described above and confrrming the insert
by digestion with XbaI and SacI. This plasmid was designated pUCW200,
Figure 3. It contains the tfdA gene in the A~ iu ll binary vector
pBI121.
Expression of tfdA on plasmid pUCW200 was tesoed by first
l"...~rr,.;,~,~ the plasmid from E. coli S17-1 into P. pufida PPO300 by
.... j.. ~,.l.. andthengrowingP.putida(pUCW200)inthepresenceof2,4-D
and detecting DCP by HPLC.
PlasmidpUCW200wasmobilr~edfromE. coliS17-1 toA. tl/mofnoion~
LBA4404 as described above. The presence of plasmid pUCW200 in A.
t~ LBA4404 was confirmed by mini-prep analysis. Expression of
tfdA in this strain was corlfirmed by growing A. ~ LBA4404
(pUCW200) in the presence of 2,4-D and v ~ the ~rr~lm~ rirm of
DCP in the media by MPLC.
Example 3. 2,4-D Toxicity to Sweetgum.
The effect of 2,4-D on av~. shoot, L~j.... ,.11. ~1~ from sweetgum
leaf pieces was examined. The results of the 2,4-D toxicity oest are shown in
Table 4. Two 2,4-D . were tesoed with two sweetgum clones.
WO 95/18862 PCTIUS9SJ00284
~ 2179985 ~
-29-
The highest r~ .0 mg/L) resulted in leaf pieces which formed
callus tissue, while the lower ~.l ~..ll..l;~) - (0.1 mg/L) resulted in callus and
root formation on the leaf pieces. No shoots were obserYed on leaf pieces at
either .., - ~ 1 Thus, dL~ iLiuu~ shoot formation in the presence- of
0.1 mg/L 2,4-D may be used as an indicator of 2,4-D resistance.
TABLE 4
2,4-D To~cr~ TO SW~ETGUM
Sweetgum (2,4-D) # roots/leaf Callys
Clone mg/L piece
10204û 1.0 0.00 yes
2040 0.1 6.53 yes
2071 1.0 0.00 yes
2071 0.1 3.94 yes
Example 4. T/u~. of Sweetgum with pB1121.
A,~,u~aLlt,; .. ~ ri~n~-mediated ll.- r ~ was used to
transform sweetgum. Agrobactenum 7,.~ n~ L84404 has the ab~lity to
transfer Yector plasmid p8I121 (Figure 4) into a plant cell. Once inside the
plant cell, DNA between the right (R8) and left (L8) borders integrates
(randomly) into the plant's ullLUlllutvllle:~ It is then replicated as if it were a
part of the genome of the plant, and thus, when this cell divides and
Liir~., into a shoot, all the cells of this au~ iLiuu~ shoot contain the
gene ~ r~.. ... J into the origindl target cell.
The sweetgum ~ r~ ;" method used is outlined below:
1. The expanding leaves from a known sweetgum clone were
surface sterilize by first rinsing them with soapy water and then stirring them
in 10% bleach solution (in sterile water) for 10 minutes, followed by three
rinses (for 2 minutes each) with sterile distilled water.
WO 95/18862 I ~_I/IJ.. ,','~ 1
2~ 7~385
-30-
2. Each leaf was aseptically cut into small (5 to 10 mm) pieces.
Some of the pieces were placed on WPM 0.1/2.5 (Table ~). These pieces
acted as the ~ control.
3. A,~o~a~c~ r ~ LB4404, uull~dilLul~lr~lllidpBll21,
was grown overnight at 30C in 50 ml of L13 containing 50 mg/L kanamycin.
The next morning this culture was moculated into 500 ml of the identical
media, and the strain was grown for 4 hours at 30C. The cells were
harvested by ( r ~11 i r~-~ ,r ~ (5 min at 10,000 x g), washed once with LB, and
finally, ~ rd in 100 ml of fresh LB.
4. The leaf pieces were co-cultivated with Agro~acterium for 30
minutes at room t~ rlLulc. They were then blotted dry on sterile Whatman
No 3 filter paper and placed on WPM 0.1/2.5. The plates were sealed with
parafilm and incubated in the growth chamber.
5. After three days, the leaf pieces were transferred to WPM
15 0.112.5 which contairled 500 mg/L c.. ul,., li.,illi,. (Cb500). Carbenicillin, an
antibiotic, was used to kill the residual A~,ubGctc,
6. After two weeks, the non-control leaf pieces were transferred
to selective media. In this case, that was WPM 0.112.5 Cb500, kanamycin 75
mg/L (Km75). Plasmid pBI121 contains the kanamycin resistance gene,~PT-
II (Figure 4). Sweetgum is sensitive to kanamycm at 75 mg/L, and will not
regenerate in its presence. Therefore, adv.,.l~iLiuu~ shoots formed in the
presence of kanamycin may contain the resistance gene.
7. Shoots, Ic~, ' under selective pressure, were excised from
the leaf piece and transferred to WPM 0.01/2.0 Cb500, Km7s.
Twenty-four putatively ~ r~ " tl sweet gum shoots that grew on
WPM 0.01 mg/l NAA, 2.0 mg/l BA, Cb500, Km75 were obtained in an
following the above-described ~ r~-""~ ", protocol.
wo 9S/18862 ~ g 8 ~ PcTnJssslo
-31-
Example5. T,~ J~ " of SweetgumwithpUCW200.
A sweetgum h r ' ~ was performed with
Agrobacterium ~r ~ LBA4404, containing plasmid pUCW200, as
described above, except the selection (at step 6) was altered. Half of the leaf
pieces were placed on WPM 0.1/2.5 Cb500 with 0.1 mg/L 2,4-D, and the
other half were placed on the normal WPM 0.1/2.5 Cb500, Km75. The results
of this selection are shown rn Table 5.
TABLE 5
EFFECT OF 2,4-D SE1ECTION ON SWEETGUM TRANS~O~MATION Fh~U~._Y
10Clone Selection Media # shoots/# leaf Ratio (shoots/leaf
pieces piece)
2027 (control) Cb, control 73/18 4.06
2027 (pUCW200) Cb, 2,4-D 38/72 0.53
2027 (pUCW200) Cb, KTn 3/85 0.04
2040 (control) Cb, control 85/30 2.83
152040 (pUCW200) Cb, 2,4-D 77/66 1.17
2040 (pUCW200) Cb, i~n 16/49 0.33
A~ 5 Cb =, ~ " Km = icanamycin.
Example 6. ELISA Analysis of Tr~ ' Sweetgum Clones
An analytical method used to conflrm the transfer of selected genes
into sweetgum clones is am enzyme linked assay (ELISA) for
the detection of the NPTII protein encoded by the kanamycrn resistance gene
onplasmidPB1121. (NPTTIIELISAKit, PrimeReport3(2):3 (1991)). Plant
tissue (from 100 to 800 mg fresh weight) is placed in 3 ml of extraction buffer
(0.25 M Tris-HCI, pH 7.8, 0.1 mM 1' y~ .lhyl~.llru~lyl fluoride) and
i-.. ,.". ',.. -i using a Tekmar Tissuizer, model TR-10 (equipped with a
Iffi~,lu~ube), for the two pulses of 30 seconds each. An additional 2 ml of
WO95/18862 r~
217~8~i
-32-
extraction buffer is added and the cellular debris is removed by
.",ir"~ ;.", at 50,000 RPM for 20 minutes at 4C. The ~
is collected and 4 volumes of ice cold acetone are added, followed by
incubation at -20C for four hours, or up to overnight. The ~?ll . '
5 proteins are collected by .. . ~h i r,.~;,.1;,.,, at 4C, 10,000 RPM for 20 minutes.
The pellet is ~ lF tl in 1 ml of extract buffer. This sample is used in the
NPT-II ELISA kit purchased from S Prime - 3 Prime, Inc., Boulder, CO.
The ELISA method involves using an antibody specific for the
kanamycin resistance protein (neomycin ~ 1 " ~1 " ~`F -`¢, NPT-rl) to detect
this protem in the l,y~u~ llil, fraction of putative sweetgum L-dl~rullll,~llL~.The presence of this protein is an indication of ilt~ ru~ iul~ because the
gene encoding it is located on the Agrobaclenum I ,' LBA4404 vector
plasmid pB1121. This plasmid is transferred into the target plant cell by A.
I ~r7ri~n~ LBA4404 where it integrates mto the plant's genome and
expresses its genes. Since the genes on this plasmid are physically linked, the
presence of one of the gene products is evidence of the presence of the other
genes located on the plasmid. For example, presence of the NPT-II protein
in plant extracts i~dl~rvllll~,d with plasmid pUCW200, the 2,4-D resistmce
plasmid, which also conhins the kanamycin resistance gene, is pDSitive
evidence for the pr¢,sence of the 2,4-D resistance gene.
Example 7. ELISA Results of Sweetgum/pB1121 ~
Of the above-described 24 shoots obtained from the pBI121
Lldl~rulll..l~iull PYrPnmPnt only 15 survived further selection on kanamycin.
These 15 were tested for the presence of NPT-Ir by ELISA. Only one clone,
2040.2hr (pAG121), gave a positive result in this assay (Figure 5). The
ELISA results of Figure 5 are shown as the A40s nm vs the reciprocal of the
antigen dilution. The antigen m this case is the plant cell extract, which is
serially diluted down the microtiter plate. A positive reaction is one in which
the sample shows a decrease in absorbance m correlation to dilution of the
Wo 95/18862 ~ 4
21~B~
-33 -
amtigen. The minimum absorbance value considered to be positive, after
correction for b~,~,kælu ~, is an O.D.405 of 0.1. The udl~rullu~ uu
frequency in these ~ was 6.7%.
E~cample 8. ELISA Results of Sweetgum/pUCVV200 n ~ .
S Thel""~r",~ rl~4,~ 0fpUCW200isolatesselectedon2,4-D
were compared to those selected on kanamycin. ELISA for NPT-II was used
as a measure of L dl~rulll.~iul. frequency. Four isolates selected on 2,4-D
and four isolates selected on kanamycin were assayed. The results are shown
im Figure 6. All four of the 2,4-D selected clones (designated 2027
(pUCW200)-TA, -TC, -TD and -TE) were positiYe for NPT-II, indicating that
they are ~ However, none of the kanamycin selected clones
(designated 2027 (pUCW200)-KA, -KB, -KC, and -KD) were positive.
Plasmid pUCW200 differs from plasmid pBI121 in that pUCW200 has
the 2,4-D resistance gene substituted for the GUS gene of pBI121. Smce the
Ll~rullll~ usrng pBI121 with selection on kanamycin gave
a Lldl~rullll.liiull frequency of 6.5%, it is not surprising that none of the four
kandmycrn selected isolates assayed in this experiment were pQsitive.
However, these results establish the l"...~rll., -~;.... frequency of pUCW200
into sweetgum, with selection on 2,4-D, at 100%. It is clear that this
selection method is much better than the standard kanamycin selection.
The fact that 2,4-D selection did not yield any false positive isolates
rl~m( that it is an~ 3 selectable marker. Also, C.A~
have shown that sweetgum leaf pieces placed on 2,4-D containing media do
not regenerate adverltitious shoots. Since co-cultivated leaf pieces did
regenerate ad~,llliLiu~ shoots in the presence of 2,4-D and 100% of those
shoots were Lldl~rullu~.~, these shoots must be using the 2,4-D resistance gene
product to convert the 2,4-D in the media to 2,4-- -- uL~h~ l. This
indicates that these isolates are expressrng the resistance gene.
WO 95118862 PCTIUS95/00284
2179~38~i 34
., ~.
Example 9. Descr ption of r, ~ V Method.
Sweetgum were propagated by growing the ~d~ iLiuub shoots on
WPM containing 0.01 mg/L NAA and 2.0 mg/L BA. The original shoot
formed new shoots on this media. The new shoots were aseptically excised
S from the original shoot and grown; I~lJ ~ ly. This IJlU,U~ LiOII was
repeated until the required number of shoots were geneMted. These shoots
were then elongated by incubating them on WPM containing O.S mg/L BA.
Elongated shoots of greater than I cm in length were then incubated on root
induction media consisting of 1/3 strength WPM containing 0.1 mg/L IBA
(mdole-3-butyric acid). When roots began to form, the plantlets were
transferred to plug tMys containing a soilless mix consisting of equal parts
peat, perlite, and vermiculite amd incubated in 100% relative humidity until
new growth appeared. The plantlets were then t~ 1 to 10 cubic inch
leach tubes and grown in a greenhouse until they reach planting size. The
plants are then hardened off to outdoor conditions until dormant, removed
from tubes, and plamted at the prepared site. At a specified time before
and/or after planting, the site is spMyed with 2,4-D. The time for spraymg
the site and the ~ of 2,4-D used are readily ~IPtPrrninP~I _y one
skilled in the art. More specifically, these factors are (lPtPl'rninP~l by the
growth of plants other than the Ll~ Dru~ ,d plants at the site. The amount of
2,4-D spMyed on the site is am amount sufficient to 1) inhibit the growth of
non-~ r~ " . . d plants at the site and 2) allow the ~ " r " ", d plants to grow.
Example 10. I~ fv-, and n~ lv of Potato.
Potatoes (prefeMbly, Russet Burbank potatoes) i fvlu~d with the
~vAA gene are produced essentially as described by De Block, Theor. Appl.
Genet. 76.767-774 (1988) except that 1) the ~r, ,~ ,. media includes 0.1
mg/L 2,4-D and 2) kanamycin is not used in I All .; '~ with the tfdA gene.
wo 95118862 21~ 9 9 8 S F~ t
-35 -
Briefly, the proccdure described by De Block, supra, is as follows:
Plant materials
Sterile plants of cvs 'Bintje', 'Desiree', 'Berolina' or 'Russet Burbank'
are propagated in vitro by ~ r~ the top shoots or 1-cm-long pieces of
stem explants together with an auxillary bud to S1 medium. The shoots are
grown at 23C with a daylength of 16 h under 3000 lux light intensity (a
mixture of "iumilux white" ard "natura" from Osram, FRG).
Media
S1: B5 medium (Gamborg et al., EXp. Cell. Res. 50:151-158 (1968)) with 20
g/l sucrose and ;. I,l,i.. ~d with 150 mg/l CaC12 2H20, 0.4% agarose,
pH 5.8.
S2: MS medium (Mll~chi~e and Skoog, Plysiol. Plant 15:473-479 (1962))
with 30 g/l sucrose and ~ with 0.5 g/l MES pH 5.5, 20 g/l
mannitol.
S3: MS medium without sucrose and ~u~l ' ' with 200 mg/l glutamine,
0.5 g/l MES pH 5.7, 0.5 g/l PVP, 20 g/l mannitol, 20 g/l glucose, 40 mg/l
adenine-SO4, 0.5 % agarose, 1 mg/l trans-zeatin, 0.1 mg/l NAA, 1 g/l
lin or 0.5 g/l cPfnt~YimP
S4: S3 ~.,l,l,l .... t .I with 10 mg/l AgNO3.
S5: S3 medium without NAA and an one-half ........ ~ ;.". of antihiotirc
S6: S5 ~,~I,l,l. .". . ~. ~i with 10 mg/l AgNO3.
S7: S5 . r ' ~ with 0.01 mg/l GA3; 250 mg/l ~ ;11,-, or 150 mg/l
CPfOtl~jm~.
S8: S5 ! ,,' ' with 0.1 mg/l GA3 and 10 mg/l AgNO3; 250 mg/l
C~ Ib~.LUk~; IUI or 150 mg/l cl~ff~tS~YimP
Antibiotics, hormones, and AgNO3 were added after duLuul~vulg. Ag2S203
was added as 10 mg/l AgNO3 + 117 mg/l Na2S2O3 5H2O. Min A medium
WO 95/188~2 ,, P~ ,5.'~ ~
~17~98~ ~
-36-
is as described (Miller, J.H., F~ in molecular genetics. Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, NY (1972)).
T~..,.~f," , selection and ,.~,."~." .
Leaves (3-10 mm) from 3- to 4-week-old shoots are cut at the base.
The leaves are not further wounded. About 10 wounded leaves are floaoed
upside down on 10 ml of infection medium S2 contamed im a 9 cm Petri dish,
30 ~1 of Agrobacterium tr~ nri~15 LBA4404 containing plasmid pUCW200,
which had been grown in Luria broth medium to late log, is added. The
plaoes are then incubated at low light intensity (500 lux). After 2 days, the
leaves are washed with S2 medium containing I gtl r~r~rh~nir~illin or 0.5 g/l
cefotaxime, patted dry on filter paper, and placed upside down on medium S3
containing 0.1 mg/l 2,~D. The Petri dishes are sealed with tape that allows
gas diffusion ("urgo pore" tape, Urgo, Chenove, France) and are incubated
at high light intensity (3000 lux, a mixture of "lumilux white" and "natura"
from Osram, FRG). After 1 week, the leaves are transferred to fresh
medium. After 2 more weeks, many small calli are formed at tbe wounded
edges of the leaves, and the leaves are transferred to selective medium S5 for
'Berolina', 'Bintje' and 'Desiree', and S6 for 'Russet Burbank'. After 2-3
more weeks, those leaves with calli are transferred to medium S7 for
'Berolina', 'Bintje' and 'Desiree', and S8 for 'Russet Burbank'. From this
point on, 250-ml glass jars are used.
When using cefotaxime in the medium, it is important to tr,msfer the
leaves to fresh medium every 10 days. Afoer 2 weeks, the f~rst shoots (0.5
cm high) are isolaoed and tr,msferred to rooting medium S1 containing 100
mgll r~rb~ni~ in or rr~fo~Yi~ Normally, the shoots root m about 1 week.
When they do not root afoer 2 weeks, a tbin slice is cut away from the soem
base: most of these recut shoots root after 1 week. All shoots are harvesoed
within a period of 3 weeks. In order to avoid isolating identical shoots, two
shoots from the same or closely lirlked calli are never taken.
~ wo 9~118862 ~ 1 7 3 g 8 ~
-37-
When roots began to form! the plantlets are transferred to plug trays
containing a soilless mix consisting of equal parts peat, perlite, and
dit~, and incubated in 100% relative humidity until new growth
appears. The plantlets are then ~ to 10 cubic inch leach tubes and
grown im a greenhouse until they reach planting size. The plants are then
hardened off to outdoor conditions until dormant, removed from tubes, and
planted at the prepared site. At a specified time before and/or after planting,
the site is sprayed with 2,4-D. The time for spraying the site and the
of 2,4-D used are readily determined by one skilled in the art.
More specifically, these factors are dPtPnninPd by the growth of plants other
than the l . ,...~r~.. ,....l plants at the site. The amount of 2,4-D sprayed on the
site is an amount sufficient to 1) inhibit the growth of non-l ~ r ~- ., . d plants
at the site and 2) allow the ~"..,~r,."... ;1 plants to grr~w.
Example 11. Ih ~ . and r~ r ~ of Soy~ean.
Soybeans (preferably, Winchester soybeans) 1. ,. . ~r - . . . 1 with the ~d 9
gene are produced essentially as described by Hinchee et al., Bio/Technology
6:917-921 (1988) except that 1) the ~.... ,-~;.-.. media includes 0.1 mg/l 2,4-
D and 2) kanamycin is not used in I~ with the tfd~ gene.
Briefly, the procedure described by Hinchee et al., supra, is as
follows:
l~g_,.." Soybean (Glycine max (~.) Winchester) seedlings are
aseptically germinated 4-10 days on 0.8% Difco purified agar at 25C under
a 1' , ' of 16:8 (cool white fluorescent light at 40 ,uEn/s). After
washing in soapy water, rinsing in distilled water, and placing in 70% ethanol
for 2 min; seeds are transferred to 50% Chlorox for 10-13 minutes followed
by rinsmg 5 times with sterile distilled water. Seeds are soaked m an aqueous
Captan solution for 1 hour before transfer to the ~ . medium. After
~.. ;.. -~;.. cotyledons are removed and placed adaxial side down on B5BA
mediurn. B5BA medium is composed of B5 salts (Gamborg et al., Exp. Cell
W095~18862 P~ll-J~.. 'C- 1
` ~17998~ ~
-38-
Res.50:152-158(1968)),20mg/lsucrose,1.15mg/ll,~..L~ylad~ lc(BA),O.l
mg/l 2,4-D, and 8 g/l Difco purified agar, at pH 5.8 prior to au~ucL~villg.
Cotyledon explants are transferred to BsO medium (identical to B5BA but
without the BA) after 3-4 weeks. These and all other cultured explants are
5 maintained under the same ,.. Vilu~ll~ ~l conditions as the ~
seedlings. Cotyledon explants producing shoots are ~ cd every 4 weeks
onto fresh BSO medium. Elongating shoots are removed and placed on 1/2B50
medium (half the major and minor salts of B50 medium) in capped glass vials
or in sterile 50 ml disposable plastic centrifuge tubes. Plantlets (rooted
shoots) are moved to ~.. lllliLuli.a. in 2" pots after several new leaves are
produced. These plantlets are then placed in a plastic container which the lid
is gradually opened to harden them off prior to growing in the ~ hUU:IC;.
Plantlets that produce new leaves after hardening off are ~ d into soil
and grown in the greenhouse for flûwermg and seed set.
Cultivar screen. Soybean seeds are aseptically germinated for 5 days
on 0.8% Difco purified agar. Hypocotyls are cut into 5 mm segments, amd
imoculated (Horsch et al., Science 227:1229-1231 (1985)) with A. ~ ~'
LBA4404 containing plasmid pUCW200. The hypocotyl segments are co-
cultured With A~l L~IiaLlc~ ... for 2 days on 1/10 SH medium (1/10 the major
and mmor salts of SH (Schenk and T~ hrPn~t Can. J. Bo~. 50:199-204
(1972))) prior to being placed on MS NAA/K medium containing 500 mg/l
c~ub~.~illi l with or without 100 mg/l kanamycin. MS NAA/K medium is
composed of MS salts amd organics (Murashige and Skoog, P)ysiol. Plant.
15:473-497 (1962)) with 2.15 mg/l kinetin amd 4.68 mg/l napthalene acetic
acid (NAA). Each cultivar sample is l~ll I by 20-40 segments. The
hypocotyl segments remain on MS NAA/K for 4 weeks prior to scoring The
number of hypocotyls which produced callus is counted as well as the number
of i...l ~ calli per explamt.
Cotyledon expl~nt llun~fL Cotyledon explants of soybean
cultivar Winchester are prepared as for 1~ ~ " T"" ~r~ . with A.
t ~,;~t-i~tt~ LBA 4404 containing plasmid pUCW200 is carried out as
~ wo 95/18862 ~ 1 7 ~ 9 8 ~
-39-
described for the hypocotyls in the cultivar screen. Inoculated cotyledons are
cultured as described for cotyledon ~ except that the B5BA
medium contained 500 mg/l r~rl~Pnirillin and 100 mg/l r~-for~jm~ Elongated
shoots are cultured in B50 medmm.
When roots began to form, the plantlets are transferred to plug trays
containing a soilless mix consisting of equal parts peat, perlite, and
iuuli~e and incubated in 100% relative humidity until new growth
appears. The plantlets then ~ 1 to 10 cubic inch leach tubes and
grown in a greenhouse until they reach planting size. The plants are then
hardened off to outdoor conditions until dormant, removed from tubes, and
planted at the prepared site. At a specified time before and/or after planting,
the site is sprayed with 2,4-D. The time for spraying the site and the
c~,.,. . ~.1."1;.. of 2,4-D used are readily deter~nined by one slcilled in tbe art.
More ~,~,ir~.,all.y, these factors are ~' ' by the growth of plants other
15 than the l"."~r,.".. ~ plants at the site. The amount of 2,4-D sprayed on the
site is an amount sufficient to 1) ir~ubit the growth of non-LI~u~rul.llcd plants
at the site and 2) allow the l~, ~r-~ plamts to grow.
* * ~ *
All ~ mentioned ~ àlJuve are hereby ;"~ ".~ 1 by
reference in their entirety.
While the foregoing invention has been described in some detail for
purposes of clarity and L ~ ;l~, it will be a~ ' by one skilled m
the art from a reading of this disclosure that various changes in for~n and
- detail can be made without departing from the true scope of the invention and
appended ~lai~ns.
