Note: Descriptions are shown in the official language in which they were submitted.
62957-211
Back~round of the Invention
It i5 well known that when different plant lines
are cross-pollinated one can achieve in the offspring a highly
desirable heterosis or hybrid vigor which advantageously
provides increased yields of the desired crop.
Soybeans (l.e., seeds of Glycine ~ax plants) are
recognized to be an important crop in many parts of the world.
For instance, approximately 65 to 75 million acres of soybeans
are planted annually in the United States which establi3hes
this to be the largest seed crop presently grown in the United
States. Various approaches to the production of hybrid
soybeans are disclosed in United States Patent Nos. 3,903,645
and 4,077,157, and in my copending Canadian Serial No. 475,718.
Also, technical articles which discuss the existance of some
degree of sterility in soybeans and the formation of hybrid
soybean seeds are identified in my copending Canadian Serial
No. 475,718.
It is an object of the present invention to provide
an improved process for producing seeds capable of forming Fl
hybrid soybean plants wherein pollen-carrying bees are employed
to accomplish the required pollen transfer.
It is an object of the present invention to provide
an improved process for maintaining male sterile soybean plants
useful in the production of male fertile Fl hybrid soybean
:
plants wherein pollen-carrying bees are employed to accomplish
the required pollen transfer.
,,
:~;
.
It is an object of the present invention to
provide an improved process for producing seeds capable of
forming Fl hybrid soybean plants wherein the required
visitatlon between the parent plants by pollen-carrying bees
is promoted on a highly effective basis.
It is another object of the present invention to
provide an improved process ~or maintaining male sterile
soybean plants useful in the production of male ~ertile F
hybrid soybean plants wherein the re~uired visitation
between the parent plants by pollen carrying bees is
promoted on a highly effective basis.
It i~ a further ob;eck of the present invention to
provide an improved process for producing seeds capable of
forming Fl hybrid ~oybean plants wherein the seed product is
caused to set in increased yields.
These and other objects as well as the scope,
nature, and utilization of the claimed invention will be
apparent to those skilled in the art from the following
detailed description and appended claims.
Summary of the_Invention
It has been ~ound that an improved process for the
efficient production of seeds capable o~ growing Fl hybrid
Glycine max comprises:
(a) growing at a location which normally experi-
ences limited natural rainfall when soybean
flowering occurs during the summer a substan-
tially uniform population of male sterile
soybean plants in pollinating proximity to a
ubstantially uni~orm population of male
fertile soybean plants which when crossed
with the male sterlle soybean plants enable
the ~ormation o~ seeds on the male sterile
~oybean plants which are capable of growing
male fertile Fl hybrid soybean plants,
(b) applying water via irrigation to the substan-
tially uni~orm populations of soybean plants
of step (a) as required to promote normal
~ plant growth up to at least the time of the
onset of ~lower formation within each of the
populations,
(c) withholdin~ irrigation water from the plants
: of the substantially uni~o~m populations at a
~ime when flowers are present within each of
the substantially uniform population~ ~or a
period of time during which no appreciable
natural rainfall occurs so as to induce
enhanced nectar flow within the flowers which
serves to render the ~lowers mora a~tractive
: : ~ to bees,
(~d? crossing the male sterile soybean plants and
the male ~ertile soybean pla~ts with the aid
:~ . of pollen-carrying bees which are attracted
;:
to the enhanced nectar ~low whereby seeds are
; .
:: ~ormed on the male sterile soyb~an plants,
and
(e) selectively recoveriny the seeds which have
~ormed on the substantially uniform popula-
tion o~ male sterile soybean plants.
,:
3- -
~,
It has been found that an improved process for the
afficient production of a binary seed blend containing a
substantial proportion of seeds capable of growing male
fertile Fl hybrid Glycine max plants comprises:
(a) growing at a location which normally experi-
ences limited natural rainfall when soybean
flowering occurs during the su~mer a sub~tan-
tially random population of male sterile
soybean plants and male ~ertile soybean
plant~ which when crossed with the male
sterile soybean plants enable the ~ormation
of seads on the male sterile soybean plants
which are capable of growing male fertile F
hybrid soybean plants,
(b) applying water via irxiyation to the substan-
tially random population of soybean plants of
step (a) as required to promote normal plant
growth up to at least the time of the onset
of ~lower formation,
(c) withholding irrigation water from the plants
of the substantially xandom population at a
time when flowers are present for a period of
time durin~ which no appreciable natural
' rainfall occurs so as to induce enhanced
nectar flow wikhin the ~lowers which serves
to render the flowers more attractive to
bees,
(d) pollinating the substantially random popula-
tion of soybean plants with the aid o~
: pollen-carrying bees which are attracted to
~4-
the enhanced nectar ~low whereby seedR are
formed on the male sterile plants which are
capable of growing male fertile Fl hybrid
soybean plants and saeds are formed on the
male fertile soybean plants as a result of
self-pollination, and
(e) recovering seeds which have ~ormed on the
subs~antially random population of soybean
plants.
It has been found that an improved process for
maintaining male sterile ~ B~ ~3~ plants useful in the
production of male fertile Fl hybrid soybean plant~ compri-
ses:
(a) growing at a location whlch normally experi-
ences limited natural rainfall when soybean
flowering occurs during the summer a ~ubstan-
tially uniform population of cytoplasmically
, male sterile soybean plants in pollinatlng
i proximity to a substantially uniform popula-
ion of male fertile main~ainer soybean
plants which when cros~ed wlth the cytoplas-
mically mal~ sterile soybean plants enable
the forma~ion of seeds on the cytoplasmically
male sterile plants which are capable of
: growing addi~ional cytoplasmlcally male
~ .
sterile pl2nt5,
~; ~ (b~ applying wa~er via irriga~ion to the ~ubstan-
tially uniform populations of soybean plants
: - of step (a) as required to promot~ normal
:~
_5_
.
plant growth up to at least the time of the
onset of ~lower formation within each of the
populations,
(c) withholding irrigation water from the plants
o~ the substantially uniform populations at a
time when flowers are present within each of
the substantially uniform populations for a
period of time during which no appreciable
natural rainfall occurs so a~ to induce
enhanced nectar flow within the flower~ which
serves to render the flowers more attractive
to bees,
(d) crossing ~ha cytoplasmically male sterile
soybean plants and the male fertile main-
tainer soybean plants with the aid of
pollen-carrying bees which are attracted to
the enhanced nectar flow whereby seeds are
formed on the male sterile soybean plants,
and
(e) selectively recovering the seed~ whioh have
formed on the substantially uniform popula-
tion of cytoplasmically male ~terlle soybean
plants.
,
;~ Detailed Descripti n of the Inventi_n
In accordance with the con~ept of the present
in~ention it is essential tha~ the parent ~oybean plants be
: gxown at a location which normally experiences limited
natural rainfall during the summer months when soybean
flower~ normally are ~ormed and cross-pollination is carried
-6~
~2~ i75
out with the aid of pollen-carrying bees. Natural rainfall
when soybean flowers are preaent can adversely impact upon
the enhanced nectar flow within the soybean flowers made
possible in the improved process of the present invention.
Accordingly, the location where the process of the present
invention is carried out generally will be dif~erent than
those areas where soybean plants customarily have been grown
in the past. For instance, the bulk of those areas of the
United States pr~viously devoted to the growing of soybeans
for commercial crops and seeds will be unsuitable for hybrid
seed production in accordance with the concept of the
pre~ent invention. If the planting area experiences
significant rainfall during the summer months when soybean
~lowers are present, this will introduce an element of
unpredictability which will make it infeasible to attempt to
implement the concept o~ the present invention. Such
rain~all at an inappropriate time will destroy the highly
e~fective enhanced pollen transfer among soybean plants as
provided herein.
The location selected for growing the soybean
plants may be one which experiences (1) limited natural
rainfall throughout the entire year, or (2) li~ited natural
rainfall during the summer months when soybean flowering
capable of producing cross-pollination occurs ~i.e., soybean
flowers are present on each parent~ and more plentiful
xainfal1 at other times during the year.
In a preferred embodiment of the present invention
the location selected to grow the soybean paxent plants will
normally experience les~ than 4 inches o~ rainfall when
soybean flowering occurs during the summer ~ e., the
--7~
~25~7~7~
62957-211
average natural rainfall for the past 10 years will be less
than 4 inches when soybean flowering simultaneously occurs on
each parent during the 3 summer months). In a particularly
preferred embodiment of the present invention the location
selected to grow the soybean parent plants will normally exper-
ience less than 2 inches of rainfall (e.g., less than 1 inch of
rain) when soybean flowering occurs during the summer (i.e.,
the average natural rainfall for the past 10 years will be less
than 2 inches when soybean flowering simultaneously occurs on
each parent during the 3 summer months).
In the United States the improved process of the
present invention commonly will be carried out in relatively
dry areas which are generally west of those where soybean
plants customarily are grown. For instance, limited areas from
within the Great Plains may be selected extending from South
Dakota, eastern Wyoming, Nebraska, eastern Colorado, western
Kansas, the Oklahoma panhandle, eastern New Mexico and western
Texas which commonly are irrigated from the vast underground
Ogallala Aquifer. See, "Plant Stress and Water Conservation
Research Program: A Progress Report" published by the College
of Ayricultural Sciences, Texas Tech University, Lubbock, Texas
(1984) at Page 7 for a map which illustrates the location of
the Ogallala Aquifer. The drier areas of Nebraska, Kansas, and
western Texas are particularly suited for carrying out the
improved process of the present invention.
Representative counties served by the Ogallala
Aquifer where the process of the present invention can be
; - 8 -
g~
carried out to particular advantage includa the South Dakota
counties o~ Shannon, Bennett, Todd, Tripp, Gregory, and
Gillette; the Wyoming counties of Converse~ Laramie, Platte,
Goshen, and Niobrara; the N~braska counties of Dawes, Box
Butte, Garden, Kimball, Morrill, Cheyenne, Sheridan, Banner,
Scotts Bluff, Keith, Arthur, McPherson, Grant, Cherry,
Hooker, Thomas, Deuel, Keya Paha, Perkins, Chase, Dundy,
Hitchcock, Red Willow, Hayes, Frontier, Lincoln, Logan,
Loup, Garfield, Holt, Rock, Valley, Greely, Sherman, Howard,
Buf~alo, Hall, Gosper, Phelps, Kearney, Furnas, Harlan,
Franklin, Webster, Adams, Wheeler, Antelope, and Boone; the
Colorado counties oE Sedgwick, Phillips, Logan, Weld,
~organ, Washington, Yuma, Kit Carson, Cheyenne, Kiowa,
Prowers, Baca, Las Animas, Bent, Otero, Crowley, and
Lincoln; the Kansas counties of Cheyenne, Rawlins, Sherman,
Thomas, Decatur, Sheridan, Norton, Phillips, Graham,
Wallace, Logan, Greeley, Wichita, Scott, Ness, Hamilton,
Kearney, Finney, Stanton, Grant, Ha~kell, Gray, Ford,
Hodgman, Morton, Stevens, Seward, Mead, Clark, Kiowa,
Comanche, Edwards, Pawnee, Pratt, Staf~ord, Barton, Rice,
Reno, Kingman, McPhereson, Harvey, and Sedgwick; the
Oklahoma counties of Cimarron, Texas, Beaver, Harpar,
Woodward, Roger Mills, and Dewey; the New Mexico counties of
Curry, Roosevelt, Chaves, L~a, and Quay; and the Taxas
countie~ o~ Hartley, Dallam, Sherman, ~oore, Hansford,
Hutchinson, Carson, Roberts, Hemphill, Gray, Wheeler, Deaf
Smith, Randall, Armstrong, Donley, Parmer, Castro, Swisher,
Briscoe, Bailey, Lamb, Hale, Floyd, Motley, Cochran,
Hockley, Lubbock, Crosby, Dickens, Yoakum, Terry, Lynn,
_g _
7~7~
Garza, Gaines, Dawson, Andrews, Martin, Mitchell, Ector,
Midland, and Glasscock.
The thirteen counties of northeastern Arkansas
(i.e., Clay, Craighead, Cross, Crittenden, Greene,
Independence, Jackson, Lawrence, Mississippi, Poinsett,
Randolph, White and Woodruf~) are well suited for carrying
out the process of the present invention since rainfall
normally is limited at that location during the summer
months.
Other illustrative areas in the United States
where the process of the present invention conveniently can
be practiced include the irrigated areas of Idaho, Oregon,
Washington, California, etc.
Illustrative areas outside of the United States
where the process of the present invention conveniently can
be practiced inalude Argentina, Brazil, Chile, Mexico,
Canada, Egypt, South Africa, Sudan, Turkey, U.S.S.R., India,
People~' Republic o~ China, Australia, New Zealand, etc.
~ It should be understood, however, that the process
:~ of the present invention can be carried out at any location
in the northern or southern hemisphQres where the ~oil will
support the growth o~ soybean plants, limited rainfall
occurs during the summer, and irrigation water is available
to promote normal plant growth~
It is preferred that the area selected be one in
which.th~re is a relatively low pestlcide usage, honeybees
customarily are kept and are available, and a wild be~
population is available.
The key parent soybean plants whi~h are grown in
accordance with the concept of the present invention are
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62957-211
male sterile soybean plants that serve as the seed parents and
male fertile soybean plants that serve as the pollen parents.
The male sterile soybean plants are fully female fertile, but
yield no viable pollen that produces unwanted self-pollination.
These male sterile soybean plants serve as the female parents
following the required cross-pollination. Accordingly, all of
the seed formed on the male sterile soybean plants following
pollination will be capable of forming the desired soybean
plants. The male sterilit~ of the male sterile soybean plants
can be of varied causation. For instance, such plants may be
cytoplasmically male sterile or genetically male sterile.
Alternatively, such plants may be rendered male sterile through
the application of a chemical which destroys the ability of the
plants to yield viable pollen (l.e., a gametocide). Represent-
ative gametocides include FW45~ available from Rohm & Haas Co.,
I'D-1123 available from Pennwalt Corp., potassium 3,4-dichloro-
5-isothiazolecarboxylate, 2,3-dichloroisobutyric acid and the
water soluble salts thereof (e.g., sodium 2,3-dichloroisobuty-
rate available from Rohm & Haas Co.), etc.
In accordance with a preferred embodiment of the
present invention, the male sterile plants are cytoplasmically
male sterile plants as described in my copending Canadian
Serial No. 475,718. As described in such copending Applica-
tion, three factors found to exist in available sources of
Glycine max plants, when properly combined in a single plant by
the intervention of man, provide an effective starting plant to
accomplish the hybrid soybean production. Such factors (as
described hereafter)
L` '' t-
have heretofore existed separately while dispersed in
soybean plants from widely differing sources. The female
fertile male sterile soybean plants possess (1) a Cms
cytoplasm, ~2) a distinct pair of recessive rlrl genes in
the cell nucleus, and (3) a distin~t pair o~ recessive r2r2
genes in the ce'l nucleus, which in combination render the
plant incapable of producing viable pollen while otherwise
carrying out the usual plant functions required to produce
soybeans if viable pollen is provided ~rom another soybean
plant.
Glycine max plants are self-destructing annuals
which cannot be satisfactorily propagated by asexual means
since if new plant~ are formed by cuttings, the new plants
ars of a progressively smaller size. The preferred female
fertile male sterile soybean plants can be successfully
propagated (i.e., maintained) by sexual means as described
hereafter. Also, these male sterile plants unlike soybean
plants which rely exclusively on nuclear genes for ~terility -
can be conveniently perpetuated or maintained without
unwanted segregation with respect to sterility, as described
herea~ter.
The atypical Cms cytoplasm of the preferred female
fertile male sterile soybean plants can be derived through
the female parent ~rom an appropriate cytvplasmic sour~e.
For instance, i~ has been found that the Cms cytoplasm
r $uired in the female ~ertile ~ully male sterile soybean
plants can b~ con~aniently derived from a Mandarin cytoplas-
mic ~ource through the female parent. Many Northern ~oybean
varieties are derived from this cytoplasmic source. Plants
of this origin have been found inherently to po ~eR~ an
-12-
~ ~ 'S~`? ~
atypical cytoplasm of the type required to practice thepresent invention. Since this required ~actor is not
contributed by nuclear genes and is not transmitted through
the pollen, it can be considered cytoplasmic, non-
Mendelian, extrachromosomal, uniparental, and maternal.
Representative commercially available soybean plants which
are derived maternally from a Mandarin cytoplasmic source
are ~delphia, Chippewa, Chippewa 64, Clark, Classic I,
Classic II, Columbus, Cutler, Disoy, Elf, Ford, Grant,
Harosoy, Harosoy 63, Hobbitt, Kent, Lincoln, Lindarin,
Lindarin 63, Magna, Prize, Provar, Rampaga, RA 203, RA 402,
RA 4~1, RAX 56, RAX 57, RAX 61, RAX 62, RAX 66, SB 27,
Shelby, Traverse, Wayne~ Wirth, Williams, etc. A particu-
larly good source for the re~uired Cms cytoplasm has been
~ound to be the El~ variety which was introduced during 1977
by AR-SEA-USD~, the Ohio Agric. Res. and Dev. Center, and
the U. of Illinois Agric. Res. Station. In 1981 this
variety was registered by the Crop Sci. Soc. of Am. as Reg.
No. 150.
It should be emphasized that when plants of the
above-identified varieties are inspected for the possible
absencQ o~ viable pollen production, that male aterile
plants (either partially male sterile or completely male
sterile~ wherein the sterility is attrikutable to the
cytoplasm are not observed. It has been found that such
sterility is not expressed even though the required atypical
Cms cytopla m is presen~ because it is not in combination
with the requirad recessive genss di cussed hereafter.
Instead such varieties can be shown to posses~ at least one
pair o~ dominant RlRl or R2R2 genes (usually both pairs)
-13-
-~$ ~,Y~ ~
which always leads to the expression of the usual viable
pollen production even in the presence o~ the Cms cytoplasm.
The pair of recessive genes rlrl for male ster-
ility present in the preferred female fertile fully male
sterile plants employed in the process o~ the present
invention can be derived through its male parent from a
~irst gene source which possesses such genes. Unlike the
male ~terile plants, the ~irst gene source may possess a
usua~ N cytoplasm which can be termed a "normal" or "neu~
tral" cytoplasm. When such cytoplasm is present, cytoplas-
mically controlled male sterility is not ~xhibited regard-
less o~ the nuclear genes which are present.
It has been found that the requisite pair of rlrl
rQcessive genes ln tha cell nucleus of the ~emale fertile
fully male sterile soybean plants conveniently can be
derived ~hrough the male parent from a Dunfield germplasm
base. Many Southern soybean variQties are derived from this
germplasm base. Plants of this origin hav~ been found
inherently to possess the required pair of recessive genes
which has been designated rlrl. Representative commercially
available soybean plants ~rom which the r1rl recessive genes
may be derived are Bedford, Bethel, Centennial, Dare, Dyer,
Forrest, Hill, Klrby, RA(d)41, RA 581, RA 603, RA 60~, RA
606, R~ 680, Tracy, Wabash, York, etc. A particularly good
source for the rlrl recessive genes has been found to be the
Bedford variety which was introduced during 1978 by
FR-SEA-USDA, and the Tennessee and ULssissippi Agric. ~xpt.
Stations. This variety was re~istered by the Crop Sci. Soc.
of Am. as Reg. No. 118.
-14-
b ~
It further should be emphasized that when plants
of the above-identified varieties having rlrl genes are
inspected for the possible absence of viable pollen produc-
tion, that male sterile plants (either partially male
sterile or completely male sterile) wherein the sterility is
attributable to the cytoplasm are not observed. It has been
found that such sterility will not be expressed unless tha
atypical Cms cytoplasm is present along with recessive genes
r2r2. Instead such varieties can be shown to possess
dominant R~R2 genes which restore male fertility.
The pair of recessive genes r2r2 for male steri-
lity present in the preferred female ~ertile fully male
sterile plants can be derived through its male parent from a
second gene sourca which possesses such genes. Such r2r2
genes are present as a distinct gene pair apart from the
rlr1 genes in the female fertile fully male sterile plants
(i.e., they are present at di~ferent loci). Unlike the male
sterile plants, the second gene source may possess a usual N
cytoplasm which can be termed a "normal" or "neutral"
cytopl~sm. As previously indicatad, when such cytoplasm is
present oytoplasmically controlled male sterility is not
exhibited regardless of the nuclear genes which are pres nt.
It has been found that the requisite pair of r2r2
recessive genes in the cell nucleus of the perferred female
fextil~ ~ully male sterile soybean plants conveniently can
be derived through the male parent from a Tokyo germplasm
base. ~any Southern soybean varietie~ arQ derived from this
germplasm basa. Plants of this origin have been ~ound
inherently to possass the required pair of recessive genes
which has been designated r2r2. Represantative commercially
-15-
~ 2~ 5
available soybean plants from which the r2r2 recessive genes
may be derived are Bragg, Braxton~ Cobb, Govan, Hampton,
Hampton 266, Hardee, Hutton, Jackson, Kirby, Majos, Ogden,
RA 604, RA 701, RA 800, Volstate, Wright, etc. A particu-
larly good source for the r2r2 recessive genes has been
found in thQ Braxton variety which was introduced during
1979 by the USDA and various state Agric. Expt. Stations.
It additionally should be emphasized that when
plants of the above identified varieties having r2r2 genes
are inspected ~or the possible absence of viable pollen
production, that male sterile plants (either partially male
sterile or completely male sterile) wherein the sterility is
attributable ta the cytoplasm are not observed. It has been
~ound that such sterility will not be expressed unless the
atypical Cms cytoplasm is present along with recessive genes
rlrl. In~tead such varieties can be shown to possess
dominant RlR~ gene~ which restore male fertility.
The pre~erred female fertile fully male sterile
plants can be maintained or perpetuated in spite of the male
sterility by crossing with pollen ~rom a soybean plant which
posse~ses an N oytoplasm and the two distinct pairs of
recessive genes rlrl and r2r2. Such preferred maintainer
plants are formed by the intervention of man through the
comblnation of the required ~ackors and are not found in
nature. The progeny sf this cross will again be ~emale
fertile and fully male sterileO Also, should the perferred
female ~rtile fully male sterile plants be crossed with
pollen from a male fertility restorer (i. , having domin~nt
RlRl genes and/or dominant R2R2 genes), then the progeny
will be fully fertile Fl hybrid soybean plants. Suitabla
-16-
male fertility restorer plants are readily available withoutmodification. For instance, any of the varieties heretofore
named can perform this function. The only requirement is
that plants which supply the pollen possess at least one
pair o~ the required dominant fertility restoring genes.
The development of preferred f~male fertile fully
male sterile soybean plants for use in the present inven-
tion, as well as preferred maintainex plants for the same,
can be exempli~iad through a plant breeding program employ-
ing plants of the Elf, Bed~ord, and Braxton varieties. It
should be understood, however, that the preferred embodiment
of the present process can be equally well practiced through
the utilization of soybean plants of other varieties
provided the essential criteria set forth herein neverthe-
less arè met. Initially plants of the Bedford variety
(i.e., having an rlrl gene source) are crossed by hand with
pollen from plants of the Braxton variety (i.e., having an
r2r2 gen~ source). The progeny of thls cross are f-llly
female fertile and male fertile and serve as a pollen source
~or plants of the Elf variaty (i.e., having a Cms cy~oplas-
mic source). Such crossing to the Elf variety is again
carried out hy hand under controlled conditions in the
absence of Elf self-pollination. When the Fl seed which has
fo~med on the Elf female parent is grown, it will be noted
that all of the resulting plants are fully female fertile
and male fertile. Each of these Fl plant~ is next
se}f-pollinated through succeedin~ generations to form F2,
F3, and F4 controlled populations which are inspected ~or
the absence of viable polLen. It i5 observed that some
plants are female fer~ile fully male fertile, som plants
-17-
are female fertile partially male fertile (i.e., produceonly a limited quantity of viable pollen), and some plants
are ~emale fertile fully male ~terile (i.e., produce no
viable pollen).
The fact that none of the F1 plants were male
sterile in this preferred embodiment aonfirms that the
stsrility subsequently observed was not controlled solely by
nuclear genes. The ratio~ in which the plants segregate in
the F2, F3 and F4 generations with respect to male sterility
confirm that the sterility is cytoplasmic in nature and the
result o~ a more complex cytoplasmic/genetic system in which
the genetic aspect is bifackorial (i.e., two distinct gene
pairs at different loci are operative and are interacting
with the cytoplasm). The ~ully male sterile plants possess
the Cms cytoplasm and the rlrl and r2r2 genes. The par-
tially male sterile plants possess the Cms cytoplasm and (l)
Rlrl and r2r~ genes or (2) rlrl and R2r2 genes. The f`ully
male fertile plants possess the Cms cytoplasm and RlRl genes
and/or R2R~ genes. When the ~ully male sterile plants are
crossed with pollen from the Elf, Bed~ord, and Braxton
varieties, all F1 progeny are fully male fertile~
Accordingly, this indicates that no single parent variety
(l.e., Elf, Bed~ord, or ~raxton) possesses sufficient genes
to create male sterile Fl plantq.
Once the preferred mals st~rile plants are on
hand, suitable maintainer plant~ (i.e~, those having an N
cytoplasm in combination with rlrl and r2r2 genes) can be
devèloped by ~tandard plant bre~ding techniques lnvolving
intercros~ing and introgre sion. For instance, tha required
rlrl and r2r2 g~nes can be provided in existing soybean
-18--
~ ~,4~ $
varieties of agronomic importance having khe usual Ncytoplasm by intercrossing and possible backcrossing by hand
with the pollen derived from female fertile partially male
fertile plants obtained from the F2, F3 and F4 controlled
populations obtained during or subse~uent to the development
of the male sterile plants (described above). The Fl plants
from this cross are grown and are self~pollinated to form F2
plants. Test cros~es of the fully male sterile plants
pre~iously developed with pollen derived from the F2 plants
are made and those plants are identified and preserved which
are capabls of yielding fully male sterile Fl progeny. Such
plants possess the full complement of recessive rlrl and
r2r2 genes. Once identified such preferred homozygous
maintainer plants can ~Q perpetuated by self-pollination.
When producing seeds capable of growing male
fertile Fl hybrid soybean plants in accordanc~ with one
embodiment of the process of the present invention, the
required male sterile soybean plants are grown at an
appropriate location as a substantially uniform population
in pGllinating proximity to a substantially uniform popula-
tion of male fertile soybean plants. In the context of the
present invent~on "pollinating proximity" specifies tha~ the
two types o~ parent plants are sufficiently near that pollen
can be tran~fexred by pollen-carrying bees without loss of
its viability. The male ~ertile soybean plants conveniently
can be a pure line variety. For instance, the two types of
plants can be grown adjacent to ~ach other as alternating
strips. In a preferred embodiment approximately 2, 4, or 6
rows o~ the male sterile soybean plants form a substantially
uni~orm population and alternate with a substantlally
--19--
uniform population of approximately 2 rows of the male
fertile soybean plants. Following pollen transfer from the
male ~ertile soybean plants to the male sterile plants (as
described hereafter3, seeds are formed on the male sterile
soybean plants. The male fertile soybean plants commonly
are self-pollinated and seeds also form on them. At harvest
time the seeds are selectively recovered from each of the
substantially uniform plant populations. Accordingly, the
seeds which are recovered from the male sterile soybean
plants are a substantially homogeneous assemblage of saeds
which upon growth yield male fertile Fl hybrid soybean
plant~.
In accordance with another embodimenk of the
process of the present invention, a substantially uniform
binary seed blend is formed containing a substantial
proportion (e.q., at least one-half by number) of seeds
capable of growing male fertile Fl hybrid soybean plants.
Such blend can be formed by growing at an appropriate
location a substantially random population of male sterile
soybean plants together with the male fertile soybean
plants. For instance, approximately 75 to 95 percent (e.a.,
approximately 90 percent) of the plants in the random
population can be male sterile plants and approximately 5 to
25 percent (e.g~, approximately 10 percent~ of the plants in
the random population can be the male fertilQ soybean
plants. Following pollen trans~er the seeds formed on the
mal~ stsrile soybean plants are capable of forming the male
fertil~ Fl hybrid soybean plants, and the seede formed on
the restorer soybean plants are the result of self-pollina-
tion. The resulting seeds formed on the subs~antlally
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76~
random population of soybean plants next are harvested in
bulk and can be planted in bulk by the grower.
The level of cross-pollination occurring in the
random population of plants (discussed hereafter) can be
visually observed by inspecting the resulting seeds or the
plants produced when the resulting seeds are grown if one
incorporates an appropriate genetic marker system into the
parent plants which gives one appearance upon cro~s-pollina-
tion and another appearance upon self-pollination. The
genetic marker can take the form o~ a recessive gene which
expresses itself upon self-pollination, but which is
dominated by another gene giving a different appearance
when cross-pollination takes place. Under such circum-
stances the male ~ertile plants could be homozygous rece~-
sive for such trait and the male sterile plants homozygous
dominant for such trait. For instance, the genetic marker
can be a distinctive pubescence color (e.q., gray pubescence
vs. tawny pubescence~, ~lower color (e.q., white flowers vs.
purple flowers~, seed pod color (e.q., tan vs. brown pods3,
hilum appearance (~, yellow vs. black hilum or bu~f vs.
black hilum), etc.
Alternatively, cytoplasmically male sterile
soybean plants use~ul in the production of male fertile F
hydrid soybean plants can be maintained, perpetuated/ and
multiplied by planting a substantially uniform population of
th~ same in pollinating proxim~ty to a substantially uniform
population o~ the maintainer soybean plants which when
crossed with the male sterile ~oybean plants enable the
formation o~ seeds on the cytoplasmically male st~rile
~oybean plants which are capabl~ of growing additional
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7 ~
cytoplasmically male sterile plants. For instance, the twotypes of plants can be grown adjacent to each other as
alternating strips as described earlier with respect to the
production of male ~ertile Fl hybrid seed. Following pollen
transfer (as described hereafter) from the maintainar
soybean plantq to the male sterile soybean plants seeds ~re
formed on the male sterile ~oybean plants. The maintainer
plants are self-pollinated and seeds also ~orm on them. At
harvest time the seeds are selectively harvested from each
of the substantially uniform plant populations.
Accordingly, the seeds which are recovered from the male
sterile soybean plants are a substantially homogeneous
assQmblage of seeds which upon growth yield cytoplasmically
male sterile soybean plants. The seeds which are selec-
tivaly recovered ~rom the maintainer plant~ can be planted
to produce additional maintainer plants or sold as a
commercial soybean product.
Since the photosensitivity of soybean plants tends
to vary among soybean varieties, it is important for best
results that the parent soybean plants (i.e., the male
sterile soybean plants and the male fertile soybean plant~)
each possess a day length sensitivity ~i-e-, a photoperiod
response) which generally corresponds to that of the
location (i.e., the latitude or di~tance ~rom the equator)
where the soybean plants are grown when carrying out the
proces~ o~ the present invention as well as to the area
wh~re the seed product i5 ultimately to be grown. Fox
instance, the parent plant~ and the ultimate seed product
preferably should posses~ a photo~ensitivity within plus or
minus one maturity group unit o~ the locations where grown.
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62957-211
Additionally, for optimum results the locations where grown
should correspond exactly -to the maturity groups of the plants
involved. See, Chapter 6 by Edgar E. Hartwig of "Soybeans:
Improvement, Production, and Uses", American Society of
Agronomy, Inc., Pages 189 to 190 (1973), for a discussion of
soybean maturity group units and their significance. For in-
stance, if a soybean variety which grows well at a southern
latitude is grown at a northern latitude, the longer days may
cause the soybean plants to grow excessively tall and to tend
to lodge. Alternatively, if a soybean variety which grows well
at a northern latitude is grown at a southern latitude, the
shorter days may cause limited plant growth (i.e., heigh-t) and
result in poor yields.
For best results soybean parent plants also are
selected which inherently exhibit a propensity to flower during
an overlapping time period at the location where grown. Such
soybean plant parents may be either of the determinate type
(i~e., flower over a period of approximately 20 to 25 days) or
of the indeterminate type (i.e., flower over a period of
approximately 35 to 50 days) so long as they flower simultane-
ously at a period of time during the summer. In a particularly
preferred embodiment the flowering period for the male fertile
soybean plant parent commences before the flowering period for
the male sterile soybean plant parent and ends after the flow-
ering period for the male sterile plant parent. Accordingly,
the male fertile parent may be from a longer flowering indeter-
minate variety and the male sterile parent may be from a short-
er flowering determinate variety. This will provide added
6~
insurance that pollen ~or the required cross-pollination
will be available when the male sterile seed parent is ready
to receive pollen.
In order to promote visitation by pollen-carrying
bees (as discussed herPafter) the male sterile soybean plant
parents can be selected for large blossom size to aid insect
entry. Parent plants having flowers which are tightly
cleistogamous are to be avoided sinc~ in such plants the
flower keel tends not to open or the stigmas tend not to be
exerted thereby making pollination difficult. Also, in
order to promote visitation by pollen-carrying bees the
flowers of both parents should be as attractive as possible
to bees. Since, bees sometimes have a tendency to preferen-
-tially visit soybean flowers o~ a given color, it is
preferred that the plants of each parent possess substan-
tially the same flower coloration (e~., be all purple or
all white). In a particularly preferred embodiment the
flowers of each parent are purple in coloration since this
coloration often is found to be preferred by ~he bees which
are relied upon to accomplish pollen transfer.
In accordance with the concept of the pxesent
inven~ion, water is applied via irrigation to the seeds
which produce the male sterile and male fertile soybean
plants fGllbwing planting as required to accomplish seed
germination and normal plant growth up to at least the time
o~ the onset of ~lower forma~ion on each o~ the two plant
types. The customaxy soybean planting times commonly are
employed. The quantity of water applied will be influenced
by the ~requency and extent of nàtural rainfall (if any).
The manner in which the irrigation water is applied may be
--~4-
~7~7"5;
varied and commonly w~ll correspond to the irrigationtechnique that may be most conveniently implement~d at the
particular location which normally experiences limited
natural rainfall. As will be apparent to those skilled in
agronomic technology, the particular irrigation technigue
selected will also be influenced by the soil type encoun-
tered and its inherent water-holding capabilities. Light
soils will inherently re~uire lesser irrigation times and
more frequent water applications. ~epresentative irrigation
technigues that may be selectPd include (1) sprinkler
systems whereby water is sprayed and impacts upon the
planting area ~rom overhead through the air, (2) flooding
systems whereby water confined by a levy or other means is
caused to flow upon the sur~ace of the soil and to substan-
tially completely engul~ the planting area, (3) furrow
systems whereby a furrow is mechanically cut in the soil
ad;acent to the locations where the soybean plant~ are grown
and is filled with water, etc. Sprinkler systems commonly
have the advantag~ of using less water. In northeastern
Arkansas commonly a flooding system will be employed. In
western Texas commonly a furrow system or a sprinkler system
will be employed. Also commonly, the water is applied via
irrigation ~or a period of approximately 0.1 to 15 days (or
more) prior to the withholding of irrigation water (as
de~cribed hereafter). The duration of the time in which
water is applied by irrigation will primarily be in~luenced
by the natural rainfall (i~ any), the okher weather condi-
tions (e.q., heat and humidity) encountered, ~nd the a~ility
of the soil to hold water once irrigat'on is commenced. In
any event, water is always appl:Led as required Yia irriga-
-2~-
~7~5
tion in sufficient quantities and at sufficient intervals to
insure normal soybean plant growth up to at least the time
when flowers are present on each of the parent plant types.
The application of water via irrigation promotes
the normal vegetative growth of the soybean plants and
flower formation. The nectar exuded by the resulting
soybean flowers, when irrigation and/or natural rainfall is
taking place, tends to be more dilute and is considerably
less attrac~ive to bees than that formed in the subsequent
step o~ the present process (described herea~ter) when
irrigation water is withheld from the planting area.
The process o~ the present invention utilizes
pollen-carrying bees to bring about the required cross-
pollination of the parent soybean plants. At most growing
areas honeybees are particularly e~fective in bringing about
the desired cross-pollination. However, bees other than
honeybee~ can alternatively be employed so long as they will
reliably visit the soybean flowers at the appropriate time.
For instance, leaf-cutter bees (i.e., Meqachile rotundata)
can be used. Also, naturally occurring bees other than
~oneybees and leaf-cutter bees (l.e., wild bees) advanta
geously may supplement the le~el of sross-pollination. Bees
appear to visit soybean plants primarily in search of nectar
and to a lesser extent for pollen that erves as a protein
sourae for the bees. As bees collect nectar, they concomit-
antly serve to pick up and carry pollen from one soybean
plant to another.
In a preferred embodiment of the process of the
pre ent invention, one or moxe honeybee hives ars situatad
in pollinating proximity to the location where the parent
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7~
62957-211
soybean plants are being grown in order to insure the ample
presence of sufficient pollen vectors. Eor instance, in a
particularly preferred embodiment, honeybee hives are provided
in pollinating proximity to the location where the parent soy-
bean plants are being grown at a rate of at least 2 hives per
acre (e.g., 2 to 3 hives per acre) of the parent so~bean
plants. When irrigation is accomplished by flooding, the hives
may be situated on a small terrace above the water level. It
~urther is preferred that a route be provided in the planting
area to provide ready ingress and egress for the beekeepers
having the responsibility of servicing the beehives. Eor best
results, it is recommended that the honeybee hives be positio-
ned so that the honeybees are not required to travel more than
approximately one-quarter of a mile to visit the parent soybean
plants. This tends to improve the foraging efficiency.
Since pollen serves as a protein source required
by bees and soybean plants tend not to produce pollen in pro-
fuse quantities, it is preEerred that a supplemental protein
source for the pollen-carrying bees be provided in addition to
the pollen formed on the male parent soybean plants in order to
more fully support the pollen requirements o~ the pollen-carry-
ing bees. Such supplemental protein source for the enrichment
of the bee diet may take various forms. For instance, plants
kno~n to form pollen in relatively copious quantities te.g.,
sorghum, sudan, pearl millet, etc.) can be grown nearby.
Alternatively, a concentrated pollen sourcel such as pollen
cakes available to beekeepers, can be placed in the vicini~y of
or within the honeybee hives.
- 27 -
~2~7~7~
Insecticides must be used with care in the
planting area since adult bees may be killed and sometimes
bees will refuse to visit fields which have been sprayed
with insecticides. Accordingly, i~ insecticides are used
they should possess a low killing potential for bees, and
preferably be applied during the night or some other time
when any beehives in the area ar~ closed.
The overall process of the present invention
provides a highly effective technique to bring about a high
level of cross-pollination among the parent soybean plants.
~t an appropriate time when flowers are present on the
parent soybean plants, irrigation water is withheld for a
period of time during which no appreciable natural rainfall
occurs in order to induce enhanced nectar flow within the
flowers, which serves to render the flowers more attractive
to bees. When irrigation water is withheld, the nectar flow
within the soybean flowers increases substantially and the
sugar component of the nectar becomes more concentrated and
more aromatic. Such enhanced nectar flow is readily
perceived by the bees that forage in the area and the bee~
are strongly attracted to the soybean flowers at a critical
time in the life cycle of the soybean plants. Such
increased bee visitation results in higher levels of the
desired cros -pollination and seed set.
It is important that the period of time during
which irrigation water is withheld not exceed that wh~ch can
be well tolerated by the parent plants involved (i.e., not
significantly impair th~ plant metabolism in a way which
would interfere with the desired seed set and seed forma-
tion). The duFation o~ the period in which lrrigation water
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:
7675
is withheld at a time when no appreciable natural rainfall
occurs will be influenced by the environmental conditions
(~g~, temperature, humidity, wind velocity, etc.) and the
water-holding ability o~ the soil. In a preferred embodi-
ment, the irrigation water is withheld for a period of at
least 8 days (e.q., 10 to 15 days).
At the conclusion of th period during which
irrigation water is withheld, the paxent soybean plants may
again be watered via irrigation to promote normal plant
growth in order to insur~ the formation of the desired seeds
that subse~u~ntly are harvested at the appropriate time in
their maturity cycle. However, th~ proce~s steps optionally
may be repeated at least one time (e.q., 1 or 2 more times3
during which irriyation water is applied, irrigation water
is withheld, and additional cross-pollination by pollen-
carrying bees is accomplished. Such repetition of the
process step~ may be carried out to particular advantage
when the parent soybean plants are selected that inherently
~lower over an extended period of time (e.g., when both of
the soybean parent have indeterminate flowering character-
istics). For instance, when both soybean plant parents have
indeterminate flowering characteristics, irrigation water
commonly is withheld for a total of 2 or 3 times (or more)
during the flowering period Howeve1, when both soybean
plant parents have determinate ~lowering characteristics,
irrigation water commonly is withheld for a total of only 1
or 2 times during the flowering period.
The concept of the pxesent invention provides a
highly efficient technique for aiding the accomplishment of
the desired cross-pollination of male sterile soybean plants
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~ ~9~;75
with the aid o~ bees. The enhanced level of seed set made
posslble by the improved process of the present invention
translates into greater and more reliable yields o~ the
desi~ed seeds (e. q., seeds capable of growing male fertile
Fl hybrid soybean plants or seeds capable o~ growing
additional cytoplasmically male sterile soybean plants that
are useful in the production o~ male fertile Fl hybrid
soybean plants).
Although the invention has been described with
pre~erred embodiments, it is to be understood that varia-
tions and modi~ications may be resorted to as will be
apparent to those ~killed in the art. Such variations and
modifications are to be considered within the purview and
scope of the claims appended hereto.
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