Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02544248 1994-02-22
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PATENT
ATTORNEY DOCKET NO: BB-1048-A
07148/007W01
TITLE
LOW LEVEL GLUCOSINOLATE BRASSICA
The present invention relates to 8rassica seeds,
meal, plant lines and progeny thereof having a reduced
level of glucosinolates.
Canola meal is widely employed as a protein
_0 supplement in animal feed. The feeding value of canola
meal is reduced due to the anti-nutritive effects of the
breakdown products of the glucosinolates, which reduce
feed intake and growth in non-ruminant animals.
Glucosinolates in the seed are broken down during the
L5 extraction process by the enzyme myrosinase to form
isothiocyanates and nitriles. These breakdown products
may also inhibit thyroid function, leading to goiter.
The feed value of canola meal can be improved by
reducing or eliminating glucosinolates from canola seeds.
Z0 Typical glucosinolate levels in.canola meal and seed are
disclosed in the following references: 1) Shahidi et al.
Journal of Food Quality, 11, 421-431 (1989), and 2)
Lichter et al., Plant Breeding 100, 209-221 (1988) and 3)
Kraling et al., Plant Breeding, 105, 33-39 (1990). The
25 typical range for the glucosinolates content of
conventional B.~ napus double low canola varieties in
~mol/g of seed at 40%. oil content and 8.5% moisture is as
follows:
2-hydroxy 3-butenyl glucosinolate 2.40-7.32
30 allyl glucosinolate 0-1.16
2-hydroxy 4-pentenyl glucosinolate 0-0.43 ~ '
3-butenyl glucosinolate 1.65-3.44
4-hydroxy 3-indolymethyl glucosinolate 2.60-4.40
4-pentenyl glucosinolate 0-1.14
CA 02544248 1994-02-22
2
3-indolylmethyl glucosinolate 0-4.18
Total glucosinolates 12.06-18.23
By creating specific mutations in the glucosinolate biosynthetic
pathway, mutations at various steps in the pathway may be combined to
reduce the total glucosinolate levels in finished varieties. A. B. raps line
(BC86-18) with low glucosinolates levels has been identified via selection by
Bell et al., Can: J. Animal Sci., 71, 497-506 (1991 ). However, the
variability
in glucosinolates content of B. napus germplasm is limited. The present
invention provides B. napus lines, seeds, and meal having a reduced level of
glucosinolates.
SUMMARY OF THE INVENTION
In accordance with an aspect of the invention, plant cell line comprising
a Brassica napus canola variety which produces seeds having a maximum
content of glucosinolates of about 3.4,~moles per g seed and in which said
glucosinolates content is stabilized for both the generation to which the seed
belongs and its parent generation.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides seeds, meal and plant lines having a
reduced level of glucosinolates generated by creating specific mutations in
the
glucosinolate biosynthetic pathway. Scheme I depicts the
CA 02544248 1994-02-22
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biosynthetic pathway of major aliphatic glucosinolates in
erassica.
CA 02544248 1994-02-22
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CA 02544248 1994-02-22
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As used herein, a "line" is a group of plants that
display little or no genetic variation between
individuals for at least one trait. Such lines may be
created by several generations of self-pollination and
selection, or vegetative propagation from a single parent
using tissue or cell culture techniques. As used herein,
the term "variety" refers to a line which is used for
commercial production.
As used herein, "mutation" refers to a detectable
and heritable genetic change not caused by segregation or
genetic recombination. "Mutant" refers to an individual,
or lineage of individuals, possessing a genetic mutation.
The term "Mutagenesis" refers to the use of a mutagenic
agent to induce random genetic iautations within a
population of individuals. The treated population, or a
subsequent generation of that population, is then
screened for usable traits) that result from the
mutations. A "population" is any group of individuals
that share a common gene pool. As used herein "MD" is
untreated seed. As used herein, "M1" is the seed (and
resulting plants) exposed to a mutagenic agent, while
"M2" is the progeny (seeds and plants) of self-polinated
M1 plants, "M3" is the progeny of self-pollinated MZ
plants, and "M4" is the progeny of self-pollinated M3
i plants "M5" is the progeny of self-pollinated M4 plants.
"M6", "M~", etc. are each the progeny of self-pollinated
plants of the previous generation.
The term "progeny" as used herein means the plants
and seeds of all subsequent generations resulting from a
7 particular designated generation.
The term "selfed" as used herein means self-
pollinated.
"Stability" or "stable" as used herein means that
with respect to a given component, the component is'
maintained from generation to generation for at least two
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. generations and preferably at least three generations at
substantially the same level, e.g., preferably ~ 15%~,
more preferably ~ 10%, most preferably ~ 5%. The method
of invention is capable of creating lines with improved
glucosinolate compositions stable up to ~ 5% from
generation to generation. The above stability may be
affected by temperature, location, soil fertility, stress
and time of planting. Thus, comparison of glucosinolate
profiles should be made from seeds produced under similar
growing conditions. Stability may be measured based on
knowledge of prior generation.
Intensive breeding has produced Brassica plants
whose seed oil contains less than 2% erucic acid. The
same varieties have also been bred so that the defatted
meal contains less than 30 ~Cmol glucosinolates/gram.
"Canola seed" is referred to herein as the seed of the
genus Brassica which shall contain less than 30 moles of
total glucosinolates per gram of whole seed at a moisture
content of 8.5%; and the oil component of such seed shall
contain less than 2% of all fatty acids as erucic acid.
The term "canola meal" is used herein to describe
a protein meal derived from seeds of the genus 8rassica
containing less than 30 ,moles of total glucosinolates
per gram of defatted meal at a moisture content of 8.5%.
i The term"canola oil" is used herein to describe
an oil derived from the seed of the genus Erassica, with
less than 2% of all fatty acids as erucic acid.
The glucosinolate contents for the present
invention are reported as ~,mol/gm seed at 40% oil content
and at a moisture content of 8.5%. To compare, the
reported values to older references ~cmol/gm of seed can
be converted to ~mol/gm of oil free meal, at 8.5%
moisture using the following conversion: (~tmol/gm~
seed)/(1.0-0.4). A comparison of this conversion from
~mol of glucosinolates/gm of seed to ~mol of
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7
glucosinolates/gm of defatted meal at 8.5% moisture using
field grown IMC 129 seed is presented in Table V in
Example 1 hereinafter.
The general experimental scheme for developing
lines with stable lowered glucosinolate mutations is
shown in Scheme II hereinafter. ,
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scxEME Ii
IMC 129 (Mp)
Treatment with N-methyl-N'-
nitrosoguanidine
M1
Greenhouse grow out
Self-pollination
M2
Greenhouse grow out
Self-pollination
0 M3
~-.---- Chemical analysis (Total
glucosinolates)
~---- Select mutants based on statistical
analysis of control population
~--- Grow out select mutants in
greenhouse
~---- Self-pollination
0
M4
Chemical analysis (Individual
glucosinolates)
5 ~--.- Select mutants based on statistical
analysis of control population
E--- Confirm altered glucosinolate
~---- Composition
.,. , ,
0 STABLE GLUCOSINOLATE MUTANTS
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IMC 129 seeds (MO) were mutagenized with N-
methyl-N'-nitro-N-nitrosoguanidine (MNNG). IMC 129 is a
canola quality commercial Spring variety with high oleic
acid grown by InterMountain Canola Inc. in the
northwestern United States registered under U.S. Plant
Variety Protection Certificate 9100151. The glucosinolate
composition of field-grown IMC 129 has remained stable
under commercial production. The glucosinolate
composition as moles per gram of whole seed at a
moisture content of 8.5% and 40% oil content is as
follows: 8.77 of total glucosinolates, 3.97 of 2-hydroxy-
3-butenyl glucosinolate, 0.38 of allyl glucosinolate,
0.35 of 2-hydroxy-4-pentyl glucosinolate, 1.84 of 3-
butenyl glucosinolate, 1.66 of 4-hydroxy-3-indolyl methyl
glucosinolate, 0.22 of 4-pentyl glucosinolate, and 0.32
of 3-indolylmethyl glucosinolate.
The disclosed method may be applied to all
oilseed Brassica species, and to both Spring and Winter
maturing types within each species. Physical mutagens,
including but not limited to X-rays, W rays, and other
physical treatments which cause chromosome damage, and
other chemical mutagens, including but not limited to
ethidium bromide, nitrosoguanidine, diepoxybutane etc.
may also be used to induce mutations. The mutagenesis
treatment may also be applied to other stages of plant
development, including but not limited to cell culture,
embryos, microspores and shoot apices. Once the mutation
has been identified it can be transferred into other B.
napus varieties by cross pollination. The present
invention includes such cross-pollinated species.
The M1 seeds derived from the mutagenesis
treatment were planted in the greenhouse and M1 plants
were individually self-pollinated. M2 seed was harvested
and planted in the greenhouse, and individually self-
pollinated to advance to the next generation. M3 seeds
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were screened for total glucosinolate content using a
TruBluGlu meter. This meter is available from Dr. R.J.W.
Truscott at Systrix Pty. Ltd., University of Wollongong,
P.O. Box 1144, Wollongong, N.S.W. 25000, Australia. The
analytical procedure employed is detailed in Truscott et
al. Proceedings of the Eighth International Rapeseed
Congress 1991, McGregor, D., Ed., Vol. 5, pp. 1425-1427.
Further details are provided in Example 1 herein after.
M4 seeds were analyzed by high pressure liquid
chromatography to determine specific alterations in the
glucosinolate composition. Those lines which remained
stable in glucosinolate content were regarded as stable
mutations.
M4 seeds were evaluated for mutations on the basis
of a Z-distribution. An extremely stringent 1 in 10,000
rejection rate was employed to establish statistical
thresholds to distinguish mutation events from existing
variation. Mean and standard deviation values were
determined from the non-mutagenized IMC 129 control
population. The upper and lower statistical thresholds
for each glucosinolate were determined from the mean
value of the population ~ the standard deviation,
multiplied by the Z-distribution. Based on a population
size of 10,000, the confidence interval is 99.99%.
"Stable mutations" as used herein are defined as
M3 or more advanced lines which maintain a selected
altered glucosinolate profile for a minimum of three
generations, and exceeding established statistical
thresholds .for a minimum of two generations, as
determined by liquid chromatographic analysis of a
minimum of 200 mg (approximately 50 seeds) of randomly
selected seeds. Alternatively, stability. may be measured
in the same way by comparing to subsequent generations.
In subsequent generations, stability is defined as having
similar glucosinolate profiles in the seed as that of the
CA 02544248 1994-02-22
., ,
11
prior-. or subsequent generation when grown under
substantially similar conditions.
The seeds of several lines having lowered
glucosinolate content have been deposited with the
American Type Culture Collection and have the following
accession numbers.
Line Deposit Date Accession No.
3Q0211 Feb. 25, 1993 75419
3Q1552 Feb. 25, 1993 75420
3Q0811 Feb. 25, 1993 75421
3Q1273 Feb. 25, 1993 75422
Stable mutants having reduced levels of
glucosinolates were obtained. Seeds and plant lines
producing such seeds, having a maximum glucosinolate
content of 3.4 moles per g seed were obtained.
Generally, the total glucosinolate as ~mol/g of seed
ranged from 2.01 to 3.41. The maximum content and range
for the individual glucosinolates is as follows as
mol/g seed at 8.5% moisture and 40% oil content:
. ~ moles/g seed
Glucosinolate Ranae Maximum
2-Hydroxy-3-butenyl 0.26-1.50 1.50
2-Hydroxy-4-pentenyl 0.03-0.13 0.13
3-Butenyl 0.15-1.10 w 1.10
4-Hydroxy-3-indolylmethyl 0.67-1.05 1.05
4-Pentenyl 0.01-0.06 0.06
3-Indolylmethyl 0.06-0.15 0.15
Total Glucosinolates 2.01-3.41 3.41
The glucosinolate content of the canola meal
derived from the seeds of these stable mutants was
calculated as previously described,from the seed ,
glucosinolate content. The meal has a maximum total
glucosinolate content of 5.7 ~ mol/g on an oil free basis
at 8.5% moisture. Generally, the total glucosinolate
CA 02544248 1994-02-22
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content ranged from 3.4 to 5.7 ;t mol/g. The individual
glucosinolate levels are as follows in ~ mol/g of oil
free meal at 8.5% moisture.
Glucosinolate umoles,/q meal
2-Hydroxy-3-butenyl 0.43 - 2.50
2-Hydroxy-4-pentenyl 0.05 - 0.22
3-Butenyl 0.25 - 1.83
4-Hydroxy-3-indolylmethyl I 1.12 - 1.75
4-Pentenyl 0.02 - 0.10
3-Indolylmethyl 0.10 - 0.25
Total Glucosinolates 3.35 - 5.68
While the invention is susceptible to various
modifications and alternative forms, certain specific
embodiments thereof are described in the general methods
and examples set forth below. For example the invention
may be applied to all Brassica species, including B.
rape, B juncea, and B. hirta, to produce substantially
similar results. It should be understood, however, that
these examples are not intended to limit the invention to
the particular forms disclosed but, instead the invention
is to cover all modifications, equivalents and
alternatives falling within the scope of the invention.
This includes the use of somaclonal variation; antisense
expression; transposon mutagenesis; physical or chemical
mutagenesis of plant parts; anther, microspore or overy
culture followed by chromosome doubling; or self- or
cross-pollination to transmit the glucosinolate trait,
alone in combination with other traits, to develop new
Brassica lines.
EXAMPLE 1
.,. IMC 129 seeds (MD) were mutagenized,with N-methyl-
N~-nitro-N-nitrosoguanidine (MNNG). IMC 129 is a canola
quality commercial Spring variety with high oleic acid
grown by InterMountain Canola Inc. in the northwestern
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United States. The glucosinolate composition of field-
grown IMC 129 is a described above and has remained .
stable under commercial production.
Prior to mutagenesis, 45,000 seeds of IMC129 were
i preimbibed in 300 seed lots for two hours on wet filter
paper saturated in 0.05 M Sorenson's buffer (pH 6.1).
The preimbibed seeds were placed in 1.0 mM MNNG for three
hours. Following mutagenesis, the seeds were rinsed
three times in Sorenson's buffer. The Ml seeds were sown
in 4 inch pots containing Pro-Mix. Approximately 21% of
the mutagenized seed survived. The plants were
maintained at 25°C/15°C, 14/10 hour day/night conditions
in the greenhouse. At flowering, each plant was
individually self-pollinated.
The M2 seed from individual plants were catalogued
and stored. Approximately 9,500 individual M2 lines were
sown in the greenhouse in 4 inch pots containing Pro-Mix
soil.. The plants were maintained at 25°C/15°C, 14/10
hour day/night cycle in the greenhouse. At flowering,
7 the terminal raceme was self-pollinated by bagging. At
maturity, selfed Mg seed was individually harvested from
each plant and catalogued.
The M3 seed was screened using the TruBluGlu meter
available from Dr. R.J.W. Truscott at Systrix Pty, Ltd.,
University of Wollongong, P.O. Box 1144, Wollongong,
N.S.W. 2500, Australia. The details of the method used
are as follows:
Two hundred milligrams (200 mg) of whole seeds
were placed in a 15 mL polypropylene centrifuge tube.
0 The seeds were crushed with a glass rod manually. Then
1.0 mL of 5o mM glycine-sodium hydroxide buffer, pH 9.0,
was added and mixed well by using the glass rod. Thd rod
was rinsed with 4.0 mL of glycine-sodium hydroxide~buffer
and filled to the 5 mL mark on the tube. The sample was
5 incubated at room temperature for 10 min. Then 1.0 mL of
CA 02544248 1994-02-22
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chloroform was added, the tube capped and shaken vigorously. 50 mL of 10%
chlorohexidine/methanol solution was added and mixed well. 1.0 mL of 100
mM sodium citrate buffer, pH 5.0, was added and mixed. Approximately 250
mg of activated carbon was added, shaken vigorously, and centrifuged for 15
min. using a bench-top centrifuge at 2000 rpm. One Clinistix~ (NDC
0193-2844-50, Miles) was immersed into the clean supernatant for 5 sec.,
excess supernatant shaken off and set aside for exactly 2 mm. The color
change was read by a TruBIuGlu meter. Triplicate determinations were made
for each sample. The TruBIuGlu meters were calibrated using water and 1
mM glucose solution before reading samples.
Selected M3 lines with less than 3~ mol/g of seed of total
glucosinolates were sown in the greenhouse in 4 inch pots containing ProMix
soil. The plants were maintained at 25°C/15°C under 14/10 hour
day/night
conditions in the greenhouse. At flowering, each plant was individually self-
15 pollinated. At maturity, the N4 seed was harvested from each plant and
catalogued.
Bulk seed of selected M4 lines was analyzed for glucosinolate
composition via high pressure liquid chromatography. The method developed
by Daun and McGregor (1991 ) was used with modification to analyze the.
2o seeds as follows.
Extraction of glucosinolates: Ground seed (200 mg) was weighed into a
test tube. The tube containing ground seed was placed in a water bath at
95°C and left for 15 mm. One mL boiling water was added to the tube and
mixed well with the ground seed. After 3 mm. the tube was withdrawn and
25 cooled for 30 mm. One mL of 1 mM benzyl glucosinolate solution was added
into the tube as the internal standard and then mixed by vortexing. 100.1 of
lead acetate and barium acetate solution were added
CA 02544248 1994-02-22
and then centrifuged at 5000 xg for 20 mm. The supernatant was retained as
the glucosinolate extract.
Preparation of desulfoglucosinolates: One mL of glucosinolates
extract was transferred to a micro column containing DEAE-Sephadex~ A-25
ion exchange resin. The extract was run into the column and washed with 3
mL of 0.02M pyridine acetate solution. Purified sulfatase solution (0.5 mL)
was added into the column. When the enzyme solution had completely
entered the column, the flow, was stopped, the column capped, and allowed
to stand at room temperature overnight. The desulfoglucosinolates were
1o eluted with 2mL of water into vials for HPLC analysis.
Profile analysis by HPLC: Fifty microliters (501) of the
desulfoglucosinolates preparation were injected into a high performance liquid
chromatograph (Hewlett-Packard 1050) equipped with a C-18 column (Pierce,
220 x 4. 6 mm). The run was carried out with a solvent gradient program of 1
15 mm. at 100% water, linear gradient over 15 mm. to 75% water and 25%
acetonitrile, held for 5 Thin., linear gradient over 5 mm, to 100% water, held
for 5 mm. The flow rate was 1.5 ml/mm. and the column temperature was
30°C. A UV detector was used at 227 nm.
The means and standard deviations were calculated from an external
2o control population of non-mutagenized IMC 129 grown next to the
mutagenized lines. Statistical thresholds were established for the
glucosinolates using the control populations. The lower thresholds were
determined to be 3.6 standard deviations below the IMC 129 control means
with a 99.99% confidence interval. The lower thresholds used in the
greenhouse selection are listed in Table IV. Selected M3 lines with altered
glucosinolate compositions were planted in the greenhouse to advance the
next generation.
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Table I summarizes the reduced level of
glucosinolates for the mutant lines generated.
Significant reductions in the levels of 2-hydroxy-3-
butenyl glucosinolate, 3-butenyl glucosinolate, and 4-
hydroxy-3-indolylmethyl glucosinolate contributed to the
overall low lvels of total glucosinolates.
Fifteen lines with low progoitrin (2-hydroxy-3-
butenyl glucosinolate) of less than 1.5 ~mol/g seed have
been produced via mutagenesis (Table II). The range of
progoitrin in the mutations is 0.26-0.97 ~mol/gm of seed.
These lines also exceed the lower statistical threshold
for 3-butenyl glucosinolate. Five of the low progoitrin
selection were also below the lower selection for 4-
hydroxy-3-indolylmethyl glucosinolate.
Six additional lines with low 4-hydroxy-3-
indolylmethyl glucosinolate of less than 1.05 ~,mol/g seed
have been produced via mutagenesis (Table III). The
individual mutants range from 0.75-0.84 ~mol/g seed. All
of these mutations are below the statistical threshold
for low total glucosinolates.
Table I presents the resulting overall
glucosinolate compositions for the mutant lines of the
present invention. Table II presents the resulting
glucosinolate compositions of mutants having low 2-
hydroxy-3-butenyl glucosinolate for several indicated
lines. Table III presents the glucosinolate compositions
of selected mutants with low 4-hydroxy-3-indolylmethyl
glucosinolate for the lines indicated. Table IV
illustrates the statistical thresholds employed for
selection of the low glucosinolate mutations. Table V
presents glucosinolate values for the control IMC 129.
All values represent moles glucosinolate per gram of
seed at 8.5% moisture and 40% oil content or per gram of
oil free meal at 8.5% moisture as indicated.
CA 02544248 1994-02-22
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The following abbreviations are used in Tables I
through V for the various glucosinolates:
PROG (Progoitrin) 2-Hydroxy-3-butenyl glucosinolate
ALLYL (Sinigrin) Allyl glucosinolate
NAPOL (Napoleiferin) 2-Hydroxy-4-pentenyl glucosinolate
GLUCO (Gluconapin) 3-Butenyl glucosinolate
4-OH (4-OH 4-Hydroxy-3-indolylmethyl
Glucobrassicin) glucosinolate
GLUCOBN (Gluco- 4-Pentenyl glucosinolate
brassicanapin)
GLUCOBB (Gluco- 3-Indolylmethyl glucosinolate
brassicin)
Table I
Glucosinolate Compositions of All Mutant Lines
~unoles glucosinolate/g seed
LINES PROG ALLYL NAPOL GLUCO 4-ON GLUCOBN GLUCOBB TOTAL
max. 1.50 - 0.13 1.10 1.05 0.06 0.15 3.41
observed
min. 0.26 - 0.03 0.15 0.67 0.01 0.06 2.01
observed
Table
II
Glucosinolate onQositions Low Progoitrin
C of
Mutants
with
pnolesucosinolate/g
9l seed
LINESPROD ALLYLNAPOLGLUCO4-ON GLUCOBNGLUCO88 TOTAL
3015530.26 tr tr 0.15 1.85 0.01 0.15 2.42
3002110.46 tr 0.06 1.10 0.92 0.02 0.09 2.64
3x15520.47 tr 0.05 0.27 1.38 0.01 0.09 2.27
3613140.65 tr 0.05 0.40 1.37 0.02 0.08 2.57
3614790.65 tr 0.08 0.38 1.43 0.02 0.13 2.69
3x05170.72 tr 0.06 0.46 0.67 0.02 0.08 2.01
3013620.79 tr 0.06 0.45 0.88 0.02 0.09 2.29
3008370.81 tr 0.05 0.38 1.33 0.03 0.10 2.69
3010500.85 tr 0.08 0.48 1.01 0.03 0.07 2.52
3612830.88 tr 0.06 0.41 0.92 0.03 0.11 2.40
30A2080.89 tr 0.08 0.54 1.06 0.03 0.09 2.69
3613870.91 tr 0.10 0.55 0.74 0.05 0.09 2.45
3604820.95 tr 0.09 0.32 1.17 0.04 0.12 ~.~
3089300.96 tr 0.03 0.57 1.20 0.03 0.12 2.91
3092890.97 tr 0.09 0.52 1.34 0.03 0.09 3.05
'
Table III
CA 02544248 1994-02-22
_ 1~ _
Glucosinolate Glucobrassicin
Compositions
of
Selected
Mutants
with
Low
4-OH
~unoles glucosinolate/g
seed
LINES PR ALLI'LNAPOLGLUCO 4-ONON GLUC N GLUCOBB TOT
L
301273 1.32 tr 0.090.72 0.75 0.03 0.09 2.99
300811 1.03 tr 0.090.54 0.79 0.03 0.09 2.58
361269 1.50 tr 0.110.86 0.80 0.04 0.09 3.40
341372 1.36 tr 0.100.74 0.80 0.03 0.11 3.14
361367 1,47 tr 0.130.86 0.81 0.06 0.06 3.41
301350 1.32 tr 0.070.56 ,0.84 0.03 0.07 2.89
Table IV
Sta tisticalThresholds
for
Selecting
Low
Glucosinolates
Mutations
Wnoles glucosinolate/g
seed
LINES PROG ALL'fLNAPOLGLUCO 4-OH GLUCOBN GLUC B TOTAL
Average 2.71 tr 0.221.49 1.3T 0.11 0.40 6.23
Std. DevØ13 tr 0.010.06 0.09 0.01 0.02 0.24
Loner 2.24 tr 0.181.2T 1.05 0.07 0.33 5.37
Threshold
Table V
Control: IRC 129 Glucosinolate
values
1 Wholes
glucosinolate
per
g
seed
o~
per
g
meat
LINES PROG ALLTLNAPOLGLUCO 4-OH GLUCOBN GLUCOBB TOTAL
Hhole 3.9T 0.38 0.351.84 1.66 0.22 0.32 8.7T
Seed
Oil Free 6.43 0.61 0.552.9T 2.69 0.35 0.515 14.14
i Meel
