Note: Descriptions are shown in the official language in which they were submitted.
Wo gs/3ooos 2 1 9 9 ~ 9 7 PCT/US95105366
n~rPTPI~ION
GENE~ REGULATING T~E RF~ OF
ZEA MAYS ~0 llATER DEFICIT
5 ~ U~ OF T~E lNVJ:.l.. l~N
A. Fi~l~ of the Inv~ntion
The present invention relates generally to the f ield
lO of maize growth and the response to varying conditions of
water avA;lAh;l;tyr the use of water, and resistance to
water deficit. The invention also c~nrP~nc the genes
involved in plant resistance to water def icit and
avA; lAh; l ;ty of water. More particularly, it concerns
15 the identif ication and various uses of genes involved in
the process of wilting during periods of drought.
B. Description o~ the Rel~td Art
Drought is a significant problem in agriculture
today. Over the last 40 years, drought accounted for 74%
of the total U. s . crop losses of corn (Agriculture,
1990). It is not only; Lan~ for plants to withstand
water deficit conditions, but also to grow and yield
under these conditions. Thus, it is important to
understand the flln~l Lal process by which plants
respond and adapt to water deficits. Identifying key
genes in this process will permit a ~PtA; 1 Pd
characterization of this process at the rPl l ~ and
whole plant level. This basic knowledge would allow
informed strategies to be developed which potentially
would enable plant:s to adapt to less than ideal
envi. - Lcll conditions.
An important agricultural problem is the protection
of plants from water deficits. When the rate of
transpiration exceeds that of absorption, a water deficit
Wo 95l30005 2 1 9 0 5 9 7 r~ J~66
occur6 and wilting symptoms appear. The responses of
plants to water deficits include leaf ch~AAin~, stomata
closure, leaf temperature increases, and leaf rolling and
wilting. Cells of a wilted leaf have reduced turgor
5 ~JLe5``U' a and plant growth is impaired. Metabolism is
also profoundly affected. General protein synthesis is
inhibited and significant increases in certain amino acid
pools, such as proline, become apparent (Barnett & Naylor
1966). During these water deficit periods,
10 photosynthetic rate decreases with the ultimate result of
1055 in yield (80yer, 1976). If carried to an extreme,
severe water def icits result in death of the plant .
Many studies have attempted to unravel the complex
15 array of physiological and morphological changes that
occur during water def icits . Abscisic acid (ABA) is
known to regulate stomata opening and perhaps signal
other r~ P~ (Milborrow, 1981). Comparison of
drought-resistant and drought-sen5itive lines of Zea mays
20 indicate that higher ABA levels are correlated with
resistance. In addition, ABA-in5ensitive mutants
(Koorneef et al., 1984; Finkelstein & Somerville 1990)
and A3A-def icient mutants (Koorneef et al ., 1982 ) of
ArAhiA~psic are prone to wilting. Other biochemical
25 differences have been implicated in drought tolerance.
Quaternary ~ illm, _ '-, such as glycine-betaine,
have been implicated (McCue & Hanson, 1990), as has
proline which is thought to play an important role in
e:yulation in plants (Delauney & Verma, 1993).
30 IIJL~:uv~:~, drought-re5istant maize lines have been shown
to contain thicker veins, larger epidermal cells, and a
greater vessel cross sPrti~n~l area per vascular bundle
than drought sensitive lines (Ristic & Cass, 1991).
These dif f erences at the cellular level may indicate that
35 resistant plant5 have a greater potential to transport
water Which may allow faster adaptation to, and IeCuv~y
from, water deficit5.
WO 95/30005 2 1 9 0 5 9 7 PCI/US95/0536C
-- 3 --
The mol~cular biology of r eyulation is less
understood. Genes involved in seed desiccation have been
implicatêd in this process. For instance, LEA (late
embryogenic abundant) and RAB (Eesponsive to absci6ic
5 acid) genes are induced by water stress or by exogenous
application of abscisic acid (ABA) (Dure et al., 1989;
Skriver & Mundy, 1990). The LEA and RAB proteins are
highly conserved and have been f ound in several plant
species. These proteins contain conserved amino acid
lO sequence motifs that are thought to form; ,h;rh;1 jr
alpha helical ~LLU~ U1eS which may have a functional role
in preventing cellular damage during desiccation.
Cis-acting seguence motifs, ABREs (ABA responsive
elements) have been identified in RAB promoters which are
15 involved in transcriptional activation in response to ABA
(Mundy et al., 1990). While these studies 6uggest an
association of specif ic genes with drought and
desiccation, further direct evidence is needed.
A genetic approach to dissecting developmental and
physiological process is beginning to revolutionize our
understanding of plant biology. Using mutagens, genes
required for plant processes can be identified and their
function inferred by pllello~y~e. A good example of this
application is the current understanding of f loral
deVPl~ L t made poscihlP by mutational analysis of
~loral homeotic genes in Ar~hi~ psi~ and Antirrhinum
(coen & ~l-yeL~ tz~ 1991; Coen, 1991). This general
approach is now being extended to studies of many
different plant processes ;nr~ ;ng enViL. ~ ~CI1 signal
transduction pathways.
A mutational approach has a distinct advantage over
conventional physiological and molecular studies. Rather
3 5 than a descriptive approach, mutations are used to def ine
genes that are functionally implicated in this process
based on mutant phenotypes. By relying on phenotypes,
, . _ _ . .. . _ .. _ _ _ . .
Woss/3ooos 21 9 0597 r~ Y_
characterizing genes or gene products that are simply
correlated with the process can be avoided. Noreover, by
characterizing mutant phenotypes at the physiological and
c~ ]lAr levels, it is possible select those that are
5 potentially more interesting. This provides a direct
link between genes and biological r~p~ at the
cellular and whole plant levels.
Three mutants have been identif ied in maize which
0 a LLate the wilt phenotype. Plants ~ ~ LLIItillg the
wilt phenotype show 108s of turgidity in leaf ti6sues,
Ar- -n;~a by drooping and t ;-~~ shrivelling of the
leaf . wil is a recessive mutant mapped to _IIL ~ ~ ~ 6L
and characterized by chronic wilting (Postlethwait &
15 Nelson, 1957). Leaves are not as cool as normal and
there is delayed differentiation of the metaxylem
vessels. W12 and Wi3 are dominant mutations that
demonstrate wilting of the top leaves under moisture and
Lu~ stress (Neuffer, 1989; Neuffer, 1990). W12
20 maps to ..l-~ ~~ 3. The Wi3 mutation is nonallelic to
Wi2 and probably llnl ;nk~d, but i8 not mapped.
Mutations, however, are only one A~t of an
approach to understanding wilt-related genes. Besides
25 the re~uisite physiological and biorhPm;c~l technique6,
cloned genes are needed for fllnrt;on~l studies of genes
and the products they encode. In complex genomes, such
as maize, chemical L~y~lls can be effective at defining
loci but these alleles have little value in obtaining
30 molecular probes. Nevertheless, both goals (mutant
alleles and cloned genes) can be achieved using
n~.l c ~.Iv~llLional mutagenesis terhn;qn~ such as gene
tagging methods. Maize, in particular, is well suited
for such studies given the dearth of well-characterized
35 ~ OCAh1~ element systems and genetic strategies for
gene tagging tChomet, 1994; Dellaporta & Moreno, 1994;
Cone, 1994). TrAn~rOSAhle ~ L~i have enabled the
wo gs/3ooos 2 1 9 0 5 9 7 r~ c~S66
isolation and characterization of numerous genes in
several biosynthetic pathways, including, anthocyanins
(Fedoroff et al., 1984; Cone et al., 1986; Dellaporta et
al., 1988; O'Reilly et al., 1985; Wienand et al., 1986;
5 Ml-T.~ ghl ;n & Walbot, 1987), carotenoids (Buckner et al.,
1990), caLl,o~lydL~te metabolism (Sullivan et al., 1991),
and the regulation seed storage protein synthesis
(Schmidt et al., 1987) to name a few. Moreover, genes
involved in disease resistance (Johal & Briggs, 1992) and
10 in plant development (Hake et al., 1989; NartiPncs~n et
al ., 1989; DeLong et al ., 1993 ) have also been isolated
by gene tagging strategie6 in maize.
Maize is an ideal organism for these studies for
15 many reasons. Maise is a model genetic system for plant
studies because of well developed technologies for formal
genetic and molecular analyses (Freeling & Walbot, 1994).
Because maize is an important agricultural plant, the
results obtained will have direct relevance to an
20 important agricultural problem.
The identif ication and characterization of plants
that are resistance to water deprivation has long been
sought . However, the identif ication of genes that are
25 directly involved in resistance to water 6tress has been
lacking. The isolation and characterization of maize
genes that provide for an i ~ v~d resistance to water
stress have the potential for the long term i uv,
in and sustAin~hi l ity of agriculture world-wide.
~lrQlARy OF THE
The present invention, in a general and overall
35 sense, c ~ plant genes, and particularly maize
genes, that are involved in the maintenance of water
relations in a plant. Such genes are herein termed wilt
wo 9s/3000s r ~ ~ . C~J6G
2 1 90597
genes (wilt), due to the fact that disruption of these
genes impairs a plant ' 5 normal balance of turgor
potential and hence causes the plant to wilt.
A wilt gene in accordance with the invention is a
gene that, when present within a plant, allows the plant
to maintain proper internal water balance under normal
conditions or under low water av~ h; l; ty or contributes
to such a response and a drought-resistance phenotype.
It i5 envisioned that a wilt gene will encode one or more
proteins or polypeptides that function in the r- ` -n; I
of drought-resistance or that stimulate the production
of, or regulate, other proteins involved in drought-
resistance. A ready means to assess whether a gene or
cDNA falls within the wilt gene group is to disrupt,
inactivate or otherwise mutate the gene, and to assess
whether plants bearing such a disrupted gene wilt under
normal water level conditions. The precise - -hAn;, - by
which a wilt gene achieves its plle,luLy~ic effect are not
relevant to the practice of the invention, given that the
isolation and various uses of are wilt genes is disclosed
herein .
The invention provides, in certain ~ Ls,
purified DNA segments that include a maize wilt gene. As
used herein, the term "DNA segment" refers to a DNA
molecule t_at has been isolated free of total genomic DNA
of a given plant species, particularly maize. Therefore,
a purified maize wilt gene DNA segment refers to a DNA
segment that contains maize wilt gene coding sequences
yet is isolated away from, or purified îree from, total
genomic DNA of Zea mays . Tnrl ~ within the term "DNA
segment", are DNA segments and smaller rL ts of such
segments, and also ~ n~nt vectors, ;nrlll-l;n~, for
example, plasmids, cosmids, phage, viruses, and the like.
WO 95l30005 2 1 9 0 5 ~ 7 P~
-- 7 --
The term "gene" iB used herein for simplicity to
refer to DNA coding unit that encodes a functional
protein, polypeptide or peptide, which coding unit is
isolated substantially away from other genes or protein
5 ~nroAin~ sequences. As will be understood by those in
the art, this functional term includes both genomic
sequences and cDNA sequences. "Isolated substantially
away from other coding sequences" means that the gene of
interest, in this case a wilt gene, forms the significant
10 part of the coding region of the DNA segment, and that
the DNA segment does not contain large portions ( i . e.,
around 40-50 kb or larger) of naturally-occurring coding
DNA, such as large ~ 1 rL _ Ls or other
functional genes or cDNA coding regions. Of course, this
15 refers to the DNA segment as originally isolated, and
does not exclude genes or coding regions later added to
the segment by the hand of man.
A particular example of a DNA segment of the
invention is that termed wilt-1256. Wilt-1256 is a 18
kilobase Sal I rI ~ ~ isolatable by the methods
Ai c~ c~A herein in Examples I through III.
R~ in:-nt vectors that include a wilt gene DNA
25 segment form important further aspects of the present
invention. Particularly useful vectors are contemplated
to be those vectors in which the wilt DNA segment is
positioned under the control of a promoter. In preferred
' i r Ls, the promoter will be one that is operable in
30 maize plants. As used herein, the term "operable in
maize plants" is used to describe a promoter that is
capable of directing the expression of a gene or
LL~ils~;Liption unit in a maize cell or maize plant.
However, the use of other yL Lt:Lrj not operable in maize
35 is also contemplated, such as may be employed in
directing the expression of a recombinant protein in a
bacterial host cell, as is commonly performed in the art.
WO 95130005 2 1 9 0 5 9 7 PCT111595/05366
-- 8 --
A pref erred Pmho~i i r L of the present invention is
the use of a promoter that is operable in maize plants,
with the promoter that is naturally associated with a
wilt gene in ZQa mays being particularly preferred. One
5 may obtain such a promoter by isolating the 5 ' non-coding
se~ut .Ices located upstream of a transcribed coding
segment or exon, for example, using recombinant cloning
and/or PCR technology, in t ~nnPctir~n with the
compositions disclosed herein, such as the 18 kb Sal I
10 fragment including wilt-1256. Also Pn~ -cl=pd within
naturally occurring promoters are regions such as
pnhAnrprsl F:; l Pn~prs and transcriptional activation
sequences that may also be used, and are Pr ~sPd by
the present invention .
In other ~ 5, it is contemplated that
certain advantages will be gained by positioning the
coding DNA 6egment under the control of a recombinant, or
heterologous, promoter. As used herein, a r~ ' inAnt or
20 heterologous promoter is intended to refer to a promoter
that is not normally associated with a wilt gene in its
natural environment. Such promoters may include
promoters normally associated with other maize genes,
and/or promoters isolated from any other bacterial,
25 viral, eukaryotic, or even a 1 ;An cell.
Naturally, it will be i L~IlL to employ a promoter
that effectively directs the expression of the DNA
segment in the cell type chosen f or expression . In terms
30 of expression in maize cells or plants, one may mention,
by way of example, promoters such as the Cauliflower
Mosaic Virus 35S, rice actin, rbcS, ~-tubulin and ocs
promoters. The use of promoter and cell type
combinations for protein expression is generally known to
35 those of skill in the art of molecular biology, for
example, see Sambrook et al. (1989). The promoters
employed may be constitutive, or in~ hlP, and can be
. . . _ _ _ _ _ _ _ _ _ _ _ _ _
WO gs/3ooo5 2 1 9 0 5 9 7 PCT/US9S/05366
used under the appropriate conditions to direct high
level expre66ion of the introduced DNA 6egment, such as
i6 advantageou6 for inducing an increased re6istance to
water deprivation in a plant or simply to provide high
5 level production of ~ in:~nt protein.
Further ~ Ls of the invention concern methods
for preparing a wilt gene DNA segment. Certain preferred
methods are tho6e involving a technique known in the art
10 a6 tr;~nRpo6nn tagging. To obtain a wflt gene DNA segment
in this manner one would generally, first, obtain a
c~llectinn of maize plant6 bearing trRn~rn6sn induced
mutation6 and 6creen the group of plant6 to identify one
or more plants that eYhibit a wilt phel~c,Ly~e. The
15 process of obtaining or preparing a collection of maize
plants bearing LL~ OS~ induced mutations involved
identifying plants that harbor an active trRn~posnn
system. With some trAn~posnn 6ystems (i.e., activator)
it i5 po6sible to select individual plants in which the
20 LL,.- -I,nRn~ has moved to different locations in the genome
(Chomet, 1994; ~-~llRrnrta & Moreno, 1994, Cone, 1994,
ins~L~L-ted herein by reference~. Such plants are then
either selfed or crossed to plant6 carrying a previou61y
identified wilt mutation. The selfed progeny or te6t
25 crossed progeny are then screened for the wilt phenotype.
Plants that exhibit the wi 7 t phenotype are readily
identifiable by simply growing the collection, or
population, of plants under normal water levels and
30 detecting tho6e plant6 that exhibit decreased turgor,
l.e., vi6ibly wilt, leaf roll, or droop, under such
conditions or by identifying those plants that show
siqnif icantly decreased turgor in comparison to other
plants in the collection. The precise - ~ni, by
35 which the mutations that affect water balance achieves
the identified phenc Ly~ic effects are not relevant to the
practice of the invention, it is clear, however, that
WO 95/30005 2 1 9 0 5 9 7 PCT/US95/05366
-- 10 --
leaf roll precedes wilting. Leaf roll was effective in
providing an early indication of wilting, for example, as
early as the third leaf 6tage.
After identifying one or more plants that exhibit a
wilt phenotype one would next prepare a genomic library
from such a plant or plant6. That is, one would obtain
DNA from the nuclei of cells from such a plant and
f ormulate the DNA so that it cont~ ins smaller, distinct
pieces of DNA that can identified as distinct from other
pieces - using any one of a variety of ~LLUVLUL~1 and
functional characteri6tics. The library of DNA thus
formed would than be screened to identify a genomic clone
that comprises the wilt gene in as~ociation with a
trAncr~C~n. This screening proces~ will generally
involve using a probe directed to a LL..11~-L~OC~II sequence
to identify the LL"~ J~C~JI- in combination with the wilt
gene. A number of trA~ os~ c that induce mutations may
be used with the present invention, with the Ac and Ds
20 trAncroconC being particularly preferred.
Further advantageou6 ~L UCedUL :s that may be used in
connection with the preparation of wilt gene DNA s~ L6
are those that concern isolating a wilt gene cDNA clone.
25 Such methods generally comprise identifying a DNA
6e~ e that f lanks the trAncrocAhl~ element located
within the genomic clone f irst isolated and using the
flanking sequPn~e in another round of molecular
screening. In particular, the flanking sequence would be
30 employed to screen a cDNA library prepared from a wild
type plant and, by identifying SeSrl~nr~C that hybridize,
to thus identify one or cDNA clones that include a wilt
gene. Suitable methods for preparing and screening cDNA
libraries will be generally known to those of skill in
35 the art of molecular biology in light of the present
;cclosllre and published references such as, for example,
Sambrook et al. (1989; incvL~vLelted herein by reference).
WO 95130005 2 1 9 0 5 9 7 ~ .'^'7~
Yet further aspects of the present invention concern
plants, particularly maize plants, that have been stably
transformed with a purified wilt gene DNA segment, such
as the wilt-1256 18 kb DNA segment. In preferred
5 PmhO~li L-;, the stably transformed plants will include a
recombinant vector that comprises a wilt gene that is
positioned under the control of a promoter that is
operable in maize plants and will thus direct the
expression of the wil t gene in the plant . Exemplary
10 promoters that may be used in this regard include the
Cauliflower Mosaic Virus 355, rice actin, rbcS, ~-tubulin
and oc~ promoters.
The present invention thus also provides methods for
15 preparing plants, particularly maize plants, that have
increased turgor, i.e., exhibit an increased resistance
to water deprivation or drought. Such methods involve
stably introducing a purified wilt gene DNA segment, such
as the 18 kb wil t-1256 DNA segment, into the genome of a
20 host plant in a manner effective to result in expression
of the wil t gene . In general, the gene will be
introduced in the form of a ~ inAnt vector and will
be under the control of a promoter operable in maize
plants. Any means of introducing the DNA segment into
25 the host plant may be employed ;nrll-Ain~, for example,
ele~ L ~,yoL~tion and microprojectile bombardment.
Another : ' '; L of the present invention is a
method for the selectivc breeding of plants that are
30 resistant to water deprivation. The method ;nclllAPC~
generally, obtaining a collect;~-n of maize plants and
screening for increased expression, copy number, or
message stability of a wil t gene DNA segment . Those
isolates having a favorable genotype, that i5 an increase
35 in expression, copy number or message stability are
tested for increased resistAnce to water deprivation
followed by breeding of those isolates having increased
_ _ . . . . . _ _ _ _ _ _ _ _ _
Wo gsl3000s r~l,.a.~ s~66
2 1 90597 12 -
resistance to water deprivation. The progeny are then
screened with a wilt gene DNA 6egment in accordance with
the present invention, and the f avorable genotypes are
tested resistance to water deprivation and once again
5 bred.
The DNA segments of the present invention ~n- -r:5
biologically functional equivalent wilt genes and their
C~LL.~ ;n~ proteins and peptides. Such sequences may
10 arise as a consequence of codon r~AIlnflAn~ry and functional
equivalency that are known to occur naturally within
nucleic acid sequences and the proteins thu6 encoded.
Alternatively, functionally equivalent proteins or
peptide6 may be created via the application of
15 1~ ' in~nt DNA technology, in which changes in the
protein ::.LLU~:LUL~ may be engineered, based on
ronci~F-rationS of the properties of the amino acids being
exchanged. Changes ~l~cign~l by man may be introduced
through the application of site-directed mutagenesis
20 techniques, e.g., to introduce i ~ L~ to the
stability or specif ic expression of the resultant protein
or to test wilt mutants in order to examine the
- ' An; crc underlying wilting activity at the molecular
level .
T~ L~E.e_Kl~ OF T~E D.~aWING~
The following drawings form part of the present
30 sp~r;f;cAtion and are ;nrl~ to further ~ LL~te
certain aspects of the present invention. The invention
may be better understood by ref erence to one or more of
these drawings in combination wit~ the detailed
description of specif ic ~ - - i r ~S presented herein .
FIG. 1. Restriction enzyme digestion map of A5Sal clone
containing the Ac trAncros~hl e element inserted in the
wo 95t3000s PCTlUsgs/os366
2 1 90597
-- 13 --
wilt-1256 gene. The activator is indicated by the
crosshatched line. The hybridization probes for (0.9H/E)
and flanking genomic DNA probes (0 . 5 and 0 . 4B/S) are
shown. A portion of the DNA sequence of the genomic DNA
5 flanking the Ac insertion in wilt-1256 has been
det~rminod. This sequence is a 247 base pair open
reading frame which has shown homology to 2 rice cDNA
sequences of unknown function. S=SalI, B=BamHI, E=EcoRI,
H=HindIII, P=PstI.
DF!T~II.ED IJ~ OF T~E ~ h~J l M ~ A
A. 8cr~ening for Ac-induced mut~tions
TrAncpoc~n tagging has several benefits over
conventional gene cloning strategies. No prior knowledge
of the gene product or even previous identif ication of
the gene is a prerequisite for trAncps6-n tagging. It
20 can be used to clone and study genes whose products are
rarely ehl~Lc:SSed. Insertional mutations often completely
or partially block gene function, resulting in phenotypic
alterations. This phe~ y~ic alteration often implicates
the gene or its product in a particular developmental or
25 bio~ h~ A l pathway. TrAncpo~on tagging strategies can
operate efficiently in organisms with large genomes, even
with great amounts of repetitive DNA. Genes for disease
resistance, nutritional iualities, and dev~ Lal
E~ vCeDDeS can thus be tagged and cloned. Nutant alleles
30 can also serve as the basis for saturation mutagenesi6
and fine structure analysis of complex loci.
`.n essential feature for de-~eloping an efficient
LL A I '-~JOS~ tagging system is the ability to ef f iciently
35 recover transposition events. Two genetic features of Ac
transposition were exploited to develop an efficient
Lc:~;UVe:Ly strategy. First, because ~LAIlD~osition is
wo 9s/3ooo~ r~l,u. ,~
2190597 14_ 1--
coupled to DNA replication, tr~nC~c~o~ Ac elements are
~ecuv~L,ad within cell lineages that contain an both empty
and filled donor site (see Chen et al., l990, 1992~. The
inventors ' strategy relies on detecting the lineage
5 containing the f illed donor site and tr-Ac element . This
lineage contains two Ac element6 (the original donor Ac
and the tr2~n~pofied Ac element). The increase in Ac copy
number results in a dosage effect - transposition is
dev~l~ L -t~llly delayed. We exploited this second
10 feature (Ac dosage effect) by using a Ds-induced reporter
allele [r-sc:m3). In practice, any Ds reporter allele
will suffice, however. By monitoring the developmental
pattern of Ds transposition (e.g. early verses late), we
have the ability to select progeny that contain more than
15 one Ac. Since the parent line carries only one Ac (donor
element), progeny kernels showing a late transposition
pattern will carry not only the original Ac element but
also a tri~ncposo~ element.
In these studies, Ac elements located at two
~ L~ ~ 1 positions were used to as6ess the folcih;l;ty
of our strategy to recover LL ana~ositions . The donor Ac
elements inserted into the P gene on ~ r -- - lS and
the liguleless-1 gene on ~ L~ ~~ 2 were employed for
these purposes. Females h~ _Y~UUS for the Ac-induced
mutations were crossed to males ~ YYUUS for the Ds
reporter allele, r-sc:m3. All Fl progeny are expected to
receive a single Ac (donor element) and the r-sc:m3
reporter allele except in cases where transposition of Ac
had oc~iuLL~d. These kernels of the wilt-1270 mutation
isolated from Ac-Ds tagging lines show the expected
coarse variegated aleurone pattern typical of L n~lo7pD~o
that contain two maternally-transmitted Ac ~ . In
cases where ~c transposition had oc~;uLL~ d, two types of
Fl kernels are ~To~-t~d: i) in cases where the donor
element was lost, as in the wilt-lZ56 mutation isolated
from Ac-Ds tagging lines, a colorless aleurone phenotype
... _ .. _ . _ .. . . . , , _ _ _ _ _ _ _ _ _ _ _ _ _ .
WO 95/3000~ 2 ~ ~ 0 5 q 7 r~l~u. _ C~66
-- 15 --
is observed, ii) in cases where the donor and tr~ncpnced
element is ~ecuvl:Led, as in the wilt-1269 mutation
isolated from Ac-Ds tagging lines, the increase in Ac
dosage results in very late transactivation of the r-
5 sc:m3 reporter allele which gives a near colorless orvery f inely spotted aleurone . The inventors recovered
approximately 10, 000 kernels containing tr-Ac elements
(M1 kernels) by using the Ds reporter system to recover
high dose Ac kernel progeny. The efficiency of recover,
10 based on F2 and testcross data, was greater than 75~. In
control populations (coarse variegated sib kernels) the
efficiency of trAncpns~Dd Ac recover was less than 10%.
This indicates that the genetic strategy for recovering
tr~ncpnced Ac elements greatly improves the frequency of
15 recovering tr~ncposPd elements over non-cPl ~ct~Dd methods.
The clear advantage of this method over other schemes i6
that this strategy can be used with any Ac element in the
maize genome regardless of its U11L~ ~ 1 position.
The M1 kernel population was screened four times.
First, M1 kernel populations and control Cibl i n~c were
field grown and screened for dominant mutations.
Mutations involved in the f lowering process such as early
or late flowering phenotypes, floral morphological
transformations, male and female sterility and mutations
affecting vegetative characters such as ligule and leaf
development, or tillering were sought. All M1 and
control plant6 were self-pollinated to generate M2 and F2
control progeny populations. Mutants were also
testcrossed to Ac tester stocks to identify Ac-linked
phenotypes .
The second screen was for mu~ ions affecting seed
characteristics such as Dn-lncpPrm ~ orphology and
defective kernels. This screen was done directly on the
N2 and F2 ears. For instance, mutants in starch,
protein, and carotenoid y~ y~ were ~lDtect~ in
wo gsl30005 2 1 9 0 5 9 7 r~
endosperm cells. From this screen, mutations affecting
kernel development (defective and embryoless kernels)
were Ltc-~vtLed. Several mutations affecting starch
bio6ynthesis (shrunken (sh) and brittle ~bt) ) were also
5 recovered.
The third screen involved the growing the f irst seed
set in sand benches to score for percent germination and
aberrant sosrll in7 phenotypes such as chlorophyll
10 pigmentation changes and disease susceptibility. Two
sets of twenty kernels were sPl PCt'~l randomly from each
M2 and F2 family. From this screen many mutations
affecting see~l;"7 development (lethals, pale-greens,
albinos, yellow-greens, dwarfs, high chlorophyll
15 fluorescence, disease lesion mimics, and the like) were
recovered .
The fourth screen involved growing the second seed
set in field plots and scoring for segregation of
2 0 vegetative and f loral phenotypes . From this screen
mutations affecting leaf morphology tliguleless, wilts,
knotted, etc. ) and influL~ ~ctl.~e morphology ltAcs~,
were recovered.
Selected mutations were studied by genetic and
molecular analysis. For example, some Ml and ~q2 mutants
were testcrossed to a set of Ac tester lines and a
genetic segregation analysis between the mutant phenotype
and the trA"cpos~d Ac element was det~mins~l. These
tests identified which mutants were seyl~yc-ting with
tr-Ac activity and putatively Ac-induced. Southern blot
analysis was performed on sPI~r~d mutant families to
identify tr-Ac elements for cloning purposes.
WO 95/3000S r~
21 9~597
-- 17 --
B. ~r~n~:5 Tagging with Mutator
TrAncpocAhl P elements have been useful in the
identification and cloning of genes that were previously
5 inA~rPqsible by other cloning methods. In maize, three
LL ~ 0S~h1 e element systems have been utilized f or
~L. l ~l,os -" tagging: Activator/nicsQri~qtion~ Suppressor-
mutator t~nh~nrPr/~nhibitor), and Robertson's Nutator.
Each system has inherent advantages and disadvantages, and
10 the choice of system depends on the tagging approach
taken, the genomic position and the expression of the
gene sought, as well a6 the stocks available for tagging.
The Mutator trAncps6Ahle element family was
15 originally identified in lines that exhibited an
.-c-l ll ly high frequency of forward mutation tRobertson,
1978). Extensive genetic and molecular analyses have
~ L~ted that the increase in mutation frequency is
caused by a family of tr7-ncposAhle elements, designated
20 Mutator (Mu) elements. The Mutator system consists of
more than eight different classes of Mu transposable
element6 each of which can be found in multiple copies.
Each element class is def ined by a unique internal
se~u~ e flanked by inverted repeats about 200 bp long
25 common to all Mu elements. GPrminAl transposition (and
presumably forward mutation) and somatic excision of the
non-aut~n~ Mu elements are under the control of the
autonl c MuR1 element (Chomet et al., 1991), now
designated MUDR-l. Other Mu Pl' ' F have bee~ cloned
30 that are similar or identical to MuDR-1 in sequence;
these were called MuA2 (Qin et al., 1991) an~ Mu9
(Hershberger et al., l991), and are now called MuDR
LL~ LJ061~C. Lines harboring the aut-- _ element(s)
are referred to as Active Mutator lines. The Mu system
35 has been useful in cloning a number of genes, including
al (O'Reilly et al., 1985), bz2 (r'-r.All~hl ;n & Walbot,
wo 95/30005 ` 2 1 q 0 5 9 7 r~llL~ c~66
-- 18 --
1987), vpl (McCarty et al., 1989), hcflo6 (Martienssen
et al., 1989), yl (Buckner et al., 1990), and hml.
C . Th~ Mutator ~ystem f or T~gging
One of tAe advantages o~ Mu over the Ac/Ds and the
Spm familie6 lies in its non-lor~ mutagenic action.
Ac and Spm often transpose to linked regions of the
~ L~ ~~ ~ (Greenblatt & Brink, 1962; Dooner & B
1989; Nowick & Peterson, 1981). Mu lines exAibit an
increase in mutation frequency for all loci r~yAm~npd
(Robertson, 1978; 1983). This does not prove that Mu
elements readily transpose to unliked sites although
recent evidence suggests this is the case (Lioch, Choret
& Freeling, 1995).
D. T~rgete~l ~n~ ~ La~ ~,Le~S Approachss to Tagging
Two approaches to obtain mutations can be pursued
~r~r~n-l~nt on the needs of the investigator. With the
targeted (or directed) method the goal is to recover
insertions in a previously identif ied locus . TAis method
~L~ a mutant alleged of the target locus is
available in a tester line. In such a case, homozygous
wild-type Mutator stocks for the target gene are crossed
to homozygous tester lines. The F~ progeny are then
screened for tAe mutant phenotype. The reported
frequency of mutation for such an approach varies from
10~ to 10~ (Robertson, 1985; Walbot et al., 1986;
Patterson et al., 1991; Brown et al., 1989). Therefore,
if po~hl~-, at least 105 gametes should be screened for a
directed tagging study. Furthermore, it is important to
have prior knowledge of the spontaneous mutation rate f or
the targeted locus. Some loci can be highly unstable in
the absence of a trAn~p~C~hl e element system (Pryor,
1987; Stadler, 1948).
WO 95/30005 2 1 9 ~ 5 9 7 PCT/U595105366
.
-- 19 --
In the case of directed tagging of dominant loci, it
is possible to "knock out" the dominant mutant allele
with ~u (Hake, lg92). In this case, a ~utator line
h~ - yu,uu5 for the dominant allele is crossed (as a
female) to a wild-type tester. The Fi is screened for
phenotypically wild-type plants. This pLe~ a
d~-leti~n het~lu~yyu~e for the given locus is
phc~u~yl ically wild-type. Such an assumption may not
always be the case and tests should be undertaken to
determine the phenotype of the hemizygote before
procee~l i n~ with the screening of the F~ population.
The nontargeted approach allows the investigator to
identify new, uncharacterized mutations as well as to
identify new mutations with previously characterized
phenotypes. Active Nutator lines should be crossed by a
non-Mutator line (inbred) or a tester line carrying a Mu-
induced unstable marker allele. (See non-aut~ n, c
genetlc marker, below). The Fl seed is selfed to produce
F2 population6; 20-40 seeds from each F2 ear are then
screened for the mutant pllellu~y~e(s). In this case,
lethal or sterile phenotypes can be identified as
homozygotes and ~e:uuv~:red as hete:Lu~yyu~es in the
population .
E. Monitoring Mut~tor Activity
Bef ore making the f irst cross to generate either an
Fl population or the selfed populations, it is best to
~l~t~rmin~ which plants in the Nutator stock carry Nutator
activity and are mutagenic. This information can be
nh~ :-in~<~ wholly or partially by a number of tests
outlined below. Because each assay measures a different
aspect of Mu transposition, each test does not
n~ Arily match results from another test for Nutator
activity .
woss/300o~ 2190597 r~l"~ el-
-- 20 --
F . Robertson ' 8 Mutator T~st
Robertson devised an assay for Nutator activity that
give an estimate of the forward mutation frequency of
5 Mutator plants (Robertson, 1978). The test is performed
by selfing and crossing the Mutator plant as a male to a
hybrid non-Mutator line to produce Fl seed. The second
ear on the non-Mutator plant is selfed as well. F~ seed,
from plants which did not segregate for a visible mutant
10 (as tlPtorm;nPd by the parental selfs~, are sown and
plants are selfed to produce F2 ears. Kernels from these
F2 ears are planted in a sand bench and observed for new
see~ll ing traits (i.e., albino, yellow, yellow-green,
etc. ) . With active Nutator lines, Robertson reported
15 approximately 10% of the F2 familie6 exhibited a new
ceP-ll;n~ mutation ~1980).
G. r ~ ~ G~n~ltic M~rlc~r~)
Non-aut,.r c Mu insertion alleles report on the
presence or absence of MuDR, the regulator element
(Chomet et al., 1991). The presence or absence of
spotting of a Mu-induced insertion allele reports only on
the somatic reversion of one Mu element (such as Mul as
bzlmum9, or Nul at al-mum2 ~ . This is in contrast to the
Robertson test, which can measure th forward mutation or
insertion of diverse Mu into many different loci. Since
these two assays do not measure the same event, use of
one assay is not always a substitute for another.
However, there is a correlation between an increase in
forward mutation rate with lines containing multiple
regulator elements that exhibit a high fre~auency of
spotting. Plants that segregate for one MuDR-1 (position
1) usually do not exhibit a high forward mutation rate
(RobeLLz,u., & Stinard, 1989). When using a mutable Mu
allele as a marker for Mu activity, it is best to
propagate the high spotting pattern. This will likely
WO 95/30005 2 1 9 0 5 9 7 ~ PCTIIJS95/0536G
-- 21 --
select for an increased number of MuDR-l elements in the
stock (Chomet et al., 1991).
. r~-le_ l-~r N~rlc~r~
A number of studies have d LL~ted a correlation
between loss of Nutator activity and methylation of Mul
at a number of methylation-sensitive restriction enzyme
sites ;nr~ A;n~ Hinfl (~'h:~n~llPr ~ Walbot, 1986;
10 Bennetzen, 1987). ~infl sites are located in the ends of
Mul and produce a 1. 3-kb fL t upon cleavage . To
determine the status of Mul methylation and the
associated Mutator activity, the DNA from Mu lines is
cleaved with ~{infl and hybridized to a Mul internal probe
15 by Southern blot analysis. The ~L~ ' ;n~n/~e of a 1.3-kb
fragment indicates cleavage, whereas the presence of a
ladder of fragments (and a reduced amount or lack of a
1. 3-kb fragment) indicates Hinfl sites in the ends of Mul
are methylated and Nu activity is prob~bly absent. It0 should be noted that this correlation is not absolute
t~Pn, 1987; Bennetzen et al., 1988).
The generation of unique Mul ' logouc rL, ~ Ls ~
not detected in parent plants, has been correlated with
25 Mutator activity (Alleman & Freeling, 1986; Bennetzen
et al., 1987). To detect new Mul P1~ LL, DNA from
parent and progeny of a Mutator lineage should be cleaved
with an enzyme that does not cut within Mul such as
~coRl, BamHl, or ~lindIII. Southern blot analysis with
30 the Mul probe will reveal a series of Mul-hybridizing
rL Ls that represent Mul at unique genomic locations.
Nul fragments not found int he parent most likely
Lt:~Lt:S~L new insertion events, indicating the presence
of Mutator activity.
wo ss/3000~ 2 1 9 0 5 9 7 P~l/L.,.~.
I. Crosses with Newly Arisen ~utants
Once a mutant i5 identified, it is useful to have
crosse6 to a number of different lines. Therefore, it i6
wise to plant a number of tester lines along with the
population to be screened.
1. If possible, selfing, to recover h~ yuLes, is
important. A h~ yuu6 stock can be utilized to
6creen for g-~rm;n;ll reversion events; revertants are
a u6eful tool in molecular identification of a
tagged locus.
2. Outcrossing the mutation to a few different non-
Nutator (inbred) lines will be useful for subsequent
---lec~ r analyses. Identifying the original,
tagged locus of interest will be i L~..L and can
be ~ hD~l because RFLPs associated with the
locus can be associated within a given line.
Crossing to di~ferent lines will also allow the
production of F2 populations segregating for the
mutant allele. These populations are n~rD~ Iry for
subsequent molecular analyses. Furthermore,
introduction of the mutant allele into a number of
different lines will also in6ure expres6ion and
~ubsequent Lt:VvVe:~y of the mutant in later
generations because expressivity or penetrance of
the mutant can be af f ected by genetic background .
30 3. If the Nutator line is recessive for Al, Cl, Bzl or
Bz2 (or other anthocyanin loci), it is advantageous
to outcross the mutant to a line lacking Mutator
activity and h~ '~yvuS for a Mu-induced allele of
the same gene (such as oz-mum9, or aal-mum2). This
allows selection in the Fl generation of seed that
carried or lacked ~lutator activity. These seed can
then be grown, selfed, and screened for the mutant
wo 95/30005 2 1 9 0 5 9 7 r~ 5 c~66
-- 23 --
phenotype. Production of populations segregating
for the mutation of interest and lacking Mutator
activity facilitates molecular analyses. New Mu
rL_ Ls are not generated, and existing, unliked
Mu fragments segregate out of the line with
subsequent outcrosses. It is important to point out
that some ~u-induced alleles are suppressible; that
is, in the absence of Mu activity the phenotype is
wild type (Martienssen et al., 1990). In such a
case, selection of inactivity selects for
phenotypically wild-type plants.
J. DNA Analy~i~
Identifying the gene rPCp~nc;h]e for the mutant
phenotype involves screening for a Mu-homologous rL L
that cvseyLey-tes with the mutant phenotype. This is
done by PYAmin;rl~r the DNA of the segregating
population(s) (as produced above) by Southern blot
analyses. The prPl ;m;nAry screen is expedited by
PYAm;n;n~ a small population first. As many different
outcrossed segregating lines should be PYAm;nPd as
possible (See, Walbot, 1992). It is also useful to
examine the population ut; l; ~;n~ a number of different
restriction enzymes, since seyL~yating rL Ls may be
obscured by other Mu homologous bands. The population
should also be S- L c ~l.ed with probes to all known Mu
Pl- ~ L~i. Inclusion of DNA from t he parent lines on
these blots is also ; LallL. A cv6eyL-~yating rL .1~ L
should not be present in the parental plant. Once a
uoseyL~ting LL L is identified, additional analyses
with different populations and a larger population set
should be performed. FUrth~ er as noted above, Mu-
induced ~u~Le:5' ible alleles can confound the
cvseyLt~aLion analysis (MartiPncs~n et al., 1989; 1990).
For this reason, it is important to emphasize linkage of
Wo 9s/3000s r ~ l, IZ,, ~ C S~6G
21 90597 24 -
a fragment with mutant individuals may be genotypically
mutant .
Once a co6egregating band is identified, cloning or
PCR i6 used to obtain a flanking, unique sequence. This
f lanking probe i6 then used to prove the locu6 i6
responsible for the mutant phenotype. This can be
accompli6hed in a variety of ways:
10 1. Identification of DNA reaLLan~ Ls, insertions, or
deletions at the locus of ; nrl~rpnllDntly generated
alleles (O'Reilly et ~l., 1985) ~1 LLwted the
clone is (or is nearby) the locus of intere6t.
Multiple alleles generated within the 5ame tagging
study can be used f or this purpose .
2. The correlation of the 10s5 of the ~Lwl, L~ " from
the locus in ~ArminAl LeveLLal~ alleles or within
somatic sectors of reveL l_c~n~ tissue also indicates
the cloned locus is rPRrnnc;hle for the phenotype.
The generation of stocks homozygous f or the mutation
and subsequent analyses (see above) is useful here.
Mu elements transpose gDrm;nAlly from a locus at a
very low frequency as compared to other trAncrns~n
systems. It may soon be p/~cc;hlD to utilize an
early reverting line identified by V. Walbot (1991;
1992) to increase the frequency of gDrm;nAl
reYersion. It has been shown that a high frequency
(49~) of gDrm;nAl revertants of bz2-mu2 are Lec~,veLed
in this line, but work with other Nu-induced alleles
is ne~ ~C~:~ry before its generalized use can be
estAhl; ~hD~l .
3. Differential RNA hybridization oan facilitate
identification of the correct clone in 6pecial cases
where the expression of the locus is well
understood, as was done for Bz2 (MnT-AIl~hl in &
Wo 95130005 2 1 9 0 5 9 7 PCrlUss5l0s366
-- 25 --
Walbot, 1987). Hybridization were performed with
the putative clones to RNA isolated from various
tissues or allelic variants that showed a predicted
pattern of expression.
The overall goal of these studies was to understand
the f-ln~A~-I Lal ~ An;! - by which plants respond to and
protect themselves from water deficits. The approach was
to dissect the _ ~ ~s of this response process by
10 isolating mutations that fail to respond normally and
which showed spontaneous symptoms of water def icit
(classified as wilt mutations~. Several wilt genes were
identif ied and one was cloned to provide molecular probes
for functional studies. A formal genetic
15 characterization of these mutations, the molecular
biology of the genes and products they encode, and their
regulation and expression is currently underway. These
studies provide important information cnnr~rn;ng the
biochemistry, cell biology, g~n~;cs, and physiology of
20 water stress in plant6.
Even though the invention has been described with a
certain degree of particularity, it is evident that many
alternatives, modif ications, and variations will be
25 apparent to those skilled in the art in light of the
foregoing ~;~closllre. Accordingly, it is intended that
all such alternatives, modifications, and variations
which fall within the spirit and the scope of the
invention be embraced by the def ined claims .
The following examples are ;nrlll~l~d to ~ L- ~Le
pref erred ~-mho~ nts of the invention . It should be
appreciated by those of skill in the art that the
techniques (~ rlnc:ed in the examples which follow
35 rt:~JLc:s~ technigues discovered by the inventor to
function well in the practice of the invention, and thus
can be r~nn~id~red to constitute preferred modes for its
. _ _ . _ _ . . . _ _ _ _ _ _ _ _ _ _ _ . .
wo ssr3000s 2 1 9 0 5 9 7 P~ . ",~ ~ cs~66
-- 26 --
practice. However, those of skill in the art should, in
light of the present disclosure, appreciate that many
chanqes can be made in the specific PmhoAi- Ls which are
~i~rloseA and still obtain a like or similar result
5 without departing from the spirit and scope of the
invention .
EXaMPLB I
p~T~ r CT~ Tzr~TI OF wlr~q~ MUTANT8
TnAPrr-nADnt transpositions of Ac from a donor locus
are fiPl Pt~PA by PYri ci on of Ac and its reinsertion
elsewhere in the genome. Recovery of the tr~n~posed Ac
15 element in the heteLuzyuuus condition is possible in
kernel progeny using Ds-induced reporter genes. Using
this strategy, and a slightly modified version, over
10,000 ;nA~ onr~ L transpositions of Ac have been
L~uv~:red and screened for mutations. Plants carrying
20 LL~ ~lJos~cl Ac Pl~ are field-grown, screened for
dominant mutations, and self-pollinated to uncover
recessive Ac-induced mutations. The F2 progeny are
suLe~ ed for recessive mutations. By this approach,
several hundred mutations have been identif ied in
25 tr~ncrosed Ac f;~m~ 1 i P~.
In particular, four recessive mutations di~playing a
wilt phenotype were identified under normal water deficit
conditions (Table 1). In addition to these four
30 mutations, a fifth 6pontaneous allele (wiltl-l) is
currently under study.
Wo ss/30005 2 1 ~ a 5 9 7 r l,u~ J~6C
-- 27 --
Table 1. PrPl;m;nlry Characterizations of wilt Mutations
AI LELE
DE8IG. ~OURCE PO~lr
wilt-1256 Ac-Ds lS early wilting, viable
wilt-1269 Ac-Ds ? late wilting, viable
wilt-1270, Ac-Ds lS early wilting, viable
wilt-6945
wilt-113 Mutator ? early wilting, viable
10 wilt-129 Mutator ? early wilting, viable
wilt-222 Mutator ? early wilting, viable
wilt-1 spontaneous 6L late wilting, viable
15 Two of the mutants, wilt-1256 and wilt-6945 were
shown to be linked to the donor P locus (source of Ac) on
Ul1L~ ~~ ~ lS. Linkage of the Wllt mutants to the donor
P locus is expected, in some cases, since Ac usually
transposes to nearby U11L~ ~ 1 sites. The genetic
ao location of the other two mutations is unknown.
One of the more interesting aspects of the wilting
phenotype i6 it6 developmental on6et. For instance, the
wilt-1256 mutant showed the first signs of leaf roll at
25 the third leaf stage. Young PlTan~l;n~ leaves were
severely leaf rolled, yet older leaves appeared to
recover and support growth of the plant. In contrast,
wilt-1269 mutants did not show any visible signs of leaf
roll until just prior to flowering. The wilt-1 mutant
30 (obtainable from the Maize GPn~Pti~ Cooperation Stock
Center, E.B. Patterson, S-118 Turner lIall, Agronomy
Department, University of Tll;n~ , 1102 S. Goodwin
Avenue, Urbana, IL 61801) exhibited wilting in late
vegetative growth. Plants i~ yuuS for wilt-1 reached
35 maturity and produced seed, although plants were
generally reduced in stature. This previously identified
mutation (Po6tlethwait & Nelson, 1957) has been mapped to
6L.
... ...... _ .. _ .
Wo 9sl3000~ Pcr/ussslo~36lj
21 90597
-- 28 --
Besides onset of the wilting phenotype, as
identified by leaf roll, the mutations also differ in
whether wilting was lethal to the plant. For example,
wilt-1270 and wilt-6945 are ~ ;ng lethals, i.e.,
5 mutant plants grew to the 4-5 leaf stage, leaves wilted,
growth ceased, and cell necrosis eventually killed the
plant. However, this characteristic is not linked to the
early wilting effect. For example, wilt-1270 and wilt-
6945 were seedling lethals while wilt-1256 wilted during
10 this same developmental period, yet recovered. It
appeared that lethality was related to the inability of
the plant to recover from the early onset of wilting. To
date, both late wilting alleles (wiltl-l and wilt-1269)
were viable mutations. Viable wilt mutants grew to
15 maturity, flower, and were fertile, although yields were
substantially reduced. The mutations were generally
deleterious, i. e., mutant plants were smaller than wild-
type and exhibited poor leaf expansion.
Anatomical studies of wilt-6945 leaves revealed no
structural difference between mutant plants and normal
~::;hl ing~, The leaf turgor potential of wilt-6945 mutants
was near zero~ Thus, although the cPl1lllAr morphology of
wilt-6945 appears normal, it was unable to r-; nt~ i n
sllff;cjfnt leaf turgor. This aspect o~ the mutant
appeared to be the primary lesion which was responsible
for its lethal phenotype. The cellular characterization
of other wilt mutations is currently under study.
1~ nnti~ication o~ a~ition~l mutation~
The identification of additional wilt mutations may
be employed to def ine additional genes involved in the
water stress response pathway. Fifteen additional wilt
mutations were identified in Ac-Ds and Robertson's
Mutator screens. All mutants are regrown to confirm
M~n~l~l ;An inheritance patterns and dominant, co~' inAnt~
. . . _ _ _ _ _ _ _ _ .
Wo 95~3000s ~ 2 ~ 9 0 5 9 7 PCrlUSsslos366
-- 29 --
recessive relati~ln~hirs to wild-type. Pairwise
complementation tests between each mutations is
performed. In addition, a directed tagging study of
wiltl-1 using the trs~n~pos~hle element Mutator is
performed. The testcros6 population of over 100,000
kernels is screened to identify wll t-l alleles.
Several criteria are est:~hl i ~hPd to decide the
priority given to each mutation for characterization.
For example, PYogPnouc ABA application may be used to
identify mutants that can be rescued or identify mutants
that show differential sensitivity. Standard genetic
tests can be used to def ine potential mutants that are
linked to LL'.I~ 5P~1 Ac or Mutator tr:~n~pos~hle elements
(Dellaporta ~ Moreno, 1994). Complementation analysis
~PtPl-mi nP~ the number of i n~lPrPn~lPnt loci that mutate to
give the wilt phenotype. Based on this information,
selected mutations are characterized further and cloned.
For mutations that do not show linkage to
LL.... -~,o__ble elements, the loci are mapped by bulk
seyLey~l-,L analysis (Mi~'hPl- e et al., 1991).
Wilt-1270, another Ac-derived wilt phenotype mutant is
mapped in this manner. Briefly, the mutant is outcrossed
to a different genetic ba-_kyLuul.d (e.g. B73) and F1
plants are self-pollinated. DNA from normal and mutant
F2 plants are pooled and bulked DNA is hybridized with
probes from known linkage groups. TJnl inkPd probes
hybridize with equal intensity (linkage equilibrium) to
both bulked DNA samples while probes linked to the
mutation show differential hybridization siy-nals (linkage
disequilibrium). These studies help map genes on the
maize genome involved in the response to water stress
which may be important ~or future studies.
WO 95/3000~5 2 1 9 0 5 9 7 PCT/US95/05366
~ 30 ~
EXI~MPLE II
Mt1T.RrlTT.~R rT~I~R~rq'T'TITS~'rTr~N OF W~LT M1JTal~IO~8
Although tr~nCposP~l Ac Pl- Lt, were selected in
S this program to isolate wilt mutations, the mutations
were not always Ac-induced. Many of the mutations were
shown to be caused by lesions other than Ac. Therefore,
gQnetic and molecular tests were nP~C~ y to d~t~rm; np
which mutations are Ac-induced to define those alleles
10 useful for gene cloning. Confirmation of the linkage of
Ac and wilt mutations is provided by reversion analysis.
wilt plants are screened for wild type, non-wilting,
revertants. A reversion CULL-'~LJ'".~l;n~ to PYl-;cjt~n of Ac
from the wilt gene conf irms linkage of Ac and wilt. Four
15 recessive wilt mutations were s. ,e:~..ed using Southern
hybridization for linkage of the wilt pllel~uLy~ue with a
LL'~l~'-LJosed Ac element. In the case o~ wilt-1256, linkage
between the mutant allele and Ac is complete. DNA was
isolated from sibling plants obtained from a selfed wilt-
20 1256 heterozygote and digested with Eco RI or separatelywith Sal I for Southern Blot analysis. In each analysis,
this digested DNA from wilted plants and wild-type
~cihl ing5 wa6 probed with an 0.4 Kb Bam HI-Sal I wilt gene
rL ~ or separately with an Ac 0. 9 Kb EcoRI-HindIII
25 fragment. The data from the analysis using an Ac 0 . 9 Kb
EcoRI-HindIII probe showed that the wilt-1256 fragment
cu 3~,1=y~lted with an 18 kb Sal I CL L and 15 kb
EcoRI restriction CL, ' which hybridized to an Ac
probe. Both bands Lt:~res~ ed the Ac Pl~ c found in
30 this line. These bands appeared in all of the mutants
(total of 74 DNAs analyzed to date) and segregated, as
~Ypect~d, in wild-type 5iblings (2/3 of the wild type
plants were PyectPd to be het-:Lu~yyuus for the mutation
and to carry these L, L6).
The wilt-1256 gene is cloned according to the
E~r~ ure:5 di8closed by Federoff (Federoff et al., 1984;
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ , ,
W0 95/30005 2 1 9 0 5 q 7 P~I IIJ ,,_ C5~66
-- 31 --
Federoff, 1988; both reference6 incorporated herein by
reference). A Sal I genomic library was constructed in
EMBL3 using DNA i601ated from a mutant plant. One clone
was isolated which contained the Ac-hybridizing 18 kb Sal
5 I genomic rL t (~5Sal, FIG. 1). This clone was shown
by restriction mapping to contain a trAnCpns~l Ac
element. Unique probes were isolated from DNA flanking
the Ac element of thi6 clone (6hown in FIG. 1 as 0.4 B/S)
and used to probe genomic blot6. These probes detected
10 only the expected 15 kb EcoRI fragment and 18 kb Sal I
fragment in mutant plants. As expected, some normal
plants also contained the mutant band and a wild type
allele, while some have only wild-type alleles. The
f lanking DNA detects a transcript in maize shoots and
15 leaves. This probe was used to isolate seven cDNA clones
from a cDNA library constructed from shoot mRNA. These
cDNA clones have been named ~W1, AW4, )~W7, )~W8, ~W9,
)~W10, and AW11. cDNA clones are verified to contain the
wilt-1256 gene by hybridization to ~5Sal. Only those
20 clones containing the wilt gene will hybridize to ~5Sal.
In summary, the wilt-1256 mutation was tightly linked to
a tri~ncpnC~ Ac element and a genomic clone, ~5Sal (FIG.
1), was obtained that contained flanking DNA that
represented unique transcribed sequences.
E~CaMPI,E III
WIrT re~ C~aRACT~T~TION
30 A. ~enatic and mol~cular confirmation o~ clon~d
_,
Further genetic and molecular evidence is obtained
to ~1 Ll.lt.e that cloned sequences do indeed correspond
- 35 to the Wilt-1256 locu6. complementation studies with
existing Wilt mutations are used to ~l~t~rm; "~ whether
additional wilt-1256 alleles have been generated. Since
all of the mutantC are generated in near i cog~n i c
wo ss/3000s 2 1 9 ~ 5 9 7 PCT/USg5/05366
ba~:hy~uu~,ds, progenitor wild-type alleles are available
for analysis. Progenitor and mutant alleles are analyzed
by Southern hybridization using wilt-1256 sequences. If
these probes detect additional re~lL ~ 9 ( e . g .
5 insertions) this provides further evidence that cloned
6equences represent wilt-1256 DNA. The second criteria
used to establish identity is the analysis of revertant
alleles detected as Ac ~YC; ~i nn events from the wilt-1256
allele. Phenotypic reversion is ~ l; ed by PYri ~:in
lO of Ac if the cloned sequence is indeed the wilt-1256
gene. Either criteria provides the n-~ceS~ :lry data to
conf irm the identity of the clone .
B. ~ ml~ly~ i8
CDNA clones of the wilt-1256 locus are analyzed.
Clones that hybridize to DNA probes adjacent to the Ac
insertion are isolated from a CDNA library made from
maize shoot apices (leaves and shoots). These clones are
24 restriction mapped and hybridized back to genomic DNA to
conf irm their identity as wilt-1256 sequences . Part of
this genomic DNA has been sequenced and det~rm;ned to be
an open reading frame as shown below:
25 GANTCGATGG I~ L TCANTCGGTG ATCCCCr-At~A GGGGGCAGAA
GACGCTGACN AAGTTCGTCA ACGGCGGGTC CANC~AACGTN TTCTACGCGC
ACGAGTACAA CGC~.AC~;GTG GAGTTCTACT GGGCGCCCTT C~;L6~LC~GAG
TCCAACTCCG At'AA~'CCt'AA GGTTCACANC GTCCCCr-~rC GGATCATCCA
GTGGCACGCC AI~1.- ~ANC ACGCGCNCAA CTGGATCGGC GTCGACA
The position of this sequence is indicated in FIG.
1. The largest CDNA clones are sequenced and the
predicted protein product det~rm; n~ . Sequence data of
wo ss/30005 = - 2 1 9 0 5 9 7 F~~
-- 33 --
the wilt-1256 gene and its predicted protein product are
analyzed via computer algorithms such as BLAST (Altschul
et al., 1990) and BLOCKS (Henikoff & Henikoff, 1991~ to
6earch for the presence of functional domains in the
5 wilt-1256 protein. Thi6 information may be useful in the
design of future bio~ hDm;c ll and physiological studies.
For example, it i5 contemplated that this analysis may
~;uggest a regulatory (e.g. DNA binding protein) or
biochemical (e.g. enzymatic activity) role of the
10 wilt-1256 protein by its similarity to known proteins.
The wilt-1256 coding region is sllhcl ~nPcl into E.
coli expression vectors for production of protein. The
protein is tagged with a peptide (polyhistidine or
15 maltose binding protein) for affinity purification.
Protein is purif ied by metal chelation chromatography
using commercially available resins (Le Grice &
Grueninger-Leitch, 1990) or by maltose affinity
techniques (Xellermann & Ferenci, 1982; Guan et al.,
20 1988). Polyclonal antiho~liDC to the fusion protein are
produced and used in immunolocalization studies.
C. 13xpr~ion an~l rsgu~ on of the wilt-1256 gene
The temporal and spatial expression pattern of the
wllt-1256 gene i8 important in understanding the function
of this gene. It i5 contemplated that one may want to
know whether gene expression is constitutive or induced
in leaf cells. By Northern hybridization analysis
(Ausebel et al., 1992), the tissue-specific expression of
mRNA (leaf, root, stem, infl~,L~scc:.,ce, etc.) is
investigated. Polyclonal a~tiho~ C and Western analysis
(Harlow & Lane, 1988) are used to ~lP~arminD protein
expression patterns. Both water-LLL-~ssed, unstressed,
and ABA-treated tissue are analyzed by Northern
hybridization and Western analysis to determine whether
induction occurs (RNA or protein). Detailed
WO 95/3000S 2 1 9 0 5 9 7 PCTIUS951OS36G
- 34 -
characterization of the regulation of expression of
wilt-1256 regulation is the result of these studies. For
example, it is contemplated that Northern analysis will
indicate whether steady-6tate mRNA levels are a~fected by
5 water stress, yet Western analysis may indicate water
deficits affect protein levels. This result would
suggest regulation is post-transcriptional and would be
further investigated. It is further contemplated that
regulation of wilt-1256 activity may be at the level of
10 RNA transcription or protein processing.
More d~t l; 1 ed expression patterns are obtained by
in situ hybridization techniques (Jackson, 1992; Freeling
& Walbot, 199~). Both ribuuLubes (sense and antisense~
15 and antibodies ~pre-immune and post-immune) are u6ed to
localize mRNA and proteins, respectively, in particular
cell types. For example, i~ Northern and Western results
indicate that wilt-1256 is only expressed in leaves, the
cell-type specificity (mesophyll, bundle sheath,
20 ~p;tl~rr-l, veins, etc. ) of wilt-1256 mRNA and protein
oc:~1;7~tion are fl~t~rm;ned.
In sum, the molecular characterization of the
wllt-1256 gene and its expression, regulation, and
25 l o~ :~l; 7ation patterns provide important information
regarding the function of this gene. As additional
tagged mutants ~ecome available, similar studies are
undertaken to clone and characterize selected mutations.
E~NPLE IV
A~D GENETIC EPI8TA8I8
~Nr~ coMpT~ IoN 8TIJDIE8
The av~ hil ;ty of multiple wilt mutations allows
the water stress response pathway to be def ined using
formal genetic studies. For example, complementation
_ _ _ _ _ _ _ _ _ _ _ _ _
w09s/3000s . 1 2190597 1 ,~
- 35 -
studies with existing and forth~ in~ wilt mutants
detPrm;npc the number of genes that contribute to the
wilt phenotype. Each available wilt mutant is crossed
sy6tematically to all other mutants. For viable mutants,
5 h~ ~ y~uu~ lines are crossed reciprocally. For lethal
mutations, het~3L.zyyuus lines are employed.
Complementation groups are ACci~nPd. Double mùtant lines
are cc,llsLLùuLed with complementing mutations to determine
genetic epistasis. To ~ollow each allele, maize RFLPs or
10 microsatellite markers are employed for genotyping
~ oses tHosington, 1987). ~ ,us double mutants
are PY~min~d to determine whether an interaction between
gene exists. For example, it is contemplated that
mutations may be additive, synergistic, antagonistic, or
15 epistatic to one another. Nutations that define
i n~P~c~nrlPnt pathways are additive while those that lie in
a shared signal LLans-lu-;Lion pathway show epistasis.
The similarity between some mutant phenotypes may
20 preclude a formal epistasis analysis. Nevertheless,
single and double mutant lines are useful for ~lPfjn;n~
molecular interactions. For example, it is contemplated
that one can determine whether gene or protein expression
is affected by mutation(s) at other loci using wilt-1256
25 probes. This result indicâtes that certain genes lie
upstream (e.g. regulate) of wilt-1256 function.
A. ~ L '-logy an~ PhyYiology of Nut~t E' L~
The r-lPr~ r characterization of wilt-1256 will
determine the temporal and spatial pattern of gene
expression. This information indicates which tissues and
cells contain the primary defect. To further investigate
the nature of the mutant, the tissue and c~ r
morphology of wilt-1256 mutants and wild type c;hl;ng5
under normal and water deficit conditions are ~Yilm;n~d.
Histological analysis of ~,LL~ssed and unstressed tissue
_ _ _ _ _ _ _ _ _ _ , . . . , _ , . _ , _ _ _ _, _, _ _ _ _ _ _ _ _ _ _
Wo ss/30005 2 1 q 0 5 q 7 " - - -
-- 36 --
from mutant and wild-type plants are performed (Sylvester
& Ruzin, 1994). Cross sections through the appropriate
tissues (e.g. leaves or roots) reveal any ~LuuL
aberrations. Transmission and 6canning electron
5 mi~:LusuU~y are used to characterize mutant and wild-type
cell structure and epidermal surfaces. Leaf surfaces are
also ~Y~min~ for normal stomate structure and density
using epidermal peels (Ristic & Cass, 1991).
~he relat j r n~h i ~ between the wilt phenotype and the
phyt~hl - ABA is investigated. Mutant rescue studies
using ABA are performed on wilt-1256 mutants to determine
whether Ai3A can rescue the phenotype (wilting and/or
lethality) or whether the mutant is hypo- or
hyper-sensitive to ABA when _ ~t d to its normal sibs .
If wilt-1256 reveals an increased or decreased ABA
sensitivity, this suggests that an important step in Ai3A
synthesis or reception is affected. To further
investigate this relationship, the ABA content of
see~ll in~ leaves and roots is measured using a
radioi - ~sed assay (Quarrie et al., 1988). This
assay helps to identify whether the defect is in Ai3A
synthesis or reception. For example mutations in the ABA
biosynthetic pathway are expected to have reduced levels
of Ai3A in the leaves and/or roots, whereas, Ai3A rece~l.u,
mutants have normal or elevated levels.
E~NPLB V
~GENIC ~"T'rCI~ OF GEN13
A. Voctor C _- LL ~_ Lion
Vectors are constructed that drive eYpression of a
35 wilt gene in Zea mays cells. A vector is ;u~ r u- ~ed to
direct constitutive eYpression. For example, the
Cauliflower Nosaic Virus 35S promoter (Odell et al.,
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _
Wo 95/30005 2 1 9 0 5 9 7 F~~ c~66
- 37 -
1985) is placed 5 ' of the wilt gene. Alternatively the
rice actin gene promoter (Wang et al., 1992) is placed 5'
of the wilt gene. It i5 anticipated that all promoters
which direct constitutive gene expression in maize are
useful when operably linked to a wilt gene. SPql~PnrPs
which direct polyadenylation are linked 3 ' to the wilt
gene. These sequences included, but are not limited to,
DNA sequences isolated from the 3 ' region of
Agrobacterium tumefaciens nnpAl inP synthase, octopine
synthase or LL ~IIS-,iL ipt 7, or potato proteinase inhibitor
II genes. It is anticipated that constitutive expression
of the wilt gene in all tissues of a maize plant will
enhance the ability of the plant to maintain water turgor
under conditions of decreased water availability.
It is further contemplated that tissue specific
expression of a wil t gene will enhance the agronomic
performance of a maize plant. Vectors for use in tissue-
specific targeting of wilt genes in LLa.-la~e:rliC plants
will typically include tissue-specific promoters and may
also include other tis~a~ -~,ecific control Pl~ L~ such
as PnhAnrPr se~ue~ s . Promoters which direct speclf ic
or PnhAnred expression in certain plant tissues will be
known to those of skill in the art in light of the
present disclosure. These include, for example, the rbcS
promoter, specific for green tissue; the ocs, nos and mas
promoters which have higher activity in roots or wounded
leaf tissue; a truncated (-90 to +8) 35S promoter which
directs PnhAnred expression in roots, an -tubulin gene
3 0 that directs expression in roots and promoters derived
from zein storage protein genes which direct expression
in Dn~-~spPrm. It is particularly contemplated that one
may advantageously use the 16 bp ocs PnhAnrP~ element
from the octopine synthase (ocs) gene (Ellis et al.,
1987; Bouchez et al., 1989), PCper;Ally when present in
multiple copies, to achieve enhAnred expression in roots.
Wo 95/3000~ 2 1 9 0 5 9 7 P 11" --
Expression of w11t genes in transgenic plants may be
desired under speclfied conditions. For example, the
expression of wilt genes may be desired only under actual
stress conditions. It is known that a large number of
5 genes exist that respond to the environment. For
example, expression of some genes such as rbcS, Dnror~ing
the small subunit of ribulose b; ~rh~srhAte carboxylase,
is regulated by light as mediated through phytochrome.
Other genes are induced by SD~ rl~ r y stimuli. For
10 example, synthesis of ~hsci~i c acid (ABA) is induced by
certain enviL L~l factors, inr l~ ;n~ but not limited
to water stress. A number of genes have been shown to be
induced by ABA (Skriver & Mundy, 1990). Promoter
regions that regulate expression of these genes will be
15 useful when operably linked to a wilt gene.
Alternatively, one may wish to obtain novel tissue-
specific promoter sequences for use in accordance with
the present invention . To achieve this, one may f irst
20 isolate cDNA clones ~rom the tissue c~nrDrn~d and
identify those clones which are e~yL~;G3~d specif ically in
that tissue, for example, using Northern blotting.
Ideally, one would like to identify a gene that is not
present in a high copy number, but which gene product is
25 relatively abundant in speci~ic tissues. The promoter
and control Dl ' Ls of ~c,rL~ ; n~ genomic clones may
then be lor~ r9cl using the terhn;q~D~ of molecular
biology known to those of skill in the art.
3 o It is yL ~ sed that in some ' - '; L~ of the
present invention expression of a wilt gene in a
LL .~ rl~ r plant will be desired only in a certain time
period during the development of the plant.
Dev~l 1, Lal timing is frequently correlated with tissue
specific gene expression. For example, expression of
zein storage proteins is initiated in the Dn~SpDrln about
15 days after pollination.
_ _ _ _
wo ss/3000s 2 1 ~ 0 5 9 7 r~
-- 39 --
B. Initi~tion ~nd M~inten~nc~ of ~riri^~t Cell
Culture~
Nilt genes may be i.l~Lu-lu~ed into maize cells
5 ; nrl IlA; nr~, but not limited to, cultured cells or immature
embryos. Transformable cell lines of maize are developed
using the protocols familiar to one of skill in the art,
;nrll-A;n~, but not limited to the following. The
composition of each culture medium is listed in Table 2.
C . Cell Line AT82 ~
For dev~ of the transformable cell line
AT824,; ~UL~ embryos (0.5 - l.Omm) were excised from
15 the B73-derived inbred line AT and cultured on N6 medium
with 100 ~LN silver nitrate, 3 . 3 mg/L dicamba, 3% sucrose
and 12 mN proline (2004, see Table 2). Six months after
initiation type I callus was transferred to medium 2008
(see Table 2). Two months later type I callus was
20 transferred to a medium with a lower col-cc ~.~Lc.tion of
sucrose (279, see Table 2). A sector of type II callus
was identified 17 months later and was transferred to 279
(see Table 2) medium. This cell line is uniform in
nature, unorganized, rapid growing, and embryogenic.
25 This culture is desirable in the context of this
invention as it is easily adaptable to culture in liquid
or on solid medium.
The first 5~p~nc;on cultures of AT824 were
30 initiated 31 months after culture initiation. Sllcp~ c;rn
cultures were initiated in a variety of culture media
including media containing 2, 4-D as well as dicamba as
the auxin source, e.g., media designated 210, 401, 409,
279 (see Table 2). Cultures were maintained by transfer
35 of approximately 2 ml packed cell volume to 20 ml fresh
culture medium at 3 3~ day intervals. AT824 can be
routinely transferred between liquid and solid culture
media with no effect on growth or morphology.
WO 95/30005 2 1 9 0 5 9 7 PCT/US951053C6
-- 40 --
Suspension cultures of AT824 were initially
~Lyu~.eserved 33-37 months after culture initiation. The
survival rate of this culture is improved when it is
:LyuuL~served following three months in Sll~p"nc~c.n
5 culture. AT824 suspension cultures have been
uLyuul~served and re-initiated from ~Lyu~L~_erved cells
at regular intervals sinCe the initial date of freezing.
Repeated cycles of freezing have not affected the growth
or transformability of this culture.
Tablo 2: Illustrativ~ of q!issun Culturo
M~l~i21 Which ar~ Us~d for Type II C~llus
D~v~ , Dcval~, ' of 8~p~ni~l~ Cultur~s
nnd r- tion of Plant C-~lls t8p-~cifically
Maiz~ Colls)
MBDIA BA8AZ 8~CR08B p}l OT~BR
NO. N~DIUN ~AmoUnt~L)
lO1 MS 3~ 6. 0 MS vitamins
lOOmg myo-inositol
Bactoagar
20 189 MS - 5 . 8 3mg BAP
. 04mg NAA
. 5mg niacin
8 0 Omg L-asparagine
lOOmg r~:~c~minr~
acids
20g sorbitol
1. 4g L-proline
lOOmg myo-inositol
Gelgro
201 N6 29c 5 . 8 N6 vitamins
2 mg L-glycine
1 mg 2,4-D
100 mg casein
hydrolysate
2 . 9 g L-proline
Gelgro
W09s/30005 - 41 - r~l~u~ css66
~EDIA BA~AL ~UCRO~E p~ OTIIBR ~
NO. MEDIUM ~Amount/L)
210 N6 3% 5. 5 N6 vitamins
2 mg 2,4-D
250 mg Ca
pantothenate
100 mg myo-inositol
790 mg L-asparagine
100 mg casein
hydro lysate
1. 4 g L-proline
2 mg glycine
Hazelton agar
223 N6 2% 5 . 8 3 . 3 mg dicamba
1 mg th i Am; n~
0 . 5 mg niacin
800 mg L-asparagine
100 mg casein
hydrolysate
100 mg myo-inositol
1. 4 g proline
Gelgro
3 mg b; A 1 Arh~-15
279 N6 2% 5.8 3.3 mg dicamba
1 mg th;Am;n~
0 . 5 mg niacin
800 mg L-asparagine
100 mg casein
hydrolysate
100 mg myo-inositol
1. 4 g proline
Gelgro
401 MS 39~ 6. 0 3 . 73 mg Na2EDTA
0.25 mg th;:~m;np
1 mg 2,4-D
2 mg NAA
200 mg casein
hydrolysate
500 mg K2S04
4 o 0 mg KH2PO4
100 mg myo-inositol
5409 MS 3% 6 . 0 3 . 73 mg Na2EDTA
0.25 mg thiAm;nl,
9 . 9 mg dicamba
200 mg casein
hydrolysate
500 mg K2SO
4 o 0 mg KH2PO4
100 mg myo-inositol
WO95/3000S 2 1 9~597 PCT/US95/05366
-- 42 --
llBDIA BA8AB 8UCRO8B pH OTHER . '
NO. NEDI:UN ~Amount/L)
4 2 5 MS 3 % 6 . 0 3 . 7 3 mg Na2EDTA
0.25 mg th;Am;nP
9 . 9 mg dicamba
200 mg casein
hydrolysate
500 mg ~C2SO4
400 mg KH2P0,,
lO0 mg myo-inositol
3 mg b; A 1 Arht~C
501 Clark' s 2% 5 . 7
Medium
607 0.5x MS 3% 5.8 0.5 mg th;AminP
0 . 5 mg niacin
Gelrite
734 N6 2% 5 . 8 N6 vitamins
2 mg L-glycine
1.5 mg 2,4-D
14 g Fe sequestrene
200 mg casein
hydrolysate
0. 69 g L-proline
Gelrite
5735 N6 2% 5 . 8 1 mg 2, 4-D
0 . 5 mg niacin
0 . 91 g L-asparagine
100 mg myo-inositol
l mg thiAmjnp
0. 5 g MES
o . 75 g MgCl2
100 mg casein
hydroly6ate
0 . 69 g L-proline
Gel~ro
739 N6 2% 5.8 l mg 2,4-D
0 . 5 mg niacin
0 . 91 g L-asparagine
lO0 mg myo-inositol
1 mg th;Amj~np
0. 5 g MES
0 . 75 g MgCl2
lO0 mg casein
hydroly6ate
0 . 69 g L-proline
Gelgro
l mg hi A 1 A~ho8
Wo 95/30005 2 1 9 0 5 9 7 PCTiU595l05366
-- 43 --
MEDIA BA5AL 8UCRO5B pH OTIIER ~ .
NO. MEDIUM ~Amount/L)
750 N6 2% 5.8 1 mg 2,4-D
0 . 5 mg niacin
0 . 91 g L-asparagine
100 mg myo-inositol
1 mg thiAminp
0.5 g MES
o . 75 g MgCl2
100 mg casein
hydrolysate
0 . 69 g L-proline
Gelgro
0 . 2 N mannitol
1 mg biAl Rrhl~5
758 N6 2% 5 . 8 1 mg 2, 4-D
0 . 5 mg niacin
0 . 91 g L-asparagine
100 mg myo-inositol
1 mg thiAm;np
0.5 g MES
o . 75 g NgCl2
100 mg casein
hydrolysate
0 . 69 g L-proline
Gelgro
3 mg hi:l 1 Arh-~5
2004 N6 3% 5.8 1 mg th;AminP
0 . 5 mg niacin
3 . 3 mg dicamba
17 mg AgN03
1. 4 g L-proline
0 . 8 g L-asparagine
100 mg casein
hydrolysate
100 mg myo-inositol
Gelrite
2008 N6 3% 5.8 1 mg ~hiAminp
0 . 5 mg niacin
3 . 3 mg dicamba
1. 4 g L-proline
0 . 8 g L-asparagine
- Basic NS medium described in Murashige ~ Skoog
(1962). This medium is typically modified by decreasing
the NH~NO3 from 1.64 g/l to 1.55 g/l, and omitting the
pyridoxine HCl, nicotinic acid, myo-inositol and glycine.
-
WO 95BOOOS 2 1 9 0 5 9 7 PCTIUS9S/OS366
-- 44 --
N6 medium described in Chu et al., 1975.
NAA = Napthol Acetic Acid
IAA = Indole Acetic Acid
2-IP = 2, isopentyl adenine
5 2, 4-D = 2, 4-Dichlorophenoxyacetic Acid
BAP = 6-benzyl aminopurine
ABA = Ahcr; ci r acid
*~tBasic medium described in Clark t 1982 )
10 D. Initiation of C~ll Lino~ of tho ~iII G l.~ ~
Transformable cell culture6 are routinely developed
from the ye~ y~e Hi-II using the following protocol.
The Hi-II genotype of corn wa6 developed from an A188 x
B73 cross. Thi6 genotype was developed specifically for
a high frequency of initiation of type II cultures (100%
response rate, AL~_I_Lu~lg et al., 1991). Immature embryos
(8-12 days post-pollination, 1 to 1.2 mm) are excised and
cultured embryonic axis down on N6 medium containing 1
mg/L 2,4-D, 25 mM L-proline (201, see Table 2) or N6
medium containing 1. 5 mg/L 2, 4-D, 6mM L-proline (734, see
Table 2 ) . Type II callus can be initiated either with or
without the ~Lest~ .e of 100 ,llM AgNO3. Cultures initiated
in the ~L~ ;e of AgNo3 are transferred to medium lacking
this ~ _ ' 14-28 days after culture initiation.
Callus cultures are incubated in the dark at 23-28C and
transferred to fresh culture medium at 14 day intervals.
Hi-II type II callus is maintained by manual
selection of callus at each transfer. Alternatively,
callus can be L~~ P~9P~l in liquid culture medium,
passed through a 1. 9 mm sieve and replated on solid
culture medium at the time of transfer. It is believed
that this seqllPnre of r~n;rlllAtions is one way to enrich
for recipient cell types. Regenerable type II callus
that is suitable for transformation can be routinely
developed from the Hi-II genotype and hence new cultures
,
w0 9sl30005 2 1 9 0 5 9 7 r~ c~66
are developed every 6-9 months. Routine generation of
new cultures reduces the period of time over which each
culture is maintained and hence insures repro~-lr;hl~,
highly le~l-eLc.ble, cultures that routinely produce
5 f ertile plants .
E. Microproj~ctil~ Bomb~rdm~nt
DNA is illLL-~du~;~d into cultured cells as follow6.
10 Cultured cells are subcultured to fresh medium 409 (see
Table 2 ) two days prior to particle bombardment . If
grown in liquid medium cells are plated on solid 409
(Table 2) medium 16-24 hours before bombardment (about
0.5 ml packed cell volume per filter). Tissue is treated
with 409 (Table 2) medium containing 200 mOsm sorbitol
(medium 431, see Table 2~ for 1 hour prior to
bombardment .
DNA is introduced into cells using the DuPont
Biolistics PDSlOOOHe particle 1~ ' L device.
DNA is precipitated onto gold particles as follows.
A stock solution of gold particles is ~L e~dr~d by adding
60 mg of 1 ,um gold particles to 1000 ,ul absolute ethanol
and incubating for at least 3 hours at room t~ ~LUL~
followed by storage at -20DC. Twenty to thirty five ~Ll
sterile gold particles are centrifuged in a
mi,:. OC~--Lr if uge for 1 min. The supernatant is removed
and one ml sterile water is added to the tube, followed
by centrifugation at 2000 rpm for 5 minutes.
Nicroprojectile particles are r~ L~ in 30 ~Ll of DNA
solution (30 ,ug total DNA). The DNA solution contains a
vector containing a chimeric wllt gene and a vector
containing a selectable marker gene, e.g., the bar gene
which is used for selection of transformants based on
resi~tance to the herbicide hiAl~rh~C (Gordon-Kamm
et al., 1990). Two hundred twenty microliters sterile
Wo s~/3000~ 2 1 9 0 5 9 7 ~ 'C~6G
-- 46 --
water, 250 ~1 2.5 M CaCl2 and 50 ~1 spPrm;~line are added.
The mixture is thoroughly mixed and placed on ice,
followed by vorteYing at 4C for 10 minutes and
centrifugation at 500 rpm for 5 minutes. The supernatant
5 is removed and the pellet rPc~lppn~lpd in 600 ~1 absolute
ethanol. Following centrifugation at 500 rpm for
5 minute6 the pellet is r~ n~lpcl in 3 6 /Ll of absolute
ethanol .
Ten ~Ll of the particle ~L~=~aLaLion are dispensed on
the surface of the flyer disk and the ethanol was allowed
to dry completely. Particles are accelerated by a helium
blast of approximately 1100 psi. One day following
bombardment cells are transferred to liquid medium 409
(10 ml, see Ta~le 2). Tissue is subcultured twice per
week . During the f irst week there is no selection
applied.
Alternatively i LULt: embryos may be directly
subjected to microprojectile bomL~L L. T LuLe
embryos (1.2 - 2.0 mm in length) are excised from
surface-sterilized, grPPnh~ce-grown ears of Hi-II 11-12
days post-pollination. The Hi-II ge-lc,Lyye was developed
from an A188 x B73 cross for high frêquency devPl,,
of type II callus from; LULe embryos (ALh.~LL~
et al., 1991). Approximately 30 embryos per petri dish
are plated axis side down on a modif led N6 medium
containing 1 mg/l 2,4-D, 100 mg/l casein hydrolysate, 6
mM L-proline, 0.5 g/l 2-(N-morpholino)eth~nPc~llfonic acid
(MES), 0.75 g/l MgCl2, and 2% sucro5e solidified with 2
g/l Gelgro, pH 5.8 (735, Table 2) Embryos are cultured
in the dark for two days at 24C.
Approximately f our hours prior to } , :,
embryos are transferred to the above culture medium with
the sucrose c~ cel.LL-tion increased from 3% to 12%. When
embryos are transferred to the high osmoticum medium they
WO 9s/3000~ 2 1 9 0 5 9 7 PCTlU~9slOs366
-- 47 --
are arranged in concentric circles on the plate,
starting 2 cm from the center of the dish, positioned
such that their coleorhizal end is orientated toward the
center of the dish. Usually two concentric circles are
5 formed with 25-35 embryos per plate. Preparation of
gold particles carrying plasmid DNA is described above.
The plates containing embryos are placed on the
third shelf from the bottom, 5 cm below the stopping
10 screen. The 1100 psi rupture discs are used. Each plate
of embryos is bombarded once. Embryos are allowed to
recover overnight on high osmotic strength medium prior
to initiation of selection.
Although this example describes the introduction of
DNA using particle bombardment, it is contemplated that
DNA will be inLL~-luced into cells using any one of a
number of technigues from which it is possible to recover
fertile ~L~ J~ i C plants. For example, fertile
20 trAn~oni~ plants are ~Luùuu~:d following ele,:~Lul!ulcltion
of cells as described in Krzyzek et al, incorporated
herein by reference.
~. 8~1~ction of !rL~.,sruL~Ls
Transformants are sQl~Qrt~d based on resistance to a
toxic ~ for which the introduced gene confers
resistance. For example cells transformed with a gene
Pnror~ i n~ a glyphosate resistant EPSPS protein are
30 resistant to the herbicide glyphosate. Similarly, cells
transf ormed with a gene Q~no~ i n~ neomycin
~l,o~yhuLL~ rerase II are resistant to the antibiotics
kanamycin or G418. It is ~ntir-irated that other
5~lQrtAhle marker genes will be useful for identification
35 of transformants. In this example transformants are
identified based on resistnnce to th~e hQ~hinifg~ hiAlArh~5
conferred by the i,.LLuduced bar gene. Following one week
Wo 9s/3ooo5 2 1 9 0 5 9 7 . ~ 66
culture in liquid medium 409 (Table 2) without selection
,ULC:S=.llLC:, particle bombarded tissue is transferred to
liquid medium 409 (Table 2) containing 1 mg/L b;AlArhns.
Cells are transferred twice per week into fresh medium
containing 1 mg/L b;AlArhn~ for two weeks. Tissue is
thin planted 3 weeks following bombardment at a
cul.c~l.LL~Ition of 0.1 ml packed cell volume per petri dish
containing medium 425 (Table 2). Transformants are
identified as discreet r~olnnir~: 6 weeks following
boml-ar L. It is the eYperience of the inventors that
all cell lines that grow on 3 mg/L biAlArhns contain the
o~r gene.
Alternatively, following particle bombardment cells
remain on solid 279 (Table 2) medium in the absence of
selection for one week. At this time cells are removed
from solid medium, rPr-~lcppnr~r~d in liquid 279 medium
(Table 2), replated on Whatman filters at 0.5 ml PCV per
filter, and transferred to 279 medium (Table 2)
containing 1 mg/L b;Al~rhn~:. Following one week, filters
are transferred to 279 medium (Table 2) containing 3 mg/L
hiAl~rhns. One week later, cells are L~ J- ~1Pr1 in
liquid 279 medium and plated at 0.1 ml PCv on 279 medium
(Table 2)containing 3 mg/L biAlArhns. Transformants are
ldentified about 7 weeks following bombardment.
Following particle ~ ~~ ,~ L of; LULe embryos,
transformants are ~ e:-~uv~:L~d. Embryos are allowed to
recover on high osmoticum medium (735, 12% sucrose, see
Table 2~ overnight (16 - 24 hours) and are then
transferred to sr~l Pr't; nn medium containing 1 mg/l
b;AlArhns (739 or 750, see Table 2). Embryos are
r-;ntA;nPd in the dark at 24 C. After three to four
weeks on the initial selection plates about 90% of the
embryos form Type II callus and are transferred to
selective medium rnntA;n;n~ 3 mg/l hiAlArh~.~s (758, Table
2 ) . RP~Irnnrl; n~ tissue is subcultured about every two
Wo ssr3000s 2 1 q 0 5 9 7 r~
-- 49 --
weeks onto fresh selection medium (758, Table 2).
Transformants are identified six to eight weeks after
bombardment .
5 G. Pl~nt R~ tion
Plants are regenerated from transformants. The
following protocol describes method for plant
L~gell~Lc,~ion, but one skilled in the art will be familiar
lO with other equally efficient protocols. For
L~y~ L~ltiOn tissue is first transferred to solid medium
223 (Table 2) and incubated for two weeks. Transformants
may be initially subcultured on any solid culture that
supports callus growth. Subsequently tran6formants are
15 subcultured one to three times, but usually twice on 189
medium (Table 2; first passage in the dark and second
passage in low light) and once or twice on 101 medium
(Table 2) in petri dishes before being transferred to 607
medium (Table 2 ) in Plant Cons~ . Variations in the
20 lcy~J.~Li~tion protocol are normal based on the pL-JyL~=5S of
plant regeneration. Hence some of the transformants are
first subcultured once on 425 medium (Table 2), twice on
189 medium (Table 2), once or twice on lO1 medium (Table
2) followed by transfer to 501 medium (Table 2) in Plant
25 Cons~. As shoots developed on lO1 medium (Table 2), the
light intensity is increased by slowly adjusting the
distance of the plates from the light source located
overhead. All subculture intervals are for about 2 weeks
at 24C. Transformants that developed 3 shoots and 2-3
30 roots are transferred to soil.
Plantlets in soil are incubated in an i l lllminAted
growth chamber and conditions was slowly adjusted to
adapt or condition the plantlets to the drier and more
35 ; ~ mi nAted conditions of the gre~nhr~ e . After
adaptation/conditioning in the growth chamber, plants are
transplanted individually to 5 gallon pots of soil in the
w0 9sl3000~ 2 1 9 0 5 9 7 r~ cs~66
-- 50 --
grr~nhol~ce. ransformed plants in Goil are cultivated in
the gr~Pnhouce following standard gre~nhollce protocols
and pollinated using standard plant breeding t~rhn;qllr~c.
It is the experience of the inventors that seed i5
5 r~ _uvc Led from most transgenic plants generated in
aCcuL ~ n~ with these procedures .
Progeny and sllhcr~ nt generations are grown in the
field and assayed for their performance under a range of
10 water avAilAhility conditions. Both qualitative and
quantitative measures of the plant's ability to withstand
water stre6s are made. Seeds are germinated in the
y~P~ C~ growth r~h~ `~ D and field conditions under
ample water supply. At one or Dore times during the
15 plant's life cycle, water avAilAh;lity is reduced in
order to identify plants expressing the wilt gene (s) .
Visual signs of wilting or the reduction of turgor are
noted. In addition to the visual signs of wilting,
which may only be ObDeL v~:d under more ,~L ulluullut:d drought
20 stress, meaDures of plant water relations are made.
Total water potential, osmotic potential and turgor
potential are quantitatively measured and ~q~tect i t~n of
dil'ferences in turgor or the ability of the plants not to
wilt can be made even when no signs of plant stress are
25 visible to the eye. Plants expressinq the most favorable
water status result in superior growth under water
stress. Different measures of growth are used to
dc 1. this superior performance. Measures of cell and
leaf area ~Anci t~n are used to identify superior plant
3 0 growth under stress .
The physiological and biochemical activity of the
tral.DroL ' plant tissue is indicative of its improved
stress tolerance. Such screening of plants with the
35 ~ L of photosynthetic activity or transpirational
activity are examples, but not all of the types of
L that can be done to identify the superiority
wo 9s/3000s 2 1 9 0 5 9 7 PcT/Us9s/0s36c
-- 51 --
of the wilt expres6ing plants compared to non-transformed
plants. Measurements of reproductive capacity including,
but not limited to the synchrony of pollen shed and silk
y~ are indicators of ; uve d stress tolerance
5 when the wilt gene is ~ , assed. In addition, it is
anticipated that the expression of the wilt gene will
m;nimi se kernel abortion during times of stress thereby
increasing the amount of grain to reach maturity. The
expression of the wilt gene allows for superior late
10 season plant health and development of full ears. It is
contemplated that barrenness will not be a problem.
Once the initial breeding lines are select~d by the
above, but not limited to, criteria, testcrosses are
15 made and hybrid seed is produced. The testcross hybrids
and breeding populations are planted in several different
fashions in the field. One scheme of evaluation is to
grow populations of hybrid plants c~nt:~inin~ the wilt
gene in many different locations and measure the
20 performance of the plants at these different locations.
Given the variability of rainfall distribution, the
different locations receive different quantities of
rainfall and in some locations, the plants will receive
stress. Yield information as well as measures which
25 quantify plant response to stress as described earlier,
are made. The information regarding the performance of
these hybrids along with that of the performance of non-
transformed or non-wilt containing hybrids is compared.
It is anticipated that the hybrids expressing the wilt
30 gene will be higher in yield performance at a given level
of water avA i l Ahi l i ty than the controls.
Where irrigation is available, more controlled
comparisons are made through the es~hli, ~ of
i5 differential irrigation treatments. The same entries of
hybrids or lines are grown under contrasting irrigation
treatments. Such an approach limits the number of
. _ . _ _ . . _ . _ . _ _ _ _ _ _ _ _ _ _
Wo95/30005 2 1 9 0597 r ~ 3. i~66
variables at work in the evaluation. Aside from the same
types os~ measurements as defined ~bove, differential
r~ are calculated because of the contrast in the
data. It is anticipated that wilt expressing hybrids
5 will have less yield reduction when grown under irrigated
versus non-irrigated conditions when c c:d to hybrids
without the Wll t gene .
Upon the identif ication of the superior perf ormance
lO of the wllt gene expressing plants, the parent selections
are advanced and inbred lines are produced through
conventional breeding techniques. ~Iybrid plants having
one or more parents containing the Wilt gene are tested
in commercial testing and evaluation 5~LuyLcl~.. and
15 performance 1- Led. Thi6 testing include
perf-" `~DC trials over a wide geo~La~llical area as well
~5 dedicated trials where water avA;l~hil;ty is varied to
reveal performance advantage and hence value.
An additional advantage of the expression of the
Wilt gene is the superior performance of the parental
inbred lines in production of hybrids. Less stress
related parent yield loss is associated wlth higher green
seed yield and thereby higher e i r margins .
It is anticipated that the perf ormance advantage
will not only be present under stress conditions. Given
the overall role o~ water in ~lDtDrm;n;nq yield, it is
contemplated that corn plants expressing the wilt gene
will utilize water more efficiently. This will improve
overall perrormance even when soil water av~ h; 1 ity is
not limiting. Through the is,LLv~uuLion of the Wilt
gene(s) and the; uvc:d ability of corn to maximize
water usage across a full range of conditions relating to
water av~ h; l ;ty (i .e., including normal and stressed
conditions), yield stability or consistency of yield
performance will be achieved.
WO 95/30005 2 1 9 0 5 9 7 PCT/US95/0536G
.
-- 53 --
A fl~nt9 ~al premise behind these studies is that
genes defined by wilt mutations define important steps in
the environmental signal transduction pathway that
protects plants from water deficits. An test of this
hypothesis involves assessing the effects of :
uvt~ L ession on water def icit tolerance . These studies
are c~n~ t~ in maize and heterologous dicot systems.
E~aNPLE VI
~8E OF WIrT GENE8 AS PROBES IN NARKER AS8ISTED R~ nTNG
The identification of maize that are bred for
resistance to adverse water conditions may be readily
assisted by using the wilt-related gene presently
closecl, as well as genes identif ied using the methods
of the present invention. To positively identify plants
for further crossings nucleic acids are isolated from
potential Fl progeny and screened for the uvl:L :~y~SSiOn
of mRNA or protein, amplification of the gene locus or
increased stability of the gene product.
T~rhn;ql~c for isolating nucleic acids and proteins
are well known to those of skill in the art (Maniatis
et ~l., 1991), and may be u6ed in conjunction with the
gene of the present invention to selectively segregate
plants that have increased resistance to water
deprivation, with or without, genetic alteration of the
plant. Natural variants of maize may be isolated and
s~l~cted for breeding following a screen for altered wilt
resistance gene message, product or product stability.
Accordingly, it is contemplated that wilt genes will
be useful as DNA probes for marker assisted breeding. In
the process of marker assisted breeding DNA S~qll~n~ ~fi are
used to follow desirable a~L, - ~c traits (Tanksley
et cll., 1989) in the process of plant breeding. It is
WO 95/30005 2 ~ 9 0 ~ 9 7 PCT/IJS95/05366
-- 54 --
anticipated that a wilt gene probe will be useful for
identification of plants with Anh~ncecl ability to utilize
water resuuL~es. Furthermore, through the use of more
than one wilt gene probe it will be possible to combine
genes that enhance the ability to utilize water. It is
contemplated that such a combination of genes would be
difficult to identify without marker as6isted breeding,
unless the plant is grown under conditions of limited
water availability.
Marker assisted breeding using the wilt gene is
undertaken as follows. Seed of plants with the desired
ability to utilize water resouL~es are planted in soil in
the gr~AAnh~u~e or in the field. Leaf tissue is harvested
from the plant for yL~lL~ltiOn of DNA at any point in
growth at which approximately one gram of leaf tissue can
be removed from the plant without i ~ing the
viability of the plant. Genomic DNA is isolated using a
~LV~ ~duLa modified from Shure et al . (1983) .
Approximately one gram of leaf tissue from a seedling is
lyorh; ~ overnight in 15 ml polypropylene tubes.
Freeze-dried tissue is ground to a powder in the tube
using a glass rod. Powdered tis6ue is mixed thoroughly
with 3 ml extraction buffer (7.0 M urea, 0.35 M NaCI,
0.05 M Tris-HCI ph 8.0, 0.01 M EDTA, 1~6 sarcosine).
Tissue/buffer ~ e is extracted with 3 ml phenol
chloroform. The aqueous phase is separated by
centrifugation, and precipitated twice using 1/10 volume
of 4 . 4 M i l~m acetate pH 5 . 2, and an equal volume of
isopropanol. The precipitate is washed with 7596 ethanol
and r~cllcrAn~ in 100-500 ~1 TE (0.01 M Tris-HCI, 0.001
M EDTA, pH 8.0). Genomic DNA is digested with a 3-fold
excess of restriction enzymes, ele~:~Lùphur~sed through
0 . 8% agarose (FMC), and transferred (Southern, 1975) to
Nytran using lOX SCP (20X SCP: 2 M NaCI, 0.6 M disodium
phosphate, 0 . 02 M ~l; cotl;l~m EDTA) .
wo 95/30005 2 1 9 O 5 q 7 P~
-- 55 --
One of skill in the art will recognize that many
different restriction enzymes will be useful and the
choice of restriction enzyme will depend on the DNA
se-lu~-lce of the wil t gene that is used as a probe and the
5 DNA seguence in the maize genome ~uLLuu-.ding the wilt
gene. One will 6elect a restriction enzyme that produces
a DNA fragment following hybridization that is
identifiable as that wilt gene. It is anticipated that
one or more restriction enzymes will be used to digest
10 genomic DNA either singly or in combinations. Filters
are ~ ybLidized in 6X SCP, 10% dextran sulfate, 2%
sarcosine, and 500 ~Lg/ml denatured salmon sperm DNA and
32p-lAhPllPd wilt gene probe generated by random priming
(Feinberg & Vogelstein, 1983; Boehringer-Mlnnh~lm~.
15 Hybridized filters are washed in 2X SCP, 1% SDS at 65
for 30 minutes and visualized by autoradiography using
Kodak XAR5 film. Those of skill in the art will
reco~n; 7e that there are many different ways to isolate
DNA from plant tissues and that there are many different
20 protocols for Southern hybridization that will produce
identical results. Those of skill in the art will
rDro~n; ~ that a Southern blot can be stripped of
rA~lioArt ive probe following autoradiography and re-probed
with a different wilt gene probe. In this manner one
25 identifies each of the various wilt genes that is present
in the plant.
Each lane of the Southern blot represents DNA
isolated from one plant. Through the use of a
30 multiplicity of wilt genes as probes on the same genomic
DNA blot, the wilt gene composition of each plant is
det~rmln~. Correlations are es~Ahl;~hPd between the
contrih~l~ionC of particular wilt genes to the ability of
the plant to adapt to conditions of decreased water
35 avA~lAh;lity. Only those plants that contain the desired
combination of wilt genes are advanced to maturity and
used for pollination. DNA probes C~JLL~ 1;n~ to wilt
Wo95/30005 21 9 0597 r~ 66
genes are useful markers during the course of plant
breeding to identify and combine particular wilt genes
without having to grow the plants under conditions of
decreased water ava;lAhil;ty and assay the plants for
5 a~L i ~ performance under these ~Lessed conditions .
* * *
All of the compositions and methods disclosed and
10 claimed herein can be made and executed without undue
experimentation in light of the present disclosure.
While the compositions and methods of this invention have
been described in terms of preferred ~mho~ Ls, it will
be ay~a~c:..L to those of skill in the art that variations
15 may be applied to the composition, methods and in the
steps or in the sequence of steps of the method described
herein without departing from the concept, spirit and
scope of the invention . Nore specif ically, it will be
,l~y~lellL that certain agents which are both chemically
20 and physiol~girAlly related may be 5ub5tituted for the
agents described herein while the same or similar results
would be achieved. All such similar substitutes and
modifications ~a~ellL to those skilled in the art are
deemed to be within the 5pirit, 5cope and concept of the
25 invention as def ined by the Arp~nrle~rl claims .
Wo 95/30005 2 1 9 0 5 9 7 r~l/u~ cs~66
-- 57 --
R~;~
The following references, to the extent that they
provide exemplary ~ceduLcll or other details
5 suppleDentary to those set forth herein, are specifically
incc,L~uLated herein by reference.
Agriculture, U. S. Dt~aL; L of. 1990. Agricultural
Statistics. US GUV~L ~ Printing Office, Washington,
D.C.
Alleman & Freeling. 1986. The Mu tr;~nRros~hl e elements
of maize: evidence for LLa~ osition and copy number
regulation during devPll L, Genetic, 112:107-119.
Altschul, et al. 1990. Basic local ~ L search
tool. J. Mol. Biol. 215: 403--410.
LLUj~ et al. 1991. Maize GPnPtiCF Coop N~ lettPr
65:92-93.
Ausebel et al. 1992. Current Protocols in Nolecular
Biology. Current Protocols, Boston.
8arnett & Naylor. 1966. Amino acid and protein
metabolism in Bermuda grass during water stress. Plant
Physiol. 41: 1222.
Bennetzen. 1987. Covalent DNA modification and the
regulation of Nutator element LLa~lDuosition in maize,
Nol. Gen. Genet., 208:45-61.
Bennetzen et al. 1988. The state of DNA modification
within and flaking maize trAnRpnCAhlP Pl~ - L:,. In
Nelson OE (ed) Plant TrAnRp~s~hle ~l- Ls, Plenum Press,
New Yûrk, pp . 2 3 7 -2 5 0 .
wo95/30005 21 90597 r~lluv~ 66
-- 58 --
Bevan. 1984. Binary Agrobacterium vectors for plant
transformation. Nuc. Acid. Res. 12: 8711-8721.
Bouchez et al. 1989. EMB0 Journal 8(13) :4197-4204.
Boyer. 1976. Water deficits and photosynthesis, pp.
154-190 -in Water deficlts and plant growth, edited by T.
T. Kozlowski. A~'A~1Pm;~ Press, New York.
Brown et al. 1989. Molecular analysis of multiple
Mutator-derived alleles of the Bronze locus of maize,
Genetics, 122:439-443.
Buckner et al. 1990. Cloning of the yl locus of maize,
a gene involved in the biosynthesis of carotenoids.
Plant Cell 2: 867-876.
ohAn~lPr & Walbot. 1986. DNA modification of a maize
Lr~ sAhle element correlates with loss of activity,
Proc. Natl . Acad. sci . USA, 83 :1767-1771.
Chomet et al. 1987. EMB0 J 6:295-3~2.
Chomet et al. 1991. Identification of a regulatory
25 trAnCpnFon that controls the Mutator trAn~p~sAhle element
system in maize, Genetics, 129:261-2~0.
Chomet. 1994. I~ ,ofi~n tagging with Mutator, pp.
243-249 in The Maize ~TAn~honl~, edited by M. Freeling and
30 V. Walbot. Springer-Verlag, New York.
Chu et al . 1975 . Scientia Sinica 18: 659-668 .
Clark. 1982. J. of Plant Nutrition 5: 1039.
Wo ss/30005 2 1 q C 5 q 7 Pcrlu59~10~366
-- 59 --
Coen & Meyerowitz. 1991. The war of the whorls: genetic
interactions controlling flower development. Nature 353:
31-37 .
5 Coen. 1991. The role of homeotic genes in flower
devPl L and evolution. Annu. Rev. Plant Physiol .
Plant Mol. Biol. 42: 241-279.
Cone. 1994. Tr~ncro~on tagging with Spm, pp. 240-242 in
10 The Maize ~n~hon~, edited by ~. Freeling and V. Walbot.
Springer-Verlag, New York.
Cone et al. 1986. Molecular analysis of the maize
anthocyanin regulatory locus C1. Proc. Natl. Acad. Sci.
USA 83: 9631-9635.
Delauney & Verma. 1993. Proline biosynthesis and
-- ~tyulation in plants. The Plant Journal 4: 215-223.
20 ~PllAr~rta et al. 1988. M~lecl~lAr cloning of the R-nj
gene by LL,.n-l,os~ tagging with Ac, pp. 263-282, in
~11L- ~ SLLucLuLe: and Function: Impact of New
Concepts, edited by J . P . Gustaf son and R. Appels .
Plenum Press, New York.
Dellaporta ~ Moreno. 1994. Gene tagging with Ac/Ds
in maize, pp. 219-239, in The Maize ~An~lhoo~
edited by M. Freeling and V. Nalbot. Springer-Verlag,
New York.
DeLong et al. 1993. Sex Determination gene Tassleseed2
of Maize encodes a short-chain alcohol de~.y-lL ~ nllse
required ~or stage-specific floral organ abort~n. Cell
74: 757-768.
Wo 95/3000~ r~
21 90591
Dooner & ~al~rhaw. 1989. Transposition pattern of the
maize element Ac from the bz-m2 (Ac ) allele, Genetlcs,
122: 447-457 .
5 Dure et al. 1989. Common amino acid sequence domain6
among the LEA proteins of higher plants. Plant Mol Biol
12: 475-486.
Ellis et al. 1987. ENB0 Journal 6(11) :3203-3208.
Federoff. 1988. TrAncpncJ~hle a~ ~s and process for
using same. United State6 Patent No. 4,732,856.
Fedoroff et al. 1984. Cloning of the bronze locus in
15 maize by a simple and generA l; 7z-hle pLc ceduLe using the
LLAI,~l,o--hla controlling element Activator (Ac). Proc.
Natl. Acad. Sci. USA 81: 3825-3829.
Feinberg & Vogelstein. 1983. Anal Blochem 132:6-13.
Finkelstein & Somerville. 1990. Three classes of
Abscisic Acid (ABA)-insensitive mutations of Arabidopsis
def ine genes that control overlapping subsets of ABA
L~ cac. Plant Physiol. 94: 1172-1179.
Freeling & Walbot. 1994. The Naize T~;ln~honk.
Springer-Verlag, New York.
Gordon-Kamm et ~l. 1990. Transformation of maize cells
30 and ~g.~ LatiOn of fertile trAncga-A;r plants. Plant
Cell 2: 603-618.
Greenblatt & Brink. 1962. Twin mutation~ in medium
variegated pericarp maize, Genetics, 47:489-501.
Guan et al. 1988. Vectors that facilitate the
expression and purification of foreign peptides in
w095~3000s 2 1 9 0 5 9 7
-- 61 --
Escherichia coli by fusion to maltose binding protein.
Gene 67: 21-3 0 .
Hake et al. 1989. Cloning Knotted, the dominant
5 morphological mutant in maize using Ds2 as a transposon
tag. EMBO J. 8: 15--22.
Hake. 1992. Unraveling the knots in plant development,
~rrends Genet ., 8 :109-114 .
Harlow & Lane. 1988. Ant;hs~ : A Laboratory Nanual.
Cold Spring Harbor Press, Cold spring Harbor, NY.
Henikoff & Henikoff. 1991. Automated assembly of
15 protein blocks for database searching. Nucl. Acids Res.
19: 6565-6572.
Hershberger et al. 1991. Mutator activity in maize
correlates with the ~LdSell~.t and expression of the Uu
20 tr?.n~pssAhle element Mu9, Proc. Natl. Acad. Sci. USA,
88: 10198-10202 .
Hosington. 1987. Maize (Zea mays L. ) RFLP clones and
linkage map - a public set. Genetics 116: 827.
Jackson. 1992 In situ hybridization in plants, pp.
163-174, in Plant Mnler--lAr Pathology: A Practical
Approach, edited by S. J. Gurr, M. J. McPherson and D. J.
80wles. Oxford University Press, oxford.
Johal & Brigg~. 1992. Reductase activity encoded by the
Hml Disease Resistance Gene in Maize. Science 258:
985-987 .
35 ~ rr-nn & Ferenci. 1982. Maltose-binding protein
from Escherichia coli. Nethods in Enzymol. 90: 459-463.
w095/3000s 2 1 9 05 97 ~ s
Koorneef et al. 1982. The isolation of Ahcc;ci~ acid
(ABA)-deficient mutants by selection of induced
revertants in non-germinating gibberellin sensitive lines
of Arabidopsis thAliAnA (L.) Heynh. Theoret Appl Genet
61: 385-393.
Krzyzek et al. United States Patent Application
07/635, 279 .
Le Grice & Grueninger-Leitch. 1990. Rapid purification
of ~ - HIV-1 reverse transcriptase by metal chelate
affinity chromatography. ~ur. J. Biochem. 187: 307-314.
Lloyd et al . 1992 . Ari~hi ~1npci C and nicotiana
anthocyanin production activated by maize regulators R
and C1. Science 258: 1773-1775.
Mart;PnccPn et al. 1989. Mnlpr~ r cloning of a nuclear
gene in maize involved in thylakoid organization, and its
regulation by Robertson's mutator. EMBO J. 8: 1633-1640.
Mar~iPnccpn et al. 1990. Somatically heritable switches
int he DNA modification of Mu tr~ncposAhle Pl~ LD
monitored with a Duy~L~ssible mutant in maize, Genes
Dev., 4: 331-343 .
McCarty et al. 1989. Molecular analysis of viviparous-
1, an ~heCieir acid-insensitive mutant of maize, Plant
Cell, 1: 523-532 .
McCue & Hanson. l99O. Drought and salt tolerance:
towards understanding and application. TIBTECH 8:
358-362 .
MrT.All~hl in & Walbot. 1987. Cloning of a mutable bz2
allele of maize by tranCposnn tagging and dif f erential
hybridization, G-- P~ir5, 117:771-778.
WO95/30005 - 63 - r~ c~66
M;rhPl ~ et al. 1991. Markers linked to
disease-resi6tance genes by bulked segregant analysis: a
rapid method to detect markers in specif ic genomic
regions by using segregating populations. Proc. Natl.
Acad. Sci. USA 88: 9828-9832.
Milborrow. 1981. Ahccil::;r acid and other hormones, pp.
348-388, in The physiology and biorhPmi ctry of drought
resistance in plants, edited by L. G. Paleg and Aspinall.
10 Academic Pre66, New York.
Mundy et al. 1990. Nuclear protein6 bind conserved
elements in the Ah5~; cic acid-respon6ive promoter of a
rice rab gene. Proc. Natl. Acad. Sci. USA 87: 1406-1410.
Mura6hige T., Skoog F. (1962). Phy6iol Plant 15:473-497.
Neuffer. 1989. Designation of four dominant mutant6.
Maize Genetic6 Cooperation New6 Letter 63: 62-63.
Neuffer. 1990. Location, description and notes on other
dominant mutants. Maize Genetics cooperation News Letter
64: 51-52.
25 Nowick & Peterson. 1981. I~ osition of the ii~nhi~-nrclr
controlling element sy6tem in maize, Mol. Gen. Genet.,
183: 440-448 .
O'Reilly et al . 1985 . M~1PCI11 Ar cloning of the al locus
of Zea mays using the tr1~ncp~cAhle ~ En and Mu.
EMBO J. 4: 591--597.
Odell et al . 1985 . Nature 313: 810-812 .
Orton & Brink. 1966. Recon6titution of the variegated
pericarp allele in maize by tranposition of Modulator
back to the P locu6. Genetics 53: 7-16.
wo ss/3000s 2 1 ~ O ~ q 7 PCT/US95/05366
Patterson et al. 1991. Genetic analysis of B-peru, a
regulatory gene in maize, Genetics 127: 205-220 .
Postlethwait & Nelson. 1957. A chronically wilted
mutant of maize. Amer. J. Bot. 44: 628-633.
Pryor. 1987. Stability of alleles of Rp. Maize Genetics
Cooperation News Letter, 61: 37-38 .
10 Qin et al. 1991. Cloning of the Nutator trAncpnc~hle
element MuA2: a putative regulator of 60matic mutability
of the al-Mum2 allele in maize, Genetic6, 129:845-854.
Quarrie et al . 1988 . A l r~n~ l antibody to
15 (S)-abscisic acid: its characterization and use in a
radio; cs~y for measuring abscisic acid in crude
extracts of cereal and lupin leaves. Planta 173:
330-339 .
Ristic & Cass. 1991. Leaf anatomy of Zea may6 L. in
e to water shortage ~nd high t~ .ILUL_: A
comparison of drought-resistant and drought-sensitive
lines. Bot. Gaz. 152: 173-185.
Robf L Lsu.l. 1978 . Characterization of a mutator system
in maize, Mut. Res., 31:21-25.
Robertson. 1980. The timing of Mu activity in maize,
Gonot;r~c~ 94:969--978.
Robertson. 1985. Differential actiYity of the maize
mutator Mu at different loci and in different cell
1 ;no~ c~ Mol . Gen. Genet., 200:9-13.
RO~C:L L~u~. & stinard. 1989 . Genetic analyses of putative
two-element system regulating somatic: ~Itability in
Wo 9S/30005 2 1 9 0 5 9 7 PcrluS9SlOS366
Mutator-induced aleurone mutants of maize, Dev. Genetics,
10: 482-508 .
Sambrook et al. 1989. Molecular Cloning: A Laboratory
5 Manual, Cold Spring Harbor Laboratory, Cold Spring
Harbor, NY.
Schmidt et al. 1987. Tr~n~pnRnn tagging and molecular
analysis of the maize regulatory locus opaque-2. Science
238:960-3.
Shure et al. 1983. Cell 35:225-233.
Sinha et al. 1993. Ove~ Lession of the maize h~ -ohn~r
15 gene, KNOTTED-l, causes a switch from determinate to
indeterminate cell fates. Genes and Dev. 7: 787-795.
Skriver & Mundy. 1990. Gene expression in Le:~uullSe to
~hc:r; F:; r acid and osmotic stress. Plant Cell 2: 503-512 .
Southern. 1975. J. Mol Biol 98:503-517.
Stadler. 1948. Spontaneous mutation at the R locus in
maize I. The aleu~ us-color and plant-color effects,
Genetic6, 31:377-394.
Sullivan et al. 1991. Analysis of maize brittle-l
alleles and a defective ~u,u~, c ~L-mutator induced
mutable allele. Plant Cell 3: 1337-1348.
Sylvester & Ruzin. 1994. Light mi~L~scu~y I: dissection
and microtechnique, pp. 83-95, in The Maize ~ ntlhonk,
edited by M. Freeling and V. Walbot. Springer-Verlag,
New York.
Tanksley et al. 1989. Bio/Technology 7: 257-264
Wo ss/3000~ 2 ~ ~ 0 5 q 7 PC~IU595105366
-- 66 --
Walbot. 1991. Early event sin Mutator lines. I. Germanl
revertants, IL somatic reversion, Maize Genetics
Cooperation News Letter 65:95-96, cited by permission.
5 Walbot. 1992. Strategies for mutl~n~cic and gene
cloning using trAnCposnn tagging and r-DNA insertional
mutagenesis , Armu . Rev. Plant Phys . Plant Mol . Biol ., in
press .
10 Walbot. 1992. Early sectoring and germinal reversion in
a Nutator line with the bs2-mU2 reporter allele. Maize
Genetics Cooperation New Letter, 66:101, cited by
p~rm; CC~
15 Walbot et al. 1986. Properties of mutable alleles
~ecuv~ d from mutator stocks of Zea ~aya L. In
Guatafaon JP, Stabbins GL, Ayals FJ (eds) Genetics,
Dev~l~, L and Evaluation, Planum Pre6s, New York, pp.
115-142 .
Wang et al. 1992. Nolecular and Cellular Biology 12:
3399-3406 .
Wienand et al. 1986. Molecular cloning of the c2 locus
25 of Zea mays, the gene coding for r-h~lcnn~ synthase. Mol
Gen Genet 203: 202-207.
Wo ss/3ooos PCT/US95/0536
~ 2~ 905q7
-- 67 --
SEQUENCE LISTING
( 1 ) GENERAL INFORMATION:
( i ) APPLICANT:
(A) NAME: DEKALB GENETICS CORPORATION
(B) STREET: 62 MaRITIME DRIVE
(C) CITY: MYSTIC
(D) STATE: CC/NN~ . lClJT
(E) COUNTRY: UNITED STATES OF AMERICA
(F) POSTAL (ZIP) CODE: 06355--1959
and
(A) NAME: YALE UNIVERSITY
(B) STREET: 451 COLLEGE STREET
(C) CITY: HEW HAVEN
(D) STATE: ~:VNN~ lCUT
(E) COUNTRY: UNITED STATES OF AMERICA
(F) POSTAL (ZIP) CODE: 06520
(ii) 1NVk~O~S: CHOMET, Paul
DELLAPORTA, Stephen L.
ORR, Peter
KRUEGER, Roger
2 5 LOWE, Brenda
(iii) TITLE OF INVENTION: GENES REGULATING THE
~ OF ZEA MAYS TO
WATER DEFICIT
(iv) NUMBER OF ~;yu~;N~ ;N: 1
(V) ~;OKKE~ ..JL _I!; ADDRESS:
(A) Anm7~ ARNOLD, WHITE & DURKEE
(B) STREET: P.O. BOX 4433
tc) CITY: HOUSTON
(D) STATE: TEXAS
WO 95/3000~ 2 1 9 0 5 9 7
-- 68 --
(E) COUNTRY: UNITED STATES OF AMERICA
(F) ZIP: 77210
(Vi) ~:U_~Ulc,l~ RT~AnAl~T,T~. FORM:
(A) MEDIUM TYPE: Floppy disk
(B) Cu..~ul~: IBN PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS/ASCII
(D) SOFTWARE: PatentIn Release #1.0, Version
#1.30
(vii) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: UNKNOWN
( B ) FILING DATE: 2 7 -APR--19 9 5
(C~ CLASSIFICATION: UNKNOWN
(viii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: USSN O8/235, 060
(B) FILING DATE: 29-APR--1994
(C) CLASSIFICATION: UNKNOWN
( ix) ATTOPNEY/AGENT INFORNATION:
(A) NAME: HIt'.~T.ANnT.'R, STEVEN L.
(B) REGIS~RATION NUMBER: 37, 642
(C) REFERENCE/DOCKET NUMBER: DEKM087P--
(X) TT~'T.T~`C~- .. IN I CATION INFORMATION:
(A) TELEPHONE: (512) 418-3000
(B) TELEFAX: (713) 789--2679
(C) TELEX: 79--0924
(2) INFt)RM~'rTN FOR SEQ ID NO:l:
U~;NCI~; CHARACTERISTICS:
(A) LENGTH: 247 base pairs
(B) TYPE: nucleic acid
(C) STRANr~T~nNT~: single
W095/30005 21 9 05q7 r~ J~66
--69--
O O O O ~`
N 'D N ~ N N
t~
t
N
t~
~`
o
C~
'
1i3
` O
C) ~1
U: ~ tJ O
Id ~ N 52 2:
~ a ~ ~ ~1 a ~ '
.. : cq ~r o
11 R _ 0
u ~r tq
h c~ ~ _
h ~ ' O
C) _ I :
.~ S ' ~ E~
.. ..
;'- ~ E-~ ` -
Z ~ O ~
a
-- -- -- Z ~ ~
~î X ~ ~î ~
_ o
-
U~ o
~1 ~I t`~
.
